TaqMan-MGB荧光定量RT-PCR快速检测副流感3型病毒

TaqMan-based real-time PCR assay for quick detection of parainfluenza type 3 virus

目的:建立特异、快速、灵敏的荧光定量RT-PCR方法用于副流感3型病毒核酸检测。方法:对副流感3型病毒的N基因应用C lustalW软件进行序列同源性比对,在保守区设计特异性引物和TaqM an-MGB探针,并对荧光RT-PCR反应条件进行优化,验证方法的特异性,敏感性和稳定性,同时应用于疑似患者临床标本检测。结果:该方法对副流感3型病毒核酸检测有高度特异性,与副流感1、2、4型,甲型、乙型流感病毒,麻疹病毒,风疹病毒,腮腺炎病毒,呼吸道合胞病毒和腺病毒等均无交叉反应。检测的灵敏度达0.1TC ID50,可从疑似患者含漱液标本中直接检出,从病毒核酸提取至完成检测仅需3 h左右。结论:本研究建立的TaqM an荧光定量RT-PCR是一种快速检测副流感3型病毒特异、敏感的方法,适用于临床早期诊断。

Objective： To establish a TaqMan - based real - time PCR assay for detection of parainfluenza type 3 virus. Methods：The gene sequences of parainfluenza type 3 virus downloaded from the genbank was aligned using the biologic software and the specific primers and probes were designed in the conserved region of the N gene for parainfluenza type 3. The PCR reactive condition was optimized to improve the sensitivity and specificity of the assay. The clinical specimens collected from the patients were detected by this assay. Results：The specificity of the assay was high and there were no cross reactions with PIV1 ,PIV2, PIV4,Influenza virus A and B, measles, adenovirus, mumps virus, respiratory syncytial virus. The sensitivity of the assay was 0. 1TCID50 and the viral RNA was directly detected from the clinical specimens by this assay. It took only three hours to extract viral RNA and do the real -time PCR. Conclusion：This TaqMan -based real -time PCR assay is a quick,sensitive and specific tool for molecular diagnosis of parainfluenza type 3 virus.