Potential sources of contamination include: Cross contamination between specimens. Amplification product contamination: Repeat amplifications of the same target sequence in PCR techniques lead to the accumulation of amplification products in the lab. A typical PCR generates about 109 copies of the target sequence. Aerosols from pipettes may contain as many as 106 amplification products. Hence, the buildup of aerosolized amplification products may contaminate lab reagents, equipment, and ventilation systems. The work environment, including laboratory work surfaces. Laboratory technicians’ work habits: Without the appropriate protective gear, lab users can transfer amplification products from contaminated rooms to clean rooms via their hair, skin, saliva, or clothing. The main ways to control contamination can be achieved through a good lab floor plan and design. A layout physically separating lab areas for sample preparation, reagent preparation, amplification, and post-amplification analysis is ideal. Cross contamination can also be controlled by minimizing sample manipulation, putting in place positive controls, and following good laboratory practices. Mechanical barriers to prevent contamination Some mechanical barriers that help prevent the carryover of amplification products include: Ensuring the strict separation of sample and reagent preparation areas from the amplification and post-amplification areas. Facilitating unidirectional workflow traffic from the reagent preparation area to the sample preparation area to the amplification area and finally to the detection area. Equipping each area with the necessary instruments, disposable devices, laboratory coats, gloves, aerosol-free pipettes, and ventilation systems. Practicing good laboratory habits, such as always wearing protective gear. Practicing good laboratory habits, such as always wearing protective gear and using color-coded lab coats for different spatially separated areas. Features of the three areas Area 1: Reagent preparation. This area is usually one of the cleanest areas in a molecular testing laboratory because it is typically free of biological material, such as DNA/RNA. To reduce the possibility of contamination, extracted DNA/RNA or PCR products should never be present in the reagent preparation room. This area is dedicated to the preparation and aliquoting of reagent stock. Preparation of reaction mixes may also be done here. Equipment found in this room includes pipettes, vortex mixers, spindown centrifuges, a refrigerator, a freezer, and laminar hoods. Area 2: Sample preparation. This area may comprise two rooms, one for nucleic acid extraction and the other for nucleic acid addition to PCR reaction mixes. If there is insufficient space for two rooms, the two steps can be done in the same room but in different areas or compartments. Lab workers should conduct PCR reactions in biosafety cabinets to protect the user, the product, and the environment from hazardous materials. Standard equipment for the sample preparation rooms includes highspeed centrifuges, vortex mixers, pipettes, autoclaves, and refrigerators. Area 3: Amplification and post-amplification analysis. These two comprise the contaminated (dirty) rooms. Therefore, no equipment or material used in these rooms can be transferred to or used in other rooms. This space is where amplification steps occur, using instruments such as a thermocycler. A laminar flow hood is recommended for any steps requiring the opening of tubes containing amplicons. To control contamination further, it is also critical that laboratory technicians performing post-PCR analysis do not return to pre-PCR work areas on the same day. Equipment Purpose of use Refrigerator For storage of chemicals and PCR products at an average temperature of +40C. Freezer For storage of frozen tissue cultures and samples at temperatures ranging between -200C and -800C. Vortex mixer For mixing small vials quickly in an oscillating circular motion and for the resuspension of cells. Vortex mixers are available with variable speeds and the option for continuous function or on-demand function. Centrifuge To separate fluids, gases, or liquids based on density. There are various types of centrifuge, which are classified by either the intended use or the rotor design. Equipment Purpose of use Thermal cycler To amplify DNA and RNA samples by PCR. It is also used for DNA sequencing, cloning, generation of probes, quantification of DNA and RNA, studying patterns of gene expression, detection of sequencetagged sites, and many more techniques. Spectrophotometer To measure light absorption in a sample, which can be used to determine nucleic acid quantitation or the number of chemicals in a solution. DNA sequencer For the observation of DNA’s molecular sequence. Microscope For the observation and evaluation of samples. Autoclave To sterilize waste and lab equipment. Laminar flow and biosafety cabinets To prevent contamination. Electrophoresis system For conducting PCR analysis. Incubator To maintain optimal conditions, such as temperature and humidity. pH meter For measuring hydrogen-ion activity in water-based solutions. Water bath For incubating samples at a constant temperature. Gel imaging system For analysis of proteins, nucleic acid, and antibodies. Unidirectional workflow The laboratory floor plan should be designed to increase work efficiency. It must also be unidirectional (i.e., continue in one direction only) from the clean area to the dirty area. Whenever researchers or laboratory technicians move from one room to another, lab coats, gloves, and other protective equipment must be changed, and hands must be washed to prevent contamination. Ventilation and air pressure requirements Air circulation between pre- and post-amplification areas can be a source of contamination in labs. So, each room should be ventilated separately, and air pressure adjusted independently. Positive air pressure means that the air pressure inside the room is higher than the one outside the room, which prevents the transport of unwanted substances from outside. Negative air pressure allows air to enter the room and prevents air migration to the surrounding rooms. In these rooms, doors must be kept closed to maintain the negative pressure. The pre-PCR rooms should have a slight positive pressure to prevent the entrance of contaminated air from outside. In contrast, the post-PCR rooms should have a slight negative pressure to keep the air in and prevent the escape of amplicons from the completed PCR samples. The ventilation of pre-PCR and post-PCR spaces should be open to different air channels and should pull air from separate locations. Other requirements Provide an office area that is separate from the laboratory rooms. It should have adequate office furniture where staff can take breaks with access to safe drinking water. Provide locked storage spaces for clothing, personal items, and protective equipment. Consider temperature and humidity requirements, ergonomic assessments, electric outlets, backup power systems, and other relevant utilities.
