Cloning And Application Of RpoB And KatG Mutation Points In Mycobacterium Tuberculosis

Objectives:Cloning common mutation sites of rpoB and katG in Mycobacterium tuberculosis and application of tuberculosis diagnoses in clinic using the low-density oligonucleotide arrays flow-through hybridization.Methods:In the previous studies, we measured and analyzed rpoB and katG gene mutations and found four common mutation sites and six mutation types. In this study, rpoB and katG gene from six clinically-isolated mutant strains were amplified by PCR and cloned into pMD19-T Vector by T-A cloning. Successful transformants were screened by double restriction endonuclease digestion and PCR amplification, and confirmed by DNA sequencing. The segments of rpoB and katG from six clinically-isolated strains were detected by DNA microarray.Results:The sequence containing mutant sequence of rpoB and katG from6mutant strains were successfully cloned and confirmed by sequencing. The rpoB and katG gene of six clinical strains gave positive spots in predictive positions in the oligonucleotide array test.Conclusions:The cloning common mutation sites of rpoB and katG gene may provide a positive control for clinic diagnoses of tuberculosis through oligonucleotide arrays.