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. 2004 Oct 13;74(4):619–628. doi: 10.1002/jmv.20221

Development and evaluation of a real‐time nucleic acid sequence based amplification assay for rapid detection of influenza A†

Catherine Moore 1,✉, Sam Hibbitts 2, Neil Owen 1, Sally A Corden 1, Graham Harrison 3, Julie Fox 4, Colin Gelder 5, Diana Westmoreland 1
PMCID: PMC7166529  PMID: 15484279

Abstract

The development and introduction of effective treatment for influenza A in the form of neuraminidase inhibitors have made the rapid diagnosis of infection important especially in high‐risk populations. The aim of this study was to develop a real‐time nucleic acid sequenced based amplification (NASBA) using a molecular beacon that could detect a wide range of influenza A subtypes and strains in a single reaction by targeting a conserved region of the influenza genome, and to evaluate its sensitivity and specificity against traditional laboratory techniques on a range of clinical samples usefulness during the 2003/2004 influenza season. The results demonstrated the assay to be highly sensitive and specific, detecting <0.1 TCID50 of virus stock. Three hundred eighty‐nine clinical samples were tested in total from two patient groups. Overall, the real‐time NASBA assay detected 64% (66/103) more influenza positive samples than cell culture and direct immunofluorescence (IF) and, therefore, was shown to be more sensitive in detecting influenza A in a wide range of respiratory samples than traditional methods. In conclusion, the real‐time influenza A assay demonstrated clinical usefulness in both hospital and community populations. J. Med. Virol. 74:619–628, 2004. © 2004 Wiley‐Liss, Inc.

Keywords: influenza A, NASBA, real‐time NASBA, molecular beacon, rapid diagnosis

†

Catherine Moore and Sam Hibbitts are joint first authors.

REFERENCES

  1. Boom R, Sol C, Salimans M, Jansen C, Wertheim‐van Dillen P, van der Noordaa J. 1990. Rapid and simple method for purification of nucleic acids. J Clin Microbiol 28: 495–503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Chan KH, Maldeis N, Pope W, Yup A, Ozinskas A, Gill J, Seto WH, Shortridge KF, Peiris JSM. 2002. Evaluation of the directigen FluA+B test for rapid diagnosis of influenza virus type A and B infections. J Clin Microbiol 40: 1675–1680. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Collins PL, Chanock RM, McIntosh K. 1996. Parainfluenza viruses In: Fields BN, Howley PM, Griffin DE, Lamb RA, Martin MA, Roizman B, Straus SE, Knipe DM, editors. Fields virology. 3rd edition Philadelphia: Lippincott‐Raven; pp 1205–1243. [Google Scholar]
  4. Cox NJ, Subbarao K. 1999. Influenza. Lancet 354: 1277–1282. [DOI] [PubMed] [Google Scholar]
  5. Deiman B, Van Aarle P, Sillekens P. 2002. characteristics and applications of nucleic acid sequence‐based amplification (NASBA). Mol Biotech 20: 163–217. [DOI] [PubMed] [Google Scholar]
  6. Ellis JS, Zambon MC. 2002. Molecular diagnosis of influenza. Rev Med Virol 12: 375–389. [DOI] [PubMed] [Google Scholar]
  7. Fox JD, Song H, Samuelson A, Zhang Y, Neale ML, Westmoreland D. 2002. Development and evaluation of nucleic acid sequence based amplification (NASBA) for the diagnosis of enterovirus infections using the Nuclisen™ Basic Kit. J Clin Virol 24: 117–130. [DOI] [PubMed] [Google Scholar]
  8. Greene SR, Moe CL, Jaykus LA, Cronin M, Grosso L, Van Aarle P. 2003. Evaluation of the Nuclisens™ Basic Kit assay for detection of norwalk virus RNA in stool specimens. J Virol Methods 108: 123–131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gubareva LV, Kaiser L, Hayden FG. 2000. Influenza virus neuraminidase inhibitors. Lancet 355: 827–835. [DOI] [PubMed] [Google Scholar]
  10. Harnden A, Brueggemann A, Shepperd S, White J, Hayward AC, Zambon M, Crook D, Mant D. 2003. Near patient testing for influenza in children in primary care: Comparison with laboratory test. BMJ 326: 480. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hayden FG, Palese P. 2002. Influenza virus In: Richman DD, Whitely RJ, Hayden FG, editors. Clinical virology. 2nd edition Washington, USA: ASM press; pp 891–920. [Google Scholar]
  12. Hibbitts S, Fox JD. 2002. The application of molecular techniques to diagnosis of viral respiratory tract infections. Rev Med Microbiol 13: 177–185. [Google Scholar]
  13. Hibbitts S, Fox JD. 2004. Real‐time NASBA In: Saunders N, Logan J, Edwards K, editors. Methods in real‐time PCR. Horizon Scientific Press; p 225–241. [Google Scholar]
  14. Horimoto T, Kawaoka Y. 2001. Pandemic threat posed by avian influenza A viruses. Clin Microbiol Rev 14: 129–149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kievits T, van Gemen B, van Strijp D, Schukkink R, Dircks M, Adriaanse H, Malek L, Sooknanan R, Lens P. 1991. NASBA isothermal enzymatic in vitro nucleic acid amplification optimized for the diagnosis of HIV‐1 infection. J Virol Methods 35: 273–286. [DOI] [PubMed] [Google Scholar]
  16. Kilbourne ED, Smith C, Brett I, Pokorny BA, Johansson B, Cox N. 2002. The total influenza vaccine failure of 1947 revisited. Major intrasubtypic antigenic change can explain failure of vaccine in a post‐World war II epidemic. Proc Natl Acad Sci 99: 10748–10752. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lau LT, Banks J, Aherne R, Brown IH, Dillon N, Collins RA, Chan KY, Fung YW, Xing J, Yu ACH. 2004. Nucleic acid sequence‐based amplification methods to detect avian influenza virus. Biophy Res Comm 313: 336–342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Leone G, Van Schijndel H, Van Gemen B, Kramer F, Schoen C. 1998. Molecular beacon probes combined with amplification by NASBA enable homogenous, real‐time detection of RNA. Nuc Acids Res 26: 2150–2155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Moore C, Clark EM, Gallimore CI, Corden SA, Gray JJ, Westmoreland D. 2004. Evaluation of a broadly reactive nucleic acid sequenced based amplification (NASBA) assay for the detection of noroviruses in faecal material. J Clin Virol 29: 290–296. [DOI] [PubMed] [Google Scholar]
  20. National Institute for Clinical Excellence (NICE) . 2003. Guidance on the use of zanamivir, oseltamivir and amantadine for the treatment and prophylaxis of influenza. Technology appraisal guidance, no 58, February. http://www.nice.org.uk/pdf/58_flu_fullguidance.pdf (accessed December 2003).
  21. Nicholson KG, Aoki FY, Osterhaus ADME, Trottier S, Carewicz O, Mercier CH, Rode A, Kinnersley N, Ward P. 2000. Efficacy and safety of oseltamivir in treatment of acute influenza: A randomized controlled trial. Lancet 355: 1845–1850. [DOI] [PubMed] [Google Scholar]
  22. Nicholson KG, Wood JM, Zambon M. 2003. Influenza. Lancet 362: 1733–1745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Palese P, Garcia‐Sastre A. 2002. Influenza vaccines: Present and future. J Clin Invest 110: 9–13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Reed L, Muench H. 1938. A simple method of estimating fifty percent endpoints. Am J Hygiene 27: 493–497. [Google Scholar]
  25. Schweiger B, Zadow I, Heckler R, Timm H, Pauli G. 2000. Application of a fluorogenic PCR assay for typing and subtyping of influenza viruses in respiratory samples. J Clin Microbiol 38: 1552–1558. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Shen KG, Zhaori B, Zweygberg‐Wirgari M, Ying M, Grandien B, Wahren B, Linde A. 1996. Detection of respiratory viruses in nasopharyngeal secretions with immunofuorescence technique for multiplex screening—An evaluation of the chemicon assay. Clin Diagn Virol 6: 147–154. [DOI] [PubMed] [Google Scholar]
  27. Stiver G. 2003. The treatment of influenza with antiviral drugs. CMAJ 168: 49–56. [PMC free article] [PubMed] [Google Scholar]
  28. Stockton J, Ellis JS, Saville M, Clewley JP, Zambon MC. 1998. Multiplex PCR for typing and subtyping influenza and respiratory syncytial virus. J Clin Microbiol 36: 2990–2995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Stohr K. 2003. Preventing and treating influenza. BMJ editorial 326: 1223–1224. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Treanor J. 2004. Influenza vaccine—Outmaneuvering antigenic shift and drift. N Engl J Med 350: 218–220. [DOI] [PubMed] [Google Scholar]
  31. Van Elden LJR, Nijhuis M, Schipper P, Schuurman R, Van Loon AM. 2001. Simultaneous detection of influenza viruses A and B using real‐time quantitative PCR. J Clin Microbiol 39: 196–200. [DOI] [PMC free article] [PubMed] [Google Scholar]

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