Establishment And Evaluation Of Visual Detection Method For Listeria Monocytogenes

Listeria monocytogenes is a common food-borne pathogen.In recent years,food safety incidents caused by this bacteria have occurred frequently,which seriously threatens human health and social-economic development.The bacterium infects humans and animals to cause listeriosis.The clinical symptoms are encephalitis,meningitis,sepsis,etc.,and the mortality rate is high.Therefore,the rapid and accurate detection of on-site monitoring and sampling has become one of the hot issues in the field of public health detection.At present,the laboratory detection methods of Listeria monocytogenes mainly include traditional biochemical identification methods,immunological methods and molecular biological methods.All of which have long time-consuming,low sensitivity,high requirements for experimental conditions.Therefore,the development of a simple,rapid and sensitive detection method for Listeria monocytogenes is of great significance for the prevention and control of listeriosis.The research of this subject is based on the structural properties and pathogenic mechanism of Listeria monocytogenes,combining molecular biology technology and nanotechnology to construct a rapid,sensitive and simple visual detection method for Listeria monocytogenes in food.Provide new ideas and new methods for environmental biotoxin research,and provide technical support for on-site testing of food safety.The main research contents include the following four parts:Part Ⅰ Preparation and identification of Listeria monocytogenes chicken egg yolk antibody IgY（1）The Freund’s complete adjuvant vaccine and the Freund’s incomplete adjuvant vaccine were separately prepared by inoculating the Listeria monocytogenes inactivated suspension.The vaccination was inoculated under the skin of the chicken chest by subcutaneous multi-point injection.The eggs were collected before and after immunization.The yolk antibody IgY was extracted by polyethylene glycol precipitation method.IgY was separated and purified by gel filtration chromatography and identified purity by polyacrylamide gel electrophoresis（SDS-PAGE）.The results showed that the IgY before purification showed multiple bands outside the target chain.The purified IgY only showed the target chain,indicating that the purified IgY was of higher purity.（2）The BCA protein quantification kit was used to determine the egg yolk antibody protein content.The content of IgY extracted ranged from 5.51 to 22.89 mg mL^-1,and the highest protein content of the extracted IgY was 22.89 mg mL^-1 at the 15th week.（3）The titer and specificity of IgY antibody were determined by routine indirect ELISA.The results showed that the antibody titer increased gradually with the increase of immunization time.At the 9th week,the antibody titer reached the maximum,namely 1:512000,then stabilized and decreased after the 15th week,but still remained at a high level.The specificity results showed that the prepared IgY of yolk antibody did not cross-react with five common food-borne pathogenic bacteria and had high specificity.Part Ⅱ Research on visual detection method of Listeria monocytogenes based on OPD-AuNPs detection system（1）Fe₃O₄ magnetic nanoparticles were synthesized by solvothermal method,Listeria monocytogenes aptamer labeled immunomagnetic nanoprobes were prepared as capture probes,BSA as template,nanoenzyme IgY-BSA-AgNCs immunoprobes were prepared as"signal transducers"of detection system,and AuNPs were prepared by sodium citrate reduction method,which was used as a"coloring signal"of the detection system.Based on OPD-AuNPs reaction and IgY-BSA-AgNCs mediated catalytic oxidation of OPD,a visual detection system for Listeria monocytogenes was established.The results show that when the capture probe is 0.6 mg,the capture efficiency was as high as 96.1%,10.0μg mL^-1 for IgY-BSA-AgNCs,and the concentration of OPD is 40 nM,the detection system achieves the best results.（2）The effect of the detection system was evaluated.The sensitivity results showed that the detection time for Listeria monocytogenes was 40minutes,the visual detection limit was 1×10 CFU mL^-1,The linear fitting curve established by UV absorption value at A525,A525 nm=0.102 lgC+1.014,R²=0.998.The results showed that the established detection method had a good linear relationship.Specificity results showed that there was no cross-reaction between the detection system and other four common foodborne pathogens,and the specificity was strong.The results of food simulation samples showed that the visual detection limit was 5×10 CFU mL^-1,and the linear fitting curve was A525 nm=0.144 lgC+0.726,R²=0.914.The results showed that the food matrix had little interference to the detection method and could be used to detect target bacteria sensitively in food matrix.The standardized recovery experiment showed that the recovery rate of the detection system was 97.4%.-101.3%and RSD less than 10%.It shows that the detection system established in this experiment has strong stability and good precision.Compared with similar colorimetric detection methods,the detection system has the advantages of high efficiency enrichment and rapid and sensitive detection.Part Ⅲ Research on visual detection method of Listeria monocytogenes based on CB7-AuNPs detection system（1）In this part,Listeria monocytogenes aptamer and urease double-labeled immunomagnetic nanoprobe was prepared,which as capture probes for detection system.AuNPs were used as the"signal"for detection system.A visual detection system for Listeria monocytogenes was established based on CB7-AuNPs self-assembly and urease hydrolysis urea reaction.The results showed that when the immunomagnetic nanoprobe was 0.5 mg,the enrichment efficiency of the target bacteria was 90.9%,and the detection system was the best when the concentration of0.86μM CB7 was 10μL.（2）To evaluate the effect of establishing visual detection system for Listeria monocytogenes.The results showed that the visual detection limit for Listeria monocytogenes was 1×10 CFU mL^-1 and the detection time was 50 min.The data were processed by A700/A525 ratio.The linear regression curve was y=0.0903 lgC+0.06798,R²=0.986 in the concentration range of 1×10-1×10⁶ CFU mL^-1.The results showed that the detection method established in this experiment had good linear relationship.the specific results show that the detection system will cause the target bacteria and other four common food-borne causes.Simultaneous detection of pathogens showed positive results only when the target bacteria were present,and the specificity was strong.Food simulation results showed that the visual detection limit was 1×10² CFU mL^-1,the linear regression curve was y=0.1048 lgC+0.5541,R²=0.940.The results showed that the detection method could detect target bacteria sensitively in food matrix.The recovery rate of the system was 97.2%-99.6%.RSD is less than 10%,which shows that the detection system established in this experiment has good stability and high precision.Compared with other colorimetric detection methods,this detection system has the unique advantages of simple operation,fast and sensitive.Part Ⅳ Study on rapid detection method of Listeria monocytogenes based on multicolor colorimetry（1）In this part,immunomagnetic nanoprobe was used as"separator",using BSA as template,IgY-BSA-MnO₂ immunoprobe was prepared as a"signal converter",and AuNRs synthesized as multi-color"signaler",Based on IgY-BSA-MnO₂ catalytic oxidation of TMB reaction and TMB²⁺-AuNRs etching reaction,a multi-colorimetric visual detection system was established.The optimized conditions showed that 0.6 mg of immunomagnetic nanoprobe and 28 mg of IgY-BSA-MnO₂ immunoprobe made the multi-color display effect of the detection system the best.（2）The evaluation results of the detection system showed that the detection time of Listeria monocytogenes was 45 min,and the visual detection limit was 1×10 CFU mL^-1.The standard curve was made by the difference of blue shift peak value of AuNRs longitudinal LSPR,andΔλ=61.364 lgC-88.539,R²=0.932.The evaluation of specificity showed that the multi-color semi-quantitative detection system was highly resistant to the interference of impurities.The results showed that within the range of 5×10-10⁵ CFU mL^-1,the linear regression curve wasΔλ=25.038 lgC-31.388,R²=0.992,which indicated that the detection method could detect the target bacteria in food matrix quickly and sensitively.The standard-added recovery rate of the detection system was 97.4%-102.4%,RSD was below 10%,and the precision was good.Firstly,based on OPD-AuNPs reaction and IgY-BSA-AgNCs mediated catalytic oxidation of OPD reaction,a visualized detection system for Listeria monocytogenes was established,which had the advantages of high capture efficiency,low detection limit,strong specificity and short detection time.In order to achieve the goal of simple,sensitive and rapid detection of Listeria monocytogenes,this study further focused on the self-assembly of CB7-AuNPs and urease activity.A visualized detection system for Listeria monocytogenes was established by hydrolysis urea reaction,which was simple,rapid and sensitive.Based on this work,a multi-color visualized detection system was established by IgY-BSA-MnO₂ catalytic oxidation of TMB reaction and TMB²⁺-AuNRs etching reaction in order to realize the visualized semi-quantitative detection of target bacteria.Multicolor color change and semi-quantitative determination of the properties of target bacteria make this detection system have the characteristics of visualization and semi-quantitative detection of target bacteria in other colorimetric detection methods,and have absolute advantages in semi-quantitative detection of target bacteria.