Development And Analysis Of A Technical System For Rapid Detection Of Vibrio Parahaemolyticus By Aptamer-Nano-Gold Colorimetry

Oligonucleotides,are also known as aptamers,which are small pieces of ss DNA or RNA molecule.Their traditional screening method is Systematic evolution of ligands by exponential enrichment(SELEX),which can specifically and tightly bind to the corresponding ligands.Aptamers have the advantages of wide targets,high affinity,and strong specificity.They are widely used in basic research,proteomics research,clinical diagnosis and drug therapy.However,there are still relatively few researches in the field of food-borne pathogen detection.This study takes the food-borne pathogen Vibrio parahaemolyticus(VP)as the research object,and is based on the X-Aptamer screening kit to obtain high affinity,highly specific binding aptamer.Using it as a recognition element,combined with nanoparticles to construct a specific,sensitive,rapid,visual detection method of VP.It provides new ideas and new methods for enriching and improving the detection technology of food-borne pathogens.VP is a common marine halophilic food-borne pathogen,mainly found in high-salt foods such as seafood and pickled vegetables.Eating foods infected with VP may cause gastrointestinal reactions such as diarrhea,headache,vomiting,nausea and abdominal cramps.The purpose of this study is to screen for specific recognitions of VP with high affinity.First,X-aptamer screening kit is used to quickly screen the aptamers of VP.The first round of negative screening uses the counter-screening bacteria Staphylococcus aureus to release a large sample of enriched VP aptamers from the microsphere library;eliminate"false positive"aptamers through the second round of positive screening;design a second pull-down screening to exclude non-specific aptamers;aptamers sequence are amplified by PCR,the sequence and abundance of the aptamers are obtained by agarose gel electrophoresis and high-throughput sequencing methods.Export aptamers sequence through cloud intelligent software,synthesize and modify them.Based on QGRS software and real-time PCR technology,the screened aptamers and VP are specifically analyzed,and aptamer ID12 with the highest specificity is selected.Secondly,in order to further obtain the binding site of Aptamer ID12 and VP,extract the outer membrane protein of VP,and further qualitative proteome by SDS-PAGE and the whole protein Label-free scientific analysis,we obtain 66 kinds of outer membrane protein information.P51002 outer membrane protein is outer membrane protein K(OmpK),a conservative sequence of VP,and its COG description shows‘Nucleoside-binding outer membrane protein'.P51002 three-dimensional model is obtained by Modeller 9.20 Structure:Barrel-shaped protein.The center of the protein is a hydrophobic cavity composed of?sheets;The secondary structure of aptamer ID12 is predicted by Mfold server,and the bases T modified by W,Y,X are simulated by DS viewer,the optimal scoring conformation is selected,and the three-dimensional structure of aptamer ID12 is constructed through RNA composer:it is in a looped state,partially similar Hairpin structure,different from DNA double-stranded,is a reverse double helix structure;The aptamer ID12 and P51002 are docked through the ZDOCK program to obtain important binding sites:the action sites on the outer membrane protein P51002 are:Phe182,Tyr183,Phe184,Asn187,Ser189;The action sites involved in the interaction of aptamer ID12 are:W19,Y20,T37,G38,C39,G40,A41;The interaction forces are hydrophobic,van der Waals,hydrogen bonding,and electrostatic.Among them,the W19 and Y20 of aptamer ID12 are closer to Tyr183 and Phe184 of outer membrane protein P51002.The hydrophobic effect is dominant.The research results show that the modified aptamer is easier to bind to the target and the binding site is the outer membrane protein K,which has positive significance for further elucidating the binding mechanism of the aptamer and the target bacteria.Finally,the wine-red nano-gold of specific size was prepared by chemical reduction method,and the experimental results were characterized by UV-vis absorption spectrum and transmission electron microscope(TEM).Due to the negative charge on the surface of the gold nanoparticles,the gold particles maintain the stability of the gold nanoparticles through electrostatic repulsion.Add high concentration of salt to the reaction system,the reaction system to destroy the stability of the gold nanoparticles,resulting in the condensation of the gold nanoparticles from red to blue;when the aptamer is present,the aptamer is directly adsorbed to the gold surface through its exposed positively charged bases in the Free State.Nano-gold adsorbed with the aptamer can still be maintained with a high concentration of salt solution,and the solution is still red.Based on the above principles,this study simulates the different pollution of VP in common seafood.Place the liquid sample of seafood contaminated with a certain number of bacteria,nano-gold solution and a certain concentration of aptamer in a 96-well plate.Adding a high-concentration salt solution to the above system,the liquid sample of seafood containing different pollution degrees shows different color changes.This method is called aptamer-nano-gold colorimetry.The results show that with trisodium citrate as the reducing agent,the additive amount of trisodium citrate was 10 m L.In the case of no protective agent,the diameter of gold nanoparticles ranged from 13 to 17 nm when heated at 100?for10 min.Under these preparation parameters,the gold nanoparticles had good dispersibility and uniform size.There is an optimal chromogenic concentration between Aptamer ID12 and VP(1×10~8CFU/m L),the color change is most obvious when the aptamer is 100 nmol/L.The control group is selected to contain Salmonella,Listeria monocytogenes and Escherichia coli.Aptamer ID12 could specifically bind to VP,only the color changes occurred in the reaction system species containing VP.The lowest detection limit of Aptamer-nano-gold colorimetric method for VP is OD 0.1(1×10~4CFU/m L),and the highest detection limit is OD 1.2(1×10~9CFU/m L).The best color development time is in 5 min.In conclusion,this paper screens the food-borne pathogenic bacterium VP aptamer ID12.Using its high affinity and specificity with the target,combined with gold nanoparticles,a sensitive,rapid and visual detection method is constructed.It provides a good technical support and a theoretical basis for the new technology of detecting food-borne pathogens based on new biosensors.