A novel detection method for the pathogenic Aeromonas hydrophila expressing aerA gene and/or hlyA gene based on dualplex RAA and CRISPR/Cas12a

Abstract

<i>Aeromonas hydrophila</i> is an emerging waterborne and foodborne pathogen with pathogenicity to humans and warm water fishes, which severely threatens human health, food safety and aquaculture. A novel method for the rapid, accurate, and sensitive detection of pathogenic <i>A. hydrophila</i> is still needed to reduce the impact on human health and aquaculture. In this work, we developed a rapid, accurate, sensitive, and visual detection method (dRAA-CRISPR/Cas12a), without elaborate instruments, integrating the dualplex recombinase-assisted amplification (dRAA) assay and CRISPR/Cas12a system to detect pathogenic <i>A. hydrophila</i> expressing <i>aerA</i> and/or <i>hlyA</i> virulence genes. The dRAA-CRISPR/Cas12a method has high sensitivity, which can rapidly detect (about 45 min) <i>A. hydrophila</i> with the limit of detection in 2 copies of genomic DNA per reaction, and has high specificity for three pathogenic <i>A. hydrophila</i> strains (<i>aerA⁺hlyA^-</i> , <i>aerA^-hlyA⁺</i> , and <i>aerA⁺hlyA⁺</i> ). Moreover, dRAA-CRISPR/Cas12a method shows satisfactory practicability in the analysis of the spiked human blood and stool and fish samples. These results demonstrate that our developed pathogenic <i>A. hydrophila</i> detection method, dRAA-CRISPR/Cas12a, is a promising potential method for the early diagnosis of human <i>A. hydrophila</i> infection and on-site detection of <i>A. hydrophila</i> in food and aquaculture.