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 30 s, 40 cycles of amplification (denaturation at 94°C for 30 s, annealing at 63°C for 30 s and elongation at 72°C for 30 s) and final elongation at 72°C for 7 min. The improved PCR reduced non-specific reactions and the total reaction time to almost half reguired by the original one. © 2005 The Japanese Society of Fish Pathology.