Diagnosis for COVID-19: current status and future prospects

Abstract

Introduction: Coronavirus disease 2019 (COVID-19), a respiratory illness caused by novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), had its first detection in December 2019 in Wuhan (China) and spread across the world. In March 2020, the World Health Organization (WHO) declared COVID-19 a pandemic disease. The utilization of prompt and accurate molecular diagnosis of SARS-CoV-2 virus, isolating the infected patients, and treating them are the keys to managing this unprecedented pandemic. International travel acted as a catalyst for the widespread transmission of the virus.Areas covered: This review discusses phenotype, structural, and molecular evolution of recognition elements and primers, its detection in the laboratory, and at point of care. Further, market analysis of commercial products and their performance are also evaluated, providing new ways to confront the ongoing global public health emergency.Expert commentary: The outbreak for COVID-19 created mammoth chaos in the healthcare sector, and still, day by day, new epicenters for the outbreak are being reported. Emphasis should be placed on developing more effective, rapid, and early diagnostic devices. The testing laboratories should invest more in clinically relevant multiplexed and scalable detection tools to fight against a pandemic like this where massive demand for testing exists.

Keywords: Biosensing; COVID-19; SARS-CoV-2; diagnosis; point of care.

Figure 1.

The schematic illustration of emergence of SARS-CoV, MERS-CoV, and SARS-CoV-2 and their diagnosis, treatment, and prevention mechanism. The deadliest human coronaviruses are SARS-CoV, MERS-CoV and SARS-CoV-2, all of which likely have evolved from a common origin in bats. Interestingly, all three of them were likely introduced into human populations through different intermediate zoonotic hosts, for example, SARS CoV via palm civets, MERS CoV via camel, and SARS-CoV-2 via pangolins. The spreading of the virus is related to close contacts with infected patients, nosocomial transmission in healthcare providers as well as fast global spread by intercontinental travel

Figure 2.

Genome and structure of SARS-CoV-2 revealing mechanism of SARS-CoV-2 infection via its spike protein (S). A) The SARS-CoV-2 genome is a non-segmented, positive single-stranded RNA genome, with an approximate size of 30 kb. The full genome arranged in an order of 5ʹ UTR, replicase genes, four structural proteins including spike glycoprotein (S), envelope protein (E), membrane protein (M), and nucleocapsid protein (N) with a 3′ poly-A tail. B) The spike protein of the SARS-CoV-2 consisting of two subunits S1 and S2 mediates membrane fusion by binding primarily to angiotensin-converting enzyme 2 (ACE2) receptor of the host cells. (Figure 2 (A,B)) are created with Biorender.com)

Figure 3.

Summary of immune response timeline to the infection of the SARS-CoV-2 virus

Figure 4.

Schematic workflow of COVID-19 Diagnostic Test using RT-PCR. A) Nasal or oral swabs are collected from infected patients containing upper and lower respiratory tract fluids. The collected samples are preserved on specific temperatures and send to labs for testing. Viral samples are deactivated by heat, RNA is isolated using RNA extraction kit and amplified using specific primers targeting segments of different genes (E, N, ORF1ab, and RNase P). Based on the amplified fluorescent signal, test results are interpreted. B) Correlation of a high number of viral loads present in sputum samples than throat swabs on different stages of infection for 30 hospitalized patients (Reprinted from [86])

Figure 5.

Detection of SARS-CoV-2 using the RT-LAMP method. A) Respiratory fluids are collected by using a nasal or oral swab from the infected patients, and RNA of SARS-CoV-2 are collected from the specimen, and LAMP primer and reaction agents are added, followed by amplification at 60–65°C. The RT-LAMP product’s final outcome represents positive reaction changed color from pink to yellow due to the decrement of pH of target DNA polymerase reaction, which is easily confirmed by naked-eye detection. Detection of the amplified samples was visualized using gel electrophoresis (modified from [115]) B) Design of a set of RT-LAMP primers targeting the RdRp gene of the SARS-CoV-2. Primers sets include two inner primers containing sequence complementary, two outer primers, and two loop primers. (Reprinted from [110])

Figure 6.

Detection strategy of SARS-CoV-2 amplicons using the CRISPR

Figure 7.

Detection of SARS-CoV-2 antibody by indirect ELISA (A) and antigen by sandwich ELISA (B)

Figure 8.

Paper-based LFAs for COVID-19 detection caused by SARS-CoV-2 coronavirus. A) Paper-based LFAs are composed of an in-line sample pad, conjugate pad, incubation and detection pad (test and control line), and absorbent pad. B) A picture of IgM/IgM COVID-19 detection kit based on paper-based LFA [193]. C) Sample loading, processing, and colorimetric detection of IgG/IgM antibody from paper-based LFAs. D, E) HybriDetect Strip based COVID-19 detection and further quantification of the limit of detection [158]

Figure 9.

Surface plasmon resonance based COVID-19 detection. A) Experimental setup of the dual-functional PPT enhanced LSPR biosensing system for SARS CoV2 detection [204]. B) Schematic illustration of the hybridization of RdRp-COVID and its cDNA sequence (RdRp-COVID-C) and their plasmonic response with/without thermal activation. C) SARS-CoV-2 viral oligos concentrations variations and their corresponding plasmonic resonance shifting. D, E) SPR sensorgram and resonance wavelength shift of functionalized AffiCoated surface with nucleocapsid protein of SARC-CoV-2 (rN) [205]. F, G) Conceptual block-diagram of AuNP functionalized colorimetric SARS-CoV-2 detection. Normalized optical response of AuNP absorption before and after adding RNA SARS CoV-2 viral load (a), their hydrodynamic diameters variations (b), TEM images of Au-functionalized nanoparticles after addition of SARS-CoV-2 viral RNA [207]