title: 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.
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 (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.
authors.affiliation: Division of Infectious Disease, Department of Medicine and the Ruy V. Lourenço Center for the Study of Emerging and Reemerging Pathogens, New Jersey Medical School, Newark, NJ 07103, USA.
authors.forename: Soumitesh
authors.initials: S
authors.lastname: Chakravorty
authors.forename: Bola
authors.initials: B
authors.lastname: Aladegbami
authors.forename: Kimberley
authors.initials: K
authors.lastname: Thoms
authors.forename: Jong Seok
authors.initials: JS
authors.lastname: Lee
authors.forename: Eun Gae
authors.initials: EG
authors.lastname: Lee
authors.forename: Vignesh
authors.initials: V
authors.lastname: Rajan
authors.forename: Eun-Jin
authors.initials: EJ
authors.lastname: Cho
authors.forename: Hyunchul
authors.initials: H
authors.lastname: Kim
authors.forename: Hyunkyung
authors.initials: H
authors.lastname: Kwak
authors.forename: Natalia
authors.initials: N
authors.lastname: Kurepina
authors.forename: Sang-Nae
authors.initials: SN
authors.lastname: Cho
authors.forename: Barry
authors.initials: B
authors.lastname: Kreiswirth
authors.forename: Laura E
authors.initials: LE
authors.lastname: Via
authors.forename: Clifton E
authors.initials: CE
authors.lastname: Barry
authors.forename: David
authors.initials: D
authors.lastname: Alland
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001431:Bacteriological Techniques / Q000379:methods*; D027081:DNA Gyrase / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D024841:Fluoroquinolones / Q000494:pharmacology*; D006801:Humans; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000302:isolation & purification; D015345:Oligonucleotide Probes / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D044366:Transition Temperature; D018088:Tuberculosis, Multidrug-Resistant / Q000175:diagnosis* / Q000382:microbiology*
chemical_list: D000900:Anti-Bacterial Agents; D017931:DNA Primers; D004269:DNA, Bacterial; D024841:Fluoroquinolones; D015345:Oligonucleotide Probes; D027081:DNA Gyrase