The Future of Point-of-Care Nucleic Acid Amplification Diagnostics after COVID-19: Time to Walk the Walk

Abstract

Since the onset of the COVID-19 pandemic, over 610 million cases have been diagnosed and it has caused over 6.5 million deaths worldwide. The crisis has forced the scientific community to develop tools for disease control and management at a pace never seen before. The control of the pandemic heavily relies in the use of fast and accurate diagnostics, that allow testing at a large scale. The gold standard diagnosis of viral infections is the RT-qPCR. Although it provides consistent and reliable results, it is hampered by its limited throughput and technical requirements. Here, we discuss the main approaches to rapid and point-of-care diagnostics based on RT-qPCR and isothermal amplification diagnostics. We describe the main COVID-19 molecular diagnostic tests approved for self-testing at home or for point-of-care testing and compare the available options. We define the influence of specimen selection and processing, the clinical validation, result readout improvement strategies, the combination with CRISPR-based detection and the diagnostic challenge posed by SARS-CoV-2 variants for different isothermal amplification techniques, with a particular focus on LAMP and recombinase polymerase amplification (RPA). Finally, we try to shed light on the effect the improvement in molecular diagnostics during the COVID-19 pandemic could have in the future of other infectious diseases.

Keywords: CRISPR; RT-qPCR; SARS-CoV-2; isothermal amplification; point-of-care diagnostics; variants.

Figure 1

Requirements of the (RE)ASSURED [17,18], CLIA [19] and STARLITE [14] criteria for point-of-care diagnostics. Created with Biorender.com (accessed on 10 October 2022).

Figure 2

Workflows of the RT-LAMP assays approved by the Food and Drug Administration for home use. (A) Detect COVID-19 test workflow. A nasopharyngeal swab is self-collected by the patient and introduced in the test tube. Then, the test tube is closed with a cap containing reagent bead. Reagents are mixed with the sample and incubated for 55 min in the Detect hub to allow LAMP amplification. Finally, results are detected via lateral flow after another 10 min. (B) Lucira COVID-19 All-in-One test workflow. A nasal swab is self-collected by the patient and placed in the Lucira hub. The test tube is pushed down to initiate the LAMP amplification reaction. A positive or negative result is obtained after 30 min of incubation. Created with Biorender.com (accessed on 12 October 2022).