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

ABSTRACT Tracking novel influenza viruses which have the potential to cause pandemics, such as the pandemic (H1N1) 2009 virus, is a public health priority. Pandemic (H1N1) 2009 virus was first identified in Mexico in April 2009 and spread worldwide over a short period of time. Well-validated diagnostic tools that are rapid, sensitive, and specific for the detection and tracking of this virus are needed. Three real-time reverse transcription PCR (RT-PCR) assays for the amplification and detection of pandemic (H1N1) 2009 virus were developed, and their performance characteristics were compared with those of other published diagnostic assays. Thirty-nine samples confirmed to be positive for pandemic (H1N1) 2009 virus from Alberta, Canada, and six additional samples that were positive for influenza A virus but that were not typeable by using published seasonal influenza H1/H3 virus assays were available for this validation. Amplification and direct sequencing of the products was considered the “gold standard” for case identification. The new assays were sensitive and able to reproducibly detect virus in a 10−6 dilution of 4 × 106 50% tissue culture infective doses/ml when 5 μl was used as the template. They showed 100% specificity and did not cross-react with other respiratory viruses or seasonal influenza A virus subtypes. The coefficient of variation in crossing cycle threshold values for the detection of different template concentrations of pandemic (H1N1) 2009 virus was ≤3.13%, showing good reproducibility. The assays had a wide dynamic range for the detection of pandemic (H1N1) 2009 virus and utilized testing platforms appropriate for high diagnostic throughput with rapid turnaround times. We developed and validated these real-time PCR procedures with the goal that they will be useful for diagnosis and surveillance of pandemic (H1N1) 2009 virus. These findings will contribute to the informed management of this novel virus.

[1]  Albert D. M. E. Osterhaus,et al.  Detection of Influenza A Viruses from Different Species by PCR Amplification of Conserved Sequences in the Matrix Gene , 2000, Journal of Clinical Microbiology.

[2]  J. Fox,et al.  Development and evaluation of a real‐time nucleic acid sequence based amplification assay for rapid detection of influenza A† , 2004, Journal of medical virology.

[3]  Influenza Outbreaks , 1965, British medical journal.

[4]  Gavin J. D. Smith,et al.  Molecular Detection of a Novel Human Influenza (H1N1) of Pandemic Potential by Conventional and Real-Time Quantitative RT-PCR Assays , 2009, Clinical chemistry.

[5]  T. Tumpey,et al.  Resurrected pandemic influenza viruses. , 2009, Annual review of microbiology.

[6]  W. Gallaher,et al.  Towards a sane and rational approach to management of Influenza H1N1 2009 , 2009, Virology Journal.

[7]  Emmie de Wit,et al.  Emerging influenza. , 2008, Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology.

[8]  L. Finelli,et al.  Emergence of a novel swine-origin influenza A (H1N1) virus in humans. , 2009, The New England journal of medicine.

[9]  E. Lyons,et al.  Pandemic Potential of a Strain of Influenza A (H1N1): Early Findings , 2009, Science.

[10]  N. Bastien,et al.  Swine Influenza (H3N2) Infection in a Child and Possible Community Transmission, Canada , 2007, Emerging infectious diseases.

[11]  R Rabadan,et al.  The origin of the recent swine influenza A(H1N1) virus infecting humans. , 2009, Euro surveillance : bulletin Europeen sur les maladies transmissibles = European communicable disease bulletin.

[12]  Edward C. Holmes,et al.  Different Evolutionary Trajectories of European Avian-Like and Classical Swine H1N1 Influenza A Viruses , 2009, Journal of Virology.

[13]  Jonathan E. Allen,et al.  Conserved amino acid markers from past influenza pandemic strains , 2009, BMC Microbiology.

[14]  D. Fleming,et al.  Update: novel influenza A (H1N1) virus infections - worldwide, May 6, 2009. , 2009, MMWR. Morbidity and mortality weekly report.

[15]  S. Rewar,et al.  Outbreak of swine-origin influenza A (H1N1) virus infection - Mexico, March-April 2009. , 2009, MMWR. Morbidity and mortality weekly report.

[16]  Gregory C Gray,et al.  Cases of swine influenza in humans: a review of the literature. , 2007, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[17]  Kanti Pabbaraju,et al.  Comparison of the Luminex xTAG Respiratory Viral Panel with In-House Nucleic Acid Amplification Tests for Diagnosis of Respiratory Virus Infections , 2008, Journal of Clinical Microbiology.