Clean, Controlled Lab for IVD and Research on SARS-CoV-2

Eurofins Genomics operates 6 facilities worldwide including operations in Europe, United States, Japan, and India. Each production facility is producing SARS-CoV-2 material for IVD and research use under stringent quality controls. Eurofins Genomics is accredited under ISO 13485, ISO 9001, and GMP, and it has additional CLIA and CAP accredited labs for clinical projects. In addition, all gene and gene fragment orders are produced in completely separate facilities, isolating those sequences from DNA oligo production to reduce the risk of cross contamination. Lastly, Eurofins Genomics oligo production facilities have taken extra, precautionary measures to segment the downstream workflow and create different tracks for any product that could potentially cross over, so that these tracks never cross or use the same equipment. Eurofins Genomics is proud to be a leading supplier of testing material during the SARS-CoV-2 crisis.

The coronavirus SARS-CoV-2 and resulting disease COVID-19 have reached almost every country on Earth… but compared with other infectious diseases such as the flu, there are still many unknowns about SARS-CoV-2. Scientists around the world are tirelessly researching the virus in order to understand it better and endeavor to find treatments. What is currently known about the genomics of SARS-Cov-2 and how is the information used in diagnostics?

What kind of virus is SARS-CoV-2?

SARS-CoV-2 is a RNA virus and belongs to the family of Coronaviridae and the genus Betacoronavirus. Its genome consists of a single-stranded positive-sense RNA. The peculiarity of single-stranded positive-sense RNA is that it can directly function as mRNA in the host cell and, therefore, is directly translated. In contrast, the RNA of a negative-sense single-stranded RNA virus first needs to be converted into positive-sense RNA in order to be translated (Figure 1).

What are the genes of SARS-CoV-2?

The RNA genome of SARS-CoV-2 is approximately 30,000 nucleotides long and encodes 27 non-structural proteins and 4 structural proteins. The 27 non-structural proteins include RNA-dependent RNA polymerase (RdRP) that was found to be highly similar to parts of the RdRP gene of coronavirus RaTG13 in bats. Other non-structural proteins are proteases and helicases for instance. The 4 genes encoding structural proteins that essentially form the envelope of the virus are: 1. the spike surface glycoprotein of the virus (S), 2. the matrix protein (M), 3. the nucleocapsid protein (N), and 3. the envelope protein (E) (Figure 2).

The spike proteins on the surface of the virus seem to mediate the binding of the virus to the angiotensin converting enzyme 2 (ACE2) receptor of host cells and the entry into the host cells. The M protein seems to be involved in formation of the spherical shape of the virus. The N protein also called nucleoprotein is associated with the single-stranded RNA and together they form the nucleocapsid. The E protein has been found to be the smallest of the 4 structural proteins. The genomic composition of SARS-CoV-2 has been analyzed and the sequence is publicly available at the GenBank sequence repository. As of now, 104 strains of SARS-CoV-2 have been isolated and sequenced.

How is SARS-CoV-2 identified?

Molecular techniques have been shown to be highly suitable for the identification of SARS-CoV-2 infections. Specifically real-time RT-PCR (qRT-PCR) is utilized for the analysis of respiratory samples. Here, the RNA of SARS-CoV-2 is extracted from the respiratory samples and subsequently reverse transcribed into complementary DNA (cDNA). The cDNA is then used for real-time PCR amplification with specific primers and probes that target three regions of the viral genome:

• The RdRP gene that is situated in open reading frame ORF1ab.
• The E gene
• The N gene

It has been suggested that the RdRP gene and the E genes show slightly higher analytical sensitivity than the N gene.
For identification purposes, it has been suggested to use assays that target two regions. One primer pair for the identification of a broader range of coronaviruses including SARS-CoV-2, and one primer pair that is specific for SARS-CoV-2:

• The recommendation by a research group at Charité Universitätsmedizin Berlin, Germany, that is the basis of the WHO recommendation, is to first utilize a primer pair that targets different regions of the E gene to detect all SARS-related viruses. In case this leads to amplification, a second analysis with a primer pair that targets different regions of the RdRP gene is performed as confirmatory testing.
• The utilization of a primer pair that targets the N gene and one that targets the ORF1b/RNAse P gene to confirm the results have also been proposed and are recommended by Centers for Disease Control and Prevention (CDC) (Figure 3).

Figure 3: Genomic organization of SARS-CoV-2

qRT-PCR can be performed in a one-step or a two-step assay. The one-step assay with reverse transcription and PCR amplification in one reaction is fast and gives reproducible results for high-throughput analysis:

• The CDC utilizes a one-step qRT-PCR assay to detect a SARS-CoV-2 in samples. Here, primers and a fluorophore-quencher probe Black Hole Quencher-1 (BHQ1) and fluorescein amidite (FAM) are utilized. During amplification the fluorophore-quencher probe is cleaved, which results in the emission of a fluorescent signal.
• Charité Universitätsmedizin Berlin, Germany reported to also utilise a one-step qRT-PCR assay. Here, the probes for the assay contained Blackberry Quencher (BBQ) and 6-carboxyfluorescein (6-FAM).

The two-step assay, where reverse transcription and PCR amplification are performed in different tubes, is generally more sensitive but requires more time.
The utilization of qRT-PCR assays requires positive controls for valid and reliable analyses.

What are the positive controls for SARS-CoV-2 identification by qRT-PCR?

A range of validated positive controls as plasmids that include all genes of SARS-CoV-2 as recommended by the CDC and WHO for example is also provided: Control Plasmids for Coronavirus Research.

References

Andersen, K.G., Rambaut, A., Lipkin, W.I., Holmes, E.C., Garry, R.F. (2020) The proximal origin of SARS-CoV-2. Nat Med. 26:450-2.
Corman VM, Landt O, Kaiser M, Molenkamp R, Meijer A, Chu DKW, Bleicker T, Brünink S, Schneider J, Schmidt ML, Mulders DGJC, Haagmans BL, van der Veer B, van den Brink S, Wijsman L, Goderski G, Romette JL, Ellis J, Zambon M, Peiris M, Goossens H, Reusken C, Koopmans MPG, Drosten C. (2020) Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Euro Surveill. 25(3):2000045.
Phan, T. (2020) Genetic diversity and evolution of SARS-CoV-2. Infect Genet Evol. 81:104260 Udugama, B., Kadhiresan, P., Kozlowski, H.N., Malekjahani, A., Osborne, Li, M., V.Y. C., Chen, H., Mubareka, S., Gubbay, J.B., Chan, W.C.W. (2020) Diagnosing COVID-19: The Disease and Tools for Detection. ACS Nano [Online ahead of print].