产肠毒素大肠杆菌快速检测方法的建立和评价

Establishment and evaluation of the loop-mediated isothermal amplification ( LAMP) assay in detection of enterotoxigenic Escherichia coli

目的利用环介导等温扩增(LAMP)技术,建立产肠毒素大肠杆菌(ETEC)的快速、便捷、敏感、特异的检测方法,并对该方法的特异性和敏感性进行评价,为实验动物检测和细菌性腹泻的诊断提供技术支持。方法根据GenBank公布的产肠毒素大肠杆菌的LT毒素基因序列(S60731.1)设计外引物和内引物进行LAMP扩增,对LAMP特异性和敏感性与PCR方法做比较。结果建立的LAMP方法检测最低浓度为100 pg/μL,灵敏度是PCR的10倍以上并具有较高的特异性,利用该方法对27份猴腹泻样品进行LAMP和PCR方法检测,发现PCR检出率为33.3%,LAMP(60 min内)结果与PCR相同,而LAMP(90 min内)检出率为92.6%,约是PCR检出率的3倍。结论建立了一种用于检测肠毒性大肠杆菌(ETEC)的LAMP检测方法,该方法特异性强,灵敏度高,方便快捷,适合于ETEC临床快速检测。

Objective Using loop-mediated isothermal amplification （LAMP） assay to develop a rapid, convenient, sensitivity and specificity of enterotoxigenic Escherichia coli （ETEC） detection method, and evaluate the specificity and sensitivity of this method, for detection and diagnosis of bacterial diarrhea in laboratory animals. Methods According to the published E. coli LT toxin gene sequences （S60731.1） to design PCR and LAMP primers, and to compare the specificity and sensitivity of LAMP with that of PCR. Result The detection limit of the established LAMP method were 100 pg, its sensitivity was 10 times higher than that of PCR, and had a high specificity. Using PCR and LAMP to detect 27 feces samples of monkey diarrhea, the results of LAMP （within 60 min） were the same as that of PC R, with a positive rate of 33.3%. But the positive rate of LAMP （within 90 min） was 92.6% , about 3 times higher than that of PCR. Conclusions We have established an ETEC LAMP detection method, which is highly specific, sensitive, convenient, suitable for rapid detection of ETEC clinical samples.