Extractionless Nucleic Acid Detection: A High Capacity Solution to COVID-19 Testing

Baksh, Shairaz; Volodko, Natalia; Soucie, Merle; Geier, Sheena Brandon; Diep, Anthony; Rozak, Kallie; Chan, Tak Yin; Mustapha, Jelili; Lai, Adjunct Professor Raymond; Estey, Associate Clinical Professor Mathew; Verity, Associate Clinical Professor Bob; Lee, Associate Clinical Professor Mao-Cheng

We describe an extractionless real-time reverse transcriptase-PCR (rRT-PCR) protocol for SARS‐CoV‐2 nucleic acid detection using heat as an accurate cost-effective high-capacity solution to COVID‐19 testing. We present the effect of temperature, transport media, rRT-PCR mastermixes and gene assays on SARS‐CoV‐2 gene amplification and limits of detection. Utilizing our heated methodology, our limits of detection were 12.5 and 1 genome copy/reaction for singleplex E‐ and N1‐gene assays, respectively, and 1 genome copy/reaction by utilizing an E/N1 or Orf1ab/N1 multiplex assay combination. Using this approach, we detected up to 98% of COVID‐19 positive patient samples analyzed in our various cohorts including a significant percentage of weak positives. Importantly, this extractionless approach will allow for > 2‐fold increase in testing capacity with existing instruments, circumvent the additional need for expensive extraction devices, provide the sensitivity needed for COVID‐19 detection and significantly reduce the turn‐around time of reporting COVID‐19 test results.