Current trends in COVID-19 diagnosis and its new variants in physiological fluids: Surface antigens, antibodies, nucleic acids, and RNA sequencing

Abstract

Rapid, highly sensitive, and accurate virus circulation monitoring techniques are critical to limit the spread of the virus and reduce the social and economic burden. Therefore, point-of-use diagnostic devices have played a critical role in addressing the outbreak of COVID-19 (SARS-CoV-2) viruses. This review provides a comprehensive overview of the current techniques developed for the detection of SARS-CoV-2 in various body fluids (e.g., blood, urine, feces, saliva, tears, and semen) and considers the mutations (i.e., Alpha, Beta, Gamma, Delta, Omicron). We classify and comprehensively discuss the detection methods depending on the biomarker measured (i.e., surface antigen, antibody, and nucleic acid) and the measurement techniques such as lateral flow immunoassay (LFIA), enzyme-linked immunosorbent assay (ELISA), reverse transcriptase-polymerase chain reaction (RT-PCR), reverse transcription loop-mediated isothermal amplification (RT-LAMP), microarray analysis, clustered regularly interspaced short palindromic repeats (CRISPR) and biosensors. Finally, we addressed the challenges of rapidly identifying emerging variants, detecting the virus in the early stages of infection, the detection sensitivity, selectivity, and specificity, and commented on how these challenges can be overcome in the future.

Keywords: Biosensors; COVID-19 virus diagnosis; Clustered regularly interspaced short palindromic repeats; Enzyme-linked immunosorbent assay; Lateral flow immunoassay; Loop-mediated isothermal amplification; Microarray assays; Reverse transcriptase-polymerase chain reaction.

Graphical abstract

Fig. 1

Timeline of identified infectious human coronaviruses (HCoVs) with the symptoms they cause and their natural and intermediate hosts. The image was created with Biorender.

Fig. 2

Schematic representation of (a) cartoon model showing the structure of SARS-CoV-2, (b) genome structure and encoded proteins, and (c) mutation in spike proteins. The image was created with Biorender.

Fig. 3

Schematic representation of the spread, transmission, and life cycle of SARS-CoV-2 virus in a human host cell. Viral spikes bind to their receptor human ACE2 (hACE2) via their receptor-binding domain (RBD) and are proteolytically activated by human proteases. The image was created with Biorender.

Fig. 4

Schematic representation showing the structure of SARS-CoV-2 virus and various detection methods: (a) viral components and human response; (b) biomarkers for detection; and (c) common laboratory tests. The image was created with Biorender.

Fig. 5

Timeline for SARS-CoV-2 infection and COVID-19 positivity tests versus the molecular diagnostic assays (PCR). The image was created with Biorender.

Fig. 6

Schematic representation of colorimetric LFIA of SARS-CoV-2 virus upon loading the sample and buffer solution to the nitrocellulose pad: (a) The antibodies specific to the virus (IgG, IgM) bind to the viral antigens and form antigen-antibody complexes. (b) When the antigen-antibody complexes flow to the secondary antibodies (antihuman IgG and antihuman IgM antibodies), the antigen-antibody complexes bind to the secondary antibodies and form sandwiches of antibodies (IgG, IgM) between the viral antigen and the secondary antibodies. (c) In a negative sample without SARS-CoV-2 specific IgG and IgM, only the control line is stained. The image was created with Biorender.

Fig. 7

Sandwich ELISA for the detection of SARS-CoV-2 antigens. (a) Microwell Plate coated with the capture antibody, (b) Addition of the patient sample containing the viral antigens, (c) Washing to remove bound antigens, then add primary antibodies, (d) Washing to remove the unbound primary antibodies, then the addition of the enzyme-bound secondary antibodies, (e) After washing to remove the unbound secondary antibodies, the substrate is added and converted by the enzyme into a detectable form by assuming a color that depends on the presence and concentration of the viral antigen; then the stop solution is added to terminate the enzyme-substrate reaction, and (f) The ELISA reader is used to detect the presence and concentration of the viral antigen in the sample. The image was created with Biorender.

Fig. 8

Schematic representation of COVID -19 in the context of biosensor technologies: (a) sample collection, (b) extraction of antigens and antibodies; and (c) biosensor detection methods. The image was created with Biorender.

Fig. 9

Single-molecule detection of SARS-CoV-2 and MERS antigens using nanobody functionalized organic electrochemical transistors: (a) The electrode is exposed to a sample (saliva) in a buffer solution. (b) Functionalization of the gold electrode surface and bio-recognition of the SAM layers (Chem-SAMs and Bio-SAMs) bind to the antigens on the virus surface. (c) Molecular architecture of the composite layers and binding of the antigen to the modified gold electrode surface. © Nature, 2021 [88].

Fig. 10

Schematic representation of nucleic acid detection of SARS-CoV-2 by RT-PCR assay. (a) RNA extraction, (b) reverse transcription, (c–e) PCR amplification by (c) c-DNA denaturation, (d) primer annealing, (e) primer elongation by DNA polymerase enzyme, (f) detection steps with TaqMan probe, (g) RT-qPCR instrument, (h) signal results, and (i) primers and probes for screening. The image was created with Biorender.

Fig. 11

An illustration shows the RT-LAMP procedure and results. (1) Collection of nasopharyngeal swabs or saliva samples, (2) extraction of viral RNA in 10–30 min, (3) RNA amplification, (4) addition of reagent and incubation at 65 °C for 30 min. Depending on the reagent and reaction conditions, different colors are observed: (5a) pH change due to phenol red, the medium is acidified after DNA amplification, (5b) hydroxy naphthol blue varies from purple to sky blue as a result of reduced Mg²⁺ in the amplified DNA, (5c) intercalating dyes such as SYBR green and displacement probes can be used as fluorescent indicators. The image was created with Biorender.

Fig. 12

Nucleic acid hybridization using DNA microarray. Fluorescent labeled viral and reference cDNA are placed into the microarray wells functionalized with specific DNA probes. (a) COVID-19 cDNA is indicated by the red fluorescence, (b) Overlaid fluorescence pattern, and (c) Reference cDNA is indicated by the green fluorescence. The image was created by Biorender.

Fig. 13

Schematic representation of nucleic acid detection of SARS-CoV-2 using CRISPR/Cas assays. (a) RNA is extracted from patient spacemen. (b) DNA must be amplified from the extracted nucleic acid. (c) Construct the guide RNA. (d) Cas13 uses the guide RNA to find its target. (e) Label the target RNA by reporter molecules that fluoresce when cleavage occurs between fluorescence and quencher. (f) The detection of nucleic acid using agarose gel, lateral flow strips, and fluorescence visualization. The image was created with Biorender.

Fig. 14

Detection of SARS-CoV-2 RNA by antisense oligonucleotide (ASO)-capped Au NPs which allow the viral detection via naked-eye [159]. © American Chemical Society, 2021.