TITLE: Multiplex real-time PCR assay for the detection of SARS-CoV-2 variants
AUTHORS: Jianfa Bai, Cori Ondrashek

[DESCRIPTION]
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.

[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. 

[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%.

[STEPS]
SECTION: Probe preparation
1. Prepare 100 µM individual probe stock solution with 1 × TE buffer.

SECTION: Probe preparation
1.1. Centrifuge the lyophilized probe tube at 6000 × g (8,000 rpm) for 5 min .

SECTION: Probe preparation
1.2. 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.

SECTION: Probe preparation
1.3. 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.
SECTION: Probe preparation
1.4. Label the tube with probe name and concentration, you initials and date of preparation. Store the stock solution in a designated -20 °Cfreezer.
SECTION: Probe preparation
2. Prepare 100 µL of probe working solution (10 µM) for each probe 
SECTION: Probe preparation
2.1. 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). 
SECTION: Probe preparation
2.3. Invert the tube 3-5 times. Do not vortex.
SECTION: Probe preparation
2.4. Briefly centrifuge. This is the probe working solutions to be used in PCR reaction described below.
SECTION: Probe preparation
2.5. Store in designated -20 °C  freezer. 

SECTION: Primer preparation
3. Prepare 100 µM individual primer stock solution with 1 × TE buffer.
SECTION: Primer preparation
3.1. Centrifuge the lyophilized primer tube at 6000 × g (8,000 rpm) for 5 min.
SECTION: Primer preparation
3.2. Dissolve lyophilized primer based on its molarity, and not molecular weight. 
i.e., add 455 µLof 1 × TE buffer to a tube containing 45.5 nM primer.

SECTION: Primer preparation
3.3. 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.
SECTION: Primer preparation
3.4. Label the tube with primer name and concentration, your initial and date of preparation. Store the stock solution in a designated  -20 °C  freezer. 

SECTION: Primer preparation
4. Prepare100 µL of 18S (10 µM) primer working solution:

SECTION: Primer preparation
4.1. Add 80 µLof nuclease-free water into a 1.5 ml sterile tube labeled with primer name and working solution concentration (10 µM).

SECTION: Primer preparation
4.2. Add 10 µL of each 100 µM primer stock solution into the 80 µL water. Pipette up- and-down 2-3 times.

SECTION: Primer preparation
4.3. Invert the tube 3-5 times.
SECTION: Primer preparation
4.4. Briefly centrifuge.
SECTION: Primer preparation
4.5. Store in a designated -20 °C  freezer. 

SECTION: Primer preparation
5. Prepare 100 µLof SARS dF/R & Omicron Primer Mix (10 µM) primer working solution:

SECTION: Primer preparation
5.1. Add 50 µLof nuclease-free water into a 1.5 ml sterile tube labeled with primer name and working solution concentration (10 µM). 

SECTION: Primer preparation
5.2. Add 10 µL of each 100 µM primer stock solution into the  50 µL water. Pipette up- and-down 2-3 times.

SECTION: Primer preparation
5.3. Invert the tube 3-5 times.
SECTION: Primer preparation
5.4. Briefly centrifuge
SECTION: Primer preparation
5.5. Store in a designated -20 °C  freezer.

 

SECTION: Real-time PCR reaction preparation
6. 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%.
SECTION: Real-time PCR reaction preparation
6.1. Master mix

       COVID19 Delta Omicron qPCR_QuantiTect                      Component         Per   rxn (μl)            2X QuantiTect Multiple RT-PCR   Master Mix         10            QuantiTect Multiplex RT Mix         0.2            SARS dF/R & Omicron Primer   Mix (10 µM)         1            18S Primer Mix (10 µM)         0.65            SARS-dPr (10 µM; FAM)         0.45            OmNm-Pr (10 µM; TxRed)         0.55            SARS-wPr (10 µM; VIC)         0.75            OmNw-Pr (10 µM; VIC)         0.75            18S-Pr (10 µM; Cy5)         0.65            Template         5            Total   reaction volume         20      

SECTION: Real-time PCR reaction preparation
6.2. 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. 

SECTION: Real-time PCR reaction preparation
6.3. 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. 
SECTION: Real-time PCR reaction preparation
6.4. Cover the reaction tubes with optically clear caps or cap strips. If using a plate, cover the plate with optically clear PCR sealing film. 
SECTION: Real-time PCR reaction preparation
6.5. Briefly centrifuge, and put the tubes or plate onto the machine with correct orientation.
SECTION: Real-time PCR reaction preparation
7. 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:
 
     50°C      20 min        95°C      15 min        45 cycles of:          95°C      45 sec        60°C      75 sec   

SECTION: Real-time PCR reaction preparation
8. 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. 
 


 

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             
In these cases, switch to “log view”, and slide the threshold bar to the middle of the linear range (Fig. 2).

 

 
SECTION: Real-time PCR reaction preparation
9. Data to be Recorded/Interpretation
SECTION: Real-time PCR reaction preparation
9.1. Positive Result: A positive sample is identified if Ct value is ≤37, and 18S Ct is ≤31.
SECTION: Real-time PCR reaction preparation
9.2. Negative Result: A negative sample is defined if there is no Ct, or Ct >39, and 18S Ct is ≤31
SECTION: Real-time PCR reaction preparation
9.3. Other Possible Results (if applicable): A weak positive/suspect/inconclusive sample is defined if Ct is >37 and ≤39, and 18S Ct is ≤31. 
SECTION: Probe preparation
2.2. Add 10 µL of the 100 µM probe stock solution and pipette up-and-down 2-3 times.