Preparation Of Immunomagnetic Complex And The Application In Detection Of Vibrio Parahaemolyticus

ObjectiveVibrio parahaemolyticusa(V.parahaemolyticus)has been identified as a major food-borne pathogen throughout the world.Given the harmful effects and prevalence of V.parahaemolyticus,establishing a rapid,specific,and sensitive detection method for V.parahaemolyticus would be valuable to the seafood industry and improving public health.The existing detection methods are always limited by the complex food material and low content of pathogen.Pretreatment and pre-enrichment are requisite.The purpose of this study was to establish and optimize an efficient sample preconcentration method for V.parahaemolyticus and combine it with polymerase chain reaction technique(PCR)to establish a rapid detection system for V.parahaemolyticus.MethodWe prepared rabbit polyclonal antibody(IgG),chicken yolk antibody(IgY)and phage display polypeptide,which could specifically recognize Vibrio parahaemolyticus.They were coated on the surface of the magnetic beads(MBs)as biometric probesto form three different kinds of immunomagnetic complexes,which were used in immunomagnetic separation method(IMS).According to the amount of immunomagnetic beads,enrichment time and enrichment efficiency,the best immunomagnetic complex was evaluated.A rapid detection system for V.parahaemolyticus was established by combining the best IMS and PCR technique,and the detection system was evaluated.Results(1)We collected eggs from the SPF Laiheng laying hen which was immunized by inactive Vibrio parahemolyticus.The eggs before immunization were the negative group and the eggs after 8 months immunization were the positive group.We used polyethylene glycol precipitation method to exact IgY.The BCA method,Enzyme linked immunosorbent assay(ELISA)and SDS-PAGE was used to characterize.The results showed that the titer of IgY was 1:128000,the protein content of antibody was7.318 mg/mL and has high purity.It could specifically identify V.parahaemolyticus and couldn’t binding Enterococcus faecali、Enterobacter sakazakii、Enterobacter cloacae、Klebsiella、K.peneumoniae、Citrobacter braakii、Listeria monocytogenes、Escherichia coli、and Bacillus mirabilis.(2)The phage display-derived peptide,which could specifically bind to V.parahaemolyticus,was screened by bio-panning procedure.After three rounds 10phage clones were selected randomly.The phage recovery was calculated and the binding ability was detected by ELISA.The positive phage clones’DNA were extracted and sequenced.Combining with the results of prevalence and ELISA,the best phage display polypeptide with the strongest binding affinity to V.parahaemolyticus were selected and its specificity was detected by biomolecular interaction system.The phage biopanning experiment’s screening effect was good.The phage recovery rate increased gradually with the rounds of biopanning,10 phage clones were all positive,and phage clone C1’s ability was strongest.The sequencing analysis results and the ELISA results iddentied peptide V1 had the highest affinity for binding V.parahaemolyticus.V1 was synthesized and evaluated by the biological interaction system.It could specificly recognize of V.parahaemolyticus and couldn’t binding Escherichia coli、Serratia marcescens、Listeria monocytogenes and Klebsiella.(3)There three biomaterial were used to coated magnetic beads to product different IMBs,and the three different IMS were compared.As for the dosages,IgG-IMB needed 1.4 mg,IgY-IMB needed 1.2 mg and peptide V1–IMB needed only0.3 mg;As for the time-taken,both IgG-IMS and IgY-IMS needed 60 min,but peptide V1-IMS only needed 30 min;As for the capture effeiciencies,the results were 73.20%and 79.25%respectively for IgG-IMS and IgY-IMS.However it could 90.49%for peptide V1-IMS,which was the highest.In terms of the dosages,detection time and capture efficiency,the peptide has been testified to have absolute superiority to IgG and IgY in IMS method.(4)The peptide V1-IMS was the best and choosed to combine with PCR to eatablish detection method for V.parahaemolyticus.This system of peptide V1-IMB-PCR could detect V.parahaemolyticus with a limit of detection(LOD)of 10~2cfu/mL without pre-enrichment in 180 min.For the whole system,30 min was for magnetic sepraretion of the target bacteria and 150 min for PCR.ConclusionIn conclusion,based on high specificity,sensitivity technique peptide reported in this research using in IMS and combined with PCR,we developed an efficient and specific pretreatment method for food samples.The convenient and rapid detection method of Vibrio parahaemolyticus could provide another view for the development of detection of Vibrio parahaemolyticus and other foodborne pathogens.