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目的建立特异、快速、灵敏的、用于人博卡病毒核酸检测的荧光定量PCR方法。方法对博卡病毒的ns1基因应用Clustal W软件进行序列同源性比对,在保守区设计特异性引物和TaqMan探针,并对荧光PCR反应条件进行优化,验证方法的特异性,并将该方法与国际上通用的针对人博卡病毒ns1基因和np基因的普通PCR方法进行灵敏度比对;同时将该方法应用于疑似患者临床标本检测。结果该方法针对ns1基因进行扩增,对人博卡病毒核酸检测有高度特异性,与副流感1、2、3型,甲型、乙型流感病毒,呼吸道合胞病毒,腺病毒,冠状病毒229E、OC43,偏肺病毒以及鼻病毒等均无交叉反应。检测的灵敏度均比国际上通用的扩增ns1基因及np基因的PCR方法高10倍。用该方法从144份急性上呼吸道感染患者咽拭标本中检出了2份博卡病毒阳性,从病毒核酸提取至检测完成仅需3 h左右;用普通PCR仅能检出1份阳性标本,且耗时需5 h左右。结论该研究建立的TaqMan荧光定量PCR是一种快速检测人博卡病毒特异、敏感的方法,适用于临床早期诊断。
Objective To establish a specific, rapid and sensitive fluorescent quantitative PCR method for the detection of human Boka virus nucleic acid. Methods Clustal W software was used to sequence homology of ns1 gene of boca virus. Specific primers and TaqMan probes were designed in conserved regions. The PCR reaction conditions were optimized to verify the specificity of the method. Methods The sensitivity was compared with the common PCR method for human bark napsin and np gene, and the method was applied to the detection of clinical specimens in suspected patients. Results The method was based on the amplification of ns1 gene and was highly specific to the detection of human BACV nucleic acid. The method was compatible with the parainfluenza virus type 1, 2, 3, A, B, respiratory syncytial virus, adenovirus, coronavirus 229E, OC43, metapneumovirus and rhinovirus. The sensitivity of the detection is 10 times higher than that of the commonly used PCR methods for amplifying ns1 gene and np gene. Two samples of pharyngeal swabs from 144 patients with acute upper respiratory tract infection were detected positive by this method. Only 3 h was needed after the detection of the virus nucleic acid. Only 1 positive specimen could be detected by ordinary PCR, And takes about 5h. Conclusion The TaqMan fluorescent quantitative PCR established in this study is a specific and sensitive method for rapid detection of human Bocavirus and is suitable for early clinical diagnosis.