ArticlePloS one2011
Rapid detection of the H275Y oseltamivir resistance mutation in influenza A/H1N1 2009 by single base pair RT-PCR and high-resolution melting.
Article in PloS one, 2011. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 15 papers.
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15 citing papers in PubMed, 24 citations in OpenAlex.
- Development of A Novel EvaGreen-Dye Based Recombinase Aided Amplification Assay Using Self-Avoiding Molecular Recognition System Primers.China CDC weekly · 2025Article
- CRISPR-Based Assays for Point-of-Need Detection and Subtyping of Influenza.The Journal of molecular diagnostics : JMD · 2024Article
- Advances in the application of molecular diagnostic techniques for the detection of infectious disease pathogens (Review).Molecular medicine reports · 2023Review
- Development of A4 antibody for detection of neuraminidase I223R/H275Y-associated antiviral multidrug-resistant influenza virus.Nature communications · 2020Article
- Characterization of influenza A(H1N1)pdm09 isolates of Peru using HRM, a post PCR molecular biology method.Bioinformation · 2019Article
- Rapid and simple detection of Tamiflu-resistant influenza virus: Development of oseltamivir derivative-based lateral flow biosensor for point-of-care (POC) diagnostics.Scientific reports · 2018Article
- Molecular diagnosis of respiratory viruses.Pathology · 2015Review
- Development and clinical testing of a simple, low-density gel element array for influenza identification, subtyping, and H275Y detection.Journal of virological methods · 2014Article
- A multiplex real-time PCR assay for detection of oseltamivir-resistant strains of influenza virus.Central European journal of biology · 2014Article
- High resolution melting analysis: rapid and precise characterisation of recombinant influenza A genomes.Virology journal · 2013Article
- Article
- Assays for monitoring susceptibility of influenza viruses to neuraminidase inhibitors.Influenza and other respiratory viruses · 2013Review
- Microbiological applications of high-resolution melting analysis.Journal of clinical microbiology · 2012Review
- Translational research in infectious disease: current paradigms and challenges ahead.Translational research : the journal of laboratory and clinical medicine · 2012Review
- Comment on: Marquez et al. Low-frequency variants in HMGA1 are not associated with type 2 diabetes risk. Diabetes 2012;61:524-530.Diabetes · 2012Article
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Authors and funding
8 authors at 5 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
introductionWe aimed to design a real-time reverse-transcriptase-PCR (rRT-PCR), high-resolution melting (HRM) assay to detect the H275Y mutation that confers oseltamivir resistance in influenza A/H1N1 2009 viruses.
findingsA novel strategy of amplifying a single base pair, the relevant SNP at position 823 of the neuraminidase gene, was chosen to maintain specificity of the assay. Wildtype and mutant virus were differentiated when using known reference samples of cell-cultured virus. However, when dilutions of these reference samples were assayed, amplification of non-specific primer-dimer was evident and affected the overall melting temperature (T(m)) of the amplified products. Due to primer-dimer appearance at >30 cycles we found that if the cycle threshold (C(T)) for a dilution was >30, the HRM assay did not consistently discriminate mutant from wildtype. Where the C(T) was <30 we noted an inverse relationship between C(T) and T(m) and fitted quadratic curves allowed the discrimination of wildtype, mutant and 30∶70 mutant∶wildtype virus mixtures. We compared the C(T) values for a TaqMan H1N1 09 detection assay with those for the HRM assay using 59 clinical samples and demonstrated that samples with a TaqMan detection assay C(T)>32.98 would have an H275Y assay C(T)>30. Analysis of the TaqMan C(T) values for 609 consecutive clinical samples predicted that 207 (34%) of the samples would result in an HRM assay C(T)>30 and therefore not be amenable to the HRM assay.
conclusionsThe use of single base pair PCR and HRM can be useful for specifically interrogating SNPs. When applied to H1N1 09, the constraints this placed on primer design resulted in amplification of primer-dimer products. The impact primer-dimer had on HRM curves was adjusted for by plotting T(m) against C(T). Although less sensitive than TaqMan assays, the HRM assay can rapidly, and at low cost, screen samples with moderate viral concentrations.
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