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TB is a chronic zoonotic disease caused by Mycobacterium tuberculosis. It is estimated that there are around 1/3 of the population of TB infection, accurate and timely diagnosis is necessary for the treatment and control of TB in TB patients. The tuberculin experimental clinical diagnosis of tuberculosis commonly used method, but also the International Organization for Animal Health (OIE) and China approved quarantine method. However, due to the tuberculin may contain the same with other mycobacterial antigens component prone to false positive detection, and this test can not distinguish between naturally infected with BCG immunization. There are also a lot of TB diagnostic methods such as bacterial culture, smear, PCR, IFN-Y diagnostic method, but the disadvantage of these methods is to detect a long time, and cumbersome to operate, and requires expensive equipment, and have dedicated operation, etc., and therefore is not conducive to the detection of tuberculosis. For these reasons, this experimental study two new TB detection method: First, the establishment of a fast, high-sensitivity, high specificity, the method of identification of Mycobacterium tuberculosis and Mycobacterium bovis. Experiment was based on consensus sequences most pathogenic Mycobacterium tuberculosis esat-6, gyrB specific sequence of Mycobacterium tuberculosis and Mycobacterium bovis, Mycobacterium tuberculosis species-specific sequence mtp40 design six pairs of specific primers sputum Mycobacterium tuberculosis detection and differential medium for isolation and culture of results and identification of conventional PCR results were compared. Experimental results show that the established LAMP detection method has a very high specificity. Can distinguish between pathogenic Mycobacterium tuberculosis and non-pathogenic Mycobacterium tuberculosis, can also identify Mycobacterium tuberculosis and Mycobacterium bovis. LAMP detection sensitivity of the technique is about 100 times higher than the classical PCR technique can be detected 7 copies / reaction. In addition, three methods of testing samples found the LAMP bacterial culture in line with the rate of 90.91%, the LAMP and conventional PCR results was 100%. The detection method can be widely used in the epidemiological investigation, and on-site rapid diagnostic and provide the basis for clinical treatment. Second, the use of multi-antigen combination method to establish a high specificity, sensitivity and good TB serological diagnostic methods. Cloned using molecular biological methods, to express the antigen protein ESAT-6 of CFP10, ACR and Rv2626c and purified using protein purification kit. Four protein ESAT-6 of CFP10, ACR and Rv2626c 1:1:1:1 combination as a diagnostic antigen to detect serum TB antibodies. Final criteria ESAT-6, CFP10, ACR and Rv2626c as a diagnostic antigen to establish an indirect ELISA method and the ELISA diagnostic methods, namely S / P ≥ 0.235 is positive, S / P lt; 0.202 negative, 0.235 gt ; S / P ≥ 0.202 as suspicious. ELISA method, a specificity of 95.8%, a sensitivity of 65.0%. 20 smear-positive and 120 healthy human serum samples were tested and compared with the results of the sputum. 13 of 20 smear-positive serum samples were positive ELISA test positive coincidence rate was 65.0%, 120 healthy human serum, 5 were positive, the rest negative, negative coincidence rate was 95.8%, the ELISA method and sputum seized diagnosis total coincidence rate was 91.4%. The study shows that the ELISA method has good prospects for the development and application of.
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