**2. Biology of SARS-CoV-2**

SARS-CoV-2 belongs to the family *Coronaviridae*, similar to SARS-CoV-1. CoV-2 consists of a spherical protein structure of ~80–160 nm diameter [8] (**Figure 1**). This sphere is composed of two lipid layers placed face-to-face close to each other (bilayer). This bilayer is embedded with proteins, collectively referred to as the structural proteins. They include the envelope (E), membrane (M) and spike (S) proteins [2]. The E, M and S-proteins are embedded in the lipid bilayer forming the sphere [9]. The S-proteins stick out of the sphere prominently, giving a spiked crown-like appearance to the virus and thereby conferring the name 'corona' (crown) [2]. They bind to the ACE-2 (human angiotensin-converting enzyme 2) receptors on the host cell membrane to initiate fusion, and therefore are key in the invasion of host cells [10–12]. Encapsulated inside this sphere is the virus genome, which in the case of CoV-2, is a plus (or positive) single-stranded (ss) RNA (ribonucleic acid, ss RNA) [2]. This genetic material is spooled around the nucleocapsid (N) proteins, which are also accounted as structural proteins [2].

The genome of CoV-2 was successfully sequenced by January 2020 [13]. The ss RNA is ~30 kb (kilobases) in length [2], encompassing all the information for protein syntheses and assembly of the new virus particles (virions). There is another group of non-structural proteins (NSPs) which are involved in non-structural functions such as genetic material replication or the assembly of virions [2]. There are 16 such NSPs identified in the CoV-2 genome, which include an RNA-dependent RNA polymerase (RdRP), ExoN (exonuclease) and ORF proteins [2]. RdRP synthesizes new viral genetic material, while ExoN is responsible for genome stability and for removing any errors in the newly synthesized genetic RNA sequence. ORF proteins act as accessory proteins [2].

Viruses have been traditionally categorized in a separate class, from the biotic or living organisms. This is due to their inability to replicate in the absence of a host. In general, the virus invades the host cell, releases its genetic material and hijacks the host machinery for synthesizing its own macromolecules (nucleic acid and proteins) [14]. Upon assembly of virions (new viruses), the host cell is lysed (broken open) to release the new infectious particles [14]. The host cells try to combat the

#### **Figure 1.**

*Cartoon representing the human SARS-CoV-2 virus structure. (Left) Sphere showing the outside of the virus. (Right) A cross-section of the virus sphere depicting the membrane structural proteins (S, M and E), along with the ss RNA genetic material which is wound around the nucleocapsid (N) proteins.*

*Molecular Biology of PCR Testing for COVID-19 Diagnostics DOI: http://dx.doi.org/10.5772/intechopen.96199*

invasion by evoking an immune response specific to the virus (adaptive immunity) [15]. This is attempted through the presentation of fragments of the foreign macromolecules on the host cell surface. These non-indigenous particles, called antigens, are sensed by the immune cells which initiate the production of antibodies against them [15]. Thereafter, any particle resembling the antigen is attacked and cleared by the host immune cells. Antibodies are broadly classified into 5 main immunoglobulin (Ig) classes: G, A, M, E and D. They differ in their structures, capacity to recognize the antigen and occurrence in the course of immune response [15]. These antibodies encompass a structural region which is specific to binding the antigen (called the variable region). The immune response and components are much more complicated than this simple excerpt presented here, and the readers are encouraged to refer to other reviews on the immune system [15].

## **3. Detection of SARS-CoV-2**

The observation that the virus from asymptomatic carriers is equally infectious as those exhibiting clear symptoms of COVID-19 [4, 16], makes it imperative to identify the asymptomatic individuals in order to take appropriate measures for their seclusion and treatment. This is highly dependent on the reliability and accuracy of the diagnostic tests. Further, these assays also permit the recognition of patients with CoV-2 infections at hospitals where it is crucial for their segregation into the COVID-19 specific wards. This is important to prevent further transmission of the virus to admitted and highly vulnerable patient populations. Reliability of a diagnostic test depends on its specificity and sensitivity. Specificity is the ability of the test to correctly detect the negative samples as negative, thus reducing false-positive results [17]. On the other hand, sensitivity is the ability of the test to correctly identify the positive cases as positive, thereby decreasing false-negative results [17]. It is essential that a test is dependable for both these features. False results, either way, will aid in the spread of the virus, and misdirect contact tracing.

#### **Figure 2.**

*Categorization of available CoV-2 diagnostic tests. The present diagnostic assays for CoV-2 can be segregated into two broad classes- molecular and serological. Molecular tests determine the presence of the virus by detecting its genome. For molecular assays, specimens can be collected from multiple relevant areas, such as the nasal swab, BAL, etc. Serological tests detect either the presence of the antigen (a protein that is only expressed by the virus) or the antibodies (generated by the host's immune system in response to the infection) in blood samples. While PCR (molecular) and antigen tests detect the presence of the virus at the time of testing, antibody assays mainly determine previous infection. All these tests can identify both, symptomatic and asymptomatic carriers of the virus. The PCR assays include reverse transcription-PCR (RT-PCR), recombinase polymerase amplification (RPA), reverse transcription-loop mediated isothermal amplification (RT-LAMP), and CRISPR-Cas based tests. These PCR assays will be discussed in this chapter.*

In the face of a pandemic, such as the one we face currently, a diagnostic assay should have:


Present diagnostic assays for CoV-2 have been categorized as shown in **Figure 2**.
