title: Improved detection of genetic markers of antimicrobial resistance by hybridization probe-based melting curve analysis using primers to mask proximal mutations: examples include the influenza H275Y substitution.
abstract: OBJECTIVE
  Numerous real-time PCR assays have been described for detection of the influenza A H275Y alteration. However, the performance of these methods can be undermined by sequence variation in the regions flanking the codon of interest. This is a problem encountered more broadly in microbial diagnostics.
  
  
  METHODS
  In this study, we developed a modification of hybridization probe-based melting curve analysis, whereby primers are used to mask proximal mutations in the sequence targets of hybridization probes, so as to limit the potential for sequence variation to interfere with typing. The approach was applied to the H275Y alteration of the influenza A (H1N1) 2009 strain, as well as a Neisseria gonorrhoeae mutation associated with antimicrobial resistance. Assay performances were assessed using influenza A and N. gonorrhoeae strains characterized by DNA sequencing.
  
  
  RESULTS
  The modified hybridization probe-based approach proved successful in limiting the effects of proximal mutations, with the results of melting curve analyses being 100% consistent with the results of DNA sequencing for all influenza A and N. gonorrhoeae strains tested. Notably, these included influenza A and N. gonorrhoeae strains exhibiting additional mutations in hybridization probe targets. Of particular interest was that the H275Y assay correctly typed influenza A strains harbouring a T822C nucleotide substitution, previously shown to interfere with H275Y typing methods.
  
  
  CONCLUSIONS
  Overall our modified hybridization probe-based approach provides a simple means of circumventing problems caused by sequence variation, and offers improved detection of the influenza A H275Y alteration and potentially other resistance mechanisms.
authors.affiliation: Queensland Paediatric Infectious Diseases Laboratory, Queensland Children's Medical Research Institute, Children's Health Service District, Brisbane, Queensland, Australia. d.whiley@uq.edu.au
authors.forename: David M
authors.initials: DM
authors.lastname: Whiley
authors.forename: Kevin
authors.initials: K
authors.lastname: Jacob
authors.forename: Jennifer
authors.initials: J
authors.lastname: Nakos
authors.forename: Cheryl
authors.initials: C
authors.lastname: Bletchly
authors.forename: Graeme R
authors.initials: GR
authors.lastname: Nimmo
authors.forename: Michael D
authors.initials: MD
authors.lastname: Nissen
authors.forename: Theo P
authors.initials: TP
authors.lastname: Sloots
mesh_terms: D024881:Drug Resistance, Bacterial; D024882:Drug Resistance, Viral; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000235:genetics; D008826:Microbial Sensitivity Tests / Q000379:methods; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D009344:Neisseria gonorrhoeae / Q000235:genetics; D009693:Nucleic Acid Hybridization / Q000379:methods*; D015345:Oligonucleotide Probes / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D044366:Transition Temperature
chemical_list: D015345:Oligonucleotide Probes