In the multiplex PCR system, preferential amplification
of one target sequence over another frequently occurs,
which could lead to uneven amplification products. In order to amplify all the specific PCR products with equal
efficiency, the concentration of individual primer pairs
was optimized. Primer concentration was increased when
intensity of the corresponding band was too low or absent,
and decreased when intensity of the bands was too high.
When the multiplex reaction is performed for the first
time, it is useful to add the primers in equimolar amounts.
The results will suggest how the individual primer concentration
and other parameters need to be changed. The
concentrations of three primer pairs in different proportions
were examined in this study. When the concentration
of primer pairs of E. coli, A. hydrophila and
Y. enterocolitica was 0.166 μM of each of the primers respectively,
primer specific for uid R gene of E. coli
excessively amplified while there was no amplification
for primer specific for A. hydrophila and Y. enterocolitica
(Figure 3 a). So the concentration of primers specific for
A. hydrophila and Y. enterocolitica was increased to
0.333 μM, keeping the concentration of the primer specific
for E. coli constant. Amplification of species specific
for 16SrRNA gene of Y. enterocolitica was significantly
favoured. Under the same primer concentration there was
no amplification for A. hydrophila (Figure 3 b). Hence the
concentration of primer specific for A. hydrophila was
increased to 0.416 μM. The experiment demonstrated that
the optimal concentration of primer pairs of E. coli,
Y. enterocolitica and A. hydrophila was 0.166, 0.333 and
0.416 μM respectively (Figure 3 c)