Diagnostic PCR
Diagnostic primers were designed at non-overlapping COI areas
for the six species (Table 1). A diagnostic PCR reaction mixture
(20μl) consisted of 0.8μl of diluted mtDNA template, 2μl of 10× PCR
buffer, 2μl of dNTP, 0.8 μl each of forward and reverse diagnostic
primer, 0.4μl of Taq DNA polymerase and 13.2 μl of deionized water.
Amplification was carried out for 35 cycles of 1 min at 94 °C for
denaturation, 1 min of species-specific annealing temperatures
(Table 1) and 1 min 30 s at 72 °C for extension.