Detection of the pandemic H1N1/2009 influenza A virus by a highly sensitive quantitative real-time reverse-transcription polymerase chain reaction assay
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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.
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- Detection of the pandemic H1N1/2009 influenza A virus by a highly sensitive quantitative real-time reverse-transcription polymerase chain reaction assay
Volume 28, Issue 1 , pp 24-35
- Cover Date
- Print ISSN
- Online ISSN
- SP Wuhan Institute of Virology, CAS
- Additional Links
- Quantitative real time reverse-transcription polymerase chain reaction (qRT-PCR)
- Influenza A virus
- Author Affiliations
- 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