Development Of Immunochromatography For The Detection And Serotyping Of Salmonella

Foodborne diseases caused by pathogens pose a significant threat to human health and the global economy.Salmonella is one of the common foodborne pathogens and is widely found in a variety of foods,such as poultry,eggs,milk,and beef.About 115 million people were infected with Salmonella each year,causing370,000 deaths.More than 2650 Salmonella serotypes have been identified,divided into approximately 50 serogroups.Different serotypes showed different pathogenic mechanisms,pathogenicity,and toxicity.Therefore,broad-spectrum detection and serotype classification of Salmonella are of great significance in food safety,public health surveillance,and poultry disease prevention and control.The traditional detection assay is the standard assay for Salmonella detection and serotype classification,but it is tedious and time-consuming.Therefore,it is urgent to develop rapid and accurate broad spectrum detection and high throughput typing assays for Salmonella.In this study,two lateral flow immunoassays（LFIA）were established based on the broad spectrum antibody strategy and spatial color corecognition strategy,respectively.Aggregation-induced emission fluorescent microsphere（AIEFM）was used as a signal probe to realize the fluorescent detection of Salmonella.High throughput typing of Salmonella was achieved by using multi-colored Latex microspheres（LMs）as signal probes.The first chapter systematically introduces the biological characteristics,serotype characteristics,harm and clinical characteristics,limitation standard,and current research progress of detection and typing of Salmonella.In addition,the structure and the detection principle of LFIA,and strategies to improve the performance of multiple detection are also summarized.In the second chapter,aggregation-induced emission（AIE）-LFIA based on a broad-spectrum antibody was established.AIEFM,which has strong fluorescence properties,were coated with antibody-friendly polydopamine（PDA）to form AIEFM@PDA as the fluorescent probe.The AIE-LFIA was used to rapidly and efficiently detect five Salmonella serotypes with high pathogenicity,including Salmonella Typhimurium variant（STV）,Salmonella Enteritidis（SE）,Salmonella Typhimurium（ST）,Salmonella Derby（SD）,and Salmonella Paratyphi B（SPB）.AIEFM@PDA has superior performance in dual-antibody sandwich-type LFIA.The LOD of STV,SE,ST,SD,and SPB in this assay was 2.3×10⁴ CFU m L^-1,8.9×10³CFU m L^-1,1.1×10⁴ CFU m L^-1,3.6×10⁴ CFU m L^-1,and 4.6×10⁴ CFU m L^-1,respectively.This study provides an efficient and convenient assay for the broad-spectrum detection of Salmonella.In Chapter 3,a spatial color corecognition LFIA was established.By setting two detection lines and using red and blue LMs as signal probes,the high-throughput typing of four common serogroups of Salmonella O:2,O:3,O:7,and O:9 was implemented.In this assay,key parameters such as p H,labeled antibody amount,blocking agent amount,and captured antibody concentration were studied.The red-green-blue（RGB）and hue-saturation-brightness（HSB）color space were introduced to quantify the color difference on the detection line,and the HSB color space demonstrates better performance in the recognition of multicolor immunochromatographic strips.The LOD of four standard strains were calculated by the saturation,and an Android application,which was used to recognize the multicolor results of spatial color co-recognition LFIA,was developed based on HSB analysis.In the assay,the LOD of Salmonella Paratyphi A（SP）on the O:2 serogroup,Salmonella Anatum（SA）on the O:3 serogroup,Salmonella Choleraesuis（SC）on the O:7 serogroup,and Salmonella Enteritidis（SE）on the O:9 serogroup,was 1.4×10⁵CFU m L^-1,1.8×10⁵ CFU m L^-1,6.5×10⁴ CFU m L^-1,and 7.6×10⁵ CFU m L^-1,respectively.Forty-four strains were tested to verify the sensitivity and specificity of the assay.The sensitivity and specificity of the assay for four serogroups were 100%.This study provides a rapid and accurate assay for serotyping of Salmonella and improves the application of HSB color space in the analysis of multicolor results.In Chapter 4,the established two detection strategies in LFIA based on the broad spectrum antibody strategy and spatial color corecognition strategy for Salmonella are systematically summarized.The potential assays to enhance the multiplexing detection performance of LFIA in the future was put forward.In summary,aggregation-induced luminescence LFIA based on the broad-spectrum antibody was established to detect the Salmonella genus,which can simultaneously detect five Salmonella serotypes with high pathogenicity.This assay can be applied to the broad spectrum detection of Salmonella.In addition,LFIA based on spatial color corecognition was proposed for rapid and accurate high-throughput classification of Salmonella by introducing HSB color space to analyze the multicolor results of immunochromatographic strips.