| Vibrio parahaemolyticus(V.parahaemolyticus)and Vibrio vulnificus(V.vulnificus)are commonly known as foodborne pathogenic microorganisms that pose serious public health problems due to their ability to cause food poisoning incidents.Current detection methods for these pathogens,which include traditional culture-based methods,immunological assays,and nucleic acid detection methods,have limitations such as being time-consuming,expensive,having low sensitivity,and poor specificity.Recently,the CRISPR/Cas system,comprising clustered regularly interspaced short palindromic repeats(CRISPR)and CRISPR-associated proteins(Cas),has been increasingly applied to nucleic acid detection.The Cas 12a protein,guided by CRISPR-related RNA(crRNA),can selectively recognize and cleave target DNA and non-specifically cleave single-stranded DNA(ssDNA).Consequently,this study aims to combine recombinase aided amplification(RAA)technology and the CRISPR/Cas 12a detection system with fluorescent signal output and enzyme-catalyzed color signal output methods to develop a new detection approach for V.parahaemolyticus and V.vulnificus.Firstly,a fluorescent detection platform based on the CRISPR/Cas12a system was developed to detect the tlh,tdh,and trh genes of V.parahaemolyticus and the vvhA gene and 16S rDNA of V.vulnificus.The platform demonstrated high sensitivity,with a limit of detection of 1 CFU/reaction for V.parahaemolyticus and V.vulnificus bacterial solutions and 2×102 CFU/mL for V.parahaemolyticus and V.vulnificus in seafood samples.This assay overcame the limitations of traditional detection methods for these pathogens and reduced the detection time to only 60 minutes.This assay exhibited good sensitivity,specificity,and versatility,making it highly applicable for detection purposes.To address the limitations of the fluorescent detection platform in practical application scenarios,this research further developed a point-of-care testing(POCT)platform for V.vulnificus based on the CRISPR/Cas12a system,namely the ssDNA between β-galactosidase and sepharose beads-CRISPR(GBs-CRISPR)detection platform.The sensitivity and specificity of the GBs-CRISPR platform were comparable to those of the fluorescent detection platform,and the signal output was achieved through a color-catalyzed reaction,eliminating the demands of specialized operations or large instruments.The GBs-CRISPR platform was user-friendly,directly applicable,and suitable for POCT of foodborne pathogenic microorganisms,which is essential for food safety management and the prevention of foodborne diseases. |
