Laboratory Testing Methods for Novel Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2)

Abstract

Following the first reports of coronavirus disease-19 (COVID-19) by China to the World Health Organization (WHO) on 31st December 2019, more than 4,302,774 novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) cases have been reported by authorities in 212 countries and territories by 12th May 2020. The outbreak and spread of COVID-19 worldwide, highlights the critical need for developing rapid and accurate diagnostic testing methods for emerging human coronavirus (CoV) infections. Testing is crucial to track the spread of disease during a pandemic, and to swiftly permit public health interventions including isolation, quarantine, and appropriate clinical management of afflicted individuals. The key components of viral diagnostic tests are (1) collection of the appropriate sample (blood, nasal swab, and throat swab), (2) availability of the genetic and proteomic sequences of the novel virus for analysis, and (3) rapid and accurate laboratory testing methods. The current gold standard for the molecular diagnosis of SARS-CoV-2 infection is the real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for the qualitative and quantitative detection of viral nucleic acids. Other relevant laboratory methods include enzyme-linked immunoassays (EIA) for viral antibody and antigen detection, and serum viral neutralization (SVN) assays for antibody neutralization determination. The challenges faced in developing a diagnostic test for a novel pathogen are the ability to measure low viral loads for early detection, to provide low or no cross-reactivity with other viral strains and to deliver results rapidly. Several point-of-care molecular devices are currently being integrated for fast and accurate diagnosis of SARS-CoV-2 infections. This review discusses the current laboratory methods available to test for coronaviruses by focusing on the present COVID-19 outbreak.

Keywords: EIA; RT-PCR; convalescent plasma; coronavirus; lateral flow diagnostics.

Figure 1

Schematic representation of SARS-CoV-2 genome. SARS-CoV-2 contains a positive-sense, positive-stranded mRNA genome with a 5′ capped mRNA sequence (C) and a 3′ poly-A tail. The coding genes are: ORF1a, ORF1b, Spike (S), ORF3a, ORF3b, Envelope (E), Membrane (M), ORF6, ORF7a, ORF7b, ORF8, ORF9b, ORF14, Nucleocapsid (N), and ORF10.

Figure 2

Molecular structure of SARS-CoV-2 and summary of the available laboratory tests and their target molecules. SARS-CoV-2 has a lipid bilayer membrane that contains Envelope (E) and Membrane (M) proteins that make up the envelope. Spike (S) glycoproteins project from the surface of the virion. Nucleocapsid protein (N) is composed of the protein that is associated with the viral genetic material. RT-PCR, reverse transcriptase polymerase chain reaction; EIA, enzyme immunoassay; LFIA, lateral flow immunoassay; SVNA, serum virus neutralization assay; INAA, isothermal nucleic acid amplification; CRISPR, clustered regularly interspaced short palindromic repeats; NGS, next generation sequencing; RT-LAMP, reverse transcriptase loop-mediated isothermal amplification; RPA, recombinase polymerase amplification.