实时荧光定量PCR快速检测大肠埃希菌的方法研究

目的基于TaqMan探针建立产肠毒素大肠埃希菌实时荧光定量PCR检测方法。方法针对编码大肠埃希菌耐热肠毒素STI、不耐热性肠毒素LTI基因片段设计引物、探针,采用外标法,绘制标准曲线,进行荧光定量PCR检测。结果引物、探针特异性良好,该方法的灵敏度可达到每反应1DNA拷贝,特异性强,检测范围在100～107拷贝每反应,重复性及稳定性好,整个过程只需要2h。结论该方法能快速、灵敏、特异检出产肠毒素大肠埃希菌。

Study of rapid method on enterotoxigenic Escherichia coli detected by real-time fluoresenee quantitative PCR

Objective To develop a real-time PCR for detecting enterotoxigenic Escherichia coli(ETEC) based on TaqMan technology.Methods Primers and probes were designed in the coding region of heat-stable enterotoxin,heat-labile enterotoxin of ETEC.ETEC were detected by real-time fluoresence quantitative PCR,making use of the exterior standard curve which was described by several different concentration.The speciality,sensitivity,accuracy,repetition,and stability of real-time fluoresence quantitative PCR system were evaluated.Results Primers and TaqMan probes were suited to the real-time fluoresence quantitative PCR.The assay showed that the method could be rapid,special,sensitive and stabile.The real-time PCR system could detect ETEC between 100-107DNA copies/reaction.The assay should be finished in two hours.Conclusion It was suggested that real-time fluoresence quantitative PCR based on TaqMan probe could be a rapid,sensitive and special method.It is significant that the excellent method could control diarrhoea caused by ETEC.