A Rapid and Visual Method for Nucleic Acid Detection of Escherichia coli O157:H7 Based on CRISPR/Cas12a-PMNT

Abstract

Rapid, accurate and visual point-of-care testing (POCT) methods for pathogenic bacteria detection are essential for avoiding foodborne diseases caused by pathogens or their toxins. In this study, we proposed a rapid and visual detection method that we named “Cas12aVIP”. By combining recombinase polymerase amplification (RPA), a CRISPR/Cas12a system and a cationic-conjugated polythiophene derivative (poly[3-(3′-N,N,N-triethylamino-1′-propyloxy)-4-methyl-2,5-thiophene hydrochloride] (PMNT) mixed with single-stranded DNA (ssDNA)), the solution turned red in the absence of the target DNA based on conformational modifications of the conjugated backbone of PMNT, whereas it displayed yellow, thus realizing the colorimetric detection of DNA. The Cas12aVIP method yielded high specificity and no interference from other nontargeted bacteria. The detection was accomplished in 40 min and the signal could be observed by the naked eye under natural light, presenting great potential for a variety of rapid nucleic acid detection applications without requiring technical expertise or ancillary equipment.

Sensing, Imaging, and Therapeutic Strategies Endowing by Conjugate Polymers for Precision Medicine

“…Recently, a CRISPR/Cas12a‐assisted colorimetric method based on CPs was developed for the detection of pathogens. [ 89 ] Pathogenic bacterial dsDNA was amplified by the recombinase polymerase amplification (RPA) reaction, and then DNA was cleaved via reaction with Cas12a. Finally, PMNT was added to the reaction solution and mixed with lysed DNA.…”

Polyphenols and CRISPR as Quorum Quenching Agents in Antibiotic-Resistant Foodborne Human Pathogens (Salmonella Typhimurium, Campylobacter jejuni and Escherichia coli 0157:H7)

“…They finally reported that the Cas12aVIP method produced high specificity and did not interfere with other non-target bacteria. This method ensures that the detection was performed within 40 min and that the signal can be observed with the naked eye under natural light, which presents great potential for multiple applications of rapid detection of nucleic acids without the need for technical expertise or auxiliary equipment [90].…”