The Kit contains (the quantity will depend on specification):
One sealed package with Novel Coronavirus Fluorescence Detection Reagent（freeze-dried reaction pellets）
One tube of Rehydration Buffer DA (White Cap)
One tube of Magnesium Acetate MC (Green Cap)
One tube of Novel Coronavirus Positive QC (Red Cap)
0.2 mL Reaction Tubes (8 wells)
l ERA Quick Operation Guide
Equipment and Reagents to be provided by user
Double-distilled water
GenDxGS8 constant temperature fluorescence detector or other constant temperature fluorescence detector
Miniature vortex
Miniature centrifuge for 0.2 mL and 1.5 mL microfuge tubes
1.5 mL centrifuge tubes
RNase inhibitor
Stability and Storage
Novel Coronavirus Fluorescence Detection Reagent: 8-well reaction tubes (with own packaging) and other kit components are contained in a sealed package.
l 12 months from date of receipt, -20 °C or lower as lyophilized powder.
l 2-5 days, room temperature with optimal activity.
Rehydration Buffer and Magnesium Acetate: Store at -20℃ to maintain optimal activity.
Positive QC: Store at -20℃ immediately from date of receipt to maintain optimal activity and avoid excessive freeze-thaw cycles.
Experimental procedures: 50 μL (reaction volume)
1. Prepare a premix for each sample as follows:
Components | Volume per tube (μL) |
Rehydration Buffer (White Cap) | 20 |
Template RNA+ water + RNase inhibitor | 28 |
Total Volume | 48 |
Mix well and centrifuge briefly.
Note: The Rehydration Buffer should be completely thawed and well mixed as failure to do so will impact the results.
2. For each sample, aliquot 48 μL of the premix to the 0.2 mL reaction tube containing the fluorescence detection reagent. Vortex to mix well until the detection reagent is fully re-suspended and then centrifuged briefly.
3. Remove the lid from each 0.2 mL tube and add 2 μL of Magnesium Acetate (Green Cap) to each lid. Carefully cap the tubes and centrifuge briefly to allow the Magnesium Acetate to combine with the premix. Vortex briefly and immediately centrifuge again. (*)
* Note: Addition of the Magnesium Acetate will initiate the ERA reaction. It is recommended to pre-set the fluorescence detector before starting the experiment and once the Magnesium Acetate is added, immediately place the tubes in the fluorescence detector to start florescence quantitation.
4.Put reaction tubes in fluorescence detector (must be able to maintain constant temperature of 40℃). Start the program to detect continuously for 20 minutes and collect the fluorescence value of the FAM channel every 30 seconds.
5. Upon completion, save the data and discard the sample tubes appropriately.
Positive Control Reaction:
The novel Coronavirus Nucleic Acid Detection Kit contains a positive QC sample which can be used to validate the activity of kit components and the equipment used. When using the positive QC sample, the procedures are identical to that described previously. The detection reagent contains a FAM-labeled positive probe, with optimal excitation wavelength of 492 nm and maximum emission wavelength of 520 nm.
1. Pipette 20 μL of Rehydration Buffer into a 1.5 mL centrifuge tube. Add 26 μL of water and vortex briefly. Place tube in a centrifuge for a quick spin.
2. Add 2 μL of the Novel Coronavirus-Positive QC (Red cap) to Step 1. Repeat vortex and quick spin. The tube is now the ‘premix’.
3. Add 48 μL of the premix to the 0.2 mL reaction tubes. Vortex to resuspend the detection reagent completely. Quickly spin the tubes. Repeat steps 3 to 5 from the above procedures for fluorescence quantitation.
Note: If using an ABI series PCR instrument, make sure to select "NONE" for both passive reference and quencher.
Important notes to consider:
1. This kit is only used for non-medical testing. Please store at-20℃ and limit light exposure.
2. Aerosols of the ERA amplification products can cause false positive results. To avoid cross-contamination, the reagent preparation zone should be separated from the amplification analysis zone.
3. A negative control (no template) should be included at all times to confirm if there were contamination of the nucleic acid to be amplified.
4. Depending on the extraction method, the RNA quantity and purity from extracted samples can vary and these factors can contribute to inconsistent amplification efficiency (for details, please refer to PCR inhibitors: ethanol, phenol, heme, EDTA, etc.)
5. Please refer to manufacturer's instructions for the unit definition of the RNase inhibitor. The recommended amount for this protocol is 5U. However, if pancreatic tissue sample is used, 40 to 80U is recommended.
6. For premix preparation, 2 μL of Positive QC is recommended while the amount of test samples can range from 2 to 5 μL. The maximum volume should not exceed 10 μL.
7. If the template RNA copy number is low, take out the reaction tube after 5 minutes of reaction, vortex to mix well, centrifuge briefly, and then put it back in the florescence detector.
8. The entire procedure from resuspending the lyophilized detection reagent to sample incubation in the florescence detector should be accomplished in approximately 5 minutes.
9. The template RNA should be separated from the Magnesium Acetate when adding to the reaction tube cap, to prevent the RNA tertiary structure from being stabilized after being mixed with the Magnesium Acetate, resulting in the inhibition of reverse transcriptase.
10. Any fluorescence detector that can excite and detect the selected fluorophore and stabilize the temperature at 40℃ is compatible for use with the novel Coronavirus Nucleic Acid Detection Kit. These include the GS8 constant temperature fluorescence detector, ABI 7500, LightCycler480, CFX 96, and other fluorescence quantitative PCR instruments.
FAQ
Q1: What service can you provide?
A:We can provide very competitive prices and very good service to our clients all over the world.
Q2: Can you do customized?
A: We can make the products as per customers' requested products and quantity
Q3:Can we have samples for development?
A: Yes we can provide free samples (small quantity) to you, but the freight cost on buyer's account.
Q4:What is the normal lead time?
A. For stock products, we will send goods to you within 2-3 days after we receive your payment.
B.For customized products, generally the delivery time is around 30 days after we receive your payment.
Contact Information
Contact person: chen mingfu
Whatsapp/Wechat: +8613489146595
E-mail: percomal@163.com
