How to monitor PCR laboratory contamination

A qualified PCR laboratory should always do a good job of monitoring contamination. Consider whether contamination is present and what causes the contamination. In order to take timely measures to prevent and eliminate pollution.

1.Set up a negative control.

Be sure to do a negative control every time you perform a PCR experiment, which includes a specimen control, a reagent control, and a blank control. Specimen control: Specimen control should use specimens of the same tissue structure whenever possible.

Reagent control: PCR amplification is performed without template DNA or RNA in the PCR reagent to monitor whether the reagent is contaminated. Blank control:

Double-distilled water can be used to monitor the entire experimental environment for contamination.

2.Set up a positive control.

A PCR positive control should be provided for each PCR experiment, which is an important reference marker for the success of the PCR reaction. For the positive control, specimens with medium amplification and good reproducibility should be selected. And be aware of the potential for cross-contamination.

After the experiment is stable, the positive control can be dispensed with.

3.Repeatability trials are conducted.

4.Primers from different regions are selected for PCR amplification.

Causes of contamination:

1) Cross-contamination between specimens: Specimen contamination mainly includes contamination of the container in which the specimen is collected, or cross-contamination between specimens due to poor sealing and overflow outside the container when the specimens are placed, or specimens sticking to the outside of the container; During the extraction process of the nucleic acid template of the specimen, the contamination between the specimens was caused by the contamination of the sampling gun. Some microbial specimens, especially viruses, can spread with aerosols or form aerosols, leading to contamination with each other.

2) Contamination of PCR reagents: Mainly due to the contamination of PCR nucleic acid templates by the sampling gun, container, double distilled water and other solutions during the preparation of PCR reagents.

3.PCR amplification product contamination: This is the most common and common contamination problem in PCR reactions.

Because the amount of copies of PCR products is large (generally 1013 copies ML), which is much higher than the limit of PCR detection of several copies, a very small amount of PCR product contamination can cause false positives and form false positives.

4) It is easy to ignore that the most likely form of PCR product contamination is aerosol pollution; Aerosols can be formed when the air and liquid surfaces are rubbed, and the reaction tube can be shaken vigorously during operation, and aerosols can be formed and contaminated by repeated sampling when the lid is opened, when the sample is suctioned, and when the sample is contaminated. It has been calculated that an aerosol particle can contain 48,000 copies, so the contamination caused by it is a problem that deserves special attention.

After contamination, we first considered that it might be contamination of the reagent, and after retesting with a new lot of HBV reagent, the result was still positive for all specimens.

The HBV specimen was taken to other regular PCR laboratories for testing, and the results showed that the specimens had negative and positive results, the negative control was negative, and the positive control was positive, indicating that the specimen was not contaminated, and the HBV reagent with two batches was taken to other regular PCR laboratories for testing, and it also showed that there was no problem with the reagent.

After wiping the work surface with disinfectant in my laboratory, I turned on all the ultraviolet lamps for one day, replaced the microsamplers, centrifuge tubes, pipette heads, gloves, markers, record books, etc., and then tested the HBV specimens, and the results were still positive.

After wiping the work surface with disinfectant, turn on all the ultraviolet lamps for one day, re-test the HBV specimens, and set up two negative controls (A and B) at the same time, a negative control is to take the reaction tube of the reagent from the kit, open the lid of the reaction tube, expose it to the air for 10 min, close the lid, and test on the machine; b Negative control is to take the reaction tube of the reagent from the kit, without opening the lid, directly on the machine for detection. The result was that all specimens and a negative control were positive, while a negative control b was negative.

So far, although it is not possible to find out what the source of the contamination is, one thing is certain, the PCR laboratory contamination is caused by the diffusion of aerosols, and after investigation, it was found that the contamination was caused by the sanitation of the specimen preparation area with a broom and a mop in the amplification area when cleaning the laboratory.

After identifying the cause of the contamination, we set out to eliminate the contamination by opening the doors, windows, and exhaust fans of the entire PCR lab every day, ventilating the entire lab, wiping the entire PCR lab with disinfectant, and turning on all UV lamps.

The test was carried out every 3 days according to 3 5 steps, and this treatment was continued for one month, and the A negative control turned negative. Three consecutive tests of the A and B negative controls, each time with negative results, confirmed that the PCR laboratory was back to normal.

Usually our prevention of contamination in PCR laboratories is to isolate different operation areas, divide reagents, improve experimental operations, etc., and the treatment of contamination is to wipe or soak with dilute hydrochloric acid; Ultraviolet irradiation method, etc.]. In order to prevent mutual contamination between the experimental compartments, each experimental compartment is as closed as possible, which increases the probability of residual contamination of PCR products in the laboratory. From the perspective of the process of contamination removal in the PCR laboratory, I think one of the keys to the elimination of contamination is ventilation.

Proper ventilation of the laboratory can play a role in air dilution, which is conducive to the reduction of PCR product residues and the elimination of laboratory contamination.

Illuminate the laboratory with a UV lamp and scrub the instrument with alcohol.

What are the most feared problems in PCR experiments? Of course, it was a false positive for the hard-earned results.

When there is a false positive in PCR, we often think about whether it is primer mix * and other contaminated templates, and then check them one by one.

Here I think we should think about it extensively, why are these reagents and instruments contaminated? In fact, it is nucleic acid aerosol contamination.

Centrifuge Centrifugation Shake the reaction tube vigorously Crack the lid of PCR When aspirating and repeating the sample of the pipette, etc., there will be friction between the air and the liquid surface, which will produce nucleic acid aerosols, that is, the laboratory (or pipette) is filled with small water droplets or small particles containing nucleic acid fragments of different lengths, and these small particles settle (hit) into the blank sample and finally amplify the band.

It is conceivable that these small nucleic acid droplets will also fall on the experimental surface, the PCR machine, the pipette *head*box, etc., if we do not pay attention, these nucleic acid droplets will accumulate over time, and there will be special problems in the experiment, such as PCR false positives.

So what should we do if the nucleic acid aerosol (that is, the small droplets of nucleic acid) in our usual experiments or laboratory management produces huge pollution?

Large-area nucleic acid contamination removal operation method:

1) Use nucleic acid aerosol pollution scavenger (PCR cleaner) to treat a large area of the contaminated area, including: floors, walls, ceilings, doors, windows and other places, and increase the ventilation volume (internal circulation is useless, as far as possible external circulation);

2) Repeated treatment of equipment in the contaminated area with nucleic acid aerosol contamination scavenger (PCR cleaner); For equipment containing air filtration systems, such as ultra-clean benches, replace the filters; Nucleic acid aerosol contamination scavenger is used to clean and sterilize contaminated equipment such as pipettes with PCR cleaner;

3) After the cleaning of the contaminated area and equipment and devices is completed, use ultraviolet lamp irradiation for more than 4 hours;

4) You can ask the cleaner to clean up for three consecutive days in accordance with the above requirements (1), (2) and (3);

5) Design the experimental control group (according to the gene fragment of the pollution source), carry out the yin and yang control experiment, and confirm the pollution according to the negative control, and use 8 consecutive rows of PCR tubes (2 yang ginseng + 6 yin ginseng); If the yang ginseng jumps cautiously and the yin ginseng jumps, the pollution needs to be cleaned up; If the yang ginseng jumps and the yin ginseng does not jump at all, it means that the pollution is under control and the normal experiment can be resumed;

Nucleic acid aerosol contamination scavenger (PCR cleaner) box of 500 ml; A 10-square-meter room requires 2 boxes to complete the cover; Its equipment and devices are purchased according to the situation;

Large-scale nucleic acid aerosol contamination is very difficult to remove, and if it is not cleaned up immediately, the contamination will be more solid, and the contaminated nucleic acid will be released for a long time, leading to laboratory shutdown.

1) Strict partitioning of the laboratory (including the partitioning of the preparation area, extraction area, amplification area and product analysis area; also includes partitions for DNA and RNA extraction), as well as strictly controlling the direction of logistics. For example, the things in each guessing area are not cross-used (including white coats, hats, gloves, plate racks, pipettes, extraction equipment, data and extraction reagents, etc.);

2) Strictly control the environment: try to prevent the diffusion of amplifications during the working process; Pollutants such as garbage and tubers should be cleaned up in a timely manner; Eliminate long fragments of pollutants with ultraviolet light; The experimental void is ventilated to reduce the pollutants in the unit space; Laminar flow is strictly controlled in the experiment; Hats and masks were used in the experiment.

3) Set up quality control, and the positive and negative control of the experimental process is a good way to find the pollution source;

4) Divide the reagents, and the reagents in large packages should be divided into small packages before use;

5) the use of UDG enzyme, which can effectively control pollutants;

6) Other effective measures.

Add positive control and negative control to monitor.

Of course, prevention is to be careful......For example, the tube is centrifuged first and then sampled, I think the current experimental conditions are not so easy to contaminate.

1。All kits and utensils are dedicated.

2.The dosing is done in a fume hood to prevent contamination by aerosols in the air3Pipette tips are used with filters to prevent cross-contamination caused by pipettes4The analysis results should not be re-sampled, preferably not in a room, to prevent contamination of the amplification product.