QC | Variants detected: Through in silico analysis, our GenomeCoV19 Detection Kits (Cat. No. G628 and G628.v2) are able to detect the following new variants of the SARS-CoV-2 virus:
SARS-CoV-2 Variant Detected | As Defined By |
Alpha / UK variant B.1.1.7 |
Defined by the following mutations of the virus's spike proteins: deletion of 69-70, deletion of 144, N501Y, A570D, D614G, P681H, T716I, S982A, and, D1118H. |
Beta / South African variant B.1.351 |
Defined by the following mutations of the virus’s spike proteins: L18F, D80A, D215G, R246I, K417N, E484K, N501Y, and A701V. |
Brazilian variant B.1.1.28 |
Defined by the following mutations of the virus's spike proteins: L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, H655Y, and, T1027I. |
Gamma / Brazilian variant P.1 (a branch off of the B.1.1.28 lineage) |
Defined by the following mutations of the virus's spike proteins: K417T, E484K, and, N501Y. |
Delta / Indian variant B.1.617.2 |
Defined by the following mutations of the virus’s spike proteins: L452R, T478K, D614G, P681R, D950N, and deletion of 156 -157. |
Delta Plus (a branch off of the B.1.617.2 lineage) |
Defined by the following mutations of the virus’s spike proteins: L452R, T478K, D614G, P681R, D950N, deletion of 156 -157, and K417N. |
Omicron / Variant B.1.1.529 |
Defined by the following mutations of the virus’s spike proteins: A67V, Δ69-70, T95I, G142D, Δ143-145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493K, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F. |
Limit of Detection (LoD):
Our LoD study established the lowest concentration of SARS-CoV-2 RNA, as genome copies (cp) per reaction, which can be detected by the GenomeCoV19 Detection Kit at least 95% of the time. This was established by testing serial dilutions of SARS-CoV-2 genomic RNA (source: ATCC-VR1986D) into nasopharyngeal clinical matrix, verified to be COVID-19 negative, at the following concentrations: 100 cp/µl, 50 cp/µl, 5 cp/µl, 4 cp/µl, 3 cp/µl, 2 cp/µl, 1 cp/µl and 0.5 cp/µl. RNA extraction was performed with the Qiagen QIAamp DSP Viral RNA Mini Kit and tests were run on the Bio-Rad CFX96 qPCR instrument. The LoD was determined to be 5 cp/reaction and results are shown in the table below.
Cp/Reaction | Samples Tested | N-gene (FAM) | Average Ct (FAM) | S-gene (HEX) | Average Ct (HEX) | % Match |
500 |
4 |
4 |
27.29 |
4 |
27.75 |
100% |
250 |
4 |
4 |
28.35 |
4 |
28.84 |
100% |
25 |
4 |
4 |
31.65 |
4 |
32.19 |
100% |
20 |
4 |
4 |
31.33 |
4 |
31.10 |
100% |
15 |
4 |
4 |
31.72 |
4 |
31.25 |
100% |
10 |
4 |
4 |
32.64 |
4 |
32.24 |
100% |
5 |
24 |
24 |
32.99 |
24 |
33.36 |
100% |
2.5 |
4 |
3 |
37.15 |
4 |
36.12 |
75% |
Clinical Performance (Contrived Samples):
A contrived clinical study was conducted with a total of 30 negative and 30 contrived positive samples. Positive samples were prepared by spiking ATCC-VR1986D SARS-CoV-2 reference genome RNA into nasopharyngeal matrix mixed with lysis buffer from QIAamp DSP Viral RNA Mini Kit at 1X, 10X, and 100X LoD. Tests were run on the Bio-Rad CFX96 qPCR instrument and results showed 100% Positive Percent Agreement (30/30 positive results) and 100% Negative Percent Agreement (30/30 negative results).
Clinical Performance (Remnant Samples):
A blinded clinical study was conducted using a SARS-CoV-2 Validation Panel with 60 nasopharyngeal/oropharyngeal/nasal swab remnant clinical specimens. These 30 SARS-CoV-2 positive and 30 SARS-CoV-2 negative remnant specimens were verified using a FDA EUA-authorized COVID-19 detection kit. RNA extraction was performed with the Qiagen QIAamp DSP Viral RNA Mini Kit and tests were run on the Bio-Rad CFX96 qPCR instrument. GenomeCoV19 Detection Kit test results showed 100% Positive Percent Agreement (30/30 positive results) and 100% Negative Percent Agreement (30/30 negative results).
Inclusivity (Analytical Sensitivity):
The primers and probes sequences were blasted against SARS-CoV-2 genomes publicly available as of June 29th, 2020. Results showed the sequences had 100% homology to all SARS-CoV-2 isolates analyzed, with two exceptions of MT451113.1 and MT534319.1 (homology of 95.2% in both cases). Both exceptions occur at the 5'end of the primer, and thus are unlikely to cause the failure of qPCR, and would not affect the test performance under specified annealing temperature.
Cross-Reactivity:
Cross-reactivity studies using a panel of organisms were conducted by both in silico analysis and wet-testing of whole organisms or purified nucleic acids. The GenomeCoV19 Detection Kit primers and probes were first analyzed in silico against each of the organisms listed in the table below using BLASTn. No cross-reactivity with >80% homology was found for any of the primer and probe sets. Additionally, the bacteria, viruses and pooled human wash described in the table below were included for wet-testing. Concentrations of 106CFU/mL (for bacteria) and 105pfu/mL (for viruses) were used to purify the nucleic acids using the Qiagen QIAamp DSP Viral RNA Mini Kit. There was no cross-reactivity observed for any of the organisms tested using the GenomeCoV19 Detection Kit on Bio-Rad’s CFX96 qPCR instrument. Cross-reactivity was defined as Ct < 40.
Organism Name | N gene | S gene | Source |
Human Coronavirus-229E |
ND |
ND |
ATCC VR-740 |
Human Coronavirus-OC43 |
ND |
ND |
Zeptometrix NATCOV(OC43)-ST |
Human Coronavirus-HKU1 |
ND |
ND |
ATCC VR-3262SD |
Human Coronavirus-NL63 |
ND |
ND |
ATCC VR-3263SD |
SARS-coronavirus |
ND |
ND |
BEI NR-3882 |
MERS-coronavirus |
ND |
ND |
ATCC VR-3248SD |
Adenovirus |
ND |
ND |
abm 000047A |
Human Metapneumovirus |
ND |
ND |
ATCC VR-3250SD |
Parainfluenza virus 1 |
ND |
ND |
ATCC VR-94D; C35 |
Parainfluenza virus 2 |
ND |
ND |
ATCC VR-92D; Greer |
Parainfluenza virus 3 |
ND |
ND |
ATCC VR-1782; ATCC-2011-5 |
Parainfluenza virus 4 |
ND |
ND |
ATCC VR-1377; CH 19503 |
Influenza A |
ND |
ND |
ATCC VR-1679D; H3N2, A/Hong Kong/8/68 |
Influenza B |
ND |
ND |
ATCC VR-1735D; B/Taiwan/2/62 |
Enterovirus |
ND |
ND |
ATCC VR-1377; CH 19503 |
Respiratory syncytial virus |
ND |
ND |
ATCC VR-1580; 18537 |
Rhinovirus |
ND |
ND |
ATCC VR-1171; 6669-CV39 |
Chlamydia pneumoniae |
ND |
ND |
ATCC 53592D; AR-39 |
Haemophilus influenzae |
ND |
ND |
ATCC 51907D |
Legionella pneumophila |
ND |
ND |
ATCC 33152D-5; Philadelphia-1 |
Mycobacterium tuberculosis |
ND |
ND |
ATCC 25177; H37Ra |
Pneumocystis jirovecii |
ND |
ND |
Zeptometrix- 0801698DNA-1UG |
Streptococcus pneumoniae |
ND |
ND |
ATCC 33400D-5 |
Streptococcus pyogenes |
ND |
ND |
ATCC 12344D-5; T1 |
Streptococcus salivarius |
ND |
ND |
Zeptometrix - 0801896 |
Mycoplasma pneumoniae |
ND |
ND |
ATCC 15531D; FH of Eaton Agent |
Pooled human nasal wash - representing diverse microbial flora in the human respiratory tract |
ND |
ND |
- |
Candida albicans |
ND |
ND |
ATCC 10231 |
Bordetella pertussis |
ND |
ND |
Zeptometrix 0801459 |
Pseudomonas aeruginosa |
ND |
ND |
ATCC 9027 |
Staphylococcus epidermis |
ND |
ND |
ATCC 12228 |
*ND = Not Detected
Precision: CV < 5% (Between and within batches). |