Study And Application Of ELISA Detection Technology For Shallot Latent Virus In Tiller Onion

Tillering onion is a characteristic economic crop in northern of China.Shallot latent virus(SLV)is one of the main viruses that infect tillering onion.Although it does not show obvious symptoms after infection,it still seriously affects the yield and quality of Allium such as tillering onion.At present,no effective virus control agents have been found.Preparation and production of high quality virus-free seedlings is an effective means of preventing and controlling SLV.Efficient and sensitive virus detection technology is the key to the quality assurance of virus-free seedlings.In this study,Kunming mice were immunized with the capsid protein of the tillering onion isolate of SLV to obtain the polyclonal antibody against SLV.On this basis,the indirect ELISA detection technology for SLV was established.Combined with the established detection technology,the tillering onion virus-free seedlings were successfully obtained through shoot tip tissue culture,which provided technical support for high-throughput detection of SLV and large-scale production of virus-free seedlings.1.SLV isolates were obtained from the leaves of onion tillers suspected to be infected with SLV,and the SLV CP gene was successfully cloned.The recombinant expression vector was constructed by homologous recombination method and induced to E.coli.BL21(DE3)After purification,high-purity recombinant protein of SLV CP was obtained.The recombinant protein was used as antigen to immunize Kunming mice,and the polyclonal antibody was successfully prepared.The titer of antibody to the crude extract of onion leaf virus infected with SLV was256000 times diluted,and SLV could be specifically recognized.2.The indirect ELISA method for SLV detection was successfully established and optimized.The crude extracts of tillering onion leaves infected with SLV were used as antigen to coat the ELISA plate overnight at 4°C,then ELISA plate wells were blocked with 5% fat-free milk for 60 min.The working concentration of antibody to SLV was 125 ng/m L,and the incubation time is 1 h at 37°C AP-labeled antibody of 500 ng/m L was incubation for 1 h.The detection limit of this method was 9.5 ng/m L;the coefficient of variation within the inter-plate was less than 5%;the established indirect ELISA and conventional RT-PCR were used to simultaneously detect 50 tillered onion samples suspected to be infected with SLV in the field.The detection results of 47 samples were consistent,and the coincidence rate was 94%.3.The tissue culture seedlings of tillering onion were obtained by direct shoot tip induction and dedifferentiation and redifferentiation through callus induction.This study optimized the disinfection method of explants,and the optimal disinfection treatment was 75% ethanol soaking for 30 s and 2% sodium hypochlorite solution soaking for 15 min;The optimal shoot tip induction medium MS + NAA0.5 mg/L + 6-BA 1.0 mg/L was determined by screening hormone ratio,bud induction rate was 85.5 %,with an average of 3.3 induced buds;The best callus medium MS + NAA 0.4 mg/L + 2,4-D 0.5 mg/L,the callus rate was 71.7 % and the callus was in good condition;The optimal bud formation medium was MS + NAA 0.4 mg/L + 6-BA 1.0mg/L,germination rate 82.2 %,average number of differentiated buds 9.23;optimum rooting medium 1/2 MS,rootability 100%.The SLV indirect ELISA method was used to detect the virus in the tissue culture vaccine.The results showed that 0.3-0.4 mm stem tip was selected for tissue culture detoxification of tillering onion,and the detoxification rate was up to 92%.Virus-free seedlings with complete detoxification and robust growth were selected for transplanting,and the survival rate was 90%.