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Assessment of Specimen Pooling to Conserve SARS CoV-2 Testing Resources
Abstract
Objectives
To establish the optimal parameters for group testing of pooled specimens for the detection of SARS-CoV-2.
Methods
The most efficient pool size was determined to be five specimens using a web-based application. From this analysis, 25 experimental pools were created using 50 µL from one SARS-CoV-2 positive nasopharyngeal specimen mixed with 4 negative patient specimens (50 µL each) for a total volume of 250 µL. Viral RNA was subsequently extracted from each pool and tested using the CDC SARS-CoV-2 RT-PCR assay. Positive pools were consequently split into individual specimens and tested by extraction and PCR. This method was also tested on an unselected group of 60 nasopharyngeal specimens grouped into 12 pools.
Results
All 25 pools were positive with cycle threshold (Ct) values within 0 and 5.03 Ct of the original individual specimens. The analysis of 60 specimens determined that 2 pools were positive followed by identification of 2 individual specimens among the 60 tested. This testing was accomplished while using 22 extractions/PCR tests, a savings of 38 reactions.
Conclusions
When the incidence rate of SARS-CoV-2 infection is 10% or less, group testing will result in the saving of reagents and personnel time with an overall increase in testing capability of at least 69%.
Journal Article
Fast coronavirus tests: what they can and can’t do
Rapid antigen tests are designed to tell in a few minutes whether someone is infectious. Will they be game changers?
Fast coronavirus tests: what they can and can’t do
Rapid antigen tests are designed to tell in a few minutes whether someone is infectious. Will they be game changers?
Journal Article
Massive and rapid COVID-19 testing is feasible by extraction-free SARS-CoV-2 RT-PCR
Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is commonly diagnosed by reverse transcription polymerase chain reaction (RT-PCR) to detect viral RNA in patient samples, but RNA extraction constitutes a major bottleneck in current testing. Methodological simplification could increase diagnostic availability and efficiency, benefitting patient care and infection control. Here, we describe methods circumventing RNA extraction in COVID-19 testing by performing RT-PCR directly on heat-inactivated or lysed samples. Our data, including benchmarking using 597 clinical patient samples and a standardised diagnostic system, demonstrate that direct RT-PCR is viable option to extraction-based tests. Using controlled amounts of active SARS-CoV-2, we confirm effectiveness of heat inactivation by plaque assay and evaluate various generic buffers as transport medium for direct RT-PCR. Significant savings in time and cost are achieved through RNA-extraction-free protocols that are directly compatible with established PCR-based testing pipelines. This could aid expansion of COVID-19 testing.
SARS-CoV-2 infection is widely diagnosed by RT-PCR, but RNA extraction is a bottleneck for fast and cheap diagnosis. Here, the authors develop protocols to perform RT-PCR directly on heat-inactivated subject samples or samples lysed with readily available detergents and benchmark performance against 597 clinically diagnosed patient samples.
Journal Article
Disentangling primer interactions improves SARS-CoV-2 genome sequencing by multiplex tiling PCR
Since December 2019, the coronavirus disease 2019 (COVID-19) caused by a novel coronavirus SARS-CoV-2 has rapidly spread to almost every nation in the world. Soon after the pandemic was recognized by epidemiologists, a group of biologists comprising the ARTIC Network, has devised a multiplexed polymerase chain reaction (PCR) protocol and primer set for targeted whole-genome amplification of SARS-CoV-2. The ARTIC primer set amplifies 98 amplicons, which are separated only in two PCRs, across a nearly entire viral genome. The original primer set and protocol showed a fairly small amplification bias when clinical samples with relatively high viral loads were used. However, as sample's viral load become low, rapid decrease in abundances of several amplicons were seen. In this report, we will show that dimer formations between some primers are the major cause of coverage bias in the multiplex PCR. Based on this, we propose 12 alternative primers in total in the ARTIC primer set that were predicted to be involved in 14 primer interactions. The resulting primer set, version N1 (NIID-1), exhibits improved overall coverage compared to the ARTIC Network's original (V1) and modified (V3) primer set.
Journal Article
The explosion of new coronavirus tests that could help to end the pandemic
Researchers are scrambling to find other ways to diagnose the coronavirus and churn out millions of tests a week — a key step in returning to normality.
Researchers are scrambling to find other ways to diagnose the coronavirus and churn out millions of tests a week — a key step in returning to normality.
A health official collects a swab sample from a woman to test for coronavirus at a temporary free testing facility in New Delhi
Credit: Xavier Galiana/AFP via Getty
Journal Article