Molecular and Immunological Diagnostic Tests of COVID-19: Current Status and Challenges

Abstract

Rapid spread of coronavirus disease 2019 (COVID-19) is ravaging the globe. Since its first report in December 2019, COVID-19 cases have exploded to over 14 million as of July 2020, claiming more than 600,000 lives. Implementing fast and widespread diagnostic tests is paramount to contain COVID-19, given the current lack of an effective therapeutic or vaccine. This review focuses on a broad description of currently available diagnostic tests to detect either the virus (SARS-CoV-2) or virus-induced immune responses. We specifically explain the working mechanisms of these tests and compare their analytical performance. These analyses will assist in selecting most effective tests for a given application, for example, epidemiology or global pandemic research, population screening, hospital-based testing, home-based and point-of-care testing, and therapeutic trials. Finally, we lay out the shortcomings of certain tests and future needs.

Keywords: Analytical Chemistry; Diagnostic Equipment; Infection Control in Health Technology; Virology.

Graphical abstract

Figure 1

SARS-CoV-2 Virus and COVID-19 (A) The virus is enveloped and spherical (~120 nm in diameter), with petal-shaped surface spikes (~20 nm long). Key structural proteins, spike (S), envelope (E), and membrane (M), are anchored on the viral envelop. The nucleocapsid (N) protein, together with the genomic RNA, forms a helical nucleocapsid inside the envelop. The virus enters host cells through the binding of S protein to angiotensin-converting enzyme 2 (ACE2) on the cell surface. (B) SARS-CoV-2 virus contains a positive-sense, single-stranded RNA genome. The organization of genome is 5′-leader-UTR (untranslated region)-replicase-S-E-M-N-3′-UTR-poly (A) tail. The open reading frame (ORF) 1a and 1b encode the replicase. Location of target genes for select COVID-19 RT-PCR tests are shown. Many tests target N gene, because sequence conservation of this gene within the coronavirus genus is low. (C) Patients with COVID-19 display symptoms similar to those of common cold and influenza, and in some cases are asymptomatic. Diagnostics must be confirmed through highly specific molecular tests. US-CDC, United States Center for Disease Control and Prevention; C-CDC, Chinese Center for Disease Control and Prevention; HKU, Hong Kong University; NIID, National Institute of Infectious Diseases (Japan). (A) Adapted with permission from Ref (Graham et al., 2013). Copyright 2013 Nature Publishing Group. (B) Adapted with permission from Ref (Jung et al., 2020).

Figure 2

LAMP-Based COVID-19 Test (A) LAMP mechanism. (i) The reaction mix contains dNTPs, DNA polymerase with high displacement activity, and pairs of primers (F1-F1c; F2-F2c; F3-F3c; B1-B1c; B2-B2c; B3-B3c). Forward inner primer (FIP) binds to the F2c region in the target and is extended. F3 primer also hybridizes to the F3c region and is extended, displacing the FIP-linked complementary strand. (ii) The displaced single-stranded DNA serves as a template for extension reactions by backward inner primer (BIP) and B3 primer. (iii) The BIP-linked DNA self-anneals and forms a dumbbell-like structure that initiates subsequent rounds of amplification. FIP binds and opens up the loop at the 3′-end converting the dumbbell shape into a stem-loop structure through its extension toward 5′ end. (iv) The extended strand from (iii) forms a new loop and serves as a template for BIP binding and extension. Both these products become the seed for the exponential amplification. (B) RT-LAMP-based detection of ORF1ab gene. The amplification process monitored via turbidimeter readings at 650 nm or visual observation of calcein-mediated color change from orange to green. The target concentration is inversely proportional to detection time and the color change. Reproduced with permission from Ref (Yan et al., 2020a). Copyright 2020 European Society of Clinical Microbiology and Infectious Diseases.

Figure 3

NEAR-Based COVID-19 Test (A) Left, NEAR duplex formation. (i) The reverse primer (P1) binds to the target region and is extended. (ii) A second P1 binds to the same target and is extended, displacing the first extended strand. (iii) Primer P2 binds to the released strand and is extended, creating a double-stranded nicking enzyme recognition site. (iv) Nicking enzyme (indicated by scissors) binds and nicks the downstream of the recognition sequence. (v) Polymerase synthesizes complementary sequence off the cleaved site. (vi) The final product is a double-stranded DNA with restriction sites at both ends. Right, exponential amplification. (vii) Nicking enzyme binds and nicks the NEAR duplex at both restriction sides, making two templates (T1, T2). (viii) Free ends of templates are extended. (ix) Repeated nicking and polymerization steps start. (x) Cleaved complexes are regenerated, whereas amplified products (A1, A2) anneal to primers (P2, P1), resulting in bidirectional extension and creating duplexes. (B) ID NOW COVID-19 system by Abbott. Disposable tools (left) minimize hands-on processes. Patient swab (nasal, nasopharyngeal, or throat) is eluted in the sample receiver containing elution/lysis buffer. After 10 s mixing, the mixture is manually transferred to the test base holder (via transfer cartridge) that contains lyophilized NEAR agents. Heating, agitation, and detection by fluorescence are performed automatically by the instrument. The assay detects SARS-CoV-2 RdRp gene. Adapted with permission from Ref (Nie et al., 2014). Copyright 2014, American Society for Microbiology.

Figure 4

RPA-Based COVID-19 Test (A) RPA mechanism. RPA reaction mix contains recombinase, primers, loading factors, and single-stranded binding proteins. (i) The recombinase binds to primers in the presence of loading factors, forming nucleoprotein filaments. (ii) This complex binds to complementary sequences in the target DNA, forming D loop structure, and initiates strand exchange. Single-stranded binding proteins stabilize the displaced DNA stands. (iii) Recombinase disassembles from the nucleoprotein filament to be re-used for subsequent amplification cycles. (iv) DNA polymerase extend primers, separating parallel strands to form duplexes. Repeated cycle of this process enables exponential amplification. (B) RT-RPA assay developed for COVID-19 diagnostics. Extracted RNA sample is mixed with RT-RPA reaction mixture. RPA activator (Mg²⁺) is loaded inside the lid of the vial. RT is performed at 37°C (1 min), and then the vial is spun to introduce Mg²⁺ into the reaction mixture. The reaction vial is heated to 40°C (4 min) for initial RPA activation. After shake and spin, the reaction is let to proceed for additional 26 min at 40°C. The reaction product is then detected via green fluorescence excited by blue light. Reproduced with permission from Ref (Xia and Chen, 2020).

Figure 5

CRISPR-Based COVID-19 Test (A) Schematic of DETECTR coupled with lateral flow readout. RNA targets extracted from nasopharyngeal swabs are amplified by RT-LAMP. Cas12a complexes, pre-incubated with guide RNAs (gRNAs), recognize target DNA and cleave single-stranded DNA (ssDNA) probes for signal generation. (B) The intact ddDNA reporters are captured on the control line, whereas the cleaved reporter is captured on the test line. Lateral flow results for the DETECTR are shown for N gene at 0 and 10 copy/μL. (C) Schematic of SHERLOCK Testing in One Pot (STOP) test. A nasopharyngeal swab or saliva is transferred to the lysis buffer. Lysate is then added to SHERLOCK master mix, and the mixture is heated for 60 min at 60°C. Test results are read out using lateral flow strips (2 min). (D) Twelve positive and five negative nasopharyngeal (NP) swab samples were analyzed by STOP. The assay made correct diagnosis of these samples. The data were displayed as mean ± standard deviation from three independent experiments. (A and B) Adapted with permission from Ref (Broughton et al., 2020). Copyright 2020 Nature Publishing Group. (C and D) Adapted with permission from Ref (Joung et al., 2020).

Figure 6

Immunoassay Design for COVID-19 Detection (A) Principles of different types of immunoassays. Antigen tests directly capture viral proteins or the whole virus, whereas in antibody tests, viral antibodies (e.g., IgG, IgM) generated from host immune response are captured by synthetic viral antigens or anti-human antibodies. Both tests use a reporter probe for signal generation. Virus neutralization tests check whether a specimen contains effective antibodies that can prevent viral infection on cells. (B) Positive rates of viral RNA and antibodies (IgG or IgM) were detected in 238 patients with COVID-19 who were at different disease stages. Note that the antibody positive rates were low in the first 5 days after initial onset of symptoms and then rapidly increased as the disease progressed. Adapted with permission from Ref (Liu et al., 2020a). (C) Similarity of coronavirus S and N proteins. Protein domains from different coronaviruses were compared with those of SARS-CoV-2 (top row, 100% concordance). S1 and S2 are subunits of S. Note that S1 has the least degree of similarity. (D) Evaluation of S1 ELISA. SARS-CoV-2 S1 protein was used as a capture agent. Serum samples from healthy donors and patients either with non-CoV respiratory, HCoV, MERS-CoV, SARS-CoV, or SARS-CoV-2 infections were analyzed. S1 ELISA showed no cross-reactivity with non-SARS serum samples. The dotted horizontal line indicates ELISA cutoff values, and the sample numbers are inside shaded rectangles. OD, optical density. (C and D) Adapted from Ref (Okba et al., 2020).

Figure 7

Examples of COVID-19 Immunoassays (A) Schematic of a rapid detection test (RDT) device. The sample, dropped on a loading pad, flows through the device via capillary effect and wet colloidal gold nanoparticles (AuNPs) loaded in the conjugation pad. AuNPs tagged with viral antigen bind to IgM and IgG antibodies, and the complexes are captured in the downstream by pre-spotted anti-human IgM and IgG antibodies. AuNP conjugated with non-human IgG antibodies are captured by appropriate antibodies to generate a control signal. (B) Two types of ELISA were compared. One used recombinant SARS-CoV-2 N protein as a capture antigen (N-ELISA), and the other, recombinant SARS-CoV-2 S protein (S-ELISA). Serum samples from 214 patients with COVID-19 were tested. Overall, S-ELISA showed higher detection rate than N-ELISA. (C) Plasma samples from patients (n = 5) who recovered from COVID-19 were used for virus neutralization tests. In a concentration-dependent manner, all five plasma inhibited the infection of 293T/ACE2 cells by SARS-CoV-2 pseudo virus. Plasma from a healthy donor was used as a negative control. The median percentage of neutralization is shown from duplicate measurements. Data points represent the median percentage of neutralization. Error bars indicate standard deviation from duplicate measurements. (B) Adapted with permission from Ref (Liu et al., 2020b). Copyright 2020 American Society for Microbiology. (C) Reproduced with permission from Ref (Wu et al., 2020).