Research On Detection Method Of Vibrio Parahaemolyticus Based On CRISPR/Cas Protein

Vibrio parahaemolyticus is a halophilic,gram-negative bacterium,widely distributed in water bodies,underwater sediments,and aquatic organisms.It frequently causes food poisoning around the world,mostly in coastal areas in summer,and is a global food-borne disease.An important pathogen.In recent years,the incidence of food poisoning due to V.parahaemolyticus has increased year by year.The main pathogenic factors of V.parahaemolyticus are Thermolabile hemolysin(TDH)and Thermostable direct hemolysin-related hemolysin(TRH).V.parahaemolyticus infection can mainly cause gastroenteritis and wound infection,and in rare cases can lead to sepsis,gastroenteritis caused by V.parahaemolyticus is an important public health event,and the rapid detection of V.parahaemolyticus is to deal with these the premise of a public health event.CRISPR(clustered regularly interspaced short palindromic repeats)is an acquired immune system that exists in archaea and bacteria,and plays an important role in preventing the invasion of foreign pathogens by bacteria and archaea.The CRISPR immune function involves the identification and digestion of specific nucleic acid fragments,which play a role in pathogen detection.CRISPR detection system requires Cas(crispr associate protein),gRNA(guide RNA)and target sequence(target).The purpose of this study is to establish a CRISPR/Cas based rapid detection method for V.parahaemolyticus,including the extraction and purification of Cas protein,the design and extraction of gRNA,and the design and detection of a fluorescent colorimetric system.In this study,Cas12a protein was selected.First,the expression sequence of Cas 12a protein was obtained by searching in NCBI,synthesized and constructed into an expression vector,transferred into competent cells for expression under suitable conditions,and then extracted and purified.The second part is the design of gRNA.Through the comparison analysis of the existing specific genes of V.parahaemolyticus tdh,trh and the specific gene of vibrio toxR,we selected the most conservative,specific and length 20-30 bp,combined with Cas12a,needs to specifically recognize PAM(prospacer adjacent motif),that is5'-TTTA-3',construct the sequence on a vector for in vitro transcription and extract gRNA.The last part is to connect the chromogenic group FAM and the quenching group BHQ I to the two ends of the single-stranded DNA.When V.parahaemolyticus is present,the CRISPR-VP system will cut the target sequence and will also develop fluorescent color The system's single-stranded DNA cuts off and releases a fluorescent signal,which makes it more intuitive for the detection of Vparahaemolyticus.In the experiment,the components were matched in proportion to the CRISPR-VP reaction system,and the Roche fluorescence PCR instrument was used to detect the fluorescent signal at a constant temperature of 37?,and the identification of V.parahaemolyticus was realized by the fluorescence value and the fluorescence curve.The CRISPR-VP detection method initially established in this study is based on the CRISPR system-specific shearing principle,which makes the method highly specific and sensitive.At the same time,the application of the fluorescent color rendering system makes the judgment of results more intuitive and overcomes traditional detection The existence of human error makes the detection result more accurate.