Rapid, sensitive and simple detection method for koi herpesvirus using loop-mediated isothermal amplification

Abstract

New methods were developed for the detection of koi herpesvirus (KHV, CyHV-3) by LAMP, which were compared with the PCR for specificity and sensitivity. We designed two primer sets targeting a specific sequence within the 9/5 PCR amplicon (9/5 LAMP) and the upper region of the SphI-5 PCR amplicon (SphI-5 LAMP), including a sequence highly conserved among the strains. The amplification was monitored in real-time based on the increase in turbidity, with magnesium pyrophosphate as the by-product. The reactionswere carried out under isothermal conditions at 65°Cfor 60 min. Thedetection limit of both LAMP was six copies, equal to the modified SphI-5 PCR. No cross-reactivity with other fish pathogenic viruses and bacteria was observed. SphI-5 LAMP was found to have a quicker response in terms of the reaction velocity than 9/5 LAMP. Therefore, we consider SphI-5 LAMP to be superior for routine use. Additionally, LAMP was found applicable to crude extract from gills and other organs. LAMPmethods are superior in termsof sensitivity, specificity, rapidity and simplicity, and are potentially a valuable diagnostic tool for KHV infections. © 2009 The Societies and Blackwell Publishing Asia Pty Ltd.