A novel two-step, direct-to-PCR method for virus detection off swabs using human coronavirus 229E

Abstract

Background: 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. Methods: Using Human Coronavirus 229E (HCoV-229E) as a model system, we spiked swabs in vitro for proof-ofconcept testing. Swabs were spiked in serial dilutions from 1.2 × 10 6 to 1.2 × 10 1 copies/mL and then placed in 2 mL 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. Results: HCoV-229E in vitro 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 1.2 × 10 3 viral copies/mL with 96.30% sensitivity. Conclusion: 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.

The Utility of Mechanical Homogenization in COVID-19 Diagnostic Workflows

“…Traditionally the process of mechanical homogenization has been employed in the laboratory setting for the disruption of animal or plant tissues in preparation for downstream molecular applications [1]. However, in the face of a global pandemic this technology has been adapted to increase efficacy and efficiency in viral detection in a variety of COVID-19 diagnostic workflows [2][3][4].…”

“…During the development of these novel testing protocols laboratory safety, diagnostic assay sensitivity and specificity became top priority [5][6][7]. In an attempt to utilize common laboratory equipment to safely speed up testing efforts, the use of mechanical homogenization was proposed to inactivate the SARS-CoV-2 from nasopharyngeal swabs as a method of increasing safety during processing [2][3][4]8].…”

“…The parameters at which a sample is processed will impact the degree to which it is dissociated and the quality of the targeted product for downstream applications [1]. In the case of SARS-CoV-2, the goal of mechanical homogenization was to disrupt the viral envelope while still maintaining the integrity of its RNA [2,3]. This allowed for a reduction in infective potential in the laboratory setting, while preserving the accuracy of polymerase chain reaction (PCR) based diagnostic assays [2,3].…”

“…In the case of SARS-CoV-2, the goal of mechanical homogenization was to disrupt the viral envelope while still maintaining the integrity of its RNA [2,3]. This allowed for a reduction in infective potential in the laboratory setting, while preserving the accuracy of polymerase chain reaction (PCR) based diagnostic assays [2,3].…”

“…Following the initial application of mechanical homogenization to COVID-19 swab-based PCR protocols, this technology was adapted to process saliva samples for both antigen and PCR detection workflows [2][3][4]. Through mechanical homogenization, high viscosity saliva samples were sufficiently processed to allow for automation integration, paving the way for the widespread application of this novel methodology [4,9].…”