A Simple LDT-PCR Method for Detection of Various Nucleic Acid Sequence Changes in a Small Region of Genes—Application for the Identification of Gene Mutations in MTB irpoB/iGene Associated with Drug Resistance

摘要

A modified low denaturing temperature PCR (LDT-PCR) method combined with DNA microarray technique is developed in our lab for quick and effective identification of various mutations in an 81 base pair region of Mycobacterium Tuberculosis (MTB) ribosome RNA polymerase subunit B (rpoB) gene associated with rifampin resistance. By incurporation of wild type (wt) allele fragments that had been PCR amplified previously, the target PCR fragments coming from mutant clinical MTB samples were codenaturized with incorporated wt type allele fragment at 94°C and then let them randomly form matched structures (homoduplex) and allele mismatch-containing structures (heteroduplex), respectively, when the temperature cooled down to 70°C. After the temperature was raised to 80°C, the heteroduplex double stranded fragments were preferentially denatured and resulted in PCR amplification as well as fluorescence incurporation. Since the homoduplex fragments need a higher temperature to be denatured, they were kept in double-stranded status at that temperature and failed to be PCR amplified. By hybridization of LDT-PCR products with the probes spotted on microarray slides, the fluorescent signals representing the presence of gene mutations were detected. We have tested this method on 35 clinical MTB samples and obtained satisfied results.