马达里亚加病毒TaqMan RT-PCR检测方法的建立

Establishment of a TaqMan RT-PCR assay for the detection of Madariaga virus

目的建立马达里亚加病毒(Madariaga virus,MADV)早期且快速的TaqMan反转录聚合酶链式反应(RT-PCR)检测方法。方法从GenBank中下载MADV基因序列,根据多序列比对结果选择MADV的NSP1基因中的高保守序列片段设计引物与探针,利用体外转录的RNA标准品建立了MADV的绝对定量分析模型,并进行灵敏度和特异性评价以及重复性验证。结果该检测方法的灵敏度为1.0×10^(2)拷贝/反应,与其它虫媒病毒无交叉反应,重复性检测变异系数均小于1.5%。通过本研究建立的快速检测方法对源自宁夏回族自治区、浙江省和云南省的共计81份不明原因脑炎病例血清标本及161批库蚊标本进行了测定,检测结果均为阴性。结论成功建立了MADV TaqMan RT-PCR的快速检测方法,可应用于实验室的早期检测。

A rapid assay for the Madariaga virus(MADV)was established by using TaqMan reverse transcription-polymerase chain reaction(RT-PCR).The MADV gene sequences were downloaded from GenBank,and the highly conserved region of the NSP1 gene of MADV was selected to design specific primers and a probe according to the results of multiple sequence alignment.The sensitivity,specificity and stability of this assay were evaluated with an absolute quantitative analysis model of MADV by using in vitro transcribed RNA standards.The sensitivity of this assay was 1.0×10^(2) copies/reaction,and no cross reaction with other arboviruses was observed.The coefficients of variation were all below 1.5%.A total of 81 serum samples from cases of encephalitis with unknown causes,and 161 batches of Culex samples collected from the Ningxia Hui Autonomous Region,Zhejiang and Yunnan Province,were tested with the established assay.The results were negative in all samples.Thus,a rapid TaqMan RT-PCR assay for MADV was established,which can be used for early detection of MADV in laboratory settings.