Real-Time PCR Assay for Detection of Fluoroquinolone Resistance Associated with
            <i>grlA</i>
            Mutations in
            <i>Staphylococcus aureus</i>

Lapierre, Pascal; Huletsky, Ann; Fortin, Véronique; Picard, François J.; Roy, Paul H.; Ouellette, Marc; Bergeron, Michel G.

doi:10.1128/jcm.41.7.3246-3251.2003

Cited by 21 publications

(11 citation statements)

References 32 publications

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“…With widespread use of nucleic acid amplification tests (NAATs), antimicrobial resistance detection will require molecular methods, as has been described for other organisms (1,7,14,(16)(17)(18)25). QRNG detection is a good model for this approach since the resistance mechanisms are based on stepwise accumulation of point mutations correlating with increased MICs (20,22,23).…”

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confidence: 99%

Use of Applied Biosystems 7900HT Sequence Detection System and Taqman Assay for Detection of Quinolone-Resistant Neisseria gonorrhoeae

et al. 2004

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Mutations in quinolone resistance-determining regions (QRDRs) have been associated with quinoloneresistant Neisseria gonorrhoeae (QRNG). Since diagnostic nucleic acid amplification tests for gonococci are now in frequent use, molecular detection of QRNG could facilitate surveillance in the absence of culture. Here we describe a real-time molecular assay for detecting QRDR mutations in gonococci.Quinolone-resistant Neisseria gonorrhoeae (QRNG) strains are rapidly emerging. Well-characterized quinolone resistancedetermining region (QRDR) mutations correlate with decreased gonococcal antimicrobial susceptibility to fluoroquinolones (MIC, Ն1 g/ml) (2-6, 8-10, 13, 19, 21, 24, 26-28).Using well-characterized isolates, we developed a method for detecting QRDR mutations utilizing Taqman chemistry and the ABI 7900HT Prism sequence detector (Applied Biosystems, Foster City, Calif.).N. gonorrhoeae strains. We evaluated 80 isolates collected in 2000 to 2001 in Israel that were characterized previously (9, 29).DNA isolation, QRDR amplification, and direct sequencing. Genomic DNA was purified (Promega Wizard, Promega Corp., Madison, Wis.), and QRDRs were amplified (11, 12). The forward primers used were GyrA Forward (NG-GYRA-Z; 5Ј-CAAATTCGCCCTCGAAACCCT-3Ј; nucleotides [nt] 30 to 50 of the gyrA gene, 368-bp product) and ParC Forward (NG-PARC-Z; 5Ј-GCCCGTGCAGCGGCGCAT-3Ј; nt 138 to 155 of the parC gene, 219-bp product). Reaction mixtures had a 50-l total volume: 25 l of PCR Master mix, 22 l of sterile water, 1 l each of forward and reverse primers, and 1 l of DNA template. PCR products were sequenced with forward primers, and data were aligned with QRDR sequences for amino acids (aa) 91 to 95 of gyrA (GenBank accession no. U08817) and aa 86 to 92 of parC (GenBank accession no. U08907). The three mutation patterns identified are shown in Table 1.ABI primer and probe design. Primers and probes (Table 2) were developed by using Primer Express 2.0 software (Applied Biosystems, Foster City, Calif.). Probes encompassed aa 91 and 95 of gyrA and aa 86, 87, and 88 of parC, the loci most often associated with resistance (2-6, 8-10, 13, 19, 21, 24, 26-28). Using National Center for Biotechnology Information BLAST analysis, primers and probes were designed to match only gonococcal target sequences.ABI real-time PCR. Diplex PCRs were performed in 96-well plates with the following per well: 25 l of Promega PCR Master mix, 21 l of sterile water, 0.2 l of each set of forward and reverse primers (0.2 M final concentration of each), 0.5 l of each probe (0.2 M final concentration of each), and 1 l of DNA template, for a total reaction volume of 50 l. Cycles included one 2-min hold (50°C); a 10-min denaturation (95°C); and 40 cycles of denaturation (95°C for 30s), annealing (60°C for 30s), and extension (72°C for 30s). Control wells included blanks, wild-type (WT) strains, strains with one mutation, and strains with multiple mutations, as determined by sequencing. The lower-level detection limit was established at five genome copies by using publi...

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Use of Applied Biosystems 7900HT Sequence Detection System and Taqman Assay for Detection of Quinolone-Resistant Neisseria gonorrhoeae

et al. 2004

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“…Real-time PCR using the evaluation of melting curve analyses for point mutations is easy, fast, less costly, and comprehensive (5,14); however, it could have artifacts due to the melting curve profile. This method suits mutation analysis of a single amplicon or of a few amplicons.…”

Section: Discussionmentioning

confidence: 99%

“…Eighteen common genotypes in the QRDRs for our microarray analysis were selected based on our previous report and those of others (4,11,14,26,27,28). Sense and antisense oligonucleotide probes from 20 to 26 bases were used to determine specific point mutations in the QRDRs of the gyrAB and grlAB genes and to detect mecA-specific DNA sequences ( Table 2).…”

Section: Methodsmentioning

confidence: 99%

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Use of a Three-Dimensional Microarray System for Detection of Levofloxacin Resistance and the mecA Gene in Staphylococcus aureus

et al. 2005

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We evaluated a novel three-dimensional microarray (PamChip microarray) system to detect the presence of levofloxacin-related resistance mutations and the mecA gene. The results were compared to those obtained for 27 Staphylococcus aureus isolates by conventional DNA sequencing or PCR methods. Hybridization and fluorescence detection were performed using an FD10 system designed for PamChip microarray under conditions optimized for each target/probe on the array. In dilution series analysis using multiplex PCR samples, the sensitivity of the microarray was about 10 times greater than that of conventional PCR methods. A high level of data reproducibility was also confirmed in those analyses. Various point mutations in quinolone resistancedetermining regions detected by our system corresponded perfectly to the results obtained by conventional DNA sequencing. The results of the mecA gene detection using our system also corresponded to the PCR method; that is, signal/band was detected in all isolates of methicillin-resistant S. aureus, and no signal/band was detected in any isolate of methicillin-susceptible S. aureus. In conclusion, our novel three-dimensional microarray system provided rapid, specific, easy, and reproducible results for the simultaneous detection of levofloxacin resistance and the mecA gene in S. aureus.

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“…Recent advances in PCR have made it possible to use clinical samples to characterize both the pathogenic agent and its susceptibility to antimicrobial drugs (7)(8)(9)(10). A recently validated real-time PCR assay (10 ) has been used for rapid detection of a ponA variation associated with a decreased susceptibility to penicillin.…”

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A Novel Real-Time Duplex PCR Assay for Detecting penA and ponA Genotypes in Neisseria gonorrhoeae: Comparison with Phenotypes Determined by the E-Test

Vernel-Pauillac

Mérien²

2006

Clinical Chemistry

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Background: For many years, the pathogenic bacterium Neisseria gonorrhoeae, the etiologic agent of gonorrhea, was generally susceptible to penicillin, until the emergence of resistant strains. Well-characterized genetic variations in the penicillin resistance-determining region correlate with decreased susceptibility to penicillin. At least 5 genes (penA, penB, mtrR, ponA, and penC) are involved in the chromosomally mediated resistance to this antibiotic. To date, no development of multiplex PCR assays targeting a range of gonococcal genes and variations as a means of predicting antibiotic resistance has been reported. Methods: The aim of this study was to develop a duplex assay using DNA from isolated strains. We describe the development and evaluation on the LightCycler platform of a real-time duplex PCR assay (hybridization probe format) for rapid and specific detection of ponA and penA variations, predicting penicillin susceptibilities. Results: The real-time duplex PCR assay successfully detected variations in ponA and penA genes by use of distinct melting temperatures from a total of 120 Neisseria gonorrhoeae isolates. Moreover, the variation profiles obtained with the real-time PCR and the melting analysis showed good correlation with the pattern of penicillin susceptibility generated with classical antibiograms. Nucleotide sequencing data were in complete agreement with multiplex assay results. Conclusions: The presented assay is suitable for the detection of chromosomally mediated resistant strains of Neisseria gonorrhoeae in genotyping studies and could be valuable in the effective antimicrobial strategy to gonococci.

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Real-Time PCR Assay for Detection of Fluoroquinolone Resistance Associated with grlA Mutations in Staphylococcus aureus

Cited by 21 publications

References 32 publications

Use of Applied Biosystems 7900HT Sequence Detection System and Taqman Assay for Detection of Quinolone-Resistant Neisseria gonorrhoeae

Use of Applied Biosystems 7900HT Sequence Detection System and Taqman Assay for Detection of Quinolone-Resistant Neisseria gonorrhoeae

Use of a Three-Dimensional Microarray System for Detection of Levofloxacin Resistance and the mecA Gene in Staphylococcus aureus

A Novel Real-Time Duplex PCR Assay for Detecting penA and ponA Genotypes in Neisseria gonorrhoeae: Comparison with Phenotypes Determined by the E-Test

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