Recombinase Polymerase Amplification Assays For Rapid Detection Of Three Fish Viruses CyHV-2、KHV、GCRV

Herpesviral haematopoietic necrosiss disease,Koi herpesvirus disease,and grass carp hemorrhage are three viral diseases that have caused serious harm in large-scale freshwater fish farmed in China.The pathogens are Cyprinid herpesvirus 2(CyHV-2)and koi herpes virus(KHV)and grass carp reovirus(GCRV).The rapid detection methods of CyHV-2 KHV and GCRV real-time fluorescent recombinase polymerase amplification(RPA)established in this paper provide theoretical and technical support for the safe production control of aquatic food ingredients.The main findings are as follows:1.Select the conservative gene fragment of CyHV-2,design the RPA primer probe based on it,and synthesize the recombinant plasmid,optimize the reaction conditions,determine the sensitivity and specificity of the RPA method,and use the qPCR method in the literature to detect sensitivity and specificity at the same time,and make a comparative analysis with the RPA method.Finally,the actual samples were detected.The results showed that:the CyHV-2-RPA detection method established in this study,with a portable PCR instrument as real-time monitoring for real-time fluorescence RPA reaction,the method can detect a minimum of 10~3copies/μL of viral DNA within 10 minutes at a constant temperature of 39°C.This method has good specificity.2.Design the RPA primer probe according to the Sph gene fragment of KHV,and synthesize the recombinant plasmid,optimize the reaction conditions,determine the sensitivity and specificity of the RPA method,and compare the sensitivity and specificity of the RPA method with the qPCR method in the literature.The results showed that:the KHV-RPA detection method established in this study,with a portable PCR instrument as real-time monitoring for real-time fluorescence RPA reaction,the method can detect a minimum of10~3copies/μL of viral DNA within 10 minutes at a constant temperature of 39°C.This method has good specificity.3.Design the RPA primer probe according to the conserved gene fragment of GCRV,and synthesize the recombinant plasmid,optimize the reaction conditions,determine the sensitivity and specificity of the RPA method,and compare the sensitivity and specificity of the RPA method with the qPCR method in the literature.At a constant temperature of 40°C,a minimum of 10~2 copies/μL of Viral nucleic acid can be detected within 10 minutes.This method has good specificity4.The virus DNA magnetic beads rapid extraction method was established,and the extraction conditions and extraction reagents were optimized.The results showed that the rapid extraction of virus DNA beads could complete the extraction of virus DNA within 15min,combined with real-time fluorescence RPA,and complete the whole process from sample processing to nucleic acid amplification within 40 min.