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

As an initial demonstration of qualitative detection of COVID-19 by qSanger, we designed PCR primers and synthetic DNA spike-in to target SARS-CoV-2 N protein (Fig. 1B) . A one-step RT-PCR mix (NEB) containing both was used to perform reverse-transcription of SARS-CoV-2 RNA and subsequent PCR amplification in one pot. In each RT-PCR, we added either nuclease-free water as a no-template control (NTC) or 100-5000 GCE of synthetic SARS-CoV-2 RNA (Twist Biosciences). All reactions also contained~200 GCE of spike-in in the RT-PCR master mix. After RT-PCR and Sanger sequencing, we observed a qualitatively clean chromatogram for the spike-in sequence for the NTC condition in which no SARS-CoV-2 RNA was added ( Fig. 2A ). At the 100 GCE level, the Sanger chromatogram showed clear mixed bases corresponding to approximately equal abundance of spike-in DNA and SARS-CoV-2 RNA. At 5000 GCE SARS-CoV-2 RNA, the chromatogram exhibited a relatively pure trace for the SARS-CoV-2 target sequence, suggesting that the SARS-CoV-2 signal overwhelmed the spike-in signal when it was present at 50-fold greater abundance.