Study On High-Sensitive Immunochromatographic Assay Detection Of Salmonella Enteritidis

Salmonella Enteritidis（S.Enteritidis）has emerged as one of most common zoonotic agents,leading a major cause of gastroenteritis,vomiting,typhoid fever,watery diarrhea and eventually death worldwide.Therefore,the early identification of bacterial infections via point-of-care testing（POCT）is critical to the safety of food industry and clinical diagnosis.Up to now,a variety of methods,including conventional microbiological culture and colony counting,enzyme-linked immunosorbent assay,polymerase chain reaction and electrochemical biosensor,have been well developed and applied to the detection of foodborne pathogens.Yet,these methods are not suitable for real-time screening,as usually take a few days to get results and cause complicated operations.The immunochromatographic assay（ICA）has been widely noted due to its outstanding advantages such as fabrication,convenient operation,effective cost,disposable feature,short assay time and facile analytical procedure,and become a well-established diagnostic tool for various POC testing and rapid detection.In this work,three sensitive and efficient immunochromatographic assays to detect S.Enteritidis.The results of this paper are as follows:1.Development of immunochromatographic assay（ICA）based on gold nanoparticles（GNPs）growth and accumulation for rapid detection of S.Enteritidis:The signal was amplified on account of the high catalytic activity of GNPs toward the reaction between HAu Cl₄ and NH₂OH·HCl,which could produce new GNPs on the surface of the initial GNPs.The experimental conditions were optimized and the optimal proportion between HAu Cl₄ and NH₂OH·HCl was selected as 1:10.The lowest visible detection limit of the signal enhanced ICA for S.Enteritidis was as low as 10⁴CFU/m L,achieving a 100-fold improvement in sensitivity compared to the traditional GNP-based ICA.In addition,the test strips had no cross-reaction with other Salmonella and non-Salmonella strains,showing its excellent specificity.Finally,to demonstrate the applicability of this method,test strip was applied to（25 m L）milk for（8 CFU）S.Enteritidis analysis.Except for incubation time,the detection process takes only 6 hours,which was obvious shorter than that of many reported methods.2.Construction and characterization of LFA based on dual recognition for rapid detection of S.Enteritidis:In this work,a novel and robust ICA that combined strong affinity of antibiotic and antibody agents based dual recognition strategy and magnetic enrichment was designed to achieve specific and sensitive determination of pathogenic bacteria.The system is used to overcome the limitations of antibodies’pairing difficulties.Moreover,the experimental conditions were optimized,the optimal concentration of Amp-MNPs was selected as 0.8 mg/m L,the optimal incubation time between Amp-MNPs and S.Enteritidis was 30 min.Under optimal conditions,S.Enteritidis can be detected as low as 10² CFU/m L with the naked eyes in milk samples with excellent specificity.Therefore,this creative antibody-and antibiotic-based dual recognition biosensor,shows great application potential in monitoring of food contamination and infectious diseases caused by bacteria.3.Construction and characterization of lateral flow assay（LFA）based on crystal violet staining for rapid detection of S.Enteritidis:In this study,target bacteria can be directly marked with crystal violet by one-step staining and the selectivity can be guaranteed by high-specificity monoclonal antibody.Through the optimization of experimental conditions,the optimal concentration of crystal violet was selected as 0.001%,the volume of crystal violet solution was 90μL,the optimal incubation time was 1 min and the reaction time was set at 10 min to apply in the subsequent experiment.Under optimal conditions,this protocol can selectively detect 80 CFU/m L S.Enteritidis with excellent specificity.Finally,our test strip for detecting S.Enteritidis can be well applied in real food samples,such as drinking water,tomatoes,and meat with sensitivity of 80,10²,10³ CFU/m L,respectively.