Process design

The PCR laboratory reasonably sets up various working areas according to the functions of the instruments used, such as polymerase linked reaction: four separate working areas are set up: reagent storage and preparation area, specimen preparation area, amplification area, and amplified product analysis area, which is the most commonly used partitioning method. If the sample needs to be crushed, it is also necessary to add a sample crushing area. If real-time fluorescent PCR method is used, the gene amplification area and the gene product analysis area can be combined in one room; Using a fully automated PCR analyzer integrating specimen processing, nucleic acid extraction and amplification detection, the specimen preparation area, amplification area and amplification product analysis area can be combined into one area, so the PCR laboratory is in principle set up five areas or four areas or three areas or two separate working areas.

The nucleic acid pre-amplification area and the nucleic acid post-amplification area can be located in the same room, but only if the following requirements are met:

1. Two experimental areas in different locations are set up in the laboratory before nucleic acid amplification, the reagent is configured in the ultra-clean workbench, and the sample processing is operated in the biosafety cabinet.

2. Use fully enclosed amplification and detection systems, such as real-time fluorescent PCR, in the post-nucleic acid amplification area.

3. Each experimenter uses its own reagents, consumables, pipettes and containers to hold contaminants.

4. Clean and disinfect the operation area and shared utensils before and after the experiment.

5. Reagents, utensils, instruments and equipment in each region are dedicated to the area and shall not be used cross-over.

Second, air flow and pressure difference requirements

The air flow of the PCR laboratory must be carried out in strict accordance with the gradual decrease of air pressure in the reagent storage and preparation area → specimen preparation area → amplification area → amplification product analysis area to prevent the amplification product from entering the pre-amplification area along with the air flow. Wind direction must not be confused.

In order to ensure the pressure difference in the room and avoid pollution, the opening and closing sequence of the fan and exhaust fan should be clearly written on the air conditioning panel. The sequence must not be confused.

The static pressure difference between adjacent rooms with different air cleanliness levels should be greater than 5 pa, the static pressure difference between the clean room (area) and the outdoor atmosphere should be greater than 10 pa, and the equipment to monitor the static pressure difference should be equipped and monitored regularly.

Under normal circumstances, the reagent preparation room and sample processing room should be micro-positive pressure to prevent the outside air containing nucleic acid aerosols from entering, causing pollution; The positive pressure effect can be achieved by controlling the inlet air volume to be greater than the exhaust air volume. The nucleic acid amplification room and product analysis room should be slightly negative pressure to prevent the aerosol containing nucleic acid from spreading out to contaminate reagents and samples, and the negative pressure effect can be achieved by controlling the exhaust air volume greater than the intake air volume. In an ideal situation, a positive pressure can be set in the buffer room of the PCR laboratory so that indoor air does not flow outdoors and outdoor air does not flow indoors. The air inlet of PCR laboratory is installed to the specified point by the original central air conditioning control requirements.

3. Laboratory area and equipment spacing

Generally speaking, the area of each room is not strictly required, as long as the required instruments and equipment can be placed, and it is easy for personnel to operate, but the room area of the biosafety cabinet can not be less than 10 square meters, and each additional biosafety cabinet, the room will be increased by 10 square meters.

The area of each laboratory buffer room is generally 1300* (1300 or 1500) mm, and the area of the buffer room can not be greater than 1/8 of the area of the laboratory room.

When doing graphic design, the first factor to consider is "safety", the laboratory is the most prone to explosion, fire, gas leakage and other places. When we do graphic design, we should try to keep the ventilation of the laboratory smooth and the escape channel smooth. According to international ergonomic standards. We make the following division for reference:

Test bench and test bench channel division standard (channel interval is represented by L)

When L>500mm, one side can stand human operation;

When L>800mm, one side can be operated by a person;

L>1200mm, one side can sit people, one side can stand people, the middle can not be people;

When L>1500mm, people can sit on both sides and people can sit in the middle;

When L>1800mm, people can sit on both sides, and people can pass the instrument in the middle

Balance table, instrument table should not be too close to the wall, 400mm away from the wall is appropriate. In order to be easy to evacuate in case of danger at work, the passageway between the test bench should all lead to the corridor. In addition: the height of the laboratory building should be 3.7-4.0 meters, the net height should be 2.7-2.8 meters, and the net height of the laboratory with special requirements such as cleanliness, pressure gradient, constant temperature and humidity should be 2.5-2.7 meters (excluding the ceiling); The clear width of the laboratory corridor should be 2.5-3.0 meters. General laboratory double door width of 1.1 meters -1.5 meters (asymmetric door) is appropriate, single door width of 0.8 meters -0.9 meters is appropriate.

Fourth, clean decoration requirements

1. The inner surface of the clean room (area) should be smooth, no cracks, tight interfaces, no particles falling off, and resistant to cleaning and disinfection, and the junction between the wall and the ground should be curved or other measures should be taken to reduce dust accumulation and facilitate cleaning.

2. Various pipelines, lamps, tuyere and other public facilities in the clean room (area) should be considered in the design and installation to avoid parts that are not easy to clean.

3, clean room (area) Windows, ceiling and access to the indoor pipes, tuyere, lamps and walls or ceiling connection parts should be sealed.

Five, work clothes requirements

Personnel working in the purification workshop should wear work clothes that meet the requirements. The material selection, style and way of wearing the work clothes should be adapted to the requirements of the production operation and air cleanliness level, and should not be mixed. The texture of clean work clothes should be smooth, do not produce static electricity, and do not shed fibers and granular substances. Sterile clothing must cover all hair, beard and feet, and prevent human shedding. Work clothes used at different air cleanliness levels should be cleaned, sorted out, and disinfected or sterilized if necessary. Additional particulate matter should not be brought into the work clothes when washing and sterilizing. Work clothes should be set cleaning cycle.

Access to each area must be strictly carried out in a single direction, and different work areas use different overalls (such as different colors). When leaving, the staff shall not take out the work clothes.

The laboratory shall establish and implement the cleaning procedures and management system for personnel entering and leaving the clean area, and the personnel cleaning procedures shall be reasonable.

6. People flow path and logistics path

To enter the laboratory area, staff should follow the following path:

Public cleaning area -- changing room (change of clean clothes) -- buffer room -- contamination test area

The path for staff to exit the laboratory is:

Pollution test area -- buffer room -- shower room (can be set up if conditions exist) -- changing room -- public cleaning area

All items entering the laboratory area must pass through the double-leafed interlocking transfer window, which can be disinfected before entering. All items in the laboratory must also pass through the transfer window before being transferred to the clean public area outside the laboratory.

Seven, the opening direction of the door

The height of the laboratory building should be 3.7-4.0 meters, and the net height of the PCR laboratory should be 2.5-2.7 meters (excluding the suspended ceiling). The clear width of the laboratory corridor should be 2.5-3.0 meters. General laboratory double door width of 1.1 meters -1.5 meters (asymmetric door) is appropriate, single door width of 0.8 meters -0.9 meters is appropriate.

Generally, the laboratory door is mainly open to the inside, but if there is a dangerous room, the door should be opened outward, and the door material is best to choose pressure glass. For rooms with differential pressure gradients, the opening direction of the door should be toward the positive pressure direction; In consideration of fire safety, the opening direction of those main escape doors should be opened in the direction of the cleaning area.

Eight, basic equipment consideration

1. Reagent storage and preparation area

The main operations in this experimental area are the preparation of stored reagents, the packaging of reagents and the preparation of main reaction mixture. Reagents and materials for sample preparation shall be transported directly to this area and shall not pass through other areas. Reagent raw materials must be stored in the area and prepared into the required storage reagents in the area. For the control of airflow pressure, the area should maintain a small positive pressure.

Reagent preparation area equipment should be sample feeder, refrigerator, balance, low speed centrifuge, mixer, mobile UV lamp and so on. An ultra-clean table can be used as a reagent preparation operating table.

2. Specimen preparation area

The main operations in this area are the preservation of samples, extraction of nucleic acids (RNA, DNA), storage and addition to amplification reaction tubes, and the determination of DNA synthesis. The pressure gradient in this area is required to be positive relative to the neighboring area to avoid aerosol pollution entering this area from the neighboring area. In addition, unnecessary movement in the area should be avoided due to the possibility of contamination from aerosols during sampling operations.

The equipment in the specimen preparation area should mainly have a biosafety cabinet (preferably B2), which can avoid the repeated circulation of extracted nucleic acid in the cabinet, resulting in cross-contamination between specimens and false positive results. In addition, sample feeder, table high speed centrifuge (frozen and normal temperature), table low speed centrifuge, constant temperature equipment (water bath and/or dry bath), refrigerator, mixer and mobile UV lamp should be equipped.

3, amplification and amplification product analysis area

The main operations in this region 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 the reaction mixture can also be carried out in this area. The pressure gradient in this area is required to be negative relative to the adjacent area to avoid the leakage of aerosols from this area. To avoid pollution caused by aerosols, unnecessary movement within the area should be minimized. Individual operations such as adding samples should be carried out in the ultra-clean table.

The main instrument in the amplification area is the nucleic acid amplification thermal cycle instrument (PCR instrument, real-time fluorescence or ordinary). The power supply of the thermal cycler should be dedicated and equipped with a regulated power supply or UPS to prevent the influence of the amplification measurement due to voltage fluctuations. In addition, according to the needs of the work, it can also be equipped with sample feeder, super clean table, etc.

Product analysis area: The instruments and equipment used in this area may be sampler, electrophoresis instrument (tank), electric transfer instrument, hybridization furnace or hybridization box, water bath, DNA sequencer, enzyme label instrument and plate washing machine.