Pre-PCR processing of samples.

Peter Rådström, Rickard Knutsson, Petra Wolffs, Maria Dahlenborg, Charlotta Löfström

Research output: Chapter in Book/Report/Conference proceedingBook chapterResearchpeer-review

Abstract

Diagnostic polymerase chain reaction (PCR) is an extremely powerful rapid method for diagnosis of microbial infections and genetic diseases, as well as for detecting microorganisms in environmental and food samples. However, the usefulness of diagnostic PCR is limited, in part, by the presence of inhibitory substances in complex biological samples, which reduce or even block the amplification capacity of PCR in comparison with pure solutions of nucleic acids (1). Thus, the presence of substances interfering with amplification will directly influence the performance of diagnostic PCR and, in particular, the assay’s sensitivity of detection. Some inhibitors may dramatically interfere with amplification, even at very small amounts. For example, PCR mixtures containing the widely used Taq DNA polymerase are totally inhibited in the presence of 0.004% (v/v) human blood (2). Consequently, sample processing prior to PCR is required to enable DNA amplification of the target nucleic acids in the presence of even traces of PCR-inhibitory substances. To improve diagnostic PCR for routine analysis purposes, the processing of the sample is crucial for the robustness and the overall performance of the method. In general, diagnostic PCR may be divided into four steps: (i) sampling; (ii) sample preparation; (iii) nucleic acid amplification; and (iv) detection of PCR products .
Original languageEnglish
Title of host publicationPCR Detection of Microbial Pathogens
Number of pages20
PublisherSpringer
Publication date2003
Pages31-50
Publication statusPublished - 2003
Externally publishedYes
SeriesMethods in Molecular Biology
Volume216
ISSN1064-3745

Keywords

  • bacterial DNA
  • DNA directed DNA polymerase
  • virus DNA
  • culture medium
  • density gradient centrifugation
  • human
  • immunomagnetic separation
  • isolation and purification
  • laboratory diagnosis
  • metabolism
  • methodology
  • polymerase chain reaction
  • review
  • Centrifugation, Density Gradient
  • Culture Media
  • DNA, Bacterial
  • DNA, Viral
  • DNA-Directed DNA Polymerase
  • Humans
  • Immunomagnetic Separation
  • Polymerase Chain Reaction
  • Specimen Handling

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