Skip to main content
SpringerLink
Log in
Book cover

Animal Influenza Virus pp 105–118Cite as

Avian Influenza Virus Detection and Quantitation by Real-Time RT-PCR

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1161))

Abstract

Real-time RT-PCR (rRT-PCR) has been used for avian influenza virus (AIV) detection since the early 2000s for routine surveillance, outbreak assessment and for research. Some of the advantages of rRT-PCR are: high sensitivity, high specificity, rapid time-to-result, scalability, cost, and its inherently quantitative nature. Furthermore, rRT-PCR can be used with numerous sample types, is less expensive than virus isolation in chicken embryos, and since infectious virus is inactivated early during processing, biosafety and bio-security are also easier to maintain. However, the high genetic variability of AIV may decrease sensitivity and increases the chances of a false negative result. This chapter provides an overview of the USDA validated rRT-PCR procedure for the detection of type A influenza.

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   109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   139.00
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. Spackman E, Senne DA, Myers TJ, Bulaga LL, Garber LP, Perdue ML, Lohman K, Daum LT, Suarez DL (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

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  2. Spackman E, Suarez DL (2005) Use of a novel virus inactivation method for a multicenter avian influenza real-time reverse transcriptase-polymerase chain reaction proficiency study. J Vet Diagn Invest 17:76–80

    Article  PubMed  Google Scholar 

  3. Akey BL (2003) Low-pathogenicity H7N2 avian influenza outbreak in virgnia during 2002. Avian Dis 47:1099–1103

    Article  CAS  PubMed  Google Scholar 

  4. Cattoli G, Drago A, Maniero S, Toffan A, Bertoli E, Fassina S, Terregino C, Robbi C, Vicenzoni G, Capua I (2004) Comparison of three rapid detection systems for type A influenza virus on tracheal swabs of experimentally and naturally infected birds. Avian Pathol 33:432–437

    Article  CAS  PubMed  Google Scholar 

  5. Slomka MJ, Densham AL, Coward VJ, Essen S, Brookes SM, Irvine RM, Spackman E, Ridgeon J, Gardner R, Hanna A, Suarez DL, Brown IH (2010) Real time reverse transcription (RRT)-polymerase chain reaction (PCR) methods for detection of pandemic (H1N1) 2009 influenza virus and European swine influenza A virus infections in pigs. Influenza Other Respi Viruses 4:277–293

    Article  CAS  Google Scholar 

  6. Lee CW, Suarez DL (2004) Application of real-time RT-PCR for the quantitation and competitive replication study of H5 and H7 subtype avian influenza virus. J Virol Methods 119:151–158

    Article  CAS  PubMed  Google Scholar 

  7. Holland PM, Abramson RD, Watson R, Gelfand DH (1991) Detection of specific polymerase chain reaction product by utilizing the 5′–3′ exonuclease activity of Thermus aquaticus DNA polymerase. Proc Natl Acad Sci U S A 88:7276–7280

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  8. Wittwer CT, Herrmann MG, Moss AA, Rasmussen RP (1997) Continuous fluorescence monitoring of rapid cycle DNA amplification. Biotechniques 22(130–1):134–138

    Google Scholar 

  9. Kim LM, Afonso CL, Suarez DL (2006) Effect of probe-site mismatches on detection of virulent newcastle disease viruses using a fusion-gene real-time reverse transcription polymerase chain reaction test. J Vet Diagn Invest 18:519–528

    Article  PubMed  Google Scholar 

  10. Das A, Spackman E, Senne D, Pedersen J, Suarez DL (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  CAS  PubMed Central  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Southeast Poultry Research Laboratory, US Department of Agriculture, ARS, 934 College Station Rd., Athens, GA, 30605, USA

    Erica Spackman

Authors
  1. Erica Spackman
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Erica Spackman .

Editor information

Editors and Affiliations

  1. Exotic and Emerging Avian Viral Diseases Unit, Southeast Poultry Research Laboratory, US Department of Agriculture, Agricultural Research Service, Athens, Georgia, USA

    Erica Spackman

Rights and permissions

Reprints and permissions

Copyright information

© 2014 Springer Science+Business Media New York

About this protocol

Cite this protocol

Spackman, E. (2014). Avian Influenza Virus Detection and Quantitation by Real-Time RT-PCR. In: Spackman, E. (eds) Animal Influenza Virus. Methods in Molecular Biology, vol 1161. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0758-8_10

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-0758-8_10

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-0757-1

  • Online ISBN: 978-1-4939-0758-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation