3.3. Optimization of multiplex PCR
Multiplex PCR was established to produce eight bands in a
single tube and the conditions were optimized with temperature,
primer concentrations and extension time to prevent non-specific
reaction. Furthermore, each primer pair concentration was
adjusted to produce similar band intensity. The concentration of
primer pairs and expected products size are shown in Table 2. The
sensitivity and specificity of the reaction were assayed with the
reference strains. Fig. 1 shows multiplex PCR results using six Listeria
species. A gel electrophoresis confirmed that all the primer
pairs specifically amplified the desired PCR products. This implied
that the primers were specific for detection of the Listeria species
used in this study and would not exhibit false-positives on account
of the PCR reaction. The results from the multiplex PCR yielded
simultaneous amplification of all the seven targets with comparable
band intensities using the PCR cycling parameters and an