A Highly Scalable and Rapidly Deployable RNA Extraction-Free COVID-19 Assay by Quantitative Sanger Sequencing

Devon Chandler-Brown; Anna M. Bueno; Oguzhan Atay; David S. Tsao

Since a major limitation for increasing testing capacity has been supply chain and lab workflow bottlenecks related to RNA extraction, we next attempted to detect SARS-CoV-2 directly from the specimen matrix (viral transport medium). There has been a previous report that RT-qPCR can be successfully performed when up to 3-7ul (total reaction volume of 20ul) of VTM without extraction is used as the template for RT-PCR [9] . We hypothesized that qSanger could be a more reliable method for detection of COVID-19 without RNA extraction because of i . increased robustness against PCR inhibitors in the specimen matrix since quantification of SARS-CoV-2 is performed via comparison with the spike-in internally control; ii. an improved limit of detection by adding more VTM to a correspondingly larger reaction size; and, iii.