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| Development of a Rapid Detection Method for Listeria monocytogenes Based on Recombinase Polymerase Amplification Combined with CRISPR-Cas12a Technology |
| CHEN Dawei, LI Bingbing, WEI Mingyue, LI Shuangshu, YANG Pengfei, LIU Liang |
| Huai’an Center for Disease Control and Prevention, Jiangsu Provincial Key Laboratory for Food Safety Risk Monitoring (Pathogenic Bacteria), Huai’an 223003, China |
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Abstract Objective: To develop a rapid detection method for Listeria monocytogenes based on clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 12a (Cas12a) combined with recombinase polymerase amplification (RPA). Methods: The virulence gene hly (GeneID: 987033) of L. monocytogenes was selected to design RPA primers, and a two-step rapid detection kit for L. monocytogenes was developed using RPA combined with CRISPR-Cas12a. The sensitivity, specificity, and reaction rate of the kit were analyzed. Results: This method was rapid and sensitive; it could detect L. monocytogenes within 30 minutes, and the minimum level of the target nucleic acid of 0.0015 ng was detected using 20 μL of the system. Furthermore, when this kit was applied to detect artificially contaminated salmon fillets, the limit of detection was 10 CFU/mL, and the results for 30 actual samples were consistent with the positive detection rate obtained by fluorescence quantitative PCR. Conclusion: RPA combined with CRISPR-Cas12a is a feasible method for rapid detection of foodborne L. monocytogenes.
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2023, Vol. 37 
