COVID-19 diagnostics: Molecular biology to nanomaterials

Abstract

The SARS-CoV-2 pandemic has claimed around 6.4 million lives worldwide. The disease symptoms range from mild flu-like infection to life-threatening complications. The widespread infection demands rapid, simple, and accurate diagnosis. Currently used methods include molecular biology-based approaches that consist of conventional amplification by RT-PCR, isothermal amplification-based techniques such as RT-LAMP, and gene editing tools like CRISPR-Cas. Other methods include immunological detection including ELISA, lateral flow immunoassay, chemiluminescence, etc. Radiological-based approaches are also being used. Despite good analytical performance of these current methods, there is an unmet need for less costly and simpler tests that may be performed at point of care. Accordingly, nanomaterial-based testing has been extensively pursued. In this review, we discuss the currently used diagnostic techniques for SARS-CoV-2, their usefulness, and limitations. In addition, nanoparticle-based approaches have been highlighted as another potential means of detection. The review provides a deep insight into the current diagnostic methods and future trends to combat this deadly menace.

Keywords: Diagnosis; Immunological detection; Molecular detection; Nanoparticles; Radiological approach; SARS-CoV-2.

Fig. 1

Life cycle of SARS-CoV-2. The SARS-CoV-2 virus recognizes the ACE-2 receptor present on the host surface. Post-receptor interaction, the virus particles enter the cell via endocytosis. The envelope of the virus fuses with the cell membrane releasing the viral RNA into the cell. The viral genome is translated to produce polyproteins. Self-proteolysis of polyproteins forms non-structural proteins. These non-structural proteins amalgamate to form a replicase/transcriptase complex (RTC) which facilitates the replication of genomic RNA. Transcription of the viral genome generates sub-genomic RNA that is translated to produce the S, M, E, N, and ORF1a proteins by the ERGIC (Endoplasmic reticulum-Golgi intermediate compartment) complex. The newly assembled viral particles are released out of the cell through exocytosis.

Fig. 2

Detection of SARS-CoV2 by RT-PCR. The patient sample is collected as a nasopharyngeal swab (1). The sample is then transported to the viral transport medium for RNA extraction (2, 3). Reverse transcription of viral RNA into cDNA and amplification is done using a thermocycler (4). The result can be determined by observing the fluorescence v/s copies per reaction curve. Positive results cross the detection threshold (5).

Fig. 3

Principle of SARS-CoV-2 diagnostic kits based on RT-LAMP assay. Mechanism of RT-LAMP (a): The reaction mixture consists of dNTPs, Bst DNA polymerase, primer pairs, and magnesium ions, incubated at an isothermal condition. SARS-CoV-2 RNA is converted into cDNA by the process of reverse transcription (1, 2). Forward inner primer (FIP) extends the target sequence. The complementary strand linked to FIP is displaced, which serves as the template for backward inner primer (BIP) (3). A Dumbbell-shaped structure is formed as a result of the self-annealing of BIP-linked DNA, leading to subsequent amplification (4). As a result of the 3′ end opening by FIP and its extension up to the 5′ end, a stem-loop structure from the dumbbell-shaped structure is formed. Extended strand formed from BIP-linked DNA forms a new loop and serves as a template for binding and extension of BIP (5). These products proceed for exponential amplification. The result of RT-LAMP (b) can be analyzed either by 1. Turbidity assay, 2. Fluorescence detection or 3.Colorimetric analysis.

Fig. 4

CRISPR-Cas methods for RNA Detection. The method proceeds with the conversion of SARS-CoV-2 RNA into dsDNA by reverse transcription. Cas13 recognizes SARS-CoV-2 ssRNA, the dsDNA is converted to ssRNA. The binding of the target sequence to the Cas enzyme activates its catalytic activity which further cleaves fluorescent ssRNA reporter for detection (Sherlock CRISPR SARS-CoV-2 Kit) (A). Cas12 identifies SARS-CoV-2 dsDNA, thus the binding of a target sequence to Cas12 activates the cleavage activity of the enzyme and cleaves fluorescent ssDNA reporter for visualization and detection (SARS-CoV-2 DETECTR Reagent Kit) (B).

Fig. 5

SARS-CoV-2 antibodies detection by lateral flow immunoassay. The blood sample is collected from the patient and loaded on the test strip (1). A buffer solution is then added to the test strip and incubated (2). The sample flows through the strip via capillary action and Antigen-Antibody interaction takes place (3). SARS-CoV-2 specific antigens are immobilized on nitrocellulose membrane along with gold rabbit conjugated antibodies. The test sample upon contact with the membrane forms a complex with SARS-CoV-2 specific antigens, that are further captured by immobilized anti-human antibodies that give a positive test line. The gold rabbit conjugate antibodies are captured further by anti-rabbit antibodies that give a control line (4). The results of lateral flow can be analyzed visually where the appearance of both the test line and control line indicates a positive test whereas the appearance of only the control line indicates a negative test (5).

Fig. 6

Chest X-ray showing progression in COVID-19 pneumonia. (a) A normal chest X-ray of a woman with COVID-19 infection at day 0. (b) Chest X-ray of the same patient on day 8 of COVID- 19 infection displaying ground glass opacification (white arrows). The black outlined arrows point to the consolidation at the periphery of the left upper and mid zones. The progression of glass ground opacification to consolidation can also be observed in the periphery of the right upper zone (black outline arrow). The image is adapted from Cleverley et al., 2021 with permission from BMJ).

Fig. 7

Naked eye detection of SARS-CoV2 using gold nanoparticle agglomeration-based approach. (Reproduced from Moitra et al. with permissions from the American Chemical Society[114]).