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.

FlashPCR: Revolutionising qPCR by Accelerating Amplification through Low ∆T Protocols

“…The substantial reduction of processing time, whilst maintain the performance characteristics of conventional protocols, will enable timely decision-making by healthcare professionals, facilitate prompt treatment initiation, and help implement efficient infection control measures. Moreover, incorporation into PoC procedures will enhance the responsiveness of public health agencies in managing outbreaks and instituting preventive measures, potentially limiting disease transmission [ 37 , 38 ].…”

Advancements in CRISPR-Based Biosensing for Next-Gen Point of Care Diagnostic Application

“…The CRISPR-Cas technology has progressed from a simpler cleaving mechanism to integrating the CRISPR protein for gene editing, developing a simple biosensor, and diagnostics methods, and this suggests CRISPR as the future of biotechnology and its allied areas due to its properties, as it can provide an answer for fundamental and applied research areas [156]. It is worth noting that CRISPR-Cas sensing technology has solved the low sensitivity issue that is faced by current detection methods [157]. For instance, a CRISPR-based diagnostic was able to detect attomolar quantities of foreign particles, showing their higher sensitivity when compared with PCR-based methods with a femtomolar range of detection.…”

Latest Advances in Arbovirus Diagnostics

“…These systems are all-in-one devices that simultaneously extract and purify nucleic acids from clinical samples, perform PCR amplification and yield a positive/negative read-out at the end of the reaction. Typical run times are 30–60 min, and sensitivity can range from as low as 12 to 6.4 × 10 3 copies/mL [ 55 ]. Veredus laboratories (Science Park Drive, Singapore) have developed a RT-PCR-based POC system for research use only, VereFever™, which can diagnose the presence of CHIKV, DENV 1–4, JEV, WNV, YFV and ZIKV on a single chip, making it the most comprehensive commercially developed system for arbovirus diagnostics.…”