Overview: Automated RT-PCR set-up

The ASSIST PLUS pipetting robot is used to automate testing of suspected COVID-19 positive cases in a 384 well plate. The pipetting robot operates a VOYAGER 12 channel 50 μl electronic pipette with 125 μl Sterile, Filter, Low Retention GRIPTIPS. To double the available testing capacity and, concurrently, decrease the cost per test of expensive One-Step RT-PCR reagents of dwindling availability, the total PCR reaction volume was miniaturized, reducing it to 10 μl – inclusive of 7.5 μl Onestep RT-PCR Mastermix and 2.5 μl of nucleic acid template. The templates were extracted from combined nasopharyngeal/ oropharyngeal flocked swabs or sputum samples. The following procedure is based on the protocol used by the Microbiology and Molecular Pathology Department at Sullivan Nicolaides Pathology (SNP) – part of the Sonic Healthcare Group – in Brisbane, Australia.

The protocol is divided into two parts:

• Program 1: Add the Mastermix (1-COVID-19)
• Program 2: Add the nucleic acid template (2-COVID-19)

Downloads: Protocols and reports for automated RT-PCR testing for COVID-19 with ASSIST PLUS

Here you can download the complete protocols and reports used in this application and use them on your ASSIST PLUS. The protocol can be customized with the VIALAB software.

Experimental set-up: Program 1

Step-by-step procedure

1. Add the Mastermix

Fill the 384 well plate with the One-step RT-PCR Mastermix.

Place the One-step RT-PCR Mastermix in a 10 ml sterile, polystyrene reagent reservoir with INTEGRA’s SureFlo anti-sealing array. Set up the deck with the required labware, as indicated in Figure 1. Select the VIALAB program 1-COVID-19. The VOYAGER pipette automatically transfers the Mastermix from the reservoir into the 384 well plate (LightCycler® 480 Multiwell Plate, Roche) using the Repeat Dispense Mode with tip touch. Each well of the plate is filled with 7.5 μl of Mastermix.

Tips:

• Using a 10 ml reagent reservoir with SureFlo anti-sealing array allows a very low dead volume (<20 μl) to minimize the loss of expensive reagent of dwindling availability (see Figure 2).
• The combination of a low pipetting speed – set at 2 – and Low Retention GRIPTIPS shows excellent results when pipetting the viscous and foamy Mastermix.
• Pre- and post-dispense settings, together with the tip touch option, guarantee reproducible, precise and accurate pipetting results (see Figure 2).
• The PCR cooling block is used as a support to fix the position of the 384 well plate on the deck, ensuring exact tip positioning when pipetting. The cooling block also helps to keep samples and reagents cool if required by the protocol.

Experimental set-up: Program 2

Step-by-step procedure

2. Add the nucleic acid templates

Transfer the samples from four 96-format tube racks to the 384 well plate.

Nucleic acid templates extracted from combined nasopharyngeal/oropharyngeal flocked swabs or sputum samples are stored in FluidX Cluster 0.7 ml tubes placed in a 96-format rack. The VOYAGER pipette transfers 2.5 μl of template from the tubes to the 384 well plate, automatically changing the GRIPTIP pipette tips after each dispense. Both Position A and B are used to house the samples on the deck (see Figure 3). The pipette prompts the user when it is time to replace the tube racks on the deck. After user confirmation, the VOYAGER pipette continues reformatting the samples from tubes to the plate.

Tips:

• The VOYAGER pipette’s tip spacing capability combined with automatic Tip Change ensures easy and rapid sample transfer without risk of contamination or reformatting errors.
• Using an air gap of 1.5 μl when aspirating the viral nucleic acid template eliminates the risk of contamination risk during pipette tip travel.

Note: Automated RT-PCR testing for COVID-19 with the ASSIST PLUS can also be performed using a VOYAGER 8 channel 50 μl electronic pipette (see Figure 5).

Remarks

4 Position Portrait Deck:

If your process allows, the protocol can be compiled in one simple program using the 4 Position Portrait Deck option of the ASSIST PLUS (see Figure 6).

96 well plates:

The protocol can be readily adapted to 96 well format.

VIALAB software:

The VIALAB programs can be easily adapted to your specific labware and protocols.

For more information about the use of the ASSIST PLUS for COVID-19 sample testing and RT-PCR set-up at the Microbiology and Molecular Pathology Department at Sullivan Nicolaides Pathology, please click here.