Oil immersed lossless total analysis system for integrated RNA extraction and detection of SARS-CoV-2

Abstract

The COVID-19 pandemic exposed difficulties in scaling current quantitative PCR (qPCR)-based diagnostic methodologies for large-scale infectious disease testing. Bottlenecks include lengthy multi-step processes for nucleic acid extraction followed by qPCR readouts, which require costly instrumentation and infrastructure, as well as reagent and plastic consumable shortages stemming from supply chain constraints. Here we report an Oil Immersed Lossless Total Analysis System (OIL-TAS), which integrates RNA extraction and detection onto a single device that is simple, rapid, cost effective, and requires minimal supplies and infrastructure to perform. We validated the performance of OIL-TAS using contrived SARS-CoV-2 viral particle samples and clinical nasopharyngeal swab samples. OIL-TAS showed a 93% positive predictive agreement (n = 57) and 100% negative predictive agreement (n = 10) with clinical SARS-CoV-2 qPCR assays in testing clinical samples, highlighting its potential to be a faster, cheaper, and easier-to-deploy alternative for infectious disease testing.

Ultrafast and absolute quantification of SARS-CoV-2 on food using hydrogel RT-LAMP without pre-lysis

“…Hence, 10 min was the most appropriate reaction time to monitor SARS-CoV-2 quantitatively in gel system. This is an ultrafast amplification for the SARS-CoV-2 testing compared with other methods ( Broughton et al, 2020 , Juang et al, 2021 , Manzanas et al, 2021 , Zhu et al, 2020 ). Similarly, we selected different concentrations of reagents (total Mg 2+ , loop primer, and reverse transcriptase) and temperature gradients to investigate the highest single-molecule amplification efficiency within 10 min (see Fig.…”

“…However, a long detection time of SARS-CoV-2 is a limitation for rapid acquisition of screening results. As for illustration, virus lysis, RNA purification, inhibitor removal, RNA reverse transcription, and cDNA amplification are vital steps for SARS-CoV-2 nucleic acid detection ( Juang et al, 2021 , Manzanas et al, 2021 , Zhang et al, 2021 , Zhu et al, 2022 ). Usually, 30-60 minutes are required for RNA reverse transcription and cDNA amplification process.…”

Magnetofluid-Integrated Multicolor Immunochip for Visual Analysis of Neutralizing Antibodies to SARS-CoV-2 Variants

“…Recently, Juang et al developed an oil-immersed lossless total analysis system for the detection of SARS-CoV-2 virus. 18 In this study, they constructed discrete hydrophilic sites on the chip surface to load microdroplets. The enrichment, amplification, and analysis of RNA was realized by the shuttle of magnetic beads in the peripheral oil phase.…”

“…As a total analysis system, microfluidic technology has unique advantages in terms of precise fluid control, reagent transfer and mixing, and system automation, which has good prospects in POCT. − For instance, Qin’s group developed an integrative volumetric bar-chart chip for quantitative immune-analysis . The chip realizes step-by-step reactions and visual output of air pressure signals by movement of different slices of the chip.…”

Application of under-oil open microfluidic systems for rapid phenotypic antimicrobial susceptibility testing

“…In addition, access to these automated systems and platforms can be limited for new antibiotics and settings 11 with low laboratory resources such as research labs, outpatient clinics, point of care, and middle-and low-income countries. Recently, there has been renewed interest in the development of multi-liquid-phase microfluidics, named UOMS [12][13][14][15][16][17][18][19][20][21][22][23][24][25] . In UOMS, cell culture is implemented with the culture media and cells contained under an oil overlay, separating the cell culture/detection microenvironment from the ambient with an immiscible liquid (i.e., oil) rather than the closed chambers/channels of traditional microfluidic devices.…”