Point-of-care detection of SARS-CoV-2 in nasopharyngeal swab samples using an integrated smartphone-based centrifugal microfluidic platform

Abstract

With its origin estimated around December 2019 in Wuhan, China, the ongoing 2020 SARS-CoV-2 pandemic is a major global health challenge, resulting in more than 45 million infections and 1.2 million deaths. The demand for scalable, rapid and sensitive viral diagnostics is thus particularly pressing at present to help contain the rapid spread of infection and prevent overwhelming the capacity of health systems. While high-income countries have managed to rapidly expand diagnostic capacities, such is not the case in resource-limited settings of low- to medium-income countries. Aiming at developing cost-effective viral load detection systems for point-of-care COVID-19 diagnostics in resource-limited and resource-rich settings alike, we report the development of an integrated modular centrifugal microfluidic platform to perform loop-mediated isothermal amplification (LAMP) of viral RNA directly from heat-inactivated nasopharyngeal swab samples. The discs were pre-packed with dried n-benzyl-n-methylethanolamine modified agarose beads used as a versatile post-nucleic acid amplification signal enhancement strategy, allowing fluorescence detection via a smartphone camera and simple optics. The platform provided sample-to-answer analysis within 1 hour from sample collection and a detection limit between 100 and 1000 RNA copies in 10 μL reaction volume. Furthermore, direct detection of non-extracted SARS-CoV-2 RNA in nasopharyngeal swab samples from patients with Ct values below 26 (n=25 plus 6 PCR negative samples) was achieved with ~94% sensitivity and 100% specificity, thus being fit-for-purpose to diagnose patients with a high risk of viral transmission. These results show significant promise towards bringing routine point-of-care COVID-19 diagnostics closer to resource-limited settings.

Rapid molecular diagnostics of COVID-19 by RT-LAMP in a centrifugal polystyrene-toner based microdevice with end-point visual detection

“…This detection limit is significantly lower than recent published studies describing LAMP in microdevices for the diagnosis of COVID-19. 20,43,44 The low detection limit found in this study is due to the fact that the use of SYBR Green leads to increased sensitivity when compared with others detection method. 47 However, the inhibitory effect of SYBR Green on the LAMP reaction is well established.…”

“…Currently, some studies have reported the molecular detection of SARS-CoV-2 by LAMP in tubes with a limit of detection of 0.1-10 copies per µL. 41,42 A recently published study by Soares et al 20 reported a LAMP assay performed on a PMMAbased microfluidic platform with a detection limit of 10 2 -10 3 copies per reaction and an analysis time of 30 min. Rodriguez-Manzano and coworkers 43 described a LAMP assay in a cartridge microfluidic device for the detection of SARS-CoV-2 RNA samples showing a limit of detection of 10 RNA copies per reaction in under 20 minutes.…”

“…The test was performed within 1.5 hours and detected SARS-CoV-2 with 2 × 10 1 copies per reaction. Soares et al 20 developed a cost-effective integrated modular PMMA centrifugal microfluidic platform to perform a 30 min LAMP assay for SARS-CoV-2 detection. The authors reached a limit of detection of 10 2 -10 3 copies per reaction.…”

Loop-Mediated Isothermal Amplification Detection of SARS-CoV-2 and Myriad Other Applications

“…Two microfluidic systems based on centrifugal liquid flow have been reported. With the objective of simplifying the sample processing step, Soares et al (2020) developed an integrated smartphone-enabled, centrifugal, microfluidic platform for a sample-to-result fluorescence-based RT-LAMP test. Using modified agarose beads, their device enhances the signal specificity and mitigates the effect of collection medium on weakly buffered colorimetric LAMP assays ("Considerations for pH-Based Colorimetric RT-LAMP").…”

Automated sample-to-answer centrifugal microfluidic system for rapid molecular diagnostics of SARS-CoV-2

“…Soares et al have used LAMP for the detection of SARS-CoV-2 on a centrifugal platform using agarose beads for signal enhancement after nucleic acid amplification. 38 Their system can detect 100 to 1000 RNA copies in a 10 μL reaction volume based on fluorescence read-out using a simple optical set-up and a smartphone camera. More recently, de Oliveira et al have developed a low-cost centrifugal device made of polystyrene-toner that is operated on a fidget-spinner for molecular diagnosis of SARS-CoV-2 RNA (prepared off-chip) using RT-LAMP.…”