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. 2024 Feb 13;13(3):837–850. doi: 10.1021/acssynbio.3c00655

Figure 6.

Figure 6

CRISPRD-based multiplex detection of HPV16, HPV18, and RNase P. (a) Schematic of the triplex CRISPRD reaction using HheCas13a (targeting HPV18 and trans-cleaving FAM reporters), TccCas13a (targeting RNase P and trans-cleaving HEX reporters), and AapCas12b (targeting HPV16 and trans-cleaving ROX reporters). (b) Possible outcomes of the triplex reaction, demonstrating that the cross-reactivity of AapCas12b with TccCas13a reporters will not affect the practical application. (c) Proof of concept of the triplex reaction. One master mix containing all reagents for a triplex reaction was prepared without the targets. Targets were then added in a final concentration of 1 ng/μL to each tube, and the reaction was allowed to proceed for 1 h (d) LoD of the triplex reaction. One master mix containing all triplex reagents was prepared. RNase P concentration was fixed to 1000,000 copies per microliter. The two other targets (HPV16 and HPV18) were mixed in one tube from which multiple serial dilutions were made. The reaction proceeded for 1 h; n = 3. Normalized background-subtracted fluorescence: background fluorescence (from tubes containing no target) was subtracted from the fluorescence signal (tubes containing targets) and then normalized to the highest signal in the corresponding reporter.