An updated review of SARS-CoV-2 detection methods in the context of a novel coronavirus pandemic

Abstract

The World Health Organization has reported approximately 430 million confirmed cases of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), worldwide, including nearly 6 million deaths, since its initial appearance in China in 2019. While the number of diagnosed cases continues to increase, the need for technologies that can accurately and rapidly detect SARS-CoV-2 virus infection at early phases continues to grow, and the Federal Drug Administration (FDA) has licensed emergency use authorizations (EUAs) for virtually hundreds of diagnostic tests based on nucleic acid molecules and antigen-antibody serology assays. Among them, the quantitative real-time reverse transcription PCR (qRT-PCR) assay is considered the gold standard for early phase virus detection. Unfortunately, qRT-PCR still suffers from disadvantages such as the complex test process and the occurrence of false negatives; therefore, new nucleic acid detection devices and serological testing technologies are being developed. However, because of the emergence of strongly infectious mutants of the new coronavirus, such as Alpha (B.1.1.7), Delta (B.1.617.2), and Omicron (B.1.1.529), the need for the specific detection of mutant strains is also increasing. Therefore, this article reviews nucleic acid- and antigen-antibody-based serological assays, and compares the performance of some of the most recent FDA-approved and literature-reported assays and associated kits for the specific testing of new coronavirus variants.

Keywords: SARS‐CoV‐2; nucleic acid molecular test; serological test; test kit evaluation; viral variants; virus detection.

FIGURE 1

Biology and serology of SARS‐CoV‐2 infection (a) Structure and infection: SARS‐CoV‐2 is an RNA virus that consists of four structural proteins, the Spike (S) protein, Nucleocapsid (N) protein, Membrane (M) protein, and Envelope (e), together with many non‐structural proteins to maintain the biological traits of the virus. Step 1–3: S protein allows the virus to bind and enter human cells and consists of S1 and S2 subunits. S1 can bind the angiotensin‐converting enzyme 2 (ACE2) receptor. After S1 binds to ACE2, S protein is hydrolyzed by the action of TMPRSS2 protease. The activated S2 subunit can then further mediate the fusion of membranes between the host cell and the virus, allowing the virus to enter the host cell. (b) SARS‐CoV‐2 variants: S protein of the first Wuhan‐Hu‐1 strain consisted of 1273 amino acid residues, in which the S1 and S2 fragments are linked by amino acid bridges, S1 includes the N‐terminal domain (NTD) and receptor‐binding domain (RBD), and S2 includes the fusion peptide (FP), heptad repeat 1 (HR1), heptad repeat 2 (HR2), and other structures. Since the start of the outbreak, many strongly infectious SARS‐CoV‐2 mutant strains have emerged, such as B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), B.1.617.2 (Delta), and B.1.1.529 (Omicron), among which mutations are particularly common in the S protein and have a substantial effect on the infectivity of the virus. (PDB ID:7DDD) (c) Immunity: Following viral infection in humans, specific antibody reactions often appear between days 5 and 15 after infection, with the IgM response lasting 3–6 weeks and the IgG response lasting several months.

FIGURE 2

Nucleic acid‐based detection of SARS‐CoV‐2 (a) qRT‐PCR: Step 1–4: SARS‐CoV‐2 RNA in different collected samples, such as nasopharyngeal swabs, can be extracted and purified using an RNA extraction kit, and complementary DNA (cDNA) for amplification and detection can be obtained by reverse transcriptase; Step 5–9: template cDNA undergoes denaturation, primer annealing, and extension in the real‐time PCR instrument The fluorescence signal is released when the fluorescence molecule is no longer inhibited by the quenching molecule, and the instrument can convert the fluorescence signal in the cycle into the cycle threshold (CT) value, which can be expressed as the quantified viral load data, and the validity of SARS‐CoV‐2 infection is verified by comparison with negative controls and threshold lines. (b) CRISPR/Cas system: Based on reverse transcription recombinant polymerase amplification (RT‐RPA) and reverse transcription loop‐mediated isothermal amplification (RT‐LAMP), purified RNA can be amplified in an isothermal instrument, and the amplified product can be reported both by the chromogenic substances in the amplification system and by the CRISPR/Cas system for further specific cleavage of nucleic acids and determination of virus infection. The CRISPR‐associated Cas protein then binds to the guide RNA, forming a complex that can target cleavage of the viral nucleic acid sequence, and the result can be reported by the fluorescence quenching molecules in the reaction, by reporting the fluorescence signal, or by the side stream chromatography color development strip of the cleaved nucleic acid fragment.

FIGURE 3

Serological detection of SARS‐CoV‐2 (a) Lateral flow assay: Quantum dots/colloidal gold can couple antibodies via specific labeling (using agent Maleamide–polyethylene glycol–succinimide ester (SMPEG)) and nonspecific labeling (using EDC/NHS chemistry methods). The rapid quantum dot and colloidal gold immunodiagnostic method for SARS‐CoV‐2 antibody‐based on high specificity recombinant protein and quantum dot/colloidal gold immunofluorescence probes by double antibody sandwich or indirect method methodology using lateral flow assay. The patient sample added to the sample pad will move to the absorbent pad along the NC membrane by chromatography, which will form the tagged‐antibody–antigen–antibody complex. After 10–15 min, test results can be observed on the test kit and operators can get an accurate fluorescence signal by a handheld fluorescent immunoanalyzer. (b) Cloud Network Platform: Rapid test kits can be used at the point of care for suspicious population screening tests, mobile devices such as cell phones can be used for result identification, handheld fluorescent immunoassay analyzers can perform a quantitative and qualitative analysis of test results, and qualitative and quantitative data can be uploaded to the terminal database, the CDC can manage relevant infections and suspicious populations through analysis of qualitative and quantitative data, give relevant clinical diagnosis recommendations, and combine with wearable devices such as smartwatches to achieve daily monitoring of people's medication, body temperature, heart rate, and other vital signs at the point of care such as communities and families, to control the development of epidemics in a timely and effective manner.