Coronavirus update: there are total 3.66 million (data from worldometer corona) people infected and confirm coronavirus case in worldwide. It will be increasing day by day, deaths coronavirus numbers 257,301.The detection kit are very important by each country to improve and fast screen suspected case. The popular detection method is nucleic acid test, the second is covid 19 antibody test, the newest test is for covid 19 antigen detection. What’s the different 3 types test coronavirus? How they work and testing principle? This article will tell you what you concern.
Today, the National Medical Products Administration (NMPA) has approved more than 113 kinds of novel coronavirus emergency medical devices from 113 companies who has CE mark or FDA approval. Among that, 80 kinds for nucleic acid detection and 33 kinds for covid 19 antibody test. Nucleic acid and serum antibodies (or antigens) detection are main methods and test procedures of novel coronavirus confirm and patient diagnosis. What are the specimen required and operation procedure? And what troubles are we facing now?
Nucleic acid detection has the characteristics of early diagnosis supporting, high sensitivity and specificity. It is also the "gold standard" for the diagnosis of novel coronavirus-infected pneumonia. The most widely method is Real-Time Polymerase Chain Reaction (RT-PCR). In general, the positive result of SARS-CoV-2 is based on the same specimen which are on the ORF1ab and N genes and meet the dual target positive, or single target positive from repeated detection, or both samples meet the single target at the same time.
The virus gene as the detection target let the selected target DNA sequence increases exponentially by PCR amplification channel. Each amplified DNA sequence can be combined with a fluorescent probe which we added in advance. The more target genes amplified, the stronger the accumulated fluorescent color. In samples without virus, there is no amplification of the target gene, so no stronger fluorescence signal can be detected. Therefore, nucleic acid detection is actually to determine the nucleic acid in the sample by fluorescent signals accumulating.
The specimens are generally nasal swabs, pharyngeal swabs, nasopharyngeal swabs, sputum, bronchial lavage fluid, alveolar lavage fluid (BALF), etc.
There are five steps: sampling, sample retention, storage, nucleic acid extraction, and computer testing. All of these procedures require rigorous scientific experiments:
1. Sampling. Wipe the posterior pharyngeal wall and bilateral pharyngeal tonsils with a pharyngeal swab 5-10 times, and rotate the swab continuously
2. Sample retention. Immerse the head of swab into the cell preservation solution, then break the end of swab and tighten the tube cap immediately as sample retention.
3. Storage. Put the sample tube in a sealed bag and send it for inspection in time. Ensure the storage environment at 2-8 degrees℃ when transiting to inspection.
4. Nucleic acid extraction. Extract the nucleic acid from the inactivated virus sample for subsequent detection, you can use automated equipment, such as nucleic acid extraction instrument.
5. Fluorescence PCR nucleic acid detection, that is, testing on the machine, it will spend 70-80mins to finish the PCR reaction.
Therefore, adding IgM and IgG antibody when the nucleic acid test is negative, can make up for the shortcomings of missed diagnosis.
After 7 days of the coronavirus pneumonia infection, serum-specific antibodies are gradually produced, first the immunoglobulin (IgM) antibody and then IgG antibodies. Therefore, IgM antibody indicates a recent acute infection, and an increase in IgG antibody indicates an infection history.
Serum testing was edited into the Diagnostic and Treatment Program of COVID-19 (7th Trial Version), because it has the biggest advantage of rapid and convenient testing, which can break through the existing limitation of personal and place, and shorten the testing time. It can be diagnosed as COVID-19 infection if there are suspected positive of the serum-specific antibodies IgM and IgG, or the IgG antibody changes from negative to positive, or the recovery period is 4 times or higher than that in the acute period.
This test kit is based on colloidal gold immunochromatography assay, the colloidal gold-labeled recombinant novel coronavirus antigen and quality control antibody gold marker are sprayed on the binding pad; two detection lines (G and M) and one quality control line (line C) are coated on the NC membrane. These tests card runs the liquid sample along the nitrocellulose membrane, if there are COVID-19 IgM antibodies, the antibodies will combine with the marked virus antigens and become the sandwich conjugate, the positive result can be read. And if there are COVID-19 IgG antibody in the sample, the antibodies will combine with the marked virus antigens and become the sandwich conjugate, and then the positive result will appear by the same way. The test card also has a quality control line (line C) to judge whether the chromatography process is success.
The specimens are generally blood, includes serum, plasma or whole blood.
1. Open the aluminum foil bag, take out the test card and place it horizontally on the desktop;
2. Use a pipette to draw the serum / plasma / whole blood sample and add it to the sample well, and then draw the buffer solution into the sample well.
3. Wait 15 minutes, then read the results.
1. False positives result. Some blood sample of the patient has the rheumatoid factor, heterophile antibodies, autoantibodies, drugs and tumor cells, etc., which are easily cause cross-reactions in the test, and then, there will have false positive results occasionally.
2. False negative result. The window period of the serum antibody test method and the different sensitivity of the test kit will also cause a false negative result.
Therefore, the serum antibody detection is only used as a supplementary test for suspected cases of negative nucleic acid test of COVID-19, and cannot be applied as a diagnostic indicator for screening alone. The combined use of serum antibody detection and nucleic acid detection can help improve the detectable rate of the COVID-19, find out the diagnosed patients as much as possible, and be more conducive to the control of the epidemic.
The COVID-19 antigen detection can directly detect whether the human sample contains the novel coronavirus. The diagnosis is fast, accurate, and requires low equipment and personnel. The Sandwich ELISA let two antigen-specific antibodies to identify and bind with one target though different epitopes, which can greatly reduce the cross-reaction and improve the test specificity.
The antigens such as N protein, E protein and S protein of the novel coronavirus can be used as immunogens to stimulate plasma cells to produce specific antibodies after the virus infects the human body. According to the principle Sandwich ELISA, when the sample is dropped on the sample pad, it will flow through the conjugation pad, the test line (T) and the quality control line (C) on the NC membrane.
The conjugation pad contains a labeled antigen-specific antibody, which can bind to the antigen (viral protein) in the sample. When the sample reaches the test line (T line), the second antigen-specific antibody fixed on this line will combined with the antigen again, the positive result will be read. The quality control line (C line) is coated with IgG antibody and can be combined with the antibody in the sample pad to judge whether the chromatography process is success.
The specimen are generally from infection sites, such as oropharyngeal swabs, nasopharyngeal swabs, sputum, serum, plasma, etc.
1. Drop the sample processing into the sample tube;
2. Stir the sampling swab and squeeze against the tube wall, so that the specimen is fully mixed with the treatment solution;
3. Prepare the test card and add the sample to the sample well;
4. Wait 15 minutes, then read the results.
1. False negative. Antigen detection requires higher sensitivity, because the novel coronavirus always infect the lower respiratory tract such as alveoli. If you collect the sample upper respiratory tract such as nasopharynx and oropharynx may not be able to get pathogens or enough COVID-19 virus and cause missed diagnosis;
2. The preparation process is difficult and time-consuming. It takes about two or three months to prepare the recombinant antigen, and then prepare the monoclonal antibody in mice. If the prepared antibody has poor performance, it needs to be prepared for two or three months again.
Therefore, the antigen detection method is not yet mature, and currently no antigen detection kit has been approved and registered by the National Medical Products Administration
The novel coronavirus nucleic acid / antibody / antigen detection has its own points and cannot be replaced by each other. A variety of detection methods can be applied together as complement, which can combine the molecular biology with immune detection, giving full play to their respective advantages, improving sensitivity and specificity. All of these can shorten the detection window period effectively, increase the positive detection rate, and provide double protection for all possible risk groups.
Reference:
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