Establishment of CRISPR/Cas12a System Combined with Recombinase Polymerase Amplification Method for Detection of Listeria monocytogenes and Vibrio parahaemolyticus
This study established a RPA-CRISPR/Cas12a method for detecting Listeria monocytogenes and Vibrio parahaemolyticus.Primers and CRISPR RNA(crRNA)were designed and synthesized based on the hly A gene sequence of L.monocytogenes and the tlh gene sequence of V.parahaemolyticus,respectively.Fluorescently labeled ssDNA probes were synthesized to establish the RPA-CRISPR/Cas12a detection method.The specificity and sensitivity of the established method were tested,and the method was verified by artificial contaminated samples.The results showed that the established RPA-CRISPR/Cas12a method could specifically detect the target genes of Listeria monocytogenes and Vibrio parahaemolyticus without cross-reaction with common pathogens including Escherichia coli,Staphylococcus aureus,Salmonella and Listeria eiderii.Sensitivity test results showed that for plasmid standards,the detection limit for Vibrio parahaemolyticus was 27 copies/μL,and for Listeria monocytogenes,it was 21 copies/μL.Artificially contaminated sample detection results showed that the method could accurately and rapidly detect Listeria monocytogenes and Vibrio parahaemolyticus in samples.The method has good specificity,high sensitivity,does not require a temperature cycling device,and can achieve rapid detection on-site and improved detection efficiency.
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