title: High-resolution melting analysis for rapid characterization of qnr alleles in clinical isolates and detection of two novel alleles, qnrB25 and qnrB42.
abstract: OBJECTIVE
  qnr genes are plasmid-mediated quinolone resistance genes. Five qnr families have been described with several alleles (7 alleles of qnrA, 53 alleles of qnrB, 1 allele of qnrC, 1 allele of qnrD and 6 alleles of qnrS). Their detection requires a PCR specific for each qnr family and further sequencing for allele characterization.
  
  
  METHODS
  High-resolution melt curve analysis (HRMA) was coupled to multiplex and simplex real-time PCR assays for detection and characterization of qnrA, qnrB and qnrS alleles. The protocol was set using 27 reference strains harbouring the most frequent alleles and was applied to 55 clinical isolates unknown for qnr positivity.
  
  
  RESULTS
  Out of the 27 reference strains tested, 21 alleles showed distinct profiles using HRMA: 6 qnrA, 12 qnrB and 3 qnrS. For the qnrB alleles showing similar profiles, we gathered them into four groups that were easily distinguished. For the alleles that we could not test, in silico analysis showed that they would be identified using the HRMA protocol set. Among the clinical isolates, 28 qnr-positive isolates were detected and the qnr allele was characterized as 8 qnrA1, 4 qnrB1, 5 qnrB2, 3 qnrB4, 1 qnrB8, 1 qnrB5, 3 qnrS1 and 1 qnrS2, with concordant results with PCR sequencing. Two new qnrB alleles were detected and distinguished using HMRA. They were further designated as qnrB25 and qnrB42.
  
  
  CONCLUSIONS
  We developed an HRMA assay for characterizing the qnr alleles in clinical isolates. This high-throughput method can be used to screen a large number of isolates. This method allowed the detection of new qnrB alleles.
authors: CHU Reims, Hôpital Robert Debré, Laboratoire de Bactériologie-Virologie-Hygiène, F-51092 Reims, France. tguillard@chu-reims.fr
authors: Thomas
authors: T
authors: Guillard
authors: Christophe
authors: C
authors: de Champs
authors: Hélène
authors: H
authors: Moret
authors: Xavier
authors: X
authors: Bertrand
authors: Jean-Michel
authors: JM
authors: Scheftel
authors: Emmanuelle
authors: E
authors: Cambau
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001431:Bacteriological Techniques / Q000379:methods; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics*; D024881:Drug Resistance, Bacterial; D004755:Enterobacteriaceae / Q000187:drug effects / Q000235:genetics*; D004756:Enterobacteriaceae Infections / Q000382:microbiology; D006801:Humans; D008403:Mass Screening / Q000379:methods; D008969:Molecular Sequence Data; D015363:Quinolones / Q000494:pharmacology*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA; D044366:Transition Temperature
chemical_list: D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; D015363:Quinolones