2006
DOI: 10.1128/jcm.44.3.970-975.2006
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Rapid Screening of Fluoroquinolone Resistance Determinants in Streptococcus pneumoniae by PCR-Restriction Fragment Length Polymorphism and Single-Strand Conformational Polymorphism

Abstract: . SSCP analysis of restricted fragments generated patterns that were highly discriminative for mutations present in the QRDRs of gyrA, gyrB, parC, and parE. This method provides a database of high resolution profiles on these mutations and allows rapid screening for new mutations of the fluoroquinolone resistance genes.

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Cited by 13 publications
(9 citation statements)
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“…Restriction fragment length polymorphism analysis is not a new technique for the typing of S. pneumoniae (1,3,6,12,18) but has been improved here by using an Agilent 2100 bioanalyzer for the accurate sizing of the DNA fragments. In addition, a PCR product of a noncoding region was used, which is likely to vary more between strains than a gene.…”
Section: Discussionmentioning
confidence: 99%
“…Restriction fragment length polymorphism analysis is not a new technique for the typing of S. pneumoniae (1,3,6,12,18) but has been improved here by using an Agilent 2100 bioanalyzer for the accurate sizing of the DNA fragments. In addition, a PCR product of a noncoding region was used, which is likely to vary more between strains than a gene.…”
Section: Discussionmentioning
confidence: 99%
“…The mutations and the amino acid substitutions at the QRDRs in the gyrA, gyrB, parC, and parE genes were analyzed by PCR-RF-SSCP as previously described (17). Briefly, for each isolate, PCR amplicons of QRDRs of the gyrA and -B and parC and -E genes were digested with AluI, HinFI, Sau3AI, and MspI, respectively, and analyzed by SSCP.…”
Section: Methodsmentioning
confidence: 99%
“…Previously, we utilized a rapid screening method using PCR-restriction fragment (RF)-single-strand conformation polymorphism (SSCP) (17) to screen for variants/mutations at the quinolone resistance-determining regions (QRDRs) of the gyrase and topoisomerase genes in CIP-resistant S. pneumoniae (MIC Ն 4.0 g/ml). Using PCR-RF-SSCP, we identified a collection of atypical strains, presumed to be pneumococcus, with CIP MICs of Ն4.0 g/ml and unique patterns suggesting sequence variations within the QRDR in comparison with S. pneumoniae R6 and possible recombinational events with other streptococci.…”
mentioning
confidence: 99%
“…Rapid detection of QRDR mutations is required and may constitute a more reliable approach than the current phenotypic method, which can represent susceptibility but cannot detect the potential of S. pneumoniae strains to harbor resistance. PCR-based techniques, such as PCR-restriction fragment length polymorphism analysis (1,15,20), single-stranded conformational polymorphism analysis (15), and PCR-oligonucleotide ligation (2), have been developed to detect QRDR mutations; however, these techniques are not appropriate for practical use, as they are complicated and time-consuming. More recently, real-time PCR methods combined with melting curve analysis (PCR-MCA) have been reported to be successful in the detection of key gene mutations associated with drug resistance in various microorganisms (12,25,26).…”
mentioning
confidence: 99%