Tuberculosis And Non-tuberculous Mycobacteria Multiple PCR Method To Establish And Preliminary Application

To understand the prevalence of TB in dairy cattle, carrying and infection case of Mycobacterium tuberculosis and non-tuberculous mycobacterial in Shihezi recent years. Avoid detection of TB quarantine PPD false-positive cattle to be wrong killed.In this way,we can Prevention diffusion of tuberculosis epidemic in dairy farms, safeguard the public health and safety. and protect the development of animal husbandry economy. The multiplex PCR method established in this study can distinguish between tuberculous and non-tuberculous mycobacteria, moreover,we carry ed out an investigation about tuberculous and non-tuberculous mycobacteria among some cattle herds cows and farms sparrow in Shihezi with the method.First, the recovery and cultivation of mycobacterial standard strains. To meet the test needs,we made Mycobacterium tuberculosis H37Ra,BCG,Avian mycobacteria,Mycobacterium intracellulare, Occasion-al mycobacteria,Smegmatis Mycobacterium,Turtle Mycobac-terium as representative for Standard strains of Mycobacterium.The DNA of H37Ra and BCG are taken from Ethnic Diseases Laboratory in the college of Medicine.At the same time,we Completed the preparation of mycobacterial medium,the recovery and training of Turtle Mycobacterium. Moreover,we Successfully cultured avian mycobacteria, Mycoba-cerium intracellulare, Incidental Mycobacterium smegmatis Mycobacterium.Then, To establish a rapid distinguish multiplex PCR method for identification of tubercul-osis and nontuberculous mycobacteria. Genus specific genes32kD of Mycobacterium, species specific gene MTP40of Mycobacterium tuberculosis and specific genes IS6110target gene of Mycobacterium tuberculosis complex, three pairs of specific primers were designed and synthesized, the standard strain of Mycobacterium tuberculosis DNA as a template DNA template concentration in the PCR reaction system optimization of various reaction parameters, concentration of Mg2+, dNTPs, primers and Taq DNA polymerase factor orthogonal design, as well as the PCR conditions, the identification of mycobacteria multiple PCR detection methods, and the specificity of the method, the sensitivity was validated. Results:506bp,396bp and984bp fragment was amplified in the standard strain of Mycobacterium Tuberculosis H37Ra,506bp and984bp fragm ent was amplified in Guerin (BCG). The506bp fragment was amplified in Mycobacterium avian, Mycobacte-rium intracellulare, Mycobacterium fortuitum, Mycobacterium smegmatis and Mycobacte-rium chelona. The result of Escherichia coli was negative. The sensitivity minimum of multiplex PCR was6.6x10-6ng. Conclusion:The method has a high specificity and sensiti-vity, and can be used in diagnostication of Mycobacterium tuberculosis and nontuberculo-us Mycobacteria for cows.Finally, conduct a TB intradermal allergy test of cows refer to GBT/18645-2002. PPD test were detected in2624cows,among them there are35Positive reaction, Positive rate was1.33%(35/2624).We collected35samples of faeces and35samples of Nasal secretions from catties those are Skin test-positive.Then we caught21Sparrow in cattle farm.We detected the samples with the multiplex PCR method,the results showed that positive rate of faeces sample is8.57%(3/35,contain1nontuberculous Mycobacteria and2Mycobacter-ium bovis), positive rate of Nasal secretions is11.43%(4/35,contain2nontuberculous Mycobacteria and2Mycobacterium bovis),among all the positive samples,only one cattle both feces and nasal secretions were Mycobacterium test positive results.The Mycobacter-ium positive rate of Sparrow lung tissue was9.52%(2/21, Two cases are non-tuberculous mycobacteria). Carry on experiments (consist of smear, staining, microscopy, pathogen isolation and culture) with All PCR positive samples,Only two cases of sparrows sample shows a small amount of acid-fast bacilli staining,But,at last,there were no pathogens isolated from samples. The results showed that,Non-tuberculous mycobacterial infection caused by the phenomenon of false-positive cow tuberculosis quarantine,Farms ambient biological carrying the mycobacterial pathogen may be caused by cows infected with one of the factors. Therefore, the test results for the the future the cows tuberculosis quarantine TB prevention and control of great significance, and provides a reference for the develop-ment and implementation of the tuberculosis control measures for cows...