CRISPRD-based
sensitive and specific detection of synthetic HPV16
and HPV18 DNA. (a) LoD for HPV16 with primer set C′ and gRNA
#99 with AapCas12b. (b) Specificity of the CRISPRD reaction for HPV16
detection. The reaction mixture was incubated with 1 ng of HPV16,
HPV18, gDNA, or RNase P (single target detection reactions). (c) HPV16
synthetic DNA fragments were spiked into 100 ng/μL gDNA to a
final concentration of 1 ng/μL. (d) LoD for HPV18 using primer
set F and gRNA #92. (e) Specificity of the CRISPRD reaction was determined
for HPV18 detection. The reaction was incubated with 1 ng of HPV16,
HPV18, gDNA, or RNase P (single target detection reactions). (f) HPV18
synthetic DNA was spiked into 100 ng/μL gDNA to a final concentration
of 1 ng/μL. (g) LoD for RNase P with primer set Pop7 and gRNA
#99 with TccCas13a. (h) Specificity of the CRISPRD reaction for RNase
P detection. The reaction mixture was incubated with 1 ng of HPV16,
HPV18, or RNase P (single target detection reactions). (i) Detection
of RNase P in gDNA: 10 ng of gDNA was incubated in the CRISPRD reaction
for RNase P detection. NTC: No-template control, RFU: relative fluorescence
units, Cycle: 2 min (two-tailed Student t test; n.s.,
not significant; *P < 0.05 1;**P < 0.01; ***P < 0.001; ***P < 0.0001; All plots show mean ± SD for n = 3 replicates).