Commentary

C2.7.1
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.
C2.7.2
Laboratories undertaking nucleic acid detection from eukaryotic cells are generally 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.
C2.7.3
Because of the presence of high levels of cultured microorganisms in microbiology laboratories and the potential number of organisms present in aerosols, three physically separate and contained areas with known airconditioned or ventilated airflows are required in a laboratory undertaking PCR-based diagnosis of microorganisms, in order to reduce the risk of cross-contamination or carry-over contamination.

Definitions

C2.7.4
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

C2.7.5
Standards S2.7.1 to S2.7.8 are the minimum standards for a PCR laboratory working with microorganisms and using non-nested PCR techniques. Standards S2.7.9 to S2.7.13 provide additional standards and requirements for laboratories performing nested PCR techniques.

Standard

S2.7.1
Three physically separate areas are required to reduce the risk of cross-contamination or carry-over contamination.
a) A dedicated contained area shall be provided for the extraction of nucleic acid from samples. This area shall be physically separate from any region of the laboratory in which microorganisms are cultured and analysed, and shall be in a separate, contained area from the reagent and postamplification areas of the PCR facility. The normal airflow into this area shall not come from the amplification/detection area or from areas of the laboratory where the target pathogens are cultured. The exhaust air from this area shall not flow into the reagent preparation or the amplification/detection areas.
b) There shall be a dedicated, separate, clean and contained area for the preparation of reagents (including dispensing of the
master mix) that is physically separate from all other areas of the laboratory. The air to this area must not come from the sample preparation or amplification/detection areas, or from any other area where potential target organisms may be present.

c) There shall be a dedicated, separate and contained area for amplification and product detection that is physically separated from any area of the laboratory in which microorganisms are cultured, analysed or stored. The normal airflow pattern from this area shall not pass into the sample preparation or reagent preparation areas.
S2.7.2
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.
S2.7.3
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 by not allowing direct flow of air from this area back to either the reagent preparation or sample preparation area.
S2.7.4
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.
S2.7.5
Equipment from other areas shall not be taken into the reagent preparation area.
S2.7.6
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.
S2.7.7
Where equipment (such as tube racks) is returned against the flow, it shall first be decontaminated in 2–10% hypochlorite for four hours before being moved from the postamplification area.
S2.7.8
Laboratory coats and gloves shall be changed before staff move to or from each area.

Guideline

G2.7.1
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.
G2.7.2
Ideally, the airflow patterns in both the sample preparation area and the reagent preparation area should achieve a slight positive pressure so that air flows out of these areas.
G2.7.3
Normal airflow patterns to the product analysis area should achieve a slight negative pressure so that air flows into the area.
G2.7.4
Aerosol-resistant pipette tips or positive displacement pipettes are strongly recommended to minimise contamination.
G2.7.5
Work surfaces should be regularly decontaminated by wiping with 2–10% hypochlorite or another similar agent. Instrumentation such as microfuges, heating blocks and waterbaths should be cleaned regularly with 2–10% hypochlorite.
G2.7.6
Instruments capable of producing aerosols (eg vortex mixers, PCR machines and microfuges) are to be placed at as great a distance from preparation areas as possible.
G2.7.7
While not specifically dealt with in these standards and guidelines, the use of robotic equipment should adhere to the principles outlined above.

Additional standards and requirements for nested PCR Commentary

C2.7.6
The standards and requirements below apply, in addition to those outlined above, when using nested PCR techniques.

Standard

S2.7.9
Reagent preparation, specimen processing and amplification/product detection shall be carried out in three separate contained areas. Where material from the first-round PCR is aliquoted in an open environment, there shall be a fourth area.
S2.7.10
Normal airflow patterns in the laboratory shall direct air out of the reagent preparation area in order to avoid contamination of the reagents. If this cannot be achieved, reagent preparation shall be carried out in a positive-pressure hood or a Class II BSC within the contained area.
S2.7.11
Normal airflow patterns in the laboratory shall direct air out of the sample preparation area in order to avoid contamination of the samples. This is not necessary if the sample preparation area is distant from the other nucleic acid detection processing areas and has no airflow connections with the other areas.
S2.7.12
Normal airflow patterns in the laboratory must not direct air from the amplification/detection area into the reagent preparation or sample preparation areas.
S2.7.13
All manipulations of samples or of materials liable to contain amplified or unamplified nucleic acids shall be carried out in a Class I or II BSC with a HEPA filter on the exhaust.

Commentary

C2.7.7
Because of the nature of the technique, nested PCR requires the most stringent guidelines. The most important aspects are:
a) adequately trained staff
b) use of aerosol-resistant pipette tips
c) constant vigilance against methodological causes of contamination.

Guideline

G2.7.8
Where a laboratory carries out large numbers of nested PCR reactions, which increases the potential for contamination, or where there are recurring or uncontrolled contamination problems, the following measures should be implemented:
a) The air conditioning/ventilation services to the reagent preparation and specimen processing areas should be at positive pressure in relation to adjoining areas, or they should be separated from adjoining areas by an anteroom that is at negative pressure to both its adjoining areas.
b) The air conditioning/ventilation services to the amplification and product detection areas should achieve a negative pressure in relation to adjoining areas, or should be separated from adjoining areas by an anteroom that is at negative pressure to both its adjoining areas.
c) All manipulations of material liable to contain amplified nucleic acids should be done in a dedicated, externally vented Class I or Class II BSC.
d) Ceiling-mounted ultraviolet light fixtures that are operated for 20–30 minutes after hours should be used, as these will assist in reducing environmental contamination.