Ultra-rapid real-time microfluidic RT-PCR instrument for nucleic acid analysis.

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 executing PCR has advanced over the last two decades, a growing interest in point-of-need testing has highlighted the deficit that exists for 'rapid PCR' systems. Here, we describe a field-forward prototype instrument capable of ultra-fast thermal cycling for real-time PCR amplification of DNA and RNA. The custom-designed, injection-molded microfluidic chips interface with a novel mechatronic system to complete 40 cycles of real-time PCR in under 10 minutes, an 84% reduction in time compared to a standard 50 minute assay. Such rapid amplification is enabled by two thermoelectric Peltiers capable of efficiently heating and cooling the sample at 12 and 10 °C s-1, respectively. Judicious selection and strategic placement of the thermal cyclers and fluorescence detector relative to the microchip enable synchronized thermal cycling and fluorescence monitoring, further reducing time-to-result. Robust amplification and detection of DNA and RNA targets empowers laboratories to achieve rapid, actionable information in endless applications.

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.

As COVID-19 transmission control measures are gradually being lifted, a sensitive and rapid diagnostic method for large-scale screening could prove essential for monitoring population infection rates. However, many rapid workflows for SARS-CoV-2 detection and diagnosis are not amenable to the analysis of large-volume samples. Previously, our group demonstrated a technique for SARS-CoV-2 nanoparticle-facilitated enrichment and enzymatic lysis from clinical samples in under 10 min. Here, this sample preparation strategy was applied to pooled samples originating from nasopharyngeal (NP) swabs eluted in viral transport medium (VTM) and saliva samples diluted up to 1:100. This preparation method was coupled with conventional RT-PCR on gold-standard instrumentation for proof-of-concept. Additionally, real-time PCR analysis was conducted using an in-house, ultra-rapid real-time microfluidic instrument paired with an experimentally optimized rapid protocol. Following pooling and extraction from clinical samples, average cycle threshold (CT) values from resultant eluates generally increased as the pooling dilution factor increased; further, results from a double-blind study demonstrated 100% concordance with clinical values. In addition, preliminary data obtained from amplification of eluates prepared by this technique and analyzed using our portable, ultra-rapid real-time microfluidic PCR amplification instrument showed progress toward a streamlined method for rapid SARS-CoV-2 analysis from pooled samples.

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

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.

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

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.