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PLoS One. 2010 Dec 31;5(12):e15719. doi: 10.1371/journal.pone.0015719.

Amp-PCR: combining a random unbiased Phi29-amplification with a specific real-time PCR, performed in one tube to increase PCR sensitivity.

Author information

  • 1Virus Research and Development, Department of Virology, Statens Serum Institut, Copenhagen, Denmark. lao@ssi.dk

Abstract

In clinical situations where a diagnostic real-time PCR assay is not sensitive enough, leading to low or falsely negative results, or where detection earlier in a disease progression would benefit the patient, an unbiased pre-amplification prior to the real-time PCR could be beneficial. In Amp-PCR, an unbiased random Phi29 pre-amplification is combined with a specific real-time PCR reaction. The two reactions are separated physically by a wax-layer (AmpliWax®) and are run in sequel in the same sealed tube. Amp-PCR can increase the specific PCR signal at least 100×10(6)-fold and make it possible to detect positive samples normally under the detection limit of the specific real-time PCR. The risk of contamination is eliminated and Amp-PCR could replace nested-PCR in situations where increased sensitivity is needed e.g. in routine PCR diagnostic analysis. We show Amp-PCR to work on clinical samples containing circular and linear viral dsDNA genomes, but can work well on DNA of any origin, both from non-cellular (virus) and cellular sources (bacteria, archae, eukaryotes).

PMID:
21209824
[PubMed - indexed for MEDLINE]
PMCID:
PMC3013126
Free PMC Article

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