Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a zoonotic RNA virus characterized by high transmission rates and pathogenicity worldwide. Continued control of the COVID-19 pandemic requires the diversification of rapid, easy to use, sensitive, and portable methods for SARS-CoV-2 sample preparation and analysis. Here, we propose a method for SARS-CoV-2 viral enrichment and enzymatic extraction of RNA from clinically relevant matrices in under 10 minutes. This technique utilizes affinity-capture hydrogel particles to concentrate SARS-CoV-2 from solution, and leverages existing PDQeX technology for RNA isolation. Characterization of our method is accomplished with reverse transcription real-time polymerase chain reaction (RT-PCR) for relative, comparative RNA detection. In a double-blind study analyzing viral transport media (VTM) obtained from clinical nasopharyngeal swabs, our sample preparation method demonstrated both comparable results to a routinely used commercial extraction kit and 100% concordance with laboratory diagnoses. Compatibility of eluates with alternative forms of analysis was confirmed using microfluidic RT-PCR (μRT-PCR), recombinase polymerase amplification (RPA), and loop-mediated isothermal amplification (LAMP). The alternative methods explored here conveyed successful amplification from all RNA eluates originating from positive clinical samples. Finally, this method demonstrated high performance within a saliva matrix across a broad range of viral titers and dilutions up to 90% saliva matrix, and sets the stage for miniaturization to the microscale.
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.
The polymerase chain reaction (PCR) is paramount in nucleic acid amplification testing, and for many assays, the use of PCR or qPCR is considered the ‘gold standard’. While instrumentation for...
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