An ultrafast SARS-CoV-2 virus enrichment and extraction method compatible with multiple modalities for RNA detection

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 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.

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

“…The 60 °C annealing temperature was determined optimal over the manufacturer’s recommended temperature of 55 °C previously. 13 Amplification was considered successful if the signal amplitude crossed the instrument-defined threshold, providing a C t value, before the 40 cycle cutoff.…”

“… 12 Nanotrap enrichment, in conjunction with one-step enzymatic extraction, has proved successful in detecting SARS-CoV-2 from clinical nasopharyngeal swabs, surveillance sample mimics, and contrived saliva samples. 13 …”

Rapid SARS-CoV-2 Virus Enrichment and RNA Extraction for Efficient Diagnostic Screening of Pooled Nasopharyngeal or Saliva Samples for Dilutions Up to 1:100

“…Before extraction, samples were enriched via nanoparticle pre-concentration as described by Dignan et al [ 11 ]. Each dilution ratio was applied to a total volume of 500 µL; for example, 50 µL of positive pooled sample was added to 450 µL of negative clinical sample for the 1:10 dilution.…”

“…Furthermore, saliva specimens provide a more facile opportunity for pooled surveillance testing, which is a strategy proposed by several institutions for the monitoring of COVID-19 transmission [ 9 , 10 ]. Alternative viral lysis methods have also been demonstrated; notably, our group proposed a technique for SARS-CoV-2 nanoparticle-facilitated enrichment and enzymatic lysis from clinical samples that leverages existing PDQeX technology in under 10 min [ 11 ]. This method demonstrated comparable sensitivity to gold-standard methods for RNA isolation from clinical samples and provided positive diagnoses from NP specimens and saliva collections.…”

“…As such, a workflow for streamlined surveillance monitoring of SARS-CoV-2 with the potential for diagnosis from pooled clinical samples is detailed here. This methodology couples the ultrafast SARS-CoV-2 virus enrichment and extraction sample preparation technique optimized previously by our group with the downstream detection of pooled samples by rapid microfluidic RT-PCR [ 11 ]. For proof-of-principle, downstream detection is accomplished by conventional RT-PCR using gold-standard instrumentation and kit chemistries given Emergency Use Authorization (EUA) by the Centers for Disease Control and Prevention (CDC).…”