On June 2, the National Health and Medical Commission issued the “Notice on Further Strengthening the Supervision of the Whole Chain of New Coronavirus Nucleic Acid Detection”.
It is required to strictly manage the qualifications of testing institutions and personnel, standardize the management of sample collection, storage and transportation, strengthen the daily supervision and management of nucleic acid testing institutions, and strengthen the emergency state Under the supervision of nucleic acid testing institutions, improve the utilization efficiency of nucleic acid testing resources, and strictly implement the withdrawal mechanism of nucleic acid testing institutions.
In the face of the frequent explosions of “third-party inspection institutions repeatedly exploded with thunder”, “multiple laboratories frequently exploded with thunder” News such as false positives” and “deliberately making false positives in order to make money”, under the highly stringent requirements of the National Health Commission, what can our nucleic acid testing laboratory do and what should we do? On May 23, Guo Yan, Supervision Commissioner of the Medical Administration and Hospital Administration Bureau of the National Health and Health Commissionintroduced: “Although The specificity of nucleic acid detection is 100%, but in actual work, the laboratory may cause false positives due to contamination caused by the operation of the experimental process.”The accuracy of the detection is The lifeblood of all testing laboratories. The main cause of false positives is nucleic acid aerosol contamination. The specific reasons and countermeasures for false positives (nucleic acid contamination) in PCR laboratories are as follows:
I. Direct factors of nucleic acid aerosol contamination
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1Contamination of PCR products
More common, especially the first generation PCR technology, the probability of occurrence is more than 60%. The PCR product has been repeatedly amplified, and its copy number far exceeds the detection limit of PCR. The copy number contained in one aerosol can be 104~10< sup>6copies, the Ct value corresponding to this copy number is around 24! Therefore, even a very small amount of contamination is enough to cause false-positive experimental results.
However, in the second generation PCR technology, the anti-pollution UNG/UDG technology is used, which can significantly reduce the Contamination caused by the product, the probability of contamination by this factor can be reduced to less than 10%. Almost all the approved new crown detection kits in China use the UNG anti-amplification product contamination technology; because of this, the probability of contamination caused by amplification products is not as high as everyone thinks. But still need to pay attention to the tightness of PCR amplification tubes! If the sealing is not tight, there is evaporation after the amplification, the liquid level is not neat, or there is a sound of exploding tubes during the amplification process, the PCR amplification tube/sealing film should be replaced batch or supplier. 2Contamination by positive controlscommon, with a probability of 40 %~60%. Recombinant plasmids are used as positive quality control materials, and the probability of contamination is higher; when constructed pseudoviruses or recombinant viruses are used as positive quality control materials, aerosol contamination is also prone to occur in the process of nucleic acid extraction. ; During the preparation of reagents, contact with contaminated containers, pipettes, pipette tips, solutions, etc. will cause the reagents to be contaminated. The author believes that this is the main cause of nucleic acid contamination caused by the laboratory at present!
3 The sample to be tested is contaminated
The probability of occurrence is between 10% and 30%. The container in which the sample is placed is contaminated, or the contact between the sample and the outside world is contaminated due to the poor sealing of the container; the sample to be extracted contains strong positive samples, and the extracted samples contain high concentrations of positive nucleic acids, forming Aerosols cause pollution. Aerosol is generated by the friction between air and liquid surface. Opening the cap, shaking the reaction tube and repeated aspiration of the contaminated sampler can form aerosol, resulting in cross-contamination. The above nucleic acid sources all form aerosols that diffuse into the air or adsorb to the surface of objects. Therefore,particular attention should be paid to the problem of pollution caused by aerosols. The false positive ct value of heavy pollution can be around 24, the ct value of moderate pollution is around 30, and the ct value of light pollution is around 33.
From the perspective of the distribution of nucleic acid pollution, there are mainly two places:
nucleic acid gas in the air Sol: It is difficult to diffuse into all corners of the laboratory; nucleic acid contaminants adsorbed on the surface of instruments and equipment: pipettes, centrifuges, nucleic acid extractors, transfer windows, disassembled kits, pipette tip boxes, opened consumables Equal surface.
As long as there is contamination in the PCR laboratory, the previous result report becomes invalid, and other experimental operations cannot be performed. The source of contamination must be identified and thoroughly removed in order to obtain accurate and effective experimental results.
II. Indirect causes of nucleic acid aerosol pollution1No Reasonable laboratory construction
A strict PCR laboratory should have a standard positive and negative pressure system and four partitions, and the laboratory should be in accordance with the biosafety secondary laboratory and PCR laboratory Design basic requirements to build.
The standards for reference are “GB50346-2011 Construction Standard Biosafety Laboratory Building Technical Specifications” and “Medical Institutions” issued by the National Health Commission Guidelines for Clinical Gene Amplification Testing Laboratory Work.
In addition, let’s talk about some risk points that are not mentioned in the current technical guidelines, but are also the most easily overlooked!
2Biosafety Cabinets
The existing technical guidelines only require the selection of BSL-II biological safety cabinets, but do not specify specific models. Considering the negative pressure of zone 2, it is recommended to configure the A2 type. However, from clinical observations, the probability of contamination of non-B2 type biological safety cabinets is much greater than that of B2 type, so it is recommended to use B2 type biological safety cabinets plus nucleic acid templates.
Type A1, Type A2, Type B1 is 70% gas recirculation to the work area through HEPA filter; Type B2 is 100% full emission type, no Internally circulated airflow. Therefore only type B2 can be used for loading (positive control). Because HEPA filters are not effective in filtering aerosol particles. If the biosafety cabinet is configured incorrectly, the circulating air can easily cause the formation of nucleic acid aerosols during sample addition, resulting in pollution, which is difficult to remove.
not different from the same type of biological safety cabinet 3Common disinfection measures cannot effectively degrade nucleic acidsUV lamps can kill viruses, but ” The Working Guidelines for Clinical Gene Amplification Testing Laboratories of Medical Institutions pointed out that short fragments of genetic nucleic acid are not sensitive to UV damage. Conventional disinfectants such as alcohol, iodide, glutaraldehyde, and quaternary ammonium salts can kill bacteria, but they cannot degrade nucleic acids, or the degradation efficiency is poor. Especially alcohol, which is actually a component of nucleic acid extraction reagents. Among the known disinfectants, hypochlorite disinfectants can degrade nucleic acids to a certain extent, but are highly corrosive and cannot handle precision instruments and metal products. 4Those easily overlooked operational detailsWhen processing samples, The glove does not fit well enough; when handling samples, forget to replace the tip, resulting in cross-contamination of samples; when handling positive samples, it causes contamination of other samples, resulting in false positives. Aspirate the positive control (plasmid) without following the principle of “slow suction and fast attack” (this requires practice to form muscle memory), and it is often “fast suction and fast attack”, causing the positive sample to contaminate the pipette tip; or Under the influence of the circulating air of the biological safety cabinet, it will cause aerosol to splash and contaminate the sampling area. It is best to use a pipette tip with a filter element.
There is no desktop trash can in the sample loading area: if the tip after adding the sample may contain a high concentration of positive nucleic acid targets, Discarded indiscriminately may cause aerosol contamination. The tip should be thrown into a tabletop trash can containing a hypochlorous acid solution. Because hypochlorous acid is easy to volatilize, the chlorine-containing disinfectant should be replaced every day!
5Contamination high risk areasIn the author’s clinical practice, the following areas are found to have a higher probability of positive contamination:
p When contamination occurred in a laboratory, the author collected 10 samples from different areas, and after PCR amplification analysis, the positive detection rates of different areas were compared. It was found that the nucleic acid extractor and centrifuge in the sample processing area had the highest degree of contamination (8/10). Pipettes are also a high-risk item for contamination in clinical practice. The pollution situation in different laboratories is different. Just like in this world, happy families are all the same, but unfortunate families have their own misfortunes. In the same way, standard laboratories are the same, and each polluted laboratory has its own pollution situation. The above data is for reference only. Three. Monitoring of PCR contamination In the daily operation of the laboratory, we should always pay attention to monitoring the pollution of the laboratory, and understand whether the laboratory is polluted, the cause of pollution, and the degree of pollution, so as to take effective measures to prevent and eliminate pollution. Contamination is generally monitored by setting negative controls. 1trial process monitoring< span>At present, the negative control of most kits is to configure the PCR reaction solution and carry out quality control in the process of on-board amplification, but there is no quality control for nucleic acid extraction.
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Laboratory equipment, air, object surface;
- environmental monitoring
every Place 10ml of purified water or physiological saline (open) in each experimental area, and sample 200μl after 1 hour as a sample, participate in extraction and amplification, and analyze whether there is aerosol pollution in the laboratory environment.
- Surface Monitoring
Surface Wiping Sampling: Dip a cotton swab in physiological saline, apply samples on the laboratory table (especially the surrounding dead corners), the surface of the instrument and the inner hole, put it in about 0.5ml of physiological saline for sampling, and use it as a sample to participate in extraction and amplification. Analyze the surface of objects for aerosol contamination.
Fourth, the method of solving PCR laboratory contamination
A solution to PCR laboratory contamination: a second-generation PCR CLEANER 2.0 strategy.
1person, machine, material, method, ring< /span>First of all, we will analyze the causes of the above-mentioned aerosol pollution, and carry out careful review and rectification from five aspects: personnel, hardware configuration, materials, methods (SOP), and environment. This is the basic premise to ensure long-term safety and no pollution in the laboratory. 2Treatment in the event of contamination
In the event of contamination , the treatment measures are based on the regional source of pollution: nucleic acid aerosol pollution in the air and nucleic acid pollutants adsorbed to equipment.
Nucleic acid aerosol pollution in the air Treatment
UV irradiation cannot degrade short nucleic acid fragments below 200bp, the action distance and effect are very Limited and prone to processing dead spots.
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The second-generation non-atomization principle “PCR CLEANER” developed by nanobio Nucleic acid aerosol pollution remover”Remove aerosols in the air!
High removal efficiency, it can solve the problem of aerosol pollution in the air as fast as 2 hours! The greater value of “her” is that at the end of the experiment every day, turning on the instrument for 60 to 120 minutes can greatly reduce the probability of contamination in the laboratory. After all, for nucleic acid aerosol pollution, prevention is greater than control! “No pollution” is the best governance!
Treatment of nucleic acid contamination adsorbed on the surface of instruments, equipment and consumables
Replace consumables (mainly tips and centrifuge tubes) in the contaminated area; Lab coats and work clothes should be soaked in hypochlorite solution for 1 hour before washing; try to use disposable biosafety level II protective clothing.
1Nucleic acid scavengers that are non-corrosive, friendly to the test personnel and the environment, and non-irritating can be selected, such asnanobio launched The second-generation nucleic acid contamination removal solution: “PCR nucleic acid plasmid aerosol contamination remover”. The main principle is that through enzymatic hydrolysis, the scavenging effect is strong, and the degradation effect is better than the common strong oxidant (hypochlorite)! safer! noneCorrosive, non-teratogenic and carcinogenic. The reagent can handle corrosive areas such as spray removal in areas such as pipettes, biological safety cabinets, centrifuges, transfer windows, etc.
nucleic acid scavenger (yeast thermostable nucleic acid degrading enzyme)sterilized with hypochlorite such as 84 (It is corrosive and cannot be used for metal products) spray on laboratory desktops, floors, and walls, and wipe it off with disposable absorbent paper or a clean rag after 20 minutes. The available chlorine content should not be less than 6g/L. Turn on all UV lamps in the laboratory (biological safety cabinets, transfer windows, laboratories, etc.), and turn on the ventilation system after 12 hours to decontaminate. Note: The test consumables, work clothes, etc. are subjected to high pressure at 121°C for 30 minutes. Although it has a certain effect, it cannot completely remove the contamination of plasmid nucleic acid. It is strictly forbidden to perform high pressure on PCR amplification products, especially in zone 2. High pressure steam is very likely to cause aerosol diffusion of amplification products.
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www.zznkbio.comEditor:yeah Reviewer:Xiao Ran /img>