693 - Shaking Things Up: Direct-to-PCR Viral Detection off Swabs Using Shaker-Mill Homogenization

Background: As the number of viral diseases are on the rise, it is critical to continue to innovate and advance diagnostic, treatment, and surveillance methods surrounding viral infections. Currently, one of the most reliable methods for viral infection detection are polymerase chain reaction (PCR) based assays. These assays often involve procedures of swabbing a patient, processing the sample to lyse the virus, extract, and purify it’s nucleotides, and then run the purified genetic material via PCR for detection of a gene product needed to confirm the patient’s suspected diagnosis. This process requires time to complete and is dependent on the availability of the reagents and plastics required to complete the lysis, extraction, purification, and amplification procedures. Herein, we have developed a method to detect virus off a swab using solely shaker-mill based mechanical lysis and the transfer of the viral lysate directly to a PCR based assay, bypassing the reagent heavy and time consuming extraction and purification steps.

Methods: Using Human Coronavirus 229E (HCoV-229E) as a model system, we spiked swabs with clinically relevant levels of the virus for proof-of-concept testing. Swabs were spiked in serial dilutions from 1.2e7 copies/mL to 1.2e1 copies/mL. The swabs were 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 uL of viral lysate was run in RT-qPCR for amplification of the nucleocapsid (N) gene, qualifying viral detection from the sample.

Results: HCoV-229E spiked swabs were run through the two-step process of homogenization direct to RT-qPCR for viral detection. After running 54 swabs, we confidently determined our limit of detection to be 1.2e3 viral copies/mL with 96.30% sensitivity in vitro.

Conclusion: We have successfully proven that shaker-mill homogenization provides sufficient viral lysis off swabs, where the resulting lysate can be used directly in PCR based assays for the detection of virus. This finding allows for decreased run time in traditional PCR based diagnostics and reduces the reagents and plastics required for each sample, ultimately reducing the cost and time of each viral test when compared to traditional PCR based methods.