RT-PCR检测CPIV方法的建立与应用

【目的】建立CPIV的RT-PCR检测方法。【方法】从临床上疑似犬副流感(CPI)的犬鼻腔分泌物中提取CPIV总RNA。根据Genebank中收录的CPIV(序列号AY581307)中NP基因保守序列,设计一对特异性引物,扩增出667 bp片段,以此建立CPIV的RT-PCR检测方法。【结果】特异性试验表明该引物不能扩增犬其它常见的传染病病毒基因。敏感性试验表明该引物能检测到10~(-6)的CPIV总RNA量。重复性试验表明该引物具有良好的稳定性和重复性。对临床采集的32份样品进行RT-PCR检测时,阳性率为53.12%。【结论】实验建立了CPI的RT-PCR检测方法,可适合于临床CPIV的早期快速诊断和小规模的分子流行病学调查。

Foundation and Application of RT - PCR Method to Detect Canine Parainfluenza Virus in Pet Dogs

【Objective and Method】The total RNA template was isolated from the nasal secretion of clinically infected dogs with canine parainfluenza virus(CPIV).According to the sequence of CPIV published in GenBank,a pair of primers were designed in NP gene region and the specific 667bp nucleotides band was found on agarose electrophoresis.The RT-PCR method for detection of CPIV was established by this way.【Result】The specific test found the primers could only amplify the genome of CPIV,rather than canine distemper virus,canine parvovirus,canine adenovirus and rabies virus.As little as 10~(-6) total RNA template of CPIV could be detected in the sensitive test.The reproducibility for the RT-PCR was highly stable.53.12% of positive cases were found in 32 samples by RT-PCR.【Conclusion】The established RT-PCR method in this paper could be used in the early clinical diagnosis and the small-scale of epidemiological survey for CPIV infections in dogs.