Development And Application Of Rapid Detection Technology For Citrus Huanglongbing

Citrus Huanglongbing(HLB)is a devastating disease in the world's citrus production.At present,there is no effective agent in China to cure HLB,and no reliable disease-resistant varieties and rootstocks have been reported.Mainly to remove diseased trees,prevent and control citrus psyllids,use disease-free seedlings to prevent and control HLB.Among them,the rapid detection of citrus seedling disease and field plant susceptibility is particularly important for the effective prevention and control of HLB.The detection methods of HLB mainly include routine PCR,nested PCR,quantitative PCR,microscopic observation,serological diagnosis,spectral detection,etc.Due to the diversity of HLB pathogen,most of the antibodies reported by predecessors can only bind to similar antigens and cannot be widely applied to the detection of HLB in different regions and varieties.Compared with traditional detection methods,loop-mediated isothermal amplification(LAMP)technology is more time-saving and labor-saving and saves detection cost.Therefore,this thesis has carried out the research of polyclonal antibody against Guangxi HLB pathogen.At the same time,a visual LAMP system for HLB was established and citrus seedlings and field plants in some areas of Guangxi were tested for HLB.(1)According to a plurality of outer membrane protein omp genes of HLB pathogenic bacteria in NCBI database,specific primers are designed to clone target fi-agments and prokaryotic expression vector pET32a-omp1 is constructed.The plasmid was transformed into Escherichia coli expression strain BL21(DE3)to induce expression of recombinant protein.Under the optimized conditions(adding inducer IPTG 0.6 mM,inducing expression at 37? for 5 h),a large number of recombinant proteins expressed in the form of inclusion bodies were induced.After Western Blotverification,about 55 kDa of recombinant protein was successfully obtained.The recombinant protein was purified(purity>90%)and used as antigen to prepare polyclonal antibody of HLB pathogen onnp,and finally polyclonal antibody with titer over 1:512 K was obtained.(2)Using PrimerExplorer V5 on-line software,LAMP primers were designed at 6 sites in the conserved region of the 16SrDNA sequence of HLB pathogen.The LAMP reaction system and conditions were optimized to obtain Mg'+ 6 mM,dNTP 1.2 mM,FIP/BIP 1.6.?M,F3/B3 0.2 ?M,Bst DNA polymerase large fragment 0.24 U/?L,10×ThermoPol 2.5 ?L,0.2%Tween-20 as the optimal reaction system,65? reaction for 50 min,80? inactivation for 7 min as the optimal reaction conditions,adding 1?L of 10 times diluted SYBR Green I nucleic acid dye to observe the reaction results.The LAMP system only amplified the HLB gene and had high specificity.The total DNA ratio of vein of infected HLB plant was diluted as template detection sensitivity.The lower limit of LAMP detection can reach 2.18 ng/?L,which is 100 times higher than conventional PCR detection,and is equivalent to fluorescence quantitative PCR.Conventional PCR and established visual LAMP detection methods were used to detect HLB in different citrus seedlings and field plants in some areas of Guangxi.No HLB pathogen was detected in 25 C.junos Sieb.ex Tanaka seedlings and 25 grafted seedlings in Citrus jinqiushatangju in the net room.The positive rate of LAMP detection method for citrus yellow dragon disease is 13.6%,the positive rate of conventional PCR detection method is 11.6%in 155 plants in the field.Samples with positive PCR results were all positive in LAMP results.The established LAMP system can be used for rapid detection of HLB field samples and seedlings in Guangxi.