The CRISPR–Cas12a system is a powerful tool for nucleic acid diagnostics, yet its canonical requirement for a protospacer adjacent motif (PAM) limits target selection for mutation detection. While strategies like heat-denaturation or artificial PAM insertion have been proposed to bypass this constraint, they add procedural complexity or restrict assay design. In this study, we demonstrate that loop-mediated isothermal amplification (LAMP) amplicons enable robust, PAM-independent Cas12a activation without additional processing. Using rifampin resistance-associated SNPs in the Mycobacterium tuberculosis rpoB gene as a model, we compared Cas12a activity triggered by LAMP and PCR products. While linear double-stranded DNA from PCR failed to activate Cas12a without a PAM, LAMP amplicons consistently induced strong trans-cleavage signals under identical conditions. Mutation discrimination analysis confirmed that specificity is preserved, with clear differentiation achieved when mismatches were positioned within seven nucleotides of the guide-proximal region. Collectively, LAMP–Cas12a format expands the practical design space of CRISPR–Cas12a diagnostics and offers a streamlined approach for the molecular detection of clinically relevant single nucleotide variants.