Rapid Detection Of RpoB Mutations In Mycobacterium Tuberculosis By Gene Chip

Rapid detection of rpoB mutations in Mycobacterium tuberculosis by gene chipObjective To understand the mutations of rpoB genes in M. tuberculosis isolates, and study the correlation of rpoB mutations with the minimal inhibitory concentrations (MICs) of rifampicin. To develop a new method, gene chip, to be used for rapid detection of rpoB mutations in Mycobacterium tuberculosis.Method The traditional culture, species indentification of mycobacteria and drug susceptibility testing were performed in the clinical specimens. The MICs of rifampicin in M. tuberculosis isolates were mensurated. The concentrations of rifampicin were 0, 25, 50, 100, 150, 200, 250ug/ml. 138 clinical isolates were identified their mycobacterial species with 16S rDNA gene by PCR-SSCP. The rpoB genes in M. tuberculosis clinical isolates were analyzed by PCR-SSCP, gene chip and PCR-DNA sequencing.Results Of 138 mycobacterial clinical isolates, 133 isolates had the same 16S rDNA SSCP profiles as the standard strain of M. tuberculosis, 5 isolates had different 16S rDNA SSCP profiles and were identified as non-tuberculous mycobacteria. 133 M. tuberculosis isolates were performed traditional drug susceptibility testing. The result showed that there were 52 rifampicin-susceptible strains and 81 rifampicin-resistant strains.We amplified the 240bp fragments of rpoB genes from 133 M. tuberculosis isolates and analyzed the amplified products by PCR-SSCP. Of 81 rifampicin-resistant M. tuberculosis isolates, 76 isolates had different SSCP profiles from that of the standard strain H37Rv. No difference from the standard strain was found in 52 rifampicin-susceptible and 5 rifampicin-resistant isolates.We also analyzed their rpoB genes by gene chip. Of 133 M. tuberculosis clinicalisolates, the results of membrane chip in 52 drug-sensitive strains were similar to that of M. tuberculosis H37Rv. 92.6% (75/81) rifampicin-resistant strains had rpoB gene mutation. 56.8% (46/81) rifampicin-resistant strains had base mutations at codon 531. 18.5% (15/81) strains had base mutations at codon 526. 16.7% ( 14/81 ) strains had base mutations at other codons. Meanwhile, DNA direct sequencing of rpoB gene was performed in 1 rifampicin-susceptible and 25 rifampicin-resistant clinical isolates of M. tuberculosis. The sequencing results from 25 of 26 clinical isolates correspond with that of gene chip. 1 rifampicin-resistant isolate was detected two base mutations at codon 517 and codon 518 by DNA sequencing, but the result of gene chip was negative.We had mensurated the rifampicin MICs in 54 M. tuberculosis clinical isolates. There were 2, 2, 3, 6, 8 and 28 strains which their rifampicin MICs were 25, 50, 100, 150,200,250ug/ml, respectively. 7 strains with rifampicin MICs of < 50ug/ml showed no rpoB mutation, and 43 Of 47 strains with rifampicin MICs of = 50ug/ml had rpoB mutations.Conclusion Gene chip might become a rapid, simple, sensitive and specific method to detect rpoB mutations in the most rifampicin-resistant Mycobacterium tuberculosis. It could be used for clinical detection of rifampicin-resistance.