About nucleic acid detection，how much do you know?
Since the outbreak of COVID-19, nucleic acid testing has become a high-frequency word. A positive nucleic acid test is an important criterion for diagnosing COVID-19. WST-8 has become the API for nucleic acid testing kits. How much do you know about nucleic acid testing? What is the principle of nucleic acid detection? Why do nucleic acid tests have false negatives? In addition to nucleic acid testing, what other technologies can detect the new coronavirus?
Table of Nucleic Acid
Demystifying the principle of nucleic acid detection of new coronavirus
Nucleic acid testing is actually to detect whether there is nucleic acid (RNA) of the new coronavirus in the body of the test subject. The nucleic acid of each virus contains ribonucleotides, and the number and order of ribonucleotides contained in different viruses are different, making each virus specific.
The nucleic acid of the new coronavirus is also unique, and nucleic acid detection is the specific detection of the nucleic acid of the new coronavirus. Before nucleic acid testing, it is necessary to collect samples of the subject’s sputum, throat swab, bronchoalveolar lavage fluid, blood, etc., and by testing these samples, it can be found that the subject’s respiratory tract is infected with bacteria. New coronavirus nucleic acid detection is commonly used for throat swab sample detection. The sample is split and purified, and the possible new coronavirus nucleic acid is extracted from it, and the preparations for the test are ready.
New coronavirus nucleic acid detection mainly uses fluorescence quantitative RT-PCR technology, which is a combination of fluorescence quantitative PCR technology and RT-PCR technology. In the detection process, RT-PCR technology is used to reverse transcribe the nucleic acid (RNA) of the new coronavirus into the corresponding deoxyribonucleic acid (DNA); then fluorescence quantitative PCR technology is used to replicate the obtained DNA in large quantities. The replicated DNA is detected and labeled with a sex probe. If there is a new coronavirus nucleic acid, the instrument can detect the fluorescent signal, and, as the DNA continues to replicate, the fluorescent signal continues to increase, thus indirectly detecting the presence of the new coronavirus.
Why do false negatives occur?
A false negative means that the person being tested has been infected with the new coronavirus, but the nucleic acid of the virus has not been detected, that is, the nucleic acid test is negative. If a subject has no relevant symptoms and the nucleic acid test is negative, it is easy to let him relax his vigilance, which may lead to a wider spread of the virus, which is also the cunning of the new coronavirus.
There are three main reasons for false negatives:
First, the nucleic acid content of the new coronavirus in the original test specimen is too low, below the lower limit of detection. This is related to the type of sample collected and the timing of collection.
Second, if the site where the new coronavirus nucleic acid binds to the probe mutates, it may affect the binding efficiency of the probe in the detection, resulting in the undetectable fluorescent signal.
Third, false negatives are also related to the sensitivity of the detection technology. In the early stage of the outbreak, the clinical kits were not mature enough and their sensitivity was not high enough, and there were frequent reports of false negatives. Later, with the improvement of the technical level, the accuracy rate of detection increased rapidly, and the false negative rate continued to decrease.
Is a positive nucleic acid test a diagnosis?
A positive nucleic acid test result can confirm the presence of viral nucleic acid in the upper respiratory tract of the subject, but it cannot be directly diagnosed as a patient with new coronary pneumonia. In the case of a positive nucleic acid test, the subject should be further diagnosed based on the epidemiological history. Contact history, etc., can be diagnosed as a confirmed case.
If the subject does not have any relevant symptoms, it is generally diagnosed as an asymptomatic infection. Asymptomatic infections are also contagious, and some asymptomatic infections later began to show symptoms and became confirmed patients.
Other technologies to detect the new coronavirus
For the subjects, the isolation of the virus from the specimen is the most authoritative basis for diagnosis. However, the isolation of the virus is a relatively dangerous operation, which needs to be carried out in a laboratory with a high level of protection, and is time-consuming and costly. . General medical institutions do not have this condition. Most of them will use the method of detecting whether there are virus antibodies in the blood to determine whether the subject is infected with the virus. Hepatitis B, AIDS, etc. are all tested in this way.
The new coronavirus can also be detected by this immunological detection method. In the face of the invasion of the new coronavirus, the human body will “resist” and produce specific antibodies IgM and IgG (immunoglobulin M and immunoglobulin G). If these two specific antibodies are detected in the blood of the test subject, it can indicate that the test subject is infected with the new coronavirus.
How to detect antibodies? The commonly used method is chemiluminescence immunoassay, that is, the use of chemiluminescence reagents or specific enzymes to label antibodies or antigens, the formation of complexes by immune reaction, and then the addition of luminescence reagents after washing. Species-specific antibodies, the chemiluminescence instrument can detect the luminescent signal. This chemiluminescence immunoassay is one of the most advanced immunoassay techniques at present. It has high sensitivity, strong specificity, wide detection range and fast results, but the detection cost is high.
In addition, because it takes a certain time for the human body to produce antibodies after being infected with the virus, it can only be detected after the antibodies are produced, which also leads to the poor timeliness of antibody detection. It takes 2 to 3 weeks after infection such as hepatitis B and AIDS to produce antibodies. It takes more than 3 days to detect IgM antibodies and more than a week to detect IgG antibodies after new coronavirus infection.
Therefore, in order to catch the tail of the new coronavirus as soon as possible, more nucleic acid detection is used, which can detect the traces of the new coronavirus earlier.