A Rapid and Visual Method for Nucleic Acid Detection of <i>Escherichia coli</i> 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.

Keywords

• foodborne pathogenic bacteria
• <i>Escherichia coli</i> O157:H7
• nucleic acid detection
• POCT
• visual method
• CRISPR-Cas12a