Colorimetric Detection Of Salmonella In Food Based On Gold Nanoparticle Probes Combined With SRCA

Salmonella is a major cause of diarrheal disease in humans worldwide and is a serious health risk for humans and animals.Human infection may cause diarrhea,typhoid fever,abdominal cramps and other symptoms,and may even be life-threatening when the disease is severe.Traditional methods for Salmonella detection are time-consuming and cumbersome,so it is important to carry out a rapid,simple and sensitive colorimetric detection method.In this study,designed probes that bind to the target sequences and bound them to gold nanoparticles(AuNPs).The target sequence was amplified by Salatory rolling circle amplification(SRCA),and it became single chain after high temperature denaturation,and after binding to the probe on the gold nanoparticles,the gold nanoparticles were released,which were aggregated under the action of MgSO4 and shovred a blue color visible to the naked eye,which was judged as positive,otherwise,it was purple and judged as negative.Accordingly,the reaction conditions were optimized and the visualization assay was constructed;in addition,the UV absorption spectra were measured at A620 and A530 respectively using UV spectrophotometer and the ratio between them is calculated,or to measure its RGB values(red,green and blue values)using smartphone software and the B(blue)/R(red)ratio was analyzed,according to which the reaction conditions were optimized and the quantitative assay was constructed.The established qualitative and quantitative detection methods were evaluated.The main findings are as follows.(1)The optimized SRCA reaction system and conditions were:Mg2+ addition was 3.0μL,dNTPs addition was 5.0 μL,63℃ was the reaction temperature,and 40 min was the reaction time.The optimized colorimetric reaction system and conditions were:the concentration of MgSO4 was 6.7 mmol/L,the concentration of the probe was 5 μmol/L,the incubation time and incubation temperature were 15 min and 50℃,respectively,and the stable UV absorption spectrum values were presented after 4 min of reaction with the addition of MgSO4 solution.(2)The results of the specificity evaluation experiment showed that 8 strains of Salmonella had obvious color change and absorbance change,which showed positive results;8 strains of non-Salmonella had no obvious color and absorbance change,which showed negative results,indicating that the method has good specificity;the results of the sensitivity evaluation experiment for the detection of pure bacterial solution showed that the sensitivity of qualitative detection by visualization with naked eyes was 5.5 CFU/mL.The linearity of A620/A530 values,B/R values and logarithm of bacterial solution concentration were good for Salmonella concentrations between 5.5 to 5.5×106 CFU/mL.The linear equation for the quantification by UV-absorption spectroscopy was y=0.011x+1.081(R2=0.9824)with a sensitivity of 4.59 CFU/mL.The linear equation for the quantification by smartphone RGB was y=0.0106x+1.0134(R2=0.9876)with a sensitivity of 5.50 CFU/mL(LOD=3S/k).(3)Salmonella spiked in sterile milk at concentrations of 3.7～3.7×108 CFU/mL was tested for artificial contamination to determine the detection limit for qualitative and quantitative detection,and the detection limit for qualitative detection by visualization with the naked eye was 3.7 CFU/mL.The linear equation for quantitative determination by UV absorption spectroscopy was y=0.0168x+1.0893(R2=0.9877)with a detection limit of The linear equation for the quantitative determination of smartphone RGB was y=0.0201x+1.215(R2=0.9859),and the limit of detection was 3.02 CFU/mL.(4)The actual samples were tested using the method established in this study on 50 actual commercially available samples and compared with the method of GB 4789.4-2016,the sensitivity of the method established in this study was 100.00%,the specificity was 97.87%and the compliance rate was 98.00%.In this study,a gold nanoparticle probe combined with SRCA amplification colorimetric method was established for the qualitative and quantitative detection of Salmonella in food.It is more specific,more sensitive,less time-consuming and easy to operate.The visualization of qualitative detection was achieved by visual observation of color change,and quantitative detection was achieved by UV absorption spectroscopy or RGB analysis,which is suitable for application in grassroots units or testing institutions.