Detection Of Pyrazinamide-Resistance Mutation In Mycobacterium Tuberculosis And Mechanism Research

Pyrazimamide(PZA)is a first-line anti-tuberculosis drug,which causes drug resistance due to improper use,and thepncA is the gene associated with PZA resistance.Mutations in pncA lead to a decrease or loss of enzyme activity,but not all mutations lead to changes in enzyme activity.Therefore,the pncA poLlymorphism site is important for PZA resistance detection.In this thesis,the PZase was expressd,and the enzyme activity was determined by Wayne method.The existing PZA resistance mutation detection system was improved based on the enzyme activity.In the first chapter,the background of tuberculosis,the drug resistance of tuberculosis and the resistance of pyrazinamide were introduced.The common diagnostic methods of pyrazinamide resistance were compared.Finally,the purpose,content and significance of this paper were proposed.In the second chapter,the experimental methods and materials were described,including the construction of plasmids by ligation independent cloning(LIC)technology,protein expression,determination of PZase activity,and experiments on the expression of pncA-11 site mutation by real-time quantitative PCR(QPCR).In the third chapter,the results of the experiment were given.Using LIC technology,we constructed 75 plasmids containing different mutations,which was used for protein expression.The polymorphic site of the pncA was determined by Wayne test,and the probe was modified from the existing detection system.The detection limit was 50 copies/reaction,the sensitivity was 75.89%and the specificity of was 96.00%when compared with the drug-susceptibility test(DST).The concordance between the detection system and the DST was 85.38%.The concordance between the detection system and the sequencing was 98.58%.