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Journal of Clinical Microbiology, November 2009, p. 3454-3460, Vol. 47, No. 11
0095-1137/09/$08.00+0 doi:10.1128/JCM.01103-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Design and Validation of Real-Time Reverse Transcription-PCR Assays for Detection of Pandemic (H1N1) 2009 Virus 
Kanti Pabbaraju,1*
Sallene Wong,1
Anita A. Wong,1
Greg D. Appleyard,1,3
Linda Chui,2,4
Xiao-Li Pang,2,4
Stephanie K. Yanow,2,5
Kevin Fonseca,1,3
Bonita E. Lee,2,6
Julie D. Fox,1,3 and
Jutta K. Preiksaitis2,7
Provincial Laboratory for Public Health (Microbiology), Calgary Site, Calgary, Alberta, Canada,1 Provincial Laboratory for Public Health (Microbiology), Edmonton Site, Edmonton, Alberta, Canada,2 Department of Microbiology and Infectious Diseases, University of Calgary, Calgary, Alberta, Canada,3 Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada,4 Department of Public Health Sciences, School of Public Health, University of Alberta, Edmonton, Alberta, Canada,5 Department of Pediatrics, University of Alberta, Edmonton, Alberta, Canada,6 Department of Medicine, University of Alberta, Edmonton, Alberta, Canada7
Received 5 June 2009/ Returned for modification 26 July 2009/ Accepted 25 August 2009
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 x 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.
* Corresponding author. Mailing address: Provincial Laboratory for Public Health (Microbiology), 3030 Hospital Drive, Calgary, Alberta, Canada T2N 4W4. Phone: (403) 944-8621. Fax: (403) 283-0142. E-mail: K.Pabbaraju{at}provlab.ab.ca
Published ahead of print on 2 September 2009.
Journal of Clinical Microbiology, November 2009, p. 3454-3460, Vol. 47, No. 11
0095-1137/09/$08.00+0 doi:10.1128/JCM.01103-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
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