Nipah病毒一步法Real-time RT-PCR检测方法的建立

Development of one step real-time RT-PCR method for detection of Nipah virus

目的建立针对Nipah病毒N基因的一步法Real-time RT-PCR检测方法,用于Nipah病毒感染样本的快速准确检测和定量。方法针对Nipah病毒的保守基因N设计引物和探针,建立一步法Real-time RT-PCR反应方法并分析敏感性和特异性。结果所设计的引物经Blast检索可以用于检测所有已知的Nipah病毒株。本研究建立的一步法Real-time RT-PCR方法可以特异性检测出Nipah病毒,不与Hendra病毒产生交叉反应。检测灵敏度为1.1×100～1.1×101copies/μl。标准曲线的线性范围为1.1×102～1.1×106copies/μl。结论本研究建立的一步法real-time RT-PCR方法敏感性和特异性较高,且不易出现污染引起的假阳性结果,适合用于Nipah病毒感染样本的检测。

Objective To develop one step real-time RT-PCR（Taqman） assays of Nipah nucleoprotein so that Nipah virus RNA in field specimens or laboratory material can be characterized and quantitated rapidly specifically.Method A pair of specific oligonucleotide primers was designed.The linearity of the standard curve allowed quantification of 102 to 106 RNA transcripts/μl.Result The sensitivity of the test was close to 1～10 transcripts/μl.The assays were highly specific without cross-reaction with Hendra virus transcrips or other virus.Conclusion This standardized technique is sensitive and reliable and allows rapid detection and quantitation of Nipah RNA in both field and experimental materials used for the surveillance and specific diagnosis of Nipah virus.