Recently, with the resurgence of the Delta strain epidemic, high-intensity nucleic acid testing tasks have become the normal work of our inspectors, and the pollution of nucleic acid testing laboratories is also a topic of more concern in the near future.
This article summarizes the causes of pollution in nucleic acid testing laboratories, anti-fouling measures, pollution monitoring and solutions, etc.
01
▶Potential source of contamination in PCR laboratory◀
①A large number of microorganisms to be tested or target nucleic acids to be tested are present in clinical specimens
② Plasmid clones obtained in scientific research
③Specific microorganisms or target nucleic acids previously analyzed and studied
④ A large number of specific microorganisms or target nucleic acids present in the experimental environment
⑤Residual contamination of the previous amplified products is also the "contamination" that is most likely to cause false positives in PCR laboratories.
PCR amplification can cause the accumulation of amplified products in the laboratory. Usually a typical PCR amplification can produce 109 copies of the target sequence. If aerosolized, even the smallest aerosol will contain 106 copies of the amplified product. If not controlled, the aerosolized amplification products accumulated in a short period of time will contaminate the reagents, instruments and ventilation systems in the laboratory, causing serious laboratory "contamination" and a large number of false positive results.
02
▶Anti-pollution measures in PCR laboratory◀
When performing PCR operations, operators should strictly abide by some operating procedures to minimize possible PCR contamination or prevent the occurrence of contamination.
(1) Dividing the operation area
At present, ordinary PCR can not achieve single-person single-tube and complete closed-tube operation. However, whether it can achieve single-person single-tube or not, the experimental operation is required to be carried out in three different areas. The pre-processing and post-processing of PCR are required. In different isolation areas:
1. Reagent preparation area.
2. Specimen preparation area.
3. PCR amplification area and analysis area.
Each work area must have a certain degree of isolation, special operation equipment, and certain directionality. Such as: reagent preparation → standard preparation → PCR amplification area and analysis area.
Remember: Do not take the products and equipment in any one room to the other two working areas.
(2) Distributed reagents
The reagents required for PCR amplification should be prepared and dispensed in a biological safety cabinet, an ultra-clean workbench equipped with a UV lamp, or a negative pressure workbench. All pipettes and pipette tips must be fixed in it, and cannot be used to absorb amplified DNA and DNA from other sources:
1. PCR water should be high-pressure double distilled water;
2. The primers and dNTPs are prepared with high-pressure double-distilled water in the area without PCR amplification products;
3. The primers and dNTP should be stored separately, and the time should be marked when aliquoted, so as to find the cause in case of contamination.
(3) Strict zoning of laboratories and strict compliance with working procedures
The reagent preparation area, sample preparation area, amplification area, detection area, etc. must be equipped with their own necessary equipment, work clothes, gloves, sample dispensers and ventilation systems. The reagents and wastes used in each area must be divided or packaged directly in their respective areas. Operators must take care to prevent the possibility of carrying amplification products from contaminated areas to clean areas through their hair, glasses, jewelry, and clothing.
(4) Chemical methods
The test bench can be cleaned with 10% sodium hypochlorite (bleach), and then washed with 70% ethanol or water to remove the sodium hypochlorite. Sodium hypochlorite has the effect of oxidative damage to nucleic acid, so that possible amplification products left on the experimental table are destroyed. It should be noted that sodium hypochlorite cannot distinguish between the extracted target DNA and PCR amplification products, and samples treated with sodium hypochlorite cannot be used for nucleic acid amplification detection. In actual work, some items, such as the tray on which the amplification reaction tube is placed, must be transferred from the contaminated area to the clean area. Before returning, they should be placed in a 2%-10% sodium hypochlorite solution overnight and rinsed thoroughly. .
(5) Modification of amplified products
If the previous amplification product is properly modified, it should be able to eliminate its pollution and the new amplification test later, but in order to ensure that it does not affect the amplification test of the target nucleic acid, the amplification product modification method must follow two basic principles:
One is that after the amplified product is modified, it should be distinguishable from the subsequently amplified target sequence
The second is that the modification cannot interfere with normal amplification detection.
Comparison of advantages and disadvantages of different methods to eliminate contamination of amplified products
Picture from "Real-time PCR Technology"
(6) Precautions for experimental operation
Although the residual contamination of the amplified sequence is mostly the cause of false positive reactions, cross-contamination between samples is also one of the reasons. Therefore, it is not only necessary to be cautious and conscientious in performing amplification reactions, but also to pay attention to all aspects of sample collection, extraction and amplification:
1. Wear disposable gloves. If you accidentally spill the reaction solution, replace the gloves immediately;
2. Use disposable tips. It is strictly forbidden to mix the tips with the tips of the PCR product analysis room. Do not expose the tips to the air for a long time to avoid aerosol pollution;
3. Avoid splashing the reaction liquid. To avoid this situation when opening the reaction tube, centrifuge a little before opening the cap to collect the liquid at the bottom of the tube. If accidentally spilled on the gloves or the desktop, replace the gloves immediately and wipe the desktop with dilute acid;
4. When working with multiple samples, prepare the reaction mixture. First mix the dNTP, buffer, primers and enzymes, and then divide them into aliquots. This can reduce operations, avoid contamination, and increase the accuracy of the reaction;
5. Finally add the reaction template, and close the reaction tube after adding;
6. Set up negative and positive control and blank control during operation, which can verify the reliability of PCR reaction and help judge the credibility of the amplification system;
7. Use replaceable or high-pressure processable sample injectors as much as possible. Since the sampler is most susceptible to contamination by product aerosols or sample DNA, it is best to use replaceable or high-pressure sample injectors. If there is no such special sampler, at least the sampler should be dedicated during the PCR operation and cannot be used interchangeably, especially the sampler used for PCR product analysis cannot be taken to the other two areas;
8. Repeat the experiment, verify the results, and draw conclusions carefully.
03
Monitoring of laboratory contamination
A good laboratory must always pay attention to pollution monitoring and consider whether there is any pollution caused by pollution, so as to 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 of whether the PCR reaction is successful or not, and whether the position and size of the product band meets the theoretical requirements. The positive control should be a sample with moderate amplification and good reproducibility, which is identified as the product by various identifications. If a recombinant plasmid is used as a positive control, its content should be low but not high (below 100 copies). However, positive controls, especially recombinant plasmids and high-concentration positive samples, are highly likely to contaminate the detection or amplification samples. Therefore, when a certain PCR reagent is stable after its own use and the tester knows it, it can be used in future experiments. No positive control is required.
2. Negative control: a negative control must be used for each PCR experiment. It includes ① specimen control: if the tested specimen is serum, use the identified normal serum as a control; if the tested specimen is a tissue cell, use the corresponding tissue cell as a control. ②Reagent control: Do not add template DNA or RNA to the PCR reagents and perform PCR amplification to monitor whether the reagents are contaminated.
3. Repeatability test
4. Select primers in different regions for PCR amplification
04
Elimination of laboratory contamination
Once the laboratory is contaminated, the testing work should be stopped immediately, and testing cannot be restarted until it is confirmed that the contamination is eliminated. First, find the source of pollution and clarify the scope of pollution. Then, take effective decontamination measures according to the pollution source and pollution scope, and combine various methods to achieve the best results.
1. When specimens contaminate the operating platform of the biological safety cabinet and cause limited contamination:
Cover with absorbent paper immediately, and spray and disinfect with 0.55% chlorine disinfectant. The disinfectant needs to be prepared for immediate use and used within 24 hours.
2. When the specimen is overturned and the laboratory is contaminated:
Keep the laboratory space closed to avoid the spread of pollutants. Immediately use a towel moistened with 0.55% chlorine disinfectant to cover the contaminated area. When necessary (such as large spills), the laboratory can be heated and fumigated with peracetic acid at a dose of 2g/m³ and fumigated overnight; or 20g/L peracetic acid disinfectant can be sprayed with an aerosol sprayer, with a dosage of 8ml/m³, effect 1- 2 hours; if necessary, or use potassium permanganate-formaldehyde fumigation: potassium permanganate 8g/m³, put it in a heat-resistant and corrosion-resistant container (terrain pot or glass container), then add formaldehyde (40%) 10ml/m³, fumigate More than 4 hours. The indoor humidity during fumigation is 60%-80%.
3. When cleaning up pollutants:
Strictly follow the biological safety operation requirements of live viruses, use pressure steam sterilization treatment, and conduct laboratory ventilation, etc. to prevent secondary hazards.
05
▶Frequently asked questions about COVID-19 nucleic acid testing◀
1. What should I do if the COVID-19 reagent has a tail lift?
Answer: If it is a single channel tail-lifting, whether it is a FAM or a VIC channel, first review as required. Re-sampling is recommended for re-sampling. If it is not convenient to re-sampling, it is recommended to re-extract nucleic acid to verify the original sample. If the review is negative, the judgment is negative. If the re-examination is still the same channel, it is necessary to carefully review the results. One is to eliminate laboratory contamination, and the other is to understand the patient`s information clearly. If it cannot be confirmed, it is recommended to re-sampling and re-examination. If necessary, another reagent can be used for verification. If there is a large number of weakly positive amplified tails, the primary reason is to consider laboratory contamination, which can be excluded through verification by environmental samples and negative controls.
2. Are the N gene and 1ab gene unique to the new coronavirus, and which one is more sensitive? Will it overlap with other coronavirus segments?
Answer: N gene and 1ab are both specific and will not cross-react with other respiratory pathogens. When designing, the sequences of the two genes are different, resulting in different sensitivity. The sensitivity of the N gene is higher than that of 1ab.
3. How to interpret the COVID-19 test results and clinical diagnosis inconsistent?
Answer: Because COVID-19 is an RNA virus, it is easy to degrade. In addition, the test results are also related to sampling, nucleic acid extraction and the nucleic acid concentration of the sample itself. It needs to be analyzed one by one, and can be interpreted in conjunction with clinical symptoms.
4. Using normal saline as a negative control, the internal standard will also increase, how to explain?
Answer: Because the reagent uses an endogenous internal standard, the endogenous internal standard is marked with a human-derived gene. This gene exists in human epidermal cells. Human-derived pollution may occur during the operation, and environmental pollution may also occur. It may also lead to an increase in internal standards, which is also the aforementioned recommendation for environmental sample quality control.
5. What should I do if the internal standard of the COVID-19 sample is not displayed?
Answer: If the internal standard of the COVID-19 sample cannot be found, the sample must be re-examined, and the sample can be re-mixed to extract nucleic acid. If the internal standard of the sample still cannot be extracted, the sample is not qualified if the extraction problem is ruled out. To notify the clinical re-sampling and review.
6. Collect environmental samples to detect COVID-19. Why can't most of the samples have internal standards, and occasionally individual samples have internal standards?
Answer: Because the COVID-19 reagent detects human endogenous internal standards, environmental samples generally do not contain human cells, so internal standards cannot be detected; if internal standards are occasionally detected, it may be that human cells adhere to the environmental samples. , So the internal standard was detected.
Finally, there are two useful tips:
● For multiple samples, when preparing the reaction mixture, first mix the dNTPs, buffer, primers and enzymes, and then use the liquid separation function for aliquots.
● When adding the template, follow the adding sequence of "negative control-sample to be tested-positive control", and the operation should follow the principle of "open tube cap-add template-cover tube cap".
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