Biplex quantitative real-time PCR for detection of six common rodent-borne pathogens

CHEN Min, LIANG Ying, LIU Qi-yong, LI Dong-mei

doi:10.11853/j.issn.1003.8280.2025.01.018

Chinese Journal of Vector Biology and Control >

2025 , Vol. 36 >Issue 1: 99 - 105

DOI: https://doi.org/10.11853/j.issn.1003.8280.2025.01.018

Technology and Method

Biplex quantitative real-time PCR for detection of six common rodent-borne pathogens

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Department of Vector Biology and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China

Received date: 2024-06-12

Online published: 2025-03-05

Supported by

Vector Surveillance and Control Project (No. 102393220020020000012)

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Abstract

Objective To establish biplex quantitative real-time polymerase chain reaction (qPCR) methods for rapid detection of six rodent-borne pathogens, including Francisella tularensis, Bartonella spp., Leptospira interrogans, Rickettsia typhi, Anaplasma phagocytophilum, and Orientia tsutsugamushi. Methods Three biplex qPCR methods were established based on the reported qPCR assays for detection of F. tularensis and O. tsutsugamushi, Bartonella spp. and L. interrogans, R. typhi and A. phagocytophilum, respectively. Standard curves were plotted, and the sensitivity, specificity, and repeatability of these methods were determined. Meanwhile, simulated nucleic acid samples of rodent tissues infected with six rodent-borne pathogens and positive samples were detected to validate the reliability of the method. Results The three established biplex qPCR methods amplified only the target pathogens, and other pathogens and negative controls did not show fluorescence signals. The lower limit of detection for all six pathogens was 1×10¹copies/μl; the coefficient of variation of cycle threshold values ranged from 0.04% to 1.20%. The results of the simulated and positive samples were positive, with 100% consistency. Conclusion These biplex qPCR methods have good specificity, high sensitivity, and good repeatability, which can be used to rapidly and accurately detect F. tularensis, Bartonella spp., L. interrogans, R. typhi, A. phagocytophilum, and O. tsutsugamushi.

Key words： Rodent-borne pathogen; Biplex quantitative real-time PCR; Detection

Cite this article

CHEN Min, LIANG Ying, LIU Qi-yong, LI Dong-mei . Biplex quantitative real-time PCR for detection of six common rodent-borne pathogens[J]. Chinese Journal of Vector Biology and Control, 2025 , 36(1) : 99 -105 . DOI: 10.11853/j.issn.1003.8280.2025.01.018

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