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

李德, 殷启凯, 侯泽英, 王瑞晨, 张维嘉, 付士红, 何英, 聂凯, 梁国栋, 许松涛, 李樊, 李兴洲, 王环宇

doi:10.11853/j.issn.1003.8280.2024.01.013

中国媒介生物学及控制杂志 >

2024 , Vol. 35 >Issue 1: 74 - 78

DOI: https://doi.org/10.11853/j.issn.1003.8280.2024.01.013

技术与方法

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

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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

李德，男，在读硕士，主要从事虫媒病毒的分离鉴定，E-mail：2695586450@qq.com；殷启凯，男，硕士，助理研究员，主要从事虫媒病毒及人畜共患病传染病研究，E-mail：yinqk@ivdc.chinacdc.cn

收稿日期: 2023-08-31

网络出版日期: 2024-03-05

基金资助

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

收起

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

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1. School of Public Health, Jiamusi University, Jiamusi, Heilongjiang 154002, China;
2. 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

Received date: 2023-08-31

Online published: 2024-03-05

Supported by

National Key R&D Program of China (No. 2022YFC2302700); Science & Technology Fundamental Resources Investigation Program (No. 2022FY100904)

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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。

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

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李德, 殷启凯, 侯泽英, 王瑞晨, 张维嘉, 付士红, 何英, 聂凯, 梁国栋, 许松涛, 李樊, 李兴洲, 王环宇 . 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[J]. 中国媒介生物学及控制杂志, 2024 , 35(1) : 74 -78 . DOI: 10.11853/j.issn.1003.8280.2024.01.013

Abstract

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.

Key words： Alongshan virus; Songling virus; Multiplex real-time fluorescent quantitative reverse transcription polymerase chain reaction; Nucleic acid detection

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