Das Bild zeigt rechts ein Trichterglas mit blauer Flüssigkeit und Glasstab zum Umrühren. Daneben steht ein Reagenzglasständer mit Reagenzgläsern, die ebenfalls blaue Flüssigkeit enthalten. Im Hintergrund ist ein Forscher zu sehen, der in der rechten Hand eine Pipette hält.

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).