Rapid detection of fluoroquinolone-resistant and heteroresistant Mycobacterium tuberculosis by use of sloppy molecular beacons and dual melting-temperature codes in a real-time PCR assay

Soumitesh Chakravorty, Bola Aladegbami, Kimberley Thoms, Jong Seok Lee, Eun Gae Lee, Vignesh Rajan, Eun Jin Cho, Hyunchul Kim, Hyunkyung Kwak, Natalia Kurepina, Sang Nae Cho, Barry Kreiswirth, Laura E. Via, Clifton E. Barry, David Alland

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Abstract

Fluoroquinolones (FQ) are important second-line drugs to treat tuberculosis; however, FQ resistance is an emerging problem. Resistance has been mainly attributed to mutations in a 21-bp region of the Mycobacterium tuberculosis gyrA gene, often called the quinolone resistance-determining region (QRDR). We have developed a simple, rapid, and specific assay to detect FQ resistance-determining QRDR mutations. The assay amplifies the M. tuberculosis gyrA QRDR in an asymmetrical PCR followed by probing with two sloppy molecular beacons (SMBs) spanning the entire QRDR. Mutations are detected by melting temperature (Tm) shifts that occur when the SMBs bind to mismatched sequences. By testing DNA targets corresponding to all known QRDR mutations, we found that one or both of the SMBs produced a Tm shift of at least 3.6°C for each mutation, making mutation detection very robust. The assay was also able to identify mixtures of wild-type and mutant DNA, with QRDR mutants identified in samples containing as little as 5 to 10% mutant DNA. The assay was blindly validated for its ability to identify the QRDR mutations on DNA extracted from clinical M. tuberculosis strains. Fifty QRDR wild-type samples, 34 QRDR mutant samples, and 8 heteroresistant samples containing mixtures of wild-type and mutant DNA were analyzed. The results showed 100% concordance to conventional DNA sequencing, including a complete identification of all of the mixtures. This SMB Tm shift assay will be a valuable molecular tool to rapidly detect FQ resistance and to detect the emergence of FQ heteroresistance in clinical samples from tuberculosis patients.

Original languageEnglish
Pages (from-to)932-940
Number of pages9
JournalJournal of Clinical Microbiology
Volume49
Issue number3
DOIs
Publication statusPublished - 2011 Mar 1

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All Science Journal Classification (ASJC) codes

  • Microbiology (medical)

Cite this

Chakravorty, S., Aladegbami, B., Thoms, K., Lee, J. S., Lee, E. G., Rajan, V., Cho, E. J., Kim, H., Kwak, H., Kurepina, N., Cho, S. N., Kreiswirth, B., Via, L. E., Barry, C. E., & Alland, D. (2011). Rapid detection of fluoroquinolone-resistant and heteroresistant Mycobacterium tuberculosis by use of sloppy molecular beacons and dual melting-temperature codes in a real-time PCR assay. Journal of Clinical Microbiology, 49(3), 932-940. https://doi.org/10.1128/JCM.02271-10