Study On Detection Of Vibrio Parahaemolyticus In Seafood By Fluorescence Sensor Based On SRCA-CRISPR/Cas12a

Vibrio parahemolyticus(V.parahaemolyticus)as a food-borne pathogen is ubiquitously found in various seafood.In the coastal areas of China,V parahaemolyticus is the major pathogen of bacterial foodborne diseases.Food poisoning incidents caused by V.parahaemolyticus have been increasing gradually.Human consumption of raw or undercooked seafood contaminated by V.parahaemolyticus may cause gastrointestinal diseases and even death in severe cases.Therefore,the development of a rapid and sensitive detection method for V.parahaemolyticus has important practical significance for ensuring food safety.Saltatory rolling circle amplification(SRCA)does not require the preparation of circular templates,it only needs a pair of primers to complete the amplification of linear DNA under isothermal conditions.It is a new type of nucleic acid isothermal amplification technology.The CRISPR/Cas12a system can target double-stranded DNA(dsDNA)under the guidance of CRISPR RNA(crRNA)to produce non-specific cleavage activity for single-stranded DNA(ssDNA).It shows a broad application prospect in the field of nucleic acid detection.Combining the CRISPR/Cas12a system with nucleic acid amplification technology can improve the sensitivity and specificity of detection.In this study,a fluorescent sensor was constructed based on the SRCA reaction and the CRISPR/Cas 12a system for detecting V.parahaemolyticus.The main contents and results of this research are summarized as follows:The toxR gene of V.parahaemolyticus was used to screen the region containing protospacer adjacent motif(PAM),the SRCA reaction primer was designed according to this region,and the crRNA was designed according to the amplified sequence.Design a ssDNA probe labeled with a fluorescent group(FAM)at 5’ end and a quenching group(BHQ1)at 3’ end as a fluorescent reporter.Then the fluorescence sensor based on SRCA reaction and CRISPR/Cas 12a system was established.The optimal reaction system of the fluorescence sensor was determined by optimizing the SRCA reaction conditions and the Cas12a cleavage reaction conditions.The results showed that the optimal temperature for SRCA reaction was 62℃ the optimal addition amount of dNTPs was 4 μL,the optimal concentration of Mg2+was 20 mM,the optimal concentration of ssDNA probe was 1.5 μM,the optimal concentration ratio of Cas12a and crRNA was 1:1,and the optimal time for Cas12a reaction was 15 min.The fluorescence sensor was used to detect different concentrations of V.parahaemolyticus genomic DNA,the results showed that the fluorescence signal change value and the logarithm of the genomic DNA concentration of V.parahaemolyticus showed a good linear relationship in the concentration range of 66.8 fg/μL～6.68 ng/μL.The linear equation is y=2.6054x-0.3549(R2=0.9837),and the detection limit is 2.2 fg/μL.The fluorescence sensor was used to detect different dilutions of pure V.parahaemolyticus liquid,the results showed that the fluorescence signal change value and the logarithm of the number of colonies of V.parahaemolyticus showed a good linear relationship in the concentration range of 1.7×101～1.7×107 CFU/mL.The linear equation is y=1.8472x+2.4738(R2=0.9866),and the detection limit is 4 CFU/mL.The fluorescence sensor was used to detect V.parahaemolyticus and non-V.parahaemolyticus,V.parahaemolyticus showed a positive result and non-V.parahaemolyticus showed a negative result.The results showed that the fluorescence sensor has excellent specificity.Shrimp and oyster samples artificially contaminated with V.parahaemolyticus were used for the spike test,and the results showed that the recovery rate of artificially contaminated samples was between 95.5 and 104.4%.The national standard(GB 4789.7-2013)method and the fluorescence sensor were used to detect 36 seafood samples,and the two methods were compared.It is calculated that the relative sensitivity of the fluorescence sensor is 100.0%,the relative specificity is 95.2%,and the relative coincidence rate is 97.2%.Therefore,this study combined the SRCA amplification reaction and the CRISPR/Cas12a system to establish a fluorescence sensor for detection of V.parahaemolyticus.The fluorescence sensor in this study has the advantages of high sensitivity and excellent specificity.It provides a new strategy for the rapid quantitative detection of V.parahaemolyticus,and provides a new idea for the detection of other food-borne pathogens.It has important practical significance and reference value for ensuring food safety.