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

Finally, we sought to demonstrate that omitting RNA extraction does not adversely affect qSanger sensitivity. We added 20 GCE (corresponding to 2x the LOD in Fig. 1 ) of viral particles in VTM containing either negative control or SARS-CoV-2 RNA (Fig. 4) . Sanger chromatograms clearly showed the absence and presence of SARS-CoV-2 signal in the negative and positive controls, respectively (Fig. 4A ). The qSanger assay correctly identified the negative and positive samples in 39 out of the 40 samples tested, with 1 negative control specimen returning an undetermined result due to sequencing failure (Fig. 4B) . Overall, the excellent performance shown by our qSanger results on unextracted VTM vs. purified RNA with respect to absolute quantification accuracy, Poisson-limited coefficient of variation, and limit of detection that is comparable to gold-standard RT-qPCR, suggests that qSanger can be performed on unprocessed specimen matrix without any loss in performance. In fact, it might be possible that RNA-extraction free methods are more reliable because it eliminates the carryover risk of high-salt, PCR-inhibiting buffers used in RNA extraction procedures.