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Competitive PCR–ELISA protocols for the quantitative and the standardized detection of viral genomes

Nature Protocols volume 2, pages 2511–2519 (2007)Cite this article

Abstract

Competitive PCR–ELISA combines competitive PCR with an ELISA to allow quantitative detection of PCR products. It is based on the inclusion of an internal standard competitor molecule that is designed to differ from the target by a short sequence of nucleotides. Once such a competitor molecule has been designed and constructed, target and competitor sequences are concurrently PCR-amplified, before hybridization to two different specific probes and determination of their respective OD values by ELISA. The target can be quantified in relation to a titration curve of different dilutions of the competitor. The competitor can alternatively be used at a unique optimal concentration to allow for standardized detection of the target sequence. PCR–ELISA can be performed in 1 d in laboratories without access to a real-time PCR thermocycler. This technique is applied in diagnostics to monitor the course of infections and drug efficacy. Competitive PCR–ELISA protocols for the quantitative and for the standardized detection of parvovirus B19 are detailed here as an example of the technique.

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Figure 1: Scheme of competitive PCR–ELISA.
Figure 2: Scheme of the construction of a competitor molecule by recombinant PCR.
Figure 3: Example of competitive titration for two serum samples.

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Authors and Affiliations

  1. Department of Clinical and Experimental Medicine, Division of Microbiology, University of Bologna, Via Massarenti 9, Bologna, 40138, Italy

    Monica Musiani, Giorgio Gallinella, Simona Venturoli & Marialuisa Zerbini

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  1. Monica Musiani
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  2. Giorgio Gallinella
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  3. Simona Venturoli
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  4. Marialuisa Zerbini
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Correspondence to Monica Musiani.

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Musiani, M., Gallinella, G., Venturoli, S. et al. Competitive PCR–ELISA protocols for the quantitative and the standardized detection of viral genomes. Nat Protoc 2, 2511–2519 (2007). https://doi.org/10.1038/nprot.2007.312

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