A Novel Two-Step, Direct-to-PCR Method for Virus Detection off Swabs using Human Coronavirus 229E

Abstract

<jats:title>Abstract</jats:title> <jats:p><jats:bold>Background </jats:bold>Currently, one of the most reliable methods for viral infection detection are polymerase chain reaction (PCR) based assays. This process is time and resource heavy, requiring multiple steps of lysis, extraction, purification, and amplification procedures. Herein, we have developed a method to detect virus off swabs using solely shaker-mill based mechanical lysis and the transfer of the viral lysate directly to a PCR assay for virus detection, bypassing the substantial reagent and time investments required for extraction and purification steps.<jats:bold>Methods </jats:bold>Using Human Coronavirus 229E (HCoV-229E) as a model system, we spiked swabs <jats:italic>in vitro</jats:italic> for proof-of-concept testing. Swabs were spiked in serial dilutions from 1.2x10<jats:sup>6</jats:sup> to 1.2x10<jats:sup>1</jats:sup> copies/mL and then placed in 2mL tubes with viral transport media (VTM) to mimic the specimen collection procedures in the clinic prior to processing via shaker-mill homogenization. After homogenization, 1 µL of lysate was processed using RT-qPCR for amplification of the nucleocapsid (N) gene, qualifying viral detection. <jats:bold>Results </jats:bold>HCoV-229E <jats:italic>in vitro</jats:italic> spiked swabs were processed in a novel two-step, direct-to-PCR methodology for viral detection. After running 54 swabs, we confidently determined our limit of detection to be 1.2x10<jats:sup>3</jats:sup> viral copies/mL with 96.30% sensitivity. <jats:bold>Conclusion </jats:bold>We have proven that the shaker-mill homogenization-based two-step, direct-to-PCR procedures provides sufficient viral lysis off swabs, where the resulting lysate can be used directly in PCR for the detection of HCoV-229E. This finding allows for reductions in the time and resources required for PCR based virus detection in comparison to the traditional extraction-to-PCR methodology.</jats:p>

Speeding Up COVID-19 Detection Using Shaker-Mill Homogenization and a Direct-to-PCR Workflow

“…19-040E) for processing. The samples were run at 4.2 m/s for 30 seconds and removed from the device [9]. The samples were permitted to sit for 1 minute after processing to allow for any froth that formed in the tubes during shaker-mill homogenization to settle [9].…”

Smart materials-integrated sensor technologies for COVID-19 diagnosis

“…To curb these pandemics, early diagnosis is the key step to initiate the most effective therapy and also, to manage the spread of infections [11,12]. Enzymelinked immunosorbent test (ELISA) [13], RT-PCR [14], polymerase chain reaction (PCR) [15], and a variety of biosensor [16] are employed to detect viruses. Virus detection platforms reliant on antigen-antibody or receptor-ligand-based are also available to the point-of-need settings [17,18].…”