Clinical specimens. De-identified frozen specimens were shared with the Cornell COVID-19 Testing Laboratory (CCTL) by other COVID-19 testing laboratories in the United States. A total of 201 nasopharyngeal (NP) swabs and 24 anterior nares (AN) swabs were included in this study. Sixty NP swabs were tested in the originating laboratory with the Xpert® Xpress SARS-CoV-2 assay (Cepheid, Sunnyvale, CA) and of these, 30 had SARS-CoV-2 positive results and 30 had SARS-CoV-2 negative results. The other 141 NP swabs were tested in the originating laboratory with the TaqPath COVID-19 Combo Kit Multiplex Real-Time RT-PCR assay (Thermo Fisher Scientific Inc., Waltham, MA); 70 were SARS-CoV-2 positive and 71 were determined as SARS-CoV-2 negative. The AN swabs were originally tested with the New York State Department of Health (NYSDOH) SARS-CoV-2 RT-PCR assay, 12 had SARS-CoV-2 positive results and 12 were determined as negative.
Sample pooling. Pool sizes of 5 or 10 samples were contrived from one SARS-CoV-2 positive sample combined with 4 or 9 negative samples, respectively. Two hundred µl of each sample were pooled. Twenty individual positive NP swab samples with original Ct values of 28 or greater were selected to generate the pools. Twelve individual positive AN swab samples with original Ct values between 19 and 34 were selected to generate the pools.
Nucleic acid extraction. Nucleic acid was extracted from 200 ml of each individual swab supernatant or pooled samples using the MagMAX Viral/Pathogen II Nucleic Acid Isolation Kit (Applied Biosystems, Foster City, CA) and the KingFisher Flex Magnetic Particle Processor following the manufacturer’s procedures (Thermo Fisher Scientific Inc.). Prior to extraction each sample was vortexed at 100 × g for 10 sec. Nucleic acid was eluted in 50 ml of elution buffer and used for SARS-CoV-2 detection as described below.
SARS-CoV-2 RT-PCR assays. Two assays for SARS-CoV-2 real-time RT-PCR were used in this study, the newly developed EZ-SARS-CoV-2 Real Time RT-PCR assay (Tetracore, Inc., Rockville, MD) and the TaqPath COVID-19 Combo Kit Multiplex Real-Time RT-PCR assay (Thermo Fisher Scientific Inc.). The EZ-SARS-CoV-2 assay was evaluated and compared to the TaqPath COVID-19 Combo Kit Multiplex Real-Time RT-PCR assay which served as a reference standard assay with a current EUA from FDA. Negative extraction, negative amplification, and positive amplification controls were included on each assay run. RT-PCR assays were performed in two detection systems: the ABI 7500 Fast Real-Time PCR System (with SDS v1.5.1 software, Applied Biosystems, Waltham, MA) and the QuantStudio 5 Real-Time PCR System (with QuantStudio Design and Analysis Desktop Software v1.5.1, Applied Biosystems).
The EZ-SARS-CoV-2 Real-Time RT-PCR (Tetracore, Inc.) is a multiplex assay targeting the SARS-CoV-2 N gene (two target regions, with both probes labeled with FAM reporter dye), the human RNase P gene (CY5 reporter dye), and an inhibition control (IC, TAMRA reporter dye). The amplification cycling conditions were 48°C for 15 minutes; 95°C for 2 minutes; 45 cycles of 95°C for 10 seconds, 60°C for 40 seconds using 18 ml of combined reagents and 7 ml of nucleic acid template. RT-PCR data was analyzed for SARS-CoV-2 and IC targets by setting the threshold at 3% of the final normalized fluorescence of the positive amplification control. For the human RNase P gene, the threshold was set at 3% of the approximate average of the final normalized fluorescence of all samples in a single plate run. The baseline was set automatically by the software for all reporter dyes.
The TaqPath COVID-19 Combo Kit Multiplex Real-Time RT-PCR assay (Thermo Fisher Scientific Inc.) targets three regions of the SARS-CoV-2 genome (ORF1ab, N, and S genes with FAM, VIC, and ABY reporter dyes, respectively) and an inhibition control target (MS2 bacteriophage, JUN reporter dye). The amplification cycling conditions were 25°C for 2 minutes; 53°C for 10 minutes; 95°C for 2 minutes; 40 cycles of 95°C for 3 seconds, 60°C for 30 seconds using 15 ml of combined reagents and 10 ml of template. RT-PCR data was analyzed for SARS-CoV-2 targets by setting the threshold at 10% of the final normalized fluorescence of the positive amplification control. For MS2, the threshold was set at 10% of the approximate average of the final normalized fluorescence of all samples in a single plate run. The baseline was set automatically by the software for all reporter dyes. For result interpretation, TaqPath RT-PCR data was analyzed with auto settings and imported to the Applied Biosystems COVID-19 Interpretive Software SOP version 1.2.
Analytical sensitivity. The limit of detection (LoD) of the EZ-SARS-CoV-2 RT-PCR was determined by preparing two-fold serial dilutions in VTM and AN sample matrix ranging from 1,000 to 62.5 copies/ml of a commercial standard containing the full length SARS-CoV-2 genomic RNA (AccuPlex SARS-CoV-2 Verification Panel – Full Genome, Seracare Life Sciences, Inc., Milford, MA, catalog number 0505-0168). Each serial dilution was tested three times independently using the ABI 7500 Fast and the QuantStudio 5 detection systems. A preliminary LoD was defined as the lowest concentration in which 100% of the replicates were positive. The final LoD of the assay was determined by testing 20 replicates containing 250 and 500 viral genome copies/ml and the LoD was defined as the lowest concentration in which at least 19/20 (95%) of the replicates were detected.
Analytical specificity. Initial cross-reactivity analyses of the EZ-SARS-CoV-2 RT-PCR primers and probes were performed in silico. Primer and probe sequences were blasted against public domain nucleotide sequences. The database search parameters were as follows: 1) The nucleotide collection consisted of GenBank NT and RefSeq sequences, but excluded EST, STS, GSS, WGS, TSA, and patent; 2) The database was non-redundant. Identical sequences were merged into one entry, while preserving the accession, GI, title and taxonomy information for each entry; 3) Database was updated on 02/11/2020; 4) The search parameters automatically adjusted for short input sequences and threshold was set at 1000; 5) The match and mismatch scores were 1 and -3, respectively. Additionally, Needleman–Wunsch alignments [23] were performed against a defined set of sequences in Supplementary Table 1. Each primer and probe of the EZ-SARS-CoV-2 PCR assay was aligned to the sequences listed in Supplementary Table 1. No gaps were allowed in the alignment and a match matrix was used. The matrix scored the alignment with a 1 for match and a 0 for anything else. The alignment score was the number of matches between the primer or probe and the pathogen. The frequency of the alignment was the number of matches divided by the length of the primer or probe.
To exclude cross-reactivity of the EZ-SARS-CoV-2 RT-PCR assay against other human pathogens and confirm its specificity in the wet test condition, the assay was tested against 40 non-target organisms known to infect humans (Supplementary Table 2). Nucleic acid from each organism (concentration of > 106 CFU/ml or > 104 TCID50/ml, when available from the vendor) was extracted using the Qiagen QIAamp Viral RNA Mini Kit and subjected to real-time PCR using the EZ-SARS-CoV-2 assay on the ABI 7500 Fast real-time PCR System. Each pathogen was tested in triplicate. SARS-CoV-2 positive and negative controls were included in each assay run.
Reproducibility. The intra- and inter-run reproducibility of the EZ-SARS-CoV-2 PCR assay were evaluated. For this, 10-fold serial dilutions containing between 100 and 10,000 genome copies/ml of SARS-CoV-2 (AccuPlex SARS-CoV-2 Verification Panel – Full Genome, Seracare Life Sciences, Inc., catalog number 0505-0168) were prepared in viral transport media (VTM, Corning product number 25-500-CM, Corning, NY) and tested in triplicate and in three independent runs.
Data analysis. Microsoft Excel was used to calculate the mean and variation of replicate Ct values, including standard deviation [SD (Ct)], coefficient of variation [%CV (Ct)], and coefficient of variation based on linearized Ct values [%Ct (2-Ct)]. Contingency (2x2) tables were analyzed in Microsoft Excel to determine the diagnostic sensitivity, diagnostic specificity, positive predictive value, and negative predictive value of the assays evaluated herein. Results provided by the originating laboratory were considered to be the reference standard for these calculations and comparisons of the assays evaluated here.
The degree of agreement between assay results was measured by Cohen’s kappa using formulas from Watson and Petrie, 2010 [24]. The plot was generated with GraphPad Prism (version 9.0.0 for Windows, GraphPad Software, San Diego, California USA, www.graphpad.com).