Simple tips on how to stay away from contamination during PCR

PCR technology is a nucleic acid amplification technique that mimics the process of DNA replication in vitro. It mainly repeats three cycles: high-temperature denaturation, low temperature annealing and extension, and then amplifies the target DNA millions of times. Thus, PCR technology has the advantage of being extremely sensitive. However, there are two sides to every coin, and the biggest disadvantage of PCR technology is that it is very susceptible to contamination, and a very small amount of contamination can lead to false positives.

Once laboratory nucleic acid contamination occurs, normal experiments must be stopped. Significant material and human resources are then required until the source of contamination is found, or the cleanliness of the laboratory is up to standard. In addition, the lab report must be invalidated. The results are reliable only after re-experimentation.

In order to effectively avoid contamination and obtain stable and reliable test results, we often need to take certain measures to prevent or eliminate contamination in practice.

PCR plate

Sources of contamination. Reagent contamination reagent containers, vessels, water and other solutions can be contaminated with nucleic acids during the preparation of PCR reagents.

Equipment contaminationSemi-automatic nucleic acid extractors and fully automatic nucleic acid extractors can cause samples or nucleic acid templates to spill during extraction and contaminate the machine. Or the bin inside the equipment is not cleaned thoroughly and residual liquid or crystals evaporate to form aerosols, which in turn can cause contamination.

Specimen cross-contaminationSpecimen cross-contamination can be caused by a variety of factors: contamination of the specimen collection container; spillage of the container due to poor sealing when the specimen is placed; specimens sticking to the outside of the container; contamination of the inhaler and aerosols in the air. In particular, viruses can spread through aerosols or by forming aerosols, resulting in mutual contamination.

PCR product contaminationThis is the most common contamination problem in PCR. It is commonly found in assays that require opening after amplification or improper handling of the product after amplification. This is because the copy size of the PCR product is much larger than the maximum of a few copies detected by PCR. Therefore, a very small amount of PCR product contamination can result in a false positive.

Cloning Plasmid ContaminationPositive references are commonly used in laboratory operations, and most of these positive references come from certain cloned plasmids. The concentration of cloned plasmids per unit volume is very high and can be easily contaminated if not used carefully.

Some laboratories clone their own plasmids as quality control products. However, after preparation, the waste is not handled strictly, leading to contamination of the laboratory by the plasmids and thus the specimens.

Measures to control contaminationExperimental compartmentalizationRational separation of laboratories. Divide or partition the operation of the following steps: specimen processing, PCR reaction solution preparation, PCR cycle amplification, PCR product identification, etc. In addition, special attention should be paid to the fact that specimen processing and PCR product identification must be separated from other steps.

It is best to divide them into specimen processing area, PCR reaction solution preparation area, PCR cycle amplification area, and PCR product identification area. And their lab supplies and aspirators should be used exclusively.

UV used in the laboratory should be sterilized before the experiment to destroy residual DNA or RNA.

Articles for each zone should not be mixed each laboratory area should be marked with different equipment and articles in a conspicuous color to avoid mixing equipment and articles from different work areas. For example, work clothes, work shoes, trash cans, mops, rags, pens, etc. used exclusively in each area.

Use disposable pipette tips, centrifuge tubes, dust-free gloves, masks, etc.

Establish standard air conditioning and ventilation systems full delivery and exhaust air conditioning systems whenever possible. A strict single flow direction is required during access to each operation area, i.e. reagent preparation and storage area → sample preparation area → PCR amplification area → amplification product analysis area, without retrograde flow.

Contamination monitoring is carried out by taking the following measures: establishing positive and negative controls, repeating tests, selecting primers for different regions for PCR amplification, etc., thus taking measures to prevent and eliminate contamination.

Regular laboratory cleaningPrepare disinfectant solution containing 0.5% active chlorine to wipe equipment surfaces, countertops, floors, article surfaces, pass boxes, doors, and windows, and soak trash cans inside the equipment.

Alcohol-to-air spraying is performed according to the top-down, inside-out principle.

Wipe down pipettes, eight-tube centrifuges, mixer shakers, and other small equipment with alcohol (75%).

Wipe equipment surfaces, countertops, and floors with purified water and rinse equipment waste tanks with distilled water.

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