title: H5N1 Oseltamivir-resistance detection by real-time PCR using two high sensitivity labeled TaqMan probes.
abstract: A single amino acid substitution, from histidine to tyrosine at position 274 of the neuraminidase gene has converted Oseltamivir sensitive H5N1 influenza A virus into a resistant strain. Currently, Oseltamivir is being stockpiled in many countries potentially affected by the influenza A virus subtype H5N1 epidemic. To identify this change in Oseltamivir-treated patients, a method based on real-time PCR using two labeled TaqMan probes was developed for its rapid detection. In order to validate the method, Oseltamivir specimen from treated (Oseltamivir-resistant strain from a Vietnamese patient, two Oseltamivir-treated tigers) and untreated subjects have been used for this study. The results thus obtained as well as those derived from clone selection and sequencing showed that TaqMan probes could clearly discriminate wild type H274 from the mutant 274Y variant. The sensitivity of this assay was as low as 10 copies/microl and allowed the detection of the mutation in a mixture of wild type and mutant. Overall, the assay based on real-time PCR with two labeled TaqMan probes described here should be useful for detecting Oseltamivir-resistant H274Y H5N1 influenza A virus in many species and various sources of specimens with high sensitivity and specificity. Such studies can address potential differences in the diagnostic outcomes between patients who develop detectable Oseltamivir resistance and those who retain only the wild type strain of H5N1.
authors.affiliation: Center of Excellence in Viral Hepatitis Research, Chulalongkorn University, Bangkok 10330, Thailand.
authors.forename: Salin
authors.initials: S
authors.lastname: Chutinimitkul
authors.forename: Kamol
authors.initials: K
authors.lastname: Suwannakarn
authors.forename: Thaweesak
authors.initials: T
authors.lastname: Chieochansin
authors.forename: Le Quynh
authors.initials: le Q
authors.lastname: Mai
authors.forename: Sudarat
authors.initials: S
authors.lastname: Damrongwatanapokin
authors.forename: Arunee
authors.initials: A
authors.lastname: Chaisingh
authors.forename: Alongkorn
authors.initials: A
authors.lastname: Amonsin
authors.forename: Olfert
authors.initials: O
authors.lastname: Landt
authors.forename: Thaweesak
authors.initials: T
authors.lastname: Songserm
authors.forename: Apiradee
authors.initials: A
authors.lastname: Theamboonlers
authors.forename: Yong
authors.initials: Y
authors.lastname: Poovorawan
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology*; D024882:Drug Resistance, Viral; D005456:Fluorescent Dyes; D053124:Influenza A Virus, H5N1 Subtype / Q000235:genetics / Q000302:isolation & purification*; D053139:Oseltamivir / Q000494:pharmacology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D000998:Antiviral Agents; D005456:Fluorescent Dyes; D053139:Oseltamivir