Rapid visual nucleic acid detection of Vibrio alginolyticus by RPA
combined with CRISPR/Cas13a
Vibrio alginolyticus ( V. alginolyticus) is a common pathogen that
infects humans and animals. In addition to causing serious economic
losses in aquaculture, it can also infect humans. The rapid detection of
nucleic acids of V. alginolyticus with high sensitivity and specificity
in the field is very important for the diagnosis and treatment of
infection caused by V. alginolyticus. Here, we established a simple,
fast and effective molecular method for the identification of V.
alginolyticus that does not rely on expensive instruments and
professionals. The method integrates RPA technology with CRISPR
technology in a single PCR tube. Using this method, the results can be
visualized by lateral flow dipstick in less than 50 minutes. The method
was confirmed to achieve high specificity for the detection of V.
alginolyticus with no cross-reactivity with similar Vibrio and common
clinical pathogens. This diagnostic method shows high sensitivity; the
detection limit of the RPA-CRISPR/Cas13a-LFD is 10 copies μL -1. The
results for 55 wild strains were consistent with TaqMan-qPCR, and it can
be concluded that the methods have 100% sensitivity and 100%
specificity. In conclusion, RPA-CRISPR/Cas13a offers great potential as
a useful tool for reliable and rapid diagnosis of V. alginolyticus
infection, especially in limited conditions.