Skip to main content
SpringerLink
Log in
Book cover

Diagnostic Virology Protocols pp 161–181Cite as

Simultaneous Molecular Detection and Confirmation of Influenza AH5, with Internal Control

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 665))

Abstract

Influenza viruses continue to be a major cause of respiratory tract infection, resulting in substantial morbidity and mortality throughout the world. Accurate and rapid differential diagnosis of influenza virus infections, particularly associated with zoonotic infections, is important for public health actions, patient management, and treatment. Real-time PCR is widely considered the gold standard for molecular detection of influenza viruses owing to its high assay specificity, extreme detection sensitivity, and wide linear dynamic range. This protocol describes the use of a real-time RT-PCR assay for identification of influenza A and B viruses, detection of H5 subtype viruses, and an internal control, in a multiplexed, single-tube format. The inclusion of an internal bacteriophage control allows the efficiency of the extraction and amplification process to be monitored, so that false-negative results may be avoided. The primers and probe sets in this multiplex assay have been validated with a panel of influenza A viruses of different subtypes (including swine influenza viruses), and influenza B viruses, and specificity further confirmed with non-related respiratory viruses.

This is a preview of subscription content, log in via an institution.

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Osterhaus, A.D., Rimmelzwaan, G.F., Martina, B.E., Bestebroer, T.M., and Fouchier, R.A. (2000) Influenza B virus in seals. Science 288, 1051–1053.

    Article  PubMed  CAS  Google Scholar 

  2. Guo, Y.J., Jin, F.G., Wang, P., Wang, M., and Zhu, J.M. (1983) Isolation of influenza C virus from pigs and experimental infection of pigs with influenza C virus. J Gen Virol 64, 177–182.

    Article  PubMed  Google Scholar 

  3. Fouchier, R.A., Schneeberger, P.M., Rozendaal, F.W., Broekman, J.M., Kemink, S.A., Munster, V., Keiken, T., Rimmelzwaan, G.F., Schutten, M., Van Doornum, G.J., Koch, G., Bosman, A., Koopmans, M., and Osterhaus, A.D. (2004) Avian influenza A virus (H7N7) associated with human conjunctivitis and a fatal case of acute respiratory distress syndrome. Proc Nat Acad Sci USA 101, 1356–1361.

    Article  PubMed  CAS  Google Scholar 

  4. http://www.who.int/csr/disease/avian_influenza/ai_timeline/en/index.html.

  5. Peiris, J.S.M., Yuen, K.Y., Leung, C.W., Chan, K.H., Ip, P.L., Rai, R.W., Orr, W.K., and Shortridge, W.F. (1999) Human infection with influenza H9N2. Lancet 354, 916–917.

    Article  PubMed  CAS  Google Scholar 

  6. Butt, K.M., Smith, G.J.D., Chen, H., Zhang, L.J., Leung, C.Y.H., Xu, K.M., Lim, W., Webster, R.G., Yuen, K.Y., Peiris, J.S.M., and Guan, Y. (2005) Human infection with an avian H9N2 influenza A virus in Hong Kong in 2003. J Clin Microbiol 43, 5760–5767.

    Article  PubMed  CAS  Google Scholar 

  7. Showronski, D.M., Tweed, S.A., Petric, M., Booth, T., Li, Y., and Tam, T. (2006) Human illness and isolation of low-pathogenicity avian influenza virus of the H7N3 subtype in British Columbia, Canada. J Infect Dis 193, 899–900.

    Article  Google Scholar 

  8. Editorial team. (2007) Avian influenza A (H7N2) outbreak in the United Kingdom. Euro Surveill 12, E070531.2.

    Google Scholar 

  9. Taubenberger, J.K., Reid, A.H., Lourens, R.M., Wang, R., Jin, G., and Fanning, T.G. (2005) Characterization of the 1918 influenza virus polymerase genes. Nature 437, 889–893.

    Article  PubMed  CAS  Google Scholar 

  10. Glezen, W.P. (1982) Serious morbidity and mortality associated with influenza epidemics. Epidemiol Rev 4, 25–44.

    PubMed  CAS  Google Scholar 

  11. Fleming, D.M. (2000) The contribution of influenza to combined acute respiratory infections, hospital admissions, and deaths in winter. Commun Dis Public Health 3, 32–38.

    PubMed  CAS  Google Scholar 

  12. Murphy, B.R., Baron, S., Chalhub, E.G., Uhlendorf, C.P., and Chanock, R.M. (1973) Temperature-sensitive mutants of influenza virus. IV. Induction of interferon in the nasopharynx by wild-type and a temperature-sensitive recombinant virus. J Infect Dis 128, 488–493.

    Article  PubMed  CAS  Google Scholar 

  13. Kandun, I.N., Wibisono, H., Sedyangingsih, E.R., Yusharmen, Hadisoedarsuno, W., Purba, W., Santoso, H., Septiawati, C., Tresnaningsih, E., Heriyanto, B., Yuwono, D., Harun, S., Soeroso, S., Giriputra, S., Blair, P.J., Jerimijenko, J., Kosasih, H., Putnam, S.D., Samaan, G., Silitonga, M., Chan, K.H., Poon, L.L., Lim, W., Klimov, A., Lindstrom, S., Guan, Y., Donis, R., Katz, J., Cox, N., Peiris, M., and Uyeki, T.M. (2005) Three Indonesian clusters of H5N1 virus infection in N Engl J Med 2000 355, 286–2194.

    Google Scholar 

  14. Ungchusak, K., Auewarakul, P., Dowell, S.F., Kitphati, R., Auwanit, W., Puthavathana, P., Uiprasertkul, M., Boonnak, K., Pittayawon­ganon, C., Cox, N.J., Zaki, S.R., Thawatsupha, P., Chittaganpitch, M., Khotong, R., Simmerman, J.M., and Chunsutthiwat, S. (2005) Probable person-to-person transmission of avian influenza A (H5N1). N Engl J Med 352, 333–340.

    Article  PubMed  CAS  Google Scholar 

  15. Du Ry van Beest Holle, M., Meijer, A., Koopmans, M., and de Jager, C.M. (2005) Human-to-human transmission of avian influenza A/H7N7, The Netherlands, 2003. Euro Surveill 10, 264–268.

    Google Scholar 

  16. Beigel, J.H., Farrar, A.M., Han, A.M., Hayden, F.G., Hyer, R., de Jong, M.D., Lochindarat, S., Nguyen, T.K., Nguyen, T.H., Tran, T.H., Nicoll, A., Touch, S., and Yuen, K.Y. Writing Committee of the World Health Organization (WHO) Consultation on Human Influenza A/H5. (2005) Avian influenza A (H5N1) infection in humans. N Engl J Med 353, 1374–1385.

    Article  PubMed  Google Scholar 

  17. Yuen, K.Y., Chan. P.K., Peiris, M., Tsang, D., Que, T., Shortridge, K., Cheung, P., To, W., Ho, E., and Sung, R. (1998) Clinical features and rapid viral diagnosis of human disease associated with avian influenza A H5N1 virus. Lancet 351, 467–471.

    Article  PubMed  CAS  Google Scholar 

  18. Chotpitayasunondh, T., Ungchusak, K., Hanshaoworakul, W., Chunsuthiwat, S., Sawanpanyalert, P., Kijphati, R., Lochindarat, S., Srisan, P., Suwan, P., Osotthanakorn, Y., Anantasetagoon, T., Kanjanawasri, S., Tanupattarachai, S., Weerakul, J., Chaiwirattana, R., Maneerattanaporn, M., Poolsavathitikool, R., Chokephaibulkit, K., Apisarnthanarak, A., and Dowell, S.F. (2005) Human disease from influenza A (H5N1), Thailand, 2004. Emerg Infect Dis 11, 201–209.

    Article  PubMed  Google Scholar 

  19. Tran, T.H., Nguyen, T.L., Nguyen, T.D., Luong, T.S., Pham. P.M., Nguyen, V.C., Pham, T.S., Vo, C. D., Le, T.Q., Ngo, T.T., Le, P.P., Nguyen, K.T., Le, H.S., Le, V.T., Christiane, D., Tran, T.T., Menno de J., Schultsz C., Cheng, P., Lim, W., Horby, P., and Farrar, J. World Health Organization International Avian Influenza Investigative Team. (2004) Avian influenza A (H5N1) in 10 patients in Vietnam. N Engl J Med 350, 1179–1188.

    Article  PubMed  Google Scholar 

  20. Writing Committee of the Second World Health Organization Consultation on Clinical Aspects of Human Infection with Avian Influenza A (H5N1) Virus. (2008) Update on avian influenza A (H5N1) virus infection in humans. N Engl J Med 358, 261–273.

    Article  Google Scholar 

  21. de Jong, M.D., Simmons, C.P., Thanh, T.T., Hien, V.M., Smith, G.J., Chau, T.N., Hoang, D.M., Van Vinh Chau, N., Khanh, T.H., Dong, V.C., Qui, P.T., Van Cam, B., Ha, D.Q., Guan, Y., Peiris, J.S., Chinh, N.T., Hien, T.T., and Farrar, J. (2006) Fatal outcome of human influenza A (H5N1) is associated with high viral load and hypercytokinemia. Nat Med 12, 1203–1207.

    Article  PubMed  Google Scholar 

  22. The Writing Committee of the World Health Organization (WHO) Consultation on Human Influenza A/H5. (2005) Avian influenza A (H5N1) infection in humans. N Engl J Med 353, 1374–1385.

    Article  Google Scholar 

  23. Gu, J., Xie, Z., Gao, Z., Liu, J., Korteweg, C., Ye, J., Lau, L.T., Gao, Z., Zhang, B., McNutt, M.A., Lu, M., Anderson, V.M., Gong, E., Yu, A.C., and Lipkin, W.I. (2007) H5N1 infection of the respiratory tract and beyond: a molecular pathology study. Lancet 370, 1106–1108.

    Article  Google Scholar 

  24. Anon (2007) Recommendations and Lab­oratory Procedures for Detection of Avian Influenza A (H5N1) Virus in Specimens from Suspected Human Cases. http://www.who.int/csr/disease/avian_influenza/guidelines/RecAIlabtestsAug07.pdf.

  25. Collins, R.A., Ko, L.S., So, K.L., Ellis, T., Lau, L.T., and Yu, A.C. (2002) Detection of highly pathogenic and low pathogenic avian influenza subtype H5 (Eurasian lineage) using NASBA. J Virol Methods 103, 213–225.

    Article  PubMed  CAS  Google Scholar 

  26. Moore, C., Hibbitts, S., Owen, N., Corden, S.A., Harrison, G., Fox, J., Gelder, C., and Westmoreland, D. (2004) Development and evaluation of a real-time nucleic acid sequence based amplification assay for rapid detection of influenza A. J Med Virol 74, 619–628.

    Article  PubMed  CAS  Google Scholar 

  27. Poon, L.L., Leung, C.S., Chan, K.H., Lee, J.H., Yuen, K.Y., Guan, Y., and Peiris, J.S. (2005) Detection of human influenza A viruses by loop-mediated isothermal amplification. J Clin Microbiol 43, 427–430.

    Article  PubMed  CAS  Google Scholar 

  28. Imai, M., Ninomiya, A., Minekawa, H., Notomi, T., Ishizaki, T., Tashiro, M., and Odagiri, T. (2006) Development of H5-RT-LAMP (loop-mediated isothermal amplification) system for rapid diagnosis of H5 avian influenza virus infection. Vaccine 24, 6679–6682.

    Article  PubMed  CAS  Google Scholar 

  29. Ellis, J.S. and Zambon, M.C. (2002) Molecular diagnosis of influenza. Rev Med Virol 12, 375–389.

    Article  PubMed  CAS  Google Scholar 

  30. Wallace, L.A., Collins, T.C., Douglas, J.D., McIntyre, S., Millar, J., and Carman, W.F. (2004) Virological surveillance of influenza-like illness in the community using PCR and serology. J Clin Virol 3, 40–45.

    Article  Google Scholar 

  31. Vernet, G. (2007) Use of molecular assays for the diagnosis of influenza. Expert Rev Anti Infect Ther 5, 89–104.

    Article  PubMed  CAS  Google Scholar 

  32. Ellis, J.S., Fleming, D., and Zambon, M. (1997) Multiplex reverse transcription-PCR for surveillance of influenza A and B viruses in England and Wales in 1995 and 1996. J Clin Microbiol 35, 2076–2082.

    PubMed  CAS  Google Scholar 

  33. Boivin, G., Hardy, I., Tellier, G., and Maziade, J. (2000) Predicting influenza infections during epidemics with use of a clinical case definition. Clin Infect Dis 3, 1166–1169.

    Article  Google Scholar 

  34. Atmar, R., Baxter, B., Dominguez, E., and Taber, L.H. (1996) Comparison of reverse transcription-PCR with tissue culture and other rapid diagnostic assays for rapid detection of influenza viruses. J Clin Microbiol 56, 168–173.

    Google Scholar 

  35. Carrraro, E., Ferreira, N., Benfica, D., SittaPerosa, A.H., Granato, C.F.H., and Bellei, N.C.J. (2007) Applications of a duplex reverse transcription polymerase chain reaction immunofluorescence assay in comparison with virus isolation for detection of influenza A and B. Diagn Microbiol Infect Dis 57, 53–57.

    Article  Google Scholar 

  36. Ellis, J.S. and Brown, D.W. (1998) PCR for the detection of influenza viruses in clinical material. In: Stephenson, J.R. and Warnes, A. Eds. Methods in Molecular Medicine, Vol. 12, Diagnostic Virology Protocols. Humana, Totowa, NJ.

    Google Scholar 

  37. Minosse, C., Selleri, M., Zaniratti, M.S., Lauria, F.N., Puro, V., Carletti, F., Cappiello, G., Gualano, G., Bevilacqua, N., and Capobianchi, M.R. (2007) Improved detection of human influenza A and B viruses in respiratory tract specimens by hemi-nested PCR. J Virol Methods 141, 225–228.

    Article  PubMed  CAS  Google Scholar 

  38. Wright, K., Wilson, G., Novosad, D., Dimock, C., Tan, D., and Weber, J. (1995) Typing and subtyping of influenza viruses in clinical samples by PCR. J Clin Microbiol 33, 1180–1184.

    PubMed  CAS  Google Scholar 

  39. Coyle, P.V., Ong, G.M., O’Neill, J.H., McCaughey, C., De Ornellas, D., Mitchell, F., Mitchell, S.J., Feeney, S.A., Wyatt, D.E., Forde, M., and Stockton, J. (2004) A touchdown nucleic acid amplification protocol as an alternative to culture backup for immunofluorescence in the routine diagnosis of acute viral respiratory tract infections. BMC Microbiol 4, 41.

    Article  PubMed  Google Scholar 

  40. Schweiger, B., Zadow, I., Heckler, R., Timm, H., and 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.

    PubMed  CAS  Google Scholar 

  41. Templeton, K.E., Scheltinga, S.A., Beersma, M.F.C., Kroes, A.C.M., and Claas, E.C.J. (2004) Rapid and sensitive method using multiplex real-time PCR for diagnosis of infections by influenza A and Influenza B viruses, respiratory syncytial virus, and parainfluenza viruses 1, 2, 3, and 4. J Clin Microbiol 42, 1564–1569.

    Article  PubMed  CAS  Google Scholar 

  42. Brittain-Long, R., Nord, S., Olofsson, S., Westin, J., Anderson, L.M., and Lindh, M. (2008) Multiplex real-time PCR for detection of respiratory tract infections. J Clin Virol 41, 53–56.

    Article  PubMed  CAS  Google Scholar 

  43. Legoff, J., Kara, R., Moulin, F., Si-Mohamed, A., Krivine, A., Belec, L., and Lepon, P. (2008) Evaluation of the one-step multiplex real-time reverse transcription-PCR ProFlu-1 assay for detection of influenza A and influenza B viruses and respiratory syncytial viruses in children. J Clin Microbiol 46, 789–791.

    Article  PubMed  CAS  Google Scholar 

  44. World Health Organization. (2006) Collecting, Preserving and Shipping Specimens for the Diagnosis of Avian Influenza A (H5N1) Virus Infection: Guide for Field Operations. (2006) http://www.who.int/csr/resources/publications/surveillance/CDS_EPR_ARO_2006_1.pdf.

  45. Ellis, J.S., Smith, J.W., Braham, S., Lock, M., Barlow, K., and Zambon, M.C. (2007) Design and validation of an H5 TaqMan real-time one-step reverse transcription-PCR and confirmatory assays for diagnosis and verification of influenza A virus H5 infections in humans. J Clin Microbiol 45, 1535–1543.

    Article  PubMed  CAS  Google Scholar 

  46. Chen, W., He, B., Li, C., Zhang, X., Wu, W., Yin, X., Fan, B., and Wang, J. (2007) Real-time RT-PCR for H5N1 avian influenza A virus detection. J Med Microbiol 56, 603–607.

    Article  PubMed  CAS  Google Scholar 

  47. Centers for Disease Control and Prevention (CDC). (2006) New laboratory assay for diagnostic testing of avian influenza A/H5 (Asian Lineage). MMWR Morb Mortal Wkly Rep 55, 127.

    Google Scholar 

  48. Payungporn, S., Chutinimitkul, S., Chaisingh, A., Damrongwantanapokin, S., Buranathai, C., Amonsin, A., Theamboonlers, A., and Poovarawan, Y. (2006) Single step multiplex RT-PCR for H5N1 influenza A virus detection. J Virol Methods 131, 143–147.

    Article  PubMed  CAS  Google Scholar 

  49. Di Trani, L., Bedini, B., Donatelli, I., Campitelli, L., Chiappini, B., De Marco, M.A., Delogu, M., Buonavoglia, C., and Vaccari, G. (2006) A sensitive one-step real-time PCR for detection of avian influenza viruses using a MGB probe and an internal positive control. BMC Infect Dis 6, 87.

    Article  PubMed  Google Scholar 

  50. Das, A., Spackman, E., Senne, D., Pederson, J., and Suarez, D.L. (2006) Development of an internal positive control for rapid diagnosis of avian influenza virus infections by real-time reverse transcription-PCR with lyophilized reagents. J Clin Microbiol 44, 3065–3073.

    Article  PubMed  CAS  Google Scholar 

  51. The World Health Organization Global Influenza Program Surveillance Network. (2005) Evolution of H5N1 avian influenza viruses in Asia. Emerg Infect Dis 11, 1503–1656.

    Article  Google Scholar 

  52. Editorial team. (2006) More cases of avian influenza infection in humans and poultry in Turkey. Euro Surveill 11, E060112.1.

    Google Scholar 

  53. Anon. (2006) Outbreak news. Avian influenza China, avian influenza Turkey. WER 81, 13–20.

    Google Scholar 

  54. Anon. (2007) Viruses responsible for avian influenza in Suffolk and Hungary “essentially identical”. Vet Rec 160, 206.

    Google Scholar 

  55. Nguyen-Van-Tam, J., Nair, P., Acheson, P., Baker, A., Barker, M., Bracebridge, S., Croft, J., Ellis, J., Gelletlie, R., Gent, N., Ibbotson, S., Joseph, C., Mahgoub, H., Monk, P., Wreghitt, T. W., Sundkvist, T., Sellwood, C., Simpson, J., Smith, J., Watson, J. M., Zambon, M., and Lightfoot, N. Incidence Response Team. (2006) Outbreak of low pathogenicity H7N3 avian influenza in UK, including associated case of human conjunctivitis. Euro Surveill 11, E060504.2.

    PubMed  CAS  Google Scholar 

  56. Curran, M.D., Ellis, J.S., Wreghitt, T.G., and Zambon, M.C. (2007) Establishment of a UK national influenza H5 laboratory network. J Med Microbiol 56, 1263–1267.

    Article  PubMed  Google Scholar 

  57. Borst, A., Box, A.T., and Fluit, A.C. (2004) False-positive results and contamination in nucleic acid amplification assays: suggestions for a prevent and destroy strategy. Eur J Clin Microbiol Infect Dis 23, 289–299.

    Article  PubMed  CAS  Google Scholar 

  58. Burkardt, H.J. (2000) Standardization and quality control of PCR analyses. Clin Chem Lab Med 38, 87–91.

    Article  PubMed  CAS  Google Scholar 

  59. Valentine-Thon, E. (2002) Quality control in nucleic acid testing – where do we stand? J Clin Virol 25, S13–S21.

    Article  PubMed  CAS  Google Scholar 

  60. de Jong, M.D., Bach, V.C., Phan, T.Q., Vo, M.H., Tran, T.T., Nguyen, B.H., Beld, M., Le, T.P., Truong, H.K., Nguyen, V.V., Tran, T.H., Do, Q.H., and Farrar, J. (2005) Fatal avian influenza A (H5N1) in a child presenting with diarrhea followed by coma. N Engl J Med 352, 686–691.

    Article  PubMed  Google Scholar 

  61. Buchy, P., Mardy, S., Vong, S., Toyoda, T., Aubin, J.T., Miller, M., Touch, S., Sovann, L., Dufourcq, J.B., Richner, B., Tu, P.V., Tien N.T., Lim, W., Peiris, J.S., and Van der Werf, S. (2007) Influenza A/H5N1 virus infection in humans in Cambodia. J Clin Virol 39, 164–168.

    Article  PubMed  Google Scholar 

  62. Uiprasertkul, M., Kitphati, R., Puthavathana, P., Kriwong, R., Kongchanagul, A., Ungchusak, K., Angkasekwinai, S., Chokephaibulkit, K., Srisook, K., Vanprapar, N., and Auewarakul, P. (2007) Apop­tosis and pathogenesis of avian influenza A (H5N1) virus in humans. Emerg Infect Dis 13, 708–712.

    Article  PubMed  CAS  Google Scholar 

  63. Rolfe, K.J., Parmar, S., Mururi, D., Wreghitt, T.G., Jalal, H., Zhang, H., and Curran, M.D. (2007) An internally controlled, one-step, real-time RT-PCR assay for norovirus detection and genogrouping. J Clin Virol 39, 318–321.

    Article  PubMed  CAS  Google Scholar 

  64. Blow, J.A., Dohm, D.J., Negley, D.L., and Mores, C.N. (2004) Virus inactivation by nucleic acid extraction reagents. J Virol Methods 119, 195–198.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Respiratory Virus Unit, Virus Reference Department, Centre for Infections, Health Protection Agency, London, UK

    Joanna S. Ellis

  2. Health Protection Agency, East of England, Clinical Microbiology and Public Health Laboratory, Addenbrooke’s Hospital, Cambridge, UK

    Martin D. Curran

Authors
  1. Joanna S. Ellis
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Martin D. Curran
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Joanna S. Ellis .

Editor information

Editors and Affiliations

  1. , Research & Development Division, Health Protection Agency, High Holborn 330, London, WC1V 7BA, United Kingdom

    John R. Stephenson

  2. Northwick Park Hospital, Research and Development, North West London Hospitals NHS Trust, Watford Road, Harrow, HA13UJ, Middlesex, United Kingdom

    Alan Warnes

Rights and permissions

Reprints and permissions

Copyright information

© 2010 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Ellis, J.S., Curran, M.D. (2010). Simultaneous Molecular Detection and Confirmation of Influenza AH5, with Internal Control. In: Stephenson, J., Warnes, A. (eds) Diagnostic Virology Protocols. Methods in Molecular Biology, vol 665. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-817-1_10

Download citation

  • DOI: https://doi.org/10.1007/978-1-60761-817-1_10

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-60761-816-4

  • Online ISBN: 978-1-60761-817-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation