Biology Reference
In-Depth Information
either live or properly frozen specimens. These techniques are expensive, time-
consuming, and may involve dissection and preparation of specimens from live
arthropods, which can be hazardous (
Barker 1994
).
The PCR offers another approach to detecting and identifying pathogenic
microorganisms if sequence information is available to design appropriate
primers (
Wise and Weaver 1991, Higgins and Azad 1995, Crowder et al. 2010
).
The PCR can be carried out with material from dead specimens, is more sensi-
tive than most immunological techniques, and is more rapid. For example, using
primers that amplify a 434-bp fragment of a protein from
Rickettsia rickettsii
,
infected fleas and ticks have been identified (
Azad et al. 1990
). Malarial DNA
has been detected in both infected blood and individual mosquitoes (
Schriefer
et al. 1991
). Using seminested PCR, as few as three
Leishmania
parasites could
be detected in infected sand flies (
Aransay et al. 2000
). Trypanosome infec-
tions could be detected in wild tsetse flies in Cameroon (
Morlais et al. 1998
).
The heartwater fever pathogen
Cowdria ruminantium
could be detected in vec-
tor ticks (
Amblyomma
) with high levels of specificity when 10
7
to 10
4
organisms
were present. The reliability of the assay dropped when ticks had only 10
3
to 10
2
organisms, which highlights the need to conduct quantitative analyses for sensi-
tivity before using PCR assays in epidemiological studies (
Peter et al. 2000
).
West Nile virus was detected in human clinical specimens, field-collected mosqui-
toes, and bird samples by a TaqMan reverse-transcriptase PCR assay (
Lanciotti et al.
2000
). This rapid, specific, and sensitive assay can be used in the diagnostic labora-
tory for testing humans and as a tool for conducting surveillance of West Nile virus
in mosquitoes and birds in the field (
Anderson et al. 1999
). Sequencing of the West
Nile virus causing encephalitis in the northeastern United States indicated the virus
was most closely related to a virus isolated in Israel in 1998 (
Lanciotti et al. 1999
).
A quantitative PCR protocol was used to assay densities of the plague bacteria
Yersinia pestis
in fleas and mice. The assays indicated fleas needed
≈
10
6
bacteria
to be able to transmit the bacteria to mice (
Engelthaler et al. 2000
). Random
primers (hexanucleotides) were used to develop primers for a multiplex reverse-
transcriptase PCR to detect five potato viruses and a viroid in aphids, leaves, and
potato tubers (
Lie and Singh 2001
).
Hamiduzzaman et al. (2010)
developed a
multiplex PCR assay to diagnose and quantify
Nosema apis
and
N. ceranae
infec-
tions in honey bees.
8.5.9 Detecting Pesticide Resistance
The malaria vectors
Anopheles gambiae
and
An. arabiensis
were screened
for permethrin resistance (nerve-insensitivity,
kdr
-type) (
Brooke et al. 1999
).
