Hendra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 Hendra virus

目的建立针对Hendra病毒N基因的一步法RealtimeRT-PCR检测方法，用于Hendra病毒感染样本的快速检测和准确定量。方法针对Hendra病毒的保守基因N设计引物和探针，构建体外转录的RNA片段作为标准品，建立一步法Real-timeRT-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Hendra病毒株。本研究建立的一步法Real-timeRT-PCR方法可以特异性检测出Hendra病毒，不与Nipah病毒产生交叉反应。检测灵敏度为2．6×10^0～2．6×10^1 copies／μl。标准曲线的线性范围为2．6×10^1-2．6×10^7 copies／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Hendra病毒感染样本的检测。

Objective To develop one step Real-time RT-PCR （Taqman） assay of Hendra nucleoprotein so that the Hendra virus RNA in field specimens or laboratory material could be characterized rapidly and specifically and quanfitated. Method A pair of specific oligonucleotide primers was designed. Hendra RNA transcripts by transcription in vitro were used as positive standards. Result The linearity of the standard curve allowed quantification of 10^1 to 10^7 RNA transcripts/μl. The sensitivity of the test was close to 1 ～ 10 transcripts/μl. The assay was high specific without cross-reaction to Nipah virus transcrips or other virus. Conclusion This standardized technique is sensitive and reliable and allows rapid detection and quantitation of Hendra RNA in both field and experimental materials used for the surveillance and specific di- agnosis of Hendra virus.