PCR Detection of Microbial Pathogens PCR Detection of Microbial ...

Standardization of PCR 61
specified in the SOP). This will provide a demonstration of the ultimate
robustness of the PCR. If it works as well with other reagents as with those
used in the originating laboratory, this should reduce the logistic burden of
the next phase of the validation, as the organizing laboratory need not supply
all the reagents to each participant.
The final PCR-based detection protocol should contain a simple and universal
sample treatment of the matrix in combination with a PCR-compatible
(pre)enrichment broth prior to amplification. This will give a degree of
familiarity to the method, which should encourage end users to replace conventional
methodology with it. Pre-enrichment is commonly employed in
several microbial detection techniques associated with foods and is used for
several reasons. It can ensure the presence of target cells in numbers above
the detection limit of the technique and either dilutes out food-derived
inhibitory substances or allows their digestion through bacterial growth
(12,13). The pre-enrichment step of the PCR-based method should be similar,
if not identical, to the conventional method, and in the case of food-borne
pathogens, there are several standard pre-enrichment methods currently available
(14–16). The simplest way of using a pre-enrichment culture would be
to put an aliquot directly into the PCR as target. This, however, may result in
inhibition of the PCR, which should be carefully ascertained. Some form of
secondary treatment may be necessary, ranging in complexity from simple
dilution of the enrichment culture, to DNA extraction, to the use of PCR
facilitators (17) (see Chapter 2). The most straightforward method should
be chosen, as it should always be remembered that the final method is
intended for use by routine high-throughput laboratories and general technical
staff.
The next and final phase, therefore, involves validation of the complete
PCR-based method as a comparison with the equivalent conventional
method. Again, this is done as an inter-laboratory trial involving 10–12 partners.
Samples of matrix spiked at various target cell densities are sent to each
participant by the organizing laboratory. In the Nordval guideline (18), these
levels should be zero, 1–10 cells, and 10–100 cells /25-g sample. The participants
should then incubate (pre-enrich) the samples, apply the PCR according
to the SOP, and record the results. Concurrently, they perform a
conventional detection procedure (plating, etc.) after the pre-enrichment and
record the results. The organizing laboratory should compare the results, or
responses, of each method. Results from at least eight laboratories with valid
results must be available, if the comparison is to be thorough (7). The diagnostic
specificity, diagnostic sensitivity, and the overall accuracy of each
method should be determined and compared. A robust and efficient PCRbased
method should be at least as accurate as the conventional method.