A Recent Update on Advanced Molecular Diagnostic Techniques for COVID-19 Pandemic: An Overview

Abstract

Coronavirus disease 2019 (COVID-19), which started out as an outbreak of pneumonia, has now turned into a pandemic due to its rapid transmission. Besides developing a vaccine, rapid, accurate, and cost-effective diagnosis is essential for monitoring and combating the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its related variants on time with precision and accuracy. Currently, the gold standard for detection of SARS-CoV-2 is Reverse Transcription Polymerase Chain Reaction (RT-PCR), but it lacks accuracy, is time-consuming and cumbersome, and fails to detect multi-variant forms of the virus. Herein, we have summarized conventional diagnostic methods such as Chest-CT (Computed Tomography), RT-PCR, Loop Mediated Isothermal Amplification (LAMP), Reverse Transcription-LAMP (RT-LAMP), as well new modern diagnostics such as CRISPR-Cas-based assays, Surface Enhanced Raman Spectroscopy (SERS), Lateral Flow Assays (LFA), Graphene-Field Effect Transistor (GraFET), electrochemical sensors, immunosensors, antisense oligonucleotides (ASOs)-based assays, and microarrays for SARS-CoV-2 detection. This review will also provide an insight into an ongoing research and the possibility of developing more economical tools to tackle the COVID-19 pandemic.

Keywords: CRISPR-Cas; LAMP; RT-PCR; SARS-CoV-2; biosensors; diagnostics; point of care.

Graphical Abstract

Conventional laboratory-based techniques for point-of-care detection of SARS-CoV-2.

Figure 1

Structure and mechanism of action/pathogenesis of SARS-CoV-2: (A) 3D model of SARS-CoV-2 with surface spike glycoprotein. (B) Different structural and non-structural proteins of SARS-CoV-2, as a target biomarker for the development of diagnostics. (C) SARS-CoV-2 binds to ACE2 and Transmembrane protease serine 2 (TMPRSS2) receptors present on lung cells using its surface spike glycoprotein and internalised. (D) The virion further releases the viral genetic material into the cytoplasm. (E) Cytokine storm induction inside the cells. (F) Inflammatory response cause tissue/organ damage in the lungs.

Figure 2

Detection of SARS-CoV-2 by conventional RT-PCR: (A) Nasopharyngeal swab samples collection in media and virus isolation. (B) RNA extraction from isolated virus using an RNA extraction kit. (C) Conversion of RNA to cDNA using reverse transcriptase. (D) cDNA PCR amplification with specific primers and fluorescent hydrolysis probes followed by analysis.

Figure 3

Selective naked eye SARS-CoV-2 detection: (A) Nasopharyngeal swab sample collected and virus isolated. (B) Viral RNA extraction. (C) Addition of AuNPs conjugated with thiolated ASOs specific for N gene. (D) Aggregation of ASO-AuNPs when target N-gene is present. (E) Addition of RNaseH to cleave RNA from RNA–DNA conjugate resulted in aggregation.

Figure 4

SPR-based sensor: Peptide monolayer functionalized with N protein coated onto gold base of SPR sensor. Addition of the clinical serum samples led to Ag–Ab interaction and change in SPR signal was recorded to detect SARS-CoV-2 Ab.

Figure 5

DETECTR LFA for SARS-CoV-2: (A) Nasopharyngeal swab viral RNA extracted from isolated virus to be used as the input sample or the sample matrix in the DETECTR assay. (B) RT-LAMP pre-amplification of E, N, and RNase P gene using ssDNA probes followed by Cas12-based detection. (C) Visual readout of the results on lateral flow strip by a fluorescence reader.

Figure 6

SERS-based LFA: (A) Preparation of double-layered DTNB-SiO₂@AgNPs by PEI-mediated absorption method followed by addition of DNTB. (B) S protein conjugation with SiO₂@Ag SERS tags. (C) LFA assembled with S protein-SiO₂@Ag SERS tags drop casted onto the conjugate pad and 2 test lines coated with IgM, IgG, and control line coated with S protein Ab. The sample was applied on the sample pad and SERS signal intensities were recorded.

Figure 7

FET-based graphene sensor: (A) Collection of nasopharyngeal swab. (B) Immobilization of spike Ab via 1-pyrenebutyric acid N-hydroxysuccinimide ester on graphene FET sensor. (C) Analysis of binding events of Ag–Ab through electrical signals obtained for detection of spike Ag.

Figure 8

Nanoplasmonic Sensor for label-free detection of COVID-19: (A) Sensor chip coated with SARS-CoV-2 mAbs. Colorimetric detection with AuNPs conjugated with ACE2 protein or mAb. (B) Au nanocup array chip with a drop of water on top. (Left) Zoomed SEM image of the nanocup array. (Right) TEM image showed air and water interfaces exhibiting green and far red pink colors, respectively.