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Establishment of a Rapid Detection Method for Vibrio parahaemolyticus Based on CRISPR-Cas13a Combined with Recombinase-Aided Amplification—Jiangsu Center for Disease Control and Prevention
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Abstract: Objective: To combine recombinase-mediated amplification (RAA) technology with the clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 13 (CRISPR-Cas13a) detection system, thereby establishing a rapid, sensitive, and specific method for detecting Vibrio parahaemolyticus (RAA-Cas13a). Methods: After RAA amplification of sample DNA, the resulting products were detected using the CRISPR-Cas13a detection system. The sensitivity of CRISPR-Cas13a was compared with that of agarose gel electrophoresis for detecting RAA products. The method was evaluated for its sensitivity and specificity, and its consistency with the real-time PCR method was assessed by analyzing clinical samples. Results: CRISPR-Cas13a demonstrated higher sensitivity in detecting RAA products than agarose gel electrophoresis. The established RAA-Cas13a method exhibited a detection sensitivity of 10 DNA molecules per reaction for Vibrio parahaemolyticus, with no cross-reactivity against other pathogens. There was no statistically significant difference in the positive detection rates between the RAA-Cas13a method and real-time PCR when testing clinical samples (P > 0.05), and the two methods showed high agreement (kappa = 0.934). Conclusion: The developed RAA-Cas13a method offers advantages such as rapidity, simplicity, sensitivity, and specificity, providing a new tool for the rapid detection of Vibrio parahaemolyticus.
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