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Lyme disease is by hard ticks spread by Borrelia burgdorferi caused by zoonoses. The disease was distributed worldwide, currently estimated global annual incidence of about 30 million people, and there is a growing trend. In the United States, Lyme disease is the \In recent years, Lyme disease pathogen biology, pathogenesis, epidemiology, diagnosis and comprehensive prevention research has made some achievements, but because of Borrelia burgdorferi antigen structural complexity and Lyme disease clinical Symptoms diversity, so the disease diagnosis more difficult. In addition, Lyme disease as a new disease in the country of their media tick species not yet fully clear, but on its pathogen genotype and taxonomic status were not clarified, which gives the diagnosis and prevention of the disease brings great challenges. Therefore, to clarify its media type and pathogen free status will help us to establish an efficient, rapid diagnostic tools and reasonable to carry out disease prevention and control work. In this paper, the Lyme disease pathogen taxonomy and diagnostics two aspects of exploration. (A) right from our part of the forest collecting Asian Hyalomma (Hyalomma asiaticum), longicornis (Haemaphysalis longicornis) and other tick species were Borrelia burgdorferi detection; (2) on the isolated spirochetes genotyped research; (3) the obtained Borrelia burgdorferi flagellin gene and flagellar genes conserved regions were cloned in vitro Expressed by Dabo burgdorferi flagellin genes conserved regions, with the conserved regions of recombinant proteins As Lyme disease diagnostic antigen, the establishment of Lyme disease ELISA diagnostic method. The first part, with the nested PCR amplified tick body Borrelia burgdorferi flagellin gene collected from Xinjiang, Qingdao and Yanbian and other regions of Asia Hyalomma and longicornis ticks and intestinal cultures were detected, the PCR The amplified gene fragments were sequenced, sequencing results by GenBank, the blast comparative analysis. Using BSK-H solid medium isolation and culture techniques, from ticks intestines isolated and purified Borrelia burgdorferi monoclonal and using molecular biology methods for isolated monoclonal colonies were typed. First, the Xinjiang region Asia Hyalomma and Rhipicephalus ticks under the microscope anatomy, whichever intestine, with BSK-H liquid medium for culture, collecting ticks intestinal cultures were extracted genomic DNA, using nested PCR amplified flagellar genes. Then, identified with Borrelia burgdorferi intestinal culture, the use of BSK-H solid culture separation technology, its isolation and purification, the establishment of a fast and convenient separation Borrelia burgdorferi culture method. Finally, the recA gene and the 5S-23S intergenic gene as a sub-type of the target gene on isolated Borrelia burgdorferi monoclonal initial genotyping, recA gene is associated with Borrelia burgdorferi evolutionarily related genes whose GC content changes can be quantitative PCR melting curve easily reflected, according to this feature, quantitative fluorescent PCR melting curve for isolated spirochetes monoclonal initial genotyping. After initial classification, the second from the Asian Hyalomma isolated gamma burgdorferi, narrow Borrelia burgdorferi and other strains. While further determine spirochetes genotype its 5S-23S intergenic for PCR amplification, the amplified products were sequenced, according to the sequencing results further typing. Borrelia burgdorferi monoclonal Isolation and genotyping studies for Lyme disease pathogen biology research foundation. The second part of the Lyme disease serological diagnostic techniques were studied. The study of recombinant Borrelia burgdorferi flagellin protein as a diagnostic antigen, the establishment of Lyme disease ELISA diagnostic method. In this study, use of biological engineering methods selected flagellar gene sequence of amino acids 131-266 session as the fragment, its cloning, connected to PGEX-4T-1 expression vector constructed conserved gene recombinant plasmid is then transformed into BL21 cells in prokaryotic Dabo burgdorferi flagellin gene antigen and conservative strong segment truncated protein with GST.BIND purification systems purified Borrelia burgdorferi flagellin gene recombinant protein, the purified protein used as a diagnostic antigen; while Borrelia burgdorferi were inoculated mice and rabbits, two weeks after the serum was collected using the above diagnostic antigen identification, discovered by infected mice and rabbit sera were positive serum, the establishment of a sensitivity and specificity higher ELISA for diagnosis. This method is to solve Lyme disease is difficult to diagnose, difficult diagnosis provides a useful reference, especially for the Lyme disease early diagnosis provides a powerful means, and there is some clinical value. In summary, this study, simple and efficient solid culture method Borrelia burgdorferi monoclonal isolates were isolated and screened a kind of Chinese Lyme disease pathogen typing new method; established to detect specific, sensitive high Lyme disease serum antibody ELISA method. This is valid, rapid diagnosis and a reasonable control of Lyme disease provide a theoretical basis, but also for the epidemiology investigation carried out to provide a powerful tool.
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