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PCR laboratories

PCR laboratory set up is currently regulated by E1873-06 Standard Guide for Detection of Nucleic Acid Sequences by the Polymerase Chain Reaction Technique (2006) and recommended by WHO’s Establishment of PCR Laboratory in Developing Countries (2011).

A PCR laboratory must be divided into three areas, according to the number of technological operations:
1. The first area should be a laboratory for nucleic acid preparation from the test material;
2. The second area should be the work area for master mix preparation, where all the reaction components necessary for the PCR amplification (except nucleic acid) are mixed together;
3. The third area is for the detection and confirmation of PCR products.

Sample preparation area has to have its own devices, which should not leave the room. This facility should be used for aliquoting of sample and preparation of positive and negative controls. Biosafety cabinet (for pathogens), refrigerator, freezer and dry heat block/water bath are needed to be placed in this area.
• Clinical samples that arrive at the laboratory must be treated as soon as possible (DNA and RNA must be isolated).
• Amplification test tubes prepared in the clean area must have DNA samples put in inside the sample preparation area and then moved to the amplification area.
• This room should be kept under negative pressure to prevent escape of infectious agent outside of the room.

For preparation of reaction mixture filter tips, micropipettes, positive–displacement pipettes or pipettors, amplification reagent, appropriate supplies, PCR-cabinet, glows and dedicated laboratory coats are needed:
• By applying a PCR-cabinet (PCR workstation) equipped with UV irradiation, it is possible to have a safe, nucleic acid-free environment which minimizes the potential for PCR reaction
contamination, since the UV light destroys the contaminating DNA inside the cabinet.
• To prevent cross-contamination and to avoid repeated freezing and thawing, reagent-stock solutions should be aliquoted into smaller volumes.
• To deter contamination, the room should be under positive pressure.

For addition of nucleic acid to the reaction mixture, a clean area is recommended. If there is no place in the molecular laboratory for separating a room for nucleic acid addition, this procedure can be made in the area of sample preparation. It is essential that handling of post-PCR materials is not allowed in this part of the room. Amplification is preferably carried out in this room, or if it is not possible (no available area), it can be done in the area used for detection and confirmation of PCR amplified nucleic acid. Real-time PCR instrument for detection and confirmation of PCR amplified nucleic acid must be placed in the area where only PCR products are going to be handled:
• Gloves and laboratory coats should be worn at all times and removed before leaving the room to control amplicon contamination of other locations.
• All equipment used for amplification and product detection should be dedicated to this room, including adjustable pipettes with plugged, aerosol-barrier tips.
• This room should be kept under negative pressure.

The main source of contamination in the PCR laboratory is the feedback from amplicons generated during the previous PCR reactions. To prevent contamination, the following conditions must be met:
1. By separating the post-PCR area from the pre-PCR area, the potential for contamination is significantly reduced. The most appropriate condition if there are separate rooms where these activities occur. It is also important to supply the separated PCR laboratory areas with equipment, devices and reagents, which are used only in the allotted room.
2. For prevention or reduction of potential contamination during PCR detection of special nucleic acid sequences, the unidirectional workflow must be applied in the molecular laboratory. It means that during the different work phases, analysis steps have to pass from the clean (pre-PCR) to the dirty (post-PCR) areas (referred to as forward flow).
3. The closed-tube system represent an additional safe mode against cross-contamination. In this case, the reaction tubes are not opened after the PCR processing, thus reducing the risk of contamination of the molecular laboratory by the amplicons; moreover, elimination of laborious post-PCR sample real-time PCR representing a closed tube system, therefore it is less sensitive for the cross-contamination than the conventional PCR.
4. All rooms of PCR laboratory must be equipped with short wave ultraviolet lamps. Laminar airflow safety cabinets must be installed for the treatment of clinical samples. These ensure the safety of personnel working with infected material; their internal surfaces must be pretreated with ultraviolet light for prolonged periods.