Triplex DARQ-LAMP Isothermal Amplification Assay For Simultaneous Detectionof Three Pathogens In Penaeid Shrimp

Shrimp farming is developing rapidly as a major commercial activity in the world.However,viral diseases have caused huge economic losses to the shrimp farming industry.So far,white spot syndrome virus,infectious hypodermal and hematopoietic necrosis virus and enterocytozoon hepatopancreatic are the three main pathogens that cause diseases in shrimp farming.The rapid spread of diseases is one of the key factors restricting the healthy development of the aquaculture industry.Therefore,rapid and accurate multiple detection technology for pathogens in shrimp is of great significance to the aquatic industry.Although traditional polymerase chain reaction(PCR)has been widely used to detect these three pathogens,PCR requires large and expensive temperature cycling equipment and is time-consuming.With the development of technology in recent years,loop-mediated isothermal amplification technology(LAMP)has been developed.LAMP technology uses four to six sets of primers to amplify the target gene under isothermal conditions using a DNA polymerase with high displacing strand activity.LAMP has high sensitivity and high specificity.However,traditional LAMP detection methods cannot detect multiple targets at the same time.Therefore,this study combines probe-based detection amplification by release quenching(DARQ)with loop-mediated isothermal amplification combined with DARQ-LAMP to achieve simultaneous rapid detection of three shrimp pathogens.In this study,a loop-mediated isothermal amplification technique was established to simultaneously detect white spot syndrome virus,infectious hypodermal and hematopoietic necrosis virus,and enterocytozoon hepatopancreatic.In this study,the vp28 gene of white spot syndrome virus(Gen Bank accession:DQ681069.1),the cp gene of infectious hypodermal and hematopoietic necrosis virus(Gen Bank accession:KF214742.1),and the ssu r RNA gene of enterocytozoon hepatopancreatic(Gen Bank accession:FJ496356)were used as target genes to design LAMP primers.In addition,the white spot syndrome virus FD primers are labeled with carboxyfluorescein(FAM),the infectious hypodermal and hematopoietic necrosis virus FD primers are labeled with VIC dye,and the enterocytozoon hepatopancreatic FD primers are labeled with Cy5 dye.The three sets of LAMP FIP primers are all labeled with quenching dye.To Establish a multiple DARQ-LAMP reaction system.In the sensitivity experiment,the limit of detection of white spot syndrome virus is 1.90×10~3 copies/μL,the limit of detection of infectious hypodermal and hematopoietic necrosis virus is1.21×10~2 copies/μL,and the limit of detection of enterocytozoon hepatopancreatic is1.13×10~2 copies/μL.Drawing a standard curve with the initial amplification time value data as the Y axis and the logarithm of the template concentration as the X axis.Shrimp white spot syndrome virus R~2=0.9974,infectious hypodermal and hematopoietic necrosis virus R~2=0.9983,enterocytozoon hepatopancreatic R~2=0.9961.It shows that the established multiplex DARQ-LAMP method has a good correlation between the template concentration and the initial amplification time.The specificity experiment results show that the multiple DARQ-LAMP technology can only detect target pathogens,and the three shrimp pathogens have specificity,and there is no cross-amplification reaction and no amplification.The practical sample detection experiment purchased P.vannamei,shrimp larvae,and freshwater prawns from the local market or aquaculture farm in Hangzhou as samples,and compared them with the fluorescent quantitative PCR method.The results showed that the positive detection rate of the two was the same.In this study,DARQ-LAMP was used for triplex detection.The three pathogens of white spot syndrome virus,infectious hypodermal and hematopoietic necrosis virus,and enterocytozoon hepatopancreatic could all be detected under isothermal conditions within 20 min.The developed DARQ-LAMP technology has been optimized to have a faster amplification time.The amplification time of the triple reaction is almost the same as that of the single reaction.The multiplex DARQ-LAMP assay has higher sensitivity and can detect three pathogens at the same time.In short,the DARQ-LAMP assay has the characteristics of high specificity,simplicity and sensitivity.Moreover,the DARQ-LAMP technology does not require complex instruments,is simple to operate,and can be quickly detected on site,and has a wide range of application prospects.