The Nucleic Acid Detection Method Based On CRISPR-Cas13a System Applied To The Detection Of Salmonella

Food safety is an important public health issue that is deeply concerned about in the world,and diseases caused by food-borne pathogens are a continuing threat to public health.Salmonella is one of the most common food-borne pathogens,and the Salmonella infection may cause diseases such as typhoid fever,acute gastroenteritis and bacteremia.The detection and identification of Salmonella in food plays an important role in preventing foodborne diseases.At present,the gold standard for the detection of Salmonella in various countries is the traditional culture detection method.This detection method is stable and reliable but often requires more than 4 days of detection time,and it is complicated and depends on the operator.Therefore,the development of a detection method that can quickly and accurately detect Salmonella is of great significance to the detection of Salmonella in food.Now many researchers are committed to developing a sensitive and specific molecular biology detection method to detect Salmonella.The CRISPR system is a prokaryotic immune system against exogenous genetic material.The Cas13a protein belongs to the type II CRISPR-Cas system and is one of the effectors of the CRISPR system.Cas13a protein can target specific RNA sequences after combining with CRISPR RNA(cr RNA).After the Cas13a-cr RNA complex binds to the target RNA,the RNase activity of Cas13a protein is activated.The special RNase activity of Cas13a protein can be used in molecular biology detection.In this study,high-purity Cas13a protein was obtained by inducing expression and protein purification.After that,the fluorescent reporter was reacted with Cas13a protein to verify its RNase activity,and PCR was combined with Cas13a protein reaction to establish a nucleic acid detection method.The detection method was used to detect Salmonella,and the application of this detection method in the detection of different serotypes of Salmonella was also explored.The Cas13a protein was expressed in Escherichia coli by introducing the plasmid.After purification by nickel ion affinity chromatography and dialysis,the Cas13a protein with a concentration of 332?g/m L was obtained,and the purity of protein was 93.6%.Target RNA,cr RNA and fluorescent reporters were used to verify the RNase activity of the Cas13a protein.After optimization of Cas13a protein reaction system,the detection limit is 10~1 p M of target RNA.A nucleic acid detection method was established by combining PCR with the Cas13a protein reaction,and the inv A gene was used as the detection target and the Salmonella was detected by this method.The detection limit of Salmonella genome was 10~1 a M,and no fluorescence signal was detected in the non-Salmonella genomes,indicating that the detection method has good specificity.The Salmonella bacterial suspension was heated and detected by the established detection method.The results showed that the method can detect Salmonella at a concentration of 10~0 CFU/m L.The detection also has the same sensitivity in milk and juice samples.Combined with the Salmonella serogroup-specific detection target,the PCR-Cas13a detection method was used to detect Salmonella of different serogroups.The results showed that the detection has good specificity and can accurately detect Salmonella in different serogroups.Multiplex PCR was used instead of PCR to detect different Salmonella,and the results still showed good specificity.In this study,a nucleic acid detection method based on the Cas13a protein was established and it was applied to the detection of Salmonella.Through experiments,we have proved that this method is sensitive and specific.It not only can be applied to the detection of Salmonella in food,but can be used to detect Salmonella in different serogroups which combined with specific targets.This has a positive significance for the prevention,control and research of Salmonella.