KHV Detection Based on Thermal Lysis - Nested PCR (Polymerase Chain Reaction)

Abstract

In the case of common carp (Cyprinus carpio) and goldfish (Carrasius auratus) that were lightly infected with Koi Herpes Virus (KHV), the use of the one-step PCR method was insensitive to detect the presence of KHV DNA. The nested PCR method has been developed for a more sensitive detection system for KHV infection. This study aims to test the sensitivity of one-step and nested PCR methods in detecting the KHV virus in lightly infected common carp to prevent early disease attacks. DNA extraction of the test samples used thermal lysis and PBS (Phosphate Buffered Saline). DNA isolate as a KHV template was used to amplify KHV sequences using KHV-TKf: 5'-GGGTTACCTGTACGAG-3 'and KHV-TKr: 5'-CACCCAGTAGATTATGC-3' primers in the one-step PCR method. The nested PCR method uses two pairs of primers, namely the KHV-TKf: 5'-GACACCACATCTGCAAGGAG-3' and KHV-TKr: 5'-GACACATGTTACAATGGTGGC-3') and the CEFAS primer pair (5'-CGTCGTGAGGAATACGACG-3' and 5'- ACCGTACAGCTCGTACTGG-3') for amplification of KHV sequences. The one-step PCR method was insensitive for detection of KHV in common carp and goldfish infected with viruses without clinical symptoms, and was sensitive enough to detect viruses with mild symptoms with a DNA fragment size of 409 bp using TK primers. The nested PCR method was more sensitive in detecting KHV DNA in fish without clinical symptoms with a DNA fragment size of 348 bp using CEFAS primers.