Implementation of a Recombinase Polymerase Amplification‐Driven Duplex Lateral Flow Diagnostic Kit for Concurrent Detecting Infectious Pancreatic Necrosis Virus and Viral Hemorrhagic Septicemia Virus: A Comparative Analysis of RPA and PCR Methods

ABSTRACT Diagnosis time of infectious diseases in aquaculture is an important issue, and local and commercial enterprises generally do not prefer laboratory diagnosis of infectious diseases due to the long lead time and cost. This leads to the under‐reporting of outbreaks in aquaculture farms and the recognition of infectious agents when they spread to many farm systems and cause significant economic loss. In this study, we aim to develop a fast and simple diagnostic test that can easily be applied under field conditions against Infectious pancreatic necrosis virus (IPNV) and Viral hemorrhagic septicemia virus (VHSV Ie), which are two crucial viral pathogens that cause significant problems in trout farming in Türkiye, both in hatcheries and farms. For this reason, we developed a lateral flow test strip for the duplex diagnosis of VHSV Ie and IPNV sp. based on Recombinase polymerase amplification (RPA), one of the molecular tests developed in recent years. We also compared Quantitative RT‐PCR (q RT‐PCR), Quantitative RT‐RPA (rtRT‐RPA) and lateral flow RT‐RPA (lfRT‐RPA) methods, which optimise both singleplex and duplex. As a result of this stage, the minimum detection limits of both rtRT‐PCR, rtRT‐RPA, and lfRT‐RPA methods were determined. The lfRT‐RPA test is almost as sensitive as rtRT‐PCR and rtRT‐RPA for the detection of duplex VHSV Ie, but for the detection of the IPNV sp., the lfRT‐RPA test was found to be approximately 10‐fold less sensitive than rtRT‐PCR and rtRT‐RPA. It is thought that the duplex lateral flow detection kit will allow timely diagnosis in the field for detecting infectious diseases of other aquaculture farms.