1 For Laboratory Use.

appliedbiosystems White Paper The Critical Function of Mixing RT-PCR Reaction Plates for the SARS-CoV-2 assay Date of Release July 29, 2020 Revision B.0 Purpose: The purpose of this paper is to provide detailed instructions for adequate mixing of RT-PCR reaction plates and to provide supporting data demonstrating the value of these instructions. Introduction: To ensure proper analysis of SARS-CoV-2 research samples, it is essential to mix the RT-PCR reaction properly by vortexing the plate, as mandated in the Instructions for Use. Failure to do so can result in Optical Mixing, a phenomenon that is likely to occur when the sample volume exceeds 20% of the PCR reaction volume. Optical Mixing can lead to RT-PCR baseline instability, resulting in QC failure of entire plates and potential false classification of samples. An example of Optical Mixing leading to false classification of samples is presented in Figure 1. Mixing Protocol: 1. After master mix, assay, water, samples, and controls have been added to the RT-PCR

reaction plate, seal the plate well with a MicroAmp Optical Adhesive Film (4311971, 4360954). Use the MicroAmp Adhesive Film Applicator (4333183) to make sure all wells are sealed completely. The MicroAmp Optical Adhesive Cover uses a pressure-sensitive adhesive backing to adhere the cover to the optical 96- or 384-well plate. It is imperative that you use enough force to activate the pressure-sensitive adhesive to prevent evaporation from the wells.

2. Set the speed of a vortex mixer, such as the Vortex-Genie 2 from Scientific Industries (shown in Figure 8), to the highest setting and set mode to “Touch.” Ensure that the vortex mixer has a platform rather than a tube cup installed.

3. Place the plate in contact with the vortex mixer and hold with medium pressure for 10 –

30 seconds. Ensure that the vortex platform is able to move vigorously and that the reaction mix moves freely in the wells; too much or too little pressure applied to the vortex platform will reduce mixing efficiency. Move the plate around during vortexing to ensure that contact with the platform has been made at all four quadrants and the center of the plate for equal time.

4. Centrifuge the reaction plate for 1–2 minutes at ≥650 × g (≥650 RCF) to remove bubbles and to collect the liquid at the bottom of the reaction plate.

Experimental Design In Experiment 1, two identical 96-well plates were created; one plate was vortexed for 30 seconds at maximum speed on a Vortex-Genie 2, and the other plate was not mixed at all. Each plate contained triplicate reactions of extracted contrived positive samples and negative samples. Contrived positive samples consisted of pooled nasopharyngeal specimens spiked with SARS-CoV-2 viral RNA at 9X, 3X or 1X the Limit of Detection (2,250

The Critical Function of Mixing RT-PCR Reaction Plates for the SARS-CoV-2 assay

2 For Laboratory Use.

GCE/mL, 750 GCE/mL and 250 GCE/mL, respectively). Samples were extracted with either the MagMAX Viral/Pathogen Nucleic Acid Isolation Kit and a 400-µL specimen volume or the MagMAX Viral/Pathogen II Nucleic Acid Isolation Kit and a 200-µL specimen volume, and both extraction workflows were run on the same RT-PCR plate. In Experiment 2, two identical 384-well plates were created; one plate was vortexed for 10 seconds at maximum speed on a Vortex-Genie 2, and the other plate was not mixed at all. Each plate contained 48 replicate reactions of extracted SARS-CoV-2 viral RNA at 10 GCE/reaction plus the MS2 Internal Control. RT-PCR runs were performed on an Applied Biosystems QuantStudio 7 Flex system with a 384-well block.

Results As shown in Figures 2, 4 and 6, the 96-well and 384-well plates that were not mixed demonstrated steep downward slopes in the fluorescent signal during the early cycles of the thermal protocol. By contrast, Figures 3, 5 and 7 reveal that proper mixing produces flatter baselines for the same conditions. Conclusion Compared with no mixing, vortexing for 10 – 30 seconds produces flatter baselines irrespective of extraction protocol, plate type or sample type. Because falling baselines can produce plate failures or inaccurate results, vortex mixing is vital for achieving reliable, specific results.

Figure 1. Example of Optical Mixing causing negative targets to cross the threshold. Confidential samples from a plate that was not mixed properly. In this case, the lack of mixing caused these four negative samples to be called Inconclusive.

The Critical Function of Mixing RT-PCR Reaction Plates for the SARS-CoV-2 assay

3 For Laboratory Use.

Figure 2. MagMAX Viral/Pathogen 400-µL protocol without mixing. Contrived positive samples at 9X, 3X and 1X LoD and negative samples were extracted in triplicate and used as templates for RT-PCR with no mixing.

Figure 3. MagMAX Viral/Pathogen 400-µL protocol with vortex mixing. Contrived positive samples at 9X, 3X and 1X LoD and negative samples were extracted in triplicate and used as templates for RT-PCR with 30 seconds of vortex mixing.

The Critical Function of Mixing RT-PCR Reaction Plates for the SARS-CoV-2 assay

4 For Laboratory Use.

Figure 4. MagMAX Viral/Pathogen II 200-µL protocol without mixing. Contrived positive samples at 9X, 3X and 1X LoD and negative samples were extracted in triplicate and used as templates for RT-PCR with no mixing.

Figure 5. MagMAX Viral/Pathogen II 200-µL protocol with vortex mixing. Contrived positive samples at 9X, 3X and 1X LoD and negative samples were extracted in triplicate and used as templates for RT-PCR with 30 seconds of vortex mixing.

The Critical Function of Mixing RT-PCR Reaction Plates for the SARS-CoV-2 assay

5 For Laboratory Use.

Figure 6. SARS CoV-2 viral RNA without mixing. Purified SARS-CoV-2 viral RNA at LoD was used as a template for RT-PCR in 48 replicate reactions without mixing. The run was performed on a QuantStudio 7 Flex system with a 384-well block.

Figure 7. SARS CoV-2 viral RNA with vortex mixing. Purified SARS-CoV-2 viral RNA at LoD was used as a template for RT-PCR in 48 replicate reactions with 10 seconds of vortex mixing. The run was performed on a QuantStudio 7 Flex system with a 384-well block.

The Critical Function of Mixing RT-PCR Reaction Plates for the SARS-CoV-2 assay

6 For Laboratory Use.

Figure 8. Photo of a Vortex-Genie 2 with recommended settings. This vortex model was used in the studies presented in this paper.

Revision history: Pub.No. MAN0014339

Revision Date Description A.0 July 24, 2020 New document. B.0 July 29, 2020 Updated intended use statement to “For Laboratory Use.”

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