Recombinase Polymerase Amplification Assays For Rapid Detection Of Shrimp WSSVAnd IHHNV

White spot syndrome virus(WSSV) and Infectious hypodermal and hematopoietic necrosis virus(IHHNV) are lethal viruses to the shrimp farming., which had caused large economic losses to the shrimp aquaculture industry.As they are strong virulence, fast copying, widely spreading, both of them had been listed as severe pathogens in OIE. Unfortunately, thus far, there are no efficient therapeutic treatments available against these lethal viruses. Hence, early d iagnosis is one of the most efficient strategies to monitor WSSV and IHHNV outbreak in shrimp farming facilities.In this study, we focused on the detedtion methods and presented the development of a novel real time isothermal recombinase polymerase amplification(RPA) assay for WSSV and IHHNV detection on a small ESEQ uant Tube Scanner device. The constructed RPA detecton systems showed superiou detection performance, which could provide technocal support for WSSV and IHHNV in site detection. The research in this study includes the following points:(1) The real time and isoethermal WSSV-RPA detection system had been constructed in this study. The detection features including sensitivity, specificity and rapidity had been evaluated both in WSSV-RPA and q PCR-WSSV detection systems by using a plasmid standard as well as viral and shrimp genomic DNAs. Compared with the q PCR-WSSV results, WSSV-RPA detection system revealed much more satisfactory performance. The WSSV-RPA detection system had reached a detection limit up to 10 molecules in 95% of cases as determined by probit analysis of 8 independent experiments within 6.4±0.17 min at 39℃.(2) Combined with a small ESEQ uant Tube Scanner device, an IHHNV-RPA detection system had been constructed, and the detectio n performance had been compared with that of the q PCR suggested in OIE for IHHNV. The redults showed great advanteges on the sensitivity, reaction time and temperature. The IHHNV-RPA detection system had achived sensitive to detect as few as 4 copies of the IHHNV genome within 7 min at 39℃with 95 % reliability.(3) By analysis the RPA primers and probe used in this study and others; we suggested some rules for the probe and primer design. The rules showed as followed: pyrimidines(T/C) instead of guanine(G) at the 5’ end(first 1-3 nt) may promote the primer and recombinase to form a recombinase-primer complex; G and C at the 3’ end of the primer are suggested.;the primers and probe may be modestly apart to achieve the desired amplification efficiency, but overlap should be avoided.;the background signals are related to the overlap degree between the probe and primers, especially the primer binding to the same strand as the probe does. The higher overlap degree of the primer with the exo probe is, the higher initial amplification signals generate.Consequently, thses rapid WSSV-RPA and IHHNV-RPA detection systems have great application potential for field use or point of care diagnostics.