Current Status of Diagnostic Testing for SARS-CoV-2 Infection and Future Developments: A Review

Abstract

The coronavirus disease 2019 (COVID-19) caused by a novel coronavirus, SARS-CoV-2, has infected more than 50.6 million individuals and caused over 1.2 million deaths globally, raising a major health concern. To date, no specific antiviral treatment or vaccine for COVID-19 has been approved by the Food and Drug Administration (FDA). Highly sensitive and specific laboratory diagnostics are therefore critical for controlling the rapidly evolving COVID-19 pandemic and optimizing clinical care, infection control, and public health interventions. The FDA has issued emergency use authorization (EUA) for hundreds of COVID-19 diagnostic tests of different classes. Whereas nucleic acid testing (NAT) such as RT-PCR remains the criterion standard for COVID-19 diagnosis, serological antibody and antigen tests are increasingly being developed. Tests based on the novel RNA sensing techniques (e.g., SHERLOCK, DETECTR, and Toehold Switch) are promising due to their relatively low cost, high accuracy, and rapid detection time. Diagnostic testing results for SARS-CoV-2 should be interpreted with caution, since they depend heavily on factors such as viral load, virus replication, the source and timing of sample collection, sample extraction, and characteristics of various testing methods. This review aims to present the current status of common diagnostic testing for SARS-CoV-2 infection, review the current regulatory requirements, and identify future directions in the development of improved diagnostics that are more accurate, accessible, and rapid.

Figure 1

Novel RNA sensing techniques and their potential applications for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) infection. On-site and rapid detection of emerging pathogens is feasible with the recent advances in molecular diagnostics. In a Toehold Switch system, virus in the breathing vapor of patients is attached by porous materials embedded with cell-free gene expression systems. The secondary RNA structure repressing the expression of LacZ consists of a sensing sequence (red), a linker sequence (black), a ribosome-binding domain (RBD, green), and an antisense sensoring sequence (blue). Sequence-specific binding of viral RNA to the sensoring sequence releases the secondary structure and lead to expression of LacZ (blue squares), which cleaves the substrate chlorophenol red-b-D-glucopyranoside (S, yellow) to produce a substrate of a visible purple color (S, purple). On a specific high-sensitivity enzymatic reporter unlocking (SHERLOCK) platform, clinical samples are collected by trained professionals or patients themselves following simple instruction. Collected samples are lysed (and therefore sterilized) and stabilized, amplified through an optional isothermal amplification process, and tested by a recently developed in vitro assay. Viral RNA can pair with the CRISPR RNA (crRNA) in a sequence-specific manner to activate robust and nonspecific single-stranded DNA trans-cleavage, which releases fluorescent signals (F) from their quenchers (Q) linked by single-stranded DNA. The fluorescent signal can be also replaced with a colorimetric visual readout on a lateral flow strip. Potential applications of these novel techniques for the on-site and rapid detection of emerging novel pathogens such as SARS-COV-2 are also depicted.