Centrifugal Microfluidic Method for Enrichment and Enzymatic Extraction of Severe Acute Respiratory Syndrome Coronavirus 2 RNA

Abstract

The diversification of analytical tools for diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is imperative for effective virus surveillance and transmission control worldwide. Development of robust methods for rapid, simple isolation of viral RNA permits more expedient pathogen detection by downstream real-time reverse transcriptase polymerase chain reaction (real-time RT-PCR) to minimize stalled containment and enhance treatment efforts. Here, we describe an automatable rotationally driven microfluidic platform for enrichment and enzymatic extraction of SARS-CoV-2 RNA from multiple sample types. The multiplexed, enclosed microfluidic centrifugal device (μCD) is capable of preparing amplification-ready RNA from up to six samples in under 15 min, minimizing user intervention and limiting analyst exposure to pathogens. Sample enrichment leverages Nanotrap Magnetic Virus Particles to isolate intact SARS-CoV-2 virions from nasopharyngeal and/or saliva samples, enabling the removal of complex matrices that inhibit downstream RNA amplification and detection. Subsequently, viral capsids are lysed using an enzymatic lysis cocktail for release of pathogenic nucleic acids into a PCR-compatible buffer, obviating the need for downstream purification. Early in-tube assay characterization demonstrated comparable performance between our technique and a “gold-standard” commercial RNA extraction and purification kit. RNA obtained using the fully integrated μCDs permitted reliable SARS-CoV-2 detection by real-time RT-PCR. Notably, we successfully analyzed full-process controls, positive clinical nasopharyngeal swabs suspended in viral transport media, and spiked saliva samples, showcasing the method’s broad applicability with multiple sample matrices commonly encountered in clinical diagnostics.

2023 National Institute of Justice Forensic Science Research and Development Symposium: American Academy of Forensic Sciences 75th Annual Scientific Conference

“…To increase the analytical range associated with forensic epigenetic analysis, the presenter has developed a microfluidic system for the automated sample preparation that includes both DNA extraction and chemical conversion. The extraction step employs a thermophilic enzyme for lysis, producing an eluate in PCR-compatible buffer; this step omits the use of any solid phase materials to immobilize and wash the extract to conserve template (Turiello et al, 2022). Released DNA proceeds through a miniaturized, dynamic solid phase BSC (dSP-BSC) process that has been previously optimized to conserve NA templates and maintain high conversion efficiency.…”

Recent advances in microchip electrophoresis for analysis of pathogenic bacteria and viruses

“…The extraction is based on immiscible filtration augmented by surface tension while RNA amplification was performed using reverse‐transcription loop‐mediated isothermal amplification. The developed microdevice can extract and detect 470 SARS‐CoV‐2 copies/ml within 1 h. Landers and co‐workers developed a multiplexed centrifugal microfluidic device for automated nanotrap viral enrichment and enzymatically based extraction of SARS‐CoV‐2 RNA from clinical samples [133]. Magnetic nanoparticles were utilized to capture virion and isolate it from clinical samples.…”

MALDI-TOF Mass Spectrometric Detection of SARS-CoV-2 Using Cellulose Sulfate Ester Enrichment and Hot Acid Treatment

“…Compared to bacteria identification, virus identification using MALDI-TOF MS faces critical challenges that hamper its application in clinical laboratories, including the difficulty of acquiring an enriched virus sample and the scarcity of characteristic mass peaks related to characteristic viral proteomes. − Molecular docking studies showed that spike proteins of SARS-CoV-2 interact with cellular heparin sulfate and angiotensin-converting enzyme 2 (ACE2) to initiate cellular entrance. , It was also shown that heparin binds spike proteins in vitro, suggesting that cellulose sulfate functional groups can be used to enrich SARS-CoV-2 . In this study we hypothesized that cellulose sulfate ester columns could be used as an enrichment method for SARS-CoV-2, and a rapid detection approach can be developed on the basis of peptide profiles from the enriched sample provided by MALDI-TOF MS.…”