摘要
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.
作者
殷启凯
刘文婧
王国玮
张维嘉
付士红
韩坤
许崇晓
许松涛
李樊
刘亚宁
何英
王振海
聂凯
王环宇
YIN Qi-kai;LIU Wen-jing;WANG Guo-wei;ZHANG Wei-jia;FU Shi-hong;HAN Kun;XU Chong-xiao;XU Song-tao;LI Fan;LIU Ya-ning;HE Ying;WANG Zhen-hai;NIE Kai;WANG Huan-yu(National Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China;State Key Laboratory of Infectious Disease Prevention and Control,National Health Commission Key Laboratory of Biosafety,Beijing 102206,China;Ningxia Medical University,Yinchuan 750004,China;Ningxia Hui Autonomous Region Center for Disease Control and Prevention,Yinchuan 750004,China;Department of Neurology,General Hospital of Ningxia Medical University,Engineering Research Center for Diagnosis and Treatment of Ningxia Nervous System Diseases,Yinchuan 750004,China)
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2022年第5期428-432,共5页
Chinese Journal of Zoonoses
基金
宁夏回族自治区重点研发计划(No.2019BCG01003)
传染病预防控制国家重点实验室重点项目(No.2015SKLID505)联合资助。