肠毒性大肠埃希菌肠毒素LTa基因特异性EMA-PCR检测方法的建立

Establishment of Method for Detecting Heat-labile Enterotoxin Subunit A of Enterotoxigenic E. coli by EMA-PCR

由于传统PCR技术无法区分样品中的死细菌与活细菌,为避免因样品中含有死细菌而造成的假阳性检测结果,本研究根据肠毒性大肠埃希菌(ETEC)不耐热肠毒素LTa基因设计特异性引物,利用叠氮溴乙锭(EMA)处理菌液,沸水浴法制备细菌裂解液,优化PCR条件,建立一种检测肠毒性大肠埃希菌活菌肠毒素基因的EMA-PCR方法。肠毒性大肠埃希菌CVCC196、CVCC197、CVCC200均能扩增出大小为438bp的特异性条带,而其他对照菌株未扩增出条带。经EMA处理,除了完全死菌组外,含有10mL/L～1 000mL/L活菌的混合悬液均可扩增出目的片段。因此,成功建立了一种快速、有效的检测肠毒性大肠埃希菌活菌肠毒素基因的EMA-PCR方法,该方法较传统PCR大大提高了检测的准确性,灵敏度可达19cfu/mL。

Since the traditional PCR technique can not distinguish dead and live bacteria in samples,in order to avoid the false positive test result caused by the samples containing dead bacteria,the primers were designed according to the LTa gene of the enterotoxigenic Escherichia coli（ETEC）,the bacteria were treated with EMA,the bacterial lysates were prepared through the boiling water bath method,and PCR conditions were optimized,and a rapid and effective detection method of EMA-PCR for enterotoxin gene of live ETEC was established in this study.The 438 bp amplicon was specifically detected in ETEC CVCC196,CVCC197 and CVCC200,and none in control strains.By EMA treatment,the target fragment was amplified in the mixed suspension containing 10 mL/L to 1 000 mL/L live bacteria and none in that containing 100% dead bacteria.In this study,the EMA-PCR method for detecting enterotoxin gene of live ETEC was successfully established.Compared with conventional PCR,the sensitivity of this method is up to 19 cfu/mL that greatly improves the accuracy of detection.