Establishment And Application Of RT-LAMP For The Detection Of Infectious Pancreatic Necrosis Virus

Infectious pancreatic necrosis virus(IPNV)is the pathogen responsible for infectious pancreatic necrosis of salmon and trout.IPNV belongs to the family Birnaviridae and is the archetypical species of the genus Aquabirnavirus.IPNV infection is highly contagious,with mortality rates of up to 90%.Fish that survive carry the virus for their whole life,causing a huge threat to the salmon and trout farming industries.IPNV is the first type of quarantine object for quarantine of fish ports.However,there are no effective drugs for the prevention and control of IPNV in China.Therefore,pathogen monitoring of aquatic products is the preferred way to control the spread of the disease.The immunological detection methods require antibodies against different serotypes of IPNV,which can be difficult and time-consuming to obtain,and the molecular biological detection methods require specialized equipment and operators,limiting on-site or large-scale detection of pathogens.Loop-mediated isothermal amplification is a new isothermal nucleic acid amplification technique in vitro.It can amplify the target sequence by Bst 2.0 DNA polymerase with chain replacement characteristic,and the reaction can be completed within 1 hour under constant temperature.The results of RT-LAMP can be detected not only by agarose gel electrophoresis,but also directly using fluorescence detection reagent SYBR Green I stain for visual inspection.It is suitable for large-scale inspection on site.The purpose of this study was to establish a method for the rapid detection of infectious pancreatic necrosis virus(IPNV,Jasper serotype)using reverse transcription loop-mediated isothermal amplification(RT-LAMP).In this experiment,the whole genome of IPNV Ch Rtm213(Genbank accession number KX23491 and KX23490)isolated and preserved in the laboratory was used as the target gene,and four sets of primers were designed by using Primer Explorer V5 primer design software.the primers were screened strictly according to the principles of primer design and matters needing attention.The optimum primer combination was obtained by comparing the amplification efficiency of four sets of primers.The best primers were used to extract IPNV(Jasper)RNA for RT-LAMP reaction,and the reaction conditions were optimized.The sensitivity test,specificity test and preliminary clinical application test of the optimized reaction system were carried out.Four groups of specific primers were designed according to the genome sequence of a Chinese IPNV isolate Ch Rtm213.The results showed that primer set B2 had the best amplification effect.When the final concentration of Mg2+ was 4 mmol/L~8 mmol/L,d NTPs were 0.5 mmol/L~1.5 mmol/L,and Betaine was 0.4 mol/L~1.2 mmol/L,the reaction couldbe completed in a 63℃ water bath within 60 min.This RT-LAMP assay for the detection of IPNV had no cross-reactivity with infectious hematopoietic necrosis virus or viral hemorrhagic septicemia virus.The detection limit was 0.0008 fg/25 μL.This method is more suitable for IPNV(Jasper)detection than the published IPNV(Sp)RT-LAMP detection method in foreign countries.The IPNV RT-LAMP detection method established in this experiment was used for clinical preliminary application.Seven liver and kidney samples from different regions were tested.The gel electrophoresis results of the test results were consistent with the color detection results.In summary,this test is aimed at the domestic popular IPNV serotype Jasper,established IPNV RT-LAMP method.It is more suitable for detecting domestic epidemic strain IPNV(Jasper)than the published IPNV(Sp)RT-LAMP detection technology.It can pass color change to determine the result of nucleic acid changes.Positive sample is fluorescent green,negative sample is reddish brown This experiment is aimed at the RT-LAMP method established by the current domestic IPNV.It has the characteristics of good specificity,strong sensitivity,direct detection by visual observation,and high accuracy in practical application.This established IPNV RT-LAMP assay is suitable for the rapid and large-scale detection of IPNV in China.