Design and Validation of Real-Time Reverse Transcription-PCR Assays for Detection of Pandemic (H1N1) 2009 Virus

Swine Outbreak of Pandemic Influenza A Virus on a Canadian Research Farm Supports Human-to-Swine Transmission

“…First, only 2 humans had throat swab specimens that were positive for pH1N1 virus. The TAG RVP, used to screen throat swab specimens, has a sensitivity of 90.2% and a specificity of 100% for pH1N1, so it is possible that some positive samples were not detected [11]. Throat swab specimens, although acceptable, are not optimal to detect pH1N1 by RT-PCR [30].…”

“…Swab specimens were tested as per manufacturer's instructions for influenza A and B virus; respiratory syncytial virus (RSV); human coronaviruses 229E, OC43, NL63, HKU1; parainfluenza virus; enterovirus or rhinoviruses; and adenoviruses by using the TAG Respiratory Virus Panel (RVP) (Luminex 97 Molecular Diagnostics) nucleic acid amplification assay with suspension microarray detection at the Alberta Provincial Laboratory for Public Health. The pH1N1 subtype was confirmed using primers and probes targeting the hemagglutinin (HA) gene [11]. Samples positive for pH1N1 were sent for complete molecular characterization to the World Health Organization (WHO) Collaborating Center for Studies on the Ecology of Influenza in Animals and Birds, St. Jude Children's Research Hospital (St. Jude), in Memphis, Tennessee.…”

Clinical presentation, microbiological features and correlates of disease severity of 2009 pandemic influenza A (H1N1) infection

“…Amplicons for sequencing were generated by reverse transcription, followed by PCR amplification to generate overlapping double-stranded DNA amplicons covering HA and NA segments of the influenza virus genome [3], the products were purified with QIAquick columns The data were collected by using ABI software (version 2.0) [6]. The sequences were analyzed by using Seqman software (Lasergene package).…”

Evaluation of Three Influenza A and B Real-Time Reverse Transcription-PCR Assays and a New 2009 H1N1 Assay for Detection of Influenza Viruses

“…The nonsubtypeable influenza A virus-positive specimens were further confirmed to be 2009 H1N1 strains by the CDC 2009 H1N1 real-time PCR assay (18). Other investigators have used a similar approach to identify 2009 H1N1 strains and differentiate them from seasonal influenza viruses using Luminex RVP assay results (6,10). Even though the CDC-developed real-time PCR protocol was used for detection of 2009 H1N1 strains, both the SW Inf A and SW H1 assays have been reported to have some drawbacks.…”

“…Most of these assays were developed to detect only seasonal influenza viruses and were not tested against recently emerged 2009 H1N1 strains. A few endpoint and real-time PCR assays have been specifically developed to detect 2009 H1N1 strains (8,10,16,18). However, these assays either use more than one gene target for the detection of H1N1 strains or were developed when a limited number of H1N1 sequences were available in the GenBank database.…”