General recommendations for quality assurance

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General recommendations for quality assurance in PCR analysis

Laboratory requirements

  • Need of 3 independent areas or rooms:
    Area 1: Handling of reagents and preparation of amplification mixture ("Clean-room")
    Area 2: Sample work-up and preparation of nucleic acid, setting up PCR reactions ("Pre-amplification area")
    Area 3: Amplification and detection of nucleic acids ("Post-PCR analysis")

General rules

  • Unidirectional workflow from area 1 and 2 to 3 ("clean" work and pre-amplification first).
  • All rooms should have assigned equipment and reagents for the respective areas (e.g. refrigerator, vortex mixer, centrifuges, tube racks, pipette, filter tips holders etc.).
  • Reduction of the risk of contamination, decontamination of working areas; use of gloves, plugged pipette tips etc.
  • Use of DNase-free materials.
  • Set up the PCR under a fume hood or laminar air flow box.
  • Change the gloves between the working areas.

Template preparation

  • Blank extraction of DNA as negative control.
  • Estimation of nucleic acid concentration and purity (ratio OD260/280 nm).
  • Extraction controls (recovery of template-positive specimens).

Amplification controls

  • Blank extraction and water as negative controls.
  • Inclusion of specimens (pathogen DNA or infected tissue sample) as positive controls.
  • Standardisation of DNA amount needed for positive amplifications (100 ng for the control gene and 400 ng for the detection of mycobacteria, respectively, as determined by spectrophotometry).
  • PCR of human control genes as control for quality of preparation and to exclude endogenous PCR inhibitors (small fragments, preferably the ß-globin primer set described).