Frontiers in Microbiology (Oct 2022)

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

  • Ziqin Lin,
  • Jinfang Lu,
  • Sihong Wu,
  • Xi Lin,
  • Laibao Zheng,
  • Yongliang Lou,
  • Xingxing Xiao

DOI
https://doi.org/10.3389/fmicb.2022.973996
Journal volume & issue
Vol. 13

Abstract

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Aeromonas hydrophila 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 A. hydrophila 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 A. hydrophila expressing aerA and/or hlyA virulence genes. The dRAA-CRISPR/Cas12a method has high sensitivity, which can rapidly detect (about 45 min) A. hydrophila with the limit of detection in 2 copies of genomic DNA per reaction, and has high specificity for three pathogenic A. hydrophila strains (aerA+hlyA−, aerA−hlyA+, and aerA+hlyA+). 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 A. hydrophila detection method, dRAA-CRISPR/Cas12a, is a promising potential method for the early diagnosis of human A. hydrophila infection and on-site detection of A. hydrophila in food and aquaculture.

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