| Abstrakt: |
ABSTRACTA multiplex real-time PCR assay was developed for the simultaneous detection of Anaplasma phagocytophilumand Borrelia burgdorferi. The assay was tested on various Anaplasma, Borrelia, Erhlichia, and Rickettsiaspecies, as well as on Bartonella henselaeand Escherichia coli, and the assay was found to be highly specific for A. phagocytophilumand the Borreliaspecies 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, flagene), 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. phagocytophilumand B. burgdorferiwas =4 logs of magnitude. Purified DNA from A. phagocytophilumand B. burgdorferiwas 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 scapularisticks 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. phagocytophilumand Borreliaspecies, two of the most common tick-borne infectious agents in the United States. |
