Biology Reference
In-Depth Information
Viral targets
For direct surveillance of viral pathogens with PCR, the DNA sequences
that have been used for pathogen detection usually target a small portion of
the viral genome, such as the 145 bp fragment in the 5′ non-coding region
(5′ NCR) of Enterovirus,
44
the vp7 gene of Rotavirus,
45
and the hexon
and fiber genes of adenovirus,
46
among others.
47
Longer targets are being
studied in the hope that the length of the amplicon can be correlated to
the infectivity of the virus. This technique is based on amplification of large
parts or in some cases the entire genome, which if damaged would not
produce amplicons because the primers would be attached to different frag-
ments. The detection of damaged genome would indicate noninfectivity
of the virus.
35
In reverse transcriptase PCR (RT-PCR), damaged genomes
would also not be polymerized and fail to be detected.
Bacterial targets
The most common DNA sequences that have been targeted for detec-
tion of bacteria have been the conserved regions of the 16S rRNA
gene.
48,49,50
These genes contain nine hypervariable regions that consti-
tute useful targets for all types of diagnostic testing. Because not enough
variability exists between strains of the same species, other targets have
also been studied such as the 23S rRNA gene
51,52
and the internal tran-
scribed spacer (ITS) between the 16S rRNA and 23S rRNA genes.
49,53
With enrichment, Lin and Tsen
54
were able to detect
E. coli
down to
1 cfu in 100 mL of water. Aside from these general targets, specific species
targets have also been tested to confirm that positive testing reflects the
specific organism in question. Therefore, for
E. coli
the
uidA
gene that
codes for the β-D-glucuronidase enzyme
53-59
and the
tir
gene that tar-
gets the translocated intimin receptor
60
have been tested. Although
E. coli
was detected at very low levels with the
uidA
target, it was also found to
cross-react with
Shigella
species and other
uidA
-carrying organisms.
59,61
The
tir
gene has shown good specificity; however, whether single-copy
genes targets are sensitive enough for routine monitoring practices still
needs to be studied.
Genetic determinants encoding virulence-associated traits, such as the
shiga toxin (
stx
) and the intimin producing attaching and effacing (
eae
)
genes of diarrheagenic
E. coli
O157:H7 and non-O157 strains,
58,62
the inva-
sion associated gene A (
invA
) of
S. typhimurium
8,63,64
and the invasion plas-
mid antigen H (
ipaH
) gene of
Shigella flexneri
65,66
involved in the entry into
intestinal epithelial cells have also been examined.
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