Serving the Society Since 1986
Advanced Search
Volume 28 Issue 1
February 2013
    Citation: Zhu Yang, Guoliang Mao, Yujun Liu, Yuan-Chuan Chen, Chengjing Liu, Jun Luo, Xihan Li, Ke Zen, Yanjun Pang, Jianguo Wu, Fenyong Liu. Detection of the Pandemic H1N1/2009 Influenza A Virus by a Highly Sensitive Quantitative Real-time Reverse-transcription Polymerase Chain Reaction Assay .VIROLOGICA SINICA, 2013, 28(1) : 24-35.  http://dx.doi.org/10.1007/s12250-013-3290-0
    shu

    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

    • Corresponding author: Fenyong Liu, liu_fy@berkeley.edu
    • These authors contributed equally to this work
    • Received Date: 30 October 2012
      Accepted Date: 23 January 2013
      Available online: 01 February 2013

      Fund Project: National Mega Project on Major Drug Development 2009ZX09103-678National Small Business Innovation and Research (SBIR) Program of China, the Technology R & D Program of Jiangsu Province, China BG20077035National Natural Science Foundation of China 81030031NIH RO1-AI050468National Small Business Innovation and Research (SBIR) Program of China, the Technology R & D Program of Jiangsu Province, China BG2008662This research has been supported by grants from National Basic Research Program of China 2011CB504800NIH RO1-DE014842NIH RO1-AI041927National Natural Science Foundation of China 31100128NIH RO1-DE014145

    • 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.
    • 加载中

      1. Amano Y and Cheng Q. 2005. Detection of influenza virus: traditional approaches and development of biosensors. Anal Bioanal Chem, 381: 156-164.
          doi: 10.1007/s00216-004-2927-0

      2. Bennett S, Gunson R N, MacLean A, Miller R and Carman W F. 2010. The validation of a real-time RT-PCR assay which detects influenza A and types simultaneously for influenza A H1N1 (2009) and oseltamivir-resistant (H275Y) influenza A H1N1 (2009). J Virol Methods, 171: 86-90.

      3. Burns N, Grimwade B, Ross-Macdonald P B, Choi E Y, Finberg K, Roeder G S and Snyder M. 1994. Large-scale analysis of gene expression, protein localization and gene disruption in Saccharomyces cerevisiae. Genes Dev, 8: 1087-1105.
          doi: 10.1101/gad.8.9.1087

      4. Cao B, Li X W, Mao Y, Wang J, Lu H Z, Chen Y S, Liang Z A, Liang L, Zhang S J, Zhang B, Gu L, Lu L H, Wang D Y and Wang C. 2009. Clinical features of the initial cases of 2009 pandemic influenza A (H1N1) virus infection in China. N Engl J Med, 361: 2507-2517.
          doi: 10.1056/NEJMoa0906612

      5. Chiu C Y, Urisman A, Greenhow T L, Rouskin S, Yagi S, Schnurr D, Wright C, Drew W L, Wang D, Weintrub P S, Derisi J L and Ganem D. 2008. Utility of DNA microarrays for detection of viruses in acute respiratory tract infections in children. J Pediatr, 153: 76-83.
          doi: 10.1016/j.jpeds.2007.12.035

      6. Dawson E D, Moore C L, Dankbar D M, Mehlmann M, Townsend M B, Smagala J A, Smith C B, Cox N J, Kuchta R D and Rowlen K L. 2007. Identification of A/H5N1 influenza viruses using a single gene diagnostic microarray. Anal Chem, 79: 378-384.
          doi: 10.1021/ac061920o

      7. Garten R J, Davis C T, Russell C A, Shu B, Lindstrom S, Balish A, Sessions W M, Xu X, Skepner E, Deyde V, Okomo-Adhiambo M, Gubareva L, Barnes J, Smith C B, Emery S L, Hillman M J, Rivailler P, Smagala J, de Graaf M, Burke D F, Fouchier R A, Pappas C, Alpuche-Aranda C M, Lopez-Gatell H, Olivera H, Lopez I, Myers C A, Faix D, Blair P J, Yu C, Keene K M, Dotson P D, Jr., Boxrud D, Sambol A R, Abid S H, St George K, Bannerman T, Moore A L, Stringer D J, Blevins P, Demmler-Harrison G J, Ginsberg M, Kriner P, Waterman S, Smole S, Guevara H F, Belongia E A, Clark P A, Beatrice S T, Donis R, Katz J, Finelli L, Bridges C B, Shaw M, Jernigan D B, Uyeki T M, Smith D J, Klimov A I and Cox N J. 2009. Antigenic and genetic characteristics of swine-origin 2009 A(H1N1) influenza viruses circulating in humans. Science, 325: 197-201.
          doi: 10.1126/science.1176225

      8. Ghedin E, Laplante J, DePasse J, Wentworth D E, Santos R P, Lepow M L, Porter J, Stellrecht K, Lin X, Operario D, Griesemer S, Fitch A, Halpin R A, Stockwell T B, Spiro D J, Holmes E C and St George K. 2011. Deep sequencing reveals mixed infection with 2009 pandemic influenza A (H1N1) virus strains and the emergence of oseltamivir resistance. J Infect Dis, 203: 168-174.
          doi: 10.1093/infdis/jiq040

      9. Gu H, Qi X, Li X, Jiang H, Wang Y, Liu F, Lu S, Yang Y and Liu F. 2010. Rapid and specific detection of H3 swine influenza virus using reverse transcription loop-mediated isothermal amplification method. J Appl Microbiol, 108: 1145-1154.
          doi: 10.1111/jam.2010.108.issue-4

      10. Gunson R, Maclean A, Davies E, Bennett S, Miller R and Carman W F. 2010. Development of a multiplex real-time RT-PCR that allows universal detection of influenza A viruses and simultaneous typing of influenza A/H1N1/2009 virus. J Virol Methods, 163: 258-261.
          doi: 10.1016/j.jviromet.2009.10.006

      11. Hata M, Tsuzuki M, Goto Y, Kumagai N, Harada M, Hashimoto M, Tanaka S, Sakae K, Kimura T, Minagawa H and Miyazaki Y. 2007. High frequency of amantadine-resistant influenza A (H3N2) viruses in the 2005-2006 season and rapid detection of amantadine-resistant influenza A (H3N2) viruses by MAMA-PCR. Jpn J Infect Dis, 60: 202-204.

      12. Hoffmann E, Stech J, Guan Y, Webster R G and Perez D R. 2001. Universal primer set for the full-length amplification of all influenza A viruses. Arch Virol, 146: 2275-2289.
          doi: 10.1007/s007050170002

      13. Jiang T, Kang X, Deng Y, Zhao H, Li X, Yu X, Yu M, Qin E, Zhu Q, Yang Y and Qin C. 2010. Development of a real-time RT-PCR assay for a novel influenza A (H1N1) virus. J Virol Methods, 163: 470-473.
          doi: 10.1016/j.jviromet.2009.09.021

      14. Klungthong C, Chinnawirotpisan P, Hussem K, Phonpakobsin T, Manasatienkij W, Ajariyakhajorn C, Rungrojcharoenkit K, Gibbons R V and Jarman R G. 2010. The impact of primer and probe-template mismatches on the sensitivity of pandemic influenza A/H1N1/2009 virus detection by real-time RT-PCR. J Clin Virol, 48: 91-95.
          doi: 10.1016/j.jcv.2010.03.012

      15. Li X, Qi X, Miao L, Wang Y, Liu F, Gu H, Lu S, Yang Y and Liu F. 2009. Detection and subtyping of influenza A virus based on a short oligonucleotide microarray. Diagn Microbiol Infect Dis, 65: 261-270.
          doi: 10.1016/j.diagmicrobio.2009.07.016

      16. Lorusso A, Faaberg K S, Killian M L, Koster L and Vincent A L. 2010. One-step real-time RT-PCR for pandemic influenza A virus (H1N1) 2009 matrix gene detection in swine samples. J Virol Methods, 164: 83-87.
          doi: 10.1016/j.jviromet.2009.12.002

      17. Nicholls H. 2006. Pandemic influenza: the inside story. PLoS Biol, 4: e50.
          doi: 10.1371/journal.pbio.0040050

      18. Pabbaraju K, Wong S, Wong A A, Appleyard G D, Chui L, Pang X L, Yanow S K, Fonseca K, Lee B E, Fox J D and Preiksaitis J K. 2009. Design and validation of real-time reverse transcription-PCR assays for detection of pandemic (H1N1) 2009 virus. J Clin Microbiol, 47: 3454-3460.
          doi: 10.1128/JCM.01103-09

      19. Palese P and Shaw M L. 2007. Orthomyxoviridae: The Viruses and Their Replication, p. 1648-1689. In Knipe D M, Howley P M, Griffin D E, et al. (ed.), Fields Virology. Lippincott-William & Wilkins, Philadelphia, Pa.

      20. Qi X, Li X, Rider P, Fan W, Gu H, Xu L, Yang Y, Lu S, Wang H and Liu F. 2009. Molecular characterization of highly pathogenic H5N1 avian influenza A viruses isolated from raccoon dogs in China. PLoS One, 4: e4682.

      21. Salomon N and Perlman D C. 1999. Cytomegalovirus pneumonia. Semin Respir Infect, 14: 353-358.

      22. Schulze M, Nitsche A, Schweiger B and Biere B. 2010. Diagnostic approach for the differentiation of the pandemic influenza A(H1N1)v virus from recent human influenza viruses by real-time PCR. PLoS One, 5: e9966.
          doi: 10.1371/journal.pone.0009966

      23. Spackman E, Senne D A, Myers T J, Bulaga L L, Garber L P, Perdue M L, Lohman K, Daum L T and Suarez D L. 2002. Development of a real-time reverse transcriptase PCR assay for type A influenza virus and the avian H5 and H7 hemagglutinin subtypes. J Clin Microbiol, 40: 3256-3260.
          doi: 10.1128/JCM.40.9.3256-3260.2002

      24. Storch G A. 2000. Essentials of diagnostic virology, 1st Edition ed. Churchill Livingstone, New York.

      25. Vijaykrishna D, Poon L L, Zhu H C, Ma S K, Li O T, Cheung C L, Smith G J, Peiris J S and Guan Y. 2010. Reassortment of pandemic H1N1/2009 influenza A virus in swine. Science, 328: 1529.
          doi: 10.1126/science.1189132

      26. World Health Organization. 2009. http://www.who.int/csr/don/2009_11_13/en/index.html. World Health Organization.

      27. World Health Organization. 2009. CDC Protocol of Realtime RTPCR for Influenza A (H1N1). World Health Organization.

      28. World Health Organization. 2010. http://www.who.int/mediacentre/news/statements/2010/h1n1_vpc_20100810/en/index.html. World Health Organization.

      29. Wright P F, Newmann G and Kawaoka Y. 2007. Orthomyxoviruses, p. 1693-1740. In Knipe D M, Howley P M, Griffin D E, et al. (ed.), Fields Virology. Lippincott-William & Wilkins, Philadelphia, Pa.

      30. Wu C, Cheng X, He J, Lv X, Wang J, Deng R, Long Q and Wang X. 2008. A multiplex real-time RT-PCR for detection and identification of influenza virus types A and B and subtypes H5 and N1. J Virol Methods, 148: 81-88.
          doi: 10.1016/j.jviromet.2007.10.023

      31. Yang Y, Huang F, Gonzalez R, Wang W, Lu G, Li Y, Vernet G, Jin Q and Wang J. 2011. Evaluation of twelve real-time reverse transcriptase PCR primer-probe sets for detection of pandemic influenza A/H1N1 2009 virus. J. Clin. Microbiol., 49: 1434-1440.
          doi: 10.1128/JCM.01914-10

      32. Yongfeng H, Fan Y, Jie D, Jian Y, Ting Z, Lilian S and Jin Q. 2011. Direct pathogen detection from swab samples using a new high-throughput sequencing technology. Clin Microbiol Infect, 17: 241-244
          doi: 10.1111/j.1469-0691.2010.03246.x

    • 加载中

    Figures(6) / Tables(2)

    PDF

    Article Metrics

    Article views(4385) PDF downloads(15) Cited by()

    Related
    Proportional views

      Detection of the Pandemic H1N1/2009 Influenza A Virus by a Highly Sensitive Quantitative Real-time Reverse-transcription Polymerase Chain Reaction Assay

        Corresponding author: Fenyong Liu, liu_fy@berkeley.edu
      • 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
      • Received Date: 30 October 2012
      • Accepted Date: 23 January 2013
      Fund Project:  National Mega Project on Major Drug Development 2009ZX09103-678National Small Business Innovation and Research (SBIR) Program of China, the Technology R & D Program of Jiangsu Province, China BG20077035National Natural Science Foundation of China 81030031NIH RO1-AI050468National Small Business Innovation and Research (SBIR) Program of China, the Technology R & D Program of Jiangsu Province, China BG2008662This research has been supported by grants from National Basic Research Program of China 2011CB504800NIH RO1-DE014842NIH RO1-AI041927National Natural Science Foundation of China 31100128NIH RO1-DE014145

      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.

        HTML