Point-of-Care Diagnostic Tools for Surveillance of SARS-CoV-2 Infections

Abstract

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a recently emerged and highly contagious virus that causes coronavirus disease 2019 (COVID-19). As of August 24, 2021, there were more than 212 million confirmed COVID-19 cases and nearly 4.4 million deaths reported globally. Early diagnosis and isolation of infected individuals remains one of the most effective public health interventions to control SARS-CoV-2 spread and for effective clinical management of COVID-19 cases. Currently, SARS-CoV-2 infection is diagnosed presumptively based on clinical symptoms and confirmed by detecting the viral RNA in respiratory samples using reverse transcription polymerase chain reaction (RT-PCR). Standard RT-PCR protocols are time consuming, expensive, and technically demanding, which makes them a poor choice for large scale and point-of-care screening in resource-poor settings. Recently developed isothermal nucleic acid amplification tests (iNAAT), antigen and/or serological tests are cost-effective to scale COVID-19 testing at the point-of-care (PoC) and for surveillance activities. This review discusses the development of rapid PoC molecular tools for the detection and surveillance of SARS-CoV-2 infections.

Keywords: COVID-19; SARS-CoV-2; diagnostics; isothermal amplification (LAMP); point-of-care; sample types; surveillance.

Figure 1

COVID-19 diagnostic testing through isothermal NAAT. Reverse transcriptase LAMP (RT-LAMP) detection of SARS-CoV-2 RNA in nasal swab and saliva samples. These samples can be stored in the refrigerator for 3 days prior to NAAT testing. Where testing can be done immediately, sample preparation and/or RNA extraction is performed, which may take between 5 and 20 min. RNA purification is often required for RT-PCR-based testing. This is done because contaminants in crude cell lysates could potentially reduce the polymerase activity of the reverse transcriptase and DNA polymerases used in RT-PCR. In contrast, the Bacillus stearothermophilus (Bst) DNA polymerase used in LAMP is more tolerant to inhibitors. For RT-LAMP testing, a set of four to six primers targeting any of the viral genes can be designed using online programs, e.g., Eiken Primer design software. It is recommended that a primer set targeting a human endogenous gene is included as a control for sample preparation/RNA extraction and amplification efficiency. In RT-LAMP, both cDNA synthesis (reverse transcriptase) and amplification (Bst) occur simultaneously and in the same reaction tube at a constant temperature (60–65°C). A double-stranded DNA intercalating dye can be added to detect amplicons either by colorimetry (show color change), fluorescent (for real-time detection) or both. RT-LAMP is prone to false-positive amplifications and as such any assay developed using this technique needs to be standardized for each test type. *In a NAAT-based assay, two targets on N gene were included in a single reaction to increase the test sensitivity. Created with Biorender.com.