TaqMan-MGB荧光定量RT-PCR快速检测副流感3型病毒

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。

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

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 0.1TCID_(50) 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.