. 2020 Feb 19;10(2):114. doi: 10.3390/diagnostics10020114

Table 3.

Comparison of detecting EGFR mutations with CRISPR-Cas12a and ddPCR.

Detecting Method	CRISPR-Cas12a			ddPCR
DNA Preparation	20 µL of liquid materials were diluted 1:3 in PBS containing 0.53% Triton X-100 to the final volume of 60 µL, and boiled to 95 °C for 6 min.			200 µL of liquid materials were extracted by using the Qiagen DNeasy^® Blood and Tissue kit per each manufacturer’s instruction, and the final volume was 20 µL. Total processing time was 30 min.
Amplification Signal	5 µLDNA was added into the final volume of 50 µL for PCR and total processing time was 1 h and 30 min.			5 µLDNA was added into the final volume of 20 µL for droplet formation and PCR. Total processing time was 2 h.
PCR Condition	Volume of DNA		5.0	Volume of DNA		5.0
	Nuclease-free water		15.2	Nuclease-free water		4.5
	10 M EGFR F primer		2.4	40× Taqman Liquid Biopsy dPCRAssay (Probe and primers)		0.5
	10 M EGFR R primer		2.4	2× ddPCR™ Supermix for Probes, No AmpErase UNG/ dUTP		10
	TaqMan 2× Universal PCR Master Mix, no AmpErase UNG		25	Total volume per reaction		20
	Total volume per reaction		50	Total volume per reaction		20
	95.0 °C	10 min	1 cycle	95.0 °C	10 min	1 cycle
	95.0 °C	10 sec	40–45 cycles	94.0 °C	30 sec	40 cycles
	54.7 °C	10 sec		60.0 °C	1 min	40 cycles
	72.0 °C	30 sec		98.0 °C	10 min	1 cycle
	72.0 °C	10 min	1 cycle	4.0 °C		1 cycle
	4.0 °C		1 cycle	4.0 °C		1 cycle
Volume of Detection	5 µL from a 50 of µL PCR mixture was added directly to the pre-assembled mixture in total 20 µL volumes for fluorescence detection.			Whole 20 µL of ddPCR mixture was applied for copy numbers detection
Pre-Assembled Mixture	250 nM of LbCas12a, 500 nM of targeting crRNA and 500 nM of ssDNA-FQ reporter.			No.
Specificity From	1. Primer pairs. 2. LbCas12a and targeting crRNA.			1. Primer pairs. 2. Taqman probe.
	Specific amplification of the product with specific CRISPR-Cas12a target binding.			Specific amplification of the product with labeling a target-specific hydrolysis probe.
	Specific CRISPR-Cas12a target binding activates collateral activity to unleash indiscriminate single-stranded DNA (ssDNA) cleavage activity and further amplify the detection signal.			If the target sequence is present, the probe anneals and is cleaved as the primer is extended. This cleavage of the probe separates the reporter dye from quencher dye, increasing the reporter dye signal.
Time for Detection	From 30 min up to 1 h.			At least 1h and 30 min.