| At present,food poisoning caused by food-borne pathogens is an important food safety issue concerned by domestic and foreign health and safety organizations.Common foodborne pathogen detection methods have their own advantages,but with the advancement of technology,these detection methods have exposed their own shortcomings.For example,the operation is cumbersome,time-consuming and labor-intensive,requires professional instruments,etc.And is not suitable for on-site rapid testing out of the laboratory.Therefore,it is of great significance to develop a system that is suitable for different food matrices and can realize the rapid and accurate detection of food-borne pathogens in the field.In this paper,a rapid and visual detection system based on RPALFD was established,which can achieve rapid and effective detection of Salmonella and Staphylococcus aureus,two important food-borne pathogens,with high sensitivity and specificity.First,establish an RPA detection system.According to the relevant characteristic sequences of the target gene inv A of Salmonella and the target gene nuc of Staphylococcus aureus,12 pairs of primers were designed and synthesized for optimized screening.The 12 pairs of primers were selected according to the band brightness of agarose gel electrophoresis and the size of the amplified product.Evaluate.The screening results showed that primer pair F2R1 was suitable for RPA detection of Salmonella,and primer pair F1R3 was suitable for RPA detection of Staphylococcus aureus.A pair of appropriate amplification primers were selected for the optimization of the amplification reaction time and reaction temperature of the RPA reaction system.The optimal reaction temperature for RPA amplification was determined to be 40°C,and the optimal reaction time was 20 min.The specific amplification of fragments can be achieved in 7.5 min of reaction at 20°C,indicating that this method can achieve rapid detection under conditions close to room temperature.Method specificity using six different strains of Listeria monocytogenes,Escherichia coli,Escherichia coli（negative）,Bacillus pasteurii,Candida albicans,S.The results show that the established RPA detection method has strong specificity.The RPA detection method has a detection sensitivity of 20 CFU/m L for Salmonella and 30 CFU/m L for Staphylococcus aureus.On this basis,the RPA-LFD detection system is established.The nfo probe was designed according to the full-length sequence of the amplified product,and the two ends of the probe were labeled with biotin and blocking groups respectively.The 5’ end of the downstream primer is labeled with a FAM fluorescent signal group.The probe and primer are used to detect the sample.If the sample to be tested contains positive target pathogenic bacteria,the two ends of the amplified DNA double-strand carry FAM fluorescent labeling group and biotin labeling group respectively.The upflow process will be captured by the antibody immobilized on the test line（T line）to form a sandwich structure.The test line（T line）will show red due to the accumulation of Au NPs.After screening and optimization of nfo probes and downstream primers,the probe/primer combinations that can be used for the specific detection of Salmonella and Staphylococcus aureus were determined.Visual detection of Salmonella and Staphylococcus aureus.Finally,the established RPA-LFD method is used to evaluate the detection effect of actual samples.Pork,seafood,vegetables,milk,eggs were used as food substrates,and different concentrations of Salmonella bacteria liquid or Staphylococcus aureus bacteria liquid were added to the food matrix to make spiked samples,and the established RPALFD detection method was used to detect Different spiked samples were tested.The experimental results show that the method can be used for the specific and rapid detection of Salmonella and Staphylococcus aureus in food samples,and the detection sensitivities are 15 CFU/m L and 20 CFU/m L.The RPA-LFD method established in this paper can realize the visual and rapid detection of Salmonella and Staphylococcus aureus in food samples.The method is easy to operate,has rapid response,high sensitivity,does not require large-scale equipment and visualization of reaction results,and is suitable for On-site rapid detection of Salmonella and Staphylococcus aureus in different types of food samples.At the same time,the method is also expected to be popularized for the detection of other food-borne pathogenic microorganisms,providing theoretical exploration and technical support for the development of visual and rapid detection tools for food-borne pathogenic microorganisms. |
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