Multiplex Real-Time PCR for Detection of Anaplasma phagocytophilum and Borrelia burgdorferi

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Abstract
A multiplex real-time PCR assay was developed for the simultaneous detection of Anaplasma phagocytophilum and Borrelia burgdorferi . The assay was tested on various Anaplasma , Borrelia , Erhlichia , and Rickettsia species, as well as on Bartonella henselae and Escherichia coli , and the assay was found to be highly specific for A. phagocytophilum and the Borrelia species tested ( B. burgdorferi , B. parkeri , B. andersonii , and B. bissettii ). The analytical sensitivity of the assay is comparable to that of previously described nested PCR assays ( A. phagocytophilum , 16S rRNA; B. burgdorferi , fla gene), amplifying the equivalent of one-eighth of an A. phagocytophilum -infected cell and 50 borrelia spirochetes. The dynamic range of the assay for both A. phagocytophilum and B. burgdorferi was ≥4 logs of magnitude. Purified DNA from A. phagocytophilum and B. burgdorferi was spiked into DNA extracted from uninfected ticks and from negative control mouse and human bloods, and these background DNAs were shown to have no significant effect on sensitivity or specificity of the assay. The assay was tested on field-collected Ixodes scapularis ticks and shown to have 100% concordance compared to previously described non-probe-based PCR assays. To our knowledge, this is the first report of a real-time multiplex PCR assay that can be used for the simultaneous and rapid screening of samples for A. phagocytophilum and Borrelia species, two of the most common tick-borne infectious agents in the United States.