Innovation
Focusing on the research and development and application of isothermal nucleic acid amplification technology
CRISPR - Cas13a 结合重组酶介导的扩增快速检测副溶血性弧菌方法的建立-江苏疾控
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Abstract: Objective To combine the recombinase-aided amplification (RAA) technology with the CRISPR-Cas13a detection system—specifically, the clustered regularly interspaced short palindromic repeats and associated protein 13a—to develop a rapid, sensitive, and highly specific method for detecting Vibrio parahaemolyticus (RAA-Cas13a). Methods After RAA amplification of sample DNA, the resulting product was analyzed using the CRISPR-Cas13a detection system. The sensitivity of CRISPR-Cas13a in detecting RAA products was compared with that of agarose gel electrophoresis. The method’s sensitivity and specificity were evaluated, and its performance was validated by comparing it with the real-time PCR assay using clinical samples. Results showed that CRISPR-Cas13a exhibited superior sensitivity for detecting RAA products compared to agarose gel electrophoresis. The established RAA-Cas13a method achieved a detection limit of 10 DNA copies per reaction for Vibrio parahaemolyticus, with no cross-reactivity observed against other pathogens. Furthermore, there was no statistically significant difference in the positive detection rates between RAA-Cas13a and real-time PCR when analyzing clinical samples (P > 0.05), and the two methods demonstrated high concordance (kappa = 0.934). Conclusion The developed RAA-Cas13a method offers several advantages, including speed, simplicity, high sensitivity, and excellent specificity, providing a new and efficient tool for the rapid detection of Vibrio parahaemolyticus.
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