Endpoint PCR Detection of Sars-CoV-2 RNA

Abstract

Quantitative 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 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.

Evidence-based pandemic preparedness: an infrastructure for population-scale genome-based based testing for COVID-19

“…We have therefore developed a x10-x20 more efficient, highly sensitive virus-genome-based test, using PCR technologies developed by some of us as part of the Human Genome Project 8 , and used to genotype billions of samples over the last decades 3 . All equipment is commercially available.…”

“…To combat the spread of COVID-19, most countries have relied on social distancing, lockdown, limited testing and contact tracing procedures, as well as crash programs to develop vaccines, which are now increasingly contributing to the fight against the pandemic. As a complement to these two basic strategies, we and others proposed to use an alternative method, repeated population-wide tests [1][2][3] , to identify (and quarantine) the infected and break infection chains. This strategy has the potential to reduce human suffering, economic costs and restrictions on personal freedom to the inevitable minimum, since quarantine would be limited to a short time for a small fraction of the population and could be much more effective than inherently leaky lockdowns.…”

Evidence-based Pandemic Preparedness: An Infrastructure for Population-scale Genome-based Testing for COVID-19

“…We have therefore developed a x10-x20 more e cient, highly sensitive virus-genome-based test, using PCR technologies developed by some of us as part of the Human Genome Project 8 , and used to genotype billions of samples over the last decades 3 . All equipment is commercially available.…”

“…To combat the spread of COVID-19, most countries have relied on social distancing, lockdown, limited testing and contact tracing procedures, as well as crash programs to develop vaccines, which are now increasingly contributing to the ght against the pandemic. As a complement to these two basic strategies, we and others proposed to use an alternative method, repeated population-wide tests [1][2][3] , to identify (and quarantine) the infected and break infection chains. This strategy has the potential to reduce human suffering, economic costs and restrictions on personal freedom to the inevitable minimum, since quarantine would be limited to a short time for a small fraction of the population and could be much more effective than inherently leaky lockdowns.…”

CoV2-ID, a MIQE-compliant sub-20-min 5-plex RT-PCR assay targeting SARS-CoV-2 for the diagnosis of COVID-19

“…The principal aim of current SARS-CoV-2 diagnostic tests is to detect unambiguously the presence or absence of viral targets. Where viral load is not being determined, there is no requirement to quantify viral load and therefore no need to detect amplification products in real-time 16 . Instead, we have developed a multiple cycle fluorescence detection protocol that measures baseline fluorescence towards the beginning of the run and then records discrete increases after a specified number of cycles to detect target-dependent amplification, whilst at the same time saving the considerable time it takes to scan a plate at the end of each cycle.…”