Automated RT-PCR set-up for COVID-19 testing

  • Automated RT-PCR set-up for COVID-19 testing

    How to prepare RT-PCR plates for SARS-CoV-2 detection with the ASSIST PLUS

    The emergence and outbreak of the novel coronavirus SARS-CoV-2 (COVID-19) has placed unprecedented demands on laboratories testing for COVID-19, leaving scientific staff to contend with a spiraling influx of patient samples and a rapid, continuous growth in workload. Laboratories need additional automated liquid handling instruments for viral nucleic acid extraction and RT-PCR set-up – which are the most labor-intensive processes in the diagnostic testing workflow – to increase the sample throughput capacity, reduce manual intervention by laboratory analysts and fast track the development of COVID-19 assays.
    The ASSIST PLUS pipetting robot together with a VOYAGER adjustable tip spacing pipette, Low Retention GripTips and SureFlo™ 10 ml Reagent Reservoirs were successfully used for RT-PCR set-up in COVID-19 testing laboratories.

  • Table of contents

      The emergence and outbreak of the novel coronavirus SARS-CoV-2 (COVID-19) has placed unprecedented demands on laboratories testing for COVID-19, leaving scientific staff to contend with a spiraling influx of patient samples and a rapid, continuous growth in workload. Laboratories need additional automated liquid handling instruments for viral nucleic acid extraction and RT-PCR set-up – which are the most labor-intensive processes in the diagnostic testing workflow – to increase the sample throughput capacity, reduce manual intervention by laboratory analysts and fast track the development of COVID-19 assays.
      The ASSIST PLUS pipetting robot together with a VOYAGER adjustable tip spacing pipette, Low Retention GripTips and SureFlo™ 10 ml Reagent Reservoirs were successfully used for RT-PCR set-up in COVID-19 testing laboratories.

    Key benefits

    • The full automation capability of the ASSIST PLUS reduces manual intervention and frees highly valuable time for laboratory personnel in this critical COVID-19 pandemic.
    • The compact and easy-to-use ASSIST PLUS pipetting robot allows fast set-up regarding installation and programming, allowing labs to immediately increase their sample processing capacity and fast track assay development for COVID-19 sample testing.
    • VOYAGER adjustable tip spacing pipettes in combination with the ASSIST PLUS provide unmatched pipetting ergonomics by automatically reformatting patient samples from tube racks into 384 well plates.
    • Optimal pipette settings, including tip immersion depth, pipetting speeds and angles, deliver reproducible, precise and accurate results, with no contamination observed in controls or patient samples.
    • The use of INTEGRA’s low dead volume, SureFlo 10 ml reagent reservoirs, together with Low Retention GripTips, demonstrated excellent results, enabling efficient handling of the precious and expensive One-step RT-PCR Mastermix used for patient testing.

    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

    Deck position A:

    10 ml reagent reservoir with SureFlo anti-sealing array containing 3 ml of One-step RT-PCR Mastermix.

    Deck position C:

    384 well plate placed on a PCR 384 well cooling block, allowing Mastermix and samples to be kept cold, and enabling exact positioning of the PCR plate on the deck.

    Figure 1: The set-up for program 1-COVID-19
    Figure 1: The set-up for program 1-COVID-19

    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.
    Figure 2: Precise and accurate dispensing of One-step RT-PCR Mastermix from the low dead volume reagent reservoir to the 384 well plate.
    Figure 2: Precise and accurate dispensing of One-step RT-PCR Mastermix from the low dead volume reagent reservoir to the 384 well plate.

    Experimental set-up: Program 2

    Deck position A and B:

    FluidX Cluster 0.7 ml tubes containing the nucleic acid templates. The tubes are stored in a 96-format rack. A total of four sample racks are used for the protocol (two on Position A and two on Position B).

    Deck position C:

    384 well plate placed on a PCR 384 well cooling block.

    Figure 3: The set-up for program 2-COVID-19.
    Figure 3: The set-up for program 2-COVID-19.

    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).

    Figure 4: Easy and rapid transfer of patient nucleic acid templates from the tube rack to the 384 well plate using the VOYAGER adjustable tip spacing pipette together with the ASSIST PLUS pipetting robot.
    Figure 4: Easy and rapid transfer of patient nucleic acid templates from the tube rack to the 384 well plate using the VOYAGER adjustable tip spacing pipette together with the ASSIST PLUS pipetting robot.
    Figure 5: Automated RT-PCR testing for COVID-19 using the ASSIST PLUS pipetting robot together with a VOYAGER 8 channel adjustable tip spacing pipette, as performed in the Microbiology and Molecular Pathology Department at SNP.
    Figure 5: Automated RT-PCR testing for COVID-19 using the ASSIST PLUS pipetting robot together with a VOYAGER 8 channel adjustable tip spacing pipette, as performed in the Microbiology and Molecular Pathology Department at SNP.

    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.

    Figure 6: Example set-up of the 4 Position Portrait Deck when combining programs 1-COVID-19 and 2-COVID-19 in one program.
    Figure 6: Example set-up of the 4 Position Portrait Deck when combining programs 1-COVID-19 and 2-COVID-19 in one program.

    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.

    Conclusion

    • In the context of a global pandemic where laboratories are under increasing pressure to analyze more and more patient specimens to confirm COVID-19 cases, testing labs can rapidly benefit from the advantages of the ASSIST PLUS pipetting robot, allowing them to increase their sample processing capacity.
    • Pipetting results were reproducible, precise and accurate, with no contamination observed in controls or patient samples.
    • The ASSIST PLUS pipetting robot, together with the VOYAGER adjustable tip spacing pipette, increases sample processing capacity, reduces the need for manual intervention by laboratory personnel and fast tracks assay development for COVID-19 testing.
    • Low Retention GripTips and a low dead volume SureFlo Reagent Reservoir allow the use of costly reagents, such as One-step RT-PCR Mastermix, to be reduced.
    • The simple and fast ASSIST PLUS pipetting robot combined with the easy-to-use VIALAB software, offers immediate help for testing labs.
    • While the current protocol uses 384 well plates, it can be readily adapted to 96 well format to meet future needs.
    • Thanks to the VIALAB software, the pipetting programs can be easily adapted to any specific protocols and labware.

    April 2020, by

    • Jan-Peter Baldin, PhD

      Product Manager

      Email

    Any questions? I'm happy to help!

    • Stephanie Stursberg

      Product Manager

      Email

    Used instruments and accessories

    ASSIST PLUS, Pipetting Robot VOYAGER, 12 Channel, 50 µl GripTip, 125 µl, Sterile, Filter, Low retention Multichannel Reagent Reservoir, 10 ml, PS PCR 384 well cooling block GripTip, 125 µl, Sterile, Filter, Low retention Multichannel Reagent Reservoir, 10 ml, PS LightCycler® 480 Multiwell Plate 384, white FluidX 96-format 66-32034 FluidX 96-format 66-32033

    ASSIST PLUS, Pipetting Robot

    INTEGRA has developed the ASSIST PLUS pipetting robot to streamline routine pipetting tasks at an affordable price. Using INTEGRA electronic multichannel pipettes, the system:

    • automates pipetting tasks,
    • eliminates physical strain and
    • ensures superior reproducibility and
    • error free pipetting.

    Part No. 4505

    Visit product page
    ASSIST PLUS Pipetting Robot

    VOYAGER, 12 Channel, 50 µl

    VOYAGER pipettes allow the tip spacing to expand anywhere between 4.5 mm and 33 mm at the push of a button.

    • Single handed operation leaves the other hand free to handle labware.
    • On the fly access to up to 3 user-defined tip spacings.
    • These user defined tip spacings are saved. No need to memorize spacings of different labware formats.

    Part No. 4736

    See it work
    VOYAGER pipettes allow the tip spacing to expand anywhere between 4.5 mm and 33 mm at the push of a button.

    GripTip, 125 µl, Sterile, Filter, Low retention

    INTEGRA uses a unique polypropylene blend to mold Low Retention GripTips with heightened hydrophobic properties. Using Low Retention GripTips prevents low surface tension samples from spreading out and “wetting” the inner wall of the tips, allowing them to bead-up for a maximum liquid recovery.

    Part No. 6565

    Learn more about low retention pipette tips
     Low Retention GripTips are offered in 6000 series racks as non-sterile, sterile and filter options.

    Multichannel Reagent Reservoir, 10 ml, PS

    • SureFlo™ anti-sealing array prevents pipette tip seal off which can cause liquid to pop into the tip, filter or even the pipette itself
    • Unique surface treatment spreads liquid evenly offering even lower dead volumes
    • Reusable base with bold, crisp, clearly visible graduation markings
    • Nest inside each other, making it possible to get twice as many reservoirs in half the space of other products on the market
    • Reducing inventory space requirements and shipping costs
    • Available as crystal clear polystyrene reservoirs or polypropylene reservoirs for improved chemical compatibility
    Find out more about Multichannel Reagent Reservoirs
    Reagent reservoirs 10ml, 25ml, 100ml

    PCR 384 well cooling block

    Aluminium 96 and 384 well PCR cooling blocks that keep reagents chilled during sample preparation in a PCR plate. The cooling blocks feature a SBS footprint.

    Part No. 6255

    Find more labware accessories
    384-well PCR cooling plate

    GripTip, 125 µl, Sterile, Filter, Low retention

    INTEGRA uses a unique polypropylene blend to mold Low Retention GripTips with heightened hydrophobic properties. Using Low Retention GripTips prevents low surface tension samples from spreading out and “wetting” the inner wall of the tips, allowing them to bead-up for a maximum liquid recovery.

    Part No. 6565

    Learn more about low retention pipette tips
     Low Retention GripTips are offered in 6000 series racks as non-sterile, sterile and filter options.

    Multichannel Reagent Reservoir, 10 ml, PS

    • SureFlo™ anti-sealing array prevents pipette tip seal off which can cause liquid to pop into the tip, filter or even the pipette itself
    • Unique surface treatment spreads liquid evenly offering even lower dead volumes
    • Reusable base with bold, crisp, clearly visible graduation markings
    • Nest inside each other, making it possible to get twice as many reservoirs in half the space of other products on the market
    • Reducing inventory space requirements and shipping costs
    • Available as crystal clear polystyrene reservoirs or polypropylene reservoirs for improved chemical compatibility
    Find out more about Multichannel Reagent Reservoirs
    Reagent reservoirs 10ml, 25ml, 100ml

    LightCycler® 480 Multiwell Plate 384, white

    LightCycler® 480 Multiwell Plates 384, white are full-skirted, white polypropylene reaction devices supplied with a bar-coded label. Their dimensions (inner/outer well geometry) and material are designed to achieve optimized assay conditions on the LightCycler® 480 Instrument, 384-well for reaction volumes from 5 to 20 µl. 

    Source: Website Roche Molecular Systems

    Visit supplier website
    LightCycler® 480 Multiwell Plate 384

    FluidX 96-format 66-32034

    FluidX 96-format, 0.7ml Internal Thread, Non-coded Tube, V-bottom, uncapped, 10 racks per case. HighBase Rack.

    Source: Catalog Brooks Life Sciences

    Open Catalog

    FluidX 96-format 66-32033

    FluidX 96-format, 0.7 ml Internal, Thread, Non-coded Tube, V-Bottom, Uncapped.

    Source: Catalog Brooks Life Sciences

    Open Catalog

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    Download this App Note as PDF file

    Download PDFAutomated RT-PCR set-up for COVID-19 testing

    Customer’s voice

    Easy and flexible pipetting robot! The device is easy to use and therfore it saves us a lot of work. In addition, pipetting is more precise that manual pipetting and there is less risk of contamination. The software for making protocols is very inuitive and provides a lot of options, so the Assist can be easily used for different applications.

    Bianca Van Driel, Genmab