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Preparation plan of hospital PCR laboratory
2024/12/3
PCR laboratory is the basis of molecular diagnosis and the necessary place for PCR experiments. In recent years, the rapid development of the molecular diagnostics industry has brought many IVD practitioners to a new stage of understanding PCR, but the basic knowledge based on the laboratory is still a very important part.
In principle, four areas should be set up: reagent storage and preparation area, specimen preparation area, amplification area, and amplification product analysis area. Depending on the function of the instrument used, the regions may be combined appropriately. The amplification area and the amplification product analysis area can be combined by using real-time fluorescence PCR instrument. Using the automatic analyzer integrating sample processing, nucleic acid extraction and amplification detection, the specimen preparation area, amplification area and amplification product analysis area can be combined.
List of essential items for each district:1. Reagent storage and preparation area (Area 1)
Functions: preparation of stored reagents, preparation of reagent packaging and amplification reaction mixture, and storage and preparation of consumables such as centrifugal tube and suction.
Essential items: refrigerator below 2 ~ 8℃ and -20℃; Mixer; Microdoser (covering 0.2-1000µl).
2. Specimen preparation area (Area 2)
Function: Nucleic acid (RNA, DNA) extraction, storage and addition to the amplification reaction tube. Operations involving clinical samples shall comply with the requirements of Biosafety Level II laboratory protective equipment, personal protection and operational specifications.
Essential items: 2-8℃ refrigerator, -20℃ or -80℃ refrigerator; High speed centrifuge; Mixer; Water bath or heating module; Microsample feeder (covering 0.2-1000µl); Biosafety cabinet.
3. Amplification zone (Zone 3)
Functions: cDNA synthesis, DNA amplification and detection.
Essential items: Nucleic acid amplification instrument (or fluorescence quantitative PCR instrument)
4, amplification product analysis area (4 area)
Function: Further analysis of amplified fragments, such as hybridization, enzyme digestion electrophoresis, denatured high performance liquid phase analysis, sequencing, etc.
Essential items: Hybridization instrument, electrophoresis instrument, sequencer, etc. Microdoser (covering 0.2-1000µl).
General purpose items (all 4 zones may be required) : portable UV lamps (near the work surface); Consumables: disposable gloves, high-pressure resistant centrifuge tubes and sampler suction heads; Special work clothes and work shoes (sets); Special office supplies; Sample tube rack (Zone 2); Explosion-proof clamp (Zone 2); PCR tube rack (Zone 2, delivery window).
1. When entering each working area, the reagent storage and preparation area → specimen preparation area → amplification area → amplified product analysis area should be carried out in strict accordance with a single direction.
2. Each work area must be clearly marked, and equipment and items in different work areas must not be mixed.
3, different work areas use different work clothes (such as different colors). When leaving each work area, the staff shall not take out the work clothes.
4. The cleaning of the laboratory should be carried out in the direction of reagent storage and preparation area → specimen preparation area → amplification area → amplified product analysis area. Different test areas should have their own cleaning equipment to prevent cross-contamination.
5, the work area must be cleaned immediately after the end of the work. The surface of the bench in the work area should be able to withstand the disinfection and cleaning effects of chemicals such as sodium hypochlorite. Ultraviolet irradiation on the surface of the test bench should be convenient and effective. Since the distance and energy of ultraviolet irradiation are very critical to the effect of decontamination, a movable ultraviolet lamp (254nm wavelength) can be used to irradiate within 60 to 90cm of the experimental bench after the completion of the work. Since the amplified product is only a few hundred or tens of base pairs (bp) and is not sensitive to ultraviolet damage, ultraviolet irradiation of the amplified fragment must be extended for a long time, preferably overnight.
6, the laboratory safety work system or safety standard operating procedures, all operations in line with the "Laboratory biosafety general requirements" (GB19489-2008).
Matters needing attention
1, reagent storage and preparation area Storage reagents and consumables for specimen preparation and other materials should be directly transported to the reagent storage and preparation area, can not go through the amplification test area, positive control products and quality control products in the kit should not be stored in the area, should be stored in the specimen processing area.
2. Due to the possible contamination caused by aerosols during the sample mixing and nucleic acid purification in the specimen preparation area, the aerosol pollution entering the area from the neighboring area can be avoided by setting the normal pressure conditions in the area. In order to avoid cross-contamination between samples, the reaction tube containing the reaction mixture must be covered after adding the nucleic acid to be tested. Potentially infectious materials must be covered in the biosafety cabinet and have clear procedures for sample handling and inactivation.
3, the expansion area in order to avoid the pollution caused by aerosols, should minimize the movement in the area. It must be noted that all tested reaction tubes must not be opened in this area.
4. There are various methods for the analysis of nucleic acid amplified products in the analysis area of amplified products, such as on membrane or microplate or on-chip probe hybridization method (radionuclide labeled or non-radionuclide labeled), direct or enzymatic digestion agarose-gel electrophoresis, polyacrylamide gel electrophoresis, Southern transfer, nucleic acid sequencing method, mass spectrometry analysis, etc.
This area is the most important source of amplification product pollution, so care must be taken to avoid the amplification product through the articles and work clothes in this area. When using the PCR-ELISA method to detect amplified products, the board must be washed using a washing machine, the waste liquid must be collected into 1 mol/L HCl, and should not be dumped in the laboratory, but should be discarded away from the PCR laboratory.
Used suction heads must also be soaked in 1 mol/L HCl before being disposed of in garbage bags according to procedures, such as incineration.
Due to the potential use of some genetic mutations and toxic substances such as ethidium bromide, acrylamide, formaldehyde or radionuclides, attention should be paid to the safety of laboratory personnel.
