Isolation And Identification Of Mycobacterium Tuberculosis In Areas Of Shandong Province And Preliminary Development Of Indirect ELISA For Its Antibody

Tuberculosis is a chronic and zoonotic infectious disease caused by Mycobacteriumtuberculosis complex. Cross-transmission of tuberculosis between humans and animalscaused widespread tuberculosis. The outbreak of the disease in the herd fauna has seriouslyaffected the development of animal husbandry and also brought about serious economiclosses for livestock production. The prevalence of the disease poses a threat to human healthand causing widespread concern of all countries. For the control and eradicate of the disease,we need quickly and accurately make a diagnosis.After decades of research, tuberculosis diagnostic technology, from the earliestMycobacterium isolation and identification to the serological test，and then to the molecularbiology techniques, has been gradually toward the more sensitive, more specific, moreconvenient and economic direction development. Due to the emergence of resistant strains ofMycobacterium tuberculosis, the immunosuppressant, and because of the prevalence of TB ineach region is not the same, these bring a lot of problems to the diagnosis of tuberculosis. Thedifferent prevalence of TB in each region, and these has already caused a lot of problems tothe diagnosis of tuberculosis. Mycobacterium tuberculosis isolation and culture of thepatient’s sputum, serum, combined with PCR technology for identification is very necessaryindeed.Using recombinant outer membrane proteins of Mycobacterium tuberculosis (OMPA)developed a rapid, highly sensitive ELISA for comprehensive testing is also necessary.Therefore, the establishment of the rapid detection of TB diagnosis is particularly important.In this study, we use a variety of detection methods to detect suspected TB sputum andserum. The methods include isolation, culture and identification of M. tuberculosis in sputumof suspected TB patients in Shandong, and detection of Tuberculosis antibody in the serum ofpatients.In this thesis, the main contents include:1. Acid-fast staining of suspected TB sputum, microscopic examination and isolation andculture were carried. According to the outbreak of tuberculosis in part region of Shandongprovince and the prevalence of serious clinical samples collected every month.Combined withsuch methods as the patient’s clinical presentation, CT imaging features, bronchoscopy, chest X-ray and other diagnostic methods,256copies of sputum were collected from the hospitaland cultured.After10-45days, the results showed that68samples of256sputums werepositive,188were negative, the positive rate was26.56%. Based on the growth ofMycobacterium tuberculosis strains obtained, several bacteria were repeatedly cultured again.2. Using the PCR detection method, the obtained strains were identified successfully.Reference to the literature, DNA of68isolates were extracted.and identified with seven pairsof primers.The results showed that3copies of the68strains were non-mycobacteria werenon-mycobacteria,12copies strains were non-tuberculous mycobacteria (Mott),40copieswere TB mycobacteria (MTBC) or African Mycobacterium II type,5copies were M.bovis,3copies were BCG,2copies strains were voles Mycobacterium and3copies were Africabranching bacillus type I, no mycobacteria M.caprae and M.canettii mycobacteria wereobtained.The test results show that the relatively high proportion of people who were infectedwith Mycobacterium bovis was7.4%in all isolates and have important reference significancefor clinical diagnosis and treatment.3. In this study, used the recombinant protein (OMPA) as coating antigen to coatmicro-plate of96wells, an indirect ELISA method was successfully developed to detect theTB antibody in the patients serum.The results showed that the best antigen solution coating buffer is CB,the optimal (best)coating condition was4℃, the confining liquid was1%BSA-PBST, the concentration ofantigen was12μg/mL, the dilution of serum was1:200, the reaction time of serum was40min,the dilution of HRP-labled rabbit anti human IgG was1:5000and the reaction timewas40min, the reaction time of chromogenic was15min,the reaction time after chromogenicwas5min. Repeatability tests revealed that the coefficients of variation of positive serumwithin and between runs were less than10%; COMPAred with the serum of the brucelliasis,hepatitis, HIV Patients, the TB-specific was not cross-reactivity，4℃can be kept for one year.the keeping time is4℃in a year. COMPAred with the results of the clinical diagnosis andtuberculin test (PPD), the diagnostic sensitivity, specificity and accuracy of the OMPA-ELISA were98.15%,95.45%and96.30%.263copies serum of suspecting patients inShandong were detected, and the results showed241copies were positive,22copies werenegative, the positive rate was91.63%.