Study On DAS-ELISA Assay Method And Nano-agent Preparation Of R.solanacearum

In recent years,bacterial wilt has caused serious damage to the sericulture industry,and it is imperative to carry out early detection and control of its pathogenicity.In this paper,we developed a DAS-ELISA kit for mulberry blight,with a minimum detection limit of 3.13×10³ CFU/mL and a detection time of 2.5 h.The PLGA nano-agent of compound caffeic acid methyl ester and caffeic acid phenethyl ester has a EC₅₀ value of 0.285 mg/mL,and the inhibition effect is good,the dosage is reduced by 2/3;the sustained-release property is good,and the pathogenicity of Ralstonia solanacearum force-related genes phcB,egl,pehC,and phcA are down-regulated from 1/42 to 1/2.Main conclusions are as follows:（1）Construction of a monoclonal antibody with specific binding abilityUsing immunological and serological techniques,mice were immunized with live strains,and spleen cells were obtained for construction of hybridomas,and three specific monoclonal antibodies 1G9,2H5 and 9G9 were prepared.All antibodies were identified as IgG1 by antibody subtypes identification.The antibody was purified by ammonium octoate ammonium sulfate method.The indirect ELISA method showed that the purified three monoclonal antibodies had strong specific binding ability to R.solanacearum,and the titer reached 1:64000.（2）Rapid and highly sensitive DAS-ELISA detection method for R.solanacearumThe immune-PCR was employed to detect the specificity of the prepared 1G9,2H5,9G9 and Polyclonal Antibody（PcAb）,and the specific binding ability of the four antibodies to R.solanacearum was evaluated.The Hrp-labeled antibody was used to prepare the enzyme-labeled antibody for the construction of R.solanacearum DAS-ELISA method.And the type of capture antibody,enzyme-labeled antibody,blocking solution and enzyme-labeled antibody dilution ratio used were screened.It was found that 1G9 was used as the enzyme-labeled antibody,1G9 and 9G9 as well as PcAb were respectively capture antibodies,1%skim milk and 500-fold diluted enzyme-labeled antibody was used as the optimal condition.The minimum detection limit was 3.13×10³CFU/mL,and the detection time was 2.5 h.（3）Preparation of compound PLGA nano-agent particles for inhibiting R.solanacearumPreparation of compound PLGA nano-agent particles for inhibiting R.solanacearum.The compound PLGA nanoparticles of methyl caffeate and phenethyl acrylate were prepared by single emulsion solvent evaporation method.The conditions of PVA stabilizer concentration,ultrasonic power,ultrasonic time and organic phase water were optimized.FT-IR and particle size were used to analyze and characterize the agent.Under optimized conditions,the PLGA nanoparticles with high drug loading rate（28.33±0.03%）were obtained,the average hydrated particle size was 188.86 nm.The nano-medicine particles have a good inhibitory effect on R.solanacearum(EC₅₀value was 0.285 mg/mL,which can cause obvious pores on the surface of R.solanacearum),and the dosage is reduced by 2/3.The prepared nano-pharmaceutical particles have good sustained release properties.Kinetic analysis of compound nano-agents particles loaded with MC and CAPE was performed using the Fickian model.The theoretical half-lives in phosphate buffers at pH 6.5,7.4 and 9.5 were 10.0 d,5.8 d and 5.7 d,respectively.In addition,RT-PCR displayed the expression levels of the pathogenicity-related genes egl,pehC,phcA,phcB,pilT and polA-238 of R.solanacearum decreasing after treatment with compound nano-agents to 1/6,1/13,1/2,1/42,1/2 and 1/8 of the compound drug treatment group respectively.It is proved at the genetic level that the agent prepared in this study inhibits the pathogenicity of the pathogen.In this study,the early diagnosis method and the prepared nano-agents constructed by DAS-ELISA enriched the new content of the control strategy of mulberry blight.