Research On The Rapid On-site Detection Of Salmonella Typhimurium Using Colorimetric/Fluorescent Signal Assay

Background:Salmonella typhimurium（S.typhimurium）is a common zoonotic pathogen with a wide range of hosts,ingestion of food and water contaminated with this bacterium can cause gastrointestinal disease in humans and animals.The clinical symptoms range from mild enteritis to severe septicaemia,and even systemic disseminated visceral damage.It is one of the most common pathogens causing food poisoning and outbreaks of infection in hospitals,posing a serious threat to human health.Current testing methods for S.typhimurium are either complex,time-consuming or trained technical personnel demanding,making it difficult to meet the demand for rapid on-site detection.Objective:It is necessary to develop a simple,rapid and sensitive detection method for S.typhimurium to prevent and control zoonoses caused by S.typhimurium.This study aims to establish a simple colorimetric fluorescence dual signal on-site detection method,and systematically evaluate the established method,so as to provide technical reserve and theoretical support for the prevention and control of zoonoses.Methods:1.Feasibility exploration assayThe interaction between S.typhimurium-specific aptamers and poly-diallyl dimethylammonium chloride（PDDA）was determined by circular dichroism spectroscopy;transmission electron microscopy and dynamic light scattering were used to evaluate gold nanoparticles（Au NPs）in different mixed solutions;ultraviolet-visible absorption spectroscopy was used to determine the optimal fluorescent material.The fluorescence intensity of the six fluorescent materials was measured after addition to the detection system to determine the optimal fluorescent material;the selected fluorescent materials were characterized by fluorescence spectroscopy.2.Optimization and establishment of detection systemThe single-step colorimetric reagent was prepared by Au NPs,PDDA,aptamer and morpholine ethanesulfonic acid（MES）buffer.The target bacteria were added into this reagent,and color changes were observed 20 min later.By adding carboxyl Cd Se/Zn S quantum dots(QDs-_COOH)into the detection system,quantitative detection of target bacteria could realize by measuring fluorescence intensity.In order to achieve best results of this method,the concentration of PDDA,S.typhimurium-specific aptamer,QDs-_COOH,and the p H value of MES buffer were optimized.3.Evaluation of detection systemA systematic evaluation of the sensitivity and selectivity of the developed rapid on-site detection system for S.typhimurium and the use of the assay for the detection of simulated contaminated samples such as chicken,cabbage,drinking water and pet urine;and evaluation of the stability of the single-step colorimetric reagent and fluorescent reagent.4.Smartphone applications developmentThe color differences in the detection results were quantified using the R-value in the RGB color system;which was used as a data base to develop an application（APP）for semi-quantitative analysis of the results;the APP parameters were calibrated by measuring S.typhimurium at different concentrations several times;the APP was applied to test simulated contaminated samples to verify its practicality.Results:1.The result of feasibility validation assayThe results of transmission electron microscopy,dynamic light scattering and ultraviolet-visible absorption spectroscopy showed the differences states of Au NPs in different mixed solutions,which verified the feasibility of the single-step colorimetric assay.The results of fluorescence spectroscopy showed that QDs-_COOH had well response characteristics to the degree of aggregation of Au NPs,and the feasibility of the fluorescence assay had been proved.2.The result of optimization of detection systemThe concentration of PDDA,S.typhimurium-specific aptamer,QDs-_COOH,and the p H value of MES buffer were optimized.The results showed that the optimal concentration of PDDA was 2 n M,the optimal concentration of aptamer was 26.7 n M,the optimal concentration of QDs-_COOH was 5 n M and the optimal p H value of MES buffer was 4.3.The result of evaluation of detection systemAfter optimization,the visualization limit of detection（LOD）for S.typhimurium was 10³ CFU/m L.After the addition of the fluorescent reagent QDs-_COOH,the difference（?F）of the fluorescence intensity in the presence（F）and absence（F₀）of S.typhimurium was proportional to the logarithm of S.typhimurium concentration ranging from 10² to 10⁷ CFU/m L.The calibration plot fitted a regression equation of?F=88.29×lg(8-163.80,with R²=0.98,and the LOD of fluorometric assay is estimated to 10 CFU/m L;the results of several common non-target zoonotic pathogens showed that only the samples with S.typhimurium were positive,while the other samples showed the same results as the negative control group,demonstrating the good selectivity of the method.The single-step colorimetric reagent and the fluorescent reagent were still able to detect the target bacteria accurately after 30 days of storage at4°C,demonstrating the good stability.4.Result acquisition using a smartphoneAfter calibration,it is defined that when R value was 144-250,the sample status was"safe";when R value was 116-144,the sample state was"dangerous";when R value is 80-116,the sample state was"extremely dangerous".The results of simulating cabbage samples analyzed with APP showed that the APP could accurately evaluate contaminate samples and has good practicability.Conclusion:1.single-step colorimetric assay was constructed using Au NPs,PDDA,aptamer and other materials,and the single-step colorimetric assay was realized for the detection of S.typhimurium.2.The fluorescence detection method was developed,and the accurate quantification of S.typhimurium was realized.Compared with the visual detection method,the LOD was improved.3.The smartphone APP developed in this study realized semi-quantitative on-site detection of S.typhimurium.4.Through the systematic evaluation of the established colorimetric/fluorescent dual-signal method for the rapid on-site detection of S.typhimurium,it is indicated that the established method is simple,rapid and accurate,and is suitable to be popularized in the detection work of grass-roots units.