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

60 Hoorfar and Cook
The choosing and testing of primers should be the first phase in the production
of a standard PCR. Here, laboratories, which are expert in PCR methodology
for a particular pathogen, should decide collectively which primer
sets will be selected for evaluation. This will generally be done on the basis
of those laboratories’ experience of the primers in use. Next, laboratories
with particular expertise in working with the pathogen concerned should draw
up a specificity strain list. This list should contain strains that comprehensively
represent the pathogen and representatives of cross-reacting species.
These other nontarget species should be ones that are closely related to the
target species, and the list may also contain more distantly related or
nonrelated species, if it is considered that they may be encountered with the
target species in the food matrix ultimately to be examined. DNA should be
extracted from each strain and analyzed in a series of PCRs containing the
primer sets to be evaluated. Each PCR should contain reagents that are as
identical as possible (supplier, batch, etc.) and use thermal cyclers that are
routinely calibrated and checked. The initial evaluation should be performed
through a limited interlaboratory trial, including at least three expert laboratories
as partners (see Fig. 2). The criteria for successful evaluation should
be strong, with specific amplification of correct target sequences and no others.
One primer set should be chosen to take forward into the next rounds of
the standardization process. The PCR should then be optimized (see Chapter
4). The detection limit of the PCR, in terms of the number of cells it can
detect with 99 % probability (11), should be established thereafter.
The second phase should take the form of a large-scale interlaboratory
trial to confirm the specificity of the PCR. This trial should involve 10–
12 partners (7), in addition to the organizing laboratory. Each participating
laboratory receives a Standard Operating Procedure (SOP), samples of
DNA from the strain list established previously, and sufficient reagents (once
again as identical as possible, e.g., same supplier and batch) to perform PCRs
in duplicate upon the DNA. The DNA samples should be blind, i.e., their
identity known only to the organizing laboratory, and coded. The participants
perform the PCRs and report the results to the organizing laboratory.
The percentage ratio of true positive results to false positive and true negative
results to false negatives should be recorded (see Table 2). With a robust
and accurate PCR that is correctly performed, this ratio should be 100% in
each case.
A robust PCR could in addition work as well with different reagents from
different suppliers. The Phase 2 ring trial participants, this time using nonidentical
reagents, should evaluate this. This is done by repeating the specificity
evaluation, but this time using enzymes and reagents of their choice
(while still conforming to the reaction concentrations and conditions as