Establishment of a multiplex real-time fluorescence quantitative PCR method for three tick-borne bacterial pathogens

HU Wei-chao, LI Jin-yu, ZHAO Ning, LIU Qi-yong, WEN Hong-ling, WU Hai-xia

doi:10.11853/j.issn.1003.8280.2024.03.014

Chinese Journal of Vector Biology and Control >

2024 , Vol. 35 >Issue 3: 334 - 338

DOI: https://doi.org/10.11853/j.issn.1003.8280.2024.03.014

Technology and Method

Establishment of a multiplex real-time fluorescence quantitative PCR method for three tick-borne bacterial pathogens

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1. Department of Microbiological Laboratory Technology, Key Laboratory of Prevention and Control of Emerging Infectious Diseases and Biological Safety in Universities of Shandong, Department of Vector Control, School of Public Health, Cheeloo College Medicine, Shandong University, Jinan, Shandong 250012, China;
2. Department of Vector Biology and Control, National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, WHO Collaborating Centre for Vector Surveillance and Management, Beijing 102206, China

Received date: 2024-03-12

Online published: 2024-06-29

Supported by

National Science and Technology Major Project of China (No. 2017ZX10303404-006-002); Vector Surveillance and Control Project (No. 102393220020000012)

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Abstract

Objective To establish a multiplex real-time fluorescence quantitative PCR (qPCR) method for simultaneous detection of the common tick-borne pathogens Anaplasma phagocytophilum, Borrelia burgdorferi, and spotted fever group Rickettsia (SFGR).Methods Specific primers and TaqMan probes were designed against the conserved regions of pathogens,and a multiplex qPCR system was established and optimized. The specificity, sensitivity, repeatability,and accuracy for tick samples were evaluated.Results There were no cross reactivity with Escherichia coli,Leptospira interrogans, Brucella sp. in the detection of A. phagocytophilum, B. burgdorferi, and SFGR by the established multiplex qPCR method and showed high specificity. The sensitivity reached the order of 10² copies/μl, and the coefficient of variation for repeatability was less than 2.00%. In tick sample detection, the results of multiplex qPCR method were consistent with the singleplex qPCR method, with 100% consistency.Conclusion The established multiplex qPCR method for three tick-borne bacterial pathogens has high specificity and sensitivity, and can be used as a technical means for the detection of common tick-borne bacterial pathogens.

Key words： Anaplasma phagocytophilum; Borrelia burgdorferi; Spotted fever group Rickettsia; Multiplex real-time fluorescence quantitative PCR

Cite this article

HU Wei-chao, LI Jin-yu, ZHAO Ning, LIU Qi-yong, WEN Hong-ling, WU Hai-xia . Establishment of a multiplex real-time fluorescence quantitative PCR method for three tick-borne bacterial pathogens[J]. Chinese Journal of Vector Biology and Control, 2024 , 35(3) : 334 -338 . DOI: 10.11853/j.issn.1003.8280.2024.03.014

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