Development of Diagnostic Tests for Detection of SARS-CoV-2

Abstract

One of the most effective ways to prevent the spread of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is to develop accurate and rapid diagnostic tests. There are a number of molecular, serological, and imaging methods that are used to diagnose this infection in hospitals and clinical settings. The purpose of this review paper is to present the available approaches for detecting SARS-CoV-2 and address the advantages and limitations of each detection method. This work includes studies from recent literature publications along with information from the manufacturer's manuals of commercially available SARS-CoV-2 diagnostic products. Furthermore, supplementary information from the Food & Drug Administration (FDA), Centers for Disease Control and Prevention (CDC), and World Health Organization (WHO) is cited. The viral components targeted for virus detection, the principles of each diagnostic technique, and the detection efficiency of each approach are discussed. The potential of using diagnostic tests that were originally developed for previous epidemic viruses is also presented.

Keywords: COVID-19; SARS-CoV-2; diagnostic tests; disease detection; viral infections.

Figure 1

(A) The schematic of the SARS-CoV-19 virus displays a spherical structure. It contains spike surface (S) proteins, envelope (E) proteins, and matrix membrane (M) proteins on the outer surface. The association between single-stranded RNA and nucleocapsid proteins is included in the inner surface. (B) The genomic structure of SARS-CoV-2 is shown from the 5′ end to the 3′ end. The open reading frame (ORF)1ab gene, S gene, and N gene are typically used for nucleic acid testing. Some laboratories and commercial companies target RdRp and Hel enzymes. S proteins and N proteins are typical targets for serological COVID-19 tests.

Figure 2

The number of commercial companies that use different target regions for designing the SARS-CoV-2 virus sequence in nucleic acid testing. The most common genes are the nucleocapsid gene, envelope gene, spike gene, ORF1ab gene, membrane gene, replicase polyprotein 1ab gene, and non-structural protein 2 gene. Among these, the most prevalent targeted gene is the nucleocapsid gene (73/163 companies). The scientific data was collected from the FDA website on 30 September 2020.

Figure 3

The main principle of the transcription loop mediated isothermal amplification (RT-LAMP) technique. RT-LAMP starts with the reverse transcription of the backward inner primmer (BIP). The BIP primer binds to the target sequence on the 3′ end of the RNA template and synthesizes a copy of the DNA strand (cDNA). Then, by using DNA polymerase, B3 primers bind to the side of the templates, generate the new cDNA strand, and release the first cDNA strand. This single strand of cDNA is then looped at the 3′ end and binds to itself. Next, the forward inner primmer (FIP) binds to the 5′ end of the strand and synthesizes a complementary strand by DNA polymerase. Then, the F3 primer binds to the end and generates a new double strand of DNA by DNA polymerase. The loop keeps running as a dumbbell-structure when the FIP or BIP primer initiates DNA synthesis again at the next target sequence location. The cycle can start at either the forward or backward side of the strand. When it starts, the strand undergoes self-primed DNA synthesis during the elongation stage of the amplification process. This amplification can be done in a short amount of time (approximately 1 h) and occurs at a constant temperature between 60–65 °C. Adapted by permission. Copyright of Linda C. et al. [66].

Figure 4

The principle of the clustered regularly interspaced short palindromic repeats (CRISPR)-based technique for the detection of SARS-CoV-2. CRISPR/Cas12 and CRISPR/Cas13 are used to detect the viral RNA of SARS-CoV-2. The Cas13 complex binds to the target sequences. This binding activates the general nuclease enzyme activity of Cas13 to cleave the target sequence. The RNA is then detected by a fluorescence signal. The activity of Cas12 is similar to the activity of Cas13. One difference between the two is the position of the nuclease enzyme. Adapted by permission. Copyright of Barrangou R. et al. [83].

Figure 5

Lateral flow immunoassay for the detection of anti-SARS-CoV-2 antibodies [21]. The sample fluid flows laterally along the strip, which includes different zones such as conjugation pad, adhesive pad, and absorption pad. The conjugation pad includes antibodies for the target analyte and antibodies conjugated with signal molecules (fluorescent particles and gold particles). The nitrocellulose membrane contains the testing lines (IgG and IgM lines) and the control line. The last zone is the absorption pad, which prevents the backflow of the liquid. Adapted by permission. Copyright of Li Z. et al. [21].