Preparation Of Bcac And Application In Rapid Detection Of Salmonella In Foods

Salmonella is a common foodborne pathogen.Vegetables,eggs and meat products are the common targets of infection.Risks of food safety caused by salmonella are becoming more prominent.It is important for food safety to detect salmonella rapidly.Among them,in the process of detecting foodborne pathogens by molecular biological methods,there are many inhibitors that affect the sensitivity of detection methods due to the complex components in food.Preliminary studies of the project team showed that bentontite coated activated carbon(BCAC)can adsorb and remove the inhibitors in food effectively and improve the detection sensitivity of molecular biological methods.However,the characteristics and mechanism of BCAC are still unclear.This study focused on the preparation,characterization and adsorption mechanism of BCAC,and applied BCAC pretreatment to food samples to improve the sensitivity of Rti-LAMP.In this study,the materials of AC,propotion of bentontite and AC,adsorption time were optimized.Then the adsorption kinetics of BCAC were studied by adsorbed with indigo carmine,tryptophan,thiamine and tannic acid.The characteristics of BCAC were studied by four characterization methods:SEM,FTIR,Raman spectroscopy and zero charge point.The results showed that the BCAC was prepared under the condition of AC with particle diameter of 1.25-2 mm and the ratio of bentontite to AC was 1:4,adsorption was 15 min.The bacterial recovery rate was up to 94.0±8.9%and the inhibitor adsorption rate was up to 73.3±0.8%.The adsorption rate for IC can be more appropriately described using the pseudo-second order rate.The data suggested that adsorption of IC by BCAC was mainly chemical adsorption by sharing or exchanging electrons with functional groups on the surface of BCAC.The adsorption process of tryptophan was better fitting the pseudo-first order model and mainly affected by single factor.The adsorption process of thiamine by BCAC is not applicable to two adsorption models.The adsorption effect of tannic acid is poor.The characterization results showed that bentontite did not change the chemical properties of activated carbon.The reason why BCAC could retain most of the target bacteria might be that both bentonite and bacteria were negatively charged,producing mutually exclusive effects.BCAC combined with Rti-LAMP was used to detect salmonella in vegetables.This study designed specific primers for the inva gene sequence of Salmonella,25 g of vegetables as a sample,add different concentrations of salmonella to the surface of the vegetables,each 25 m L of eluent was treated with 4 g of BCAC.After centrifugation,the precipitation was suspended with 0.5 m L 1×TZ lysate,which was mixed and heated in a metal bath at 99.5℃for 10 min and centrifuged at 12000 g for 5 min.The supernatant was then used as DNA template for Rti-LAMP detection.The sensitivity of BCAC-Rti-LAMP to detect Salmonella in lettuce and bean sprout was up to 2 CFU/g,which was 10 times higher than that of Rti-LAMP.In addition,the detection limit of BCAC-Rti-LAMP for salmonella contaminated bean sprouts was up to 2 CFU/g,indicating that BCAC could effectively adsorb inhibitors in vegetables and improve the detection sensitivity.Finally,salmonella on the surface of egg shell was detected by BCAC-Rti-LAMP method.After elution for 1 min with normal saline,the precipitation was resuspened by 25m L normal saline,4 g of BCAC was added and treated in a shaking table at 150 rpm for 15min.DNA template was extracted for detection.The sensitivity of both Rti-LAMP method and BCAC-Rti-LAMP method reached 2 CFU/cm~2.Presumably the eggshells used in the experiment were relatively clean,BCAC was not required.Although there were a little of inhibitors on the surface of the egg shell,the detection process could not identify the dead and alive bacteria.The samples were treated with 4μg/m L of EMA.After dark reaction for5 min,the samples were illuminated under a halogen lamp for 15 min.The results showed that EMA-Rti-LAMP method could effectively distinguish living and dead cells.The sensitivity of detection of living cells and detection of living and dead cells is 2 CFU/reaction,and the ratio of living cells to dead cells is 2:1998/reaction.The sensitivity of EMA-Rti-LAMP was 10 CFU/cm~2 for the detection of live and dead cell contaminated eggs.The detection limit of artificially VBNC salmonella contaminated eggs was up to 4CFU/cm~2.