Instruction Manual
Table Of Contents
- Intended Use
- Summary and Explanation
- Principles of the Procedure
- Materials Required (Provided)
- DTT solution 500mM
- SARS-CoV-2 PCR Mix
- Equipment and Supplies
- Warnings and Precautions
- Specimen Collection, Handling, and Storage
- Procedure
- For viscous samples (sputum and bronchial wash): Performed inside BSC 2
- NOTE: In the event that the test system becomes inoperable, notify supervision or designee for further direction. Patient specimens must be stored in a manner that maintains the integrity of the specimen.
- Quality Control
- Limitations
- Conditions of Authorization for the Laboratory
- Performance Characteristics
- References
14
Performance Characteristics
1) Limit of Detection
The Limit of Detection (LOD) is defined as the lowest SARS-CoV-2 RNA concentration that is successfully detected
with a probability of 95% or greater. Sensitivity standards were prepared by serially diluting the SARS-CoV-2 viral
RNA transcript containing an 1100 nucleotide region from the N gene in stabilizing buffer RNA Diluent P to the
following concentrations: 2,580, 968, 363, 136, and 51 copies/mL. RNA was quantified by an RNA fluorometric
method (Qubit HS Assay). The LOD was evaluated by testing the sensitivity standards over three separate runs
using the SARS-CoV-2 RNA Qualitative RT-PCR assay. In each run, 7 replicates at each concentration level were
purified using the MagNA Pure 96 and each replicate was then tested in the ABI 7500 to yield a total of 21 replicate
results at each concentration level.
The Limit of Detection study results are shown in the table below. The concentration of SARS-CoV-2 RNA that was
successfully detected with at least a 95% detection rate was calculated as 136 copies/mL for nCoV-N1 and nCoV-
N3 primer/probe sets. The LOD of the test is established at 136 copies/mL.
Table 1. Sensitivity Results for nCoV RNA Qualitative RT-PCR
nCoV N1
nCoV N3
sample ID
nCoV copies/m L
nCoV log
copies/m L
mean Ct
detection rate*
mean Ct
detection rate*
LOD 1
2,580
3.41
30.43
100%
29.77
100%
LOD 2
968
2.99
31.95
100%
31.02
100%
LOD 3
363
2.56
33.31
100%
32.44
100%
LOD 4
136
2.13
34.88
95%
34.27
100%
LOD 5
51
1.71
35.85
81%
34.93
86%
* samples w ith nCoV Ct < 40.00 cycles are considered detected (positive), nCoV Ct > 40.00 are considered not detected (negative)
2) In silico inclusivity testing.
Quest Diagnostics is using the same sequences as CDC therefore, additional in silico studies were not performed.
CDC performed an alignment with the oligonucleotide primer and probe sequences of the CDC 2019 nCoV Real-
Time RT-PCR Diagnostic Panel with all publicly available nucleic acid sequences for 2019-nCoV in GenBank as of
February 1, 2020 to demonstrate the predicted inclusivity of the CDC 2019 nCoV Real-Time RT-PCR Diagnostic
panel. All the alignments showed 100% identity of the CDC panel to the available 2019-nCoV sequences with the
exception of one nucleotide mismatch with the N1 forward primer in one deposited sequence. Similarly, a single
mismatch is observed in the alignment of the N3 probe. The risk assessment of these single mismatches resulting
in a significant loss in reactivity, and false negative result, is low due to the design of the primers and probes with
melting temperatures > 60°C and run conditions of the assay with annealing temperature at 55°C to tolerate one to
two mismatches.
3) Cross-reactivity
Organisms in the a commercially available Respiratory Verification Panel were extracted and tested with the Quest
SARS-CoV-2 Real-Time RT-PCR assay to demonstrate analytical specificity and exclusivity. The commercially
available panel comprised of 22 individual inactivated respiratory related pathogens (purified, intact virus particles
and bacterial cells) manufactured specifically for use as positive controls in nucleic acid tests. There was no cross-
reactivity observed for any of the tested pathogens.
Pathogen
Strain
Human coronavirus 229E
229E
Human coronavirus OC43
OC43