Establishment Of Isothermal Amplification Visualization Detection Method For Giant Salamander Iridovirus

Giant Salamander Iridescent virus disease are malignant infectious diseases caused by Giant Salamander Iridovirus,GSIV infections,which spread quickly and have a high mortality rate.Bringing significant economic losses to the Andrias davidianus farming industry。Most of the existing testing methods rely on the operation of laboratory equipment and professionals,which seriously restricts the monitoring and prevention of Giant Salamander Iridovirus in daily farming.This study used Single Crossing Priming Amplification and Recombinase Polymerase Amplification technology,combined with rapid detection of nucleic acid test papers,and established two simple,fast and visually visible GSIV on-site detection methods to provide technical support for dog prevention and control.The main contents of this paper are as follows:1.Establish a Single Crossing Priming Amplification Visual Detection Method for Giant Salamander IridovirusThe primers for SCPA amplification were designed for the nucleocapsid protein MCP gene of the GSIV genome,and the concentration of each component of the SCPA basic reaction system,as well as the reaction temperature and time were optimized to determine the optimal reaction conditions.The results demonstrate that optimal concentration of primers are as follows,2R1 F is 1.0 umol/L,2R(2R-Bio)and 3R(3R-Bio)are 0.5 μmol/L separately,and 4F and 5R are 1.0 μmol/L separately.It was shown that optimum concentration of Betaine,d NTPs and Mg2+ are 0.7 mol/L,1.2 mmol/L and 8.0 mmol/L respectively.Furthermore,amplification last at 63 °C for 40 minutes.The SCPA amplification product was combined with a nucleic acid rapid test strip to establish a GSIV-SCPA method for GSIV on-site detection.The specificity of the GSIV-SCPA method was evaluated using a genomic DNA recombinant plasmid of a similar species such as Turbot reddish body iridovirus、Red sea bream iridovirus、 Tiger Frog Virus and Koi herpes virus disease,The results demonstrate that the GSIV-SCPA method can specifically amplify the MCP gene of GSIV with good specificity;Sensitivity evaluation of the GSIV-SCPA method showed that the sensitivity of the GSIV-SCPA method can reach 10-7 template dilution,the sensitivity of the PCR method is 10-6 template dilution,and the sensitivity of the GSIV-SCPA method is higher than that of the PCR 10 times,suitable for detecting trace amounts of viral DNA,can be used as an on-site detection method for GSIV.2.Establish a Recombinase Polymerase Amplification visual detection method for Giant Salamander IridovirusThe primers for RPA amplification were designed for the nucleocapsid protein MCP gene of the GSIV genome,and then the reaction time was optimized.The RPA amplification product was combined with the nucleic acid rapid detection test paper to establish an on-site detection method for GSIV.Named GSIV-RPA.The specificity of the GSIV-RPA method was evaluated using a genomic DNA recombinant plasmid of a similar species such as Turbot reddish body iridovirus、Red sea bream iridovirus、 Tiger Frog Virus and Koi herpes virus disease,The results demonstrate that the GSIV-RPA method can specifically amplify the MCP gene of GSIV with good specificity;The sensitivity evaluation of the GSIV-RPA method showed that the sensitivity of the GSIV-RPA method can reach 10-7 template dilution,the sensitivity of the PCR method is 10-6 template dilution,and the sensitivity of the GSIV-RPA method is higher than that of the PCR.10 times,a small amount of viral DNA can be detected;When used for the detection of actual samples,the GSIV-RPA method showed a consistent detection rate consistent with the PCR method,demonstrating that the newly established GSIV-RPA method is suitable for on-site detection of GSIV.GSIV-SCPA detection method and GSIV-RPA detection method are free from the constraints of expensive thermal cycle equipment and high standard laboratory conditions,the amplification time is short,the amount of amplification product is large,the reaction conditions are easy to reach,and the region is remote and the resources are underdeveloped.The region can also complete the rapid detection of GSIV,improve the monitoring efficiency of GSIV in daily breeding and introduction,and provide technical support for the rapid diagnosis of GSIV.