Recently, as the COVID-19 strain has mutated, its toxicity and infectivity have become stronger, and the global epidemic situation has become extremely serious. To prevent the spread of the virus, in addition to prevention, it is more important to control the source of infection. Nucleic acid testing can The virus lurking in the human body is detected at the first time. Nucleic acid testing is to find out whether there are nucleic acids of foreign viruses in the respiratory specimens, blood or feces of patients to determine whether they are infected by COVID-19. Therefore, once the test is “positive” for nucleic acid, it can prove that there is a virus in the patient’s body.
After COVID-19 infects the human body, it will first reproduce in the respiratory system. Therefore, the virus nucleic acid in sputum and nasopharyngeal swabs can be tested to determine whether the human body is infected with the virus. Therefore, a positive nucleic acid test can be used as a new standard for the diagnosis of COVID-19 infection.
Principles of COVID-19 nucleic acid detection
The most common method to detect the specific sequence of COVID-19 is fluorescence quantitative PCR (polymerase chain reaction). Since the PCR reaction template is only DNA, the COVID-19 nucleic acid (RNA) should be reverse transcribed into DNA before the PCR reaction. In the PCR reaction system, a pair of specific primers and a Taqman probe are included. The probe is a specific oligonucleotide sequence with a reporter fluorophore and a quencher fluorophore labeled at both ends. When the probe is complete, the fluorescent signal emitted by the reporter group is absorbed by the quencher group; if there is a target sequence in the reaction system, the probe binds to the template during the PCR reaction, and the DNA polymerase uses the enzyme’s exonuclease activity to transfer the probe along the template. Enzyme digestion and degradation, the reporter group is separated from the quenching group and emits fluorescence. Every time a DNA strand is amplified, a fluorescent molecule is produced. The fluorescent quantitative PCR instrument can monitor that the number of cycles (Ct value) at which the fluorescence reaches the preset threshold is related to the concentration of viral nucleic acid. The higher the concentration of viral nucleic acid, the smaller the Ct value. The products of different manufacturers will determine the positive judgment value of this product based on the performance of their own products.
COVID-19 nucleic acid detection process
For the nucleic acid detection of COVID-19, first according to the sample requirements of the nucleic acid extraction kit instructions, some samples are collected. The conventional sample types include throat swabs, nasal swabs, sputum, bronchial lavage fluid, alveolar lavage fluid, etc.
After obtaining the patient sample, the test should be carried out as soon as possible. If the sample that needs to be transported cannot be tested immediately, it should be packaged at a low temperature according to the instructions and sent to a special testing agency for testing. After receiving the sample, the testing institution shall perform nucleic acid extraction on the sample, and the nucleic acid extraction reagent shall use the nucleic acid extraction kit specified in the approved product manual.
Viral RNA needs to be reverse transcribed into cDNA first, and then amplified and tested. PCR amplification and detection should use the fluorescent quantitative PCR instrument specified in the approved product manual. The Ct value of the sample obtained by fluorescent quantitative PCR can be used to determine whether the patient sample contains COVID-19.
In the above-mentioned process, the collection, storage, and transportation of samples, the extraction and detection of sample nucleic acids, and the interpretation of results must all be carried out in strict accordance with the requirements of the kit instructions.
“False positive” and “false negative” in nucleic acid testing
The “false positive” of the nucleic acid test means that the patient has not been infected with the new coronavirus, but the nucleic acid test has a positive result. We have also introduced to you how to improve the accuracy of the COVID-19 nucleic acid test. The occurrence of “false positives” is usually caused by cross-contamination between specimens or laboratory nucleic acid contamination during laboratory testing. On the technical level, as long as the laboratory strictly implements the quality control work, the occurrence of “false positives” can be effectively avoided.
The “false negative” of the nucleic acid test means that the patient’s clinical symptoms, lung imaging results and even epidemiological history support the new coronary pneumonia, but the patient’s viral nucleic acid test result is “negative”, and the test result is inconsistent with the clinical.
The usual causes of “false negatives” are:
(1) In the initial stage of virus invading the human body, the amount of virus in the human body has not yet reached a detectable level. In different periods of viral incubation, mild symptoms, and severe symptoms, the viral load of different parts of the human body (such as the nasopharynx, oropharynx, trachea, bronchi, and alveoli) will vary. Therefore, different sampling timing and sampling locations may result in insufficient virus in the collected specimens;
(2) Any test reagent has its lower limit of detection (ie sensitivity). If the virus in the patient’s specimen does not reach the lower limit of detection of the reagent used, a false negative will occur;
(3) Poor performance of laboratory equipment and personnel, poor quality management, etc. will also produce “false negatives”;
(4) Irregular sampling, improper collection location, and atypical collection of specimens have resulted in too few or no virus-infected cells in the specimens. That may cause “false negatives.”
The above is an introduction to the current principles and procedures of nucleic acid detection for COVID-19. It is hoped that the source of infection can be controlled to the greatest extent through nucleic acid detection and a method to suppress the virus can be found as soon as possible.