东南亚十二节段RNA病毒荧光定量qRT-PCR和常规RT-PCR检测方法的建立

Development of Real-time Fluorescent Quantitative RT-PCR and Conventional RT-PCR for Detection of Southeastern Asian Dodeca RNA Viruses

东南亚十二节段RNA病毒属为呼肠孤病毒科中的一个新属,该属包括版纳病毒(BAV)、芒市病毒(MSV)、卡皮罗病毒(KDV)和辽宁病毒(LNV)四种病毒。为建立上述四种病毒的群特异性核酸检测方法,本研究根据BAV、MSV、KDV和LNV毒株VP12基因序列的保守区,设计特异性引物和TaqMan探针,建立了荧光定量qRT-PCR和常规RT-PCR检测方法,并分别进行了特异性、灵敏性和重复性的检测。实验结果表明,两种检测方法分别对四种病毒均有特异性的扩增和荧光信号检出,而对流行性出血病病毒(EHDV)、蓝舌病病毒(BTV)、中山病病毒(CHUV)、广西环状病毒(GXOV)、阿卡斑病毒(AKAV)、云南环状病毒(YUOV)等病毒无扩增和无有效荧光信号检出,具有较好的特异性。灵敏性实验结果显示,荧光定量qRT-PCR检测四种病毒核酸的下限可达10 copies/μL,常规RT-PCR的检测下限为10^(2) copies/μL,荧光定量方法灵敏度是常规方法的10倍。四种病毒荧光定量qRT-PCR方法的组内和组间重复试验标准差均小于0.8,变异系数均小于3%,表明该方法均具有良好的稳定性。以上结果表明,本研究建立的BAV、MSV、KDV和LNV四种病毒的荧光定量qRT-PCR和常规RT-PCR方法具有快速、准确、稳定等优点,可为四种病毒的早期诊断、快速检测、流行病学调查和实验研究等提供有效的技术方法。

Southeastern Asian dodeca RNA viruses(Seadornavirus) belong to a new genus of the Reoviridae family,which includes Banna virus(BAV),Mangshi virus(MSV),Kadipiro Virus(KDV) and Liaoning virus(LNV).In order to develop a group-specific nucleic acid detection method for BAV,MSV,KDV and LNV,specific primers and TaqMan probes were designed based on the conserved regions of the VP12 gene sequences of the 4 viruses for development of a real-time quantitative RT-PCR(qRT-PCR) and conventional RT-PCR.The results showed that both assays specifically amplified the 4 viruses but not for epizootic hemorrhagic disease virus(EHDV),bluetongue Virus(BTV),Chuzan virus(CHUV),Guangxi Orbivirus(GXOV),Akabane virus(AKAV) and Yunnan Orbivirus(YUOV).The detection limits were 10 copies/μL for qRT-PCR and 10^(2) copies/μL for conventional RT-PCR.In addition,the standard deviation and coefficient of variation of qRT-PCR were less than 0.8 and 3%,respectively,which indicated its good stability.In conclusion,qRT-PCR and RT-PCR methods developed here proved to be specific and sensitive for detection of BAV MSV,KDV and LNV.Both assays provided effective technical tools for early diagno sis,rapid detection,epidemiological investigation and experimental re search of the four viruses.