狂犬病病毒实时荧光RT-RPA等温检测方法的建立

Development of real time RT-RPA for Rapid Detection of Rabies virus

为了建立一种灵敏、特异、快捷的实时荧光重组酶聚合酶扩增(RPA)检测狂犬病病毒的方法,依据狂犬病病毒不分节段单股负链中N基因相对保守的核苷酸序列,设计了4条引物及1条探针,筛选出一套可用于RPA引物与探针组合,建立了狂犬病病毒的荧光RT-RPA检测方法,测试了该方法灵敏度和特异性,并进行了临床验证。结果显示,本研究建立的RPA方法在39℃恒温反应仅需20 min,能够特异的检测狂犬病病毒,最低检测限为1.83×101 Copies/μL,临床样品测试与标准方法结果一致。表明本研究建立的检测狂犬病病毒的荧光RT-RPA方法具有特异,灵敏,快速,简便的特点,适用于现场检测以及在基层实验室推广应用。

A sensitive,specific and rapid recombinant polymerase amplification assay(RPA)was developed for the detection of rabies virus(RV).Based on the relatively conservative nucleotide sequence of the N gene in the non segmental single strand of the rabies virus,four primers and one probe were designed and screened.Result showed that a set of RPA primers and probe combination which fit for the RPA.And then,based on this set of primers and probe,RV realtime RT-RPA detection method was established.Furthermore,the real-time RT-RPA was subjected to evaluation of sensitivity,specificity and the clinical samples.Results show that the real-time RT-RPA assay could be finished in 20 min at 39℃.The primer set used in RPA was specific and did not show any cross-amplification with other viruses.Experiments confirmed a detection limit as low as 1.83×101 Copies/μL.Results of the clinical detection showed that the developed realtime RT-RPA had 100%consistency when compared with RT-qPCR assay.The realtime RT-RPA method for detecting RV in this study is specific,sensitive,rapid and simple.It is suitable for field detection and application in the basic laboratory.