阿龙山病毒及松岭病毒多重实时荧光定量RT-PCR快速检测方法的建立  

Establishment of multiplex real-time fluorescent quantitative RT-PCR for rapid detection of Alongshan virus and Songling virus

作　　者：李德 殷启凯 侯泽英 王瑞晨 张维嘉 付士红[2] 何英[2] 聂凯[2] 梁国栋[2] 许松涛[2] 李樊 李兴洲[1] 王环宇[2] LI De;YIN Qi-kai;HOU Ze-ying;WANG Rui-chen;ZHANG Wei-jia;FU Shi-hong;HE Ying;NIE Kai;LIANG Guo-dong;XU Song-tao;LI Fan;LI Xing-zhou;WANG Huan-yu(School of Public Health,Jiamusi University,Jiamusi,Heilongjiang 154002,China;National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases,National Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China)

机构地区：[1]佳木斯大学公共卫生学院,黑龙江佳木斯154002 [2]传染病溯源预警与智能决策全国重点实验室,中国疾病预防控制中心病毒病预防控制所,北京102206

出　　处：《中国媒介生物学及控制杂志》2024年第1期74-78,共5页Chinese Journal of Vector Biology and Control

基　　金：国家重点研发计划(2022YFC2302700);科技基础资源调查专项(2022FY100904)。

摘　　要：目的 建立一种多重实时荧光定量反转录聚合酶链式反应(多重实时qRT-PCR)法,用于阿龙山病毒(Alongshan virus,ALSV)及松岭病毒(Songling virus,SGLV)核酸的快速检测。方法 针对ALSV NS 3基因和SGLV S基因保守区设计特异性引物及TaqMan探针,建立针对2种病毒的多重实时qRT-PCR检测方法,评价方法的特异性、灵敏度和重复性,并应用蜱标本对该方法进行应用评价。结果 该检测方法与森林脑炎病毒等其他6种虫媒病毒无交叉反应,灵敏度达1×101拷贝/μl,重复性检测的循环阈值(Ct)变异系数均<2.00%;运用该方法,从2019年黑龙江省采集的30组蜱标本中检出2组ALSV阳性,1组SGLV阳性。经验证,该方法与普通PCR方法检测结果的一致性为100%。结论 建立了敏感性高、特异性好的ALSV和SGLV多重实时qRT-PCR快速检测方法。Objective To establish a multiplex real-time fluorescent quantitative reverse transcription polymerase chain reaction(multiplex real-time qRT-PCR)method for rapid detection of nucleic acids of Alongshan virus(ALSV)and Songling virus(SGLV).Methods Specific primers and TaqMan probes were designed for the conserved regions of the NS 3 gene of ALSV and the S gene of SGLV.A multiplex real-time qRT-PCR method was established for the two viruses,and the specificity,sensitivity,and repeatability of the method were evaluated.Tick specimens were used to verify the method.Results The detection method had no cross-reactivity with six other arboviruses,such as tick-borne encephalitis virus,with a sensitivity up to 1×101 copies/μl,and the coefficient of variation of cycle threshold for repeatability testing was less than 2.00%.Through this method,two groups of ALSV-positive specimens and one group of SGLV-positive specimens were detected from 30 groups of tick specimens collected from Heilongjiang,China in 2019.This method was verified to be 100%consistent with the general PCR method in terms of test results.Conclusion In this study,a highly sensitive and highly specific multiplex real-time qRT-PCR method for rapid detection of ALSV and SGLV has been successfully established.

关 键 词：阿龙山病毒 松岭病毒 多重实时荧光定量反转录聚合酶链式反应 核酸检测 

分 类 号：R373[医药卫生—病原生物学]