Objective: To develop and evaluate a novel genotypic test for rapid detection of RFP and INH resistance of multidrug-resistant M.tuberculosis isolates by gene chip,gene array and multiplex probe array.
Methods: Probes were designed was fabricated according to the 30 single nucleotide polymorphisms of 11 mutations on 4 genes associated with RFP and INH resistance. Probes were designed according to the sequence of M.tuberculosis rpoB gene and the gene array was developed.The DNA fragment which contained hot mutation sites of rpoB gene was amplified with biotin-labelled primers by PCR, and then hybridized with gene array. M.tuberculosis strain H(37)Rv DNA was used as the control. The rpoB genes in M.tuberculosis clinical isolates were also analyzed by polymerase chain reaction-single stranded conformational polymorphism PCR-SSCP and PCR-DNA sequencing. A multiplex probe array was designed for genotypic test to simultaneously screen the mutations of rpoB, katG, inhA and ahpC genes, associated with INH and INH resistance in M. tuberculosis.
Results: 85 of 110 strains resistant to INH and 22 of 30 strains sensitive to INH were detected, while 77 of 94strains resistant to RFP and 40 of 46 strains sensitive to RFP were detected. The results from the gene-chip detection were consistent with the sequence information.We analyzed the rpoB genes of 111 M.tuberculosis clinical isolates by PCR-SSCP. Of 70 RFP-resistant M.tuberculosis isolates, 63 isolates had different SSCP profiles from that of the standard strain H(37)Rv.So also analyzed their rpoB genes by gene array. Of 111 M.tuberculosis clinical isolates, the results of gene array in 41 drug-sensitive strains were similar to that in M.tuberculosis H(37)Rv.63/70 RFP-resistant strains had rpoB gene mutation. The sensitivity and specificity were 88.5% and 100% for RFP resistance, and 86.5% and 100% for INH resistance, respectively
Conclusions: The gene-chip technology, a fast test with high accuracy, specificity and sensitivity Gene array might become a rapid, simple, and accurate method for detecting rpoB mutations in most of the RFP-resistant M.tuberculosis