Abstract: A fluorescent method based on polymerase chain reaction (PCR) and the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 12a (CRISPR/Cas12a) system was developed for the early and rapid detection of Cronobacter sakazakii. Specifically, PCR primers and CRISPR RNA (crRNA) guides were designed and screened based on the ompA gene of C. sakazakii. The sensitivity of the method was evaluated using serial concentrations of C. sakazakii genomic DNA. In addition, the specificity was assessed using Staphylococcus aureus, Salmonella, Enterobacter cloacae, Escherichia coli, Bacillus cereus, and Listeria monocytogenes. Furthermore, quantitative PCR (qPCR) was used to evaluate the accuracy of the fluorescent method for detecting C. sakazakii in milk and infant formula samples. The fluorescent method demonstrated a linear range from 102 to 108 CFU/mL (R2 = 0.995), with a limit of detection as low as 1 CFU/mL. This method exhibited high specificity and no cross-reactivity with any of the non-target bacterial species. Moreover, recoveries from milk and infant formula samples spiked at 104, 105 and 106 CFU/mL ranged from 89.51%­ to 104.71%. In conclusion, the CRISPR/Cas12a-based fluorescence method is highly sensitive and specific, providing a new reference for developing rapid and sensitive detection methods for C. sakazakii in dairy products.

Key words: Cronobacter sakazakii; clustered regularly interspaced short palindromic repeats/CRISPR-associated protein; polymerase chain reaction; fluorescence detection; dairy products