TITLE: Extraction and PCR for animal samples using the Thermofisher Scientific TaqPath COVID-19 Combo Kit
AUTHORS: Brianna Stenger

[DESCRIPTION]
To describe the steps and materials needed to perform a multiplex real-time reverse transcriptase PCR test intended for the qualitative detection of nucleic acid from SARS-CoV-2, the causative agent of COVID-19, in animal respiratory swab samples.

This document includes the information and procedures to comply with the ThermoFisher Scientific TaqPath™ COVID-19 Combo Kit under the Food and Drug Administration’s (FDA) Emergency Use Authorization (EUA). The TaqPath™ COVID-19 Combo Kit includes a Real Time PCR assay multiplex that contains three primer/probe sets specific to the SARS-CoV-2 and a primer probe set for bacteriophage MS2 as an internal extraction control. 

Sections of this procedure are taken directly from the TaqPath™ COVID-19 Combo Kit Instructions for Use, Publication MAN0019181, Version G.0. For additional information, please refer to these instructions. 

[GUIDELINES]
1. The TaqPath™ RT-PCR COVID-19 Kit workflow should be performed by qualified and trained staff to avoid the risk of erroneous results. Use separate areas for the preparation of patient samples and controls to prevent false positive results. Samples and reagents must be handled in a biological safety cabinet.

2. The assay is for in vitro diagnostic use under the FDA Emergency Use Authorization Only.

3. This test has not been FDA cleared or approved.

4. This test has been authorized only for the detection of nucleic acid fromSARS-CoV-2, not for any other viruses or pathogens.

5. This test is only authorized for the duration of the declaration that circumstances exist justifying the authorization of emergency use of in vitro diagnostics for detection and/or diagnosis of COVID-19 under Section 564(b)(1) of the Act, 21
U.S.C. § 360bbb-3(b)(1), unless the authorization is terminated or revoked sooner.

6. Samples and controls should always be treated as if infectious and/or biohazardous in accordance with safe laboratory procedures.

7. Follow necessary precautions when handling specimens. Use personal protective equipment (PPE) consistent with current guidelines for the handling of potentially infectious samples.

8. Always use pipette tips with aerosol barriers. Tips that are used must be sterile and free from DNases and RNases.

9. Do not eat, drink, smoke, or apply cosmetic products in the work areas.

10. Reagents must be stored and handled as specified. See manufacture instructions.

11. Components of reagent kits of different lot numbers must not be interchanged.

12. Do not use the kit after the indicated expiry date.

13. Dispose of waste in compliance with local, state, and federal regulations.

Positive results are indicative of the presence of SARS-CoV-2 RNA

[STEPS]
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1. Prepare working areas by cleaning with ~10% bleach or ~70% alcohol, followed by a rinse and use of another solution to reduce the presence of RNases such as RNaseZAP or Eliminase.
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4.1. Start the run and load prepared plates in positions when prompted by the instrument.
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4.2. Immediately remove the elution plate from the instrument and cover with film/foil. Run lasts ~24 minutes.

Seal immediately to avoid evaporation and store up to 48hrs at 4°C, -20°C, or -70°C.
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3.  Prepare Plates for extraction following table 2.

Table 2: Plate setup.
 
      Plate Position  Plate ID  Plate Type  Reagent  Volume per Well    1  Sample Plate  KingFisher™     Deepwell 96    Plate or Equivalent    10 µL Proteinase K       + 400 µLSample    + 550 µL Bead mix (new tips!)    + 10 µL MS2 Phage Control    2  Wash 1  Wash Buffer   1000 µL    3  Wash 2  80% Ethanol  1000 µL    4  Elution Plate  Elution Solution  50 µL    5  Tip Comb  Place a KingFisher™ 96 tip comb for DW magnets   in a KingFisher™ 96 KF microplate  
     
 

 

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4. Select the program MVP_2Wash_400_Flex on the KingFisher™ Flex Magnetic Particle Processor with 96 Deep-Well Head
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2.3. Mix well by inversion, then store at room temperature. 
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2. Prepare Binding Beads/Lysis mix on each day of use.

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5. If frozen, thaw the reagents on ice. Gently vortex the reagents, then centrifuge briefly to collect liquid at the bottom of the tubes.
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6. Prepare a Master Mix of the PCR reagents containing enough for all samples, controls, plus extra. Master Mix is prepared manually using the volumes in Table 3.

 Table 3. PCR Reagent Volumes.
              Nuclease-free   Water  12.5      4X   TaqPath 1-Step Multiplex MMX (No ROX)  6.25      COVID-19   Real Time PCR Assay Multiplex  1.25      µl MM per rxn  20      Template DNA  5      Total Volume  25  

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6.2. Add 5 µL of extraction RNA or negative control to corresponding well. Total reaction volume will be 25 µL

       
 
                                                                                                                                                     
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6.3. Add 2 µL of diluted positive control +3 µL of nuclease-free water for positive control well.

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6.4. Diluted positive control is made by:

Pipet 98 µL of TaqPath™ COVID‑19 Control Dilution Buffer into a microcentrifuge tube, then add 2 µL of TaqPath™ COVID‑19 Control. Mix well, then centrifuge briefly

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6.5. Pipet 87.5 µL of TaqPath™ COVID‑19 Control Dilution Buffer into a second microcentrifuge tube, then add 12.5 µL of the dilution created in substep 13.1. Mix well, then centrifuge briefly.

                                                           
 

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6.6. Once sample and controls have been added, seal plate thoroughly.
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6.1. Pipette 20 µL of master mix into each well. 

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6.7. Vortex plate at high speed for 10-30 seconds. Recommend a plate holder attachment to avoid damaging wells.
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6.8. Centrifuge the reaction plate for 2 minutes (~600 x g) to remove bubbles and collect liquid to the bottom of the reaction plate. 

            
 
                                                                                
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2.2. For the number of required reactions, prepare the Binding Bead/Lysis Mix according to Table 1
 
Table 1: Binding Bead/Lysis Mix
     Component      Volume per RNA Sample or Control      Volume for 110 rxns        Binding Solution      530 μL      58.3 mL        Nucleic Acid Magnetic Beads      20 μL      2.2 mL        Total Reaction Mix volume      550 μL      60.5 mL   
 
   
 
                                                                                                                        
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2.1. Vortex the Total Nucleic Acid Magnetic Beads to ensure that the bead mixture is homogeneous.
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3.1. Invert the Binding bead Mix (from 2.2) five times gently to mix, then add 570 µL (binding solution + beads + PK + MS2 phage control) to each sample well and negative control well in the sample plate. 


 


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3.2. Add samples and controls to the appropriate wells:
Samples:  400 µLof sample to a well.
Negative Control: 400 µLL of Nuclease-free Water (not DEPC-Treated) to the Negative Control 

 

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3.3. Seal sample plate with film. Wipe outside of plate with disinfectant and place in transport container. Wipe/disinfect transport container and carefully bring to BSC in Extraction Room.
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7. Setting up Thermal Cycler 
This protocol used the ABI 7500 Fast or ABI 7500 DX thermal cycler.
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      
Set up thermal cycler according to manufacture instruction manuals along with table 4 and 5.
Refer to https://www.fda.gov/media/136112/download in reference sections for more information
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7.1.   Table 4. Target, dyes, and quencher.
 
 
 
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7.2. Table 5. Thermal cycler conditions.
 
 
 
 
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8. Interpretation of Results 
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8.1. A minimum of one Negative Control and one Positive Control must be present for each run. Additional Negative Control wells must be run for each extraction that is represented on a real-time RT-PCR plate. All control wells must pass for the real-time RT-PCR plate to be considered valid.
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8.2. Validation of results is performed automatically by the Applied Biosystems™ COVID‑19 Interpretive Software based on performance of the Positive, Negative, and Internal Controls. See Table 6.

Table 6. Interpretation of Results