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

Whiley, David M., Jacob, Kevin, Nakos, Jennifer, Bletchly, Cheryl, Nimmo, Graeme R., Nissen, Michael D. and Sloots, Theo P. (2012) 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. Journal of Antimicrobial Chemotherapy, 67 6: 1375-1379.


Author Whiley, David M.
Jacob, Kevin
Nakos, Jennifer
Bletchly, Cheryl
Nimmo, Graeme R.
Nissen, Michael D.
Sloots, Theo P.
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
Journal name Journal of Antimicrobial Chemotherapy   Check publisher's open access policy
ISSN 0305-7453
1460-2091
Publication date 2012-06
Sub-type Article (original research)
DOI 10.1093/jac/dks040
Volume 67
Issue 6
Start page 1375
End page 1379
Total pages 5
Place of publication Oxford, United Kingdom
Publisher Oxford University Press
Collection year 2013
Language eng
Formatted abstract 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.
Keyword PCR
Influenza
Variation
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: Official 2013 Collection
School of Medicine Publications
Clinical Medical Virology Centre Publications
 
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