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.
