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

XUE Zhi-jing, ZHAO Ning, WANG Jun, SONG Xiu-ping, MENG Feng-xia, LIANG Wen-qin, ZHOU Jing-zhu, WANG Dan, ZHANG Zhong, LIU Qi-yong

doi:10.11853/j.issn.1003.8280.2021.02.003

Chinese Journal of Vector Biology and Control >

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

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

Experimental Study

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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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

Cite this article

XUE Zhi-jing, ZHAO Ning, WANG Jun, SONG Xiu-ping, MENG Feng-xia, LIANG Wen-qin, ZHOU Jing-zhu, WANG Dan, ZHANG Zhong, LIU Qi-yong . Establishment and application of RT-hemi-nested PCR assay for detection of mosquito-borne alphaviruses[J]. Chinese Journal of Vector Biology and Control, 2021 , 32(2) : 132 -138 . DOI: 10.11853/j.issn.1003.8280.2021.02.003

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