Development Of Loop-mediated Isothermal Amplification (lamp) And Inderect Elisa For Detection Of Mycobacterium Tuberculosis

Tuberculosis that caused by Mycobacterium Tuberculosis is a chronic zoonosis. One-third of the world population is estimated to have Mycobacterium tuberculosis infection. Accurate and timely identification of infected individuals is critical for treatment and control. For the detection of TB, tuberculin skin test (TST) is the only method recommended by OIE.Tubereulin is also called Purified Protein derivatives(PPD) which is a proteinmixture that has complex components.PPD includes many antigens shared with other Mycobacterium species besides specific antigens of M.bovis.So,the PPD skin test cannot avoid the cross reaction with other mycobaeterial.Moreover the method propably provides false Positive results when there is serious tubereulosis or immunosuppressive conditions exist. Moreover,the current diagnostic methods such as culture,smear,PCR and so on lack the desired sensitivity and specificity, require sophisticated equipment and skilled workforce or take weeks to yield results. Therefore, this study developed two specific and sensitive diagnostic methods for TB.1. To develop a method of high efficiency, sensitivity and specificity to detect Mycobacterium tuberculosis and Mycobacterium bovis. Specifc primers were designed by targeting the esat-6 gene, gyrB gene and mtp40 gene. The results of LAMP detection were compared with the results of bacterium culture and PCR detection. The LAMP is a high efficient and specific method which can detect Mycobacterium tuberculosis and Mycobacterium bovis. The sensitivity of LAMP that can detect 7 copies/reaction was 100 times greater than PCR.Compared with three detection methods, it showed that the results of detection between LAMP and bacterium culture were 90.91% identity;the results of detection between LAMP and PCR were 100% identity. This new method might facilitate genetic analysis applied in clinical laboratory and field surveillance. Meanwhile, it also has some potentials for rapid test.2. To develop a efficiency, sensitivity and specificity serodiagnostic methods for TB. The study cloned and expressed four proteins which include EAST-6 and CFP10 that express during the early stage of infection,and the others are ACR and Rv2626c that express during the stage of dormancy.The diagnostic antigens were shown specifically react to sera from people with tuberculosis.In this study, an indirect ELISA was established using "cocktail" proteins as the diagnostic antigen.The ELISA were nomalised with 100 serum samples from healthy people,with a S/P≥0.235 being positive and S/P＜0.202 being negative.Test of 20 serum samples from people with tuberculosis and 120 serum samples from healthy people detected 65.0% positive with a specificity of 95.8%.The ELISA results of 140 serum samples (20 from people with positive test of sputum smear; 120 from people with negative test of sputum) were compared with those of test of sputum.13 out of the 20 samples from people with positive test of sputum smear were also positive by ELISA,with a positive coincidence of 65.0%.5 out of the 120 samples from people with negative test of sputum were positive by ELISA.The total coincidence of the ELISA method and test of sputum smear was 91.4%.At the same time,the study tested 150 serum samples from bovine with negative skin test of PPD,and all of the samples were negative when tested by ELISA.