A Colorimetric Sensor Based On SRCA-Photosensitization Reaction For The Detection Of Salmonella In Food

Salmonella is a gram-negative bacterium,which widely exists in meat,eggs,milk,and its various products,and is a major foodborne pathogen.Eating food contaminated by Salmonella can cause food poisoning,lead to physical discomfort,and even life-threatening.Most foods contaminated by Salmonella will not change significantly,making consumers often inadvertently infected with Salmonella.Salmonella contamination in food has been a serious threat to consumer health and life safety.Therefore,there is an urgent need to develop a simple,rapid,and sensitive detection method to monitor Salmonella contamination in food and ensure food safety.Saltatory rolling circle amplification(SRCA)is a novel isothermal nucleic acid amplification technique developed by our research team,which can amplify the linear template with only one pair of primers and one enzyme.Photosensitized colorimetric method is a colorimetric method based on indirect oxidation of substrate by photosensitizer.SYBR Green I is selected as the photosensitizer,which can be embedded into double-stranded DNA to improve the photosensitization efficiency.Under the irradiation of cyan LED,a large amount of singlet oxygen is generated,and the substrate is oxidized to appear blue,and the solution appear stable yellow after adding the stop solution.In this study,with the combination of SRCA and photosensitive colorimetric method,a SRCA-C sensor was developed to detect Salmonella in food.SRCA was used to identify and amplify the target DNA.Double-stranded DNA signal was converted into optical signal by photosensitization reaction,and the visualization and instrumental detection of Salmonella was completed by observing the color change of the solution and measuring the absorbance of the solution,respectively.The main research contents and results are as follows:The SRCA primers were designed for the highly conserved and specific invA gene of Salmonella,the SRCA amplification reaction was validated,and after screening the suitable primers.Then,the SRCA-C sensor was constructed,and the feasibility of the sensor was verified by comparing the absorbance of positive group with that of blank control group.After that,the key conditions of this sensor were optimized,and the results showed that the optimum reaction time for SRCA was 50 min,the optimum reaction temperature was 62℃,the optimum addition amount of Mg2+ was 5.5 μL,the optimum light time was 15 min,the optimum addition amount of SYBR Green I was 2.5 μL,and the optimal pH of buffer was 5.Under optimal conditions,the detection performance of the sensor was evaluated in terms of linear detection range,detection limit,specificity,and repeatability,and its practical value was evaluated by testing artificially contaminated milk samples and actual samples.The results were as follows:The detection limit of visualized SRCA-C is 130 CFU/mL,and the detection limit of SRCA-C sensor with absorbance as the measurement index is 10 times lower than that of visualized SRCA-C.When the concentration of Salmonella solution was between 1.3× 101～1.3×106 CFU/mL,the absorbance value(y)showed a good linear relationship with the logarithm of the bacterial solution concentration(x),the linear equation was y=0.222x0.114(R2=0.9981),the detection limit was 13 CFU/mL.In the specificity validation test,only the absorbance of the Salmonella group was significantly higher than that of the blank control group,while the absorbance of the non-Salmonella group was basically the same as that of the blank control group,indicating that the specificity of the sensor for the detection of Salmonella was excellent.In the reproducibility validation test,all six groups showed relatively consistent absorbance values,and the relative standard deviation was only 3.46%,indicating that the sensor has outstanding reproducibility.In the detection of artificially contaminated milk samples,the spiked recoveries ranged from 98.4%to 118.4%,indicating that the sensor has good detection accuracy for Salmonella in food matrices.Seventy real samples were tested separately using the sensor and the standard method(GB 4789.4-2016).Compared with standard method,the sensitivity,specificity,and accuracy of the method were 100%,98.5%,and 98.6%,respectively.In summary,this study successfully constructed a colorimetric sensor based on SRCA and photosensitized reaction for the detection of Salmonella in food,which has the advantages of simple,rapid,and sensitive detection.Moreover,the sensor can realize intensive detection and improve the detection efficiency of large volume samples.It provides a new strategy for the detection of Salmonella in food,and provides new ideas for the detection of other food-borne pathogenic bacteria,and has important practical significance for the protection of consumer health and life safety.