Views: 0 Author: Site Editor Publish Time: 2022-09-15 Origin: Site
In principle, the clinical gene amplification laboratory is divided into four separate working areas: reagent storage and preparation area, sample preparation area, amplification reaction mixture preparation and amplification area, and amplification product analysis area. In order to avoid cross contamination, entering each working area must strictly follow a single direction, that is, only from the reagent storage and preparation area → sample preparation area → amplification reaction mixture preparation and amplification area → amplification product analysis area.
The transfer of reagents and samples between the experimental areas shall be conducted through the transfer window.
(3) air conditioning and ventilation system design and pressure control of PCR Laboratory
There are no strict purification requirements for PCR laboratories, but in order to avoid the possibility of cross contamination among various experimental areas, it is appropriate to adopt the air distribution form of all sending and all discharging. At the same time, the proportion of air supply and exhaust should be strictly controlled to ensure the pressure requirements of each experimental area.
Reagent storage and preparation area
The main operations in this experimental area are the preparation of storage reagents, the sub packing of reagents and the preparation of the main reaction mixture. Reagents and materials used for specimen preparation shall be directly transported to this area and shall not pass through other areas. Reagent raw materials must be stored in this area and prepared into required storage reagents in this area.
There are no strict requirements for the control of air flow pressure in this area.
Specimen preparation area
The main operations in this area are the preservation of clinical specimens, the extraction and storage of nucleic acids (RNA and DNA), and the synthesis of cDNA when it is added to the amplification reaction tube and RNA is measured.
The pressure gradient in this area is required to be positive pressure relative to the adjacent area to avoid aerosol pollution entering this area from the adjacent area. In addition, since aerosol pollution may occur during the sampling operation, unnecessary walking in the area should be avoided.
Amplification reaction mixture preparation and amplification area
The main operation in this region is DNA or cDNA amplification. In addition, the addition of the prepared DNA template and the synthesized cDNA (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. In nested PCR determination, the reaction tube must be opened after the first round of amplification, so nested amplification has a high risk of contamination, and the second sampling must be carried out in this area.
The pressure gradient in this area is required to be negative pressure relative to the adjacent area to avoid aerosol leakage from this area. In order to avoid pollution caused by aerosols, unnecessary walking in the area should be minimized. Individual operations such as sample addition shall be carried out in the ultra clean bench.
Amplification product analysis area
The main operation in this area is the determination of amplified fragments. If full-automatic closed analytical instrument is used for detection, this area may not be set.
This area is the main source of amplification product pollution, so the pressure gradient in this area is required to be negative pressure relative to the adjacent area, so as to prevent amplification product from diffusing from this area to other areas.
(4) pollution prevention and control
The core problem of PCR laboratory design is how to avoid pollution. In practical work, the following types of contamination are common: contamination of amplification products; Contamination of natural genomic DNA; Contamination of reagents and samples. Once the pollution occurs, the experiment must be stopped until the pollution source is found, and the experimental results must be invalidated and the experiment needs to be carried out again. Therefore, it is not only time-consuming and cumbersome to find the source of pollution around the laboratory after pollution occurs, which wastes manpower and material resources. Therefore, to avoid pollution, prevention should be the first step, not elimination.
