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Detection Methods of 2019 Novel Coronavirus (2019-nCoV) (I)

The detection of 2019 novel Coronavirus (2019-nCoV) is mainly divided into nucleic acid detection and antibody detection. The two detection methods are completely different, with different purposes, different sampling, different methods, different interpretations of results, and different applications.

The most common sample for nucleic acid detection is a nasopharyngeal swab, and sampling requires professional techniques. The US FDA has approved the use of saliva samples collected at home for testing.

In addition, Yale University compared the results of PCR nucleic acid detection of saliva and nasopharyngeal swabs. Experimental results show that, compared with nasopharyngeal swabs, the stability of saliva sampling is quite good, even better. This is a good starting point.

The sample for antibody detection is blood or serum, which can be tested using colloidal gold color test, similar to pregnancy test. The advantage of this method is that it is very simple, easy to use, cheap, and does not require complex equipment, and very fast.

The RNA sequencing method of nucleic acid detection greatly reduces the difficulty of the experimental operation and can make the experimental results more stable, reliable and quick to obtain. On this basis, the researchers observed interesting mutation information.

Molecular diagnosis is an important part of disease diagnosis. Disease diagnosis is a complex problem, especially when new diseases appear. For an infectious disease, molecular diagnosis becomes an extremely important indicator when the pathogen is known. Its main purpose is as follows: First, it determines the clinical symptoms, such as whether the fever is caused by the new coronavirus, to avoid confusion with other diseases. Second, qualitatively (such as positive and negative) or quantitative description of the progress of the disease, such as the amount of viral load. Third, from a more elaborate point of view, certain molecular diagnoses can also provide a treatment plan for the disease, help determine its plan, and provide evidence support. Fourthly, from the perspective of viral diseases, it is possible to track the variation, which is also very important. It is also important to study and predict the evolution of the infection and pathogenic ability of the virus during the transmission process.

What is molecular diagnosis? The narrow sense of molecular diagnosis is generally nucleic acid detection. Nucleic acid testing refers to testing whether samples contain DNA or RNA fragments of a specific sequence as evidence of the presence of pathogenic microorganisms.

In addition to this, our test for antibodies is antibody testing. Antibody testing does not look at the virus itself, but at the presence of antibody molecules in the blood or body fluids.

Another test is antigen detection, and the target is the substance on the virus.

In the final analysis, the detection is based on the measurement of molecules, and the number of molecules; this is determined by the structure and composition of the virus itself, and has its material basis. In terms of detection, in addition to nucleic acid molecules, it is one molecule in each virus, and there are many protein molecules. But the number distribution of these molecules is still different. There are probably hundreds of S proteins per virus; there are thousands of N proteins and M proteins per virus. Understanding these numbers is very helpful for us to develop specific detection techniques to determine the presence of viruses in the future.

Nucleic acid amplification testing

For nucleic acid detection, the first step is sampling.

There are two keys to ensuring successful sampling. One is that the swab used is exquisite, not just a cotton swab. At the same time, the preservation environment after swab sampling is also exquisite.

In addition, the operation of sampling itself is exquisite, and the technique is also important, and it can not be obtained with a poke in the past. For example, the most common sample is a nasopharyngeal swab. Note that the sampled swab should extend to the back of the nasopharynx, above the oropharynx. This is not an easy operation, it is very empirical and tricky, and it is difficult to do it by yourself. It is generally not recommended to sample by yourself. There are also throat swabs, and open the mouth. It is also necessary to deepen the long swab into the back of the mouth, on the side of the throat, next to the tonsils. This sampling is not something that you can simply do, you usually have to press your tongue down.

The different sampling locations are reflected in the different test results. Scientists have summarized some rules very early.

For example, as time increases, the number of nucleic acids sampled decreases. Another result is that the amount of nucleic acid collected by the nasopharyngeal swab is higher than that of the throat swab, and the stability is slightly better. The experience gained early has important implications for the subsequent sampling process, that is, how and where samples should be taken.

The characteristics of the disease lead to the characteristics of the virus infection site, which has both preferences and the influence of the time of onset. In fact, as a test, various samples can be used, not just two swabs. Clinical doctors proposed that viral nucleic acid can be detected in specimens of nasopharyngeal swabs, sputum, other lower respiratory tract secretions, blood, and feces. There are also investigations and studies showing that, after summing up so many papers, it seems that nucleic acid viruses may be detected in many samples such as tears and feces, but the sputum or lower respiratory tract secretions are the least likely to be missed. The detection rate is the highest, and the next is the nasopharyngeal swab.

After the sample is collected, the first step is usually nucleic acid extraction, which is to leave the nucleic acid part of the sample, remove other parts of impurities, human exfoliated cells, other molecules such as protein molecules and so on.

In general nucleic acid extraction, the nucleic acid molecules are first adsorbed with materials with strong affinity to it; after the other substances are separated, the adsorbed nucleic acid molecules are eluted to achieve extraction, which is also purification and enrichment. In the laboratory, either silica gel membranes are used for adsorption, or surface-modified small magnetic particles are used for adsorption. These two methods are now widely used in the detection of nucleic acids in 2019-nCoV.

To be continued in Part II…

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