Improvement of a PCR Method with the Sph I-5 Primer Set for the Detection of Koi Herpesvirus (KHV)

Abstract

Improvement of a PCR method using the Sph I-5 primer set, which has been used for detection of KHV in Japan, was carried out to reduce negative factors, such as the appearance of non-specific reactions and the comparatively long reaction time. Moreover, there was a wrong sequence in the original reverse primer. The modified PCR protocol with the corrected primer set is as follows : initial denaturation at 94°C for 30s, 40 cycles of amplification (denaturation at 94°C for 30s, annealing at 63°C for 30s and elongation at 72°C for 30s) and final elongation at 72°C for 7min. The improved PCR reduced non-specific reactions and the total reaction time to almost half reguired by the original one.