LAMP-CRISPR-Cas12-based diagnostic platform for detection of Mycobacterium tuberculosis complex using real-time...

A CRISPR-based nucleic acid detection platform, termed LACD (loop-mediated isothermal amplification coupled with CRISPR-Cas12a-mediated diagnostic) has been developed. In the LACD system, the core primer used in conventional LAMP (forward inner primer or backward inner primer) was engineered to contain a PAM (protospacer adjacent motif) site (TTTT) at the linker region. As a result, the LAMP amplicons contained a specific PAM site for CRISPR-Cas12a recognition. At the CRISPR-mediated detection stage, the resulting LAMP products can activate the corresponding CRISPR-Cas12a effector upon the formation of the CRISPR-Cas12a/gRNA/target DNA complex. The single-strand DNA (ssDNA) reporter molecules are then rapidly cleaved due to the CRISPR-Cas12a's trans-enzyme activity. The ssDNA degradation can then be visualized on a lateral flow biosensor or measured  by a real-time fluorescence instrument. Our LACD assay allows any target sequence to be detected (even targets which do not contain any PAM sites) as long as they met the design requirement for LAMP. The feasibility of the LACD methodology for nucleic acid detection was validated on the Mycobacterium tuberculosis complex (MTC). This proof-of-concept assay can be reconfigured to detect a variety of target sequences by redesigning the engineered LAMP primers.