Ứng dụng quy trình kỹ thuật multiplex – PCR để phát hiện một số nhóm escherichia coli gây bệnh trong thực phẩm

PCR ESCHERICHIA COLI Chun ngành: : 604280 Cơng trình c hoàn thành t i: Tr ng i h c Bách Khoa TP HCM Cán b h ng d n: TS Nguy n Phúc PGS.TS Nguy n Thúy H ng Cán b ch m nh n xét 1: TS Nguy n H u Phúc Cán b ch m nh n xét 2: TS Võ ình L Tâm Lu n v n th c s c b o v t i tr ng i h c Bách khoa, HQG Tp HCM ngày 24 tháng 12 n m 2013 Thành ph n h i ng ánh giá lu n v n bao g m: PGS TS Nguy n c L ng TS Lê Th Thanh Mai TS Nguy n H u Phúc TS Võ ình L Tâm PGS TS Nguy n Th Thúy H ng Xác nh n c a ch t ch h i ng ánh giá LV Tr lu n v n ã u c s a ch a CH T CH H I NG ã ký PGS.TS Nguy n ng khoa qu n lý chuyên ngành sau TR NG KHOA K THU T HÓA H C ã ký c Lu ng PGS.TS Phan Thanh S n Nam i - - NHI M V LU H tên h c viên: N Chuyên ngành: 10/12/1985 Công ngh Sinh h c MSHV: 11310606 Mã s : 604280 I nhóm Escherichia coli II DUNG: - - - nhóm Escherichia coli III IV : 24/06/2013 : 22/11/2013 V : Tp HCM, ngày CÁN B NG D N tháng CH NHI M B 13 O NG KHOA K THU T HÓA H C Lu ii L IC c Bách K Ths b cho tơi Lu iii TĨM T T Escherichia coli tron Escherichia coli gây plex bfpA, eaeA, stx1 stx Escherichia coli Salmonella, Shigella, E coli, Staphylococcus aureus, Bacillus cereus, Vibrio paraheamolyticus, Vibrio cholera, Enterobacter sakazakii, Pseudomonas aeruginosa Listeria monocytogene) - - - PCR E coli Lu iv ABSTRACT In recent years, the group of Escherichia coli pathogenic is one of the causative food poisoning agents through the food line They will cause symptoms gastrointestinal tract disorders In addition, clinical manifestations have changed from mild to severe, which can cause death depending on the degree of infection, the infectivity and ability to respond of each person Therefore, the using of Multiplex PCR method is a necessary requirement to determine Escherichia coli pathogenic groups (ETEC, EPEC and EHEC) in the food poisoning outbreaks Through the evaluation and application of Multiplex PCR method is to detect toxic genes (LT, ST, bfpA, eaeA, stx1 and stx2) of Escherichia coli in food, we have obtained the following values: the primer pairs were specific with the target strains, and very selective with the non - target strains (Salmonella, Shigella, E coli, Staphylococcus aureus, Bacillus cereus, Vibrio paraheamolyticus, Vibrio cholera, Enterobacter sakazakii, Pseudomonas aeruginosa Listeria monocytogene), the detection level of the method (1 - 3CFU/25g: milk samples; 6CFU/25g: seafood samples and - CFU/25g: meat samples), relative accuracy, relative sensitivity and relative specificity of 90 - 100% for the three of food sample matrixs above From this study, there should be large-scale studies with a huge of samples and types of samples to assess more the value of using of Multiplex PCR method Besides that, the Multiplex - PCR method should be applied to detect toxic gene of E coli (EPEC, ETEC and EHEC) in food poisoning outbreaks Lu v L - tác - trách Lu vi M CL C iii ABSTRACT .iv vi ix xi xii E coli 1.1 E coli EPEC) ETEC) .11 - EIEC) .16 Enterohemorrhagic E coli EHEC) 18 Enteroaggregative E coli EAEC) .23 tán (Diffusely Adherent E coli - DAEC) .26 1.2 Multiplex PCR .29 29 Lu vii 30 - PCR 32 32 32 33 1.4 34 36 .36 36 36 36 - 37 37 37 38 38 Multiplex - PCR .38 39 40 2.2 40 .41 41 2.2.2.2 Qui trình Multiplex - PCR 41 EC, ETEC EHEC) .42 - PCR dùng cho 43 Lu viii - PCR: g (2003) 43 Multiplex - coli 46 48 nhóm E coli 48 49 3.3 - PCR: g 50 50 (sensitivity - SE) theo ISO/TS 16140 (2003) .55 - E coli E coli 58 - 62 62 63 64 67 Lu 61 20 (-) 21 (-) 22 (-) 23 (-) 24 (-) 25 (-) 26 (-) 27 (-) 28 (+) 29 (-) 30 (-) (+) EPEC (+) (*) Hình 3.5 di Chú thích: MK: marker; (25: 24 - Lu 62 : K T LU N - KI N NGH 4.1 K t lu n E coli E coli (EPEC, - gây E coli (EPEC, ETEC EHEC) + - CFU/25g CFU/25g CFU/25g CFU/25g CFU/25g CFU/25g - PCR - Lu 63 - Multiplex - plex - PCR + PC 4.2 Ki n ngh - PCR - E coli (EPEC, ETEC EHEC) tro PCR Escherichia coli Lu 64 TÀI LI U THAM KH O [2] 2, pp 120 - 122 157 - 197 [4 - 408 [5 Tp.HCM, Vol.11-No 1-2007, pp 31-34 [6 [7] L [8 -179 [9 - 241 [10] - 55 [11] A Hegde, M Ballal, S Shenoy, Original article, Vol 30, Issue 3, 2012, pp 279 284 [12] Arthur H Bryan, Charles A Bryan, Charles G Bryan, Bacteriology Principles and Practice Sixth Edition No 3, 1966, pp 239 240 Lu 65 [13 vol 1, 2000, pp 633 - 634 [14] Chariya Chomvarin, Orn-Anong Ratchtrachenchai and et al Characterization Of Diarrheagenic Escherichia Coli Isolated From Food In Khon Kaen, Thailand Southeast Asian J Trop Med Public Health, July 2005, Vol 36, No 4, 931 [15] Deng, Ming Y.1; A Multiplex PCR for Rapid Identification of Shiga-Like Toxin-Producing Escherichia coli O157:H7 Isolated , Journal of Food Protection®, Number 6(7) , pp 570 - 673, 1996 pp 570 - 576 [16] Hala A, Hoda A Multiplex - PCR for detection of Diarrrheagenic E coli in Egyptian children, J Med.Sci 7(2): 255-262, 2007 [17 - Microbiology of food and animal feeding stuffs-Protocol for the validation of alternative met [18] James P Nataro, Cheryl A Bopp, Patricia I Fields, James B Kaper, and Nancy A Strockbine Microbiology 9th edition, vol 1(43), 2007, pp 672 [19] James P Nataro and Jame Escherichia coli Clinical Microbiology Reviews, vol 11, Jan 1998, pp 144-185 [20] Kyu H K, Joon II C Development of Multiplex - PCR assays to identify E coli pathodenic genes in food Food Sci Biotechnol 19(5): 1205-1210, 2010 [21] Marguerite A Neill, Phillip I Tarr, David N.,Taylor and Marcia Wolf Escherichia coli Foodborne Disease Handbook 2nd edition, vol 1(9), 2001, pp 170-196 [22] NordVal Protocol for the validation of alternative microbiological methods, 2009 [23] NCCLS Performance standard for Antimicrobial Disk Susceptibility Tests, vol 23(1), 2003 [24] Peter F, Stephen D W, Karen J Diarrrheagenic E coli BAM, 2011 [25 PCR Evaluation of a Multiplex System for Simultaneous Detection of Salmonella Lu spp.,Listeria 66 monocytogenes, and Escherichia coli O157:H7 in Foods and in Food Subjected to , Foodborne Pathogens And Disease, Volume 6, Number 1, pp.81-89 [26] Sehand K et al Identification of Different Categories of Diarrrheagenic E.coli in stool samples by using Multiplex PCR technique Asian Journal of Medical science 2(5): 237-243, 2010 [27] Sameera Nayani Study on the Prevalence of Virulent genes in Escherichia coli isolated from Fish using Multiplex PCR Heli, Vol.2, pp 316 319 [28] http://www.tieuchay.com/nguyen-nhan-bi-benh-tieu-chay/336-dich-tieu-chayla-do-chung-ecoli-moi-cuc-doc.html [29] http://vi.wikipedia.org/wiki/Escherichia_coli [30] http://www.lhu.edu.vn/Data/News/388/files/Chuong5.pdf [31] http://yhvn.vn/tai-lieu/escherichia-coli [32] http://www.khoahocphothong.com.vn/news/detail/21659/phat-hiennhanh-vi-khuan-gay-ngo-doc-thuc-pham.html Lu 67 PH L C 1.1 E coli 10-1 -2 10-3 E coli o C o C to E coli E coli E coli E coli E coli E coli E coli 1.2 E coli E coli o phòng - a 10 o Lu 68 o C 24 - -1 o o E coli (+) (IMViC ++ ) [8] 2.1 Lu 69 - -DNA, RNA-R -RNA [3] 2.2 linked immunosorbent assay ELISA hay enzyme immunosorbent assay Salmonella, E coli Listeria Staphylococcus CFU/ml ( ] 2.3 PCR (Polymerase chain reactio Lu 70 nhân dòng, ] kì - o C - 95oC vịng 30 giây - phút - o C 70o ng kéo dài phút o [3] Lu 71 Hình1 [8] Lu 72 Hình2: - Khn m u (Template) t c [8] n DNA c n khuy i - Các oligonucleotide primer (các m i) g i amplifier (nhân t khu ch i), oligo hay primer Lu 73 - DNA polymerase ch u nhi t (thermostable) - dNTP: lo i nucleotide triphosphate - Dung d m (buffer) thích h p MgCl2 50o Taq polymerase Thermophilus aquaticus Các primer p oligonucleotide nucleotide cao [4] Lu 74 3% agarose có µg/ml ethidium o -20o theo [10] Lu 75 H TRÍCH NGANG 10/12/1985 Mi - QUÁ TRÌNH CƠNG TÁC: Chí Minh Lu ... coli Các dòng E coli gây Lu [8] E coli không gây E coli xác E coli E coli E coli nhóm E coli gene Hi n nay, h u h t nghiên c u th gi i Vi t Nam v phát tri n k thu nh nhóm E coli gây b nh ch y... nghiên c u phát tri n k thu t Multiplex - cs gi m s E coli gây b ng u c công b [16, 20, 26] - PCR nh nhóm E coli E coli nhóm E coli ultiplex E coli (ETEC, EPEC, EHEC) Lu - iêu: E coli - PCR - +... 1.1.6 Kh nh E coli v E coli E coli E coli - k - Viêm màng não: E coli - t E coli E coli E coli E coli Enterotoxigenic E coli (ETEC), Enteropathogenic E Lu coli (EPEC), Enteroinvasive E coli (EIEC),