Detection Of KatG, InhA And RpoB Gene Mutations Of Mycobacterium Tuberculosis Through DNA Microarray And Study Their Correlations With INH And RFP Resistance

Pulmonary tuberculosis (PTB) caused due to infection of Mycobacterium tuberculosis is a major worldwide epidemic and infectious disease. Conventional effective antituberculosis drugs such as isoniazid (INH) and rifampicin (RFP) are invalid for treatment of multiple drug resistant-tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis. Early stage, rapid and sensitive diagnosis of such tuberculosis may contribute to the adoption of effective and rational clinical therapeutic drug and programs. DNA microarray being applied to diagnosis of MDR-TB is rapid and sensitive with high throughput.Lowenstein-Jensen solid culture method is adopted for conventional drug susceptibility test of Mycobacterium tuberculosis. CapitalBio Mycobacterium tuberculosis drug resistance detection kit and related instruments are adopted to conduct nucleic acid extraction, PCR amplification, microarray hybridization, cleaning and scanning so as to respectively determine the drug susceptibility of common discontinuity points of Mycobacterium tuberculosis INH and RFP resistant-genes KatG315, inhA-15and rpoB. Through extractign DNA of the genome of Mycobacterium tuberculosis, adopting PCR to amplify KatG and rpoB gene fragments and completing purification, and after nucleic acid electrophoresis identification, the gene sequence of KatG and rpoB gene were respectively gained after DNA sequencing. Based on the gained results, the correspondence between drug resistance of DNA microarray test and conventional drug susceptibility test was analyzed. Moreover, the difference of the results between DNA microarray and DNA sequencing was also compared.The results of DNA microarray test showed that:(1) with respect to inhA-15mutation (C→T), main mutation form of Mycobacterium tuberculosis INH-resistant gene KatG in Suzhou City is (AGC→ACC) Ser→Thr (S315T), accounting for96.3%of the total resistant mutants; with the INH drug-resistant test result obtained from Lowenstein-Jensen solid culture method as the standard, the accuracy rate of DNA microarray test for bacterial strain INH susceptibility test is73.8%; and with the results of DNA sequencing as the standard, the accuracy rate of DNA microarray test for KatG315mutation test is88.9%.(2) main mutation form of Mycobacterium tuberculosis RFP-resistant gene rpoB in Suzhou City is RRDR-531(TCG→TTG) Ser→Leu, accounting for76.9%of the total mutants of tested specimens rpoB-RRDR locus; with the RFP drug-resistant test result obtained from Lowenstein-Jensen solid culture method as the standard, the accuracy rate of DNA microarray test for bacterial strain susceptibility test is93.3%; and with the results of DNA sequencing as the standard, the accuracy rate of DNA microarray test for rpoB gene RRDR mutation test is82.4%.