Typing (A/B) and subtyping (H1/H3/H5) of influenza A viruses by multiplex real-time RT-PCR assays

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

“…This method can be used to quantify the virus loads in both clinical samples and samples tested in vitro containing a mixed mutant and wildtype virus population. Most protocols currently employed are based on one-step reverse transcription real-time PCR (22,33), and thus, it is common to use as the control a known concentration of RNA. However, in the cycling probe method, it is not possible to adopt a one-step real-time PCR, because it includes RNase H and the template could not achieve a sufficient length of cDNA during the reverse transcription.…”

Comparison of the BD Veritor System for Flu A+B with the Alere BinaxNOW Influenza A&B Card for Detection of Influenza A and B Viruses in Respiratory Specimens from Pediatric Patients

“…A second RT-PCR was done on influenza A-positive samples to further subtype as seasonal H1N1 or seasonal H3N2 according to methods published earlier (10,11). Briefly, real-time RT-PCR for seasonal A/H1N1 and seasonal A/H3N2 was performed on the Applied Biosystems 7500 Fast real-time PCR system (Life Technologies Corporation, Foster City, CA) in a total reaction volume of 25 l containing 12.5 l of 2ϫ reaction mixture with ROX, 0.5 l of respective forward and reverse primers (40 M stock) and probe (10 M stock) specific for seasonal HIN1 and H3N2, 0.5 l of SuperScript III reverse transcriptase-Platinum Taq mix, 4.0 l of nuclease-free water, and 5 l of template RNA.…”

Subtyping Influenza A Virus with Monoclonal Antibodies and an Indirect Immunofluorescence Assay

“…Nucleic acid from each influenza A virus culture specimen was extracted with a MagMAX-96 total nucleic acid isolation kit (Applied Biosystems, Foster City, CA) according to the manufacturer's protocol. Primers used to subtype influenza virus culture specimens originated from published reports involved in screening respiratory samples (4,12). RT-PCR was performed on each sample using the three influenza primer sets in separate reactions with a Verso one-step RT-PCR kit (Thermo Scientific) according to the manufacturer's specification.…”