Protocol Citation: Toby 2020. Mikro SARS-CoV-2 Multiplex Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.bk8dkzs6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: September 09, 2020
Last Modified: September 09, 2020
Protocol Integer ID: 41957
Disclaimer
DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK
The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with protocols.io, can be held responsible for your use of the information contained in or linked to this protocol or any of our Sites/Apps and Services.
Materials
MATERIALS
Ethanol, Absolute, Molecular Biology GradeThermo Fisher ScientificCatalog #BP2818500
Buffer AVLQiagenCatalog #19073
Buffer AVEQiagenCatalog #1020953
UltraPlex 1-Step ToughMix (4X)QuantabioCatalog #95166-01K
Nuclease-Free WaterQiagenCatalog #129117
STEP MATERIALS
Ethanol, Absolute, Molecular Biology GradeThermo Fisher ScientificCatalog #BP2818500
Buffer AVEQiagenCatalog #1020953
Buffer AVLQiagenCatalog #19073
UltraPlex 1-Step ToughMix (4X)QuantabioCatalog #95166-01K
Protocol materials
UltraPlex 1-Step ToughMix (4X)QuantabioCatalog #95166-01K
Ethanol, Absolute, Molecular Biology GradeThermo Fisher ScientificCatalog #BP2818500
Buffer AVLQiagenCatalog #19073
Buffer AVLQiagenCatalog #19073
Buffer AVEQiagenCatalog #1020953
Buffer AVEQiagenCatalog #1020953
UltraPlex 1-Step ToughMix (4X)QuantabioCatalog #95166-01K
Ethanol, Absolute, Molecular Biology GradeThermo Fisher ScientificCatalog #BP2818500
Nuclease-Free WaterQiagenCatalog #129117
Buffer AVLQiagenCatalog #19073
Ethanol, Absolute, Molecular Biology GradeThermo Fisher ScientificCatalog #BP2818500
Buffer AVEQiagenCatalog #1020953
UltraPlex 1-Step ToughMix (4X)QuantabioCatalog #95166-01K
Before start
The protocol is designed for a single sample, utilizing a single well.
It is designed for a custom RT-qPCR device, but may be adapted for other equipment.
Sample Collection
Sample Collection
Remove the lid of sample collector. Direct the sample provider to expel saliva into the collector until 0.5 mL has been collected. Replace lid.
Note
Teeth brushing, mouthwash, drinks, food, and nasal sprays should be avoided for half an hour before sample collection.
Heat Pre-Treatment
Heat Pre-Treatment
Load sample for RNA extraction.
Apply heat-treatment to sample
| Step | Action | Specification | Time | |
| 1 | Heat | 95°C | 2 min | |
| 2 | Cool | Room temperature | 2 min | |
| 3 | Centrifuge | 500 x g | 2 min | |
Collect cellular debris.
Add 100 µL of
Ethanol, Absolute, Molecular Biology GradeThermo Fisher ScientificCatalog #BP2818500
Mix for 00:00:30
RNA Extraction
RNA Extraction
Immediately proceed with RNA extraction. Do not store sample.
Prepare 500 µL of
Buffer AVLQiagenCatalog #19073
Add 100 µL of heat-treated sample to buffer.
Mix for 00:00:15
Incubate at room temperature for 00:07:00
Centrifuge at 500 x g for 00:02:00
Then remove buffer.
Proceed with RNA elution.
Add 60 µL of
Buffer AVEQiagenCatalog #1020953
Centrifuge at 1000 x g for 00:02:00
RT-qPCR
RT-qPCR
Prepare 8 µL multiplex primer-probe-water mix in advance, containing the following:
| Component | Spec | Concentration | Volume (1 reaction) | |
| 2019-nCoV_N1-F | US CDC | 200nM | 0.6 µL | |
| 2019-nCoV_N1-R | US CDC | 400nM | 1.2 µL | |
| 2019-nCoV_N1-P | US CDC | 200nM | 0.6 µL | |
| 2019-nCoV_N2-F | US CDC | 200nM | 0.6 µL | |
| 2019-nCoV_N2-R | US CDC | 400nM | 1.2 µL | |
| 2019-nCoV_N2-P* | Custom probe (JUN-QSY) | 200nM | 0.6 µL | |
| RP-F | US CDC | 200nM | 0.6 µL | |
| RP-R | US CDC | 400nM | 1.2 µL | |
| RP-P* | Custom probe (VIC-MGB) | 200nM | 0.6 µL | |
| Nuclease-free water | 0.8 µL |
Prepare 140 µL of master mix in advance, using
UltraPlex 1-Step ToughMix (4X)QuantabioCatalog #95166-01K
with the following quantities:
| Component | Volume (1 reaction) | |
| UltraPlex 1-Step ToughMix | 15 µL | |
| Primer-probe-water mix | 8 µL | |
| Nuclease-free water | 117 µL |
Add 60 µL of previously purified RNA sample to 140 µL of master mix.
Load onto qPCR device.
Proceed with thermocycling, under the following conditions:
| Step | Temperature | Time | |
| 1 | 50°C | 10 min | |
| 2 | 95°C | 1 min | |
| 3 | 95°C | 3 sec | |
| 4 | 60°C | 30 sec | |
| Repeat 40 Cycles for Step 3-4 | |||