A novel two-step, direct-to-PCR method for virus detection off swabs using human coronavirus 229E
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A novel two-step, direct-to-PCR method for virus detection off swabs using human coronavirus 229E Zachary P. Morehouse1,2* , Caleb M. Proctor2,3, Gabriella L. Ryan2,3 and Rodney J. Nash2,3,4*
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 × 106 to 1.2 × 101 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 × 103 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. Keywords: Virus detection, Viral diagnostics, PCR, Coronavirus
Introduction 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. Herein, we are proposing a novel method for viral pathogen detection off swabs as an improvement or alternative to the current PCR based assays commonly used * Correspondence: [email protected]; [email protected] 1 Michigan State University College of Osteopathic Medicine, East Lansing, MI, USA 2 Omni International Inc, Kennesaw, GA, USA Full list of author information is available at the end of the article
for viral detection [1, 2]. The traditional protocol for preparing a sample for PCR based detection often involves procedures of swabbing a patient, processing the sample to lyse the virus, extract, and purify its nucleotides, and then amplify the purified genetic material via PCR for detection of a gene product needed to confirm the patient’s suspected diagnosis [3]. In the face of the COVID-19 pandemic, attempts have been made to perform PCR bas
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