The biggest feature of the PCR reaction is its large amplification capacity and extremely high sensitivity, but the headache is the problem of easy contamination, and extremely small amount of pollution can cause false positives.

Causes of pollution:

(1) Cross-contamination between specimens: specimens are mainly contaminated by containers that collect specimens, or when specimens are placed, because the seals are not strictly outside the container, or specimens are stuck outside the container, causing cross-contamination between them; the nucleic acid template of the specimen is During the extraction process, contamination of the specimens is caused by contamination of the sample gun; some microbial specimens, especially viruses, can diffuse with aerosols or aerosols, causing contamination between each other.

(2) Contamination of PCR reagents: mainly due to contamination of the PCR reagent template by the addition of the sample gun, container, double distilled water and other solutions during the preparation of the PCR reagent.

(3) PCR amplification product contamination. This is the most common and most common contamination problem in PCR reactions. Because of the large amount of PCR product copy (usually 1013 copies/ml), it is much higher than the limit of several copies of PCR detection. A very small amount of PCR product contamination can cause false positives to form false positives.

There is also a form that is easily overlooked, and the most likely form of contamination of the PCR product is aerosol pollution; when the air and the liquid surface are rubbed, an aerosol can be formed, and the reaction tube is shaken more vigorously during operation, when the lid is opened, and when the sample is opened, Repeated aspiration of contaminated injection guns can form aerosols and pollute. It is calculated that an aerosol particle can contain 48,000 copies, so the pollution caused by it is a problem that deserves special attention.

(4) Contamination of cloning plasmids in laboratories: This problem is also common in molecular biology laboratories and some laboratories that use cloning plasmids as positive controls. Because cloning plasmids are quite high in unit volume, and are also purified. In the process, more tools and reagents are needed, and the plasmid in living cells has a high degree of vitality due to the simplicity of living cell growth and reproduction.

Monitoring of pollution: A good laboratory should always pay attention to the monitoring of pollution, consider whether pollution is caused by pollution, and take measures to prevent and eliminate pollution.

Control test:

1. Positive control: PCR positive control should be set up in the establishment of PCR reaction laboratory and general inspection unit. It is an important reference mark for whether the PCR reaction is successful and whether the position and size of the product strip meet the theoretical requirements. To select a medium degree of amplification and good reproducibility, the identification of the product is a variety of identification, such as the recombinant plasmid as a positive control, the content should be low and not high (100 copies or less). But the positive control is especially the recombinant plasmid and High-concentration positive specimens are highly likely to contaminate the detected or amplified samples. Therefore, when a PCR reagent is stabilized by its own use and the inspectors have a number of hearts, a positive control can be omitted in future experiments.

2. Negative control: Negative control must be done for each PCR experiment. It includes 1 specimen control: the specimen to be tested is serum and the normal serum after the identification is used as a control; the specimen to be examined is the tissue cells and the corresponding tissue cells are used. Control. 2 reagent control: PCR amplification was performed without adding template DNA or RNA in the PCR reagent to monitor whether the reagent was contaminated.

3. Repeatability test

4. Select primers from different regions for PCR amplification

Ways to prevent pollution:

one. Prevention of pollution:

When performing PCR operations, operators should strictly follow some operating procedures to minimize possible PCR contamination or prevent contamination.

(1) Division of operation area: At present, ordinary PCR can not achieve single-person single tube, achieving complete closed-tube operation, but whether or not a single-unit single tube can be achieved, the experimental operation is required to be carried out in three different areas, PCR Pre- and post-processing should be carried out in different isolation zones:

1. Specimen processing area, including preparation of an amplification template;

2. PCR amplification zone, including preparation of the reaction solution and PCR amplification;

3. Product analysis area, gel electrophoresis analysis, product photographing and preparation of recombinant clones.

Each work area must have a certain degree of isolation, special equipment for operation, and must have a certain direction. Such as: specimen preparation → PCR amplification → product analysis → product processing.

Remember: Do not get the other two work areas for the products and equipment in the product analysis area.

(2) Packing reagents: The reagents required for PCR amplification should be prepared and dispensed on a clean bench or a vacuum bench equipped with UV lamps. All samplers and tips need to be fixed in place and cannot be used to extract amplified DNA and DNA from other sources:

1. PCR water should be high pressure double distilled water;

2. Primers and dNTPs were prepared in a non-PCR amplified product region using high pressure double distilled water;

3. Primers and dNTPs should be stored separately, and the time should be indicated when dispensing, in case of contamination.

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