猴副流感病毒RT-PCR检测方法的建立及初步应用

Rapid Detection of Simian Parainfluenza Virus 5 by RT-PCR

为对易感动物以及相关生物材料和生物制品进行猴副流感病毒(SV5)的快速病原检测,根据SV5病毒SH基因序列,设计了1对引物,建立了检测SV5的RT-PCR方法,通过优化确定了RT-PCR体系的最佳工作条件。核酸序列分析显示,所扩增片段与GenBank数据库中报道的SV5病毒株WR-21005序列同源性达到97%,证明该方法特异。敏感性分析显示,该方法可检测的最小RNA浓度为2.1 ng/μL,能检出的最小病毒滴度为3.98CCID50/0.1 mL。初步将该方法运用于猴、地鼠、常用传代细胞和猴源生物制品的检测,在所检测的样品中均未检出猴副流感病毒核酸序列。结果表明,所建立的RT-PCR方法可用于猴副流感病毒(SV5)的诊断和分子流行病学调查。

To detect the presence of Simian parainfluenza virus 5 （SV5） in laboratory animals and related biological products or materials, a pair of primers that amplify the SH gene were designed and synthesized. Mter the best reaction conditions were tested out, RT-PCR technique was established for the detection of RNA of SV5. By nucleic acid sequence analysis, sequence homology of RT-PCR positive fragment and the reported SV5 WR-21005 strain achieved 97%, which testified that the method was specific for the detection of SV5 nucleic acid sequence. Sensitivity of the method was determined as the minimum RNA concentration of 2.1 ng/t^L and the minimum virus titer of 3.98CCID5o/0.1 mL. For initial application of the method, samples including organs of simian and hamster＇ s, biological products from simian （OPV） and five commonly used passage cells from simian kidney were tested by the RT-PCR method for the SV5 nucleotide gene. Results came out that no SV5 nucleotide gene was detected in above specimens. The results showed that the SV5 RT-PCR technique we established provided a diagnosis method of SV5, and also a technique for SV5 molecular epidemiologic survey.