双重实时荧光PCR检测肠产毒大肠埃希菌肠毒素基因方法的建立

Detection of enterotoxin genes of enterotoxigenic Escherichia coli by duplex real-time fluorescence polymerase chain method

目的:建立一种快速、准确、灵敏的检测肠产毒大肠埃希菌肠毒素基因方法。方法:于GenBank中查找ETEC肠毒素基因lt和st序列并进行比对后设计引物及TaqMan-MGB探针。对引物、探针、Mg2+、TaqDNA聚合酶及dNTPs浓度进行优化,并对方法的灵敏度、特异性、重复性进行分析。结果:建立了双重实时荧光PCR法检测ETEC肠毒素基因lt和st方法。结论:建立了一种灵敏、特异的检测ETEC肠毒素基因的方法。该法在疾病控制相关和临床实验室中具有广泛的实际应用价值。

Objective：To develop a rapid,accurate,sensitive method to detect the enterotoxin genes of enterotoxigenic Escherichia coli.Methods： The sequences of enterotoxin genes lt and st of ETEC were searched and blasted,then primers and TaqMan-MGB probes were designed.The primers,probes,concentrations of Mg2＋,Taq DNA polymerase and dNTPs level were modified.The specificity,sensitivity and repeatability were analyzed.Results： A duplex real-time PCR assay was developed for the detection of enterotoxin genes lt and st of ETEC.Conclusion： A sensitive and specific method for the detection of enterotoxin genes of ETEC was developed.This method has widely practical application value in disease prevention and clinical laboratories.