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influence the ability to produce a transmissible infection in the vector and to
disseminate from a bite site after transmission.
Many arthropod-borne viruses, bacteria, and parasites are difficult to culture
in the laboratory, and microscopic direct counts of microbes in infected
tissues are labor-intensive and relatively insensitive. The advent of quantitative
molecular tools has provided rapid and sensitive alternative means to determine
microbial numbers at different stages of infection in arthropod and mammalian
hosts. Prominent among these new tools is real-time quantitative PCR (qPCR).
Several instrumentation and detection systems devoted to real-time PCR are
commercially available and have been used widely to quantitate gene and
transcript copy numbers (reviewed in
refs.
2-5
). Although these systems differ
in their details, they share the same basic principles: The accumulation of a
target sequence is measured in real time during the exponential phase of PCR
via a fluorescent label. The number of PCR cycles needed for amplification-
associated fluorescence to reach a specific threshold level of detection (the
C
t
value) is inversely proportional to the amount of target sequence in the sample.
Therefore, the copy number of the target in an unknown sample can be deter-
mined by interpolation of its
C
t
value versus a standard curve of
C
t
values
obtained from a serially diluted solution containing known amounts of target.
A variety of real-time qPCR strategies have been successfully applied
to detect and quantify arthropod-borne pathogens in their vectors and
hosts. From these studies has come important new information on the
kinetics and progression of infection in both the arthropod and the mammal
(6,7,8,9,10,11,12)
, the number of microbes transmitted by an infected vector
during a blood meal
(13,14,15)
, the number of microbes taken up by an
uninfected vector in an infected blood meal
(13,16)
, and the comparative
competence and transmission efficiency of different arthropod vectors for an
agent
(17,18)
. In this chapter, we detail real-time qPCR protocols used to inves-
tigate flea-borne transmission dynamics and pathogenicity of
Yersinia pestis
(13,16)
. A key feature is the use of tissue-matched standards containing known
numbers of
Y. pestis
, minimizing the effect of PCR inhibitors in different sample
tissues and allowing absolute quantification of bacterial cells. We have found
that estimation of
Y. pestis
in flea, skin, and lymph node samples by qPCR
compared favorably with the “gold standard” colony-forming unit quantitation
method (
see
Fig. 1
).
2. Materials
1. ABI Prism 7700 or 7900HT Sequence Detection System (Applied Biosystems,
Foster City, CA) and associated data analysis software.
2. Primer Express software v2.0 (Applied Biosystems).
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