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

Yersinia PCR 319
Table 2
Sequences and Characteristics of Primers
for the Yersinia enterocolitica Multiplex PCR Detection Assay
GC
Size Content Location of
Primer Sequence (5'→3') (bp) (%) T M a Target gene target gene
P1 TGT TCT CAT CTC CAT ATG CAT T 22 36 60 yadA pYV plasmid
P2 TTC TTT CTT TAA TTG CGC GAC A 22 32 58 yadA PYV plasmid
Y1 GGA ATT TAG CAG AGA TGC TTT A 22 36 60 16S rRNA Chromosomal
Y2 GGA CTA CGA CAG ACT TTA TCT 21 43 60 16S rRNA Chromosomal
a The theoretical melting temperature is calculated from the formula: TM = (4 × [GC])(2 × [AT])°C.
PCR product from the chromosomal 16S rRNA gene. The location of the Y1
primer corresponds to the variable V3 region of the 16S rRNA gene, and the
Y2 primer corresponds to the variable V9 region of the same gene according to
the nomenclature (37). A mismatch (C instead of G) was introduced during the
development of the assay, at the second 3´end of the Y2 primer, to avoid detection
of Hafnia alvei. More than 30 Enterobacteriaceae strains have been tested
for specificity, and all were found to be negative. For detection to be considered
positive, the two PCR products originating from both genes must be visualized
on the gel (see Fig. 3). The 0.6-kb amplicon defines the virulence, while
the 0.3-kb amplicon confirms the Yersinia origin. The specificity of the assay
has been evaluated (29). It was found that the Y1 and Y2 primers amplified the
region of the 16S rRNA gene for some Y. enterocolitica-like organisms, such
as Y. kristensenii and Y. intermedia.
3.3.2. Preparation of PCR Master Mixture
Each 25-µl reaction will contain the following reagents: 2.5 µL PCR buffer
(10X), 2.5 µL dNTP mixture (0.2 mM of each nucleotide), 1 µL primer Y1
(0.4 µM), 1 µL primer Y (0.4 µM), 1 µL primer P1 (0.4 µM), 1 µL primer P2
(0.4 µM), 0.15 µL Taq DNA polymerase (0.75 U), and 10.85 µL doubledistilled
H 2O.
Calculate the total amounts of reagents according to the number of amplification
reactions of the series. Alternative DNA polymerases and PCR mixtures
can also be used (see Note 5).
Use of 5 µL cell suspension as PCR template.
3.3.3. PCR Program
1. The PCR program starts with a denaturation step at 94°C for 5 min.
2. The denaturation is followed by 36 cycles consisting of heat denaturation at 94°C
for 30 s, primer annealing at 58°C for 30 s, and extension at 72°C for 40 s.