DETECTION OF SOME FOODBORNE PATHOGENS USING SPECIES - SPESIFIC POLYMERASE CHAIN REACTION TECHNIQUE
A Thesis Submitted to the Council of the Faculty of Agricultural Sciences at the University of Sulaimani in Partial Fulfillment of the Requirements for the Degree of Master in Food Sciences
Biotechnology
By
Sakar Kamal Hamasalih B.Sc. Food science (2009), University of Sulaimani
Supervisor
Dr. Khlood Ibrahim Hassan Assistant Professor
2014 A D.
2714 K.
دؤزيية وة ى هة ىد يَك لة هؤكارة كاىى ىة خؤشى كة دة طويَسريَية وة لة رِيَطاي خؤراكة وة بة بة كارهيَياىي كارليَكى تة كييكي ثة ملة رة ي زجنرية يى بؤ جؤري تايبةت
ىامةيةكة ثيَصكةش كراوة بة ئةجنومةىي فاكةلَيت زاىصتة كصتوكالَيكان لة زاىكؤي شميَناىي وةك بةشيك لة ثيَداويصتيةكاىي بةدةشتويَياىي برِواىةمةي ماشتةري زاىصـتى خوَراك
بايؤ تيَكيؤلؤذى
لةاليةن
شاكار كةمال حة مة صاحل بةكالــؤريـؤس -زاىصتى خؤراك ( ،)2009زاىكؤي شميَناىي
بة شةرثةرشتى
د.خمود ابراهيه حة شة ن ثرِوَفيصؤري ياريدةدةر كاىوىي دووةو 2024ز
بة فراىبار 20024ك .
J
الكشف عن بعض المسببات المرضية المنتقلة عن طريق الغذاء باستعمال تقنية تفاعل البلمرة المتسلسل للنوع المتخصص
رسالة مقدمة الى مجلس فاكلتي العلوم الزراعية -جـامـعـة السـليمانيــة كجـزء من متـطلبات نيـل درجـة الماجستير فـي علوم األغذية
التقانات االحيائية
من قـبــل
ساكاركمال حمة صالح بكالـوريـوس علوم االغذية ( ,) 9002جامعة السليمانية
بإشـــــــراف
د .خلود ابراهيم حسن أستاذ مساعد
كانون الثاني 9024م
ربيع االول 2416ه
SUMMARY For developing a method based on the gene specific PCR, for detection of some food-borne pathogens, specific primers and PCR amplification of the target gene were used to detect five species of bacteria, including: Salmonella Typhimurium, Listeria monocytogenese, E.coli o157:H7, Staphylococcus aureus and Shigella flexneri from enrichment cultures of various types of artificially inoculated and Salmonella spp. and Staphylococcus aureus in naturally contaminated foods. The bacterial strains, used in this study were obtained from Medya Diagnostic Centre in Erbil, they were cultured in tryptic soy broth yeast extract (TSBYE) at 35°C for 6 hours before the extraction DNA of these bacteria were used in PCR reaction based on the specific primer pairs of each bacterial species, they produced specific amplicons of the expected sizes of each of them (284bp in Salmonella spp. ,691 bp in Listeria monocytogenese, 556 bp in E.coli o157:H7, 600 bp in Shigella flexneri and 108 bp in Staphylococcus aureus) their specificity were studied by using the primers that targeting specific pathogen in other four types of bacterial strains and no results were obtained in any one of them. To achieve this study, 13 food samples were collected from Sulaymaniyah market during April-October, 2012 including meats (meat of beef, sheep, goats, fresh chicken, and frozen chicken) and vegetables including (celery, tomato, cucumber, pepper, lettuce, broccoli, carrot, leek ). DNA isolated from each sample before and after enrichment using phenol -chloroform based method, positive results were obtained just after enrichment step.
I
In the naturally contaminated food, A specific PCR assay, targeting the invA gene, and Sa 442
gene combined with two step
enrichment for the detection of Salmonella and Staphylococcuse aureus respectively were developed the results indicate the detection of Salmonella spp., by generating a PCR product of 284 bp in size in three samples out of 13 food samples tested, whereas the detection of Staphylococcus aureus achieved by generating a PCR product of 108 bp in size in four samples out of 10 food samples tested. In the sensitivity experiments, food samples were spiked with reference bacteria at known concentrations and subjected to DNA extraction and PCR analysis. The detection limit of the assay for the bacterial targets was estimated 10 3 -10 4 CFU per ml of each type of food ( chicken meat for Salmonella, lettuce for Staph.aurease, celery for Shigella, beef meat for E.coli and goats meat for L.monocytogenes) . The detection limit was 3
10 CFU/ ml For Salmonella and Staphylocccus aureus and Listeria, and it was 104 CFU/ ml for Shigella and E.coli o157:H7.The overall results of this study indicate that PCR is a good way for the rapid detection of bacteria pathological transmitted through food.
II
ثوختـــــــــــة دؤزينة وة و ثةرةثيَداني ئةو رِيَطايةي كة ثشت دةبةستيَت بةبةكارهيَناني رِيَطاي كارليَكي ثة ملة رة ى زجنريةيي بةندة لةسةر جيين تايبةت ,لةم تويَذينةوةيةدا هةويَين تايبةت بؤ جينيَكي دياريكراو لة جؤرى بةكرتيا بةكارهات لة ضوارضيَوةي كارليَكي ) (PCRبؤدياريكردني ثيَنج جؤر لةبةكرتيا كةئةمانة دةطريَتةوة: o157:H7,
E.coli
monocytogenese,
Listeria
Typhimurium,
(Salmonella
)Staphilococcuse aureus and Shigella Flexner لة ناوةندي ضاندن جؤرةكاني بة كرتيا و تيكةلَكردن بةشيَوةي دةستكردبؤهة نديَك جؤري حؤراك وة دؤزينة وة ي دووجؤر لة بة كرتياكة Staph.aureus, Salmonellaبوون لة جؤرة جياوازةكاني خؤراكي ثيسبوو بةشيَوةية كي سروشيت ,جؤرةكاني بةكرتياي ثاكمان دةستكةوت بؤ ئةم تويَذينةوةية لةتاقيطةي ناوةندي ميديا ى ثزيشكي) لةشاري هةوليَر كةطةشةي ثيَكرا ,لةسةر ناوةنديَكي ضاندني طشيت ( (TSBYEلةثلةي طةرمي 53 ثلةي سةدي بؤ ماوةي 6كاذيَر ثيَش ئةوةي DNAليَ دةربهيَنريَت كة بةكارهيَنراوة لةتاقيكردنةوةي PCR ئةويش بةبةكارهيَناني هةويَين تايبةت بؤ هةر جؤرة بةكرتيايةك كةبةرهةم هيَنراوة لة ثارضة دووهيَندبووةكان بةقةبارةي ضاوةرِوانكراو بؤ هةريةكيَكيان ( 482تفيت جووت بؤ 696 ,Salmonella spp.تفيت جووت بؤ 666 , L.monocytogenesتفيت جووت بؤ
336 ,Shigella flexneriتفيت جووت بؤ E.coli
108 ,o157:H7تفيت جووت بؤ )Staph.aureusثاشان ثيَوانةى تايبةتي بؤ هةرجووتيَك لةهةويَنةكان كرا بةتاقيكردنةوةي ئةو هةويَنانة لةسةر 2جوَر بةكرتياي ديكة كة ئامانج نةبوون ,كة ئةجنامي دووهيَند بوومنان بؤ هةريةك لةوانة دةست نةكةوت . بؤ هيَنانةدي ئاماجنةكاني ئةم تويَذينةوةية 65منوونةمان لةخؤراك لةبازارةكاني سليَماني كؤكردةوة لةماوةي نيَوان مانطي نيسان بؤ تشريين يةكةمي سالَي 4664كةبريتيبوون لة طؤشيت ( مانطا ,مريشكي زيندوو و بةستوو , بزن ,بةرخ ) .سةوزة كةئةمانةي طرتةوة ( كةرةوز ,تةماتة ,خةيار ,بيبةر ,كاهو ,بروَكلي ,طيَزةر و كةوةر ). DNAكةجياكرايةوة بؤهةر منوونةيةك لة منوونةكاني خؤراك ,ثيَش زياد كردني ناوةندي خؤراكي و دواي زيادكردني ئةو ناوةندة بة بة كارهيَناني رِيَطاي فينؤأل_كلؤرؤفؤرم وةئةجنامي ئةريَين (ثؤزةتيف) دةست نةكةوت
لةكاتي جيَبةجَكردني كارليَكي ثة ملة رة دا ,تةنها دواي زيادكردني ناوةندي خؤراكي ,هةويَين تايبةت بةكارهيَنرا كةجيين ( )invAئامانج لةبةكرتياي Salmonellaو جيين ( )Sa 442لة بةكرتياي
Staph.aureus
كةهاوتاية لةطةلَ دوو هةنطاوي بةكارهيَناني ناوةندي خوَراكي طشيت و ناوةندي خؤراكي هةلَبذيَردراو بؤ هةموو بةكرتياكان ,ئةوةش بؤ دياريكردني Salmonellaو Staph.aureusلةمنوونة خؤراكيةكاندا ,وةئةجنامةكان نيشاني دةدةن كةدؤزينةوةى Salmonellaبةدروستكردني ثارضةي دووهيَندبووة لة DNAبةهؤي هةويَنيَك بةقةبارةي 482تفيت جووت لة 5منوونةي خؤراكي لةكؤي 65منوونة ,لةهةمان كاتدا Staph. aureusدياريكرا بةدروستكردني ثارضةي دووهيَندبوو لة DNAبة هؤي هةويَنيَك بةقةبارةي 668تفيت جووت لة 2منوونة لة كؤي 66منوونة . لةتاقيكردنةوةكاني هةستياريَيت دا منوونة خؤراكيةكان ثيسكران بة جؤرةكاني بةكرتيا بةخةسيت دياريكراو دواتر DNAلةو ناوةندانةدا دةرهيَنرا كة لةثاشان بةكارهيَنرا لة جيَبةجيَكردني كارليَكةكاني PCRبؤ زانيين كةمرتين خةسيت كةدةتوانريَت بةهؤيةوة ئةو كارليَكانة ئاشكرا بكريَت ,وة دؤزرايةوة كة خةسيت دياريكراو بؤ هةر جؤريَكي بةكرتيا لة منوونةي خؤراكيدا كةدةتوانريَت ئاشكرا بكريَت بةهؤي PCRلةنيَوان ( ) 662-665كولَوَني بوو بؤ هةر مليلرتيك ,ئة طةر سنوري شلؤق بؤ Salmonellaو Staph . aureusو )665( Listeriaبيَت لةكاتيَكدا 662كولَوَني بوو بؤ هةر مليلرتيك بؤ Shigellaو . E.coli بةشيَوةيةكي طشيت دةرةجنامي ئةم تويَذينةوةية دةريدةخات كة PCRرِيَطايةكي باشة بؤ دياريكردنيَكي خيَرا بؤ ئةو بةكرتيايانةى ئةبيَتة نةخؤشى كة دةطويَزريَتةوة لةرِيَطاي خؤراكةوة.
Dedication This thesis is dedicated to: My father and mother Mydearrest sister (Rozhgar) My lovely husband (Zana) My dearest brothers (Briar and Zaniar)……
Sakar K. Hmasalih
ACKNOWLEDGEMENTS First of all I would like to thank GOD for giving me patient and power to carry out this work. Special gratitude to my supervisor, Assistant Professor Dr. khlood I. Hassan , University of Sulaimani- Food Science Department who introduced me the scientific research and for his guidance during the course of this study and for his patience, support and encouragement. I am also deeply thankful to the presidency of Sulaimani University and the faculty of Agriculture science especially the dean of the faculty, the Assistant Professor Dr. Aram A. Abbas for his help an encouragement to carry out this thesis. I would like to express my deep thanks and appreciation to Dr. Rafiq M.S. Rashid, the head of Food Science Department for his help during the experiment work. Grateful thanks are due to (Dr. Muhamad O.babashekh, Dr. Peshnyar M. Atta and Dr. Zana H. Mahmood) in directory veterinary of sulaimani, Dr.Dunya S. khalaf, Dr.Taghreed A. Wahwah Alnashi, Dr.zaid Kh.Khidir, Dr. Ibrahim M. Noori and Dr.Nawroz A. Razaq. to their great help which they offered during my research work. Also I would like to thank Mr. Ali, Mr. Atta, Mr. Azad, Mr.hiwa, Mr. Karzan, Mr.Sirwan, Mr.Omer, Mr. Muhammed, Mr.Bakir, Mr. Dler, Mrs.Pari Mrs.Soz, Mrs. Paiman, Mrs. Hiran, Mrs.Shadiya, Mrs.Tazhan, Mrs. Shilan and Mrs.Nada. To my family along with all my friends that remained positive and supportive towards me during this challenging period of my life. Finally, I would like to express my deep thanks and appreciation to all who have helped in the achievement of this research.
Sakar K. Hamasalih
LIST OF CONTENTS Series
Title Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Page No. I
List of contents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
III
List of figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
VI
List of tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
VIII
List of Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII Chapter one: Introduction
1
Chapter two: Literature Review
3
2.1
Food borne disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3
2.2
Application of PCR for detection of Salmonella . . . . . . . . . . . . .
4
2.3
Application of PCR for detection of Staphylococcus aureus . . .
6
2.4
Application of PCR for detection of Shigella . . . . . . . . . . . . . . .
9
2.5
Application of PCR for detection of Escherichia coli o 157:H7.
12
2.6
Application of PCR for detection of Listeria monocytogenese.
16
Chapter three: Materials and methods
20
3.1
Materials and chemicals. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
20
3.1.1
Equipments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
20
3.1.2
Chemicals. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
21
3.1.3
Culture Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
22
3.1.3.1
Commercial dehydrate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
22
3.1.3.2
Laboratory prepared cultured media Peptone water. . . . . . . . . . .
23
3.1.3.3
TSBYE BUFFER (tryptic soy broth yeast extract) . . . . . . . . . . .
23
3.1.4
Other reagents and stains. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
23
3.1.4.1
Ethidium bromide stain (10mg/ml) . . . . . . . . . . . . . . . . . . . . . . .
23 III
3.1.4.2
Tris HCl (1 M) pH 8.0) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
23
3.1.4.3
EDTA (0.5 M) pH 8.0 500 ml . . . . . . . . . . . . . . . . . . . . . . . .. . .
24
3.1.4.4
10X Tris-Boric acid-EDTA (10X TBE) buffer . . . . . . . . . . . . . .
24
3.1.4.5
Loading buffer (10X) 1 L . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
24
3.1.4.6
Sodium Chloride (5M) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
24
3.1.4.7
SDS Solution (10%). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
24
3.1.4.8
Tris-EDTA buffer (TE) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
24
3.1.4.9
2 X CTAB/NaCl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
25
3.1.4.10 Chloroform Isoamyl alcohol (24:1) . . . . . . . . . . . . . . . . . . . . . . .
25
3.1.4.11 Phenol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
25
3.1.4.12 Phenol/ Chloroform/ Isoamyle (25:24:1) . . . . . . . . . . . . . . . . . . .
25
3.1.4.13 Washing buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
25
3.1.4.14 Ethanol 70%. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
25
3.2
Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
25
3.2.1
Bacterial strains. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
25
3.2.2
Samples collection and preparation. . . . . . . . . . . . . . . . . . . . . . .
27
3.2.3
DNA Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
27
3.2.4
Gel Electrophoresis Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . .
28
3.2.5
DNA quantification and quality determination. . . . . . . . . . . . . .
29
3.2.6
PCR analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
29
3.2.6.1.
Primer selection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
29
3.2.6.2.
PCR Reaction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
30
3.2.7
Determination of the Specifity of PCR Protocol . . . . . . . . . . . . .
32
3.2.8
Determination of detection sensitivity . . . . . . . . . . . . . . . . . . . . .
33
3.2.9
Method used for detection of Salmonella in food . . . . . . . . . . . .
33
3.2.10
Method used for detection of Staphylococcus aureus in food. . .
34
3.2.11
Method used for detection of Shigella in Food. . . . . . . . . . . . . .
34
3.2.12
Method used for detection of E.coli in Food . . . . . . . . . . . . . . . .
34
3.2.13
Method used for detection of Listeria in Food . . . . . . . . . . . . . .
35 IV
Chapter Four Results and Dissections
36
4.1
Genomic DNA Isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
36
4.2
PCR Reaction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
38
4.2.1
Primer design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
38
4.2.2
PCR analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
40
4.2.2.1
PCR analysis of Salmonella. . . . . . . . . . . . . . . . . . . . . . . . . . . . .
40
4.2.2.1.1 Optimization of PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
40
4.2.2.1.2 Detection of Salmonella in naturally contaminated samples. . . .
41
4.2.2.1.3 Detection of Salmonella in Artificially Spiked Samples. . . . . . .
44
4.2.2.2
PCR analysis of Staphylococcus aureus . . . . . . . . . . . . . . . . . . .
46
4.2.2.2.1 Detection of Staph. aureus in naturally contaminated samples . .
46
4.2.2.2.2 Sensitivity of PCR for detecting Staph.aureus. . . . . . . . . . . . . . .
47
PCR analysis of Shigella . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
49
4.2.2.3.1 Detection of Shigella in Artificially Spiked Samples. . . . . . . . . .
49
4.2.2.3
4.2.2.4
Escherichia coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
52
4.2.2.4.1 Optimization of PCR Reaction . . . . . . . . . . . . . . . . . . . . . . . . . .
52
4.2.2.4.2 Sensitivity of PCR for detection of E. coli O157:H7. . .
53
4.2.2.4.3 The effect of enrichment on PCR detection of E. coli O157:H7
55
4.2.2.5
Sensitivity of PCR for detection of Listeria monocytogenese.
57
Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
60
Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
61
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
62
Summery in Kurdish (from right cover) Summery in Arabic (from right cover)
V
LIST OF FIGURES Figure
Title of figures
No.
Page No.
1- Represent the results of the DNA extraction from ten samples of food, that was performed on (1.2%) agarose gel electrophoresis and run at 90 volt /cm for one hour, the first lane represents DNA ladder 1kb and the Lane 1, 2, 3, 4, 5, 6, 7, 8,9 and 10
37
represent the DNA extracted from beef meat, tomato, celery, chicken meat, frozen chicken meat, lettuce , meat of Sheep, cucumber , leek and green pepper, respectively. 2- Electrophoretic analysis of PCR-amplified target genes from five different bacterial pathogens. Mobilities of the different target gene amplicons are indicated on the figure, the first lane represents DNA ladder 1 kb (size marker) Lanes1, 2, 3, 4 and 5
40
represent L.monocytogenese, Shigella flexneri, E.coli o157:H7, Salmonella.Typhimurium and Staph.aureus respectively. 3- Electrophoretic analysis of 284 bp amplification product from invA gene of Salmonella spp. Isolated from some samples of food M: 1kb marker. Lane 1: represent chicken meat, Lane 4
44
represents tomato, Lane 8 represents frozen chicken. 4- Sensitivity of the PCR for the detection of Salmonella Typhimurium, the first Lane represents DNA ladder 1kb; Lanes 1 through 4: target gene isolated from 10, 102, 103, 104 CFU/
45
ml, respectively.
VI
5- Electrophoretic analysis of 108 bp amplification product from Sa442 gene of Staph.aureus isolated from some samples of food M: 1kb marker. Lane 1: represents tomato, Lane 3 represents
47
frozen chicken, Lane 6 represents lettuce and Lane 8 represents chicken meat. 6- Sensitivity of the PCR for the detection of Staphilococcuse aureus, the first Lane represents DNA ladder 1 kb; Lanes 1 through 4: target gene isolated from 10, 10 2, 103,104 CFU/ ml,
49
respectively. 7- Sensitivity of the PCR for the detection of Shigella flexneri, the first Lane represents DNA ladder 1 kb; Lanes 1 through 4: target gene isolated from 10, 102, 103, 104 CFU/ ml,
52
respectively. 8- Sensitivity of the PCR for the detection of E. coli o157:H7, the first Lane represents DNA ladder 1 kb; Lanes 1 through 4: target gene isolated from 10, 102, 103, 104 CFU/ ml,
54
respectively. 9- Sensitivity of the PCR for the detection of L .monocytogenese, the first Lane represents DNA ladder 1 kb; Lanes 1 through 4: target gene isolated from 10, 102, 103, 104 CFU/ ml,
59
respectively.
VII
LIST OF TABLES Table
Title of Tables
No.
1- The equipments used in this study and their manufacturing companies. . . . . . . . . . . . . . . . . . . . . . . . . . 2- The Chemicals used and their manufacturing companies.… . . . . 3- The media and their manufacturing companies ….. . . . 4- The type of bacterial used in this study and the number of their genus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5- The primers used in this study with their nucleotide sequence and annealing temperatures.………….. . . . . . . .
Page No.
20
21 22 27
32
LIST OF ABBREVIATIONS Abbreviation
Detail
ATP
Adenosine tri phosphate
AOAC
Association for Official Analytical Chemists
bp
Base pair
ADP
Adenosine di phosphate
BPW
Buffer peptone water
CFU
Colony forming unit
CTBA
Cetyl trimethyl ammonium bromide VIII
dATP
Deoxy adinosine triphosphate
dCTP
Deoxy cytidine triphosphate
dGTP
Deoxy guanocine triphosphate
dTTP
Deoxy thimidin triphosphate
DNA
Deoxyribo Nuclic Acid
E.coli
Escherichia coli
RTQ-PCR
Real time Quantitative PCR
EDTA
Ethylene diamine tetra acetic acid
SS agar
Salmonella Shigella agar
VBNC
viable but nonculturable
Kb
Kilo base
M
Molar mass
μg
Micro gram
ml
Milliliter
μl
Micro liter
mΜ
Micro molar
M.wt
Molecular weight
m.PCR
Multiplex Polymerase chain reaction
Ng
Nano gram
PBS
Peptone Buffer Solution
PCR
Polymerase chain reaction
Pg
Pico gram
S.
Salmonella
STEC
E.coli Shigella toxin
TE
Trise – EDTA buffer
pmol
Picomol
TBE
Trise – Boric acid – EDTA buffer
Staph
Staphylococcus
IX
X
CHAPTER ONE INTRODUCTION
Foodborne illness is defined by the World Health Organization as diseases, usually food born intoxication ,food born infection and food born toxicoinfection, caused by agents that enter the body through the ingestion of food. Bacterial contamination of food represents one of the major public health problems, Salmonella spp., Staph.aureus, E. coli O157: H7 and L.monocytogenese are the predominant bacteria species that cause public health problems worldwide (Lei et al., 2008). The detection of pathogens in food is an important component of any integrated program to ensure the safety of foods throughout the food supply chain. Microbiological analysis for each food type was used to monitor the state of contamination at all times and analyze its trends so as to detect emerging risks. These analyses are based on the detection of microorganisms by visual, biochemical, immunological, or genetic means, either before enrichment (quantitative methods) or after enrichment (qualitative methods, also known as presence/absence tests (López et al., 2012). Pathogen detection system has long been used in food production as a means of process and quality control, tracking of contamination sources, and monitoring regulatory compliance. Traditionally the method for detection and identification of bacteria based on specific microbiological and biochemical tests after their isolation in differential and selective growth media, although these methods can be sensitive, inexpensive and give both qualitative and quantitative information on the number and the nature of the microorganisms tested, they are greatly restricted by assay time, especially with the food samples of short shelf life, moreover elaborate DNA extraction methods also need 1
Introduction to be subverted, therefore, extensive research has been carried out over the years to reduce assay time through the use of alternative methods for detecting foodborne microorganisms such as DNA-based assay and there has been an explosion in recent years in the using of nucleic acid–based assays for the detection and identification of foodborne pathogens. Among these, polymerase chain reaction (PCR)has distinct advantages over traditional methods for detecting and identifying bacteria in foods, in term of specificity, sensitivity, rapidity, accuracy and capacity to detect small amounts of target nucleic acid in a sample, beside it based on DNA which is more stable than proteins to high temperatures, high pH, organic solvents, and other chemicals; hence samples can be treated in a relatively harsh manner without destroying the nucleic acid for detection (Germini et al., 2009). Many PCR tests have been validated and commercialized to make PCR a standard tool used by food microbiology laboratories to detect pathogens in foods (Jason et al., 2010 and AOAC International, 2011). Numerous studies have been published on PCR detection of foodborne pathogens including E. coli O157: H7 (Barbora et al., 2011; Jeshveen et al., 2012). Salmonella spp. (Noelle et a l., 2009; Radji et al., 2010). Shigella spp. (Fernandez-Prada et al., 2000; Jiménez et al., 2010). Staphylococcus aureus (Farber et al., 2001; Alarcon et al., 2006). and for Listeria monocytogenese (Almeida and Almeida, 2000; Gouws and Liedemann, 2005). The aim of this study was to optimize a single method based on the gene specific PCRs, for detection some pathogenic bacteria from the some food samples, which some of them were artificially contaminated with the pathogenic bacteria and then tested through PCR method to determine their sensitivity which can help to develop effective prevention and control strategies. 2
CHAPTER TWO LITERATURE REVIEW 2.1. Food borne disease Surveillance of foodborne diseases is of an increasingly high priority in the public health agenda worldwide, bacterial contamination of food represents one of the major public health problems. In the field of food protection, it is extremely important to be able to identify the type of pathogenic and spoilage microorganisms at an early stage (SanathKumar et al., 2002). About 98% of microbes found in food commodities are non-pathogenic e.g. L. monocytogenese is pathogenic but L. innocua is non-pathogenic. Therefore it is required to develop diagnostic tests that can specifically detect the target pathogen (Shabir et al., 2014). The characterization and detection of food borne pathogens continue to rely on conventional culturing techniques. Traditionally, the detection and identification of bacteria mainly rely on specific microbiological and biochemical identification. Although these methods can be sensitive, inexpensive and give both qualitative and quantitative information on the number and the nature of the microorganisms tested, they are greatly restricted by assay time, with initial enrichment needed to detect pathogens, which typically occur in low numbers in food and water (Leonard et al., 2003). Tools used for application of nucleic acid detection like DNA primers and probes are more defined entities than antibodies and antigens, and their composition can be accurately checked by sequence analysis, and produced in DNA synthesizers whenever necessary. The more commonly applied nucleic acid assay technologies in diagnostic include nucleic acid probes and polymerase chain reaction (Kwang, 2006). PCR has distinct advantages over culture and other standard methods for detecting and identifying bacteria in foods, and offers the advantages of specificity, sensitivity, rapidity, accuracy and capacity to detect small amounts 3
Literature Review of target nucleic acid in a sample (Toze, 1999). PCR have been used extensively for several years for identification and characterization of bacteria in food samples including meat and dairy products (Aslam et al., 2003; Ercolini et al., 2004 and Alarcón et al., 2006). Numerous studies have been published on PCR detection of food borne pathogens including pathogenic (E. coli O157: H7, Salmonella, Shigella, V.parahaemolyticus, E.coli, Salmonella Typhimurium, Vibrio spp., Staph. Aureus, Yersinia enteroliticas, Listeria spp., Campylobacter jejuni and Darobacter butzleri) multiple primers can be used to detect different pathogens in one multiplex reaction. Alternatively, diagnostic methods like nucleic acid probes, restriction fragment length polymorphism, or sequence analysis can also be used to confirm the identity of the PCR product and to further characterize the genome (Vogel et al., 2004). The coming years will bring the first practical benefits to the field of microbial food safety, including strategies and tools for the detection, identification and control of pathogenic bacteria species that cause public health problems worldwide.
2.2 Application of PCR for detection of Salmonella Salmonella is a facultative anaerobe, gram negative, flagellated rodshaped bacterium which is about 2-3 x 0.4-0.6 μm in size (Montville and Matthews, 2008). Salmonella is still the leading cause of food borne infections all over the world (Aoust et al., 2013). Transmission of Salmonella to humans is usually by consumption of undercooked meat, milk, eggs and other crosscontaminated foods, such as vegetables that are eaten without cooking. Animal food products are the major responsible mean for the large distribution of Salmonella and all the subsequent problems (Peresi et al., 1999). Meat products predominate among the animal foods considered as source of Salmonella, especially those obtained from poultry (Sumner et al., 2004).
4
Literature Review There are 1.3 billion annual cases of gastroenteritis and 3 million epidemiologic deaths worldwide due to Salmonella (Bhunia, 2008). Typically,
Salmonella
Enteritidis,
Salmonella
Typhimurium
and
Salmonella Heidelberg are the three most frequent serotypes recovered from humans each year (Grayand Fedorka-Cray, 2002; Boyen et al., 2008). Salmonellosis is one of the most common infectious food borne diseases in the world, both in animals and humans. It can cause a wide spectrum of illnesses, ranging from gastroenteritis to severe, life threatening enteric fever. Due to the health risks posed by Salmonella infections, detection must be rapid, sensitive, and accurate to reduce false negative and -positive results. Conventional method used for Salmonella detection is time consuming, requiring 4 days for confirming negative results and 5-7 days for positive results (FDA, 2006). The long time for final results is due to various incubation periods used in this method for pre-enrichment, selective enrichment, colony isolation in selective and differential agar and biochemical and serological confirmation. To overcome this disadvantage, several rapid and sensitive methods for the detection of Salmonella in foods have been developed to decrease the detection time (Noelle et al., 2009). For evaluation of PCR for the detection of Salmonella in food (Luciana et al., 2001). Artificially contaminated chicken meat by used different dilutions of Salmonella Typhimurium or Salmonella enteritidis cells (107, 108 or 109 CFU/ml) inoculated in chicken meat samples, in order to establish the limits of detection. In another study, species specific PCR of a 287-bp region of the invA gene was used and compared to a microbiological technique to determine the presence of Salmonella in retail beef and in cantaloupe rinse samples. Both methods showed the same level of sensitivity, detecting 1 CFU/25 g of meat after enrichment for 24 h at 42 ºC. The presence of Salmonella was also determined in 50 commercial top sirloin beef samples (Gallegos-Robles et al.,
5
Literature Review 2009). The specificity of invA primers based PCR for detection of Salmonella is also confirmed by biochemical and serological assay (Radji et al., 2010). A pre-PCR sample preparation protocol including a pre enrichment step in buffered peptone water followed by DNA extraction-purification was applied when 110 various food samples (chicken rinses, minced meat, fish, and raw milk) were investigated for Salmonella. The diagnostic accuracy was shown to be 100% compared to the traditional culture method. This methodology can contribute to meet the increasing demand of quality assurance laboratories for standard diagnostic methods (Malorny et al., 2004). A combination of immune magnetic separation and polymerase chain reaction (IMS-PCR) was used to detect Salmonella in food samples, using specific antibodies with primer specific for salmonella (Jeníková et al., 2000). Recently real-time PCR was developed and validated for the specific detection of Salmonella in food (Priya et al., 2012). This method will broaden our ability to screen large number of samples in a short time with a sensitivity of 1 CFU/g (Cheng et al., 2008), this method depended on an oligonucleotide probe that becomes fluorescent upon hybridization to the target DNA such as the sequence within the putative type III secretion ATP synthase gene (ssaN) (Jing et al., 2010). 2.3 Application of PCR for detection of Staphylococcus aureus Staphylococcus aureus is a gram-positive facultative anaerobic bacterium, recognized as one of the major bacterial pathogens which cause clinical infection and food-poisoning cases, inhabiting the skin, skin glands and mucous membranes of humans, other mammals and birds. The Staphylococcus genus includes at least forty species, among these; nine have two subspecies and one has three subspecies (Harris and Foster, 2002). Staph. aureus is one of the biochemicaliest active bacteria and produces a family of virulence factors such as adhesion proteins, enterotoxins, superantigens,
pore-forming
hemolysins, 6
ADP-ribosylating
toxins,
and
Literature Review proteases. For food industry and food safety, the main topic of interest is its ability to produce a wide spectrum of thermostable enterotoxins, which cause acute gastroenteritis after food consumption (Bhunia, 2008). It produces a large variety of enterotoxins (A, B, C, D, E, G, H, I, J, K, L, M, N, O, P, Q, R and U), but 95% of poisoning outbreaks are caused by classical enterotoxins: A, B, C, D and E) (Letertre et al., 2003). The amount of staphylococcal enterotoxins required for establishment of typical symptoms of food poisoning is very low, ranging from 20 ng to 1 μg (Normanno et al., 2007). This corresponds to approximately 105 staphylococci colony-forming units per gram of food (FDA, 2006). Foods commonly associated with staphylococcal Staph. aureus which have been isolated from several foods: meat and meat products, chicken, milk and dairy products and fish products (De Neeling et al., 2007; Lee, 2006 and Tamarapu et al., 2001). Cream-filled bakery products, and dairy products (Bennett and Monday, 2003). Among these food products there are some reports about isolation of Staph.aureus from raw chicken meat in various countries (Pesavento et al., 2007; Normanno et al., 2007). Many of these items are contaminated during preparation in homes or food service establishments and subsequently mishandled prior to consumption. In processed foods, contamination may result from human, animal, or environmental sources. In raw food, especially animal products, the presence of Staphylococcus aureus is common and the contamination are during dressed the animal carcasses which Staph. aureus is common and often unavoidable. Raw milk and unpasteurized dairy products may contain large numbers of Staph. aureus, usually a result of staphylococcal mastitis (Bennett and Monday, 2003). Staphylococcal gastroenteritis is caused by the ingestion of food that contains one or more enterotoxins, which are produced only by some stapylococcal species and strains (Cunha et al., 2006).
7
Literature Review Staph. aureus is an important human and animal foodborne pathogen and causes a wide variety of diseases ranging in severity from slight skin infection to more severe diseases such as pneumonia and septicaemia, Staphylococcal foodborne diseases are estimated to cause 6–81 million illnesses and up to 9000 deaths per year, and accounts for 14–20% of outbreaks involving contaminated food in the USA (Mead et al., 1999).They grow in a wide range of temperatures from 7 to 48.5°C with an optimum of 30 to 37°C, pH from 4.2 to 9.3, with an optimum of 7 to 7.5, and sodium chloride concentrations up to 15% NaCl. These characteristics enable Staph.aureus to grow in a wide variety of foods. Traditional methods for detection of Staph. aureus in food are based on the use of selective media for direct enumeration of isolates. This takes from five to six days, other approximations like most-probable-number (MPN) are also laborious and time-consuming (Pascual and Calderon, 2000). Recently, it has been suggested that techniques based on the Polymerase Chain Reaction (PCR) could be used for the discrimination of bacterial species (Farber et al., 2001). In the last 10 years, several PCR detection methods have been proposed for the detection of food-borne pathogens to replace the time-consuming culturebased classical techniques (Alarcon et al., 2006). They are rapid, easy to handle, sensitive and specific and constitute very valuable tools for microbiological applications. Specific primers for PCR detection of Staph.aureus have been directed to the nuc gene encoding thermostable nuclease (Brakstad et al., 1992). Enterotoxin genes (Ma¨ntynen et al., 1997 and Becker et al., 1998), tst gene shock syndrome (Johnson et al., 1991), 16S-23S rDNA spacer region (Saruta et al., 1997). Characterize Staphylococcus aureus isolated from bovine subclinical mastitis in turkey by PCR amplification of clumping factor A (clfA) and protein A (spa) genes, whereas, Rall et al.(2008) used primers targeting the genes encoding the enterotoxins for isolating Staph.aureus strains from raw or pasteurized bovine milk .
8
Literature Review To evaluate the specificity of species specific PCR for Staph.aures, Alarcón et al. (2006) used nuc targeted primers for PCR detection of Staphylococcus aureus in different food matrices and to establish a RTQ-PCR procedure suitable for the routine detection and quantification of this pathogen in food. Multiplex PCR was used to investigate the presence of enterotoxins genes (sea, seb, sec, sed and see) and femA gene (specific for Staphylococcus aureus) in coagulase-positive staphylococci (CPS) isolated from cheese and meat products (Marcia et al., 2009). The primers targeting enterotoxins genes in PCR reaction also used by (Rall et al., 2008). For detection of Staph. aureus, isolates associated with food intoxication more recently, real-time PCR approaches have been developed for quantitative detection of Staph. aureus (Hein et al., 2001). Real time PCR assay was used to provide a rapid and sensitive method for the specific detection of Staphylococcus aureus in raw milk and its product such as cheese, ice-cream (Eman et al., 2011). A real-time immunoquantitative PCR (iqPCR) method for detection of Staphylococcus aureus enterotoxin B (SEB) was developed and evaluated using both pure cultures and foods. The assay consisted of immunocapture of SEB and real-time PCR amplification of the DNA probe linked to the detection antibody. The iqPCR was approximately 1,000 times more sensitive (<10 pg ml−1) than ELISA (Andreja et al., 2006).
2.4. Application of PCR for detection of Shigella Shigella
spp.
is
a
virulent
bacterium
that
belongs
to
the
Enterobacteriaceae family. Bacteria of the genus Shigella is gram-negative, non–spore-forming, no motile, and rod-shaped. Shigella has been identified as cause of food borne diseases and one of the most important agents of diarrhea (WHO, 2005). The infectious dose of Shigella is as low as 10 bacterial cells (Germani and Sansonetti, 2006). Shigellosis is caused by Shigella spp., 9
Literature Review including Shigella dysenteriae, Shigella flexneri, Shigella boydii and Shigella sonnei (Kuo et al., 2008). Although 99% of the cases of shigellosis occur in the developing world, industrialized countries including the United States, among others, have reported outbreaks of forborne shigellosis in the last decade (Germani and Sansonetti, 2006). In the United States, Shigella is reported as the third highest cause of foodborne disease, after Salmonella and Campylobacter (UCDC, 2009). Guidelines reported that annual cases of shigellosis are around 165 million, with more than 1.1 million deaths (Silva et al., 2008). Shigellosis is currently an important health problem around the world, occurring predominantly in children younger than five years old, mainly in developing countries (Savadkoohi and Kacho, 2007). Shigella survives in various foods, under optimal temperature conditions, and without severe acidity, they may be recovered from milk, eggs, cheese. Other associated foods such as salads (potato, tuna, shrimp, macaroni, and chicken, raw vegetables, and poultry and other foods that require a lot of mixing and handling and no further heat treatment . Shigella is generally transmitted by contaminated water, uncooked food, and by contact with infected individuals (Navia and Gascón, 2005 and WHO, 2005). Food borne outbreaks occur due to the clonal propagation of one or few Shigella strains, it invades the local epithelium of the colon (large intestine) in a stepwise format: entry into epithelial cells, intracellular multiplication, intra-and intercellular spreading, and killing of the host cell (Parsot, 2005). Laboratory diagnosis is made by culturing on selective media, and then tested by established biochemical reactions as traditional microbiological assays for detection of Shigella in food, all of which are time consuming, requiring several days to obtain results. On the other hand, Shigella is considered as a fastidious pathogen for bacteriological isolation, which in the context of 01
Literature Review indigenous micro flora and other substances makes detection less feasible (Germani and Sansonetti, 2006). Shigella has been also characterized by other methods, including antibiotic resistance and enzyme immunoassay (EIA) for detection of shiga toxin rapidly (Penatti et al., 2007). Also can serve as a preliminary typing method. However, due to the low discrimination power of the antimicrobial resistance typing, other more accurate methods has been recommended, Since the advent of the biotechnological revolution, many other techniques have been developed to overcome these drawbacks, among these the most widely used method for diagnosis of Shigella spp. is PCR, which consists of the specific amplification of a small portion of the genetic material of an organism in a complex sample, such as food, using thermostable DNA polymerases. PCR has been applied with success because it is rapid to perform, presents relatively low cost and also has been used to type many other pathogens (Penatti et al., 2007 and Oliveira et al., 2007). A fast PCR was designed to detect these pathogens from food by the amplification of a 760 bp region in a Hind III fragment of the 220 kbp invasive plasmid (Lampel et al., 1990). This PCR product was easily detected by agarose gel electrophoresis. This is clearly advantageous in case of water and food samples, where the number of pathogens can be relatively low, and during culturing in broth they could be overgrown by the microflora. The application of PCR using selective and automated in vitro replication of a specified region of a known virulence gene can reveal the presence of a potentially pathogenic microorganism. As the genetic background of the virulence properties of Shigella strains have been discovered, primer targeting the invasiveness genes Shigella spp. have been developed, for example the ipaC and ipaH regions. The specificity of the primers was determined by testing DNA from other bacterial strains, showing no interference with the PCR
00
Literature Review amplification, suggesting that these regions are unique and conserved in Shigella (Bej et al., 1991). Later on other different PCR systems were developed and spread. Most of these use primers specific for the ipaH gene (Fernandez-Prada et al., 2000). Several others, Shigella
specific genes are also suitable for developing
diagnostic methods based on PCR, even in multiplex form for examples Houng and co-workers developed a Shigella serotype specific multiplex system (Houng et al., 1997) who applied PCR which amplified a species-specific target, virA, a gene present in all virulent strains of Shigella. A rapid multiplex PCR (m-PCR) method that allows the simultaneous detection, in a single tube, of six commonly encountered waterborne pathogens is developed. One of them was the target genes used were the invasion plasmid antigen H (ipaH) gene of Shigella flexneri, as the gene targets (Kong et al., 2002). To get a real number of the disease burden caused by Shigella, Real timePCR was used to detect Shigella-associate DNA. The target of the primer pair is the invasion plasmid antigen H gene sequence (ipaH) carried by all four Shigella species (Von Seidlein et al., 2007).
2.5 Application of PCR for detection of Escherichia coli o 157:H7 Escherichia coli is a facultative anaerobic bacterium commonly found in the mammalian intestinal tract and
live as a fecal-oral lifestyle and can
comprise up to 1% of the gastrointestinal population of mammals and is used as an indicator of environmental fecal contamination of water supplies (Winfield and Groisman, 2003). Some E. coli strains can cause hemorrhagic colitis in humans, but the best known enterohemorrhagic E. coli (EHEC) strain remains O157:H7 each year, more than 60 people die and 73,000 people are getting ill by this kind of bacteria (Scotland et al., 1990). 01
Literature Review Escherichia coli O157:H7 was first recognized as a pathogen in 1982 during an outbreak investigation of hemorrhagic colitis, it was associated with a multi-state outbreak of hemorrhagic colitis caused by the consumption of undercooked ground beef products (Riley et al., 1983). Since then, cattle have been identified as a principal reservoir of E. coli O157:H7 (Meng et al., 2001). As much as 28% of cattle fecal samples were positive for E. coli O157:H7 (Elder et al., 2000). Studies have shown than up to 30% of all cattle are asymptomatic carriers of E. coli O157:H7 (Callaway et al., 2006 and Reinstein et al., 2007). Manure from cattle production facilities can contain viable E. coli O157:H7 and be washed into the water supply and consumed directly in drinking water, or be used as irrigation water on crops, or transmitted by other animal vectors (Hill et al., 2006; Thurston-Enriquez et al., 2005). Therefore, methods that reduce E. coli O157:H7 populations in food animals prior to entry to the food chain have great potential to reduce human illnesses (Sargeant et al., 2007). Still, it was not until 1993, after a large multistate E. coli O157 outbreak linked to undercooked ground beef patties sold from a fast-food restaurant chain (Bell
et al., 1994). That E. coli O157 became broadly recognized as an
important and threatening pathogen. Clinical laboratories began examining more stool specimens for E. coli O157 (Boyce et al., 1995). Recently, the United States government ordered the second largest recall of beef in the U.S. history (approx. 19 million pounds of beef were recalled from 21 states) after at least 16 people infected from eating E. coli O157:H7 contaminated beef (Recall Notification Report, 2004). In addition, a variety of other foods, such as apple cider, milk, cheese, yogurt, lettuce, water and sprouts have been involved in E. coli O157:H7 outbreaks (Hilborn et al ., 2000). Escherichia coli O157:H7 is a robust pathogen that can readily adapt to and survive a wide range of environmental conditions, including shifts in 03
Literature Review temperature, low pH and desiccation (Iijima et al., 1998). Only a few cells (less than 10) are necessary to cause human illness (Warner and Oliver, 1998). A number of studies have demonstrated the ability of E. coli O157:H7 to survive for several months in food, soil, water, manure and artificial microcosms (Maule, 2000). Long-term maintenance of E. coli O157:H7 under starvation conditions (distilled water at 25°C for 12 weeks) can result in entry into a VBNC state (Wang and Doyle, 1998). Conventional enrichment and isolation methods with selective and/or indicator media, such as E. coli (EC) broth, lauryl sulfate tryptose 4methylumbelliferyβ-D-glucuronic acid broth, eosin methylene blue agar, and MacConkey sorbitol agar, are currently used to detect E. coli O157:H7 in ground beef. However, such tests often require several days to get results, during which time meat may be shipped from production plants to retail stores. These tests also lack the ability to identify E. coli O157:H7 strains definitively, thus development of detection methods requiring less time is important in preventing distribution of contaminated beef from producer to consumer (Vanderzant and Spittstoesser, 1992). E. coli 0157:H7 outbreaks are on the rise; hence it is important to develope a sensitive, rapid, and species-specific method to identify this pathogen in water and food. Commercial kits are available in the market for detection but are still deemed time consuming as they require long enrichments prior to detect microorganisms. Thus, a sensitive and rapid technique for detection of this pathogen is required (Johnson et al., 1995). Recently, traditional microbiological culturing techniques are being replaced by PCR based techniques for the identification and detection of E. coli 0157:H7 as it is less laborious and saves significant amount of time (Johnson et al., 1995). PCR requires a small amount of DNA unlike the large numbers required for genetic based molecular diagnostic methods (Feng, 1993).
04
Literature Review According to Shah et al. (2009), PCR assays are proven specific and sensitive in detecting microbial pathogens such as E. coli 0157:H7. PCR which targets genes from known virulent regions of E. coli O157:H7 is able to reach the detection sensitivity of 4 colony-forming units (CFU) from a culture 6 and 8 CFU from milk and meat samples, respectively (Barbora et al., 2011). To establish a protocol for the detection of the pathogen E. coli O157:H7 E. coli virulence genes (eaeA, rfbE, hly, stx1, and stx2) in a multiplex PCR protocol using five specific primer pairs were used. The target genes produced species-specific amplicons indicating that the established PCR protocol is suitable for a rapid and specific analysis of the pathogenic E. coli O157:H7 in environmental water samples (Jeshveen et al., 2012). A multiplex PCR based on fliC and rfbE genes could sensitively and specifically detect E. coli O157:H7 in artificially inoculated raw pork meat and ready-to-eat (RTE) meat products using two different DNA extraction procedures for application on meat products. The optimized PCR included an enrichment step in brilliant green bile 2% broth at 37 °C (Gordillo et al., 2011). To elaborate fast and sensitive method of detection of E. coli O157:H7 in food samples, Lekowska et al.( 2002) used raw ground meat obtained from retail and artificially inoculated with low numbers of E. coli O157:H7. 18 h enrichment culture allowed pathogenic bacteria to multiply to the levels detectable in multiplex PCR targeting (attaching and effacing) gene, stx shiga toxin genes. The sensitivity of E. coli O157:H7 detection method was shown to be 1 CFU per 25 g of food sample, the same rate of sensitivity was obtained by (Yanming and Zhang , 2008) using reverse transcription-PCR for detection of E. coli O157:H7 targeting the rfbE and fliC genes in diluted cultures increase to 4 CFU/liter in tap water, 7 CFU/liter in river water. Real-time PCR used for the detection of Escherichia coli O157:H7 in cattle and dairy waste water samples produced from mozzarella cheese factories (Spano et al., 2005). 05
Literature Review Pina et al. (2010), used Real-time multiplex PCR assays to detect E. coli O157:H7 in Apple cider, raw milk and ground beef and lettuce after inoculated with these bacteria and subjected to enrichment in Rapid check E. coli O157:H7 broth at 42°C.Miszczycha et al.( 2012) found that real-time PCR protocol is a useful tool for rapid, specific, and sensitive detection of E. coli O157:H7 in raw milk and raw ground meat products, and in lettuce leaves inoculated with E. coli O157:H7 (Lee and Levin,2011). E. coli O157:H7 was identified using multiplex PCR With the enrichment procedure from artificial inoculated pasteurized milk (Wei and Yan, 2004). Real-time multiplex PCR assays targeting the stx 1, stx 2, wzy O157 and fliC h7 gene (Pina et al., 2010). 2.6 Application of PCR for detection of Listeria monocytogenese Listeria monocytogenese is an opportunistic intracellular pathogen that has become an important cause of human foodborne infections worldwide (Liu, 2006). Foodborne listeriosis, caused by the pathogen Listeria monocytogenese, is a relatively rare but serious disease with high fatality rates (20–30%) compared with other foodborne microbial pathogens, such as Salmonella While L. monocytogenese causes a relatively mild gastroenteritis in healthy adults, the illness can be severe in susceptible individuals. Basically, L. monocytogenese most often affects those with a severe underlying disease or condition (such as cirrhosis that impair the immune system), pregnant women, unborn or newly delivered infants and the elderly. Symptoms range from flu-like illness to severe complications including meningitis, septicaemia, spontaneous abortion or listeriosis of the newborn (FAO/WHO, 2002). Due to its physiological characteristics, such as resistance to acidic and sodium chloride stress, ability to grow at low temperature and possibility to form biofims (Sleator et al., 2003 and Liu et al., 2005a). The Listeria species can persist and/or re-contaminate food products, thereby representing an important risk for the safety of the consumers. With globalization and increased 06
Literature Review consumption of manufactured ready-to-eat foods throughout the world, it is hardly surprising that L. monocytogenese has become recognized as an important opportunistic human foodborne pathogen. The lack of decrease in the occurrence of listeriosis, reported in the community summary report on foodborne outbreaks in the European Union in 2007, warns for the need of special attention to this foodborne pathogen in order to combat its presence in foodstuffs. Data indicate that the infective dose of L.monocytogenese in contaminated food is usually more than 100 CFU/g
(Swaminathan, 2001). But the food
vehicle responsible for the listeriosis outbreak in the United States in 1998, however, contained less than 0.3 CFU/g of the bacterium. The genus Listeria includes 6 different species (L. monocytogenese, L. ivanovii, L. innocua, L. welshimeri, L. seegligeri and L. grayi). Both L. ivanovii and L.monocytogenese are pathogenic in mice, but only L. monocytogenese is consistently associated with human illness (Seafood Network Information Center, 2007). Listeria monocytogenese has again become a major public health concern. After a series of high profile outbreaks in the 1980, some steps were taken to reduce contamination of food, and it appeared that this pathogen was under control. But a spate of recent outbreaks and recalls has demonstrated that this bacterium still poses a significant health risk. This is partly due to L. monocytogenese widespread occurrence in nature and to its ability to grow at refrigeration temperatures. The virulent nature of listeriosis, with a fatality rate of 20% or more, has particularly attracted the attention of the public and food safety professionals. Approximately 76,000,000 cases of foodborne illness are estimated to occur in the U.S.A. each year, and of these about 2,500 (<1%) are caused by L. monocytogenese. Nevertheless, Listeria
are responsible for
approximately 27.6% of the total deaths attributed to foodborne illness (Mead et al., 1999).
07
Literature Review Listeria monocytogenese is present in several foods, including corn, chocolate, milk, shrimp, and rice salad, have been reported as vehicles ( Schlech WF 3rd, 2000). Inclusion of L. monocytogenese in the list of organisms subject to HACCP has recently driven the search for detection methods suitable for online monitoring. Conventional methods for detection and identification of
L.
monocytogenese are laborious, time-consuming and are not very sensitive (Klein and Juneja, 1997). Since Kary Mullis in 1983, said that polymerase chain reaction (PCR) technology has proved to be an invaluable method for detection of pathogens in foods. PCR become one of the most promising techniques for rapid detection of microorganisms in food. This process has provided increased sensitivity for detection and therefore enhanced the likelihood of detecting bacterial pathogens (Lampel et al., 2000). PCR was used for it is ability to accurately detect and confirm the presence of L. monocytogenese in food products (Gouws and Liedemann, 2005). Other studies have conducted that depending on PCR for detection of L. monocytogenese,
targeting
specific
virulent
DNA
sequences
in
L.
monocytogenese, which do not cross-react with other bacterial genus (Fitter et al., 1992) such as the study of (Almeida and Almeida , 2000),who detected L. monocytogenese
based on PCR amplification of the iap, prfA and hly gene
sequences. Although the conventional PCR techniques could detect both viable and non-viable cells, the inclusion of an enrichment step or separation by immunomagnetic particles can solve this disadvantage (Cudjoe et al., 1993). The PCR-based method proved to be a reliable means of detecting the pathogen in food samples independently from the extraction procedure used, even for a contamination cell number of 1 CFU/g before culture enrichment. The molecular assay, showing perfect agreement with standard microbiological tests and a considerably shortened analysis time, provides a sensitive and rapid 08
Literature Review alternative for applications in the testing of foods for microbiological contamination, and high lights the potential of PCR technology in routine food control (Amagliani et al., 2007). More recently, other kinds of PCR were applied for the same purpose such as the study of a (Kalliopi et al., 2008), who described the development of a quantitative PCR (qPCR) technique to detect, quantify and determine the vitality of Listeria monocytogenese in foods. The method was based on the amplification of the intergenic spacer region (IGS) between the 16S and 23S rRNA genes.A panel of more than 100 strains of Listeria spp. and non-Listeria was used in order to verify the specificity of the primers and Taqman probe and amplification signals were obtained only when L. monocytogenese DNA and RNA were loaded in the qPCR mix. Standard curves were constructed in several food matrices (milk, meat, soft cheese, fermented sausage, cured ham and readyto-eat salad). In a study on the degree of contamination in the surroundings and contacting eggs until reaching the markets and consequently the consumers, it was concluded that egg shell was more subjected to contamination with L. monocytogenese than egg content. It can be concluded that it was uncomfortable result to find L. monocytogenese by this degree of contamination in table eggs and how extent the zoonotic view is meaningful (Mohammed et al., 2009).
09
CHAPTER THREE MATERIALS AND METHODS 3.1 Materials and chemicals 3.1.1 Equipments The equipments used in this study and their manufacturing companies are listed in table 1 below: Table 1: The equipments used in this study and their manufacturing companies.
No.
Equipment
Company
Origin
1
Autoclave
Daikyo
Japan
2
Centrifuge
Sigma
Germany
3
Vortex
Eppendorf
Germany
4
Gel document system
UVI TEC
UK
Ammersham
USA
5
Gel Electrophoresis apparatus and Power supply
6
Hotplate magnetic stirrer
Stuart
UK
7
Ice-crusher
Warring commercial
USA
8
Ice-maker
Scotman
USA
9
Incubator
Prodit
Italy
10
Laminar flow cabinet
Kojair
Finland
11
Microcentrifuge
Hettich
Germany
12
Micropipettes
Eppendorff
Germany
13
Micro shaker
Cam lab
UK
14
Oven
Memmert
Germany
15
pH-meter
Hanna
Portugal
20
Materials and methods
16
Refrigerator
Hitachi
Germany
17
Sensitive balance
Voyager
Switzerland
18
Shaker water bath
Clifton
England
19
UV. Spectrophotometer
Heiou Lnican
England
20
Water bath
Grant
England
21
Water distillate
Aquateron
France
22
Eppender tube
Eppendorff
Germany
3.1.2. Chemicals The chemicals used and their manufacturing companies are listed in table 2 below : Table 2: The Chemicals used and their manufacturing companies.
No.
Chemicals
Company
Origin
Ammersham
USA
1
Agarose
2
Boric acid
BDH
England
3
Bromophenol blue
BDH
England
4
Chloroform
α-Alpha
India
Sigma
Germany
5
Ethylene diamine tetra acetic acid (EDTA)
6
PCR Master mix
Promega
Germany
7
Ethanol 99.9%
α-Alpha
India
8
Ethidium bromide
Ammersham
USA
9
Ethanol 70%
α-Alpha
India
10
Ammonium acetate
Scharlu
European Union
11
Cetyl trimethyl ammonium bromide
Sigma
Germany
12
Phenol
Scharlu
European Union
21
Materials and methods
13
Iso amyl alcohol
α –Alpha
India
14
Isopropanol
α –Alpha
India
15
DNA-Ladder
Sigma
Germany
16
Sodium Chloride
α-Alpha
India
17
Sodium dodecyl sulfate (SDS)
Sigma
Germany
18
Tris- Base
BHD
England
19
Lysozyme
Ammersham
USA
20
Protenase K
Ammersham
USA
3.1.3 Culture media 3.1.3.1 Commercial dehydrate All media were prepared as recommended by the manufacturing company and sterilized by autoclave at 121ºC for 15 min, cooled to 50ºC before poured on the plates. The commercially dehydrated media and their manufacturing companies are listed in Table 3 below : Table 3: The media and their manufacturing companies.
No. 1 2 3 4
Origin Germany UK Germany Germany
5
Name of Media Company Tetrathionate broth Merck Brain heart infusion Lab MacCkonky agar Himedia TSB buffer and Yeast Merck extract Blood base agar Lab
6
Manitol salt agar
Lab
UK
7
Oxford
Merck
Germany
8
Salmonella Shigella Agar
Merck
Germany
9
TSA agar
Merck
Germany
22
UK
Materials and methods
3.1.3.2 Laboratory prepared cultured media Peptone water
Peptone
10g
Sodium chloride
5g
Distilled water
1 litter
Suspend of the dehydrated material in a liter of distilled water, mix thoroughly and heat with frequent agitation, boiling for one minute, sterilized by autoclaving at 121ºC for 15 minutes. 3.1.3.3 TSBYE medium (Tryptic soy broth yeast extract) It was prepared by mixing 30 g of tryptic soy broth powder, 6 g of yeast extract and 1 liter of distilled water, boiling for one minute, sterilized by autoclaving at 121ºC for 15 minutes.
3.1.4 Other reagents and stains 3.1.4.1 Ethidium bromide stain (10mg/ml) It was prepared by dissolving 0.1 g of ethidium bromide powder in 1 ml distilled water. The volume was made up to 10 ml by distilled water. Stir on a magnetic stirrer for several hours to ensure that the dye has dissolved. Wrap the container in aluminum foil or transfer the 10 mg/ml solution to a dark bottle and store at room temperature, preparing this stain extra care was taken as ethidium bromide is a powerful mutagen (Maniatis et al., 2001). 3.1.4.2 Tris HCl (1 M) pH 8.0 It was prepared by dissolving 108 g of Tris-base in 400 ml distilled water; pH was adjusted to 8.0 by adding few drops of 10N HCl. The volume was completed to 500 ml with distilled water and then autoclaved at 121ºC for 15 minutes
23
Materials and methods
3.1.4.3 EDTA (0.5 M) pH 8.0 (500 ml) It was prepared by dissolving 93.5 g of EDTA in 400 ml distilled water; pH was adjusted to 8.0 by adding few drops of 10N NaOH. The volume was made up to 500 ml by distilled water, and then autoclaved at 121ºC for 15 minutes 3.1.4.4 10X Tris-Boric acid-EDTA (10X TBE) buffer (1L) It was prepared by adding 108 g of Tris-base, 55 g of boric acid and 40 ml EDTA (0.5 M pH 8.0) then dissolved in 800 ml distilled water, pH was adjusted to 7.8 by adding few drops 10N HCl. The volume was made up to 1000 ml by distilled water. 3.1.4.5 Loading buffer (6X) It was prepared by dissolving 0.25 g of bromophenol blue and 30 ml of glycerol, in 50 ml of distilled water; pH was adjusted to 8.0 by adding few drops of 10N NaOH. The volume was made up to 100 ml by distilled water and kept at 4oC. 3.1.4.6 Sodium chloride (5M) It was prepared by dissolving 146.1 g of sodium chloride in 400 ml distilled water, the volume completed to 500 ml by distilled water. 3.1.4.7 SDS solution (10%) This was prepared by dissolving 10 g of SDS in 100 ml distilled water with heating at 55oC. 3.1.4.8 Tris-EDTA buffer (TE) This buffer consisted of 10 ml Tris-HCl (1 M) and 2 ml of EDTA (0.5 M), the pH was adjusted to 8.0. The volume was made up to 1000 ml with distilled water, then autoclaved at 121ºC for 15 minutes
24
Materials and methods
3.1.4.9 2 X CTAB/NaCl It was prepared by adding 10 g of CTAB to 14 ml of heated NaCl (5M) the volume was made up to 100 ml by distilled water. 3.1.4.10 Chloroform Isoamyl alcohol (24:1) It consists of 96 ml of chloroform and 4 ml of isoamylalcohol and kept at 4 ºC. 3.1.4.11 Phenol The crystal of phenol was milted at 68 ºC in water bath and then added the equal volume of Tris 1M that pH (8.0) and was allowed to stand overnight at 4 ºC. 3.1.4.12 Phenol/ Chloroform/ Isoamyle (25:24:1) 50 ml of phenol was mixed with 48ml of chloroform and 2ml of isoamyl alcohol to obtain 100ml of solution and stored at dark bottle until using . 3.1.4.13 Washing buffer 76 ml of absolute ethanol was added to 133 microliter of ammonium acetate and then completed to 100ml by distilled water. 3.1.4.14 Ethanol 70% It was prepared by mixing of 70 ml of absolute ethanol 99.9% concentration and 30 ml distilled water, according to (Sambrook et al., 1989). 3.1.4.15 Ammonium Acitate (5M) It was prepared by adding 38.5g of Ammonium acitate to 80 ml of distlled water and the volum was completed to 100ml
3.2 Methods 3.2.1 Bacterial strains The type of Bacterial used in this study and the number of their strains are listed in table 4, they were obtained from the Medya Diagnostic Centre (MDC) in Erbil, Kurdistan region-Iraq. Aerobic or facultative anaerobic bacteria 25
Materials and methods
including E. coli, L. monocytogenese ,Salmonella Typhimurium, Shigella flexneri and Staphylococcus aureus were grown for 24 h in 10 ml of tryptic soya broth yeast extract and incubated in water bath at 37 ºC (Wang et al., 1997). The levels of each species in broth were in the range of 108 to 10
9
CFU/ml. After incubation for 24 h, samples were centrifuged again at 4000 rpm for 15 minutes and resultant pellets were used to extract DNA. They were used as positive control in PCR reactions. For determining the number of viable cells, serial decimal dilutions of cultures in phosphate-buffered saline (PBS) were plated onto trypticase soy agar (TSA) for each bacterium. The plates were then incubated at 37 ºC for 48 hours before enumeration. For the study of the sensitivity of PCR,the cultures were then serially diluted (10-1 –10-10) in sterile peptone water and enumerated in Tryptic Soy Agar (TSA) plates. Bacterial concentration was estimated by calculating the average number of colonies on plates containing 30 to 300 colonies. Dilutions of each bacteria which containted 107CFU/ml were prepared and 1ml of each dilution was used to inoculate 25g of food sample. Then each inoculated sample was placed in 225 ml of TSBYE medium and homogenized using a stomacher at 90s. Those cultures were incubated for 6h at 36 ºC. After incubation, bacterial DNA was extracted from each culture following the method developed by (Fontana et al., 2005) and modified by (Estrada et al., 2007).
26
Materials and methods
Table 4: Represent the types of bacterial used in this study and the number of their genus.
Genus Escherichia Listeria Salmonella Shigella Staphylococcuse
Species coli O157 H7 monocytogenese Typhimurium flexneri aureus
Source Lab strain( MDC) Lab strain( MDC) Lab strain( MDC) Lab strain ( MDC) Lab strain( MDC)
Number 157 11994 14028 12022 25923
3.2.2 Samples collection and preparation Thirteen teen food samples including chicken meat, meat and vegetable were purchased, randomly from local food stores in Sulaymaniah market.25 g of each sample was aseptically removed and placed in a sterilized plastic bag and transported to the laboratory, food samples were homogenized following the standard methods
(Farber et al., 2001). Then 225 ml of TSBYE broth was
added to 25 g of food , and incubated for 6 h at 35°C. After incubation ,various cultures or food sample were centrifuged at 4000 rpm for 15 min. The pellet was then resuspended in 2ml of TE buffer (pH 7.8) and vortexed, this was used to extract DNA.
3.2.3DNA extraction 10 ml from each samples including (chicken meat, meat and vegetable) and also 10 ml of the broth media of each bacteria were used for DNA extraction according to (Sambrook and Russel ,2001) as follows: 1. The pellets of 24 hours broth media were re-suspended in 2 ml of buffer a (500 mmol Tris-HCl [pH 8.0], 100 mmol NaCl, 1 mmol sodium citrate, 5 mmol EDTA) or simply on TE buffer.
27
Materials and methods
2. Five
mg/ ml lysozyme added and incubated for 1 h at 37°C with
occasional agitation. 3. Three cycles of freezing and thawing in a 65°C water bath were included to facilitate cell wall destruction, and thus, release of nucleic acids. 4. Proteinase K was added to a final concentration of 2 mg /ml, and the mixture incubated for an additional 30 min with occasional agitation. 5. Thirty microlitre SDS, 10% were added and the solution mixed gently by inversion, incubated for 30 minutes at 37 ºC. 6. An equal volume of phenol chloroform isoamyl used then centrifuged for 5 minutes. 7. Nucleic acids were precipitated via addition of ammonium acetate and an equal volume of isopropanol alcohol, centrifuged at maximum speed in a micro centrifuge for 20 min. Pellets were washed with 70% ethanol and air-dried at 37ºC for 5 min, after which they were dissolved in 50 μl distilled water and stored at 30 º until further use. 3.2.4 Gel Electrophoresis Analysis DNA samples were monitored by using horizontal agarose gel electrophoresis, prepared as described by (Sambrook et al., 1989), indicated as the following: Agarose gel was prepared by suspending dry agarose 1.2g in 100 ml 1X TBE buffer, and then heat it until agarose melted into a clear solution. The melted agarose was allowed to cool to 60 ºC and then Ethidium bromide (5 μl /100 ml agarose gels) was added to the melted agarose. The solution was then poured into a tray about 5-7 mm thick, then the comb was inserted, the agarose gel was allowed to solidify. 28
Materials and methods
Agarose gel with tray was placed into a container, filled with 1X TBE buffer sufficient to cover entire gel.Carefully the comb was removed and then each well was loaded by mixing 2 μl of 6X loading dye (bromo phenol blue) with 5μl of DNA. The electrical power was turned on at 90 volts for 60 min, agarose gel was tray placed inside gel documentation system for visualization of DNA bands ultra violet light.
3.2.5 DNA quantification and quality determination: To determine DNA concentration and aliquot of 10 μl of DNA, sample was added to 990 μl of TE buffer mixed, then optical density (O.D) was measured by using UV spectrophotometer at wavelengths of 260 and 280 nm. The DNA concentration in the solutions was calculated according to the following formula: DNA concentration (μg/μl) = (OD260 × 100 × 50 μg/μl) / 1000 Theoretically O.D = 1 at 260 nm corresponds to approximately (50 μg/μl) for double stranded DNA. The ratio between the readings at 260 nm/280 nm provides estimate of the purity of nuclic acid (Maniatis and Russel, 2001).
3.2.6 PCR analysis 3.2.6.1 Primer selection The primers were provided by Bionerr Company (Kores) in lyophilized form, to prepare working solution, they were dissolved in sterilized deionized distilled water to obtain 10 pmol/ μl as final concentration the primer were used in this study listed in table 5 with their nucleotides sequence and annealing temperature of each primer .
29
Materials and methods
3.2.6.2 PCR reaction The reagents required for PCR reaction mixed about 20-μl reaction
mixture contained
1U of
Taq
DNApolymerase,10 mM of Tris-HCl
(pH9.0),30 mM of KCl, 1.5 mM MgCl2,each dNTP (dATP, dCTP, dGTP, dTTP ) and template DNA (25-50ng ), primer (10 pmol) .
The amplification program was run as follow: Using thirty five cycles for E.coli One cycle for 94 ºC for 2 min (Initial dinaturation) Step 1: 94 ºC for 30 sec (Dinaturation) Step 2: 55ºC for 15 sec (Annealing) Step 3: 72 ºC for 2 min (Extension) One cycle of 72 ºC for 5 min (Final extension) Then the products were running by 1.2% agarose gel electrophoresis and stained by Ehidium bromide for detection of the amplified fragments. Listeria monocytogenese primer: using thirty five cycles One cycle for 94 ºC for 2 min (Initial denaturation) Step 1: 94 ºC for 1 min (Denaturation) Step 2: 54 ºC for 1 min (Annealing) Step 3: 72 ºC for 2 min (Extension) One cycle of 72 ºC for 5 min (Final extension) Salmonella typhimurium primer : using thirty cycles One cycle for 94 ºC for 2 min (Initial denaturation) Step 1: 95 ºC for 5 min (Denaturation) Step 2: 65 ºC for 1 min (Annealing) Step 3: 72 ºC for 2 min (Extension) One cycle of 72 ºC for 5 min (Final extension) Shigella flexneri primer : using thirty cycles 30
Materials and methods
One cycle for 94 ºC for 1 min (Initial denaturation) Step 1: 94ºC for 1 min (Denaturation) Step 2: 55 ºC for 1 min (Annealing) Step 3: 72 ºC for 2 min (Extension temperature) One cycle of 72 ºC for 5 min (Final extension) Staphylococcus aureus primer : using forty cycles One cycle for 96 ºC for 3 min (Initial denaturation) Step 1:95ºC for 1 min (Denaturation) Step 2: 55 ºC for 1 min (Annealing) Step 3:72 ºC for 2 min (Extension) One cycle of 72 ºC for 5 min (Final extension) This study was monitored by the use of reaction controls. The Quality Control Positive (QCP) contained a DNA sample of each pathogen which gives a positive result across all primer sets. All target pathogens were used for the QCP. Contamination was monitored through a Quality Control Blank (QCB), which contained sterile distilled water in place of template DNA in the PCR reaction mix (Malorny et al., 2003). The reaction products were then analyzed by electrophoresis in 1.2% agarose gels stained with ethidium bromide, visualized under UV light, and recorded by using a gel documentation system.
31
Materials and methods
Table 5: The primers used in this study with their nucleotide sequence and annealing temperatures.
NO
Types of Bacteria
Primer sequence 5 3
Annealing temperature Co
M.wt pb
F/ CGA GGG CTT GAT GTC 1
E.coli O157:H7
TAT CAG
R/ TCA GTA TAA CGG CCA
55
556
54
691
65
284
55
600
55
108
Ref.
( Lampel et al., 2000).
CAG TCC
F/AGGGCTTCAAGGACTTA 2
L. monocytogenes
CCC
R/ACGATTTCTGCTTGCCA
(Doumith et al., 2004a)
TTC
F/GTGAAATTATCGCCACG 3
Salmonella spp.
TTCGGGCAA
R/TCATCGCACCGTCAAAG
(Rahn et al., 1992)
GAACC
F/GCCGGTCAGCCACCCT 4
Shigella flexneri
CTGAGACTAC
R/GTTCCTTGACCGCCTTT
(Lampel et al.,2002)
CCGTACCGTC
F/AATCTTTGTCGGTACAC 5
Staph.aurese
GATATTCACG
R/CGTAATGAGATTTCAGT
(Samadi et al., 2007)
AGATAATACAACA
3.2.7Determination of the specificity of PCR protocol In order to evaluate and verify the specificity of the primers in this study, each primer pair was tested by PCR on DNA templates prepared from a panel of five different bacterial isolates including Salmonella Typhimurium 12022,
32
Materials and methods
Shigella Flexneri 14028, Listeria monocytogenese 25923, E.coli O 157:H7 and Staphylococcus aureus 11994). 3.2.8 Determination of detection sensitivity To determine sensitivity of the PCR method, known bacterial counts (1010 4 CFU/ml) of each pathogen were added to 1 ml of food sample homogenates immediately prior to DNA extraction .To do that, all strains were grown for 24 h in 10 ml of universal culture medium called tryptone soya broth yeast extract (TSBYE ) at 37 ºC. Various concentrations (10 -10 4 CFU/ml) of each pathogen which were serially 10-fold diluted in sterile saline, was added as 1ml to 9 ml of homogenate food samples(Wang et al ., 1997). And the bacterial mixtures from each dilution were harvested by centrifugation at 4000 rpm for 20 min and analyzed by preparing DNA as described previously. Detection probability was calculated as positive PCR results corresponding to particular concentrations of bacterial suspensions determined as colony-forming unit per milliliter by the plate count method. 3.2.9 Method used for detection of Salmonella in food A modified method based on (Cui et al.,2004) was used to isolate Salmonella From meat samples (meat of beef, sheep, goats, fresh chicken and frozen chicken) and vegetables samples (celery, tomato, cucumber,pepper, lettuce, broccoli, carrot and leek). Twenty-five gram of each samples were weighed and transferred into sterile Stomacher bags, each one containing 225 ml of buffered peptone water (BPW) in 37 ºC water bath with shaking at 100 rpm/min for 6 h, then 1 ml pre-enriched rinse was transferred to 10 ml tetrathionate (TT) broth and incubated at 42ºC with shaking at 100 rpm/min for 24 h. After incubation, the TT broth was used to extract DNA of Salmonella as mentioned above 33
Materials and methods
3.2.10 Method used for detection of Staphylococcus aureus in food Based on (Alarco´ n et al .,2006)
Staph.aureus isolated from food
samples including meats (meat of sheep ,fresh chicken, frozen chicken and beefmeat ) and vegetables including( lettuc, pepper, leek, tomato, celery ,cucumber ) Twenty-five g of each sample were weighed and transferred into sterile Stomacher bags, containing 225 ml of Brain heart infusion broth in 37ºC water bath with shaking at 100 rpm/min for 6 h, and 1 ml pre-enriched rinse were transferred into to selective agar (Manitol salt agar) after incubation for 24 hours, this selective media was used to extract DNA of Staph.aureus as mentioned previously. 3.2.11. Method used for detection of Shigella in Food Based on (Kenia et al., 2010). Twenty five grams of minced food sample (Leek, Tomato, Peper, Lettuce, Broccoli, Carrot, Cucumber, Celery, meat of Beef , meat of Sheep , meat of Goats and Fresh Chicken meat ) were mixed with 225 ml of 0.1% peptone water and homogenized. 1 ml of the sample mixture was mixed with 9 ml of Salmonella -shigella agar (SS broth) and the mixture was incubated at 42°C and shaken for 24 h. the last broth used for extraction DNA and analyzed by PCR method as described above. 3.2.12. Method used for detection of E.coli in Food Based on
(Novicki et al., 2000). Twenty-five g subsamples of each
(Cucumber, Tomato, Celery, Peper, Lettuce, Broccoli, meat of Beef , meat of Goats, meat of Sheep, frozen Chicken meat and Fresh Chicken meat) were weighed and transferred into sterile Stomacher bags, one containing 225 ml of 0.1% peptone water and homogenized in 37 ºC water bath with shaking at 100 34
Materials and methods
rpm/min for 6 h, 1 ml of the homogenous sample was inoculated into 9 ml of MacConkey agar, after incubation at 35°C for 24 - 48 h, the last broth used for extraction DNA and analyzed by PCR method using the primers specific for E.coli as described above. 3.2.14. Method used for detection of Listeria in Food Based on (Amagliani et al., 2007). The samples of food used here were (frozen Chicken, Fresh Chicken, meat of Sheep , meat of Beef and meat of Goats, Cucumber, Tomato, Celery, Peper and
Lettuce) also 25 g of each
homogenized samples were mixed with 225 ml of 0.1% peptone water and kept in 37ºC water bath with shaking at 100 rpm/min for 6 h, 1 ml of each of these samples was inoculated into 9 ml of Oxford broth. After incubation at 35ºC for 24 - 48 h, the last broth used for extraction DNA and analyzed by PCR method using the primers specific Listeria for as described above.
35
CHAPTER FOUR RESULTS AND DISCUSSION 4.1 Genomic DNA Isolation At the beginning of this study DNA extracted directly from the food samples using different protocols without any enrichment steps, for instance DNA extracted from vegetables according to the method described by Weigand et al. (1993), Suitable yields of genomic DNA were obtained following these procedures, however no results were obtained in the detection of the pathogenic bacteria in natural contaminated food by application of species specific PCR, the same results obtained in other studies (Rodriguez and Hernandez, 2006; Jofré et al.,2005;Bhagwat,2003; Heller et al., 2003; Liao and Shollenberger,2003 and Shearer et al., 2001).So pre enrichment steps and selective media were applied on the homogenized food samples before extraction of DNA and positive PCR results were obtained using the protocol of (Anonymous ,2002). (Figure 1) The purity of DNA isolated in this study was found to be good ranging between 1.71.8 determined by spectrophotometer ratio A260/A280, from beef meat and chicken meat according to (Koh et al., 1998).
63
Result and Discussion
Figure 1: Represent the results of the DNA extraction from ten samples of food, that was performed on (1.2%) agarose gel electrophoresis and run at 90 volt /cm for one hour, the first lane represents DNA ladder 1kb and the Lane 1, 2, 3, 4, 5, 6, 7, 8,9 and 10 represent the DNA extracted from beef meat, tomato, celery, chicken meat, frozen chicken meat, lettuce , meat of Sheep, cucumber , leek and green pepper, respectively.
These methods were found to be very efficient for a number of reasons: rapid yielding of DNA suitable for PCR amplification, the availability of materials and apparatus needed. The advantage of this method also resided on several components in the extraction buffer that helped in isolation of a good yield and pure DNA, including: lysozymes , known as muramidase or Nacetylmuramide glycan hydrolase, these enzymes are glycoside hydrolyses, that damage bacterial cell walls by catalyzing hydrolysis of 1,4- ß linkages between N-acetyl muramic acid and N-acetyl-D-glucosamine residues in a peptidoglycan and between N-acetyl-D-glucosamine residues in chitodextrins (Yoshimura et al.,1988). Three cycles of freezing and thawing in a 65°C water bath were included to facilitate cell wall destruction, and thus, release of nucleic acids, Proteinase K is able to digest native protein, Hence the predominant site of cleavage is the peptide bond adjacent to the carboxyl group of aliphatic and aromatic amino acids with blocked alpha amino groups. It is commonly used for 63
Result and Discussion its board specificity and phenol chloroform-isoamyl used to remove residue of proteins. Saturated phenol acted as an efficient organic solvent capable of protein denaturation and other cell materials (Sambrook et al., 1989). Chlorophormisoamyl also acted as further organic solvent, but in addition to that, leaves nucleic acid in an aqueous phase as well as removing phenol residue from solution which affected DNA polymerase activity. Protein contamination is removed by chloroform extraction stages. In addition later nucleic acids were separated from other cell component by isopropanol precipitation (Weigand et al., 1993).
4.2 PCR reaction 4.2.1 Selecting primer In order to evaluate and verify the specificity of the primers in this study, five pairs of oligonucleotide primers were selected to detect five different types of foodborne pathogens by PCR .The primer pair was tested by PCR on DNA templates prepared from the different bacterial isolates. The analyses indicated that all primer pairs were specific for their corresponding target organisms. The analysis indicated that all primer pairs showed specificities only for their corresponding target organisms, it has been demonstrated in figure 2 that the primers specifically amplified 284bp, 108bp, 600bp, 556bp and 691 bP in bacterial strains belonging to Salmonella spp., Staphylococcus aureus, Shigella spp., E.coli 0157:H7 and Listeria monocytogenes, respectively. To confirm the identity of the amplicons, the size of the amplified products of each gene amplicon on agarose gel was consistent with the size predicted by the corresponding gene sequence. These results indicated that the DNA extraction method yielded sufficient DNA template to permitted PCR to detect the five target pathogens. This specificity could be developed, and in which ‘‘false positive signals would be unexpected. 63
Result and Discussion As limiting factor in detecting any bacteria from food would be isolating a template DNA from that bacterium in the food to get a suitable DNA which prepared for use as a PCR template from 25 g of food. The PCR-detection method has shown that the total DNAs extracted by a phenol-chloroform method provided a sufficient quantity of bacterial DNA to allow for a rapid and simple detection procedure however, Szakál et al. (2003) used cell lysat instead of extraction of DNA for PCR template and obtained good results, the PCR test in food samples may be limited by the presence of substances that inhibit the PCR reaction (FDA, 1995). When evaluating PCR for the detection of microorganisms, two important criteria must be satisfied: specificity and sensitivity (Malorny et al., 2003). Specificity considered as a way to detect false negatives and sensitivity as away to detect false positives. There for, our results therefore, indicated that this particular set of primers were suitable for the specific detection of these strains of bacteria by PCR.
63
Result and Discussion
Figure 2: Electrophoretic analysis of PCR-amplified target genes from five different bacterial pathogens. Mobilities of the different target gene amplicons are indicated on the figure, the first lane represents DNA ladder 1 kb (size marker) Lanes1, 2, 3, 4 and 5 represent L.monocytogenese, Shigella flexneri, E.coli o157:H7, Salmonella Typhimurium and Staph.aureur respectively.
4.2.2 PCR analysis 4.2.2.1 PCR analysis of Salmonella 4.2.2.1.1 Optimization of PCR As PCR kit used in this study, so optimization of PCR reaction included two main parameters: The concentration of primers and the annealing conditions which affect the specificity of PCR. High primer concentrations and low annealing temperature allow mis-priming, the products of which will actively compete with the target sequence for primers.
The concentration of 10pmol was used in the optimization of primer concentration, to obtain positive results, however 5 pmol of any primer give no result. Optimization of cycling conditions was performed with this concentration of primers. The annealing conditions that produced the highest yield were 65ºC for 30 sec and were therefore adopted as the optimum primer annealing conditions. In this study, amplification occurred efficiently with the 04
Result and Discussion extension phase of 240 sec, an extension phase may be helpful at late cycles when the product concentration exceeds the enzyme concentration. This resulted in a higher PCR yield. The point at which a single band was observed via 1.2% agarose gel electrophoresis following adjustments of annealing temperatures (65°C) meant that the reaction conditions were optimal. Sometimes other amplicons appear, this can be overcome by changing the annealing temperature, where the amount of non-specific amplification products decreases as the annealing temperature increases, to a point where only the desired amplicons is visualize (Malorny et al.,2004). 4.2.2.1.2 Detection of Salmonella in naturally contaminated samples While it was of the highest priority to find naturally contaminated samples for the purpose of validation, the samples collected for this study tested negative for naturally contaminating Salmonella using the PCR, without enrichment, this is in agreement with other studies (Anonymous, 2002). This may be explained by the fact that a relatively low number of Salmonella are present in these samples which could not be detected by traditional PCR. A specific PCR assay, targeting the invA gene, combined with a two step enrichment for the detection of Salmonella was developed, the results shown in figure 3 indicate the detection of Salmonella spp., by generating a PCR product of 284 bp in size in three samples from 16 samples (18.75%) of food, this is in agreement with (Wang et al., 2005) who found that the rate of contamination of Salmonella spp. detected in unspiked foods was 8.67% ,also coincide with(Moussa et al.,2010) who detected Salmonella serovars in 5.92%
of
examined poultry samples. These results indicated the health hazard of poultry as a major source of Salmonella foodborne pathogens (Humphrey, 2002 and Schlundt, 2002). No positive result obtained in beef meat samples, however the presence of Salmonella on fresh meat carcasses was addressed by the European Union and 04
Result and Discussion new regulatory microbiological criteria in two from of 50 carcasses tested (Anonymous, 2005a) . To increase the concentration of the target organism and to ensure detection of viable bacteria, samples were homogenized and pre enriched in non-selective BPW followed by selective enrichment in TT broth. Without pre enrichment on non selective media, no results obtained in this study because this step increased the detection limit of these bacteria, similar results have been reported previously by other authors (Ellingson et al., 2004; Fratamico, 2003; Myint et al., 2006 and Patel et al., 2006), who observed that at least 103 to 105 CFU/ml must be present to give positive results by PCR and this concentration not be obtained without a pre-enrichment step, The enrichment steps, also minimizes the detection of any initial non-culturable or dead cells and diluted the Possible biological contaminants such as blood and fats present on meat samples which can cause PCR inhibition are also diluted (Rodriguez-Lazaro and Hernandez, 2006). So to assure the positive PCR detection of Salmonella in various samples, especially when target cells are present in very limited numbers, enrichment leading to a predominance of bacteria was carried out (Wang et al., 2002 and Chiu et al., 2005). Some studies suggest being pre-enriched food samples in BPW for 24 h, however after the pre-enrichment for one day, the number of total bacteria 9
reaches as high as 10 CFU/ml. Although the number of the Salmonella cells also increases, it is usually outnumbered by other bacteria because they have a much higher number of cells when combined. Although selective enrichment using TT broth may inhibit the growth of many of these bacteria to a certain extent, their overgrowth likely will mask the growth of Salmonella. Consequently a large number of colonies may appear on selective agar plates which make it very difficult to isolate Salmonella. When the samples were pre-enriched in BPW for 6 h, the concentrations of bacterial cells were significantly lower than those from 24 h incubation. 04
Result and Discussion 4
However this 6h was long enough for one Salmonella cell to grow to 10 CFU in BPW. This mean there will be enough Salmonella cells to grow on selective enrichment broth. And of the low starting concentration of other bacteria in TT broth, Salmonella competed well with other bacteria in selective broths and become the dominant organism (Myint et al., 2006). In an international research project for the validation and standardization of PCR for the detection of five major food borne pathogens including Salmonella, the most selective primer set was found to be that which targets the invA gene and that which targets the hilA gene. Amplification of invA or hilA genes now has been recognized as an international standard for detection of Salmonella genus (Malorny et al., 2003). InvA gene encodes a protein in the inner membrane of bacteria which is responsible for invasion to the epithelial cells of the host (Jennifer et al., 2003). In this study, the PCR produced positive amplification of 284 bp fragments of invA (100%), specific for Salmonella species, while all other strains
were negative, including strains of E. coli, Shigella flexneri,
Staph.aureus and L.monocytogenese, it mean that they did not give any false positive results. These results were parallel to those obtained by (Oliveira et al., 2002 and Nucera et al., 2006) who reported that the primers, which target the invA gene were able to identify all the examined Salmonella strains, whereas all non Salmonella strains gave negative results, these results concluded that, all Salmonella carry the invA gene, which is not carried by any other bacterial species (Lin et al., 2007). This means that these primers are genus specific primers and the main reason of using genus specific primers rather than species specific primer in this study, due to the detection of other Salmonella species beside Salmonella spp. and Salmonella Enteritidis such as Salmonella Agona and Salmonella Newport which detected from imported frozen chickens which are
indications of the ability of the imported food to introduce different 06
Result and Discussion Salmonella species to the local area that can cause new and devastating outbreaks (Cohen et al., 1993). So PCR offers a great and rapid diagnostic tool for Salmonella detection and the 33h of this PCR assay offers a good tool for the routine monitoring of Salmonella contamination in food in contrast to the 3-4 days using culture method.
Figure 3: Electrophoretic analysis of 284 bp amplification product from invA gene of Salmonella spp. Isolated from some samples of food M: 1kb marker. Lane 1: represent chicken meat, Lane 4 represents tomato, Lane 8 represents frozen chicken.
4.2.2.1.3 Detection of Salmonella in artificially spiked samples Food samples containing natural flora were spiked with Salmonella Typhimurium strains at three inoculums levels and used for validation. The decimal serial dilution of a 12 to 16 hours culture of Salmonella Typhimurium were detected, the detection limit of Salmonella inoculated to 24 h following optimization of the PCR assay was 103CFU/ml (Fig. 4, Lane 3) Similar results have been reported previously by other authors (Ellingson et al., 2004; Myint et al., 2006 and Patel et al., 2006), who observed that at least 103 cells/ml must be present to give positive results by PCR. 00
Result and Discussion Validation of the PCR assay for the detection of Salmonella spp. in artificially contaminated food samples yielded good results as well in other studies (Malorny et al., 2003b and Malorny et al., 2004a). This detection limit may vary by the type of food as many studies found that, the detection sensitivity of the PCR process may vary with the method used for target DNA preparation (Tsen et al., 1998). And the food samples selected for assay (Dickinson et al., 1995).
Figure 4: Sensitivity of the PCR for the detection of Salmonella Typhimurium, the first Lane represents DNA ladder 1kb; Lanes 1 through 4: target gene isolated from 10, 102, 103, 104 CFU/ ml, respectively.
So this assay has the potential to become a standardized method for routine analysis of food for the presence of Salmonella. However using new methods of PCR such as real time PCR methods allowed sensitive detection of Salmonella spp. in food yielding positive results even at the lowest contamination levels tested (2.5 CFU/25 g Salmonella in minced meat or 5 CFU/25 g of chicken meats) (Perelle et al., 2004 and Wang et al., 2004). Besides, reducing the analysis time would make it a valuable asset in the future to the food testing industry. 04
Result and Discussion
4.2.2.2 PCR analysis of Staphylococcus aureus 4.2.2.2.1 Detection of Staph. aureus in naturally contaminated samples In the present work a protocol suitable for routine analysis of Staph. aureus in food with an improved sensitivity was established. The conventional PCR procedure assayed includes the use of specific primers targeted to the (Sa 442)
gene producing 108 bp amplified products which were confirmed on
Agarose gel electrophoresis (Fig. 5). This is in agreement with (Samadi et al., 2007) It was tested successfully on
high number of strains of Staph. aureus
(Pinto et al., 2005). No amplification products were observed using template DNA from other strains (Salmonella spp., E.coli O157:H7, Shigella flixneri, Listeria monocytogenese). Also negative controls, non contaminated samples never gave positive result. In the results of the PCR detection of Staph. aureus (Sa 442) different food samples analyzed showed that out of
ten food samples 4 samples were
positive for Staph. aureus including :tomato, lettuce, fresh chicken meat, frozen chicken meat using the sepsis specific PCR-based , this is in agreement with (Neeling et al., 2007) who detect Staphylococcus aureus from several foods including meat, chicken, milk and dairy products, also similar results were presented by (Alarcón et al., 2006) who found that out of 164 naturally contaminated foods tested for the presence of Staph. aureus 74 were positive by PCR different selective media for extraction the bacteria, this mean that the developed PCR-based method, which involves overnight selective enrichment under aerobic conditions, facilitated sensitive next-day Staph.aureus detection. Moreover, the method was able to overcome the problematic colony identification using standard microbiological method.
03
Result and Discussion
Figure 5: Electrophoretic analysis of 108 bp amplification product from Sa442 gene of Staph.aureus isolated from some samples of food M: 1kb marker. Lane 1: represents tomato, Lane 3 represents frozen chicken, Lane 6 represents lettuce and Lane 8 represents chicken meat.
4.2.2.2.2 Sensitivity of PCR for detecting Staph. aureus The efficacy of the developed PCR-based detection method was further evaluated using four food samples artificially contaminated with Staph. aureus, at the levels of 101to 104 CFU/ g. Two of the samples (103,104 CFU/ ml) were detected positive by the PCR-based method (Figure 6), On the other hand, two samples produced negative results (10,102 CFU/ ml), sensitivity was usually assayed in pure cultures and it was frequently reported the variation in the sensitivity when it was applied to artificially inoculated food This is agreement with (Hill, 1996). One of the reasons is variation in the procedure used for the isolation of nucleic acids from the complexes food matrices, the sensitivity of the assay was determined to be about 103 CFU/ ml . This is in agreement with (Ercolini et al., 2004) who found that at least 103 CFU /g was needed to obtained positive results for Staph.aurus in food.
03
Result and Discussion There is another method to estimate the sensitivity of PCR using the molecular weight of the DNA that used in PCR reaction as the method used by (Yang et al., 2002) who found the limit of detection of 104.5 pg of the PCR which would be equivalent to 3.64 x 104 genome. As it is well known that Staphylococcus spp. grow in clusters of cells, and it has been reported that these clusters of 10– 20 cells represent a single CFU (Duguid, 1989). This means that one CFU of Staph. aureus may actually represent 10–20 genome equivalents. This could explain the apparent 15-fold difference in PCR sensitivity when it is expressed as CFU or genome equivalents. At the present time, the standard method available for Staph. Aureus detection in food is based on selective enrichment followed by microbiological and biochemical identification, because the selective medium alone is insufficient for the precise identification of Staph. aureus colonies (Ingham and Schoeller, 2001). These procedures are labor-intensive and time-consuming and providing not always accurate and reliable results. So, molecular-based methods have a well ground potential to overcome the insufficiencies of identification procedures associated with the results based on the biochemical characteristics. As this study aimed to develop and optimize a highly sensitive method for the rapid and definite detection of Staph. aureus in food based on original species-specific PCR performed after overnight selective enrichment. In our study, PCR proved to be more sensitive for the detection and identification of Staph. aureus than classical cultural methods. The most impressive advantages of PCR-based detection method in comparison to the standard microbiological detection method are admittedly considered speed, sensitivity, and exactness of the obtained results. Implementation of these methodologies to the routine laboratory analysis still needs more data on their application in naturally contaminated food, as well as, their validation with the currently accepted procedures for any new PCR detection method. 03
Result and Discussion
Figure 6: Sensitivity of the PCR for the detection of Staphilococcuse aureus, the first Lane represents DNA ladder 1 kb; Lanes 1 through 4: target gene isolated from 10, 102, 103,104 CFU/ ml, respectively.
4.2.2.3 PCR analysis of Shigella 4.2.2.3.1 Detection of Shigella in artificially spiked samples The products from DNA amplification by PCR are shown in Figure7. In the amplification reaction, bands of 600 bp (ipaH) were observed with Shigella DNA. The ipaH products were not observed with the other bacterial strains. To test sensitivity of the PCR assay, templates were prepared by the decimal serial dilution of the bacterial 24h culture of Shigella flexneri. The results showed that after inoculation with 102, 103, 104, 105 CFU/ ml of Shigella flexneri into samples, and after enrichment with SS broth, the detection level of Shigella in lettuce by PCR was 104 CFU/ ml from each sample.
Heijnen and Medem (2006) and Theron et al. (2001) also identified
virulent Shigella flexneri and enteroinvasive E. coli (EIEC) in spiked environment water samples by using PCR method, with detection limit of 1.6 × 103 CFU /ml. PCR detection of virulence genes in Shigella such as ipaH, are considered a good and specific diagnostic tool of Shigella from clinical and food samples 03
Result and Discussion (Germani and Sansonetti, 2006; Lampel and Orlandi, 2002; Silva et al., 2008 and Thong et al., 2005). The ipaH loci comprise a multicopy antigen gene family unique to Shigella species and enteroinvasive Escherichia coli (EIEC). Because EIEC are exposure to antimicrobials it is though that most organisms detected by ipaH PCR are Shigella spp. Fernandez-Prada et al.(2000), reported that ipaH of Shigella flexneri was required for efficient escape from endocytic vacuoles, and Toyotome et al.(2001), determined that IpaH proteins are exported by type III secretion after entry into the host cell and that once secreted, ipaH accumulates in the nucleus, and inhibit nuclear factor kappa B (NFkappa B) dependent gene expression that is responsible of the immune response of the host cell (Haraga and Miller, 2003). In this way, ipaH serves to subvert host cell signaling events involved in the immune response to epithelial invasion. This study using ipaH based- PCR have been published from several Asian countries including Thailand, Bangladesh and more recently India and Vietnam (Thiem et al., 2004; Sethabutr et al., 1994; Islam et al., 1998 and Dutta et al., 2004). Gene produced a 60-kilodalton antigen and because of increased dosage of this gene and presence in multiple copies on both the plasmid and the chromosome of Shigella spp. (Sethabutr et al., 1993). A primer derived from ipaH might be less compromised by plasmid loss and selective deletion events of the invasion plasmid and perhaps be more sensitive as a diagnostic tool for Shigella spp. (Sethabutr et al., 2000; Dutta et al., 2001). It was also used by (Li et al., 2009) for detection of Shigella in food. We also obtained superior results using an enrichment step prior to PCR analysis rather than the direct PCR analysis of samples. This is in agreement with (Jiménez et al., 2010) who found that the low detection sensitivity of Shigella spp. might be conquered by enrichment to achieve successful PCR because when target cells are present in very limited numbers, enrichment led to a predominance of Shigella (Wang and Yeh, 2002).This is in coinciding with 44
Result and Discussion Song et al. (2005) who said that no Shigella can be detected by PCR before 24 h enrichment. The use of enrichment provides advantages such as multiplication of the bacterial target levels to obtain detectable concentrations, dilution of inhibitory substances present in food and dilution of dead target cells, which provides some assurance that the detected DNA belongs to viable target cells (Lee and Fairchild, 2006).It also to resuscitate physiologically stressed or injured cells (Kim and Bhunia, 2008). In this study, experiments with artificially challenged samples, without pre-enrichment, failed to detect Shigella flexneri by PCR, Such result was also reported previously by (Theron et al., 2001). For this reason, it was necessary to know the maximum volume of diluent that did not inhibit the PCR reaction. For this purpose, the bacterial strains were inoculated in the food sample and subsequently diluted in buffered peptone water. Even though conventional culture is considered the gold standard for Shigella detection but traditional culture methods take too long to get a positive result due to all the steps needed for confirmation of presumptive positives, what is not acceptable for certain products with short shelf-lifes, on the other hand, the molecular method showed a higher diagnostic sensitivity and a faster result as compared to the conventional culture among them, PCR technique can detect a small number of culturable as well as non-culturable organisms. The time requirement of this technique is lower compared to that of the culture techniques, so it can be chosen as an alternative to the culture technique, and can further be used for identification of Shigella, PCR methods have been also used to detect various species of Shigella and the gene targets include ipaH (Wen et al., 2010and Deanne and Keith, 2010), Vira gene
(Toytome et al.,
2001) and Wzx gene (Li et al., 2009). The most serious potential disadvantage of PCR-based assays is that secondary priming sites may yield a montage of amplified sequences or fragment equal in size to the expected PCR product, or if the annealing 44
Result and Discussion temperature is too low. So different temperatures are tested for annealing primers, and optimizing Taq replication efficiency. Therefore, it is not easy to carefully control hybridization stringency during the entire course of the reaction and to get accurate results, the PCR reaction must be repeated at least twice.
Figure 7: Sensitivity of the PCR for the detection of Shigella flexneri, the first Lane represents DNA ladder 1 kb; Lanes 1 through 4: target gene isolated from 10, 102, 103, 104 CFU/ ml, respectively.
4.2.2.4 Escherichia coli 4.2.2.4.1 Optimization of PCR reaction The important parameter for optimization of PCR reaction for detection of E. coli 0157:H7 was annealing temperature that should be optimized to achieve distinct bands for each primer set (Elizaquivel and Aznar, 2008). using this pair of primers that was specific to E. coli 0157:H7 and have 556 bp, it was tested in temperatures ranging from 50°C to 60°C. Amplifications were most consistent at annealing temperatures of 58°C. Annealing temperature has been identified as a crucial parameter during the optimization of PCR protocol, as nonspecific amplification can be reduced by optimizing the annealing temperature. This 44
Result and Discussion parameter is related to the melting temperature of the primers utilized as it is usually more or less than 5°C of the melting temperature of the primers. To a greater extent, high annealing temperature will result in greater specificity (Jeshveen et al., 2012). This contradicts with the results of (Sipos et al., 2007) that the better results were observed at lower annealing temperature. The second parameter was the preparation of PCR template (target DNA) using suitable method, the method applied for genomic DNA extraction in this study was phenol chloroform based method which was effective in obtaining the genomic DNA of pathogenic bacteria (Kalia et al., 1999). This method has been used previously to extract genomic DNA from E. coli O157:H7 (Atashpaz et al., 2010). The method has few steps which make it rapid and minimize the possibility of contamination, and because this method is not specific for a particular organism, thus it may be useful for detecting other bacterial pathogens in ground beef. At last, the primer sequence is an important parameter for obtaining successful result of PCR reaction. The primers that used in this study target the Stx2A gene; they were used previously by (Toyotome et al., 2001). The PCR reported here was extensively optimized to ensure that it detected only its intended target. It is evaluated by testing an assay against a number of non targets strains Staphylococcus aureus, L.monocytogenes and of Salmonella spp., and the results of the tests confirmed the selectivity of the gene (Stx2A) primer set.
4.2.2.4.2 Sensitivity of PCR for detection of E. coli O157:H7 Prior to inoculation experiments, pure cultures were tested, for E. coli O157:H7 strains used in this study. The experiment was performed to evaluate the ability of the assay to detect E. coli O157:H7 that had been inoculated into ground beef. 46
Result and Discussion To assess the effect of ground beef substances on the PCR, different concentrations of E. coli O157:H7 cells were suspended in TSBYE broth.Figure.8 shows the results of the detection limit evaluation. The detection limit for cell suspended in media in a concentration of 104 CFU/reaction gave positive results, faint bands were observed using the concentration of 103 and no band was observed in 102 CFU/ml concentrations, and this is attributable to substances in the sample inhibitory to PCR (Fode-Vaughan et al., 2001). This inhibition was relieved by dilution. So this compares with the detection of 104 cells which appeared to be the limit of the assay of (Maurer et al., 1999) whereas the detection limit for cells suspended in pure saline was 1.34 × 10
5
CFU /ml (Cui, 2004).
Figure 8: Sensitivity of the PCR for the detection of E.coli O157:H7, the first Lane represents DNA ladder 1 kb; Lanes 1 through 4: target gene isolated from 10, 102, 103, 104 CFU/ ml, respectively.
40
Result and Discussion 4.2.2.4.3 The effect of enrichment on PCR detection of E. coli O157:H7 To improve the sensitivity of detection, an enrichment step was added. Beef were enriched in TSBYE medium base for 6 h. After this step, the Stx2A gene was consistently and reproducibly detected in all samples, as determined 4
by viable counts, the concentration of E. coli O157:H7 cells were 10 CFU /g beef after 6 h enrichment. The same enrichment times as short as 6 h were applied by (Campbell et al., 2001) for simultaneous amplification of E. coli O157:H7. Broth enrichment serves two purposes: inhibitors in the sample are diluted, and bacterial growth increases the number of copies of the target sequence. The relatively short enrichment step was advantageous in that it lessened the time for normal micro biota to outcompete indicator bacteria (Clark, 1980). A problem occurs regarding E. coli O157:H7 and some pathogenic bacteria for which the infectious dose is low (Feng, 2001). Because the ground beef samples were thoroughly homogenized, use of this protocol reduced the chance that E. coli O157:H7 cells remained undetected due to protection within food matrices, the presence of rather high prevalence of E. coli O157 positive animals at abattoir is a major link in the transmission of E. coli O157 to the food chain by cross-contamination of the carcass and ground beef with feces (Richards et al., 1998 and Slutsker et al., 1998). Currently, several beef processors are employing strategies to ensure that contaminated product is not released into commerce (Brabban et al., 2004). Such a system requires rigorous testing to fulfill its objectives. The testing methods (i) must be rapid, because ground beef is a highly perishable product (ii) must have low numbers of false-positive results to ensure wholesome product is not needlessly discarded, and (iii) must have no false-negative results, which would defeat the purpose of such a system (Cui, 2004). The most
44
Result and Discussion important of these issues is that of false-negative results, which can come in one of two forms. First, a test may not detect a certain subtype of E. coli O157:H7. A wide range of genetic variability has been identified among E. coli O157:H7 strains (Barkocy-Gallagher et al.,2001 and Kim et al., 2001).Therefore, genetics-based tests may focus on a target that is present in all O157:H7 strains. The second type of false-negative result comes from testing methods in which the sensitivity is simply insufficient for the application in the method that used. Therefore, the various testing methods that based on PCR must be able to detect as least organisms per sample as possible to increase its sensitivity. As E. coli O157:H7 is a pathogenic microorganism that can cause several human foodborne illnesses and incidence rate involving it in food samples is on the rise ,so detection of this pathogen is vital, as many traditional culture based approaches for detection of this pathogen may take several days. And many of them fail to detect strains of virulent bacteria present in foods at low levels, because such pathogenic strains, do not compete effectively in physiologically demanding enrichments and can be overgrown by nonpathogenic members the same genus or species (Hill et al., 1985), besides some pathogenic Escherichia coli may lose plasmids that may harbor some virulence genes, which cause of the loss of the protein that produced by these genes which may be the target of some biochemical test for that bacteria . The PCR provides a mechanism to circumvent these difficulties by permitting the specific amplification of target genes, even that of injured cells. In contrast to culture-based methods, which may result in false-negatives as E. coli O157:H7 has been reported to enter a viable, non-cultural state under some environmental conditions (Wang and Doyle, 1998) which may lead to incorrect results, the detection system that based on PCR methods can detect cells that are non-cultural, and however it does not necessarily indicate that a sample has live pathogenic bacteria, because dead cells or free DNA would also be detected. 43
Result and Discussion Such false positives would be expected in any PCR assay, and this could be advantageous for some purposes. For example, PCR could be useful in tracing the source of an outbreak to identify STEC in a sample. Various PCR-based strategies were used for the detection of which are able to amplify a single gene related to one pathogen or single virulence factor gene of pathogens (Thomas et al., 1994 and Tornieporth et al., 1995( Through this study, a rapid, reliable and specific method were developed successfully for detect the virulence gene belonging to E. coli O157:H7 from food samples. This particular typing method that based on PCR can be used for future surveillance studies and sensitive screening in monitoring cases of outbreaks.
4.2.2.5 Sensitivity of PCR for detection of Listeria monocytogenese To determine sensitivity of the PCR protocol, for detection of L. monocytogenese, overnight- enrichment samples of L. monocytogenese in TSBYE broth were serially diluted 10-fold and DNA extracted from them by phenol– chloroform and was used as templates for PCR amplification. Each pathogen was diluted from 104, 10
3,
102 CFU of reference strains bacteria (L.
monocytogenese). The results of PCR as shown in Figure 9 represent the gel electrophoresis of the PCR products which confirmed that primer pairs specifically amplified the desired PCR products a 691 bp band specific for L. monocytogenese gene (lmo0737). The reproducibility of the 691 bp fragment was approved by a repeated -PCR assay. These results are in accordance with that of (Doumith et al., 2004a), who used the same primer pairs to specifically amplified L. monocytogenese gene (lmo0737). Other food born pathogen that investigated in this study, optimization reactions were achieved to get the accurate results. As specific and sensitive amplification of the target gene of L. monocytogenese
by PCR are dependent
on a number of some parameters like annealing temperature, primer concentration (Szabo et al., 2000).
Therefore, many experiments were 43
Result and Discussion performed to optimize the PCR conditions to obtain the results; this is in agreement with (Kong et al., 2002). Even they studied more parameters in optimization of PCR reaction, such as Mg concentration. The public health hazard view L. monocytogenese as one of the major pathogens (Doganay, 2003). Many studies reported that most of the major food borne outbreaks due to invasive listeriosis in both Europe and North America have been caused by L. monocytogenese strains (Jacquet et al., 2004 and Doumith et al., 2004 b). Suggesting that these strains may possess unique virulence properties. Human infections primarily result from eating contaminated food with L. monocytogenese may lead to serious and potentially life-threatening listeriosis (Posfay-Barbe and Wald, 2004). The frequent occurrence of L. monocytogenese in meat and chicken may pose a potential risk for consumers (Hassan et al., 2001 and Akpolat et al., 2004). So, L. monocytogenese in food is an ongoing biosafety concern worldwide. Because of its
ability to grow over wide
temperature and pH ranges, beside its ability to formation of biofims which causes L. monocytogenese to persist in the food processing line. The regulatory protocols for isolating Listeria from food samples, which based on culture methods are not totally effective, allowing approximately 30% of Listeria-positive food samples to escape detection (Ryser et al.,1996) due to the bacterium’s ability to enter VBNC (viable but nonculturable) state as a result of environmental stress which may be lethal sometimes (Oliver, 2000). This tends to give false negative results in investigation of microbiological quality of food that employs the conventional culture-dependent method only. On the other hand, molecular diagnostic techniques have greatly contributed to the detection and identification of this pathogen since it is highly sensitive, precise and fast. Among them PCR methods have superior sensitivity when compared to standard nucleic acid probes or immunoassays method (Shearer et al., 2001).
43
Result and Discussion PCR is also based on stable genotypic characteristics rather than relying on biochemical or physiological traits, which may be unstable (Lawrence et al., 1994). Beside it allow obtaining efficient reliable results and is ideal for monitoring the presence of L. monocytogenese in foods, offering results within two days after the sampling date (Janzten et al., 2006). So, it is highly recommended in order to obtain more precise and reliable result (Wong et al., 2011). However, complex sample preparation methods and the use of gel electrophoresis end point detection have hampered the transition of these methods from research to routine use in food microbiology laboratories. Therefore, some studies suggest that still the combined use of two or more procedures is generally more discriminatory and powerful than each applied alone (Jeyaletchumi et al., 2010).
Figure 9: Sensitivity of the PCR for the detection of L.monocytogenese, the first Lane represents DNA ladder 1 kb; Lanes 1 through 4: target gene isolated from 10, 102, 103, 104 CFU/ ml, respectively.
43
Conclusions
1. Successful method for genomic DNA isolation was obtained observed which based on using phenol-chloroform, from the seven types of food born bacteria has been used including Salmonella typhimurium, E. coli O157: H7, L. monocytogenes, Shigella flexneri and Staph. aureus. 2. Successful amplification product in expected size were obtained using species- specific primers in PCR reaction for identification of these types of bacteria. 3. The major contribution of this study is the establishment of a protocol for the simultaneous screening and detection of several important bacterial species which are a public safety concern in food. 4. The enrichment steps still needed for detection of this food born bacteria using this kind of PCR which called traditional PCR with sensitivity ranged between 103 to 104. 5. The PCR ability to rapidly monitor various types of microbial pathogens would be extremely useful not only for routine assessment of food hygiene to protect public health, but also for the rapid assessment of suspected food poisoning cases.
06
Recommendations 1. It can be concluded also that PCR procedure can be employed as a routine procedure for the screening of food born bacteria in a diverse range of food types, it provide a powerful supplement to conventional methods. 2. The possibilities of combining different rapid methods, including improved technologies for separation and concentration of specific bacteria, such as metal hydroxide immobilization, will facilitate the direct detection of pathogens in food. 3. The positive results still require further quantitative analysis, which can be done by performing Real-Time PCR. 4. DNA sequencing of PCR amplified product is needed in future studies to verify its identity. 5. This PCR method should be further improved for the detection of other important food and water borne pathogenic bacteria and extended to examination of many kinds of food. 6. Multiplex PCR method can be used later for identifying and simultaneous screening and detection of several important bacterial species in a single tube.
16
Examining Committee Certification We certify that we have read this thesis and discussed with the Student (Sakar Kamal Hamasalih) in its content and what is relevant to it, and in our opinion it deserved to be accepted for granting the Degree of Master of Food Science "Biotechnology".
Dr. Raad Abdul – Ghany Basheer Al - Sanjary Professor
Dr. Tagreed Abd Wahwah Al - Nashi Lecturer
College of Veterinary Medicine/ University of Mosul / / 2014 (Chairman)
Faculty of Agricultural Sciences/ University of Sulaimani / / 2014 (Member)
Dr. Dwnia Salman Khalaf
Dr. Khlood Ibrahim Hassan
Lecturer
Assistant Professor
Faculty of Agricultural Sciences/ University of Sulaimani / / 2014 (Member)
Faculty of Agricultural Sciences/ University of Sulaimani / / 2014 (Supervisor-Member)
Approved by the Council of the Faculty of Agricultural Sciences.
Dr. Aram Abbas Mohammed Assistant Professor /
/ 2014
(The Dean)
2714 K.
2014 A.D
Supervisor Certification I certify that this thesis was prepared under my supervision in the University of Sulaimani, Faculty of Agricultural Sciences, as partial fulfillment of the requirements for the degree of Master of Science in Food science –Biotechnology
Dr. Khlood Ibrahim Hassan Assistant Professor /
/2013
In view of the available recommendation, I forward this thesis for debate by the examining committee.
Dr. Rafiq Muhammad Salih Rashid Assistant Professor Head of the Food science Department Faculty of Agricultural Sciences /
/2013
Linguistic Evaluation Certification
I herby certify that this thesis prepared by (Sakar Kamal Hamsalih), has been read and checked and after indicating all the grammatical and spelling mistakes; the thesis was given again to the candidate to make the adequate corrections. After the second reading, I found that the candidate corrected the indicated mistakes. Therefore, I certify that this thesis is free from mistakes.
Dr. Ibrahim Maaroof Noori Lecturer Horticulture Department Faculty of Agricultural Sciences / / 2013
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Saccharomyces
الخــــالصـــــــــــة االجياد وتطوير طريقة معتمدة على استخدام طريقة تفاعل البلمرة متسلسل PCRاملبنية على اجلينات ااملتخصصة يف هذه الدراسة مت استخدام بادئات جلينات متخصصة من السالالت البكرتيه ضمن تفاعالت البلمرة للكشف عن عن مخسة أنواع من البكترييا واليت تشمل :
)Salmonella Typhimurium, Listeria monocytogenese, E.coli o157:H7, )Staphylococcuse aureus and Shigella Flexneri من االوساط الزرعية لتلك السالالت البكتريية و احملقونة بها بشكل صناعي لبعض انواع االغذية وللكشف عن نوعني من البكرتيا وهي Salmonella sppوال .Staphilococcuse aureusمن خمتلف انواع االغذية امللوثة بها بشكل طبيعي. مت احلصول على السالالت البكرتية النقية املستعملة يف هذه الدراسة من مركز ميديا الطبى التشخيصي يف اربيل اذمت تنميتها يف وسط زرعي عام وهو ال TSBYEعلى ̊ 35م ملدة 6ساعات قبل استخالص ال DNAمنها والذي استعمل بدوره يف جتارب ال PCRوذلك باستعمال بادئات متخصصة لكل ساللة بكتريية ,واليت انتجت قطع متضاعفة باالحجام املتوقعة لكل منها ( 482زوج قاعدي يف ال 691, Salmonella spp.زوج قاعدي يف ال Listeria 600, monocytogeneseزوج قاعدي يف ال ,Shigella flexneri
556زوج قاعدي لل E.coli
01,0157:H7و 018زوج قاعدي لل ) Staphylococcus aureusومت قياس التخصص لكل زوج من البادئات بتجربة تلك البادئات على اربعة انواع اخرى من السالالت البكتريية االخرى غري املستهدفة اذ مل يتم احلصول على نتائج متضاعفة يف اي منها . ولتحقيق اهداف هذه الدراسة مت مجع 01منوذج من االغذية من اسواق السليمانية للفرتة بني نيسان لغاية تشرين االول لعام 4104مبا يف ذلك اللحوم ( حلوم البقر وحلم الدجاج الطازج و اجملمد وحلم املاعز والضان ) .واخلضراوات واليت مشلت( الكرفس, الطماطة,اخليار,الفلفل ,اخلس ,الربوكلي ,اجلز و الكراث ) .اذ مت استخالص ال DNAمن كل منوذج من مناذج الغذاء, قبل وبعد اضافة الوسط املغذي باستخدام طريقة الفينول كلوروفورم ،مل يتم احلصول على نتائج موجبة عند تطبيق تفاعالت البلمرة اال بعد اضافة الوسط املغذي ،مت استعمال البادئ املتخصص والذي يستهدف جني ال ( )invAيف Salmonellaوجني ال Sa 442يف Staph. aureusمقرتنة مع خطوتني الستخدام الوسط الغذائي العام والوسط الغذائي االنتخابي لكل بكرتيا ,وذلك للكشف عن Salmonellaو Staph. aureusيف مناذج الغذاء و وتشري النتائج
إىل ان الكشف عن Salmonellaبتكوين قطعة متضاعفة من ال DNAبواسطة البادئ حبجم 284زوج قاعدي يف ثالثة من مناذج الغذاء من جمموع 01منوذج يف حني مت الكشف عن Staph.aurusبتكوين قطعة متضاعفة من ال DNA باستعمال البادئ املتخصص هلذه البكرتيا واليت تضاعف قطعة من ال DNAحبجم 018زوج قاعدي يف اربعة مناذج من جمموع عشرة مناذج .يف جتارب احلساسية مت تلويث العينات الغذائية بالسالالت البكتريية وبرتاكيز معينة بعدها مت استخالص ال DNAمن تلك االوساط واليت مت استعماهلا فيما بعد يف اجراء تفاعالت البلمرة ملعرفة اقل تركيز ميكن بواسطته الكشف عن تلك التفاعالت .ولقد وجد ان الرتكيز احملدد لكل نوع بكرتي يف مناذج الغذاء والذي ميكن كشفه بواسطة ال PCR ترتاوح بني 012-011مستعمرة
لكل مل لكل نوع من الغذاء(حلم الدجاج لل ,Salmonellaخس لل
,Staph.aureusكرفس لل ,Shigellaحلم البقر لل E.coliو حلم الضان لل (L.monocytogenesاذ كان احلد احلرج لل Salmonellaو Staph. aureusو Listeriaهو 011مستعمرة لكل مل يف حني كان احلد احلرج 012مستعمرة لكل مل لل Shigellaو , E.coli o157:H7عموما نتائج هذه الدراسة تشري إىل أن PCR طريقة جيدة للكشف السريع البكترييا املرضية اليت تنتقل عن طريق الغذاء .
