[Rapid detection of rpoB mutations in Mycobacterium tuberculosis by gene array].

OBJECTIVE

To develop a new method, gene array, which can be used for rapid detection of rpoB mutations in Mycobacterium tuberculosis.

METHODS

Probes were designed according to the sequence of Mycobacterium 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. Mycobacterium tuberculosis strain H(37)Rv DNA was used as the control. The rpoB genes in Mycobacterium tuberculosis clinical isolates were also analyzed by polymerase chain reaction-single stranded conformational polymorphism (PCR-SSCP) and PCR-DNA sequencing.

RESULTS

We analyzed the rpoB genes of 111 Mycobacterium tuberculosis clinical isolates by PCR-SSCP. Of 70 rifampicin-resistant Mycobacterium tuberculosis isolates, 63 isolates had different SSCP profiles from that of the standard strain H(37)Rv. No difference from the standard strain was found in 41 rifampicin-susceptible and 7 rifampicin-resistant isolates. We also analyzed their rpoB genes by gene array. Of 111 Mycobacterium tuberculosis clinical isolates, the results of gene array in 41 drug-sensitive strains were similar to that in Mycobacterium tuberculosis H(37)Rv. 90% (63/70) rifampicin-resistant strains had rpoB gene mutation. 53% (37/70) rifampicin-resistant strains had serine substitution at codon 531. 21% (15/70) strains had histidine substitution at codon 526. 16% (11/70) strains had amino acids substitution in other position. The results of gene array corresponded with that of PCR-SSCP and DNA sequencing.

CONCLUSION

Gene array might become a rapid, simple, and accurate method for detecting rpoB mutations in most of the rifampicin-resistant Mycobacterium tuberculosis.