TaqMan-rRT-PCR检测猪霍乱沙门菌方法的建立和实验室评价

Development and evaluation of TaqMan real-time reverse transcription PCR assays for detection of Salmonella Choleraesuis

目的基于猪霍乱沙门菌血清型特异性基因,建立TaqMan逆转录实时聚合酶链反应(TaqMan-rRT-PCR)检测猪霍乱沙门菌的方法。方法利用基因组序列比对筛选到猪霍乱沙门菌特有的基因SC0358,通过普通PCR及利用多种血清型沙门菌及非沙门菌菌株共计145株,评价该方法的菌株特异性,针对该基因设计TaqMan-rRT-PCR检测方法的引物,优化反应条件,建立针对该靶基因的TaqMan-rRT-PCR检测方法,以纯菌及血液模拟标本RNA为模板进行敏感性检测。结果利用该方法检测26株猪霍乱沙门菌均为阳性,其余菌株扩增均阴性,对纯菌RNA检测中,TaqMan-rRT-PCR的最低检测限度为5 fg/反应,约为10个拷贝/反应。对全血液模拟样品分析中,敏感性达25 cfu/mL。结论以猪霍乱沙门菌保守、特异基因SC0358建立的TaqMan-rRT-PCR方法,能够简便快捷区分猪霍乱沙门菌与其他血清型沙门菌,尤其能够区分与其有抗原式相同的丙型副伤寒沙门菌,此方法为猪霍乱沙门菌感染的快速诊断提供了简便的手段,可用于对猪霍乱沙门菌的早期诊断。

A TaqMan real-time reverse transcription polymerase chain reaction(TaqMan-rRT-PCR)assay was established for the detection of Salmonella Choleraesuis on the basis of the SC 0358 gene.The specific S.Choleraesuis gene SC 0358 was identified through alignment of the genome sequences of S.Choleraesuis with those of other Salmonella serotypes in GenBank and the human genome,and the specificity of SC 0358 was confirmed by PCR.The specificity and detection limit of the TaqMan-rRT-PCR assay were evaluated on pure culture strains and S.Choleraesuis simulated blood specimens.All S.Choleraesuis strains in our study were amplified and found to be positive with TaqMan-rRT-PCR assays,whereas other isolates were negative.The detection limit of total RNA from the pure cultured isolates was 5 fg/reaction,equivalent to 10 molecular copies per reaction.For RNA from stimulated blood,the sensitivity was 25 cfu/mL.The established assay based on the conserved and specific gene SC 0358 for detecting S.Choleraesuis had high sensitivity and specificity in distinguishing S.Choleraesuis from other serotypes,particularly S.Paratyphi C,with the same antigenic formula.This method should be suitable for rapid detection and diagnosis in early clinical treatment.