Commentary and definitions

    C1.9.1
    Laboratories undertaking nucleic acid amplification should be configured to minimise the risk of contamination of samples and reagents by other samples in the laboratory or by amplified material.
    C1.9.2
    Laboratories undertaking nucleic acid detection from eukaryotic cells are viewed as being at significantly lower risk of contamination than those laboratories undertaking nucleic acid detection of microorganisms. In microbiology laboratories, microorganisms are present in samples in large numbers or are cultured at high concentrations. There is also greater potential for aerosol contamination because of the small size of microorganisms compared to eukaryotic cells.
    C1.9.3
    The wording of the following sections is intended to allow flexibility of laboratory layout without compromising the guiding principle that laboratories undertaking nucleic acid amplification should be configured to minimise the risk of contamination of samples and reagents by amplified material or other samples in the laboratory.
    C1.9.4
    The term ‘separate areas’ is used in the following sections to mean laboratory spaces that are used for nucleic acid based testing and are separated from other laboratory spaces by walls, distance or strict laboratory practice, or by performance of the test within the working space of an instrument, as dictated by the methods and technology available in the laboratory. The term ‘contained area’ means a laboratory space that can be isolated from the rest of the laboratory either by walls and doors or within the working space of an instrument.

    Minimum standards for a nucleic acid amplification facility

    Commentary

    C1.9.5
    The standards listed below are the minimum standards for a PCR laboratory using exclusively eukaryotic cells, tissues or isolated DNA.

    Standard

    S1.9.1
    Three physically separate areas are required in order to reduce the risk of cross-contamination or carry-over contamination.
    S1.9.2
    The three areas required in each nucleic acid amplification laboratory are:
    a) a separate area for the extraction of nucleic acids from samples and for the addition of sample DNA to tubes containing master mix before PCR amplification
    b) a dedicated clean area for the preparation of reagents (including dispensing of the master mix)
    c) a dedicated, contained area for amplification and product detection.
    S1.9.3
    The normal airflow pattern of each of the regions shall be known and the layout of the laboratory areas designed to minimise the potential for aerosol cross-contamination.
    S1.9.4
    Where the areas for preparation of reagents and sample preparation are located within a single room, wide separation of these activities shall be maintained and appropriate procedures and controls shall be implemented to detect contamination.
    S1.9.5
    Post-PCR analysis shall not be incorporated into areas where reagent preparation or sample preparation occurs. The post-PCR area shall be positioned so as to minimise the possibility of cross-contamination of preamplification areas. Generally, this can be achieved by positioning the post-PCR area at an appropriate distance from the preamplification area.
    S1.9.6
    Reagents and equipment shall be limited to the appropriate sections. In particular, no nucleic acid samples shall be taken into the reagent preparation area. Samples shall be stored separately from reagents.
    S1.9.7
    Equipment from other areas shall not be taken into the reagent preparation area.
    S1.9.8
    The movement of specimens and equipment shall be unidirectional; that is, from preamplification to postamplification areas. Only sealed PCR amplification tubes and tube racks shall be carried between the preamplification area and the postamplification area.
    S1.9.9
    Where equipment (such as tube racks) is returned against the flow, it shall first be decontaminated in 2–10% hypochlorite or another noncorroding decontaminating agent for four hours before being moved from the postamplification area.
    S1.9.10
    Laboratory coats and gloves shall be changed before staff move to or from each area.

    Guideline

    G1.9.1
    Sample processing and reagent preparation should occur in different rooms. Where the areas for preparation of reagents and sample preparation cannot be separated, the provision of a positive-pressure hood or Class II biological safety cabinet (BSC) for reagent preparation and a Class II BSC for specimen preparation is strongly recommended. The air outflow from the sample preparation BSC must have a high- efficiency particle arrest (HEPA) filter and must be directed away from the reagent preparation area.
    G1.9.2
    Equipment designated for a particular area should be marked (eg by colour) to clearly indicate to which area they belong.
    G1.9.3
    Aerosol-resistant pipette tips or positive displacement pipettes are strongly recommended to minimise contamination, and should be used routinely.
    G1.9.4
    Work surfaces should be regularly decontaminated by wiping with 2–10% hypochlorite or another similar decontaminating agent. Instrumentation such as microfuges, heating blocks and waterbaths should be cleaned regularly with 2–10% hypochlorite or another noncorroding decontaminating agent.
    G1.9.5
    Instruments capable of producing aerosols (eg vortex mixers, PCR machines and microfuges) should be placed at as great a distance from preparation areas as possible.
    G1.9.6
    While not specifically dealt with in these standards and guidelines, the use of robotic equipment in the laboratory should adhere to the principles outlined above.