Advanced Molecular and Immunological Diagnostic Methods to Detect SARS-CoV-2 Infection

Abstract

COVID-19 emerged in late 2019 in China and quickly spread across the globe, causing over 521 million cases of infection and 6.26 million deaths to date. After 2 years, numerous advances have been made. First of all, the preventive vaccine, which has been implemented in record time, is effective in more than 95% of cases. Additionally, in the diagnostic field, there are numerous molecular and antigenic diagnostic kits that are equipped with high sensitivity and specificity. Real Time-PCR-based assays for the detection of viral RNA are currently considered the gold-standard method for SARS-CoV-2 diagnosis and can be used efficiently on pooled nasopharyngeal, or oropharyngeal samples for widespread screening. Moreover, additional, and more advanced molecular methods such as droplet-digital PCR (ddPCR), clustered regularly interspaced short palindromic repeats (CRISPR) and next-generation sequencing (NGS), are currently under development to detect the SARS-CoV-2 RNA. However, as the number of subjects infected with SARS-CoV-2 continuously increases globally, health care systems are being placed under increased stress. Thus, the clinical laboratory plays an important role, helping to select especially asymptomatic individuals who are actively carrying the live replicating virus, with fast and non-invasive molecular technologies. Recent diagnostic strategies, other than molecular methods, have been adopted to either detect viral antigens, i.e., antigen-based immunoassays, or human anti-SARS-CoV-2 antibodies, i.e., antibody-based immunoassays, in nasal or oropharyngeal swabs, as well as in blood or saliva samples. However, the role of mucosal sIgAs, which are essential in the control of viruses entering the body through mucosal surfaces, remains to be elucidated, and in particular the role of the immune response in counteracting SARS-CoV-2 infection, primarily at the site(s) of virus entry that appears to be promising.

Keywords: COVID-19; ELISA; RT-PCR; SARS-CoV-2; anti-SARS-CoV-2 antibodies; antigen-based immunoassays; secretory IgA; spike protein.

Figure 1

Progression of SARS-CoV-2 infection and in vitro diagnostics. The detection of active SARS-CoV-2 infection is performed using molecular diagnostics, i.e., real-time reverse transcription-polymerase chain reaction (RT-PCR), droplet-digital PCR (ddPCR), clustered regularly interspaced short palindromic repeats (CRISPR) and next-generation sequencing (NGS), in order to detect the viral RNA during the first weeks after the emergence of symptoms. In this period, it is also possible to detect the presence of SARS-CoV-2 components using antigen-based immunoassays, i.e., the enzyme-linked immunosorbent assay (ELISA), lateral flow immunoassay (LFIA) and immunochromatographic assay (ICA). Detection of SARS-CoV-2 antibodies (IgG and IgM) occurs through antibody-based immunoassays, i.e., ELISA, LFIA and chemiluminescence immunoassay (CLIA), from the third week after symptoms.