Page 299 - The Veterinary Laboratory and Field Manual 3rd Edition

268 Susan C. Cork and Roy Halliwell

known spot. The obtained data are semi-quanti- ers, depending on the sample type. Any DNA
tative and may be validated using the real-time extraction and purifying process is likely to expe-
PCR technique. Commercial diagnostic micro- rience some loss in total DNA from the original
arrays is a developing field. In the near future, preparation and this needs to be taken into
it is expected that this technique may become consideration.
more popular in diagnostic laboratories due to Amplification relies on the use of primers
its ability to screen for multiple microbial agents (short segments of site-specific oligonucleotides)
in a single assay. to anneal to opposite strands of the target DNA
and by utilizing PCR for synthesizing and ampli-
fying the targeted sequence many fold. Primers
DNA restriction
need to be specific to prevent cross-reactivity
PuLSEd FIELd GEL ELEctroPHorESIS (PFGE) with non-targeted sequences. Primer design
Pulsed field gel electrophoresis is a method that programmes are available on the internet and
uses enzymes that cut at infrequently found custom-made primers are manufactured com-
sites (rare-cutting restriction enzymes) to cleave mercially to order. The PCR process basically
whole genomic DNA into large fragments that involves heat-mediated denaturation of DNA
are then separated by size on an electrophore- to a single-stranded state, followed by anneal-
sis gel. SNPs in any one of these rare restriction ing of the primers to the target sequence.
sites will lead to an altered banding pattern on Annealing temperature should be such that
the electrophoresis gel and have been used to it is stringent enough to prevent non-specific
identify strain types for some organisms, for binding. This step is followed by extension
example, Escherichia coli O157:H7, Salmonella, (DNA synthesis step) at a higher tempera-
Shigella, Listeria, or Campylobacter. Differences ture. The process is repeated around 35 times,
between operator handling and in the equipment theoretically doubling the amplified fragment
used can lead to inter-laboratory variations. In (amplicon) at every cycle resulting in over 10
9
order to facilitate comparisons between strains copies. Target based amplification is therefore
from laboratories around the world, protocol generally much more sensitive than probe based
standardization and laboratory certification methods for detecting organisms in clinical
processes have been established (for example, specimens.
PulseNet). The patterns obtained are then pho- There are a number of factors that need to
tographed, standardized and the data stored be considered when using PCR based detection.
digitally which enables ready comparison to Mutations in the primer binding site resulting in
strains in other laboratories. reduced sensitivity or non-amplification and pos-
sible amplification of closely related sequences
aMPLIFIcatIon resulting in false positives are such examples.
DNA for PCR must be free of the enzymatic Given the underlying principles of PCR, one of
inhibitors that can be present in growth media the greatest problems to manage is preventing
or in specimen samples, for example, stool amplification of non-specific DNA, either in the
specimens in particular. There are many DNA target organism or through contamination of
preparation methods, some as simple as a rapid the reaction. It is therefore very important that
boil preparation in water, to sophisticated com- PCR protocols be thoroughly validated in order
mercially available DNA kits, some of which to estimate the specificity and sensitivity of any
are for use on direct tissue samples. However, test and that positive and negative controls be
some methods are more successful than oth- included in each experimental run.

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