小反刍兽疫病毒与产气荚膜梭菌双重RT-PCR检测方法的建立

Establishment of a Duplex RT-PCR for Detecting Peste Des Petits Ruminants Virus and C.perfringensin Sheep and Goats

为实现小反刍兽疫病毒与产气荚膜梭菌的鉴别诊断,采用改良RNA/DNA提取试剂提取动物组织中的细菌DNA和病毒RNA,基于2014年流行的小反刍兽疫病毒N蛋白基因和产气荚膜梭菌α毒素编码基因序列分别设计合成特异性引物,建立双重PCR方法,优化反应条件,并分析方法的灵敏度与特异性。结果表明,所建立的双重PCR检测方法可以同步检测PPRV和产气荚膜梭菌,分别扩增出406bp和272bp大小特异性条带,方法特异性好,检测灵敏度能够分别达到0.001TCID50/mL和10个CFU/mL。

In order to make the simultaneous detection of PRRV and C. perfringens, a duplex RT-PCR method was established. The RNA of PRRV and DNA of C. perfringens from animal tissues were extrac- ted by modified extraction method. Two pairs of specific primers were designed according to the N gene of PRRV and a toxin gene of C. perfringens. Based on optimizing PCR reaction condition, the duplex RT- PCR assay was developed, and two bands of 406 bp（PRRV） and 272 bp（C. perfringens） emerged on the gel simultaneously. The relative sensitivity and specificity of the duplex RT-PCR were evaluated. The re- sults showed that the method was specific, sensitive and stable, and no specific band could be amplified from other pathogens. The sensitivity of the assay was 0. 001 TCID50/mL and 1 CFU/mL, respectively.