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Molecular Biotechnology

, Volume 41, Issue 1, pp 1–7 | Cite as

Identification of Rifampin-Resistant Genotypes in Mycobacterium tuberculosis by PCR-Reverse Dot Blot Hybridization

  • Xueqiong Wu
  • Junxian Zhang
  • Lixue Chao
  • Jianqin Liang
  • Yang Lu
  • Hongmin Li
  • Yourong Yang
  • Yan Liang
  • Chuihuan Li
Research

Abstract

A PCR-reverse dot blot hybridization (RDBH) assay was developed for rapid identification of rifampin (RFP)-resistant genotypes in Mycobacterium tuberculosis clinical isolates. The assay used the rpoB gene as target and was used to evaluate 148 clinical isolates (97 RFP-resistant isolates and 51 RFP-susceptible isolates). At the same time, the isolates were subjected to DNA sequencing and conventional drug susceptibility test. One hundred and forty one (95.3%) and 136 (91.9%) of the 148 strains were correctly identified by DNA sequencing and RDBH assay, respectively. None of the 51 RFP-susceptible isolates examined had alterations in rpoB. The sensitivity and specificity of the DNA sequencing were 92.8% and 100%, and the positive predictive value (PPV) and negative predictive value (NPV) were 100% and 87.9%, respectively. The sensitivity and specificity of the RDBH assay were 87.6% and 100%, and the PPV and NPV were 100% and 81.0%, respectively. Codons 531 and 526 of the rpoB were found to be the most common sites of nucleotide substitutions. Mutations at codons 511, 513, 515, 516, 517, 518, and 533 were also found. There were two-codon mutations in four isolates. No deletion and insertion was found in the rpoB gene. These results indicate that the RDBH assay is a rapid, simple, and reliable method for routine identification of RFP resistance in M. tuberculosis.

Keywords

Rifampin Drug resistance Molecular diagnosis M. tuberculosis 

Notes

Acknowledgments

This work was supported by the Army Research Foundation for the outstanding person with ability of China (No. 01J020), and by “973” Infectious Diseases Special Foundation of China (No. 2005CB523102).

References

  1. 1.
    Ministry of Health of the People’s Republic of China. (2001). Report on nationwide random survey for the epidemiology of tuberculosis. Ministry of Health of the People’s Republic of China.Google Scholar
  2. 2.
    Lee Ann, S. G., Lim, I. H. K., Tang, L. L. H., & Wong, S. Y. (2005). High frequency of mutations in the rpoB gene in rifampin-resistant clinical isolates of Mycobacterium tuberculosis from Singapore. Journal of Clinical Microbiology, 43, 2026–2027. doi: 10.1128/JCM.43.4.2026-2027.2005.CrossRefGoogle Scholar
  3. 3.
    Hirano, K. C., Abe, A. N. D., & Takahashi, M. (1999). Mutations in the rpoB gene of rifampin-resistant Mycobacterium tuberculosis strains isolated mostly in Asian countries and their rapid detection by line probe assay. Journal of Clinical Microbiology, 37, 2663–2666.Google Scholar
  4. 4.
    Varma-Basil, M., El-Hajj, H., Colangeli, R., Hazbón, M. H., Kumar, S., Bose, M., et al. (2004). Rapid detection of rifampin resistance in Mycobacterium tuberculosis isolates from India and Mexico by a molecular beacon assay. Journal of Clinical Microbiology, 42, 5512–5516. doi: 10.1128/JCM.42.12.5512-5516.2004.CrossRefGoogle Scholar
  5. 5.
    Chinese Antituberculosis Association. (1995). Chinese laboratory science procedure of diagnostic bacteriology in tuberculosis (pp. 9–21). Chinese Antituberculosis Association.Google Scholar
  6. 6.
    Saiki, R. K., Walsh, P. S., Levenson, C. H., & Erlich, H. A. (1989). Genetic analysis of amplified DNA with immobilized sequence-specific oligonucleotide probes. Proceedings of the National Academy of Sciences of the United States of America, 86, 6230–6234. doi: 10.1073/pnas.86.16.6230.CrossRefGoogle Scholar
  7. 7.
    Telenti, A. (1998). Genetics of drug resistant tuberculosis. Thorax, 53, 793–797.CrossRefGoogle Scholar
  8. 8.
    Hillemann, D., Weizenegger, M., Kubica, T., Richter, E., & Niemann, S. (2005). Use of the genotype MTBDR assay for rapid detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis complex isolates. Journal of Clinical Microbiology, 43, 3699–3703. doi: 10.1128/JCM.43.8.3699-3703.2005.CrossRefGoogle Scholar
  9. 9.
    Mäkinen, J., Marttila, H. J., Marjamäki, M., Viljanen, M. K., & Soini, H. (2006). Comparison of two commercially available DNA line probe assays for detection of multidrug-resistant Mycobacterium tuberculosis. Journal of Clinical Microbiology, 44, 350–352. doi: 10.1128/JCM.44.2.350-352.2006.CrossRefGoogle Scholar
  10. 10.
    Ma, X., Wang, H., Deng, Y., Liu, Z., Xu, Y., Pan, X., et al. (2006). rpoB gene mutations and molecular characterization of rifampin-resistant Mycobacterium tuberculosis isolates from Shandong province, China. Journal of Clinical Microbiology, 44, 3409–3412. doi: 10.1128/JCM.00515-06.CrossRefGoogle Scholar
  11. 11.
    Guerrero, C., Stockman, L., Marchesi, F., Bodmer, T., Roberts, G. D., & Telenti, A. (1994). Evaluation of the rpoB in rifampin-susceptible and resistant M. avium and M. intracellulare. Antimicrobial Agents and Chemotherapy, 33, 661–663.Google Scholar
  12. 12.
    Dabbs, E. R., Yazawa, K., Mikami, Y., Miyaji, M., Morisaki, N., Iwasaki, S., et al. (1995). Ribosylation by mycobacterial strains as a new mechanism of rifampin inactivation. Antimicrobial Agents and Chemotherapy, 39, 1007–1009.Google Scholar
  13. 13.
    Quan, S., Venter, H., & Dabbs, E. R. (1997). Ribosylative inactivation of rifampin by Mycobacterium smegmatis is a principal contributor to its low susceptibility to this antibiotic. Antimicrobial Agents and Chemotherapy, 41, 2456–2460.Google Scholar

Copyright information

© Humana Press 2008

Authors and Affiliations

  • Xueqiong Wu
    • 1
  • Junxian Zhang
    • 1
  • Lixue Chao
    • 1
  • Jianqin Liang
    • 1
  • Yang Lu
    • 1
  • Hongmin Li
    • 1
  • Yourong Yang
    • 1
  • Yan Liang
    • 1
  • Chuihuan Li
    • 2
  1. 1.Institute of Tuberculosis ResearchThe Second Affiliated Hospital of PLA General HospitalBeijingChina
  2. 2.Thorax Disease Hospital of Hebei ProvinceShijiazhuangChina

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