title: Amp-PCR: combining a random unbiased Phi29-amplification with a specific real-time PCR, performed in one tube to increase PCR sensitivity.
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).
authors.affiliation: Virus Research and Development, Department of Virology, Statens Serum Institut, Copenhagen, Denmark. lao@ssi.dk
authors.forename: Lena
authors.initials: L
authors.lastname: Erlandsson
authors.forename: Lars Peter
authors.initials: LP
authors.lastname: Nielsen
authors.forename: Anders
authors.initials: A
authors.lastname: Fomsgaard
mesh_terms: D017103:Bacillus Phages / Q000235:genetics*; D001483:Base Sequence; D004267:DNA Viruses / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics*; D018450:Disease Progression; D005821:Genetic Techniques; D016679:Genome, Viral; D018259:Herpesvirus 1, Human / Q000235:genetics; D052162:Human papillomavirus 16 / Q000235:genetics; D007577:JC Virus / Q000235:genetics; D008969:Molecular Sequence Data; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012306:Risk; D012680:Sensitivity and Specificity
chemical_list: D004279:DNA, Viral