The core of PCR laboratory design:
1, PCR laboratory layout
PCR amplification test laboratory is divided into three separate working areas in principle: reagent storage and preparation area, specimen preparation area, amplification and amplification product analysis area. In order to avoid cross-contamination, entering each work area must strictly follow a single direction, that is, only from the reagent storage and preparation area → the specimen preparation area → the amplification and amplification product analysis area. The transfer of reagents and samples between each experimental area should be carried out through the transfer window.
2. Laboratory air conditioning and ventilation system design and pressure control
PCR laboratory does not have strict purification level requirements, but in order to avoid the possibility of cross-contamination between various experimental areas, it is advisable to adopt a full delivery and full exhaust air organization form. At the same time, the ratio of air supply and exhaust must be strictly controlled to ensure the pressure requirements of each experimental area.
(1) Reagent storage and preparation area
The main operations in this experimental area are the preparation of storage reagents, the dispensing of reagents and the preparation of the main reaction mixture. Reagents and materials used for sample preparation should be transported directly to this area and not through other areas. The reagent raw materials must be stored in this zone and prepared into the required storage reagents in this zone. For the control of airflow pressure, this area should maintain a slight positive pressure outside.
(2) Specimen preparation area
The main operations in this area are sample preservation, nucleic acid (RNA, DNA) extraction, storage and adding to the amplification reaction tube and measuring DNA synthesis. The pressure gradient in this area is required to be positive pressure relative to the adjacent area to avoid aerosol pollution entering the area from the adjacent area. In addition, since contamination caused by aerosols may occur during sample loading operations, unnecessary walking in this area should be avoided.
(3) Amplification and amplification product analysis area
The main operations in this area are DNA amplification and the determination of amplified fragments. In addition, the addition of the prepared DNA template (from the sample preparation area) and the preparation of the main reaction mixture (from the reagent storage and preparation area) into a reaction mixture can also be carried out in this area. The pressure gradient requirements in this area are: negative pressure relative to the adjacent area to avoid aerosol leakage from this area. To avoid pollution caused by aerosols, unnecessary walking in the area should be minimized. Individual operations such as sample addition should be carried out in an ultra-clean bench.
3. Pollution prevention and control
The core issue of PCR laboratory design is how to avoid contamination. In actual work, the following types of pollution are common: contamination of amplified products; contamination of natural genomic DNA; contamination of reagents and contamination between specimens. Because once pollution occurs, the experiment must be stopped until the source of the pollution is found, and the result of the experiment must be invalidated and the experiment must be repeated. Therefore, it is time-consuming and tedious to find the pollution source around the laboratory after the pollution occurs, and waste manpower and material resources. Therefore, to avoid pollution, the first priority should be prevention, not elimination.
(1) Strict division of work area
The setting of each experimental area is reasonable; each experimental area should have obvious markings (such as eye-catching house number or different ground color, etc.) to avoid confusion of equipment and reagents in different experimental areas.
(2) Reasonable system settings
Reasonable air conditioning and ventilation system settings, try to use full delivery and full exhaust air conditioning systems; strict airflow pressure control to ensure different pressure requirements in different experimental areas.
(3) Standard operation
The technicians of the amplification testing laboratory must undergo on-the-job training, and only after being qualified can they be engaged in clinical gene amplification testing; during the experimental operation, the operator must wear gloves and change them frequently. In addition, the use of disposable caps in the operation is also an effective measure to prevent pollution; the cleaning work is timely and correct. After the experimental work is over, the area must be cleaned immediately. In addition to the conventional disinfection liquid to wipe and disinfect the surface or ultraviolet light irradiation disinfection, some experimental equipment should also be autoclaved.
(4) Strict management
Strictly control personnel entering and leaving the laboratory. Personnel who have nothing to do with the experiment are not allowed to enter and leave the laboratory at will. If possible, set up independent passages and doors to enter and exit the entire experimental area; minimize unnecessary walking in the experimental area to reduce the possibility of cross-contamination; amplification products The analysis area is the main source of contamination of amplified products. Waste liquid cannot be dumped in the laboratory. It must be soaked and disinfected in a disinfectant and discarded away from the laboratory. Disposable materials such as used tips should also be disinfected. After soaking and disinfection, uniform treatment, such as incineration, etc.; the amplification product analysis area may use some genetic mutations and toxic substances, and special attention should be paid to the safety protection of experiment personnel.
(5) Complete laboratory supporting facilities
Complete laboratory supporting facilities are necessary to ensure the experimental work. Corresponding equipment and instruments should be equipped according to the different experimental contents of each laboratory, such as ultra-clean benches, centrifuges, samplers, etc.