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[Preprint]. 2020 Oct 20:2020.10.14.20212795. [Version 2] doi: 10.1101/2020.10.14.20212795

Figure 2. SARS-CoV-2 detection by SENSR via Fluorescence and Lateral Flow assay.

Figure 2.

[A] Cleavage properties of E-targeting gRNA-T in fluorescence context. Fluorescence detection of E-gene in an IVT-coupled coupled context. gRNA-T incubated in three different contexts: with E-gene template, with N-gene template, or with both E- and N-gene template. Accumulation of fluorescence occurs when gRNA-T is incubated with the E-gene target template leading to cleavage of the ssRNA probe. Collateral cleavage is not observed when gRNA-T is incubated with the non-target N-gene template. [B] Cleavage properties of N-targeting gRNA-Z in fluorescence context. Fluorescence detection of N-gene in an IVT-coupled coupled context. gRNA-Z incubated in three different contexts: with N-gene template, with E-gene template, or with both N- and E-gene template. Accumulation of fluorescence occurs when gRNA-Z is incubated with the N-gene target template leading to cleavage of the ssRNA probe. Collateral cleavage is not observed when gRNA-Z is incubated with the non-target E-gene template. [C] CasRx nucleic acid limit of detection (LOD) for gRNA-Z detection via fluorescence following cleavage of N-gene target. Total copy number of 10000, 1000, 100, 10, or 0 of viral RNA template input into initial RT-RPA reaction, followed by CasRx detection by fluorescence. Performing a one-way ANOVA followed by a Dunnett’s test call copy numbers to the NTC significance was found for 10000, 1000, and 100 copies (p<0.0001) and no significance was found for 10 copies (p=0.9999). Results shown as background-subtracted fluorescence (A.U.) following 90 minute fluorescent readout (4 technical replicates each, Mean and SD). [D] CasRx nucleic acid LOD for gRNA-T detection via fluorescence following cleavage of E-gene target. Total copy number of 10000, 1000, 100, 10, or 0 of viral RNA template input into initial RT-RPA reaction, followed by CasRx detection by fluorescence. Performing a one-way ANOVA followed by a Dunnett’s test call copy numbers to the NTC significance was found for 10000, 1000, and 100 copies (p<0.0001) and no significance was found for 10 copies (p>0.9999). Results shown as background-subtracted fluorescence (A.U.) following 90 minute fluorescent readout (4 technical replicates each, Mean and SD). [E] LOD of the gRNA-Z recognition of N-gene target by lateral flow assay. CasRx detection reaction incubated for 1h prior to lateral flow assay. [F] LOD of the gRNA-T recognition of E-gene target by lateral flow assay. CasRx detection reaction incubated for 1h prior to lateral flow assay.