Overcoming Supply Shortage for SARS-CoV-2 Detection by RT-qPCR

Abstract

In February 2020, our laboratory started to offer a RT-qPCR assay for the qualitative detection of severe acute respiratory syndrome coronavirus 2. A few months after the assay was released to our patients, some materials, reagents, and equipment became in short supply. Alternative protocols were necessary in order to avoid stopping testing to the population. However, the suitability of these alternatives needs to be validated before their use. Here, we investigated if saliva is a reliable alternative specimen to nasopharyngeal swabs; if 0.45% saline is a reliable alternative to guanidine hydrochloride as a collection viral transport media; the stability of SARS-COV-2 in guanidine hydrochloride and in 0.45% saline for 10 and 50 days at room temperature; and if the primers/probe concentration and thermocycling times could be reduced so as to overcome the short supply of these reagents and equipment, without a significant loss of the assay performance. We found that saliva is not an appropriated specimen for our method—nasopharyngeal swabs perform better. Saline (0.45%) and guanidine hydrochloride have a similar SARS-CoV-2 diagnostic capability as tube additives. Reliable SARS-CoV-2 RNA detection can be performed after sample storage for 10 days at room temperature (18–23 °C) in both 0.45% saline and guanidine hydrochloride. Using synthetic RNA, and decreasing the concentration of primers by five-fold and probes by 2.5-fold, changed the assay limit of detection (LOD) from 7.2 copies/reaction to 23.7 copies/reaction and the subsequent reducing of thermocycling times changed the assay LOD from 23.7 copies/reaction to 44.2 copies/reaction. However, using real clinical samples with Cq values ranging from ~12.15 to ~36.46, the results of the three tested conditions were almost identical. These alterations will not affect the vast majority of diagnostics and increase the daily testing capability in 30% and increase primers and probe stocks in 500% and 250%, respectively. Taken together, the alternative protocols described here overcome the short supply of tubes, reagents and equipment during the SARS-CoV-2 pandemic, avoiding the collapse of test offering for the population: 105,757 samples were processed, and 25,156 SARS-CoV-2 diagnostics were performed from 9 May 2020 to 30 June 2020.

Inactivation of SARS-CoV-2 virus in saliva using a guanidium based transport medium suitable for RT-PCR diagnostic assays

“…The safety provided using a viral inactivating buffer may simplify testing in several settings, including home sample collection and rapid assay testing outside of conventional laboratories. Recent studies have shown that buffers containing guanidinium thiocyanate can inactivate SARS-CoV-2 [ 5 , 7 , 9 , 12 , 13 , 19 , 20 ]. In this study, we conclusively demonstrated that eNAT, a guanidinium thiocyanate-based buffer can be used as a viral inactivation and stabilization media for RT-PCR based detection of SARS-CoV-2 in clinical specimens.…”

Point-of-care COVID-19 testing in the emergency department: current status and future prospects

“…Furthermore, the detection of bacterial co-infection and superinfection remains a challenge as many syndromic panels do not include many bacterial targets and lower respiratory tract specimens are more difficult to obtain than upper respiratory tract specimens. Challenges to POC testing implementation for SARS-CoV-2 testing in the ED include continued concerns for supply chain issues in the case of molecular tests, as well as issues with other supplies such as specimen collection supplies and media [ 53 ]. An additional consideration is POC testing for symptomatic versus asymptomatic screening.…”

COVIDFast™: A high-throughput and RNA extraction-free method for SARS-CoV-2 detection in swab (SwabFAST™) or saliva (SalivaFAST™)

“…(which was not certified by peer review) and human error. Furthermore, in a period of high demand, a shortage of nucleic acid extraction supplies can exacerbate the limitations of such viral detection methods (2,11).…”

“…The initial nucleic acid isolation and purification step (i.e., extraction step) required in conventional methods, prior to undergoing PCR, constitutes a major bottleneck in the diagnostic process (2), as it remains both manually laborious and expensive, and further increases the chances of accidental contamination and human error. Furthermore, in a period of high demand, a shortage of nucleic acid extraction supplies can exacerbate the limitations of such viral detection methods (2,11).…”