Page 300 - The Veterinary Laboratory and Field Manual 3rd Edition

Microbiology 269

Following PCR, the resulting amplicon can be organisms for which there is currently not
checked a number of ways to determine whether much sequence data.
it is the expected targeted sequence and the • Arbitrarily primed PCR (AP-PCR) is a related
method will depend on what type of amplifica- technique.
tion has been used. However, results need to be • Asymmetric PCR, which is used primarily
assessed in context along with other diagnostic to produce multiple copies of a single DNA
evidence as, just because DNA from an organ- strand.
ism has been successfully amplified, it does not • Reverse transcriptase PCR, where reverse
automatically follow that this organism is the transcriptase is used to produce copy DNA
cause of the disease, for example, it could be a (cDNA) from RNA (and hence, can also be
case of mixed infection. used for RNA virus detection). The cDNA
then acts as a template for conventional PCR.
convEntIonaL Pcr • Combinations of PCR plus enzyme restric-
Conventional PCR uses a pair of primers tion are also used, for example, amplified
designed to anneal to the opposite strands of fragment length polymorphism (AFLP).
DNA at either end of the targeted sequence
and the generation of multiple copies of this There are some limitations to conventional PCR
region by repeated synthesis cycles in a thermo- when compared to the real-time PCR including;
cycler (Figure 4.32). The products of all these (1) it is not quantitative and only a detection
reactions are visualized and the fragment size method, (2) it is less sensitive than real-time
estimated by running on electrophoresis gels. PCR, (3) time consuming and, (4) prone to con-
The amounts of DNA product can be estimated tamination and brings health hazards because of
against standards on gels, but it is, at best, only post-PCR processing. In fact, non-quantitative
semi-quantitative. Alternatively, the amplicons conventional PCR techniques are not recom-
can be analysed by sequencing them which then mended for viral disease diagnosis in situations
allows comparison/matching with sequences in where viruses that are found in healthy animals
databases. There are a number of variations on can cause clinical disease. For these reasons,
this basic technique. many laboratories are increasingly applying
real-time PCR for diagnostic purposes.
• Nested PCR, where specificity is increased
by using a second set of internally positioned rEaL-tIME Pcr (qPcr)
primers (or partially internal as is the case Real-time PCR assays use either fluorescent
with hemi-nested PCR) to amplify a smaller reporter probes (of which there are different
section of the original amplicon. types) that bind to the target sequence or, DNA
• Multiplex PCR, where multiple primer sets dyes that intercalate with double stranded DNA.
are included in a reaction to simultaneously Both detection methods rely on changes in the
identify a number of targeted regions, for fluorescent signal to measure double stranded
example, multiple antibiotic gene markers in DNA. Therefore, one of the main benefits of
an isolate. real-time PCR is the direct quantification of
• Random amplification of polymorphic DNA PCR product (hence the abbreviation, qPCR)
(RAPD). PCR reactions are conducted with by fluorescence and post-PCR processing is not
whole genome DNA and short random required (Figure 4.33). This technique can also
primers. The resulting pattern is used to be employed for quantification of target RNA by
characterize strains and can be useful for using reverse transcriptase to produce cDNA and

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