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

The nucleic acid extraction is followed by an amplification step, which is achieved by several tubes of reagents provided by the manufacturer. The detection process is called RT-PCR, which is reverse transcription-polymerase chain reaction. This is an operation that uses the nucleic acid replication mechanism that exists in nature to achieve the in vitro replication of DNA molecules. It has been invented for nearly 40 years. It is a basic technology for molecular diagnosis and an important foundational technology for modern biology and medical progress.

In addition, the selection of the probe sequence, that is, the primer sequence, is very particular. In the sequence of about 30,000 bases in length, it takes a lot of effort to detect segment.

A group of Korean scientists recently published a comparison result, which made a relatively objective comparison of the effectiveness of the detection sites of the nucleic acid tests. The sites identified by scientists from various countries for diagnosis are different.

Can nucleic acid testing be improved? First look at the speed, a standard nucleic acid detection process, sampling is fast, and then store it in the transport medium, when the sample is stored to a certain amount, concentrated inactivation, 56 degrees, 30 minutes, and then nucleic acid extraction. The nucleic acid extraction is about 30-90 minutes, and the PCR reaction is 40-60 minutes. This can be done with a batch of samples. After they come out, you can quickly determine the results. The whole experiment is about half a day. Can this process be accelerated? If it is a single sample, do it directly after sample collection, you can skip many steps, such as putting it directly in the lysate, and then doing rapid nucleic acid extraction, or even doing rapid PCR without extraction, which may take about 30 minutes. It can be seen that PCR detection itself can be done quickly, especially when the sample volume is small, but it is better to operate batch by batch when the samples are more efficient.

In addition to NEAR, there are many isothermal amplification reactions that can be used for nucleic acid detection. For example, a relatively well-known LAMP loop mediates isothermal amplification, which was invented by Japanese scientist. This method also uses four or six primer sequences to add to the substance to be tested.

There are other methods, one of which is RPA, which is relatively easy. It uses a combination of two enzymes, recombinase and polymerase, put together, through a very complex cycle, to achieve the amplification of nucleic acid molecules, although complex but very efficient and very fast. Based on RPA, , a Chinese scientist in Boston, United States, combined it with gene editing and grafted it to achieve RNA detection. They named it SHERLOCK.

Comparing these two methods, they can achieve 30 to 50 minutes of detection, which are relatively fast. These two methods can read the results in the form of test strips, and are easily understood by everyone, without the need for complicated instruments.

In addition to shortening the time, for large-scale testing, it is very important to increase the throughput of the test. The kit can be used to carry out multiple tests, but the samples must be tested one by one. There is also the work of nucleic acid extraction, which is particularly physically intensive and requires experience, skills, and training. It is best to leave it to a machine that is tireless and not easy to make mistakes. Therefore, we see a large number of automated nucleic acid extraction instruments appearing on the testing site. Such instruments are found in designated hospitals and disease control centers at all levels.

As the epidemic progresses, more and more testing needs emerge. As home isolation becomes the norm, can self-testing and self-sampling be feasible?

Less than a month ago, scientists at Yale University in the United States published a paper comparing the effects of PCR nucleic acid detection on saliva and nasopharyngeal swabs. Experimental results show that, compared with nasopharyngeal swabs, the stability of saliva sampling is quite good, even better. What’s more interesting is that the results seen on most patients have more viruses detected in saliva. The results may be more accurate and reduce the occurrence of false negatives. This is a good starting point, so a few days ago, Rutgers University in New Jersey and their affiliated testing agencies obtained approval from the U.S. Food and Drug Administration to start using samples collected from saliva for the detection of new coronavirus. They used an automated machine with large-scale pipeline processing, which can test 10,000 samples per day. Saliva sampling detection┬áis a very encouraging new method, and if it is promoted, it is expected to well solve the problems we faced in the past sampling.

Antibody detection

First of all, the samples are very different, they are all tested with blood or serum. Antibody detection methods are diverse; the most common are enzyme-linked immunosorbent assay and immunochemiluminescence. The principle of these two methods is very similar, and it is also the most common detection method in laboratory research. Taking the principle of enzyme-linked immunosorbent detection as an example, we first lay a layer of artificially prepared antigen on the detection substrate, and then add the serum sample. After incubation for a period of time, it specifically binds to the antibody; then we elute it, leaving specifically bound antibody; at this time, add enzyme-labeled secondary antibody, the secondary antibody can recognize immune antibody, and it also has catalytic enzyme on it; then incubate for a period of time, leaving specific secondary antibody after elution, Adding a substrate will enzymatically catalyze, which will lead to the development of color reaction, so that the color of the solution will change. Finally, we use the concentration of the substance that produces the color reaction to derive the concentration of the antibody you want to detect. This method has a simple principle and specificity is ensured by antibody recognition. It does not require particularly complicated operations. It can be automated or semi-automated, and the experimental throughput is not low.

Another method is to use colloidal gold color test paper detection method.

The reason why the colloidal gold color test is done is because this experiment can be applied to large-scale screening. Many places have to investigate the infection rate. If the infection rate is high and most people have antibodies, the virus will not spread easily. If the infection rate is very low and most people do not have antibodies, the virus will spread easily. This is to better prevent the next epidemic.