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19 October 2017

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Int J Med Sci 2012; 9(2):148-156. doi:10.7150/ijms.3605

Research Paper

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

Jun Li, Jiaojiao Xin, Liyuan Zhang, Longyan Jiang, Hongcui Cao, Lanjuan Li

State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, the First Affiliated Hospital, School of Medicine, Zhejiang University. 79 Qingchun Rd., Hangzhou, China. 310003.

Abstract

Objective: To establish a rapid detection method for identifying rpoB mutations associated with rifampin (RIF) resistance in sputum specimens.

Methods: We detected rpoB 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 rpoB sequencing and conventional drug susceptibility testing.

Results: The positive detection rate of Mycobacterium tuberculosis (M. tuberculosis) was 52.2% by Acid-Fast Bacilli staining and 72.2% by conventional mycobacterial culture. In contrast, the positive rate was significantly higher (93.3%) by PCR-based detection of the rpoB 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 rpoB mutations, were observed in the 63 abnormal patterns. The match rate of rpoB mutations detected by DGGE reached 96.9% when compared to DNA sequencing.

Conclusion: Our findings indicate that PCR-based DGGE is a rapid and reliable bio-technique for direct detection of rpoB mutations associated with RIF resistance in the sputum of suspected tuberculosis patients.

Keywords: Genotyping, Mycobacterium Tuberculosis, Resistance, Microbial Drug Resistance.

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How to cite this article:
Li J, Xin J, Zhang L, Jiang L, Cao H, Li L. Rapid Detection of rpoB Mutations in Rifampin Resistant M. tuberculosis from Sputum Samples by Denaturing Gradient Gel Electrophoresis. Int J Med Sci 2012; 9(2):148-156. doi:10.7150/ijms.3605. Available from http://www.medsci.org/v09p0148.htm