Abstract
Vibrio alginolyticus (V. alginolyticus) is a common pathogen in the ocean. 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 recombinase polymerase amplification (RPA) technology with CRISPR system in a single PCR tube. Using this method, the results can be visualized by lateral flow dipstick (LFD) in less than 50 min, we named this method RPA-CRISPR/Cas13a-LFD. 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. We successfully identified 35 V. alginolyticus strains from a total of 55 different bacterial isolates and confirmed their identity by (Matrix-assisted laser desorption ionization time-of-flight mass spectrometry, MALDI-TOF MS). We also applied this method on infected mice blood, and the results were both easily and rapidly obtained. In conclusion, RPA-CRISPR/Cas13a-LFD offers great potential as a useful tool for reliable and rapid diagnosis of V. alginolyticus infection, especially in limited conditions.
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The datasets generated during the current study are available from the corresponding author on reasonable request.
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This work was supported by the National Natural Science Foundation of China [No.81401311].
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Experimental protocol designed was performed by YW. Animal experiments was completed by YW, YH and XL. Experimental procedure was optimized by YH and XL. Environmental Vibrio alginolyticus strains identified were by NL and YD. Nucleic acid extracted from environmental Vibrio alginolyticus strains were by FL, WX and YZ. WX, JC, and CC supervised the project. YW wrote the main manuscript text. All authors reviewed the manuscript.
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Wang, Y., Hou, Y., Liu, X. et al. Rapid visual nucleic acid detection of Vibrio alginolyticus by recombinase polymerase amplification combined with CRISPR/Cas13a. World J Microbiol Biotechnol 40, 51 (2024). https://doi.org/10.1007/s11274-023-03847-2
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DOI: https://doi.org/10.1007/s11274-023-03847-2