AIEgens probes enhanced the sensitivity of CRISPR/Cas12a system for Listeria monocytogenes rapid detection
An enhanced fluorescent DNA probe based on aggregation-induced emission fluorogens(AIEgens)was devel-oped,and a new method for sensitive detection of Listeria monocytogenes(LM)was established based on recombinant polymer-ase amplification(RPA)combined with CRISPR/Cas12a.The AIEgens enhanced probe contained both single-stranded and doub-le-stranded DNA sequence,where a Black Hole Quencher group was labeled on the 5'end of probe,and the cationic AIEgens dramatically enhanced the fluorescent intensity of DNA probe via binding with the dsDNA.The fluorescent signal of the devel-oped probe was turned on by the trans-cleavage of Cas12a protein,which was trigged by the target DNA fragment of LM.Using the AIEgens enhanced probes as a signal output,the sensitivity of CRISPR/Cas12a for the dsDNA fragment was achieved at 0.37 pmol·L-1,40 times higher than that of traditional fluorescent reporter.Combined with recombinase polymerase amplifica-tion,the sensitivity of the proposed CRISPR/Cas12a system was achieved at 3×101 CFU·mL-1 for the LM determination.In-tra-and inter-assays were evaluated by determining the artificially contaminated LM milk and flammulina mushroom samples,and the average recoveries ranged from 91.14％to 108.47％with the coefficient of variation less than 12.71％,indicating an ac-cepted accuracy and precision.An AIEgens enhanced fluorescent DNA probe was constructed to improve the detection efficiency of CRISPR/Cas system,the proposed CRISPR/Cas system achieved highly sensitivity for the foodborne pathogenic detection.
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