Rapid visual detection of cyprinid herpesvirus 2 by recombinase polymerase amplification combined with a lateral flow dipstick

Abstract

Abstract Herpesviral haematopoietic necrosis ( HVHN ), caused by cyprinid herpesvirus 2 (Cy HV ‐2), causes significant losses in crucian carp ( Carassius carassius ) aquaculture. Rapid and convenient DNA assay detection of Cy HV ‐2 is useful for field diagnosis. Recombinase polymerase amplification ( RPA ) is a novel isothermal DNA amplification and detection technology that can amplify DNA within 30 min at ~37°C by simulating in vivo DNA recombination. Herein, a rapid and convenient detection assay based on RPA with a lateral flow dipstick ( LFD ) was developed for detecting Cy HV ‐2. The highly conserved ORF 72 of Cy HV ‐2 was targeted by specific and sensitive primers and probes. The optimized assay takes only 15 min at 38°C using a water bath, with analysis of products by 2% agarose gel electrophoresis within 30 min. A simple lateral flow strip based on the unique probe in reaction buffer was developed for visualization. The entire RPA ‐ LFD assay takes 50 min less than the routine PCR method, is 100 times more sensitive and displays no cross‐reaction with other aquatic viruses. The combined isothermal RPA and lateral flow assay ( RPA ‐ LFD ) provides a simple, rapid, reliable method that could improve field diagnosis of Cy HV ‐2 when resources are limited.