| Tuberculosis is a global public health problem that seriously endangers human life and health.The widespread epidemic of drug-resistant tuberculosis poses great challenges to the prevention and control of tuberculosis.The rapid detection of multi-drug resistant tuberculosis(Mycobacterium tuberculosis resistant to isoniazid and rifampicin at least)has important guiding significance for clinical use of drugs.The heterogeneous drug resistance of Mycobacterium tuberculosis means that both sensitive and resistant strains are carried in a sample of tuberculosis patients.A highly sensitive detection method is needed to diagnose the drug resistance of Mycobacterium tuberculosis.In this dissertation,a highly sensitive detection system for rifampicin resistant mutations and multidrug resistant mutations of Mycobacterium tuberculosis was established by using DeepMelt method which could detect low proportion mutations.In chapter one,the epidemic situation of tuberculosis,the prevention and control of tuberculosis,the classification of anti-tuberculosis drugs,the diagnosis and treatment of drug-resistant tuberculosis are summarized respectively.Then the characteristics and detection requirements of heterogeneous drug resistance of Mycobacterium tuberculosis were introduced and the principle of polychromatic melting curve analysis for detection of mutations and the fluorescent labeling and modification of self-quenched probes were described.Finally,the research content of this paper was proposed.In chapter two,we take rifampicin resistance gene as the research object to established a highly sensitive detection system of rifampicin resistance mutations in one-tube with four color,based on the polychromatic probe melting curve analysis technology.Then the detection of mutations in Rifampicin resistance determining region(RRDR)was realized.In order to ensure the full coverage detection of the RRDR region by four probes,we investigated the modification mode,arrangement and melting point of the fluorescence/quenched group of the probe,and proposed a novel design scheme of the probe.We used plasmid reference products to examine the analytical performance of the system,the heterogeneity resistance detection ability of rpoB 531TCG>TTG was 1%mutation,for rpoB 526CAC>GAC,rpoB 516GAC>GGC and rpoB 511CTG>CCG,the detection ability of heteroplasmic resistance was 5%,10%,and 20%respectively.Finally,450 clinical specimens were used to evaluate the clinical performance of the system.The sensitivity of the system was 93.84%,the specificity was 92.76%,and the Kappa value was 0.85,according to the results of drug sensitivity test,which indicated that the system is consistent with the results of drug sensitivity test and is suitable for clinical application.In chapter three,a highly sensitive detection system for multidrug resistant mutations of Mycobacterium tuberculosis was established by using DeepMelt technique.The detection of the drug resistance mutations(katG 315 codon,inhA promoter and ahpC promoter)and RRDR mutations were achieved.The 8 common mutation sites were detected by heterogenous reference materials.High-frequency mutation sites rpoB 531TCG>TTG and katG 315AGC>ACC detection levels as low as 1%the detection level of rpoB 526CAC>GAC,rpoB 516GAC>GGC,inhA promoter-15C>T,ahpC promoter-30C>T and ahpC promoter-10C>T is 5%,rpoB 511CTG>CCG was 10%.Finally,1230 clinical specimens from four clinical units in Shenyang,Beijing,Henan and Guangzhou were collected for multicenter clinical evaluation.According to the results of drug sensitivity test,the sensitivity for the detection of rifampicin and isoniazid resistance was 95.67%,89.66%,specificity was 94.27%,95.48%,Kappa value was 0.875,0.85,respectively.The overall multi-drug agreement rate was 91.02%.The multi-drug resistant system established in this study was simple,rapid,sensitive,specific and easy to use.It is expected to be applied to the rapid diagnosis of clinically multi-drug resistant... |
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