| M.tuberculosis infection can cause various organs diseases including tuberculosis.With the widespread epidemic of tuberculosis in the world,the number of drug-resistant tuberculosis cases increased in recent years,tuberculosis control has brought severe challenges.At present,bacteriological methods are commonly used in clinical diagnosis for tuberculosis and drug-resistant diagnosis.Bacterial culture takes a long time and could not provide diagnostic results timely,it is urgent to find other methods to accurately and quickly to diagnose tuberculosis and drug-resistant tuberculosis.Objective:(1)To understand the commonly used drugs(Rifampicin,Isoniazid,Streptomycin,Ofloxacin and Ethambutol)resistance of clinical isolates of M.tuberculosis in Kashi.(2)Investigate the relationship between resistance phenotypes of drugs and the gene mutations,initially evaluate the consistency between the drug susceptibility test(DST)and DNA sequencing,provides the basis of rapid diagnosis for the drug-resistant tuberculosis.(3)Preliminary research on the value of M.tuberculosis specific proteins in serological tests,provides ideas for the research of newly diagnosed proteins.(4)Screening out stimulating antigens which including rich T cell epitopes from M.tuberculosis proteins,initially explore the potential value as stimulatory antigens in IGRAs.Methods:(1)The drug resistant phenotypes were obtained from 113 clinical isolates of M.tuberculosis by DST.(2)Amplification and sequencing drug-resistance determinants of five drugs in the strains.the DST results were used as standards to calculate kappa values and analyze consistency.(3)Construction prokaryotic expression vectors for 18 genes,expression and purification recombinant proteins;established the indirect ELISA method to detection the tuberculosis specific antibodies,analyze the efficiency of each protein in the diagnosis of tuberculosis.(4)Using prediction software to predict T cell epitopes of proteins,select high score protein as T lymphocyte stimulator.Screening out stimulatory antigens that induce high levels of IFN-γ release IGRAs are performed by stimulating combination antigens and the results are compared with T-SPOT.TB to analyze the value of stimulator.Results:(1)The total drug resistance rates of RFP,INH,SM,EMB,and OFX were4.4%,11.5%,8.8%,2.6% and 3.5% of the clinical isolates;single drug resistance rate is66.7% and multi-drug resistant rate is 33.3%;compare with DST results,the Kappa value of DNA sequencing for RFP,INH,SM,OFX,and EMB were 1,0.955,0.721,0.796,and 1respectively.(2)Successfully construct 18 recombinant plasmids;17 proteins were expressed and purified.All of the best conditions for the detection of IgM and IgG antibodies proteins were determined.CFP-10,PPE57,and Rv3807 have higher diagnostic efficiency in detecting tuberculosis-specific IgM antibodies.The AUC are between 0.621 to 0.762,sensitivity are between 54.7% to 59.3%,the specificity are between 71.3% to 90%;PPE57,Ag85 B,CFP-10,Rv0220,and 38 kDa proteins were excellent in detection of tuberculosis IgG antibodies,the sensitivity was maintained between 65.1% to 76.7%,specificity was between 78.1% to 88.3%.(3)High T-cell epitope scores proteins were successfully predicted and established double-antibody sandwich ELISA for human IFN-γ;Rv1987,Rv3807,PPE57,Rv0220 andMPT64 proteins had higher correlations with ETC(r>0.5,p<0.01).Combined proteins Rv1987+Rv3807,16kDa+Rv0220,MPT64+Rv1986,stimulated the T cells release IFN-γ had a good correlation(r> 0.692,p<0.01)compare with control;the sensitivity of the combination protein as a IGRAs stimulated antigen for detecting tuberculosis are between 62.5% to 80%,and specificity are between 33.3% to 75%.Conclusions:(1)Analyze RobB,KatG,InhA,RpsL,rrs,gyrA,and embB genes by DNA sequencing technique have high sensitivity and specificity for the determination of drug resistance,can provides a theoretical basis for rapid diagnosis of drug resistance.(2)CFP-10,38 kDa,PPE57,and Rv3807 have better diagnostic efficiency when detecting IgM antibodies that appear in the early stage of tuberculosis infection;PPE57,Ag85 B,CFP-10,Rv0220,and38 kDa are used to detect tuberculosis-specific IgG antibodies has better diagnostic efficiency.(3)Rv1987,Rv3807,PPE57,Rv0220,and MPT64 proteins have good correlations with ECT as IGRAs stimulator,can be a candidate antigen for further research;Rv1987+Rv3807,16kDa+Rv0220,MPT64+Rv1986 has an excellent correlation(r>0.692)compare with ECT in IFN-γ release amount,can be provides a certain reference value for the screening of new stimulator for IGRAs. |
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