Rapid Detection Of Common Virus In Aquaculture Fish Based On Loop-mediated Isothermal Amplification

The study aimed to solve the above-mentioned issues in fish virus detection in order to meet the needs of aquaculture development.Spring viremia of carp virus（SVCV）,Grass carp reovirus（GCRV）and Cyprinid herpesvirus-Ⅲ（CyHV-Ⅲ）,as common virus in aquaculture,were chose to the objectives in this study.Based on their gene conserved sequence,their primers were designed.And LAMP system was optimized and compared to real-time fluorescence quantitative PCR（qPCR）.In additon,three terminal detection methods of LAMP products were investigated by their evaluating the sensitivity and specificity of detection.Finally,two sets of primers are tried to use to LAMP reaction,and their reaction time and temperature,specificity,sensitivity and other aspects of the detection were evaluated by comparing with one set of primers reaction detected by LAMP combined lateral flow dipstick（LFD）（LAMP-LFD）.The study will provide theoretical and technical guidance for the rapid detection of fish virus.The main research contents and conclusions are as follows:1.Comparison of three terminal detection methods based on LAMP assay for SVCVSVCV is an important salmonid pathogenic virus belonging to the family Rhabdoviridae which can cause hemorrhagic symptoms of carp（Cyprinus carpio）with highly contagious,and the virus outbreaks are generally in fishery banks.This study developed a LAMP assay combined with different terminal detection methods in order to achieve SVCV field-based detection.A set of six specific primers were designed based on the SVCV glycoprotein（G）gene for LAMP assay.The reaction parameters of LAMP were optimized,and three terminal detection methods（SYBR Green I staining,LFD,and agarose gel electrophoresis（AGE））were applied to the detection of LAMP products.The FIP was labeled with Biotin（BIO）and a hybrid primer（HP）labeled with fluorescein amidite（FAM）was also designed which are used in LFD detection.The results showed that 8 mmol/L Mg²⁺,320 U/mL Bst DNA polymerase,1.4 mmol/L dNTP,and 1mol/L Betaine were optimum at 63℃with an incubation time of 40 min in LAMP system.Among the three ways,LFD was preferred for on-site terminal detection of LAMP products by comparing their sensitivity and specificity.The detection limit of LAMP-LFD in this assay was 860 fg with no cross-reaction with any of the other aquaculture common viruses.The LAMP-LFD was presented for field-based detection of SVCV with its advantages of speed,simplicity,disposability and portability in on-site testing in the study.Further,the study also provides a valuable alternative to immunoassays and PCR-based tests for other virus or bacteria.2.Development of a LAMP-LFD method for detection of GCRVGrass carp reovirus（GCRV）is the most virulent strain in Aquareovirus.This study developed a novel LAMP-LFD method and compared with qPCR assay in order to achieve GCRV field-based detection.A set of six specific primers（FIP/BIP,F3/B3,LF/LB）were designed based on the GCRV S8 sequence for LAMP assay,and FIP was labeled with BIO.A HP labeled with FAM was also designed.The results showed that LAMP reaction parameter for GCRV was at 63℃for 40min.The whole detection time,from LAMP to LFD visualization was about 50min,which was nearly 1 h shorter than that of qPCR.For specificity,LAMP-LFD was similar to qPCR,which discriminated GCRV from CyHV-Ⅲand SVCV.As for pure plasmid of GCRV gene,the detection limit of LAMP-LFD was970 fg,and qPCR was 97 fg.Although detection limit of LAMP-LFD was 10 times lower that of qPCR,the presented LAMP-LFD method was suitable for field-based detection of GVRV due to its simple operation and low dependence of instrument and equipment.3.Development of a LAMP-LFD method for detection of CyHV-ⅢKoi herpes virus disease（KHVD）is of great concern due to its high degree of contagiousness and lethality.Cyprinid herpesvirus-Ⅲ（CyHV-Ⅲ）is the main pathogen causing this disease.A novel LAMP-LFD was developed by designing three sets of four specific primers（FIP/BIP,F3/B3）based on the TK gene of CyHV-Ⅲ,and FIP being labeled with BIO.After choosing the primers used in the experiment by their amplificate effects,loop primers（LF/LB）and a HP labeled with FAM were also designed for the best one to detect the LAMP products by LFD.The results showed that one set of primers LAMP reaction parameter for CyHV-Ⅲwas at 63℃for 40min,and two sets of primers LAMP reaction parameter was at 61℃for 30minin which the products could be detected in 20 min which was nearly 1h shorter than that of qPCR.Furthermore,the specificity and sensitivity were also evaluated.LAMP,LAMP-LFD was similar to qPCR,which could discriminate CyHV-Ⅲf rom GCRV and SVCV.As for pure plasmid of CyHV-Ⅲgene,the detection limit of one set of primers LAMP-LFD was 32.6 fg,and qPCR was 32.6 fg,and two sets of primers was 3.26 fg.In this study,two sets of primers LAMP-LFD had not been established because of the inappropriate primers location while the AGE method was used to detect its products.Considering the two sets of primers LAMP system established in this study owned high reaction speed,specificity and sensitivity,it was suitable for field-based detection of CyHV-Ⅲ.In summary,the LAMP-LFD detection system was determined in this study.With optimizing the reaction components and conditions,the optimal reaction system was established and successfully applied to the detection of SVCV,GCRV,and CyHV-Ⅲ.The new detection method is of great significance in rapid detection of aquatic virus on-site,prevention of viral diseases in fish,and the improvement of the economic benefits of aquaculture.