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.
Anaplasma phagocytophilumBorrelia burgdorferiSpotted fever group RickettsiaMultiplex real-time fluorescence quantitative PCR
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