Up to now, the State Administration of drug administration has approved 29 medical and emergency medical approvals for New Coronavirus, including 18 nucleic acid detection classes and 11 serum antibodies. Nucleic acid detection and antibody / antigen detection are important means to confirm COVID-19, and also one of the important processes for patients to diagnose. But do you know what their detection principle is? What are the sample types? What is the process of inspection and what are the problems? Today, I'd like to popularize science for you~~
New Coronavirus nucleic acid detection
Nucleic acid detection is novel coronavirus pneumonia with high sensitivity and specificity. It is the gold standard for diagnosing new crown pneumonia. The most widely used method is real-time fluorescence quantitative RT-PCR. Generally, two targets located in orf1ab and N genes of SARS CoV are detected. The same sample needs to meet the requirements of double target positive or repeated detection for single target positive, or two samples meet the requirements of single target at the same time to confirm the SARS cov-2 virus nucleic acid positive.
1. Principle of nucleic acid detection kit?
The unique gene sequence of the virus is used as the detection target. By PCR amplification, the selected target DNA sequence increases exponentially. Each amplified DNA sequence can be combined with a fluorescent labeled probe that we added in advance to generate a fluorescent signal. The more target genes are amplified, the stronger the accumulated fluorescent signal will be. In the samples without virus, there is no target gene amplification, so the fluorescence signal enhancement can not be detected. Therefore, nucleic acid detection is to detect the accumulation of fluorescence signal to determine whether there is virus nucleic acid in the sample.
2. What are the types of nucleic acid test samples?
Generally, nasal swab, pharyngeal swab, nasopharyngeal swab, sputum, bronchoalveolar lavage fluid, etc.
3, five steps to test New Coronavirus
The detection procedure needs to go through five steps: sampling, sample retention, preservation, nucleic acid extraction and computer detection, which requires rigorous scientific experiments to complete.
1. Wipe the posterior pharyngeal wall and bilateral pharyngeal tonsils with pharyngeal swab for 5-10 times, and rotate the swab continuously;
2. Medical staff are required to keep the sample, immerse the swab head in the cell preservation solution, and screw down the tube cover immediately after breaking the tail;
3. Preservation: put the sample tube into the sealed bag and send it for inspection in time. In the process of sending it for inspection, strict transportation environment is required, and it should be kept at 2-8 ℃.
4. Operate nucleic acid extraction, extract nucleic acid from the sample after inactivation of virus for subsequent detection, and use automatic equipment, such as nucleic acid extractor, etc.
5. Fluorescent PCR nucleic acid detection, that is, on the machine detection, the extract for fluorescent PCR amplification reaction, takes 70-80 minutes.
4. What are the problems in nucleic acid detection?
■ false negative, due to improper sampling, improper specimen preservation, different types of specimens and the use of reagents from different manufacturers, nucleic acid test results may be "false negative" and missed diagnosis;
High requirements for detection equipment or platform, high sensitivity RT-PCR instruments are expensive, and requirements for laboratory cleanliness and operators are also high;
Nucleic acid detection takes a long time, and it usually takes 4-6 hours to complete a RT-PCR detection. However, considering the sample transportation and large sample backlog, the results can be reported as soon as 24 hours.
Therefore, when nucleic acid detection is negative, adding IgM and IgG antibody detection can make up for the shortcomings of nucleic acid detection which is easy to cause missed diagnosis.
Detection of serum antibody of New Coronavirus
7 days after the onset of New Coronavirus pneumonia, the serum specific antibody was gradually produced. The first immunoglobulin IgM antibody appeared and then the IgG antibody appeared. Therefore, the increase of IgM antibody indicates recent acute infection, and the increase of IgG antibody indicates previous infection.
The biggest advantage of serological detection is that it is convenient and quick, and the detection time is short. It can effectively break the limitation of the existing detection technology to personnel and places, shorten the detection time, and is written into the New Coronavirus pneumonia diagnosis and treatment plan (Trial Seventh Edition). If the serum specific IgM and IgG antibody of the suspected case is positive, the IgG antibody changes from negative to positive, or the recovery stage is 4 times or more higher than the acute stage, it can be diagnosed that the suspected case is infected with new coronavirus.
1. Principle of serum antibody test kit?
Colloidal gold immunization technology was used to spray colloidal gold labeled New Coronavirus recombinant antigen and quality control antibody gold marker on the pad. The NC membrane was coated with two detection lines (G line and M line) and a quality control line (C line). The M-line was coated with mouse anti human IgM monoclonal antibody, the g-line was coated with mouse anti human IgG monoclonal antibody, and the c-line was coated with quality control antibody. When the sample is added to the sampling hole, the sample will move forward along the detection card under the action of chromatography. If the sample contains a new coronavirus IgM antibody, the antibody will combine with the colloidal gold labeled virus antigen to form a sandwich complex, which will present a positive result. If the sample contains New Coronavirus IgG antibody, the antibody will bind to colloidal gold labeled New Coronavirus antigen and form a sandwich complex, which will show positive results. The card also contains a quality control line (C line) to judge whether the chromatographic process is smooth.
2. What are the types of serum antibody test samples?
Generally, it is blood, including serum, plasma and whole blood.
3, three steps to test New Coronavirus
1. Open the aluminum foil bag of the test card, take out the test card and place it horizontally on the desktop;
2. Pipette the serum / plasma / whole blood sample and add it to the sample adding place, then use the same method to suck the buffer solution and add it to the sample adding hole of the test card.
3. Wait for 15 minutes to judge the result.
4. What are the problems in serum antibody detection?
False positive: some patients have rheumatoid factor, heterophilic antibody, autoantibody, drugs and tumor cells in their blood, which can easily cause cross reaction of the test, so false positive results are occasionally found;
False negative results may occur due to certain window period of serum antibody detection methods and different sensitivity of detection kits.
Therefore, serum antibody test can only be used as a supplementary test for suspected cases with negative nucleic acid test of NCV, and can not be used as a diagnostic index for screening. The combined use of serum antibody detection and nucleic acid detection is helpful to improve the detection rate of disease, find out the diagnosed patients as much as possible, and is more conducive to the control of the epidemic situation.
Interpretation of the results of nucleic acid combined with IgG and IgM antibodies
New Coronavirus antigen detection
New Coronavirus antigen detection can directly detect whether the human body contains New Coronavirus. The diagnosis is fast, accurate, and requires less equipment and personnel. Using double antibody sandwich method and using two antigen specific antibodies to identify and combine different epitopes of a target antigen, the probability of cross reaction can be greatly reduced, thus improving its specificity.
1. Principle of antigen detection kit?
New Coronavirus's N protein, E protein and S protein can be used as immunogen and stimulate the plasma cells to produce specific antibodies after the virus infects the human body. According to the principle of double antibody sandwich ELISA, the sample was dripped on the sample pad, and then passed through the binding pad, the detection line (t line) and the quality control line (C line) on the NC membrane by liquid chromatography. The binding pad contains labeled antigen-specific antibody, which can bind with the antigen (virus protein) in the sample. When the liquid flow reaches the detection line (t line), the second antigen-specific antibody fixed on this line will bind with the antigen again, which will show a positive result. The quality control line (C line) is coated with IgG antibody, which can be combined with the antibody in the sample pad to judge whether the chromatographic process is smooth or not.
2. What are the types of antigen test samples?
It is usually the sample of infection site, such as oropharyngeal swab, nasopharyngeal swab, sputum, serum, plasma, etc.
3, four steps to test New Coronavirus
1. Drop the sample treatment solution into the sample treatment tube;
2. Stir the swab and squeeze the tube wall, so that the sample can be fully eluted into the treatment solution;
3. Take out the test card and add the sample to the round hole;
4. Wait for 15 minutes to judge the result.
4. What are the problems in antigen detection?
False negative, antigen detection requires more sensitivity. Because New Coronavirus mainly invades the lower respiratory tract such as alveoli, it is not always possible to take pathogens from nasopharynx or oropharynx and other upper respiratory tract.
The preparation process is cumbersome and time-consuming. It takes about two or three months to prepare the recombinant antigen first, and then to prepare the monoclonal antibody in mice. If the prepared antibody has poor performance, it needs to be prepared again, and it takes another two or three months.
Therefore, the antigen detection method is not mature, and no antigen detection kit has been approved and registered by the State Food and drug administration.
Joint detection
New Coronavirus nucleic acid / antibody / antigen detection has different focuses, and can not be replaced each other. A variety of detection methods are applied together, complement each other, combine molecular biology and immune level detection, give full play to their advantages, improve sensitivity and specificity, effectively shorten the detection window period, improve the positive detection rate, and provide a dual guarantee for all possible risk groups.
References: ZhengTu Li, Yongxiang Yi et al. Development and clinical application of a rapid IgM ‐ IgG combined antibody test for SARS ‐ cov ‐ 2 infection diagnosis. J Med Virol. 2020; 1-7
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