Development and Application of a Triplex PCR Assay for Detection of Koi Herpes Virus (KHV) Gene Infected Common Carp (Cyprinus carpio L.)

Abstract

Koi herpesvirus (KHV) has been responsible for worldwide KHV disease (KHVD) outbreaks, resulting in high mortality rates, and has been reported in Cyprinus carpio cultivation. A Triplex Polymerase Chain Reaction (T-PCR) was developed for the detection of KHV gene to improve the diagnosis of KHV infection. Three sets of primers were designed targeting specific sequences of KHV gene which were frequently used in PCR assay for detecting KHV (modifications primer by Yuasa,290 bp; Gilad 484 bp, and TK, 409 bp) in once PCR running. The validity test was conducted by verified using DNA sample of Aeromonas hydrophila. Sensitivity test of singleplex and Triplex conducted by dilution. The result showed that T-PCR method using these pairs of primers is at 55 annealing temperature with consistency of T-PCR method has been repeated in triplicates, and with the same results. The validity test found that no band detected with A. hydrophila. This Triplex PCR-based assay may have useful for the clinical diagnosis tool of KHV disease.