Development of a novel real-time reverse-transcriptase PCR method for the detection of H275Y positive influenza A H1N1 isolates

Abstract

During the 2007-2008 influenza season global strain surveillance for antiviral resistance revealed the sudden emergence of oseltamivir resistance in influenza A H1N1 isolates. Although oseltamivir resistance rates vary from region to region, 16% of isolates tested globally were found to be oseltamivir resistant by a histidine to tyrosine mutation of residue 275 of the neuraminidase gene of influenza A. In order to implement effective resistance testing locally a novel real-time reverse-transcriptase PCR (RT-PCR) assay was developed for the detection of the H275Y mutation. To evaluate this method, 40 oseltamivir resistant and 61 oseltamivir sensitive H1N1 influenza isolates were tested using Sanger sequencing, which is the reference method for detection of resistance, pyrosequencing and the novel H275Y RT-PCR assay. In comparison to Sanger sequencing, the sensitivity and specificity of the H275Y RT-PCR assay were 100% (40/40) and 100% (61/61) respectively, while the sensitivity and specificity of pyrosequencing were 100% (40/40) and 97.5% (60/61) respectively. Although all three methods were effective in detecting the H275Y mutation associated with oseltamivir resistance, the H275Y RT-PCR assay was the most rapid and could easily be incorporated into an influenza subtyping protocol.

Neuraminidase inhibitor susceptibility surveillance of influenza viruses circulating worldwide during the 2011 Southern Hemisphere season

“…Targeted surveillance directed to the isolation and testing of influenza viruses from immunocompetent or immunocompromised individuals undergoing treatment with NAIs may allow a more focused and thorough assessment of the potential for influenza viruses to develop clinically significant resistance to these compounds. In addition, monitoring could be enhanced by detection of H275Y directly on clinical specimens using molecular methods, including pyrosequencing25, 26, 27 or real‐time RT‐PCR techniques 28, 29…”

Rapid and Specific Detection of Amantadine-Resistant Influenza A Viruses with a Ser31Asn Mutation by the Cycling Probe Method

“…Thus, our method is perhaps one of the quickest and most affordable with respect to the running cost (which is equivalent to that of the TaqMan probe method) among the methods currently available for the identification of amantadine resistance, and it is ideal for largescale screening for resistant mutants and subtyping even with specimens with low template concentrations, such as nasopharyngeal swab specimens. Of note, the TaqMan probe real-time PCR was applied for the detection of oseltamivir-resistant influenza virus A/H1N1 possessing His274Tyr (N2 numbering) in the neuraminidase gene and used for monitoring for resistant viruses (5). We are currently developing new cycling probe sets to detect other amantadine resistance mutations in the M2 gene and the oseltamivir resistance mutation (His274Tyr) in the neuraminidase gene.…”

Quantitative Analysis of Single-Nucleotide Polymorphism for Rapid Detection of TR 34 /L98H- and TR 46 /Y121F/T289A-Positive Aspergillus fumigatus Isolates Obtained from Patients in Iran from 2010 to 2014

“…The quantitative analysis of SNPs has been a reliable method in diagnostic microbiology for identification of a single nucleotide in the genomes of humans (11)(12)(13)(14)(15), viruses (16)(17)(18)(19)(20), and bacteria (18). In this assay, an extension probe can be simply designed to anneal to the template in a posi-tion that places the mutation site immediately adjacent to the 3= end of the probe, and the use of dideoxynucleoside triphosphates (ddNTPs) allows the extension of only 1 nucleotide from the 3= end of the probe.…”

“…In this assay, an extension probe can be simply designed to anneal to the template in a posi-tion that places the mutation site immediately adjacent to the 3= end of the probe, and the use of dideoxynucleoside triphosphates (ddNTPs) allows the extension of only 1 nucleotide from the 3= end of the probe. Labeling of each ddNTP with a different fluorescent dye allows the differentiation of the genotype at the SNP by the color of the extended probes (11)(12)(13)(14)(15)(16)(17)(18)(19)(20).…”