Comparison of Competitive PCR and Positive Control-Based PCR

  • François Mallet
Part of the Methods in Molecular Medicine™ book series (MIMM, volume 26)


The exponential amplification of small amounts of nucleic acids makes polymerase chain reaction (PCR) not only powerful but also challenging as a quantitative method. Variations in nucleic acid preparation, thermal cyclic performance, the choice of the polymerase, and the amplification procedure can cause large differences in final product yield. To address the challenges of quantitative PCR, the procedure has been critically examined, leading to an understanding of the critical parameters involved in quantitative amplification. Accepted parameters can be summarized as a series of choices: external vs internal standard, exogenous vs endogenous standard, competitive vs noncompetitive amplification, and exponential vs plateau amplification (1, 2, 3).


Human Immunodeficiency Virus Acceptance Criterion Capture Probe Competitive Polymerase Chain Reaction Microtiter Plate Format 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. 1.
    Clementi, M., Menzo, S., Bagnarelli, P., Manzin, A., Valenza, A., and Varaldo, P. E. (1993) Quantitative PCR and RT-PCR in virology. PCR Methods Applic. 2, 191–196.Google Scholar
  2. 2.
    Ferre, F. (1992) Quantitative or semi-quantitative PCR: reality versus myth. PCR Methods Applic. 2, 1–9.Google Scholar
  3. 3.
    Sninsky, J. J. and Kwok, S. (1993) The application of quantitative polymerase chain reaction to therapeutic monitoring. AIDS 7(Suppl.), S29–S34.CrossRefPubMedGoogle Scholar
  4. 4.
    Simmonds, P., Balfe, P., Peutherer, J. F., Ludlam, C. A., Bishop, J. O., and Leigh Brown, A. J. (1990) Human immunodeficiency virus-infected individuals contain provirus in small numbers of peripheral mononuclear cells and at low copy numbers. J. Virol. 64, 864–872.Google Scholar
  5. 5.
    Dickover, R. E., Donovan, R. M., Goldstein, E., Dandekar, S., Bush, C. E., and Carlson, J. R. (1990) Quantitation of human immunodeficiency virus DNA by using the polymerase chain reaction. J. Clin. Microbiol. 28(9), 2130–2133.PubMedGoogle Scholar
  6. 6.
    Innocenti, P., Ottmann, M., Morand, P., Leclercq, P., and Seigneurin, J. M. (1992) HIV-1 in blood monocytes: frequency of detection of proviral DNA using PCR and comparison with the total CD4 count. AIDS Res. Hum. Retroviruses 8, 261–268.CrossRefPubMedGoogle Scholar
  7. 7.
    Pannetier, C., Delassus, S., Darche, S., Saucier, C., and Kourilsky, P. (1993) Quantitative titration of nucleic acids by enzymatic amplification reactions run to saturation. Nuc. Acids Res. 21, 577–583.CrossRefGoogle Scholar
  8. 8.
    Piatak, M., Jr., Luk, K.-C., Williams, B., and Lifson, J. D. (1993) Quantitative competitive polymerase chain reaction for accurate quantitation of HIV DNA and RNA species. BioTechniques 14, 70–81.PubMedGoogle Scholar
  9. 9.
    Siebert, P. D. and Larrick, J. W. (1993) PCR MIMICS: competitive DNA fragments for use as internal standards in quantitative PCR. BioTechniques 14, 244–249.PubMedGoogle Scholar
  10. 10.
    Nedelman, J., Heagerty, P., and Lawrence, C. (1992) Quantitative PCR: procedures and precisions. Bul. Math. Biol. 54, 477–502.Google Scholar
  11. 11.
    Kellog, D. E., Sninsky, J. J., and Kwok, S. (1990) Quantitation of HIV-1 proviral DNA relative to cellular DNA by polymerase chain reaction. Anal. Biochem. 189, 202–208.CrossRefGoogle Scholar
  12. 12.
    Lee, T. H., Sunzeri, F. J., Tobler, L. H., Williams, B. G., and Busch, M. P. (1991) Quantitative assessment of HIV-1 DNA load by coamplification of HIV-1 gag and HLA-DQ-a genes. AIDS 5(6), 683–691.CrossRefPubMedGoogle Scholar
  13. 13.
    Arnold, B. L., Itakura, K., and Rossi, J. J. (1992) PCR-based quantitation of low levels of HIV-1 DNA by using an external standard. Genet. Anal. Tech. Appli. 9, 113–116.Google Scholar
  14. 14.
    Cone, R. W., Hobson, A. C., and Huang, M. L. W. (1992) Coamplified positive control detects inhibition of polymerase chain reactions. J. Clin. Microbiol. 30(12), 3185–3189.PubMedGoogle Scholar
  15. 15.
    Dyster, L. M., Abbott, L., Bryz-Gornia, V., Poiesz, B. J., and Papsidero, L. D. (1994) Microplate-Based DNA hybridization assays for detection of human retroviral gene sequences. J. Clin. Microbiol. 32(2), 547–550.PubMedGoogle Scholar
  16. 16.
    Kohsaka, H., Taniguchi, A., Richman, D. D., and Carson, D. A. (1993) Microtiter format gene quantification by covalent capture of competitive PCR products: application to HIV-1 detection. Nuc. Acids Res. 21(15), 3469–3472.CrossRefGoogle Scholar
  17. 17.
    Mallet, F., Hebrard, C., Brand, D., Chapuis, E., Cros, P., Allibert, P., Besnier, J.-M., Barin, F., and Mandrand, B. (1993) Enzyme-linked oligosorbent assay for detection of polymerase chain reaction-amplified human immunodeficiency virus type 1. J. Clin. Microbiol. 31, 1444–1449.PubMedGoogle Scholar
  18. 18.
    Mallet, F., Hebrard, C., Livrozet, J. M., Lees, O., Tron, F., Touraine, J. L., and Mandrand, B. (1995) Quantitation of human immunodeficiency virus type 1 DNA by two PCR procedures coupled with enzyme-linked oligosorbent assay. J. Clin. Microbiol. 33(12), 3201–3208.PubMedGoogle Scholar
  19. 19.
    Yerly, S., Chamot, E., Hirschel, B., and Perrin, L. H. (1992) Quantitation of human immunodeficiency virus provirus and circulating virus: Relationship with immunologic parameters. J. Infect. Dis. 166, 269–276.PubMedGoogle Scholar
  20. 20.
    Aoki, S. A., Yarchoan, R., Thomas, R. V., Pluda, J. M., Marczyk, K., Broder, S., and Mitsuya, H. (1990) Quantitative analysis of HIV-1 proviral DNA in pheripheral blood mononuclear cells from patients with AIDS or ARC: decrease of proviral DNA content following treatment with 2′, 3′-dideoxyinosine (ddI). AIDS Res. Hum. Retroviruses 6(11), 1331–1339.PubMedGoogle Scholar
  21. 21.
    Mallet, F., Cros, P., and Mandrand, B. (1995) Enzyme-linked oligosorbent assay for detection of PCR-amplified HIV-1, in PCR: Protocol for Diagnosis of Human and Animals Virus Diseases (Becker, Y. and Darai, G., eds.), Springer-Verlag, Heidelberg, pp. 19–28.Google Scholar
  22. 22.
    Allibert, P., Cros, P., and Mandrand, B. (1992) Automated detection of nucleic acid sequences of HPV 16, 18 and 6/11. Eur. J. Biomed. Tech. 3(14), 152–155.Google Scholar
  23. 23.
    Mabilat, C., Desvarenne, S., Panteix, G., Machabert, N., Bernillon, M.H., Guardiola, G., and Cros, P. (1994) Routine automated identification of Mycobacterium tuberculosis complex isolates with a DNA probe. J. Clin. Microbiol. 32(11), 2702–2705.PubMedGoogle Scholar
  24. 24.
    Cros, P., Allibert, P., Mandrand, B., Tiercy, J.M., and Mach, B. (1992) Oligonucleotide genotyping of HLA polymorphism on microtitre plates. Lancet 340, 870–873.CrossRefPubMedGoogle Scholar
  25. 25.
    Clouse, K. A., Powell, D., Washington, I., Poli, G., Strebel, K., Farrar, W., Barstad, P., Kovacs, J., Fauci, A. S., and Folks, T. M. (1989) Monokine regulation of human immunodeficiency virus-1 expression in a chronically infected human T cell clone. J. Immunol. 142, 431–438.PubMedGoogle Scholar
  26. 26.
    Souazé, F., Ntodou-Thomé, A., Tran, C. Y., Rostène, W., and Forgez, P. (1996) Quantitative RT-PCR: Limits and Accuracy. BioTechniques 21, 280–285.PubMedGoogle Scholar
  27. 27.
    Coutlée, F., He, Y., Saint-Antoine, P., Olivier, C., and Kessous, A. (1995) Coamplification of HIV type 1 and b-globin gene DNA sequences in a nonisotopic polymerase chain reaction assay to control for amplification efficiency. AIDS Res. Hum. Retroviruses 11, 363–371.CrossRefPubMedGoogle Scholar
  28. 28.
    Chelly, J., Kaplan, J. C., Maire, P., Gautron, S., and Kahn, A. (1988) Transcription of the dystrophin gene in muscle and non-muscle tissues. Nature 333, 858–860.CrossRefPubMedGoogle Scholar
  29. 29.
    Yamamura, M., Uyemura, K., Deans, R. J., Weinberg, K., Rea, T. H., Bloom, B. R., and Modlin, R. L. (1991) Defining protective responses to pathogens: cytokine profiles in leprosy lesions. Science 254, 277–279.CrossRefPubMedGoogle Scholar
  30. 30.
    Alard, P., Lantz, O., Sebagh, M., Calvo, C. F., Weill, D., Chavanel, G., Senik, A., and Charpentier, B. (1993) A versatile ELISA-PCR assay for mRNA quantitation from a few cells. BioTechniques 15, 730–737.PubMedGoogle Scholar
  31. 31.
    Mallet, F., Oriol, G., and Mandrand, B. (1998) Characterization of RNA using Continuous RT-PCR coupled with ELOSA in Methods in Molecular Biology, Vol. 86: RNA Isolation and Characterization Protocols (Rapley, R. and Manning, D. L., eds.), Humana Press, Totowa, NJ, pp. 161–172.CrossRefGoogle Scholar
  32. 32.
    Morrison, F. and Gannon, F. (1994) The impact of the PCR plateau phase on quantitative PCR. Biochim. Biophys. Acta 1219, 493–498.PubMedGoogle Scholar
  33. 33.
    Siebert, P. D. and Larrick, J. W. (1992) Competitive PCR. Nature 359, 557–558.CrossRefPubMedGoogle Scholar
  34. 34.
    Mallet, F., Oriol, G., Mary, C., Verrier, B., and Mandrand, B. (1995) RT-PCR using AMV-RT and Taq DNA polymerase: characterization and comparaison to uncoupled procedures. BioTechniques 18, 678–687.PubMedGoogle Scholar
  35. 35.
    Mallet, F. (1996) Continuous RT-PCR using AMV-RT and Taq in PCR: Essential Techniques (Burke, J. F., ed.), BIOS Scientific Publishers Ltd., Oxford, pp. 82–85.Google Scholar
  36. 36.
    Diviacco, S., Norio, P., Zentilin, L., Menzo, S., Clementi, M., Biamonti, G., Riva, S., Falaschi, A., and Giacca, M. (1992) A novel procedure for quantitative polymerase chain reaction by coamplification of competitive templates. Gene 122, 3013–3020.CrossRefGoogle Scholar
  37. 37.
    Sholler, R., Gervasi, G., Avigdor, R., and Castanier, M. (1987) Comparaison de deux méthodes de dosage in Colloque sur les actualités en immunoanalyse, CORATA, Université de Bordeaux II, Bergeret, Bordeaux, 2–4 Dec 1987, pp. 225–233.Google Scholar
  38. 38.
    Natarajan, V., Plishka, R. J., Scott, E. W., Lane, H. C., and Salzman, N. P. (1994) An internally controlled virion PCR for the measurement of HIV-1 RNA in plasma. PCR Methods Appl. 3, 346–350.PubMedGoogle Scholar
  39. 39.
    Van Gemen, B., Van Beuningen, R., Nabbe, A., Van Strijp, D., Jurriaans, S., Lens, P., and Kievits, T. (1994) A one-tube quantitative HIV-1 RNA NASBA nucleic acid amplification assay using electrochemiluminescent (ECL) labelled probes. J. Virol. Methods 49, 157–168.CrossRefPubMedGoogle Scholar
  40. 40.
    Vener, T., Axelsson, M., Albert, J., Uhlén, M., and Lundeberg, J. (1996) Quantitation of HIV-1 using multiple competitiors in a single-tube assay. BioTechniques 21, 248–255.PubMedGoogle Scholar
  41. 41.
    Zimmermann, K., Schögl, D., Plaimauer, B., and Mannhalter, J. W. (1996) Quantitative multiple competitive PCR of HIV-1 DNA in a single reaction tube. BioTechniques 21, 480–484.PubMedGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 1999

Authors and Affiliations

  • François Mallet
    • 1
  1. 1.Unité Mixte de Recherche, CNRS-BioMérieuxEcole Normale Supérieure de LyonFrance

Personalised recommendations