犬瘟热病毒RT-nested PCR检测方法的建立与应用

根据犬瘟热病毒(CDV)弱毒株Onderstepoort的核衣壳蛋白(NP)基因序列设计了套式引物,建立了RT-nested PCR检测方法。特异性试验表明,该法可以特异扩增出CDV NP基因片段,但从RNA病毒狂犬病病毒(RV)、犬副流感病毒(CPIV)、犬冠状病毒(CCV),DNA病毒犬细小病毒(CPV)、犬腺病毒(CAV)及正常Vero细胞中均不能扩增出条带。敏感性试验表明,RT-PCR可以扩增10-6稀释度的病毒RNA,而建立的RT-nested PCR可以扩增到10-9稀释度的病毒RNA,后者的敏感性明显高于前者。用RT-nested PCR法检测了2006年我国东北地区临床表现犬瘟热症状的病例19例,检出率达90%,明显高于RT-PCR的检出率(45%)。部分病例扩增片段的测序结果表明,CDV NP基因出现个别碱基变异。研究结果表明,建立的犬瘟热病毒RT-nested PCR方法敏感、特异,适用于动物犬瘟热的快速诊断。

Development of RT-nested PCR for detecting canine distemper virus

A RT-nested PCR assay for detecting canine distemper virus(CDV) was established using 2 pairs of primers.The primers were designed using nucleocapsid protein(NP) gene information of CDV Onderstepoort strain in GenBank.The assay amplified only a NP gene from CDV;but not from other virus such as rabies virus (RV),canine parainfluenza virus(CPIV),canine parvovirus(CPV),canine adenovirus(CAV),or Vero cell.Serial 10-fold dilutions of 10-6 TCID50 CDV3 strain were used for both PCR and RT-nested PCR.The lower detection limit for CDV RNA was 10-9 and 10-6 dilution for the RT-nested PCR and the traditional RT-PCR,respectively.Of the 19 CD samples collected in Northeast region of China in 2006,the positive ratio was up to 90% and 45% for RT-nested PCR and the traditional RT-PCR,respectively.Sequences of parts of the amplified products from viscera samples were consistent with what have been reported although minor variations may occur occasionally.The results showed that the RT-nested PCR was easy,specific and sensitive for detecting CDV.It could be used for a quick diagnosis of CD.