General recommendations for quality assurance
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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).