Duplex PCR Procedure for the Detection of EHEC O157: H7

doi:10.7668/hbnxb.2011.06.012

Abstract

Abstract: Construct a duplex PCR for the detection of E． coliO157: H7 from clinical samples． Analyses of the available sequences of the two major virulence genes listed in GenBank allowed us to develop the two-gene multiplex PCR protocol that maintained the specificity of each primer pair． Sensitivity and specificity were verified by detection of E． coliO157: H7 culture and prepared E． coliO157: H7 positive samples． Applying the duplex PCR，we detect E． coliO157: H7from bovine feces and isolate the positive samples． The resulting two bands for rfbEand fliCwere even and distinct with product sizes of 812 and 625 bp， respectively． The proportion of rfbEand fliCof PCR amplification reaction was 2． 5 ∶ 1(V /V) and the optimal PCR amplification temperature was 56℃． Sensitivity tests showed that the procedure amplified genes from a fecal sample spiked with a minimum of 104 cfu /g． After a 6-h enrichment of E． coliO157-spiked samples，a sensitivity level of 10 cfu /g was achieved． The procedure was validated with a total of 24 E． colistrains as well as 15 non-E． colistrains． Specificity of the O antigen was indicated by amplification of only E． coliO157，and not other E． coliserotypes and non-E． colistrains． Specificity of the H antigen was indicated by amplification of only E． coliO157 and E． coliO55，and not others． From 63 bovine feces，we obtained 4 isolates of E． coliO157: H7． Biochemical feature tests showed that isolated E． coliO157: H7 have the representative feature of E． coliO157: H7． Susceptibility tests indicated that the four E． coliO157: H7 have broad-spectrum drug tolerance． Sequence analyses of rfbEand fliCsugGested that the four isolates have homology of 97． 3%- 100%． The four E． coliO157: H7 isolates showed different pathogenicity， isolate 28# and 38# were high，3 # and 17 # low． We successfully developed the duplex PCR for O157: H7 for the detection of clinical samples．

Key words: EHEC O157: H7, Duplex PCR, rfbE, fliC, Detection samples

CLC Number:

LI Li, ZHANG Xue-han, HE Kong-wang, JIANG Ping, ZHAO Pan-deng, YE Qing, LUAN Xiao-ting, WEN Li-bin, NI Yan-xiu, ZHOU Jun-ming, LU Li-xin, GUO Rong-li, YU Zheng-yu, MAO Ai-hua, LI Bin, WANG Xiao-min. Duplex PCR Procedure for the Detection of EHEC O157: H7[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2011, 26(6): 58-66. doi: 10.7668/hbnxb.2011.06.012.

/ / Recommend / Download Citations

URL: http://www.hbnxb.net/EN/10.7668/hbnxb.2011.06.012

http://www.hbnxb.net/EN/Y2011/V26/I6/58

References

[1] Centers for Disease Control and Prevention(CDC). Preliminary FoodNet data on the incidence of infection with pathogens transmitted commonly through food-10 states， 2008[J]. Morbidity and Mortality Weekly Report，2009， 57(14): 366-370.
[2] Reinstein S，Fox J T，Shi X， et al. Prevalence of Escherichia coli O157: H7 in the American bison(Bison bison)[J]. Journal Food Protection，2007，70(11): 2555-2560.
[3] Doyle M P，Erickson M C. Summer meeting 2007-the problems with fresh produce: an overview[J]. Journal Applied Microbiology， 2008， 105(2): 317-330.
[4] 唐中伟，周志江. 产志贺毒素EHEC 国内分离株Stx2基因的克隆及生物信息学分析[J]. 山西农业科学，2011， 39(2): 167-159.
[5] 张雪寒，何孔旺，卢维彩，等. 出血性大肠杆菌0157:H7 Stx2B-Tir-Stx1B 多价融合蛋白表达及免疫性研究[J]. 华北农学报， 2010， 25(4): 180-185.
[6] 张雪寒，何孔旺，卢维彩，等. 出血性大肠杆菌0157:H7 的Tir 与Stx1 /2 融合基因的构建和表达[J]. 内蒙古农业科学报， 2009(3): 59-61.
[7] Marcus R. New information about pediatric foodborne infections: the view from FoodNet[J]. Journal Food Protection. 2008， 20(1): 79-84.
[8] Gannon V P，King R K，Kim J Y， et al. Rapid and sensitive method for detection of Shiga-like toxin-producing Escherichia coli in ground beef using the polymerase chain reaction[J]. Applied and Environment Microbiology， 1992， 58: 3809-3815.
[9] Fagan P K，Hornitzky M A，Bettelheim K A， et al. Detection of Shigalike toxin(stx1 and stx2), intimin(eaeA), and enterohemorrhagic Escherichia coli(EHEC) hemolysin( EHEC hlyA) genes in animal feces by multiplex PCR[J]. Applied and Environment Microbiology，1999，65: 868-872.
[10] Fratamico P M，Bagi L K，Pepe T. A multiplex polymerase chain reaction assay for rapid detection and identification of Escherichia coli O157: H7 in foods and bovine feces[J]. Journal Food Protection，2000，63: 1032- 1037.
[11] Hu Y，Zhang Q，Meitzler J C. Rapid and sensitive detection of Escherichia coli O157: H7 in bovine faeces by a multiplex PCR[J]. Journal Applied Microbiology， 1999， 87: 867-876.
[12] Chapman P A. Methods available for the detection of Escherichia coli O157 in clinical， food and environmental samples[J]. World Journal Microbiology Biotechnoogyl， 2000， 16: 733-740.
[13] Jianfa Bai，Xiaorong Shi T G，Nagaraja. A multiplex PCR procedure for the detection of six major virulence genes in Escherichia coli O157: H7[J]. Journal of Microbiological Methods， 2010， 82: 85-89.
[14] 梅明珠，杨亚贤，陆承平，等. 多重PCR 快速鉴定大肠杆菌O157[J]. 中国人畜共患病杂志，2007，3(4):351-354.
[15] 朱文冠，薛佘强，洪洁心，等. 多重PCR 检测大肠杆菌O157: H7 的初步研究[J]. 中国人兽共患病学报，2006， 22(5): 428-432.
[16] 徐晓可，吴清平，周艳红，等. 肉类中大肠杆菌O157:H7 多重PCR 检测方法的建立[J]. 微生物学通报，2008， 35(4): 619-622.
[17] Fujii J，Kita T，Yoshida S， et al. Direct evidence of neuron impairment by oral infection with verotoxin-producing Escherichia coli O157: H7 in mitomycin-treated mice[J]. Infect Immun， 1994， 62(8): 3447-3453.
[18] Belgin Sarimehmetoglu，Mihriban Hatun Aksoy，Naim Denia Ayaz. Detection of Escherichia coli O157: H7 in ground beef using immunomagnetic seperation and multiplex PCR[J]. Food Control， 2009， 20: 357-361.
[19] Mekillip J L， Jaykus L A. A comparison of methods for the detection of Escherichia coli O157: H7 from artificially contaminated dairy products using PeR[J]. Journal of Applied Microbiology， 2000， 89: 49-55.
[20] Naylor S W，Low J C，Besser T E， et al. Lymphoid follicle- dense mucosa at the terminal rectum is the principal site of colonization of enterohemorrhagic Escherichia coli O157: H7 in the bovine host[J]. Infection Immunity， 2003， 71: 1505-1512.
[21] Pearcem C，Fenlon d，Low J C. Distribution of Escherichia coli O157 in svine fecal pats and its impact on estimates of the prevalence of fecal shedding[J]. Applied and Environment Microbiology，2004，70(10): 5737- 5743.
[22] Ozgur Cadirci，Belgin Siriken，Gokhan Inat，et al. The prevalence of Escherichia coli O157 and O157: H7 in ground beef and raw meat ball by immunomagnetic separation and the detection of virulence genes using multiplex PCR[J]. Meat Science， 2010， 84: 553-556.
[23] 于风泰，赵志武，陈继永，等. 天津市首次在羊粪便中检出O157: H7 大肠杆菌[J]. 医学动物防制， 2003， 19(5): 268-270.
[24] Yukiko Hara-Kudo， Jiro Nemoto，Kayoko Ohtsuka， et al. Sensitive and rapid detection of Vero toxin-producing Escherichia coli using loop-mediated isothermal amplification[J]. Journal of Medical Microbiology，2007，56: 398-406.

Please choose a citation manager

Content to export