Skip to main content
Springer Nature Link
Log in

Apta-PCR

  • Protocol
Nucleic Acid Aptamers

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1380))

Abstract

Real-time Apta-PCR is a methodology that can be used for a wide variety of applications ranging from food quality control to clinical diagnostics. This method takes advantage of the combination of the sensitivity of nucleic acid amplification with the selectivity of aptamers. Ultra-low detection of target analyte can potentially be achieved, or, improved detection limits can be achieved with aptamers of low-medium affinity. Herein, we describe a generic methodology coined real-time Apta-PCR, using a model target (β-conglutin) and a competitive format, which can be adapted for the detection of any target which an aptamer has been selected for.

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

Access this chapter

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Sano S, Smith CL, Cantor CR (1992) Immuno-PCR: very sensitive antigen detection by means of specific antibody-DNA conjugates. Science 258:120–122

    Article  CAS  PubMed  Google Scholar 

  2. Nam JM, Stoeva SI, Mirkin CA (2004) Bio-bar-code-based DNA detection with PCR-like sensitivity. J Am Chem Soc 126:5932–5933

    Article  CAS  PubMed  Google Scholar 

  3. Mweene AS, Ito T, Okazaki K, Ono E, Shimizu Y, Kida H (1996) Development of immuno-PCR for diagnosis of bovine herpesvirus 1 infection. J Clin Microbiol 34:748–750

    PubMed Central  CAS  PubMed  Google Scholar 

  4. Niemeyer CM, Adler M, Wacker R (2005) Immuno-PCR: high sensitivity detection of proteins by nucleic acid amplification. Trends Biotechnol 23:208–216

    Article  CAS  PubMed  Google Scholar 

  5. Adler M (2005) Immuno-PCR as a clinical laboratory tool. Adv Clin Chem 39:239–292

    Article  PubMed  Google Scholar 

  6. McKie A, Samuel D, Cohen B, Saunders NA (2002) Development of a quantitative immuno-PCR assay and its use to detect mumps-specific IgG in serum. J Immunol Methods 261:167–175

    Article  CAS  PubMed  Google Scholar 

  7. Niemeyer CM, Adler M, Pignataro B, Lenhert S, Gao S, Chi L et al (1999) Self-assembly of DNA-streptavidin nanostructures and their use as reagents in immuno-PCR. Nucleic Acids Res 27:4553–4561

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  8. Fredriksson S, Gullberg M, Jarvius J, Olsson C, Pietras K, Gústafsdóttir SM et al (2002) Protein detection using proximity-dependent DNA ligation assays. Nat Biotechnol 20:473–477

    Article  CAS  PubMed  Google Scholar 

  9. Gustafsdottir SM, Schallmeiner E, Fredriksson S, Gullberg M, Söderberg O, Jarvius M et al (2005) Proximity ligation assays for sensitive and specific protein analyses. Anal Biochem 345:2–9

    Article  CAS  PubMed  Google Scholar 

  10. Gustafsdottir SM, Nordengrahn A, Fredriksson S, Wallgren P, Rivera E, Schallmeiner E et al (2006) Detection of individual microbial pathogens by proximity ligation. Clin Chem 52:1152–1160

    Article  CAS  PubMed  Google Scholar 

  11. Yang L, Fung CW, Eun JC, Ellington AD (2007) Real-time rolling circle amplification for protein detection. Anal Chem 79:3320–3329

    Article  CAS  PubMed  Google Scholar 

  12. Yang L, Ellington AD (2008) Real-time PCR detection of protein analytes with conformation-switching aptamers. Anal Biochem 380:164–173

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  13. Wang XL, Li F, Su YH, Sun X, Li XB, Schluesener HJ et al (2004) Ultrasensitive detection of protein using an aptamer-based exonuclease protection assay. Anal Chem 76:5605–5610

    Article  CAS  PubMed  Google Scholar 

  14. Zhang H, Wang Z, Li XF, Le XC (2006) Ultrasensitive detection of proteins by amplification of affinity aptamers. Angew Chem Int Ed Engl 45:1576–1580

    Article  CAS  PubMed  Google Scholar 

  15. Fischer NO, Tarasow TM, Tok JBH (2008) Protein detection via direct enzymatic amplification of short DNA aptamers. Anal Biochem 373:121–128

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  16. Pinto A, Bermudo Redondo MC, Ozalp VC, O’Sullivan CK (2009) Real-time apta-PCR for 20 000-fold improvement in detection limit. Mol BioSyst 5(5):548–553

    Article  CAS  PubMed  Google Scholar 

  17. Pinto A, Lennarz S, Rodrigues-Correia A, Heckel A, O’Sullivan CK, Mayer G (2012) Functional detection of proteins by caged aptamers. ACS Chem Biol 7:359–365

    Article  Google Scholar 

  18. Svobodova M, Miairal T, Nadal P, Bermudo Redondo MC, O’Sullivan CK (2014) Ultrasensitive aptamer based detection of β-conglutin food allergen. Food Chem 165:419–423

    Article  CAS  PubMed  Google Scholar 

  19. Pinto A, Nadal P, Henry O, Bermudo Redondo MC, Svobodova M, O’Sullivan CK (2014) Label-free detection of gliadin food allergen mediated by real-time apta PCR. Anal Bioanal Chem 406:515–524

    Article  CAS  PubMed  Google Scholar 

  20. Svobodova M, Bunka D, Nadal P, Stockley PG, O’Sullivan CK (2013) Selection of 2’F-modified RNA aptamers against prostate-specific antigen and their evaluation for diagnostic and therapeutic applications. Anal Bioanal Chem 405:9149–9157

    Article  CAS  PubMed  Google Scholar 

  21. Nadal P, Canela N, Katakis Y, O’Sullivan CK (2011) Extraction, isolation, and characterization of globulin proteins from Lupinus albus. J Agric Food Chem 59:2752–2758

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Chemical Engineering, Universitat Rovira I Virgili, Avinguda Päısos Catalans 26, Tarragona, 43007, Spain

    Alessandro Pinto, Pedro Nadal Polo, Miriam Jauest Rubio, Marketa Svobodova, Teresa Mairal Lerga & Ciara K. O’Sullivan

  2. Department of Bioengineering, Rice University, 6500 Main Street, 77030, Houston, TX, USA

    Alessandro Pinto

  3. Center for Omic Sciences (COS), Universitat Rovira I Virgili, Avinguda Universitat n° 1, 43204, Reus, Spain

    Pedro Nadal Polo

  4. Institució Catalana de Recerca I Estudis Avançats, Passeig Llúıs Companys 23, 08010, Barcelona, Spain

    Ciara K. O’Sullivan

Authors
  1. Alessandro Pinto
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Pedro Nadal Polo
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Miriam Jauest Rubio
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Marketa Svobodova
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Teresa Mairal Lerga
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ciara K. O’Sullivan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ciara K. O’Sullivan .

Editor information

Editors and Affiliations

  1. University of Bonn, Bonn, Germany

    Günter Mayer

Rights and permissions

Reprints and permissions

Copyright information

© 2016 Springer Science+Business Media New York

About this protocol

Cite this protocol

Pinto, A., Polo, P.N., Rubio, M.J., Svobodova, M., Lerga, T.M., O’Sullivan, C.K. (2016). Apta-PCR. In: Mayer, G. (eds) Nucleic Acid Aptamers. Methods in Molecular Biology, vol 1380. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3197-2_14

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-3197-2_14

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3196-5

  • Online ISBN: 978-1-4939-3197-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics