Study On SHERLOCK Detection Method Of Pathogenic Vibrio Parahaemolyticus

Vibrio parahaemolyticus is a foodborne pathogen found in shellfish and other marine products,widely distributed in coastal and marine waters,and can lead to gastroenteritis,wound infection,septicemia and other diseases.The foodborne pathogen contaminants caused by Vibrio parahaemolyticus occurs often,leading to nausea,diarrhea and death in severe cases.Studies have shown that these symptoms are mainly caused by the tlh、tdh、trh which are virulence factors of Vibrio parahaemolyticus,and are also important targets for the detection of Vibrio parahaemolyticus.Methods such as PCR,LAMP,ELISA techniques e.t.c have been used to detect virulence factors of Vibrio parahaemolyticus.This study is based on SHERLOCK detection method developed by Crispr-Cas13a system,and aimed at tlh、tdh、trh、tox R which are four specific genes of pathogenic Vibrio parahaemolyticus,studied a novel rapid,highly sensitive and simple operation detection method.The main results are summarized below:（1）Construction of standard plasmid for SHERLOCK detection reaction system.Three specific target genes tlh、tdh、tox R of Vibrio parahaemolyticus were designed and amplified through PCR technique using a Vibrio parahaemolyticus standard strain（RIMD2210633）as a template to transform into T-Vector p MD19 vector for verification and sequencing.After sequencing,the amplified fragments of tlh、tdh、tox R gene were compared with the corresponding chromosome sequences in RIMD2210633 strains.The similarities were 100%,99.5%and 99.9%,respectively.The single base mutation site of tdh and tox R gene amplification fragment is not close to the target sequence,and the detection process is not affected.The standard plasmids for trh gene detection were synthesized,and all four plasmids were used for the construction of subsequent detection reaction systems.（2）Expression and purification of SUMO proteases and Cas13a,Cas12g proteins to provide raw proteins for subsequent SHERLOCK detection and multiple detection systems.After optimizing the temperature,time and IPTG concentration of each protein,the culture was expanded for further expression.According to the isoelectric point of each protein and its own characteristics,the relevant buffers of different components and p H were selected,high purity target protein was obtained by affinity chromatography,ion exchange chromatography and gel filtration chromatography.（3）Through RPA reaction testing and primer verification,selection and detecting target sites,design and preparation of cr RNA,verification of Lw Cas13a cleavage activity,selection of reagents and fluorescent probes for detection of reactions,SHERLOCK detection reaction system of Vibrio parahaemolyticus was created,while improving the reaction efficiency and reducing the reaction cost.The specificity,sensitivity and the rapid detection time of Vibrio parahaemolyticus were tested,the seawater samples were also verified.The sensitivity of tlh,tdh,trh and tox R gene detection rates reached 100,100,1,1 copies/μl,respectively.At the nucleic acid level,tlh、tdh、trh、tox R genes of Vibrio parahaemolyticus were detected efficiently.Using plasmid sample of the genes with a minimum detection limit,the fastest detection time was determined.The rapid detection time of tdh gene was 10 min,trh genes was20 min,while tlh and tox R genes was 30 min,respectively.All four genes were detected within 30 min.In addition,the detection sensitivity of tlh genes in actual samples reached 10^-1 CFU/m L,which is higher than the national detection standards of Vibrio parahaemolyticus based on q PCR and RPA（10²CFU/m L,10³ CFU/m L）.The research results show that the SHELROCK detection method for Vibrio parahaemolyticus has been established successfully,the target genes of Vibrio parahaemolyticus tlh,tdh,trh and tox R,detection sensitivity is higher than the national standard and relative safety regulations.It has the potential for further development of products.