Background: Swift and accurate detection of , which is a prominent causative pathogen associated with seafood contamination, is required to effectively combat foodborne disease and wound infections. The gene is relatively conserved within and is primarily involved in the expression and regulation of virulence genes with a notable degree of specificity. The aim of this study was to develop a rapid, simple, and constant temperature detection method for in clinical and nonspecialized laboratory settings.
Methods: In this study, specific primers and CRISPR RNA were used to target the gene to construct a reaction system that combines recombinase polymerase amplification (RPA) with CRISPR‒Cas13a. The whole-genome DNA of the sample was extracted by self-prepared sodium dodecyl sulphate (SDS) nucleic acid rapid extraction reagent, and visual interpretation of the detection results was performed by lateral flow dipsticks (LFDs).
Results: The specificity of the RPA-CRISPR/Cas13a-LFD method was validated using strain ATCC-17802 and six other non-parahaemolytic species. The results demonstrated a specificity of 100%. Additionally, the genomic DNA of was serially diluted and analysed, with a minimum detectable limit of 1 copy/µL for this method, which was greater than that of the TaqMan-qPCR method (10 copies/µL). The established methods were successfully applied to detect wild-type , yielding results consistent with those of TaqMan-qPCR and MALDI-TOF MS mass spectrometry identification. Finally, the established RPA-CRISPR/Cas13a-LFD method was applied to whole blood specimens from mice infected with , and the detection rate of by this method was consistent with that of the conventional PCR method.
Conclusions: In this study, we describe an RPA-CRISPR/Cas13a detection method that specifically targets the gene and offers advantages such as simplicity, rapidity, high specificity, and visual interpretation. This method serves as a valuable tool for the prompt detection of in nonspecialized laboratory settings.
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| http://dx.doi.org/10.1016/j.imj.2024.100111 | DOI Listing |
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