About coronavirus detection methods

Today, a global pandemic affects the world and it is the defining global health crisis of our time. Since its emergence late last year, the virus has spread to every continent in the world. Our governments are taking stronger measures to tackle the spread of the COVID-19 pandemic. At Biofortis Mérieux NutriSciences, we are closely monitoring the COVID-19 issue and we will continue to do so (check our continuity plan). That’s why, today most of the Biofortis teams are teleworking, in order to slow down the spread, and not to complicate the work of our healthcare staff even more.

Biofortis regularly talks to you about microbiomes, NGS and other aspects of molecular biology analyzes.

Did you know that the first tests set up to detect coronavirus in patients were molecular biology tests?

Coronaviruses

Coronaviruses were discovered in the 1930s and take their name from the Latin term corona (crown) since the envelope surrounding the particle is covered with widely spaced surface projections of glycoproteins (Figure 1).

Some are pathogenic for humans and others for cats, or dogs, or pigs, or chickens, or turkeys, etc. Even if each virus is mainly related to one host, they can cross the species barrier and jump from one species to another. Understanding the pathology often requires to identify the original host of the pathogen, as for Human Immunodeficiency virus that derives from a chimpanzee virus or for avian influenza that jumps from bird to human.
Although they have been known for a long time, coronaviruses have become infamous in recent years with the epidemics of SARS (severe acute respiratory syndrome caused by SARS-CoV) in 2002-2004 and MERS (Middle East respiratory syndrome coronavirus outbreak caused by MERS-CoV) in 2012, 2015 and 2018. These recent events are found in the evolution of the number of scientific articles where the 3 epidemics clearly appear (Figure 2).

The causative agent of the COVID-19 (coronavirus disease 2019) was designated SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) by the Coronavirus Study Group of the International Committee on Taxonomy of Viruses. Since COVID-19 is a respiratory disease, viruses are mainly detected in the respiratory tract, but may be found in other biological matrices such as stool or urine less frequently. As the other coronaviruses, SARS-Cov-2 is an enveloped virus with a positive-sense single-stranded RNA genome. In vitro diagnostic (IVD) tests can target different elements of the virion (virus particle) but the genome is currently the main, by using molecular biology methods.

IVD Test

Within the whole analytical process of the IVD test, the following 3 steps will be required (Figure 3):

1 – Biological sampling of upper respiratory specimens (ex. nasopharyngeal swab) and/or lower respiratory specimens (ex. sputum). The validity of the analysis requires a standardized sample taken by a healthcare professional and then stored under conditions that guarantee the integrity of the sample.
2 – Sample lysis in order to break the capsid and the envelope and make the viral genome reachable (an optional step is to isolate the viral RNA from other molecules). Since RNA is more fragile than DNA, the extraction method must make it possible to obtain RNA of sufficient quality to allow its possible detection.
3 – Detection method

Regarding the detection way, several molecular biology methods can be used, with mainly the real-time reverse-transcription polymerase chain reaction (rRT-PCR or RT-qPCR). The principle is to use enzymes capable of first copying specific regions/genes of viral RNA as complementary DNA (cDNA), then reproducing these cDNA millions of times, transforming this increase in the number of copies into a fluorescent signal characteristic of a positive result.
The viral genes targeted so far include the N, E, S and RdRP genes and depending on the method used, one or a combination of them is amplified in the rRT-PCR reaction. As for other IVD methods, the goal is to be as sensitive and specific as possible.

• Sensitivity refers to the capability to detect coronavirus in all infected patients / No detection in an infected patient is a false negative.
• Specificity refers to the capability to give positive results that come only from SARS-CoV-2 and not from other pathogens / A positive result coming from other pathogen is a false positive.

Note that the enzyme used to produce the cDNA belongs to the reverse transcriptase enzyme family. Reverse transcriptases were discovered independently by H Temin and D Baltimore in 1970 from two RNA tumour viruses. These are the enzymes that allow certain viruses to enter the host genome during the infectious process. It is therefore viruses that have given us a tool to better detect them.

Finally, if molecular biology tests allow us to detect coronaviruses, the following tests to be carried out must be able to take a look at our immune system: to see if our immune system has already encountered the virus, and especially to see if the system has kept this experience, so making us immune to this virus.

– Thomas Carton, Scientific Operations Manager, Biofortis Mérieux Nutrisciences –