Establishment And Application Of LAMP Visual Detection Method For Avian Leukosis Virus Subgroup J

Avian leukaemia is caused by avian leukaemia virus poultry a variety of oncologic disease of the general name,although J subgroup was discovered the most late,but its infectivity and pathogenicity are higher than other subgroup,there is no effective vaccine to prevent and cure at present,can only continue to eliminate positive chicken,thus achieve the purpose of eliminating this disease.Commonly used molecular biological methods for laboratory detection of pathogens mainly include PCR technology,nested PCR technology and fluorescence quantitative PCR technology.Although these three methods have certain effects,they are time-consuming and laborious,have high requirements for operators,and cannot meet the requirements of simple and rapid diagnosis.LAMP technology is a brand new gene amplification method.The reaction can be completed by placing template,primer,enzyme and matrix together at a certain temperature through one step.The existence of target gene sequence can be judged according to the presence of amplified products.However,LAMP technology is currently difficult to be popularized due to factors such as high false positive rate,need to open the lid for result determination,low degree of visualization and poor repeatability of the operation process,which greatly limits the application scope of this detection method.This study intends to establish a LAMP visualization detection method for avian leukemia virus of subgroup J by combining the latest research trend of this technology and visualization of tube closure operation and results.In this study,the SD0101 strain of avian leukemia virus of subgroup J was firstly amplified and cultured on DF-1 cells.Meanwhile,EILSA detection method and IFA detection method were used to detect the proliferation of the virus on cells.According to the gp85 gene sequence of avian leukemia virus of subgroup J logged in Gen Bank,After artificial synthesis,the DNA plasmid template ALV-J-gp85-p MD18-T was prepared,and the concentration of the positive recombinant plasmid ALV-J-gp85-p MD18-T was determined by nucleic acid protein analyzer,and the copy number was converted by formula.The specific ALV-J LAMP primers for avian leukemia virus subgroup J were designed using online design software.The primers ratio,betaine concentration,magnesium ion concentration,d NTP concentration,Bst enzyme concentration,reaction temperature and reaction time were optimized to screen out the best ALV-J LAMP reaction conditions.Finally,anti-pollution reagent and visual reagent are added to the reaction system to facilitate the judgment of reaction results.The whole test process does not need to open the lid,which reduces the possibility of pollution and ensures the accuracy of experimental results.The feasibility of this method was tested by distinguishing avian leukemia virus from other subgroups,specificity test,sensitivity test,repeatability test and clinical sample test.The results showed that the proliferation of SD0101 strain on DF-1 cells was confirmed by IFA and ELISA.The concentration of the positive plasmid template ALV-J-gp85-p MD18-T was 166 ng/?L,and the copy number was 4×10¹⁰copies/?L.Through the optimization of ALV-J LAMP reaction system,the optimal ratio of internal and external primers for ALV-J LAMP reaction was determined to be 8:1.Betaine concentration was 4 M,magnesium ion concentration was 6 m M,d NTP concentration was 1.4 m M,Bst enzyme concentration was400 U/m L,the optimal reaction temperature was 64?,the optimal reaction time was 54 min.The pollution caused by LAMP reaction was reduced by standardized operation and adding1.4 m M d UTP and 0.01 U/?L thermosensitive UDG into the reaction system.Finally,1-3?L SYBR series fluorescent dye or NEB phenol red reagent was added into the reaction system to judge the results.When SYBR fluorescent dye was added,the positive results showed yellowish green under UV lamp,while when phenol red reagent was added to the system,the positive results showed yellow under naked eye.The ALV-J LAMP visualization method established in this study did not cross-react with other major avian leukemia subgroups except J subgroup,nor did it have non-specific reactions with other common avian tumor diseases.Sensitivity test results showed that the ALV-J LAMP visualization method was 100 times more sensitive than the traditional PCR method.The minimum number of copies that could be detected was 4×10~1copies/?L,and the limit of detection was 0.166 fg/?L.The results of in-batch repeatability test and clinical samples also showed that the method had good repeatability.ALV-J LAMP visual detection method and traditional PCR method were used to detect suspected avian leukemia virus infection respectively,and the coincidence rate of the two detection methods was 100%.To sum up,through the closed tube operation,the optimization of reaction conditions and use of visual reagent,successfully established for subgroup J avian leukosis virus LAMP visual detection method,this method has low cost,good specificity and test results for observation,in be used actually has extensive application prospect.