Loop-mediated isothermal nucleic acid amplification coupled with CRISPR-Cas12b for rapid and sensitive detection of SARS-CoV-2 and Nipah virus

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Kielich, Dominic Miguel Simard
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SARS-CoV-2 and Nipah virus are two zoonotic viruses responsible for global and local outbreaks of high consequence, respectively, putting a strain on capacity for rapid testing and diagnostic testing. Developed by Joung et al. 2020, STOPCovid.v2 combines chemical RNA extraction with loop-mediated isothermal amplification and Cas12b-mediated detection, capable of detecting SARS-CoV-2 nucleic acid. However, the assay sensitivity is not as good as the gold standard RT-qPCR. Moreover, an assay of this sort does not exist to detect Nipah virus. In this investigation, various RT-LAMP and Cas12b optimization strategies, based on extensive literature reviews, were employed to examine their effects on the assay to potentially improve kinetic and detection performance. It was found that substituting Tween-20 with Brij-35 in the reaction buffer, increasing the MgSO4 concentration, and adding dithiothreitol to the reaction only slightly improved assay characteristics. A rapid enrichment-extraction protocol using minimal commercially available reagents and resources was developed herein. Beyond this, a previously published Nipah virus RT-LAMP primer set combined with an in-house-designed sgRNA was developed and found to detect the Nipah virus N gene, improving the sensitivity 2000-fold compared to the published colorimetric RT-LAMP assay. These findings show that combining previously developed isothermal amplification with CRISPR-based detection can improve pathogen detection, paving the way for developing CRISPR-based point-of-care diagnostics.
SARS-CoV-2, Diagnostics, Molecular Diagnostics, CRISPR, Loop-mediated isothermal amplification, Nipah virus