大肠埃希菌O157:H7新型多重PCR检测方法的建立及其初步应用  

Development and preliminary application of multiple PCR for detection of E. coli O157∶H7

作　　者：李争[1] 卜昭阳[1,2,3] 卢晓冉 李诗语[2] 郎需龙[2,3] 王兴龙[2,3] 吴大成 

机构地区：[1]吉林农业大学动物科技学院,吉林长春130062 [2]军事医学科学院军事兽医研究所,吉林长春130062 [3]吉林省人兽共患病预防与控制重点实验室,吉林长春130062 [4]广东省农科院动物卫生研究所,广东广州510640

出　　处：《中国生物制品学杂志》2015年第9期956-960,共5页Chinese Journal of Biologicals

基　　金：广州市科技计划项目(201300000036)

摘　　要：目的建立一种能够满足现场快速检测、完全脱离实验室仪器设备的大肠埃希菌O157∶H7多重PCR检测方法。方法利用大肠埃希菌O157∶H7抗原基因保守序列fli Ch7和rfb E设计2对引物,并对Palm PCR G1-12退火温度、循环数、引物浓度及引物最佳比例进行优化,建立微量快速检测大肠埃希菌O157∶H7的多重PCR方法,并对该方法的特异性、灵敏性、重复性进行验证。取疑似病牛粪便共9份,利用粪便DNA提取试剂盒提取DNA,按照建立的PCR方法进行扩增,扩增产物经微型电泳仪电泳检测并照相。结果确定大肠埃希菌O157∶H7 PCR的最佳退火温度为54℃,循环数为25个循环,上下游引物浓度为10μmol/L,2对引物最适比为fli Ch7∶rfb E=1∶3。大肠埃希菌O157∶H7扩增产物可见625 bp(fli Ch7)和213 bp(rfb E)的特异性片段,而大肠埃希菌(ATCC25922)、猪链球菌2型、金黄葡萄球菌、鲍氏不动杆菌、小肠耶尔森菌、单增李斯特菌、沙门菌的PCR检测结果均为阴性,最低扩增DNA浓度为10 pg/μl,重复扩增5次均可见目的条带。9份疑似病牛粪便样品中,共检测出7份大肠埃希菌O157∶H7阳性样品。结论建立了大肠埃希菌O157∶H7微量快速多重PCR检测方法,该方法特异性、灵敏性和重复性均良好,可在现场约1 h报告结果,对突发性传染病的防治具有重要意义。Objective To develop a multiple PCR method for rapid on-site detection of E. coli O157 ： H7 in absence of laboratory instruments and equipments. Methods Two pairs of primers were designed based on the conserved fliCh7 and rfb E gene sequences of E. coli O157 ∶ H7 antigen, and the annealing temperature, cycle number as well as concentration and ratio of primers were optimized, based on which a multiplex PCR method for micro-rapid detection of E. coli O157 ∶H7 was developed and verified for specificity, sensitivity and reproducibility. Results The optimal annealing temperature,cycle number, primer concentration and ratio of two pairs of primers（fli Ch7∶ rfb E） were 54 ℃, 25, 10 μmol / L and 1 ∶ 3respectively. Specific fli Ch7 and rfb E gene fragments, at lengths of 625 and 213 bp respectively, were amplified from E. coli O157 ∶ H7, while were not amplified from E. coli（ATCC25922）, Streptococcus suis type 2, Staphylococcus aureus,Acinetobacter baumannii, Yersinia enterocolitica, Listeria monocytogenes or salmonella. The minimum DNA concentration for amplification was 10 pg / μl. Target gene bands were observed in all the five repeat amplification. E. coli O157 ∶ H7 were detected in 7 of 9 fecal samples of cattle with suspected infection. Conclusion A multiple PCR method for rapid detection of E. coli O157 ∶ H7 was developed, and showed high specificity, sensitivity and reproducibility, by which the test result was reported on-site within 1 h. It was of an important significance in prevention and therapy of emergent infec-tious diseases.

关 键 词：大肠埃希菌O157∶H7 多重PCR 

分 类 号：R378.21[医药卫生—病原生物学] Q78[医药卫生—基础医学]