Comparison Of PCR Primers For Detection Citrus Huanglongbing And Development Antibody Of Its Outer Membrane Protein

Huang Long Bing(HLB) is one of the most devastating diseases of citrus industry. It was known as "cancer" of citrus, bacause it can not be effectively cured currently. HLB is so wide range of transmission that distributed in Asia,Africa,Americas and Oceania that more than 50 countries. HLB can infect major citrus production areas, cause huge economic losses, even citrus industry is destroyed in areas. HLB is a serious threat to the citrus industry all over the world.HLB bacteria belonging to the alpha subclass, phloem bacillus genus, gram-negative bacteria,according to the difference of 16 S rDNA sequences, HLB bacteria are divided into three types:’Candidatus Liberibacter asiaticus’,’Candidatus Liberibacter africanus’, and ’Candidatus Liberibacter americanus’. Researchers were blocked to know pathogenic bacteria, Since HLB bacteria is hard to be cultivated. The main symptom of blade that diseased tree is mottled yellow and fruit "red nose", seriously affect the quality and yield of fruit. In the case of non-resistant varieties and effective cure, prevention and control of HLB mainly depends on virus-free planting seedlings, control fleas, timely excavation diseased trees. Early diagnosis of the disease is crucial, however, due to the existence of a subjective field diagnosis of miscarriage of justice,it is now the most widely used diagnostic techniques HLB is the laboratory of molecular detection techniques. Faced constantly being reported out of the new polymerase chain reaction(PCR) primers detecting HLB, screened a pair high sensitivity and specificity of the primers used in the laboratory to detect HLB is very necessary. Six pairs of detection primers that reported in the literature commonly were choosed, combining with conventional PCR and Reatime fluorescence quantitative PCR(qPCR), each pair of primers was detected the sensitivity and specificity, the following are results:1、The recombinant plasmid pT-16 S HLB bacteria containing 16 S rDNA sequence prepared and HLBarsp quantitative PCR probes corresponding to the standard, esTablelelished in our laboratory fluorescence quantitative PCR detection system. Based on real-time PCR-positive samples in the determination of the amount of content 16 S rDNA citrus greening bacteria, and then get citrus greening bacteria genome, 0.25% to 0.35% of total nucleic acids in leaf samples.2、Five pairs of conventional PCR primers that used in the literature Commonly: OI1 / OI2 c,P400, P535, A2J5, Las606/LSS and16 S primers were designed for the experimental group.Combined with quantitative PCR, six pairs of primers designed to detect the sensitivity and specificity, It is found that the sensitivity size of the smooth: Las606/LSS> 16S> P400> OI1/OI2c= P535 = A2J5, Las606/LSS has the highest sensitivity that is 1.08×10-5 ng/μL.Six pairs of primers specific were higher, in line with the detection of Huanglongbing as a primer requirements.Serological detection method is the wide use of disease detection has advantages in handling a large number of test samples, rapid detection. Faced with a large area of southern Jiangxi HLB spread, prepared for Gannan region HLB strain antibodies esTablelelish serological detection methods is necessary. The paper also uses cloning and immunological methods for preparing the antibody, achieve results is as follows:3 、 Due to the HLB full-length outer membrane protein is difficult to expression, the expression of full-length segment. ccording to forecasts NCBI daTablelease HLB outer membrane protein structure and function, 466-781 aa fragment surface antigenic sites,hydrophilic binding protein analysis, length downstream length 1071 bp fragment designated as fragment by fragment that purpose cloning, expression and immune induction experiments,ELISA titer determination, finally get titer ≥1: 512000 of citrus greening bacteria antibodies.Using this antibody samples were collected from the field for testing, and found that the antibody can detect a consistent positive samples, and the general PCR results, indicating that the expression of citrus HLB outer membrane protein fragment antibody preparation is successful,after the esTablelelishment of serological laboratory thread detection methods laid a solid foundation.