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Virologica Sinica, 28 (1) : 24-35, 2013
Research Article
Detection of the Pandemic H1N1/2009 Influenza A Virus by a Highly Sensitive Quantitative Real-time Reverse-transcription Polymerase Chain Reaction Assay
1. Institute of Virology, School of Life Sciences, Nanjing University, Nanjing 210093, Jiangsu, China

2. Taizhou Institute of Virology, Taizhou 225300, Jiangsu, China

3. Jiangsu Affynigen Biotechnologies, Inc., Taizhou 225300, Jiangsu, China

4. State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, China

5. College of Letters and Sciences, University of Chicago, Chicago, IL 60637, USA

6. Division of Infectious Diseases and Vaccinology, School of Public Health, University of California, Berkeley, CA 94720, USA
 Correspondence: liu_fy@berkeley.edu
(759.41KB)  
Abstract
A quantitative real time reverse-transcription polymerase chain reaction (qRT-PCR) assay with specific primers recommended by the World Health Organization (WHO) has been widely used successfully for detection and monitoring of the pandemic H1N1/2009 influenza A virus. In this study, we report the design and characterization of a novel set of primers to be used in a qRT-PCR assay for detecting the pandemic H1N1/2009 virus. The newly designed primers target three regions that are highly conserved among the hemagglutinin (HA) genes of the pandemic H1N1/2009 viruses and are different from those targeted by the WHO-recommended primers. The qRT-PCR assays with the newly designed primers are highly specific, and as specific as the WHO-recommended primers for detecting pandemic H1N1/2009 viruses and other influenza viruses including influenza B viruses and influenza A viruses of human, swine, and raccoon dog origin. Furthermore, the qRT-PCR assays with the newly designed primers appeared to be at least 10-fold more sensitive than those with the WHO-recommended primers as the detection limits of the assays with our primers and the WHO-recommended primers were 2.5 and 25 copies of target RNA per reaction, respectively. When tested with 83 clinical samples, 32 were detected to be positive using the qRT-PCR assays with our designed primers, while only 25 were positive by the assays with the WHO-recommended primers. These results suggest that the qRT-PCR system with the newly designed primers represent a highly sensitive assay for diagnosis of the pandemic H1N1/2009 virus infection.
  Accepted: 30 Oct 2012  Published online: 1 Feb 2013
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