key: cord-0942314-hy4pqbe4 authors: Haile, Simon; Nikiforuk, Aidan M.; Pandoh, Pawan K.; Twa, David D.W.; Smailus, Duane E.; Nguyen, Jason; Pleasance, Stephen; Wong, Angus; Zhao, Yongjun; Eisler, Diane; Moksa, Michelle; Cao, Qi; Wong, Marcus; Su, Edmund; Krzywinski, Martin; Nelson, Jessica; Mungall, Andrew J.; Tsang, Frankie; Prentice, Leah M.; Jassem, Agatha; Manges, Amee R.; Jones, Steven J.M.; Coope, Robin J.; Prystajecky, Natalie; Marra, Marco A.; Krajden, Mel; Hirst, Martin title: Optimization of magnetic bead-based nucleic acid extraction for SARS-CoV-2 testing using readily available reagents date: 2021-10-20 journal: J Virol Methods DOI: 10.1016/j.jviromet.2021.114339 sha: aac6e5f48b7c0abd4a28ce67346191d29e05a88e doc_id: 942314 cord_uid: hy4pqbe4 The COVID-19 pandemic has highlighted the need for generic reagents and flexible systems in diagnostic testing. Magnetic bead-based nucleic acid extraction protocols using 96-well plates on open liquid handlers are readily amenable to meet this need. Here, one such approach is rigorously optimized to minimize cross-well contamination while maintaining sensitivity. The COVID-19 pandemic has placed unprecedented strain on instrument and consumable supply chains for SARS-CoV-2 nucleic acid (NA) testing (1) . NA protocols involve lysis and purification of NAs on columns or magnetic beads. Bead-based protocols are amenable to automated workflows and are widely available, rendering them attractive alternatives to proprietary commercial offerings (2) . Recent reports have suggested that generic bead-based protocols can be successfully deployed on generic open-deck liquid handling instruments (3) (4) (5) ; yet lack rigorous measures of specificity and sensitivity that are required for clinical deployment. Step-by-step protocols are described in the supplementary file. We sought to establish an automated protocol to support extraction-based SARS-CoV-2 NA testing using generic reagents, automated on an open deck Hamilton NIMBUS96 liquid handler. We benchmarked this against an existing clinical NA testing workflow in place at the Figure 1A) . The SOP is designed to accept specimens aliquoted into a plate from a variety of commercial transport mediums (Copan UTM, Hologic STM, Roche cobas® PCR Media, YOCON UTM) or common laboratory buffers. Cultured Influenza A virus (Flu-A) spiked into transport medium was used for initial comparisons between the NIMBUS and MagMax protocols. Following extraction, Flu-A RNA recovery was measured using a TaqMan qRT-PCR assay, developed by BCCDC, that detects To measure specificity of our protocol we deployed a "checkerboard" input plate where Copan UTM containing Flu-A was alternated with Copan UTM alone. This test revealed a ~85% specificity; carryover into blank wells was judged to be an aggregate effect of the manual processes performed in the biosafety cabinet (BSC) and automated liquid handling. To decouple these sources of contamination, we utilized synthetic DNA (g-block) controls and matched primers and probe sets. A master checkerboard plate was first generated by aliquoting an amount of g-block DNA sufficient for an extraction-free control plate and three extraction test plates. Using a dedicated NIMBUS, the master plate was aliquoted into a control plate containing elution buffer only and into a deep-well plate that was pre-loaded with a mixture of Copan UTM, RLT Plus, beads, and isopropanol to mimic the extraction chemical milieu (extraction plates), respectively. The samples from two of the extraction plates were then purified using a second NIMBUS and those from the third extraction plate were purified on a third NIMBUS. As shown in Figure 1 , all the 40 blank wells in the control plate were determined to be negative via the qPCR assay (i.e. undetermined Ct values) (100% specificity). In contrast, the three extraction plates displayed 17, 13 and 12 false positive wells, representing 65% specificity. To improve specificity, we switched from 1.2mL to 2.2 mL deep-well plates, reduced tip mixing steps and number of washes and optimized pipetting techniques to eliminate residual droplets. We wrote new code to eliminate extraneous vertical movement of the robot head between aspirate and dispense steps and to reduce the robot gantry's lateral speed to prevent Figure 2B ). Having established comparable sensitivity of our optimized NIMBUS protocol to that of the initial version of the NIMBUS and the MagMax protocols, we next tested its specificity. We performed three independent experiments using g-block checkerboards, each including two extraction-free control plates and two extraction test plates (total of 240 positive wells and negative wells). None of the blank wells had detectable NA and all wells with g-block DNA yielded expected Ct values indicating 100% sensitivity and specificity. A Flu-A checkerboard was performed and again achieved 100% sensitivity and specificity ( Figure 2C ). contamination assessment Workflow is shown in the upper panel. This assay is designed to decouple the manual upstream BSC steps from the steps on the liquid handler (in this case a NIMBUS). A synthetic DNA fragment (g-block) is used as a starting material and a master gblock checkerboard plate is manually generated. The g-block DNA is aliquoted into a plate that contains elution buffer (control plate) and a deep-well plate that was pre-loaded with a mixture of Copan UTM, RLT Plus, beads, and isopropanol (extraction plates), respectively, using a devoted liquid handler. The samples from the extraction plates are then purified on separate liquid handlers. The g-block DNA eluates from the extraction plates and diluted g-block DNA from the control plates are subsequently used as templates in the same run of qPCR (lower panel). Step To enable extraction of total nucleic acids from nasopharyngeal swab samples for testing and other applications related to SARS-CoV-2 and Influenza A/B. Document Title Document Number Agencourt RNAdvance Tissue Protocol (Note: ALINE EvoPure RNA Isolation Kit uses the same protocol as Agencourt RNAdvance Tissue Protocol). 000473v003 All Laboratory Safety procedures will be complied with during this procedure. The required personal protective equipment includes a laboratory coat and gloves. For chemical safety, see safety data sheet (SDS). 1.9. Use appropriate positive and negative controls. The workflow of the extraction process is depicted below. The upstream part of the process (in blue; steps 1-3) is performed within a class II Biological Safety Cabinet (BSC). The downstream steps are performed on the Microlab NIMBUS (Hamilton) liquid handling platform (in grey; steps 4-7). The upstream process begins with transferring a portion of the sample from the Copan UTM or Hologic STM tubes into a 2.2 mL KingFisher Deep 96-well plate. Qiagen's RLT Plus lysis buffer is then added to the samples. This step serves to lyse the sample and contributes to viral inactivation. Subsequently, a mixture of magnetic beads in isopropanol is added to the lysate to capture of nucleic acid from the lysate on the magnetic beads for subsequent purification and the isopropanol facilitates the second part of inactivating residual (non-lysed) virus. Following these steps within the BSC, the plate with inactivated virus is taken to the NIMBUS deck outside the BSC as outlined. 3.1. Before beginning the extraction protocol: 3.9. Shake the prepared bead bind mix in the Falcon tube(s), pour into a reagent trough, and immediately transfer 400 µL to each well using a multichannel pipette one column at a time carefully without causing any splashing. Do not mix. Change tips between columns. Double check the plate to ensure that the bead mix is added to all wells and ensure the top edge of the wells is dry and clean. Seal the plate with a clear tape. Wipe the plate with bleach, incubate 10 minutes inside BSC, and wipe with 70% ethanol. Carefully transport the plate(s) to the NIMBUS workstation. 4.6. The NIMBUS allows the processing of either 1 or 2 plates at time; you will be prompted to choose. 4.7. Follow prompts on the NIMBUS for the rest of the procedure. Note that there will be two layers of the deck layout that the NIMBUS will specify. 4.8. Note that this is not a walk-away program; users will need to attend to tip needs, movement of plates to save tips, and other aspects as prompted by the NIMBUS. 4.9. Following elution of the total nucleic acids, seal the destination plate and perform a quick spin. For temporary storage, cover with clear tape seal and place the plate on ice. For longer term storage, use heavy foil tape and store at -80°C. Manual total extraction of total nucleic acid from nasopharyngeal swab specimen Integrated DNA and RNA extraction using magnetic beads from viral pathogens causing acute respiratory infections Overcoming the bottleneck to widespread testing: a rapid review of nucleic acid testing approaches for COVID-19 detection SARS-CoV-2 RNA Extraction Using Magnetic Beads for Rapid Large-Scale Testing by RT-qPCR and RT-LAMP Before beginning the extraction protocol: 1.1. Retrieve sufficient 70% Ethanol (EtOH) from 4°C and to warm up to room temperature Using a multi-channel pipettor Mix 5 times at 96% of the total volume of lysate and bead binding mix Place the sample plate onto a Magnum FLX magnet plate for 10 minutes minimum or longer if needed until the supernatant has cleared Dispense 70% EtOH into a fresh V-bottomed sterile solution basin. With the plate on the magnet, add 600 µL of 70% EtOH to each sample. Allow beads to resettle for 2 minutes Repeat the EtOH wash, for a total of 2 EtOH washes During the incubation, dispense Qiangen's EB buffer into a fresh fresh V-bottomed sterile solution basin Move the plate back onto the magnet without the spacer on for 2 minutes to clear the supernatant Transfer the supernatant (containing the total nucleic acids) to an AB1000 96-well plate, the final destination plate Customized Nimbus Code as well as .STEP or .SLT files of the plate spacer described below are available upon request 1. Mix 5x at 775 µL Incubate at room temp for 5 minutes Place on Magnum FLX magnet plate for 10 minutes Pipette off and discard all 800 µL supernatant (over-aspirate volume 820 µL is 20 µL more than the volume in the wells) With the plate on the magnet, add 600 µL of 70% EtOH. Allow beads to resettle for 2 minutes. Remove all EtOH with an over-aspirate of 613 µL Move the plate off the magnet Allow to dry for 10 minutes Pour EB buffer into a low profile reservoir and place on deck as prompted. The robot will prompt you to remove the waste plate and place this reservoir on the deck Move the plate to the magnet without spacer and clear for 2 minutes Transfer the 40 µL supernatant (containing the total nucleic acids) to an AB1000 96-well plate, the final destination plate, using an over-aspirate volume of 50 µL For temporary storage, seal with a clear tape seal and place the plate on ice. For longer term storage, use heavy foil tape and store at -80°C The method runs without an on-deck shaker so we modified beadelution to accommodate the altered shape and increased surface area of the bottom of the wells. Specifically, the elution volume (40 µL) did not cover the bead ring formed around the inner wall of the well Open lab (Nimbus) 3. 5x Mixing and bead binding 4.