蚊媒甲病毒属病毒半巢式PCR方法的建立及应用

薛志静, 赵宁, 王君, 宋秀平, 孟凤霞, 梁文琴, 周敬祝, 王丹, 张忠, 刘起勇

doi:10.11853/j.issn.1003.8280.2021.02.003

中国媒介生物学及控制杂志 >

2021 , Vol. 32 >Issue 2: 132 - 138

DOI: https://doi.org/10.11853/j.issn.1003.8280.2021.02.003

实验研究

蚊媒甲病毒属病毒半巢式PCR方法的建立及应用

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1. 中国疾病预防控制中心传染病预防控制所媒介生物控制室, 传染病预防控制国家重点实验室, 北京 102206;
2. 山东第一医科大学基础医学院, 山东泰安 271000;
3. 贵州省疾病预防控制中心, 贵州贵阳 550004;
4. 山东省新发传染病溯源与防控协同创新中心, 山东泰安 271000

薛志静,女,在读博士,主要从事病媒生物与虫媒病研究工作,E-mail:573799518@qq.com

收稿日期: 2020-11-06

网络出版日期: 2021-04-20

基金资助

国家科技重大专项（2018ZX10101002-002）

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Establishment and application of RT-hemi-nested PCR assay for detection of mosquito-borne alphaviruses

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1. State Key Laboratory of Infectious Disease Prevention and Control, Department of Vector Biology and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China;
2. Department of Pathogenic Biology, Shandong First Medical University, Tai'an, Shandong 271000, China;
3. Guizhou Center for Disease Control and Prevention, Guiyang, Guizhou 550004, China;
4. Collaborative Innovation Center for the Origin and Control of Emerging Infectious Diseases, Tai'an, Shandong 271000, China

Received date: 2020-11-06

Online published: 2021-04-20

Supported by

Supported by the National Science and Technology Major Project of China (No.2018ZX10101002-002)

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摘要

目的建立基于蚊媒甲病毒属病毒通用引物的半巢式PCR（RT-hemi-nested PCR）检测方法，用于甲病毒属病毒的实验室检测。方法根据GenBank中的甲病毒属病毒全基因组序列，在其保守区设计3条甲病毒属病毒通用引物，以辛德毕斯病毒（SINV）cDNA、基孔肯雅病毒（CHIKV）cDNA为模板优化条件建立半巢式PCR方法，验证该方法的敏感性及特异性，并对野外采集的蚊虫标本进行检测。结果敏感性检测结果显示，SINV、CHIKV的检测下限值分别为3×10³和3×10⁴拷贝/μl。特异性检测结果显示，该体系仅能扩增蚊媒甲病毒属病毒目的基因而不能扩增其他病毒。用该方法对野外采集的蚊虫标本进行检测，结果显示未扩增出阳性条带。结论建立的半巢式PCR检测方法特异性强、灵敏度高，可用于甲病毒属病毒感染的流行病学调查研究。

关键词： 蚊虫; 甲病毒属病毒; 半巢式聚合酶链式反应; 检测

本文引用格式

薛志静, 赵宁, 王君, 宋秀平, 孟凤霞, 梁文琴, 周敬祝, 王丹, 张忠, 刘起勇 . 蚊媒甲病毒属病毒半巢式PCR方法的建立及应用[J]. 中国媒介生物学及控制杂志, 2021 , 32(2) : 132 -138 . DOI: 10.11853/j.issn.1003.8280.2021.02.003

Abstract

Objective To establish a RT-hemi-nested PCR assay for laboratory detection of mosquito-borne alphaviruses based on the universal primers of mosquito-borne alphaviruses. Methods Three universal primers of mosquito-borne alphaviruses were designed in the conserved regions based on the whole genome sequence of alphaviruses in GenBank. The RT-hemi-nested PCR assay was established using the cDNAs of Sindbis virus (SINV) and Chikungunya virus (CHIKV) as templates under optimized conditions; the sensitivity and specificity of this method were validated and mosquitoes collected in the field were tested. Results The sensitivity test results showed that the lowest detection limit of SINV and CHIKV was 3×10³ and 3×10⁴ copies/µl. The sensitivity test results showed that the RT-hemi-nested PCR was only able to amplify the alphavirus. The established method was used to detect mosquito specimens collected in the field. The results showed that mosquito-borne alphavirus was not detected in all samples. Conclusion The RT-hemi-nested PCR assay established in the study is highly specific and sensitive and can be used for epidemiological survey on alphavirus infection.

Key words： Mosquito; Alphavirus; RT-hemi-nested PCR; Detection

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