Abstract
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.
Keywords: COBAS 4800; COVID-19; E-gene; Extractionless; Heat extraction; N1-gene; N2-gene; Orf1ab gene; Quantabio; RdRp gene; Roche; SARS-CoV-2.
【저자키워드】 COVID-19, SARS-CoV-2, Roche, ORF1ab gene, E-gene, RdRp gene, Heat extraction, COBAS 4800, Extractionless, N1-gene, N2-gene, Quantabio,