Here we present a rapid and versatile method for capturing and concentrating SARS-CoV-2 from contrived transport medium and saliva samples using affinity-capture magnetic hydrogel particles. We demonstrate that the method concentrates virus from 1 mL samples prior to RNA extraction, substantially improving detection of virus using real-time RT-PCR across a range of viral titers (100–1,000,000 viral copies/mL) and enabling detection of virus using the 2019 nCoV CDC EUA Kit down to 100 viral copies/mL. This method is compatible with commercially available nucleic acid extraction kits (i.e., from Qiagen) and a simple heat and detergent method that extracts viral RNA directly off the particle, allowing a sample processing time of 10 min. We furthermore tested our method in transport medium diagnostic remnant samples that previously had been tested for SARS-CoV-2, showing that our method not only correctly identified all positive samples but also substantially improved detection of the virus in low viral load samples. The average improvement in cycle threshold value across all viral titers tested was 3.1. Finally, we illustrate that our method could potentially be used to enable pooled testing, as we observed considerable improvement in the detection of SARS-CoV-2 RNA from sample volumes of up to 10 mL.

A generic method for real-time monitoring of enzyme kinetics is described in this paper. This approach enables rapid development of assays for high-throughput screening or reaction monitoring in the linear range of the enzyme kinetic curve. In this paper, we used protein kinase A and kemptide (a well-studied assay system) to demonstrate assay optimization by using micro parallel liquid chromatography. The optimal substrate and enzyme concentrations were determined rapidly and conveniently compared with the traditional methods for determining these parameters. Additionally, the data collected from the same experiment permitted calculations of K (m) for the substrate, V (max), and time-course study. In general, this approach provides two advantages. First, the broad ranges of detectable product conversions facilitate selection and implementation of assay conditions for high-throughput screening. Second, the system permits determination of 50% inhibitory concentration values at less than 1% conversion of substrate to product, thereby validating screening hits in the linear range of the enzyme kinetic curve. Overall, this optimization process can be done in less than 8 h. To demonstrate the ability to monitor a wide range of assay conditions, we varied initial concentrations over eight orders of magnitude within a single experiment. Compared with a classical enzyme kinetics study, this method significantly speeds the target validation process and reduces time associated with assay development and high-throughput screening implementation.

AbstractHere we present a rapid and versatile method for capturing and concentrating SARS-CoV-2 from transport medium and saliva using affinity-capture magnetic hydrogel particles. We demonstrate that the method concentrates virus prior to RNA extraction, thus significantly improving detection of the virus using a real-time RT-PCR assay across a range of viral titers, from 100 to 1,000,000 viral copies/mL; in particular, detection of virus in low viral load samples is enhanced when using the method coupled with the IDT 2019-nCoV CDC EUA Kit. This method is compatible with commercially available nucleic acid extraction kits, as well with a simple heat and detergent method. Using transport medium diagnostic remnant samples that previously had been tested for SARS-CoV-2 using either the Abbott RealTime SARS-CoV-2 EUA Test (n=14) or the Cepheid Xpert Xpress SARS-CoV-2 EUA Test (n=35), we demonstrate that our method not only correctly identifies all positive samples (n = 17) but also significantly improves detection of the virus in low viral load samples. The average improvement in cycle threshold (Ct) value as measured with the IDT 2019-nCoV CDC EUA Kit was 3.1; n = 10. Finally, to demonstrate that the method could potentially be used to enable pooled testing, we spiked infectious virus or a confirmed positive diagnostic remnant sample into 5 mL and 10 mL of negative transport medium and observed significant improvement in the detection of the virus from those larger sample volumes.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) RNA monitoring in wastewater has become an important tool for COVID-19 surveillance. Although many viral concentration methods such as membrane filtration and skim milk are reported, these methods generally require large volumes of wastewater, expensive lab equipment, and laborious processes. We utilized a Nanotrap&R Microbiome A Particles (Nanotrap particle) method for virus concentration in wastewater. The method was evaluated across six parameters: pH, temperature, incubation time, wastewater volumes, RNA extraction methods, and two virus concentration approaches vs. a one-step method. The method was further evaluated with the addition of the Nanotrap Enhancement Reagent 1 (ER1) by comparing the automated vs. a manual Nanotrap particle method. RT-qPCR targeting the nucleocapsid protein was used for detection and quantification of SARS-CoV-2 RNA. Different pH, temperature, incubation time, wastewater volumes, and RNA extraction methods did not result in reduced SARS-CoV-2 detection in wastewater samples. The two-step concentration method showed significantly better results (P<0.01) than the one-step method. Adding ER1 to wastewater prior to viral concentration using the Nanotrap particles significantly improved PCR Ct results (P<0.0001) in 10 mL grab samples processed by automated Nanotrap particle method or 10 mL and 40 mL samples processed by manual Nanotrap particle method. SARS-CoV-2 detection in 10 mL grab samples with ER1 and the automated method showed significantly better (P=0.0008) results than 150 mL grab samples using the membrane filtration method. SARS-CoV-2 detection in 10 mL swab samples with ER1 via the automated method was also significantly better than without ER1 (P<0.0001) and the skim milk method in 250 mL Moore swab samples (P=0.012). These results suggest that Nanotrap methods could substitute the traditional membrane filtration and skim milk methods for viral concentration without compromising on the assay sensitivity. The manual method can be used in resource-limited areas, and the high-throughput platform is appropriate for large-scale COVID-19 wastewater-based surveillance.

Presented here is a magnetic hydrogel particle enabled workflow for capturing and concentrating SARS-CoV-2 from diagnostic remnant swab samples that significantly improves sequencing results using the Oxford Nanopore Technologies MinION sequencing platform. Our approach utilizes a novel affinity-based magnetic hydrogel particle, circumventing low input sample volumes and allowing for both rapid manual and automated high throughput workflows that are compatible with nanopore sequencing. This approach enhances standard RNA extraction protocols, providing up to 40x improvements in viral mapped reads, and improves sequencing coverage by 20-80% from lower titer diagnostic remnant samples. Furthermore, we demonstrate that this approach works for contrived influenza virus and respiratory syncytial virus samples, suggesting that it can be used to identify and improve sequencing results of multiple viruses in VTM samples. These methods can be performed manually or on a KingFisher Apex system.