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Overall Planning Of PCR Purification Laboratory

Jul 22, 2020

In order to prevent cross contamination during operation, the main requirements for its design, construction and operation management are:


1. Laboratory design and construction, safe working system or safety standard operating procedures shall meet the requirements of the current "General Requirements for Laboratory Biosafety" GB19489.


  2. PCR is divided into four functional areas according to the working steps, namely reagent storage and preparation area, specimen preparation area, amplification area, and amplification product analysis area. If a real-time fluorescent PCR instrument is equipped, the amplification and product analysis areas can be combined. Each functional area must be clearly marked, and equipment and articles in different functional areas must not be mixed.


  The air flow direction of the PCR laboratory can be carried out in accordance with the reagent storage and preparation area→specimen preparation area→amplification area→amplified product analysis area to prevent the amplified products from entering the pre-amplified area along with air flow. It can be carried out in the manner of decreasing air pressure from reagent storage and preparation area → specimen preparation area → amplification area → amplification product analysis area. It can be achieved by installing exhaust fans, negative pressure exhaust devices or other feasible methods.


  3. Entering each work area should strictly follow a single direction, that is, reagent storage and preparation area → specimen preparation area → amplification area → amplification product analysis area. Different work areas (such as different colors) should be used. Workers shall not take out work clothes when they leave each work area.


  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 experimental areas should have their own cleaning appliances to prevent cross contamination.


   5. After the work is over, the work area must be cleaned immediately. The surface of the laboratory bench in the work area should be able to withstand the disinfection and cleaning effects of chemicals such as sodium hypochlorite. The ultraviolet irradiation on the surface of the laboratory bench should be convenient and effective. Since the distance and energy of the ultraviolet irradiation are very critical to the decontamination effect, a movable ultraviolet lamp (254nm wavelength) can be used, and after the work is completed, it can be adjusted to irradiate within 60-90cm on the experimental table. Since the amplified product is only a few hundred or tens of base pairs (bp), it is not sensitive to UV damage, so the UV-irradiated amplified fragment must be irradiated for a longer time, preferably overnight.


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The functions of the reagent storage and preparation area are: the preparation of storage reagents, the dispensing of reagents and the preparation of the amplification reaction mixture, as well as the storage and preparation of consumables such as centrifuge tubes and tips. Materials such as storage reagents and consumables used for specimen preparation should be directly transported to the reagent storage and preparation area, and cannot pass through the amplification detection area. The positive control materials and quality control materials in the kit should not be stored in this area, but should be stored in this area. Specimen processing area.


   The function of the specimen preparation area is: nucleic acid (RNA, DNA) extraction, storage and adding to the amplification reaction tube. For operations involving clinical samples, the requirements for protective equipment, personal protection, and operating specifications for biosafety secondary laboratories should be met. Since contamination by aerosols may occur during sample mixing and nucleic acid purification, positive pressure conditions can be established in this area to avoid aerosol contamination from neighboring areas. To avoid cross-contamination between samples, after adding the nucleic acid to be tested, the reaction tube containing the reaction mixture must be covered. For potentially infectious materials, the lid must be opened in the biological safety cabinet, and there must be clear procedures for sample handling and inactivation.


  The function of the amplification zone is: DNA synthesis, DNA amplification and detection. In order to avoid pollution caused by aerosols, walking in the area should be minimized. It must be noted that all tested reaction tubes must not be opened in this area.


  The function of the amplified product analysis area is: further analysis and determination of amplified fragments, such as hybridization, enzyme digestion electrophoresis, denaturing high performance liquid analysis, sequencing, etc. Nucleic acid amplification products have various analysis methods, such as probe hybridization methods on membranes or on microplates or chips (radionuclide labeling or non-radioactive nuclide labeling), direct or digested agarose gel electrophoresis, Polyacrylamide gel electrophoresis, Southern transfer, nucleic acid sequencing methods, mass spectrometry, etc.


  The amplification product analysis area is the main source of amplification product contamination in the PCR laboratory, so care should be taken to avoid carrying the amplification product out through the articles and work clothes in this area. When using the PCR-ELISA method to detect amplified products, a plate washer must be used to wash the plates, and the waste liquid must be collected in 1 mol/L HCl, and cannot be poured in the laboratory, but should be discarded away from the PCR laboratory. Drop. The used tip must also be soaked in 1 mol/L HCl and then placed in a garbage bag for disposal according to procedures, such as incineration. As this area may use certain genetic mutations and toxic substances such as ethidium bromide, acrylamide, formaldehyde or radionuclides, etc., the safety protection of experimenters should be paid attention to.