Analytical Sensitivity and Specificity of Two RT-qPCR Protocols for SARS-CoV-2 Detection Performed in an Automated Workflow

Abstract

WHO declared the novel coronavirus (COVID-19) outbreak a global pandemic on 11 March 2020. The establishment of standardized RT-qPCR protocols for respiratory secretions testing, as well as sharing of specimens, data, and information became critical. Here, we investigate the analytical performance of two interim RT-qPCR protocols (Charité and Centers for Disease Control (CDC)) for the qualitative detection of SARS-CoV-2 executed in a fully automated platform. Analytical specificity, PCR amplification efficiency, analytical sensitivity (limit of detection), and cross-reactivity were evaluated using contrived samples. The on-going accuracy was evaluated by retrospective analysis of our test results database (real clinical samples). N1, E, and a modified version of RdRP assays presented adequate analytical specificity, amplification efficiency, and analytical sensitivity using contrived samples. The three assays were applied to all individuals who requested the SARS-CoV-2 molecular test assay in our laboratory and it was observed that N1 gave more positive results than E, and E gave more positive results than RdRP (modified). The RdRP and E were removed from the test and its final version, based on N1 assay only, was applied to 30,699 Brazilian individuals (from 19 February 2020 to 8 May 2020). The aggregated test results available in the database were also presented.

rRT-PCR for SARS-CoV-2: Analytical considerations

“…In addition, because the N gene sequence located in most subgenomic regions, it has the high expression level and in contrast, the RdRP presents in the ORF1b region and therefor has low expression. All in all, the results of this study showed that the N1 assay has all needed diagnostic criteria to detect SARS-CoV-2, efficiently [50] .…”

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

“…The PCR amplification efficiencies using the SARS-CoV-2 diagnostic RNA (described in [ 1 ]) were 98.5% (y = −3.357x + 42.74) for [+], 97.5 % (y = −3.383x + 42.52) for [−], and 97.5% (y = −3.383x + 43.35) for [−] fast ( Figure 4 a). The probit regression analysis showed a LOD of 7.3 (95% CI 5.21–18.84) copies/reaction for [+], 23.7 (95% CI 16.1–57) copies/reaction for [−] (3.2-fold decrease versus [+]), and 44.2 (95% CI 30.2–99.4) copies/reaction for [−] fast (6-fold decrease versus [+]).…”

“…To evaluate if the primer/probe concentrations and thermocycling times could be reduced without a loss of assay performance, a previously described synthetic SARS-CoV-2 RNA fragment was introduced directly into the RT-qPCR reactions [ 1 ]. Additionally, 105 anonymized leftover RNA samples that were positive for the virus were submitted to the three tested conditions (validated primer/probe concentrations and thermocycling conditions, named (+); decreased primer/probe concentrations and validated thermocycling conditions, named [−]; and decreased primer/probe concentrations and reduced thermocycling times, named [−] fast) in order to evaluate their effect on the Cq values.…”

“…The RT-qPCR reaction (20 μL) consisted of 4 μL of LightCycler ® Multiplex RNA Virus Master (5×; Roche, Basel, Switzerland), 1500 nM of (or 300 nM for experiments with a reduced concentration) N1 SARS-CoV-2 primer and 375 nM (or 150 nM for experiments with ra educed concentration) probe (FAM), 80 nM of RPP30 primer and 40 nM of RPP30 probe (HEX), 0.1 μL of reverse transcriptase (200×) (Roche), 10 μL of extracted nucleic acid, and 3.34 μL of nuclease-free water. The primer and probe sequences were described elsewhere [ 1 ]. The thermocycling conditions were reverse transcription (10 min at 50 °C) and polymerase activation (3 min at 94 °C), followed by 45 cycles of 15 s at 95 °C and 30 s at 55 °C, with a total time of 85 min or a reverse transcription of 5 min at 50 °C; polymerase activation (30 s at 94 °C); and this was followed by 45 cycles of 5 s at 95 °C and 15 s at 55 °C, for a total time of 60 min.…”

“…In February 2020, our laboratory started to offer an RT-qPCR assay for the qualitative detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) for Brazil’s Federal District population. The validated protocol was an optimization of the assay described by the Centers for Disease Control and Prevention (CDC) [ 1 ]. A few months after the assay was released to our patients, some materials, reagents, and equipment became in short supply.…”