Rapid visual detection of cyprinid herpesvirus 2 by recombinase polymerase amplification combined with a lateral flow dipstick |
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| Authors: | D Xu P Podok J Xie Ys Jiang Lq Lu |
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| Institution: | 1. National Pathogen Collection Center for Aquatic Animals, Shanghai Ocean University, Shanghai, China;2. Key Laboratory of Freshwater Aquatic Genetic Resources, Ministry of Agriculture, Shanghai Ocean University, Shanghai, China;3. National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai, China;4. Faculty of Agricultural Technology, Phuket Rajabhat University, Phuket, Thailand;5. Shanghai Engineering Research Center of Aquatic Product Processing & Preservation, Shanghai Ocean University, Shanghai, China |
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| Abstract: | Herpesviral haematopoietic necrosis (HVHN), caused by cyprinid herpesvirus 2 (CyHV‐2), causes significant losses in crucian carp (Carassius carassius) aquaculture. Rapid and convenient DNA assay detection of CyHV‐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 CyHV‐2. The highly conserved ORF72 of CyHV‐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 CyHV‐2 when resources are limited. |
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| Keywords: | crucian carp CyHV‐2 herpesviral haematopoietic necrosis isothermal detection lateral flow dipstick recombinase polymerase amplification |
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