Endpoint PCR Detection of Sars-CoV-2 RNA

Abstract

AbstractQuantitative real-time PCR methods have been used to perform approximately 278 million tests for COVID-19 up to mid-July 2020. Real-time PCR involves a rate limiting step where the samples are measured in situ during each PCR amplification cycle. This creates a bottleneck limiting scalability and as a consequence reducing access to inexpensive reliable testing at national and international scales. We investigated endpoint PCR for the qualitative detection of SARS-CoV-2 sequences on synthetic RNA standards and hospital patient samples. The endpoint PCR detection limit is constrained only by the stochastics of low copy numbers and reliably detected single copies of synthetic RNA standards. On a set of 30 patient samples, endpoint PCR found one additional positive sample and was able to confirm an indeterminate sample as negative. These results were found using 4 μl reagent and 1 μl of sample representing an 80% reduction in required RNA extract input and PCR reagent volumes relative to the NHS protocol (20 μl reagent and 5 μl sample). These results indicate that endpoint PCR should be the method of choice for large scale testing programmes. Based on the experience from ultra-high throughput genotyping efforts a single workflow using 384-well plates has similar PCR capacity (250 Million) to that required for all testing done worldwide during the first 7 month of the pandemic.