Nucleic acid testing is the gold standard for the detection of novel coronaviruses and real-time quantitative fluorescence RT-PCR is a commonly used method for clinical testing. False positives are caused by the presence of a fluorescent signal in a sample that should not have a fluorescent signal, leading to misjudgment of positive results.
Main Causes of False Positives in PCR Tests
1. Inappropriate primer design: The selected amplification sequence has homology with the non-target amplification sequence, thus when PCR amplification is performed, the amplified PCR product is the non-target gene sequence. However, in clinical practice, as basically finished kits are used, the inappropriate primer design does not usually occur, but non-specific amplification still exists with individual manufacturers' reagents, and it is recommended that performance evaluation and comparative screening are carried out before use.
2. Cross-contamination of target sequences or amplification products: This contamination has two causes. One is cross-contamination of entire genomes or large fragments leading to false positives. The second is the pollution of small fragments of nucleic acid in the air. Although these small fragments are shorter than the target gene sequence, they have certain homology. After splicing with each other, it can be combined with primers to amplify PCR products, resulting in false positives. Under the premise of standard operation, the second situation should be avoided as much as possible.
3. Interspecimen cross-contamination: Specimen contamination is mainly caused by contamination of the container in which the specimen is collected. Cross-contamination is caused by specimens spilling out of the container when it is placed, or by specimens adhering to the outside of the container. Specimen contamination between specimens due to contamination of the pipette during extraction of the nucleic acid template. Some microbial specimens, particularly viruses, can spread with or form aerosols, leading to contamination between specimens.
4. Contamination of PCR reagents: mainly due to the contamination of the PCR nucleic acid template by the sample gun, container, distilled water and other solutions during the preparation of PCR reagents.
5. Contamination of cloned plasmids in the laboratory: the positive control in the kit participates in the nucleic acid extraction process and causes cross-contamination. In order to monitor the quality of the nucleic acid extraction process, some kits require positive control in the kit (pseudovirus containing the target gene) samples are used for nucleic acid extraction together. Due to heating, vibration and other processes in the automated nucleic acid extractor, aerosols may contaminate the extractor during operation.
Strategies for Reducing False Positives
1. Standard laboratory design: The laboratory includes a liquid preparation area, DNA extraction area, amplification area, and electrophoresis area, and different experimental operations are completed in continuous and independent spaces. If there is no professional laboratory, at least it is necessary to ensure that the preparation, sample addition and testing are in different areas.
2. Standard laboratory operation: The laboratory is regularly ventilated and disinfected, the instruments are regularly cleaned, and aerosol scavengers are used. Use disposable utensils and operate with care to avoid cross-contamination.
3. Standardize the management of reagent consumables: Newly purchased reagents need to be verified before the experiment and repackaged, and the consumables used need to be sterilized by high pressure.
4. Pre-experiment prevention: Firstly, it is necessary to ensure the specificity of the designed primers to prevent self-amplification of the primers, and secondly, UNG enzyme and dUTP anti-pollution system can also be used.
5. Post-pollution treatment: In case of pollution, the experiment should be stopped immediately, and operations such as opening windows for ventilation, ultraviolet light irradiation, and spraying with spray devices should be adopted. After that, if the negative control is normal for three consecutive days, the normal experiment can be resumed.
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