Rapid Detection of rpoB Mutations in Rifampin Resistant M. tuberculosis from Sputum Samples by Denaturing Gradient Gel Electrophoresis

• Main Author: Jun Li, Jiaojiao Xin, Liyuan Zhang, Longyan Jiang, Hongcui Cao, Lanjuan Li
• Format: Article
• Language: English
• Published: Ivyspring International Publisher 2012-01-01
• Series: International Journal of Medical Sciences
• Online Access: http://www.medsci.org/v09p0148.htm

<p><b>Objective</b>: To establish a rapid detection method for identifying <i>rpoB</i> mutations associated with rifampin (RIF) resistance in sputum specimens.</p><p><b>Methods</b>: We detected <i>rpoB</i> mutations directly in 90 sputum specimens collected from suspected tuberculosis patients using PCR-based denaturing gradient gel electrophoresis (DGGE) and compared these results with those obtained by <i>rpoB</i> sequencing and conventional drug susceptibility testing.</p><p><b>Results</b>: The positive detection rate of <i>Mycobacterium tuberculosis</i> (<i>M. tuberculosis</i>) was 52.2% by Acid-Fast Bacilli staining and 72.2% by conventional <i>mycobacterial</i> culture. In contrast, the positive rate was significantly higher (93.3%) by PCR-based detection of the <i>rpoB</i> gene in the same specimens. Furthermore, 75% of the tested specimens presented abnormal patterns compared with the wild-type pattern (standard H37Rv strain) analysed by DGGE. A total of 12 different patterns, representing 12 different <i>rpoB</i> mutations, were observed in the 63 abnormal patterns. The match rate of <i>rpoB</i> mutations detected by DGGE reached 96.9% when compared to DNA sequencing.</p><p><b>Conclusion</b>: Our findings indicate that PCR-based DGGE is a rapid and reliable bio-technique for direct detection of <i>rpoB</i> mutations associated with RIF resistance in the sputum of suspected tuberculosis patients.</p>