1.3.5 CRISPR/CAST package sensitivity test
The IS711 positive plasmid concentration was quantified by
spectrophotometer, SimpliNano (GE Health Bio-Sciences (USA)). The copy
number of the plasmid was calculated by the following equation: Copy
number =
(M×6.022×1023)/(n×650×1×109), where
M is the amount of DNA in nanograms, n is the length of the plasmid in
the base pair, and the average weight of a base pair is 650 Daltons. The
plasmids gradient dilution was from 108 to
100 copies/μl. RPA sensitivity was measured by
RPA-Cas12a mentioned in 1.3.3, with each diluted concentration positive
plasmid as a template and RNase-free water as a negative control. The
Real-Time PCR system measured the CRISPR/CAST package sensitivity by
collecting fluorescent signals, and the nucleic acid test strip observed
the color changes with naked eyes. Each concentration had three
replicates.