大肠杆菌肠毒素基因多重PCR检测方法的建立  

Multiplex PCR detection on enterotoxin genes in enterotoxigenic Escherichia coli

作　　者：孟相秋[1] 袁超文[1] 刘文鑫[1] 关玮琨[1] 杜元策[1] 李丹丹[1] 赵姝静[1] 唐杰[1] 师东方[1] 

机构地区：[1]东北农业大学动物医学学院传染病教研室,哈尔滨150030

出　　处：《中国人兽共患病学报》2015年第1期6-10,共5页Chinese Journal of Zoonoses

基　　金：国家科技支撑计划项目(2012BAD12B03;2012BAD12B05);黑龙江省科技攻关计划项目(GC12B303)~~

摘　　要：目的建立一种快速检测大肠杆菌耐热肠毒素(heat-stable enterotoxin,STa,STb)和不耐热肠毒素(heat-labile enterotoxin,LT-Ⅰ,LT-Ⅱ)基因的多重PCR方法。方法参照文献合成四对可扩增产肠毒素大肠杆菌(Enterotoxigenic Escherichia coli,ETEC)耐热肠毒素基因(estA、estB)和不耐热肠毒素基因(elt-Ⅰ、elt-Ⅱ)的特异性引物,通过反应条件的优化,敏感性、特异性试验和临床样品检测,建立检测大肠杆菌肠毒素的多重PCR方法。结果用所建立的多重PCR方法可特异性扩增出estA(229bp)、estB(480bp)、elt-Ⅰ(605bp)和elt-Ⅱ(300bp)基因片段,最低检出量分别为2.55×101 CFU/μL、2×101 CFU/μL、2×101 CFU/μL和2.47×103 CFU/μL。从22株大肠杆菌分离株中检测到estA基因(2/22),elt-Ⅱ基因(3/22),未检测到estB和elt-Ⅰ基因,检测结果与常规PCR检测结果一致。结论建立了检测大肠杆菌肠毒素基因(estA、estB、elt-Ⅰ和elt-Ⅱ)的多重PCR方法,该方法具有良好的特异性和敏感性,能够满足对细菌培养物的检测要求。To develop a rapid and specific multiplex PCR method for detection of enterotoxins genes（estA,estB,elt-Ⅰand elt-Ⅱ）in ETEC,four pairs of primers were synthesized according to the conserved sequences of estA,estB,elt-Ⅰ and elt-Ⅱgenes.The assay was tested for its optimal reaction conditions,specificity,sensitivity,detection of clinical sample,and then the multiplex PCR method were developed.Results indicated that this method had a high specificity in detecting enterotoxins genes（229bp/estA,480bp/estB,605bp/elt-Ⅰ,and 300bp/elt-Ⅱ）,and the detection limit were 2.55×10^1 CFU/μL,2×10^1 CFU/μL,2×10^1 CFU/μL,and 2.47×10^3 CFU/μL,respectively.Using this multiplex PCR,we detected 22 E.coli strains isolated from fecal swabs of calves with diarrhea,and found 2 estAgene positive strains,3 elt-Ⅱgene positive strains,while no isolated strain was detected to carry the estBor elt-Ⅰ genes,and the assay result was in agreement with that of conventional PCR.This multiplex PCR method has high specificity and sensitivity,and it can satisfy the detection of bacterial cultures.

关 键 词：产肠毒素大肠杆菌 耐热肠毒素 不耐热肠毒素 多重PCR 

分 类 号：R378.2[医药卫生—病原生物学]