Real-time Fluorescent Quantitative LAMP Method For Detection Of Pathogenic Microorganisms In Shrimps

Abstract:The aquaculture industry has developed vigorously for many years and been one of the economic pillar industries in many coastal countries.However,in the process of prawn breeding,diseases such as white shift syndrome and other prawn breeding diseases have a great impact on the prawn breeding industry and become the bottleneck hindering its development.Among them,White spot syndrome virus and Infectious hypodermal and hematopoietic necrosis virus are two major DNA viruses that infect prawn.Enterocytozoon hepatopenaei is the main pathogenic parasite and Vibrio parahaemolyticus is the common food-borne pathogenic microorganism in aquatic products.At present,there is no specific drug for the first three,and the weak immune system of prawn itself brings great difficulties in the prevention and treatment of prawn breeding diseases.It is particularly important to carry out early diagnosis and prevention for the aquatic pathogenic microorganisms mentioned above.Four aquatic pathogens including VP28 gene(GenBank Accession No.DQ681069.1),CP gene of infectious subcutaneous and hematopoietic necrotizing virus(GenBank Accession No.KF214742.1),ssu rRNA gene(GenBank Accession No.FJ496356.1)and toxR gene(GenBank Accession No.GQ228073.1)were designed respectively for specific LAMP primers and a novel LAMP assay based on SYTO-16was established.The reaction system was optimized by exploring the influence of LAMP primer concentration,Bst 2.0 enzyme concentration and temperature change on LAMP amplification system,and the efficient real-time fluorescent quantitative LAMP detection methods of WSSV,IHHNV,EHP and VP were established,respectively.When LAMP detection was performed on SYTO-16 determined by dye screening,its comprehensive amplification efficiency,signal-to-noise ratio and relative fluorescence value were more prominent,and its working concentration was10-100μM.In this study,a real-time fluorescent quantitative LAMP system was established based on 10μM SYTO-16 dye.After optimization,the optimal concentration of primers,Bst 2.0 DNA polymerase and the optimal temperature of LAMP reaction were determined to be 40μM,8 U/mL and 63-64℃,respectively.LAMP systems had good specificity,and only the corresponding positive targets were detected,but no cross-reaction was found with other aquatic pathogenic microorganisms.The detection limits of WSSV,IHHNV,EHP and VP by real-time fluorescent quantitative LAMP methods were 1.37×10~1 copies/μL,1.54×10~1copies/μL,1.78×10~1 copies/μL,and 1.68×10~1 copies/μL,respectively.The qPCR method showed that the sensitivity was consistent with LAMP results.The template with high copy concentration can be detected within 5-8 min.In the practical sample detection,we compared the real-time fluorescent quantitative LAMP assay with qPCR assay.In the detection of actual samples,the detection rate of wssv-lamp method was 22.6%,the detection rate of WSSV-qPCR method was 21.8%,and the coincidence rate of the two methods was 96.6%.The detection rate of IHHNV-LAMP method was 38.9%,that of IHHNV-qPCR method was 37.0%,and the coincidence rate of the two methods was 95.9%.The detection rate of EHP-LAMP method was 31.0%,the detection rate of EHP-qPCR method was30.2%,and the coincidence rate of the two methods was 96.9%.The detection rate of VP-LAMP method was 47.1%,that of VP-qPCR method was 46.3%,and the coincidence rate of the two methods was 97.5%.In this study,we developed a rapid real-time fluorescent quantitative LAMP assay for detecting WSSV,IHHNV,EHP and VP.The use of fluorescent nucleic acid dye(SYTO-16)reveals a more satisfactory performance.The quantitative LAMP assay can be considered as a valid tool for rapid detection of microsporidian in prawns.Our study provides a scientific basis to follow the effect of the pathogen infection on growth of cultured penaeid shrimp.