3.3 Sensitivity test of LAMP coupled with CRISPR Cas12a
When combined the LAMP and CRISPR cleavage reaction, the sensitivity of
the LAMP-CRISPR reaction was determined with a 10-fold serial diluted
template with a concentration of 7× 106 copies/μl,7×
105 copies/μl,7× 104 copies/μl,7×
103 copies/μl,7× 102 copies/μl,7×
101 copies/μl and 7× 100 copies/μl
of the template, respectively. The results showed that the developed
LAMP-CRISPR system can detect up to 7× 101 copies/μl
of the dsDNA template (Figure 3). To further improve the sensitivity of
this assay, the proportion of the complex RNP formed by crRNA and Cas12a
was further optimized with 7× 101 copies/μl and 7×
100 copies/μl of the template. The results indicated
that when the ratio of crRNA to Cas12a is 1:1 (50nM:50nM), the
LAMP-CRISPR are able to detect 7× 100 copies/μl of the
template in the reaction, which is the detection limit of this method.
Moreover, 6 out of 10 target templates with 1 copies/μl can be detected
by the LAMP-CRISPR system.