Study On The Detection Of Salmonella In Food Using Saltatory Rolling Circle Amplification Combined With FTA Card

With the improvement of people's living standards and the development of economic globalization,food safety had gradually become a topic of global concern.Food safety incidents were common,and these caused by Salmonella account for a large proportion.The traditional culture method as the "gold standard" is laborious and time-consuming,which generally takes 5?7 days and cannot meet the needs of rapid test on-site.Therefore,it is urgently to establish a simple,rapid,specific,sensitive and efficient method for detecting Salmonella.This study established a detection method using saltatory rolling circle amplification combined with FTA card(FTA-SRCA)to detect Salmonella in food.FTA card was used to adsorpt and fix the nucleic acid for quickly extracting template DNA,which was utilized for SRCA reaction.SRCA reaction was carried out in a concave plate capable of intensive detection under constant temperature with a pair of primers and an enzyme.By adding dyes to the amplified products,the results can be determined visual.Specific primers were designed and screened for the invA gene of Salmonella,and the specificity of primers of 46 strains was verified.The results showed that the 17 strains of Salmonella tested were all positive,and the 29 strains of non-Salmonella were all negative,confirming the good specificity of primers.The treatment conditions of FTA card and the reaction conditions and reaction system of SRCA were optimized.The treatment conditions for template DNA extracted by FTA card were determined as follows:drying for 10 min at 55?60?,washing twice with FTA purification reagent,washing twice with TE buffer,drying for 15 min at 55?60?.Moreover,the FTA-SRCA reaction temperature was 62? and reaction time was 40 min.The reaction system included 0.75 mM dNTPs,0.75 × Bst DNA polymerase buffer,3.0 mM Mg2+,0.5 ?M forward and reverse primers,8 U Bst DNA polymerase,and sterile deionized water made up the reaction system to 20.0 ?L.By optimizing the addition amount of SYBR Green ? dye,finally the study determined that the addition amount of SYBR Green ?(50 ×)dye was 2.00 ?L.Genomic DNA of pure culture of Salmonella at different concentrations was extracted by kit and FTA card,respectively.PCR,SRCA and FTA-SRCA tests were conducted to compare and analyze the sensitivity of three methods.The results were determined by the gel electrophoresis method and the fluorescence visualization method.For gel electrophoresis method,the sensitivity of FTA-SRCA method was 6.8 × 100 CFU/mL,which was 100 times higher than PCR method and was 10 times higher than SRCA method.For fluorescence visualization method,the sensitivity of FTA-SRCA method was 6.8 × 100 CFU/mL,which was 100 times higher than PCR method and the same as SRCA method.Genomic DNA in artificially contaminated milk samples was extracted by kit and FTA card,respectively.PCR,SRCA and FTA-SRCA tests were conducted to compare and analyze the detection limits of three methods.The results were determined by the gel electrophoresis method and the fluorescence visualization method.For gel electrophoresis method,the detection limit of FTA-SRCA method was 3.2 × 100 CFU/mL,which was 1000 times lower than PCR method and was 10 times lower than SRCA method.For fluorescence visualization method,the detection limit of FTA-SRCA method was 3.2 x 100 CFU/mL,which was 1000 times lower than PCR method and the same as SRCA method.Using PCR,SRCA and FTA-SRCA methods to detect Salmonella in 60 samples,based on the national standard GB 4789.4-2016,the sensitivity,specificity and coincidence rate of FTA-SRCA method were calculated to be 100.00%,94.64%,and 95.00%,respectively.In conclusion,the detection method of Salmonella in food established in this study using saltatory rolling circle amplification combined with FTA card was feasible.This method has the advantages of simple operation,low cost,short detection time,good specificity,high sensitivity,and low detection limit.In addition,FTA card has good nucleic acid extraction effect and can be reused without affecting the accuracy of the detection results.It achieved intensive testing for a large number of samples.FTA-SRCA method provides reliable technical support for relevant supervision enforcement agencies and food companies.