Instruction Manual

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8) Intra-assay Precision
Intra-assay precision is defined as the reproducibility of a sample within an assay run and was evaluated by testing
3 replicates of 3 separate precision standards in a single run using the SARS-CoV-2 RNA Qualitative RT-PCR
assay. The precision standards were prepared by diluting the SARS-CoV-2 viral RNA transcript containing an 1100
nucleotide region from the N gene in stabilizing buffer RNA Diluent P to final concentrations of 44,000 copies/mL
(high), 13,200 copies/mL (mid), and 5,657 copies/mL (low). Aliquots of each standard were prepared and stored
frozen until the time of testing. Precision standard replicate CV’s ranged from 0.0% to 0.4%, with mean overall intra-
assay precision equal to 0.3%. The individual precision standard replicate results were within 0.13 detection cycles
of their respective mean values. The intra-assay precision data is included in the table below.
9) Clinical Evaluation:
The clinical evaluation consisted of 30 SARS-COV-2 RNA-positives and 30 SARS-COV-2 RNA-negatives
(negatives were RNA Diluent P buffer). SARS-COV-2 RNA-positives consisted of 24 virus-positive RNA
preparations derived from clinical specimens, with 6 randomly selected and run in duplicate for a total of 30
positives. RNA preparations were obtained from a well-established clinical laboratory located in the Republic of
Korea (originating lab) and consisted of 12 paired extracted patient samples from both an upper respiratory (NP/OP
swabs) and lower respiratory source (sputum). Extraction from patient specimens was performed using the MagNA
Pure 96 system and MagNA Pure 96 DNA and Viral NA Small Volume Kit. Amplification was performed using a RT-
PCR kit commercially available in the Republic of Korea to identify the paired rRT-PCR positives.
The samples were randomized, blind-labeled and tested using the SARS-CoV-2 RNA Qualitative RT-PCR assay.
Considering the SARS-CoV-2 RNA positive RNA extracts would degrade during the nucleic acid extraction step of
the assay only the RT-PCR amplification and detection step was performed in this study. RT-PCR Mix 1 and RT-
PCR Mix 2 were formulated to include the RNA internal positive amplification control (RIPC) at a final concentration
that is comparable to the expected concentration in MP96-extracted preparations. There was 100% agreement
(30/30, 95% CI 88.7-100%) for the positive samples and 100% agreement for the negative samples (30/30, 95% CI
88.7-100%).
Comparator RT-PCR Test
Positive
Negative
Quest SARS-CoV-2
rRT-PCR Test
Positive
30
0
Negative
0
30
Positive Percent Agreement: 100% (95% CI 88.7-100%)
Negative Percent Agreement: 100% (95% CI 88.7-100%)
Overall Agreement: 100% (95% CI 93.98-100%)
10) Specificity in a Presumed Negative Population (Upper Respiratory)
Quest Diagnostics randomly selected 72 presumed-negative nasopharyngeal/throat swabs submitted for
respiratory pathogen testing in October 2019 and stored at < -10°C. One specimen was initially indeterminant, and
upon re-peat testing, RNA was not detected. SARS-CoV-2 RNA was not detected in any of the samples tested for
a specificity of 100% (72/72, 95% CI 95-100%).
11) Specificity in a Presumed Negative Population (Lower Respiratory)
Quest Diagnostics randomly selected 30 presumed-negative lower respiratory specimens (and one upper
respiratory specimen) submitted for respiratory pathogen testing during January and early February 2020, including
22 BAL specimens, 8 sputum specimen remnants, and one M4 swab specimen. The sputum and M4 swab remnants
were tested in duplicate and the first result was used for the analysis. SARS-CoV-2 RNA was not detected