GENERATION Biotech N501Y SARS-CoV-2 Variant Detection Kit Instructions
- June 13, 2024
- GENERATION Biotech
Table of Contents
SARS-CoV-2 Variant Detection Kit – “N501Y”
– For positive detection of wildtype and N501Y S gene mutation –
Multiplexed qRT-PCR Kit – For Research Use Only (RUO)
Instructions for Use
Application
The SARS-CoV-2 Variant Detection Kit is a reagent system, based on
quantitative reverse transcriptase (qRT) PCR technology, for the detection of
RNA specific to emerging viral variants of concern (Severe Acute Respiratory
Syndrome Coronavirus 2, SARS-CoV-2) that cause the Coronavirus Disease 2019
(COVID-19).
The “N501Y” multiplex assay of this kit specifically detects the viral
mutation N501Y in a separate channel as a positive signal rather than as a
negative drop-out, thereby increasing sensitivity and accuracy.
Quantitative polymerase chain reaction (qPCR) technology utilizes an enzyme
(reverse-transcriptase, RT) to convert RNA into complementary DNA (cDNA), from
which specific target sequences are then amplified and targeted with specific
probes for the detection of their copy number (concentration) in the initial
specimen. The detection probes are labelled with differently colored
fluorescent reporter dyes, which enable the direct comparison of several
genetic loci (M, N, S and RP) in a single assay.
For research use only (RUO). Not for use in diagnostic procedures.
Equipment and Consumables
Customer supplied equipment
Quantitative PCR Cycler / Digital PCR system
Calibrated single-channel pipettes
Vortex mixer
Benchtop microcentrifuge for 1.5 ml tubes
Customer supplied consumables
Pipette tips (with aerosol barriers, nuclease-free) Quantitative| General
laboratory supplier
---|---
PCR Plates / dPCR consumables| General laboratory supplier
1.5 ml tubes (low DNA binding, nuclease-free)| General laboratory supplier
Kit Components
| Component | Tube Label |
|---|---|
| Buffer Mix | BM |
| Enzyme Mix | EM |
| Primer / Probe Mix | PPM |
| Positive Control | CTR |
| Nuclease-free water | Water |
Storage
SARS-CoV-2 Variant Detection Kits are shipped on dry ice or with -80°C ice
packs. The components of the kit should arrive frozen. If one or more
components are not frozen upon receipt, or if tubes have been compromised
during shipment, contact Generation Biotech for assistance.
All components should be stored between -25°C and -15°C upon arrival.
Repeated thawing and freezing of reagents should be avoided, as this might
affect the performance of the assay. The reagents should be subdivided and
frozen in aliquots if they are to be used intermittently.
Storage between +2°C and +8°C should not exceed a period of two hours.
Protect Primer/Probe Mixes from light.
Product Description
The SARS-CoV-2 “N501Y” multiplex assay detects three SARS-CoV-2 genes (M, N
and S) with improved accuracy when compared to prior SARS-CoV-2 ‘wildtype’
assays that detect only the M and N genes.
The additional S gene assay enables the specific detection of strains carrying
the N501Y mutation that has been associated with an increased viral
infectivity. This mutation is present in the variants B.1.1.7 (“UKVariant”),
B.1.351 (“South-African Variant”) and
B.1.1.28 (“Brazilian Variant”), amongst others.
The kit further includes a primer/probe set that detects the human RNase P
gene as an internal control.
This assay detects whether the patient specimen collection was properly
carried out, thereby reducing false negative tests.
The SARS-CoV-2 “N501Y” Variant Detection Kit consists of:
- Buffer Mix
- Enzyme Mix ………………………………… (reverse transcriptase and heat-stable polymerase)
- Primer / Probe Mix……………………… (for SARS-CoV-2 genes M, N & S and for human RNase P gene RP)
- Positive Control…………………(SARS-CoV-2 genes M, N & S, including N501Y, and RP gene)
- Water………………………………..(nuclease-free)
The test consists of three processes in a single tube / single reaction assay:
- Reverse transcription of target RNA (from SARS-CoV-2) into cDNA
- PCR amplification of target cDNA (M, N & S genes) as well as of human control DNA (RP gene)
- Quantitative detection of PCR amplification products by fluorescent dye labelled probes
Procedure
6.1 Sample Preparation
RNA extracted from the specimen is the starting material for the SARS-CoV-2
Variant Detection Kit.
The quality of the extracted RNA has a profound impact on the performance of
the entire test system. The time point(s) of sampling is important for the
detection of SARS-CoV-2 RNA: the best time for an accurate identification of
positive swab samples via qRT-PCR testing is 4 – 12 days after infection.
It is recommended to ensure that the system used for nucleic acid (RNA/DNA)
extraction is compatible with the desired type of quantitative PCR technology
and instrumentation.
If using a spin column-based sample preparation procedure that includes
washing buffers containing ethanol, it is highly recommended to perform an
additional centrifugation step for 10 min. at maximum speed, using a new
collection tube and prior to the elution of the nucleic acid, to obtain
reliable results.
For additional information and technical support regarding pre-treatment and
sample preparation please contact our Technical Support (see Technical
Assistance).
6.2 Reaction Setup
All reagents and samples should be thawed completely, mixed (by pipetting or
gentle vortexing) and centrifuged briefly before use.
| Reagents | Volume per Reaction |
|---|---|
| Buffer Mix (BM) | 10 µl |
| Enzyme Mix (EM) | 0.2 µl |
| Primer / Probe Mix (PPM) | 2 µl |
| Sample or Positive Control (CTR) | 5 µl |
| Nuclease-free Water | 2.8 µl |
| Total Reaction Volume | 20 µl |
Programming the quantitative PCR Instrument
For basic information regarding the setup and programming of different
quantitative PCR instruments, please refer to the user manual of the
respective instrument.
7.1 Settings
Define the following settings as described below.
Settings
Reaction Volume| 20 µl
Ramp Rate| Default
Passive Reference| (None)
7.2 Fluorescence Detector
Define the fluorescence detectors as described below.
| Target | Reporter Dye | Quencher Dye |
|---|---|---|
| SARS-CoV-2 gene N1 | FAM | (none) |
| SARS-CoV-2 gene M | HEX | (none) |
| Human RNase P gene | ROX | (none) |
| SARS-CoV-2 gene S N501Y | CY5 | (none) |
7.3 Temperature Profile
Incubate the reaction as described below.
| Step | Stage | Cycles | Acquisition | Temp | Time |
|---|---|---|---|---|---|
| RT | Hold | 1 | – | 50 °C | 10 min |
| Denaturation | Hold | 1 | – | 95 °C | 2 min |
| Denaturation | Cycling | 50 | – | 95 °C | 5 sec |
| Amplification | YES | 60 °C | 30 sec |
Data Analysis / Interpretation of Results
SARS-CoV-2 genes M/N| Human RNase P| SARS-CoV-2 gene S N501Y| Status| Result|
Action
---|---|---|---|---|---
NEG| NEG| NEG| Invalid| NA| Repeat Test
NEG| POS| NEG| Valid| SARS-CoV-2
Not Detected| Report Results
POS| POS or NEG| POS or NEG| Valid| SARS-CoV-2
Detected| Report Results
POS| POS or NEG| POS| Valid| N501Y Mutation
Detected| Report Results
POS| POS or NEG| NEG| Valid| N501Y Mutation
NOT Detected| Report Results
For detailed instructions
regarding the analysis of the data generated with the SARS-CoV-2 Variant
Detection Kit on different quantitative PCR instruments please contact our
Technical Support.
Technical Assistance
For customer support, please contact our Technical Support: e-mail:
[email protected]
phone: 609-637-0878
Disclaimer
For Research Use Only. Not for use in diagnostic procedures. Generation Biotech does not assume any responsibility for your use of these materials, the accuracy of any documentation or the safety of user protocols. Use standard laboratory safety precautions for infectious specimens. Use at your own risk. Take appropriate precautions to avoid false positive results. Routine use of nucleic acid high amplification technologies such as PCR carries a high risk of contamination. Perform nucleic acid extraction and reaction setup in an environment free from the presence of SARS-CoV-2 genes M, N & S and RNase P gene RP.
VoXcreen SARS-CoV-2 Screening & Variant Detection Kit
Instructions for Use – 20211231
Generation Biotech
31 Airpark Road
Princeton, NJ 08540
www.gen-bio.com