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

摘要: Objectives: Numerous real-time PCR assays have been described for detection of the influenza A H275Y alteration. However, performance these methods can be undermined by sequence variation in regions flanking codon interest. This is a problem encountered more broadly microbial diagnostics. Methods: In this study, we developed modification hybridization probe-based melting curve analysis, whereby primers are used to mask proximal mutations targets probes, so as limit potential interfere with typing. The approach was applied alteration (H1N1) 2009 strain, well Neisseria gonorrhoeae mutation associated antimicrobial resistance. Assay performances were assessed using and N. strains characterized DNA sequencing. Results: modified proved successful limiting effects mutations, results analyses being 100% consistent sequencing all tested. Notably, included exhibiting additional probe targets. Of particular interest that assay correctly typed harbouring T822C nucleotide substitution, previously shown typing methods. Conclusions: Overall our provides simple means circumventing problems caused variation, offers improved potentially other resistance mechanisms.