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
Autor: | Theo P. Sloots, Cheryl Bletchly, Kevin Jacob, Michael D. Nissen, Graeme R. Nimmo, David M. Whiley, Jennifer Nakos |
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Rok vydání: | 2012 |
Předmět: |
Microbiology (medical)
Microbial Sensitivity Tests Biology Real-Time Polymerase Chain Reaction medicine.disease_cause Sensitivity and Specificity Melting curve analysis DNA sequencing law.invention Influenza A Virus H1N1 Subtype law Drug Resistance Bacterial Drug Resistance Viral medicine Humans Transition Temperature Pharmacology (medical) Typing Polymerase chain reaction Pharmacology Genetics Mutation Hybridization probe Nucleic Acid Hybridization Sequence Analysis DNA Neisseria gonorrhoeae Infectious Diseases Molecular Diagnostic Techniques Genetic marker Oligonucleotide Probes |
Zdroj: | Journal of Antimicrobial Chemotherapy. 67:1375-1379 |
ISSN: | 1460-2091 0305-7453 |
Popis: | Objectives: 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. |
Databáze: | OpenAIRE |
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