Laboratory diagnosis of SARS-CoV-2: available approaches and limitations

Abstract

The ongoing pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is one of the most devastating outbreaks witnessed in the last 100 years. The outbreak started in China and spread rapidly to almost every country, culminating in woefully overwhelmed health-care systems in most countries. The only approved diagnostic test to accompany radiographic evaluation is reverse transcription PCR. However, the applicability of this test in diagnosis and surveillance is challenged by a global shortage of reagents and the lack of well-equipped laboratories with specialized staff in several low- and middle-income countries. Loop-mediated isothermal amplification and CRISPR-based diagnostic assays have developed and expected to play a role however, their accuracy is still inferior to the recommended PCR approach. The need for the development of accurate and rapid diagnostic assays became apparent. Immunodiagnostic tests and other molecular approaches were developed and tested. Other recently developed point-of-care molecular tests are expected to be helpful in pandemic management as no particular skills are required from the operator. Fortunately, a number of serological tests have been granted authorization for use under the emergency situation by the US FDA for the diagnosis of SARS-CoV-2. The majority of recently authorized serological tests detect IgG and IgM in blood of infected individuals by on ELISA, chemiluminescence platforms or lateral flow cassettes.

Keywords: Antigen; Coronavirus disease 2019; Isothermal amplification; Nasopharyngeal swab; Real-time RT-PCR; Serology.

Fig. 1

Principle of reverse transcription loop-mediated isothermal amplification (RT-LAMP). B2 backwards inner primer (BIP) anneals to the target RNA forming a start point for reverse transcriptase to synthesize a complementary DNA strand (cDNA) (1). The outer primer B3 anneals to a sequence outside that of B2 to initiate synthesis of a new cDNA (2). During synthesis of the second cDNA, the former strand is displaced by the reverse transcriptase (3). The displaced strand is released with a loop at the 5′ end. Forward inner primer F2 anneals to the 3′ end of the newly released cDNA strand and DNA polymerase starts synthesis of a complementary DNA strand (4). Forwards outer primer F3 anneals to the 3′ end of the strand formed by the F2 primer and DNA polymerase initiates the synthesis of new strand and simultaneous displacing of the former (5). The recently displaced cDNA strand contains complementary sequences at both ends, hence it forms a dumbbell-shaped structure (6). This structure is the starting material for loop-mediated amplification cycles. It contains multiple sites for amplification initiation and is rapidly converted to stem-loop DNA structures in response to self-primed synthesis at the 3′ end (7). At the same time, backwards inner primer B2 to the single-stranded loop region to start DNA synthesis culminating in long concatemers (8 and 9). Further synthesis leads to dumbbell-shaped structures and accumulation of amplification dsDNA products (10).

Fig. 2

Principle of immunochromatography. This drawing shows lateral flow to detect an antigen. The specimen containing the antigen (Analyte) is placed on the sample pad. The antigen, with the fluid, moves to the conjugate pad where it is bound by a labelled antibody specific to the targeted antigen. The conjugate pad also contains labelled antibodies non-specific to the antigen to be detected. Antigen–antibody complexes migrate through the nitrocellulose membrane and reach the ‘Test line’ area. In this area, antigen-specific antibodies are immobilized to catch the antigen–antibody complexes. When antigen–antibody complexes accumulate in this area, the line becomes visible to the naked eye. The non-specific antibodies also migrate and pass the ‘Test line’ to reach the ‘Control line’ area and that line also becomes visible. The test is considered positive only when the two lines (T and C) are visible. (Reproduced from Paulini et al. [82]).