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).Four...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).Four groups of specific primers were designed,according to the genome sequence of a Chinese IPNV isolate ChRtm213.The results showed that primer set B2 had the best amplification effect.When the final concentration of Mg2+was 6 mmol·L-1,dNTPs were 1 mmol·L-1 and betaine was 0.4 mol·L-1,the reaction could be 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,viral hemorrhagic septicemia virus,grass carp reovirus and spring viremia of carp virus.The detection limit was 3.2×10-12 ng·μL-1.The sensitivity of this method was 10-fold higher than that of a previously published RT-LAMP assay for detecting the Spajarup(Sp)serotype of IPNV.This method,aimed at detecting IPNV isolates that were currently prevalent in China,possessed the characteristics of strong specificity,high sensitivity and direct interpretation by the naked eyes.The IPNV RT-LAMP was successfully applied to determine the clinical samples,which indicated the IPNV RT-LAMP assay was suitable for the rapid and large-scale detections of IPNV in China.展开更多
基金Supported by the National Natural Science Foundation of China(31802345)China Postdoctoral Science Foundation(2018M630893)Heilongjiang Province Postdoctoral Science Foundation(LBH-Z18275)。
文摘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).Four groups of specific primers were designed,according to the genome sequence of a Chinese IPNV isolate ChRtm213.The results showed that primer set B2 had the best amplification effect.When the final concentration of Mg2+was 6 mmol·L-1,dNTPs were 1 mmol·L-1 and betaine was 0.4 mol·L-1,the reaction could be 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,viral hemorrhagic septicemia virus,grass carp reovirus and spring viremia of carp virus.The detection limit was 3.2×10-12 ng·μL-1.The sensitivity of this method was 10-fold higher than that of a previously published RT-LAMP assay for detecting the Spajarup(Sp)serotype of IPNV.This method,aimed at detecting IPNV isolates that were currently prevalent in China,possessed the characteristics of strong specificity,high sensitivity and direct interpretation by the naked eyes.The IPNV RT-LAMP was successfully applied to determine the clinical samples,which indicated the IPNV RT-LAMP assay was suitable for the rapid and large-scale detections of IPNV in China.
