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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 
 JOURNAL OF CLINICAL MICROBIOLOGY, Vol.49(3) : 932-940, 2011 
Journal Title
Issue Date
Anti-Bacterial Agents/pharmacology* ; Bacteriological Techniques/methods* ; DNA Gyrase/genetics ; DNA Primers/genetics ; DNA, Bacterial/chemistry ; DNA, Bacterial/genetics ; Fluoroquinolones/pharmacology* ; Humans ; Mycobacterium tuberculosis/drug effects* ; Mycobacterium tuberculosis/isolation & purification ; Oligonucleotide Probes/genetics ; Polymerase Chain Reaction/methods* ; Transition Temperature ; Tuberculosis, Multidrug-Resistant/diagnosis* ; Tuberculosis, Multidrug-Resistant/microbiology*
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.
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1. College of Medicine (의과대학) > Dept. of Microbiology (미생물학교실) > 1. Journal Papers
Yonsei Authors
Lee, Eun Gae(이은계)
Cho, Sang Nae(조상래)
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