Screening And Application Of Highly Specific Aptamers Against Vibrio Parahaemolyticus

Vibrio parahaemolyticus,as a typical foodbome pathogen,is widely found in Marine products in coastal areas,and food poisoning cases caused by the use of food contaminated with Vibrio parahaemolyticus occur every year in all provinces of China.Therefore,the establishment of an efficient,rapid and specific detection method of V.parahaemolyticus is the focus of current research.Based on the urgency of the current situation,we have developed a specific method for the detection of V.parahaemolyticus food contamination,which uses innovative tools and materials,compared with the classical and up-to-date methods,it has high specificity and short time,and is very suitable for rapid detection in the field.The main findings are as follows:1.We developed an advanced cell-SELEX strategy featuring functionalized graphene oxide(GO)and isothermal rolling circle amplification(RCA)to select aptamers against a prevailing foodborne pathogen,Vibrio parahaemolyticus.Polyethyleneglycol(PEG)and chitosan(CTS)were grafted onto the sheet-like GO molecules to synthesize a PC-GO material.Functionalized graphene oxide can enhance the solubility of the buffer,thereby adsorbing non-specific ssDNA to improve the accuracy of screening.Two-round isothermal amplification is a new method for SELEX,which can greatly shorten the selecting time and improve the efficiency.TEM and FT-IR characterization demonstrated that the PC-GO composites were near-nanometric scale and could bind with ssDNAs with lower affinities to target cells,therefore the selection efficiency is greatly enhanced.The cell-binding aptamer candidates(CACs)were amplified by 107 fold using complementary ring mediated(CRM-RCA),a created amplification method that generate single-stranded products,which could be directly used in the next round selection.2.By combining the established amplification method with a new cell-SELEX method with functional graphene oxide,we were able to determine a set of reasonable and smooth aptamer screening procedures.From this we obtained four aptamers highly target to V.parahaemolyticus,and tested the specificity and affinity of these aptamers.The specific results showed that the four aptamers were highly compatible with V.narahaemolvticus.but the binding ability to other similar foodborne pathogens was weak,which indicated that the screening results were good.The Kd value analysis showed that the four aptamers had high affinity to the target and could form the corresponding secondary structure,of which Apt-2 had the highest affinity.3.And then,a biosensor using two aptamers(Dual-Apt)and cut-assisted rolling circle amplification(CA-RCA)for rapid and visualized detection of V.parahaemolyticus was established.The anchoring aptamer(A-Apt)that specifically binds to the surface of V.parahaemolyticus was applied to separate and enrich the bacterium from the food matrix with the help of streptavidin magnetic beads.While the detecting aptamer(D-Apt),binding on the different sites of the cell surface,was used as a signal reporter.CA-RCA with an enhanced amplification rate was fabricated here to amplify the D-Apt to produce the monomeric G4 sequence that catalyzes the oxidation of ABTS2-,resulting in the coloration visible to the naked eye.Free from DNA extraction,visualized signal output and no need for expensive instruments enable Dual-Apt and CA-RCA to be a promising strategy for on-spot rapid detection.After optimizing the experimental conditions and the concentration of the reagent,the method can reach the standard result,and the specificity of the V.parahaemolyticus is high,and the target can be detected accurately and sensitively,and it can be used in the detection of real food samples,the results are consistent with the classical plate culture counting method and PCR method.