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

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2. Pepsin should be stored in the dark at room temperature (20°C or less, but not
below 4°C); avoid exposure to humidity. Pepsin should be no more than 6 mo old.
3. Balancing of primers. The primer-set mixture prepared with equimolar concentrations
of all oligonucleotides generally provides good results; if results are not
optimal, and the presence of T. murrelli is suspected, the concentration of the
primer set IV can be doubled.
4. For the automatic amplification of DNA, other thermal cyclers could be used, but
it could be necessary to first determine their efficiency in amplifying DNA.
5. Collection of worms from frozen samples: larvae from frozen muscle samples
should be collected according to the protocol of Subheading 3.1., yet to avoid
DNA destruction, all procedures after digestion should be carried out very
quickly, and sedimentation should be carried out on ice.
6. Collection of worms from formalin-fixed muscle samples: formalin-fixed muscle
samples cannot be used to collect larvae, because formalin destroys the DNA.
7. Collection of worms from ethyl alcohol-fixed muscle samples: worms can be
collected as follows: using a scalpel, cut the muscle sample into grain-size pieces.
Crush the pieces between two trichinoscope slides (8-mm thick) and check for
the presence of larvae among the muscle fibres under a dissection microscope at
20–40x. Mark the position of the larva on the bottom slide. Gently remove the
upper slide and cut away the muscle surrounding the larva using a scalpel and
one or two small needles (if the larva is encapsulated, remove it from the capsule
with the scalpel and needles) under a dissection microscope at 20–40x. Place the
larva in a 0.5-mL conical vial with 400 µL of cold H 2O. Wash the larva 3–4 times
with cold H 2O, then store in 5 µL of H 2O at –20°C.
8. Pooled larvae: the protocol for the preparation of crude DNA of a single larva can
also be used for pooled larvae by simply increasing the quantity of solution (for
example, for 10 larvae, it is sufficient to double the quantity of solution used for
a single larva). When using pooled larvae, it should be kept in mind that the
presence of larvae belonging to two or more genotypes (mixed infections) could
affect the interpretation of the results.
9. Precautions for PCR: use tip with barrier and gloves.
10. If using Taq DNA polymerases other than Takara, it is important to perform specific
tests to evaluate their effectiveness.
11. Pipeting the sample for the PCR amplification: a sufficient quantity of DNA is
critical for a successful amplification, thus, be sure to pipet the sample at the
bottom of the tube and avoid collecting the mineral oil. To remove the oil from
the tube, it is best to use pipeting, because chloroform or other organic solutions
could remove part of the DNA sample.
12. If using restriction enzymes other than those of New England Biolabs for RFLP
analysis, check the optimal working temperature of the enzyme.
Acknowledgments
We are very grateful to Marco Amati and Gianluca Marucci for their help in
the preparation of figures.