Rapid visual nucleic acid detection of Vibrio alginolyticus by RPA combined with CRISPR/Cas13a

Abstract

Abstract 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.