Description:
In view of the joint global efforts of advancing collaborative research in diagnostics, therapeutics, and vaccination in
the fight against the COVID-19 (SARS-CoV-2) pandemic, Bio-Helix has specifically developed the LifeDireX.
COVID-19 RT-qPCR Detection Kit Plus for human respiratory tract specimens. The kit is characterized by:
(1) High specificity for the N target markers as recommended by WHO and US CDC;
(2) Data obtained in less than 2 hours; and
(3) Compatible with standard RT-qPCR machines (ABI 7500, Bio-Rad CFX96, QuantStudio’s 7 Flex).
Application:
A study was performed to assess the performance of COVID-19 RT-qPCR Detection Kit Plus. A testing of 20 replicates at the tentative Limit of Detection (LoD) concentration was carried out with 50 copies/reaction at 95% detection rate. Samples were spiked SARS-CoV-2 RNA in pooled negative clinical nasopharyngeal swab matrix, then extracted with QIAamp Viral RNA Mini Kit and tested on QuantStudio™ 5 Real-Time PCR System. The synthetic SARS-CoV-2 RNA was obtained from Twist Biosciences (Assay Ready Control 14,EPI_ISL_710528).
Clinical Evaluation
A study was performed to assess the clinical performance of COVID-19 RT-qPCR Detection Kit Plus. A testing of 20 replicates at low positive with 100 copies/reaction, 10 replicates at high positive with 2500 copies/reaction and 30 replicates were unspiked. Samples were spiked SARS-CoV-2 RNA in pooled negative clinical nasopharyngeal swab matrix, then extracted with QIAamp Viral RNA Mini Kit and tested on QuantStudio™ 5 Real-Time PCR System. The synthetic SARS-CoV-2 RNA was obtained from Twist Biosciences (Assay Ready Control 14,EPI_ISL_710528). The clinical samples were provide by Shuang Ho Hospital, Ministry of Health and Welfare, Republic of China (Taiwan).
Please contact us for quote: info@geneon.net
PROTOCOL | QP019T-0100_Protocol_20210911.pdf |
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Safety Data Sheet|SDS | QP019T-0100_COVID-19_RT-qPCR_Detection_Kit_Plus_SDS.pdf |
The Kit is available and is for
Research & Development only (RUO)
1. Description
SARS-CoV-2 (2019-nCoV) virus detection system is a kit for efficient in vitro detection of coronavirus SARS-CoV-2 (or 2019-nCoV) based on one-tube cDNA synthesis and Real-Time PCR. Kit includes two primers and probe set for detection and confirmation steps.
Real-time RT-PCR technology include the reverse transcription reaction (RT) stage to convert RNA to complementary DNA (cDNA) and the polymerase chain reaction (PCR) stage to amplify and detect a target sequence using specific primers and fluorescent probes.
2. Components
3. Instruments:
The kit can be used on the following PCR instruments:
• LightCycler® 96 (Roche)
· LightCycler® 480 (Roche)
· ABI Prism® 7500 SDS (Applied Biosystems)
• ABI Prism® 7500 Fast SDS (Applied Biosystems)
• Rotor-Gene® 6000 (Corbett Research)
• Rotor-Gene® Q5/6 plex Platform (QIAGEN)
• CFX96™ Real-Time PCR Detection System (Bio-Rad)
• CFX96™ Deep Well Real-Time PCR Detection System (Bio-Rad)
4. Design/Principal:
1) Initially, all samples are tested using a system of primers and probe N with the setting of the corresponding positive and negative controls.
2) Next, positive samples must be re-confirmed using a primer system and an ORF1b probe with the appropriate positive and negative controls.
3) Select the FAM channel for the detection of the amplification signal of the N and ORF1b genes.
5. Procedure
5.1 Sample preparation:
Extraction of the encapsidated RNA is mandatory before use in real time RT-PCR. The quality of the extracted RNA has a profound impact on the performance of the entire test system. It is recommended to ensure that the system used for nucleic acid extraction is compatible with real-time PCR technology.
5.2 Protocol
Screening (detection).
All reagents and samples should be completely thawed, mixed (by pipetting or gentle shaking) and discarded droplets by centrifugation.
Prepare reagent premixes with a set of primers and probe N, based on the following formula: (2+х+1) reaction mixtures, where 2 show reactions with PTC and NTC, x is the number of samples and +1 additional volume to compensate for the possible pipetting error.
Confirmation.
At the confirmation stage, prepare reagent premixes with a set of primers and an ORF1b probe, from the following formula: (2+ y +1) reaction mixtures, where 2 show reactions with PTC and NTC, y - the number of samples positive for the N gene assay, and +1 additional volume to compensate for the possible pipetting error.
Сomponent calculation for 1 and 7 reactions:
Component: 1 reaction 7 reaction
2× qPCR Master Mix: 12,5 µl 125 µl
BioMaster-mix 1,0 µl 10 µl
Primer-probe set
N/ORF1b 1,5 µl 15 µl
============================================
Total: 15,0 µl 150 µl
5.3 Reaction setup
- Take a 96-well plate, add using an automatic pipette 15.0 μl of the prepared premix for detection or confirmation stage.
- Add 10.0 μl of sample solution (solution after RNA isolation) and controls (PTC and NTC) to the appropriate cells containing premixes with different primers.
- Make sure positive and negative controls have been added.
- Gently mix sample and control solutions with premixes by pipetting. Remember to change the tip of the automatic pipette!
- Close the 96-well reaction plate with appropriate covers or optical adhesive film. If you use reaction tubes, make sure they are suitable for use in real-time amplifiers.
- Centrifuge the 96-well reaction plate in a centrifuge with a microtiter plate rotor for 30 seconds at a speed of approximately 1000 x g (~ 3000 rpm).
- For basic information on setting up and programming various real-time PCR tools, please refer to the user manual of the corresponding tool.
5.4 Suggested thermal cycling conditions
Step |
Temperature |
Time |
Cycles |
Reverse Transcription |
45 °C |
30 min |
1 |
Initial activation |
95 °C |
5 min |
1 |
Denaturation |
95 °C |
10 sec |
45 |
Annealing and extension* |
56 °C |
30 sec |
* detection in FAM channel
6. Data analysis and interpretation:
Screening
At the screening stage, the researcher identifies samples in which SARS-CoV-2 RNA is present.
PCR was successful and the data are reliable subject to the following criteria:
- in the negative control, the signal in the FAM channel is absent or Ct > 37 cycles;
- in the positive control in the FAM channel, the signal corresponds Сq 22-26.
Test on detection of HKU-N RNA positive control:
Samples in which a signal with Ct values less than 37 is detected in the FAM channel are subjected to further analysis in the Confirmation stage.
Samples where the signal is negative (Ct > 37 cycles) in the FAM channel do not contain RNA of SARS-CoV-2 virus.
Confirmation
At the Confirmation step, the researcher confirms that SARS-CoV-2 virus RNA is present in the sample.
PCR was successful and the data are reliable subject to the following criteria:
- in the negative control, the signal in the FAM channel is absent or Ct > 38 cycles
- in the positive control in the FAM channel, the signal corresponds Сq 25-29.
Test on detection of HKU-ORF1b RNA positive control:
Samples in which a signal with Cq values less than 38 is observed in the FAM channel are positive for the presence of RNA of SARS-CoV-2 virus.
Samples in which the signal is negative in the FAM channel are undefined and require confirmation by the reference system or send to the reference laboratory.
7. Storage and transport
Storage terms: in a place protected from light at +4 ° C – 1 week; at -20 ° С - 1 year; not more than 30 thawing-freezing cycles.
Transport: @ 0 - +4 ° C.
Limitation: Research Use Only!
Reference:
Chu D. K. W. et al. Molecular Diagnosis of a Novel Coronavirus (2019-nCoV) Causing an Outbreak of Pneumonia. Clinical Chemistry, hvaa029
Please request Stock-quantity info@geneon.net
SARS-CoV-2 (2019-nCoV) virus detection system is a kit for efficient in vitro detection of coronavirus SARS-CoV-2 (or 2019-nCoV) based on one-tube cDNA synthesis and Real-Time PCR. Kit includes two primers and probe set for detection and confirmation steps.
Real-time RT-PCR technology include the reverse transcription reaction (RT) stage to convert RNA to complementary DNA (cDNA) and the polymerase chain reaction (PCR) stage to amplify and detect a target sequence using specific primers and fluorescent probes
Final price excl. shipping costs3
Applications:
- RT PCR
- Synthesis of cDNA
- mRNA 5’-end Mapping by Primer Extension Analysis
- End-labeling of DNA
- Dideoxynucleotide Sequencing
Visit Webpage for more data of:
Moloney Murine Leukemia Virus (M-MuLV RT) is an RNA-dependent DNA polymerase
Tags: Corona virus test kit, Test system COVID-19