RAPID VISUAL DETECTION OF KHV BY RECOMBINASE POLYMERASE AMPLIFICATION (RPA) COMBINED WITH A LATERAL FLOW DIPSTICK (LFD)

Cyprinus carpio and Cyprinus carpio kio are important aquaculture varieties in China. Highly pathogenic and infectious of Cyprinid herpesvirus 3 (CyHV-3, KHV) has caused great losses in the culture of Cyprinus carpio and Cyprinus carpio kio. Therefore, the rapid and convenient detection technology is desperately needed to be used for non-laboratory and fast detection. Recombinase polymerase amplification (RPA) is such an Isothermal amplification technology which can amplify DNA within 30min under low reaction temperature. Then we combined RPA with lateral flow dipstick (LFD) and established a rapid and sensitive method which are suitable for field clinical detection of KHV. In this study, primers and probe sequences were designed according to the conserved fragment of SphI-5 gene of KHV. The experimental results show that the RPA technique can detect target fragment by agarose gel electrophoresis within 30min at the optimal temperature of 38℃ and the RPA results can be visualized in only 5min in combination with the LFD and the entire RPA-LFD assay takes 50min which are a lot faster than PCR method. The method established in this experiment for the detection of KHV by RPA-LFD is so simple and sensitivity that can be useful for rapid diagnosis in aquaculture with limited resources.