Detection of Mutations in RNA Polymerase Beta Subunit Gene Encoding Resistance to Rifampin in Mycobacterium tuberculosis by DNA Microarray

Abstract

Rapid and efficient diagnosis is essential in the management of drug‐resistant tuberculosis. A DNA microarray technique based on differential hybridization method was described in the present study for detecting mutations in the RNA polymerase beta subunit (rpoB) gene of Mycobacterium tuberculosis (M. tuberculosis) cultures and in clinical specimens. The mutations in rpoB confer resistance to rifampin, an important first‐line antituberculosis drug. The differential hybridization approach was mainly based on the effect of a single base mismatch on the melting temperature of the hybridized DNA; therefore, any point mutation of rpoB gene resulting in the rifampin resistance can be detected efficiently. The development of the DNA microarray involves the design of dozens of oligonucleotide probes for identifying rifampin‐resistant and ‐sensitive strains. The method comprises isolating genomic DNA from the samples containing M. tuberculosis cells, amplifying rpoB gene coding sequence to produce fluorescently labelled product, and hybridization with the oligonucleotide arrays. The results demonstrated the capability of DNA microarray to provide important clinically relevant information about the rpoB gene of mycobacterial organisms. The DNA microarray offers a reliable diagnostic test for rapidly detecting multidrug resistance caused by gene mutations of mycobacteria.

Keywords:

• Mycobacterium tuberculosis
• RNA polymerase beta subunit (rpoB) gene
• rifampin resistance
• differential hybridization
• DNA microarray

Acknowledgments

This work is supported by the Chinese Academy of Sciences and National Natural Science Foundation of China.