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A rapid fluorescence polarization-based method for genotypic detection of drug resistance in Mycobacterium tuberculosis

  • Applied genetics and molecular biotechnology
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Abstract

Rapid detection of drug-resistant Mycobacterium tuberculosis is critical to the effective early treatment and prevention of the transmission of tuberculosis. However, conventional drug susceptibility tests for M. tuberculosis require up to several weeks. In the present study, the One Label Extension genotyping method was adapted for rapid detection of drug resistance-associated sequence variations in six genes of M. tuberculosis, viz. rpoB, rpsL, rrs, embB, katG, or inhA. The method utilizes polymerase chain reaction amplified fragments of the drug resistant genes as reaction templates, and proceeds with template-directed primer extension incorporating a fluorescence-labeled nucleotide, which is then measured by fluorescence polarization. A total of 121 M. tuberculosis isolates from clinical sputum specimens were examined by this genotyping method and verified by direct sequencing of polymerase chain reaction amplicons harboring previously reported mutational sites associated with M. tuberculosis drug resistance. Based on phenotyping results obtained from microbiology-based drug susceptibility tests, the sensitivity, specificity, and test efficiency estimated for One Label Extension assays were respectively 83.9 %, 95.5 %, and 92.4 % with ropB in rifampin resistance, 67.3 %, 97.1 %, and 84.3 % with rpsL and rrs in streptomycin resistance, 60.0 %, 96.0 %, and 91.4 % with embB in ethambutol resistance, 68.4 %, 94.9 %, and 86.3 % with inhA and katG in isoniazid resistance, and 74.1 %, 98.9 %, and 93.2 % in multiple drug resistance defined as resistance to at least both isoniazid and rifampin. In conclusion, examination of clinical sputum specimens by One Label Extension based genotyping provides a valid method for the rapid molecular detection of drug-resistant M. tuberculosis.

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Acknowledgments

We thank Mr. Ka-King Chu and Ms. Peggy Lee for technical assistance. Financial support from the Pharmacogenetics Limited of Hong Kong, the National Health and Family Planning Commission (grant no. 2013ZX10003001), and Department of Science and Information Technology of Guangzhou (grant no. 2013J4500012), P. R. China are gratefully acknowledged.

Conflict of interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Division of Life Science and Applied Genomics Center, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China

    Yisuo Sun, Wai-Kin Mat, Cunyou Zhao & Hong Xue

  2. Nano Science and Nano Technology Program, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China

    Yisuo Sun & Hong Xue

  3. Department of Medical Genetics, School of Basic Medical Sciences, Southern Medical University, Guangzhou, 510515, China

    Shufen Li, Lin Zhou & Cunyou Zhao

  4. Antituberculosis Research Institute of Guangdong Province, Guangzhou, 510630, China

    Lin Zhou, Qiu Zhong & Tao Chen

  5. Division of Microbiology Test, Shenzhen NanShan Centre for Disease Control and Prevention, Shenzhen, 518055, China

    Shisong Fang

  6. National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China

    Lijun Bi

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  1. Yisuo Sun
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  2. Shufen Li
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  3. Lin Zhou
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  10. Cunyou Zhao
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  11. Hong Xue
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Correspondence to Cunyou Zhao or Hong Xue.

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Yisuo Sun, Shufen Li, and Lin Zhou contributed equally to this study.

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Sun, Y., Li, S., Zhou, L. et al. A rapid fluorescence polarization-based method for genotypic detection of drug resistance in Mycobacterium tuberculosis . Appl Microbiol Biotechnol 98, 4095–4105 (2014). https://doi.org/10.1007/s00253-013-5356-1

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