New Real-Time PCR Assay Using Locked Nucleic Acid Probes To Assess Prevalence of ParC Mutations in Fluoroquinolone-Susceptible Streptococcus pneumoniae Isolates from France

Abstract

A real-time PCR assay with locked nucleic acid probes was developed to screen mutations at codons 79 and 83 of the Streptococcus pneumoniae parC gene. Only silent mutations were detected among 236 French invasive fluoroquinolone-susceptible strains. This test could be useful for some high-risk patients or in national surveys.The worldwide spread of multidrug-resistant clones has led to the increasing use of fluoroquinolones (FQs) in the therapy of Streptococcus pneumoniae infections (21). In some countries the increase of FQ resistance (FQR) in that species and some clinical failures have been reported (5,9,13,16,18). The mechanisms of FQR mostly correspond to stepwise mutations in the quinolone resistance-determining regions (QRDR) of ParC and GyrA, two subunits of the FQ targets (DNA gyrase and the topoisomerase IV) (29). The low-level resistance firststep parC mutants (FSPC) were implicated in the selection following levofloxacin therapy of the highly resistant parC-gyrA double mutants (7,16). FSPC mutants are classified as susceptible to FQs according to the standard breakpoints (19,26,27,28). Sequencing of QRDR parC gene was considered the gold standard. We described a real-time PCR assay with TaqMan probes including locked nucleic acid (LNA) bases to detect mutations in codon 79 or 83 of parC, the two codons being mostly implicated in FQR in S. pneumoniae (5,8,9,16). The LNA base is a bicyclic RNA analogue that increases the stability of the DNA/LNA mixmer (14). When probes include LNA bases, the melting temperature of the duplex DNA target/probe and then the specificity of the test increase. This assay was tested for an epidemiological survey that included a panel of controls and S. pneumoniae invasive strains, using sequencing as control method.Control strains including wild-type and ParC mutant strains were tested repeatedly (Table 1). A sample of 236 clinical FQsusceptible strains was randomly selected from the annual survey program performed between 2000 and 2003 in 105 general hospitals as part of the Collège de Bactériologie-Virologie Hygiène des Hôpitaux (ColBVH) Study Group and screened for mutations. The MICs were determined by an agar dilution method and interpreted according to Clinical and Laboratory Standards Institute guidelines as previously described (11,12,22,23). Two specific primers and TaqMan probes were designed to detect wildtype alleles at positions 79 and 83 of the parC QRDR of S. pneumoniae (nucleotide positions 3852 to 3854 and 3864 to 3866, respectively) ( Table 2). DNA crude extracts were prepared from a loopful of overnight plate culture suspended in 200 l of distilled water, using a rapid DNA extraction kit (QIAamp DNA MiniKit; QIAGEN, Courtaboeuf, France) according to the manufacturer's instructions. The PCR experiments were performed on a Smart Cycler (Cepheid, Sunnyvale, Calif.); 5 l of DNA extract was transferred into a 20-l PCR mixture containing 0.25 M concentrations of the primers parC3 and parC4, 0.25 M concentrations of each probe, and 12.5 l of the Smart Kit (Euroge...

Correction to: Novel qPCR probe systems for the characterization of subaerial biofilms on stone monuments

“…LNAs have been inserted in order to increase melting temperature of the probes (Decousser et al 2006), which is critical for the accuracy in recognizing the correct template region. Primer and probe design is arguably the most critical factor in a multiplex assay.…”