title: Detection of influenza virus types A and B and type A subtypes (H1, H3, and H5) by multiplex polymerase chain reaction.
abstract: Infections with influenza virus type A and B present serious public health problems on a global scale. However, only influenza A virus has been reported to cause fatal pandemic in many species. To provide suitable clinical management and prevent further virus transmission, efficient and effective clinical diagnosis is essential. Therefore, we developed multiplex PCR assays for detecting influenza types A and B and the subtypes of influenza A virus (H1, H3 and H5). Upon performing multiplex PCR assays with type-specific primer sets, the clearly distinguishable products representing influenza A and B virus were separated by agarose gel electrophoresis. In addition, the subtypes of influenza A virus (H1, H3 and H5), which are most common in humans, can be readily distinguished by PCR with subtype-specific primer sets, yielding PCR products of different sizes depending on which subtype has been amplified. This method was tested on 46 influenza virus positive specimens of avian and mammalian (dog and human) origins collected between 2006 and 2008. The sensitivity of this method, tested against known concentrations of each type and subtype specific plasmid, was established to detect 10(3) copies/microl. The method's specificity was determined by testing against other subtypes of influenza A virus (H2, H4 and H6-H15) and respiratory pathogens commonly found in humans. None of them could be amplified, thus excluding cross reactivity. In conclusion, the multiplex PCR assays developed are advantageous as to rapidity, specificity, and cost effectiveness.
authors.affiliation: Center of Excellence in Clinical Virology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand.
authors.forename: Pitirat
authors.initials: P
authors.lastname: Boonsuk
authors.forename: Sunchai
authors.initials: S
authors.lastname: Payungporn
authors.forename: Thaweesak
authors.initials: T
authors.lastname: Chieochansin
authors.forename: Rujipat
authors.initials: R
authors.lastname: Samransamruajkit
authors.forename: Alongkorn
authors.initials: A
authors.lastname: Amonsin
authors.forename: Thaweesak
authors.initials: T
authors.lastname: Songserm
authors.forename: Arunee
authors.initials: A
authors.lastname: Chaisingh
authors.forename: Pornchai
authors.initials: P
authors.lastname: Chamnanpood
authors.forename: Salin
authors.initials: S
authors.lastname: Chutinimitkul
authors.forename: Apiradee
authors.initials: A
authors.lastname: Theamboonlers
authors.forename: Yong
authors.initials: Y
authors.lastname: Poovorawan
mesh_terms: D000818:Animals; D003362:Cost-Benefit Analysis; D017931:DNA Primers / Q000737:chemistry; D004285:Dogs; D004587:Electrophoresis, Agar Gel; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000378:metabolism*; D053122:Influenza A Virus, H3N2 Subtype / Q000378:metabolism*; D053124:Influenza A Virus, H5N1 Subtype / Q000378:metabolism*; D009980:Influenza A virus / Q000378:metabolism*; D009981:Influenza B virus / Q000378:metabolism*; D007251:Influenza, Human / Q000175:diagnosis; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers