尼帕病毒实时荧光RT-PCR检测方法的建立

Establishment of Real-time RT-PCR method for the detection of Nipah virus

目的建立快速检测尼帕病毒的实时荧光RT-PCR方法。方法分析尼帕病毒N基因片段核苷酸序列的保守性,设计实时荧光RT-PCR引物和探针,优化引物和探针浓度,利用尼帕病毒DNA质粒作为阳性对照,确定最佳反应体系,并进行灵敏性、重复性、特异性检测。结果该方法对尼帕病毒DNA的最适线性检测范围为102~108拷贝/μl,检测下限为102拷贝/μl。对2个不同浓度(102、104拷贝/μl)的尼帕病毒DNA进行4次重复检测,具有良好的重复性。与登革病毒、基孔肯雅病毒、黄热病毒、乙脑病毒和汉坦病毒等无交叉反应。结论建立的尼帕病毒实时荧光RT-PCR法能准确、快速地检测尼帕病毒。

Objective To estsblish a fast and sensitive method for the detection of Nipah virus(NIV)by using Real-time RT-PCR.Methods The N segment sequences of NIV strains were analyzed.A pair of primers and probe were designed in the conserved region and used for Real-time RT-PCR.The optimal reaction system,sensitivity and specificity were determined by using NIV DNA positive control.Results The optimal linear detection range of NIV-N-DNA was 102-108copies/μl,and the detection limit was 102copies/μl.Four replicates of two different concentrationsof NIV-N-DNA were tested with good repeatability,and had no cross reaction with Dengue virus,Chikungunya virus,Yellow fever virus,Japanese encephalitis virus,Hataan virus.ConclusionThe Real-time RT-PCR method with accurate,rapid for NIV detection had been established,which could be used for the rapid detection of NIV.