Nov 12, 2024
Multiplex real-time PCR assay for the detection of SARS-CoV-2 variants
- Jianfa Bai1,
- Cori Ondrashek1
- 1Kansas State University
Protocol Citation: Jianfa Bai, Cori Ondrashek 2024. Multiplex real-time PCR assay for the detection of SARS-CoV-2 variants. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5jm26l1b/v1
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: Working
We use this protocol and it's working
Created: February 21, 2023
Last Modified: November 12, 2024
Protocol Integer ID: 77377
Funders Acknowledgement:
Vet-LIRN
Grant ID: 1 U18 FD008005
Disclaimer
Reference to any commercial materials, equipment, or process does not in any way constitute approval, endorsement, or recommendation by the Food and Drug Administration.
Abstract
This document describes a real-time 4-plex, TaqMan based RT-PCR assay used for the detection of the majority of SARS-CoV-2 strains, and differentiation of both Delta and Omicron variants. 18S ribosomal RNA gene is included as an internal control for the assay quality control. The Bio-Rad CFX 96, Bio-Rad CFX Opus 96 RT-PCR systems, or other real-time PCR systems that have similar function and can generate similar results, can be used for this assay.
Method validation/evaluation/verification:
This method was evaluated through Single Randomized Method Test (SRMT) by Kansas State Veterinary Diagnostic Laboratory and Vet-LIRN. The SRMT report summarizing method performance evaluation data is available upon request.
Guidelines
- Always wear gloves.
- Put samples on ice until ready for use.
- Use aerosol pipette tips only.
- The master mix should be prepared ONLY in a clean hood designated for this purpose. Always prepare enough master mix to cover the number of samples and an additional 10-20%.
Materials
Materials
A. Reagents
- Nuclease-free water
- Tris-EDTA (TE) buffer
- Dulbecco's Modified Eagle's Medium (DMEM)
Table 1: Primers and probes used in this assay.
B. Equipment/miscellaneous
- KingFisher 96 magnetic particle processor, or equivalent (Thermo Electron Corp. Cat#5400050)
- Benchtop centrifuge
- BioRad CFX96, BioRad Opus real-time PCR machine or equivalent
- Level 2 Biological Safety Cabinet or equivalent
- AirClean 600 PCR Workstation-AirClean Systems (Model# AC648LFUVC) or equivalent
- Vortex Mixer
- Mini Plate Spinner – Labnet (Model# MPS 1000) or equivalent, or larger centrifuge capable of centrifuging 96-well plates
- Seward Stomacher® 80 Biomaster, or equivalent
- QIAvac 24 Plus vacuum manifold, or equivalent
- Freezer (-15 °C --25 °C )
C. Supplies
- 1000 μl, 200 μl, and 20 μl pipets
- 1000 μl, 200 μl and 20 μl aerosol pipet tips
- Serological pipettor
- 50 ml reservoirs
- Sterile 1.5 ml microcentrifuge tubes
- Sterile PCR reaction tubes, strip tubes, or 96-well plates
- Sterile PCR strip caps
- Optically clear adhesive seals for plates
- Gloves
- 10 ml, 5 ml serological pipets
Before start
Specimen Preparation:
Nucleic Acid can be extracted using the ThermoFisher MagMax™ Viral RNA Isolation Kit or the Qiagen QIAamp Viral RNA Mini Kit (Cat #52906). DMEM (ThermoFisher Cat. #: 11965-092) may be used to homogenize tissue prior to NA extraction.
Probe preparation
Probe preparation
Prepare 100 µM individual probe stock solution with 1 × TE buffer.
Centrifuge the lyophilized probe tube at 6000 × g (8,000 rpm) for 00:05:00 .
5m
Dissolve lyophilized probe based on its molarity, and not molecular weight.
i.e., add 455 µL of 1× TE buffer to a tube containing 45.5 nM probe.
Mix by vortexing at half-full speed 3-4 times, 4-5 sec each time and centrifuge at 6000 × g for 10 sec. Do not vortex at high speed or for prolonged periods of time.
Label the tube with probe name and concentration, you initials and date of preparation. Store the stock solution in a designated -20 °C freezer.
Prepare 100 µL of probe working solution (10 µM) for each probe
Add 90 µL of nuclease-free water to a dark 1.5 ml sterile tube labeled with probe name and working solution concentration (10 µM).
Add 10 µL of the 100 µM probe stock solution and pipette up-and-down 2-3 times.
Invert the tube 3-5 times. Do not vortex.
Briefly centrifuge. This is the probe working solutions to be used in PCR reaction described below.
Store in designated -20 °C freezer.
Primer preparation
Primer preparation
Prepare 100 µM individual primer stock solution with 1 × TE buffer.
Centrifuge the lyophilized primer tube at 6000 × g (8,000 rpm) for 00:05:00 .
5m
Dissolve lyophilized primer based on its molarity, and not molecular weight.
i.e., add 455 µL of 1 × TE buffer to a tube containing 45.5 nM primer.
Mix by vortexing at half-full speed 3-4 times, 4-5 sec each time and centrifuge at 6000 × g for 10 sec. Do not vortex at high speed or for prolonged periods of time.
Label the tube with primer name and concentration, your initial and date of preparation. Store the stock solution in a designated -20 °C freezer.
Prepare100 µL of 18S (10 µM) primer working solution:
Add 80 µL of nuclease-free water into a 1.5 ml sterile tube labeled with primer name and working solution concentration (10 µM).
Add 10 µL of each 100 µM primer stock solution into the 80 µL water. Pipette up- and-down 2-3 times.
Invert the tube 3-5 times.
Briefly centrifuge.
Store in a designated -20 °C freezer.
Prepare 100 µL of SARS dF/R & Omicron Primer Mix (10 µM) primer working solution:
Add 50 µL of nuclease-free water into a 1.5 ml sterile tube labeled with primer name and working solution concentration (10 µM).
Add 10 µL of each 100 µM primer stock solution into the 50 µL water. Pipette up- and-down 2-3 times.
Invert the tube 3-5 times.
Briefly centrifuge
Store in a designated -20 °C freezer.
Note
All probe and primer working solutions can be prepared in different volumes if proportions of each component are kept in the same ratio. For example, use half volume of each component for 50 µl preparation, or use 2 times of each component for 200 µl preparation, etc.
Real-time PCR reaction preparation
Real-time PCR reaction preparation
Master mix preparation
Prepare the master mix in a 1.5 ml microcentrifuge tube using the volumes of reagents listed. DO NOT add the nucleic acid (template) at this time. Ice or cool blocks should be used for reaction preparation. Mix the master mix by low-speed vortexing, inverting, and/or flicking the tube, and then briefly centrifuge. Always prepare enough master mix for your samples, controls and an additional 10-20%.
Master mix
| A | B | |
| COVID19 Delta Omicron qPCR_TaqPath | ||
| Component | Per rxn (μl) | |
| 4X TaqPath™ 1-Step RT-qPCR Master Mix, CG | 5 | |
| Nuclease-Free Water | 3.75 | |
| SARS dF/R & Omicron Primer Mix (10 µM) | 1 | |
| 18S Primer Mix (10 µM) | 1 | |
| SARS-dPr (10 µM; FAM) | 0.5 | |
| OmNm-Pr (10 µM; TxRed) | 0.75 | |
| SARS-wPr (10 µM; VIC) | 1 | |
| OmNw-Pr (10 µM; VIC) | 1 | |
| 18S-Pr (10 µM; Cy5) | 1 | |
| Template | 5 | |
| Total reaction volume | 20 |
Label PCR tubes, strips, worksheet or a plate map with sample IDs, negative controls, and positive controls. Add 15 µL of master mix to each well or tube.
Transfer plate or tubes with master mix to a DNA cabinet, or a clean working bench. Add 5 µL of sample (Template) to each corresponding PCR tube or well and mix by pipetting up and down several times. If the sample NA is in a plate, use extra caution when handling the uncovered NA plate to prevent any splashing. A multichannel pipet may be used to add 5μl of each sample NA to the reaction plate or tubes. Re-cover the NA plate immediately after all sample NA and control aliquots have been removed.
Cover the reaction tubes with optically clear caps or cap strips. If using a plate, cover the plate with optically clear PCR sealing film.
Briefly centrifuge, and put the tubes or plate onto the machine with correct orientation.
Machine run for the real-time PCR reactions
Run the samples, negative controls, and positive controls on a PCR machine using the protocol below
RT-qPCR Protocol:
| A | B | |
| 48°C | 10 min | |
| 95°C | 5 min | |
| 45 cycles of: | ||
| 95°C | 20 sec | |
| 60°C | 40 sec |
Reaction Analysis
Threshold checking and data generation: After a run, the threshold levels need to be checked for each channel for accuracy. If it is off (Fig. 1), data could be misinterpreted.
Incorrect threshold settings
Incorrect threshold settings
In these cases, switch to “log view”, and slide the threshold bar to the middle of the linear range (Fig. 2).
Correct threshold settings
Data to be Recorded/Interpretation
Positive Result: A positive sample is identified if Ct value is ≤37, and 18S Ct is ≤31.
Negative Result: A negative sample is defined if there is no Ct, or Ct >39, and 18S Ct is ≤31
Other Possible Results (if applicable): A weak positive/suspect/inconclusive sample is defined if Ct is >37 and ≤39, and 18S Ct is ≤31.