Available Methods to Rapid Detection of Novel Coronavirus

Posted by beauty33 on March 19th, 2020

The background

The outbreak caused by the new/novel coronavirus (2019-nCov, SARS-CoV-2, or COVID-19) has attracted the attention of people throughout the world. Among them, there is a question that has drawn widen attention: why the number of confirmed cases are increasing rapidly since January 18, 2020? How can the novel coronavirus be detected quickly?

In this article, some questions are discussed and explained.

The pathogen of this unexplained case of viral pneumonia was initially determined to be "novel coronavirus". Whether it can be detected quickly and accurately becomes the key to prevention and control.

Prior to the emergence of specific diagnostic methods, suspected cases could be found based on patient signs. Unfortunately, the specificity of this new type of coronavirus pneumonia is not strong. Compared with SARS, the onset of pneumonia was not urgent, and some patients did not even have a high fever. In the flu season, it is very difficult to distinguish patients with new coronaviruses from.

On January 16, the first batch of PCR kits for the new coronavirus was delivered to provincial CDCs, which was the direct cause of the rapid increase in the number of confirmed cases in recent days.

The Coronavirus Detection PCR Kit works roughly by extracting RNA from patient samples, performing reverse transcription-polymerase chain reaction (RT-PCR), and amplifying trace amounts of virus information in the samples by amplification reactions, and finally read the signal fluorescently. If the signal is positive after PCR, it can be said that the virus is present (infected) in the sample, otherwise it is not infected.

1. How long does a nucleic acid test take?

It takes about 16 hours at the beginning (the data in this article is estimated based on the normal laboratory operation time and does not represent the actual time).

The new coronavirus that caused this pneumonia, like its close relatives SARS and MERS, its genetic information is composed of single-stranded RNA. To complete its detection, at least two steps are required to extract viral RNA and reverse transcription PCR (RT-PCR). Extracting viral RNA itself also involves multiple steps such as lysing the sample and purifying the RNA, which can take several hours. RT-PCR generally takes at least three or four hours to complete. If the entire process line is operated, it will take about one working day, that is, about 6-8 hours. The test results need to be reviewed, and repeated experiments will take twice the time.

Based on this, some organizations have developed a simplified version of the kit, which can reduce the time of a single test to about 3 hours.

2. How to develop and produce kits?

Some people might wonder why did it take so long to develop and produce a kit after a month or so from the discovery of the first patient to the delivery of the kit?

Let's take a look at the development process of the kit.

Pathogen isolation and investigation: The pathogen that caused the outbreak is a brand new coronavirus. Before confirming that this is a pathogen that has never been seen, we need to exclude all known pathogens: including all types of influenza viruses (influenza A, avian flu, etc.), adenoviruses, rhinoviruses and Coronaviruses that cause pneumonia (SARS, MERS), Chlamydia, Mycoplasma, etc.

Design primers: This is a very critical step. Only by designing appropriate primers can the PCR reaction for virus detection be performed. Scientists need to perform genome-wide sequencing and bioinformatics analysis on this newly found virus, design primers (that is, a DNA sequence), verify its specificity and sensitivity, and ensure that it can only recognize new coronavirus genes.

Industrialized production: It can be said that all components in all viral nucleic acid detection kits are the same except for primers, so the synthesis of a large number of appropriate primers is the key to industrialized production kits.

Pathogen isolation and screening takes at least a week, and sequencing and bioinformatics analysis take about three days. Probe design and specificity checks take another two or three days. In other words, it takes almost two weeks to put the designed primers into industrial production when everything is going well.

In addition to nucleic acid testing, protein testing is also on the way.

In addition to the current nucleic acid detection methods used in health systems, detection techniques based on viral proteins can also play a significant role in the rapid detection of pathogens.

In addition to the genetic information carried by DNA / RNA, the protein shell of the virus also carries a large amount of information about the structure and pathogenic mechanism of the virus. It can also determine whether the patient is infected with the virus and whether it is immune to the virus.

Nucleic acid detection relies on primers, and detection of viral proteins mainly depends on antibodies. As long as an antibody with sufficient affinity and specificity is found, the tedious steps of extracting viral RNA can be omitted and the protein immunoassay can be directly performed using the serum or sputum of the patient at the onset of disease (generally enzyme-linked immunosorbent assay, ELISA). The results will get in 3-5 hours. If a new-generation immune reactor based on a microfluidic platform is used, the total detection time can be shortened to 30 minutes under the condition of ensuring detection sensitivity, and real-time diagnosis can be truly achieved.

The difficulty in the development of protein-based virus detection lies in the production of antibody screening. The production of experimental antibodies is highly dependent on animals (generally produced by animals such as mice, rabbits, and sheep). A few days after the injection of the antigen, the animal's immune cells need to be screened to find lymphocytes that can be used to produce highly specific antibodies and expand them. Even at full speed, it will take 2-3 weeks to get the initial available monoclonal antibodies. Compared to RNA-based PCR kits, protein detection kits require longer development time and higher production costs.