Abstract
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 min. 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.
Keywords: Affinity nanoparticles; Enzymatic extraction; Loop-mediated amplification (LAMP); Polymerase chain reaction (PCR); Recombinase polymerase amplification (RPA); Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2).
【저자키워드】 Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), Polymerase chain reaction (PCR), Affinity nanoparticles, Enzymatic extraction, Loop-mediated amplification (LAMP), Recombinase polymerase amplification (RPA), 【초록키워드】 severe acute respiratory syndrome coronavirus 2, SARS-CoV-2, Saliva, coronavirus, COVID-19 pandemic, severe acute respiratory syndrome coronavirus-2, severe acute respiratory syndrome Coronavirus, RT-PCR, Particle, Laboratory, RNA, Severe acute respiratory syndrome, polymerase chain reaction, amplification, clinical samples, Viral, Concordance, VTM, Loop-mediated isothermal amplification, nasopharyngeal swabs, LAMP, zoonotic, reverse transcription, isothermal amplification, viral transport media, RNA virus, respiratory, pathogenicity, recombinase polymerase amplification, affinity, RPA, Coronavirus-2, miniaturization, Analysis, Dilution, alternative methods, Chain Reaction, alternative method, the stage, characterization, acute respiratory syndrome, solution, acute respiratory syndrome coronavirus, diagnoses, reverse transcription real-time polymerase chain reaction, Stage, double-blind study, viral titers, positive, compatibility, dilutions, polymerase, transmission rate, viral titer, RNA isolation, polymerase chain, SARS-CoV-2 viral, clinically, form, characterized, demonstrated, comparable, RNA eluate, 【제목키워드】 SARS-CoV-2 virus, RNA,