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. 2022 Jan:299:114339.
doi: 10.1016/j.jviromet.2021.114339. Epub 2021 Oct 20.

Optimization of magnetic bead-based nucleic acid extraction for SARS-CoV-2 testing using readily available reagents

Simon Haile  1 , Aidan M Nikiforuk  2 , Pawan K Pandoh  1 , David D W Twa  3 , Duane E Smailus  1 , Jason Nguyen  4 , Stephen Pleasance  1 , Angus Wong  1 , Yongjun Zhao  1 , Diane Eisler  4 , Michelle Moksa  5 , Qi Cao  5 , Marcus Wong  5 , Edmund Su  5 , Martin Krzywinski  1 , Jessica Nelson  1 , Andrew J Mungall  1 , Frankie Tsang  4 , Leah M Prentice  6 , Agatha Jassem  7 , Amee R Manges  8 , Steven J M Jones  9 , Robin J Coope  1 , Natalie Prystajecky  7 , Marco A Marra  9 , Mel Krajden  7 , Martin Hirst  10
Affiliations

Optimization of magnetic bead-based nucleic acid extraction for SARS-CoV-2 testing using readily available reagents

Simon Haile et al. J Virol Methods. 2022 Jan.

Abstract

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.

Keywords: COVID-19; Contamination; Cross-well; Hamilton NIMBUS; Magnetic beads; Nucleic acid extraction; RNA; SARS-CoV-2.

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Conflict of interest statement

MK has received grants/contracts from Roche, Hologic and Siemens paid to British Columbia Centre for Disease Control Public Health Laboratory unrelated to this work.

Figures

Fig. 1
Fig. 1
Assessment of cross-well contamination associated with a liquid handler. The 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 g-block 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).
Fig. 2
Fig. 2
Specificity and sensitivity of the optimized NIMBUS-based protocol. (A) Workflow of the optimized NIMBUS-based protocol. Modifications included: removing the manual mixing steps in the BSC (He et al., 2017), performing all NIMBUS steps in 2.2 mL plates instead of 1.2 mL (1-8); removing the mixing step following addition of the wash buffer on NIMBUS (Klein et al., 2020), reducing the number of ethanol washes (Klein et al., 2020), and reducing the number of mixing in the elution step (7). The other changes that were implemented are summarized in Supplementary Figs. 2-3. The protocol is expected to capture host/cellular RNA and gDNA as well as viral RNA which constitute the viral genome for RNA viruses such as SARS-CoV-2. (B) Comparison of sensitivity of the NIMBUS protocol with that of the MagMax. One to five log dilutions of Flu-A virus stocks were spiked into Copan UTM and yield was measured via qRT-PCR. p = 0.000015 (paired, two-tailed t-test; n=4 for each of the dilutions except 10^5 NIMBUS; n=3) error bars=standard deviations. (C) Assessment of cross-well contamination levels associated with the NIMBUS protocol. Checkerboard pattern was set-up with alternating wells of Flu-A virus that was spiked into Copan UTM and Copan UTM without Flu-A virus. UNT = undetected. (D) Comparison of the improved NIMBUS-based protocol with the MagMax protocol using Covid-19 samples. A heatmap on Ct values obtained from qRT-PCR measurements for both the RDRP and E-gene targets is shown. These data are from two independent experiments.
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