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
| Autor: | Natalia Kurepina, Barry N. Kreiswirth, Soumitesh Chakravorty, Hyun-Kyung Kwak, Sang Nae Cho, Vignesh Rajan, Kimberley Thoms, David Alland, Hyun-Chul Kim, Jong Seok Lee, Eun Jin Cho, Bola Aladegbami, Clifton E. Barry, Eun Gae Lee, Laura E. Via |
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| Rok vydání: | 2010 |
| Předmět: |
Microbiology (medical)
DNA Bacterial Mutant medicine.disease_cause DNA gyrase Polymerase Chain Reaction law.invention Mycobacterium tuberculosis chemistry.chemical_compound Molecular beacon law Tuberculosis Multidrug-Resistant medicine Humans Transition Temperature Polymerase chain reaction DNA Primers Genetics Mutation Bacteriological Techniques biology Mycobacteriology and Aerobic Actinomycetes biochemical phenomena metabolism and nutrition biology.organism_classification Molecular biology Anti-Bacterial Agents Real-time polymerase chain reaction chemistry DNA Gyrase Oligonucleotide Probes DNA Fluoroquinolones |
| Zdroj: | Journal of clinical microbiology. 49(3) |
| ISSN: | 1098-660X |
| Popis: | 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 ( T m ) 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 T m 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 T m 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. |
| Databáze: | OpenAIRE |
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