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Amp-PCR: combining a random unbiased Phi29-amplification with a specific real-time PCR, performed in one tube to increase PCR sensitivity
- Source :
- PLoS ONE, Vol 5, Iss 12, p e15719 (2010), PLoS ONE
- Publication Year :
- 2010
- Publisher :
- Public Library of Science (PLoS), 2010.
-
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).
- Subjects :
- Bacterial Diseases
Viral Diseases
DNA polymerase
Applied Microbiology
Gene Identification and Analysis
Gene Expression
lcsh:Medicine
Bacillus Phages
Herpesvirus 1, Human
Genome
Biochemistry
law.invention
DNA amplification
Molecular cell biology
law
lcsh:Science
Polymerase
Polymerase chain reaction
Human papillomavirus 16
Multidisciplinary
biology
Reverse Transcriptase Polymerase Chain Reaction
JC Virus
Clinical Laboratory Sciences
Nucleic acids
Real-time polymerase chain reaction
Infectious Diseases
Genetic Techniques
Medical Microbiology
Disease Progression
Medicine
RNA extraction
Research Article
Risk
Infectious Disease Control
Molecular Sequence Data
Genome, Viral
Microbiology
Sensitivity and Specificity
Molecular Genetics
Diagnostic Medicine
Virology
Genetics
Sensitivity (control systems)
Biology
Detection limit
Base Sequence
lcsh:R
DNA Viruses
DNA
Molecular biology
Viral Disease Diagnosis
DNA, Viral
biology.protein
Clinical Immunology
lcsh:Q
Subjects
Details
- Language :
- English
- ISSN :
- 19326203
- Volume :
- 5
- Issue :
- 12
- Database :
- OpenAIRE
- Journal :
- PLoS ONE
- Accession number :
- edsair.doi.dedup.....0c8e624aabaa0962bc1a770d52bd128f