Electrochemical Biosensor Based On Single Primer Isothermal Amplification (SPIA) For Detection Of Salmonella In Food

Salmonella,as one of the most common foodborne pathogens,is widely found in many foods,including eggs,milk,poultry,meat and fresh vegetables.Contamination of Salmonella can cause abdominal pain,diarrhea,convulsion,fever,and even death in severe cases,which poses serious threat to global food safety and public health security.Therefore,it is urgent to establish a rapid,sensitive,accurate and feasible method for detection of Salmonella in food.In recent years,electrochemical detection method has become a research hotspot in the detection field for various biomolecular due to its advantages of high sensitivity,low cost,fast analysis speed and simple operation.In addition,single primer isothermal amplification(SPIA)is a novel linear nucleic acid isothermal amplification technique reported in recent years.Compared with other nucleic acid amplification strategies,SPIA has merits of high fidelity,low contamination and high stability.In this study,an electrochemical biosensor combined with SPIA(named SPIA-E)is first developed for detection of Salmonella in food.In order to construct the sensor,SPIA reaction was first performed targeting gene invA to obtain Salmonella amplification products.The probe P2 specifically binding to the amplification products was designed.Secondly,gold nanoparticles(AuNPs/GCE)were modified on the surface of glassy carbon electrode(GCE),and probe P2 was fixed on the surface of AuNPs/GCE(P2/AuNPs/GCE)by Au-S bond.Then the fixed electrode was immersed in MCH solution to form ordered monolayer DNA molecular structure and occupy the remaining gold binding sites,forming MCH/P2/AuNPs/GCE.Scanning electron microscope images showed that AuNPs were evenly distributed on the surface of GCE with good morphology.According to the high consistency of CV and EIS images,AuNPs,probe P2 and MCH were successively modified on the electrode.Then,the feasibility of the sensor was verified.In this study,positive and blank control experiments were conducted with and without Salmonella,respectively.The significantly difference of current response values between the two groups could clearly distinguish the positive and blank control results.The SPIA amplification results were verified by polyacrylamide gel electrophoresis and the apparent molecular weight was consistent with the expected results.Subsequently,the experimental conditions in SPIA-E system were optimized.The optimal conditions were as following:addition amount of dNTPs(4μL),addition amount of Bca DNA polymerase(1 μL)and RNase H enzyme(0.5μL),concentration of probe P2(6 μM),incubation time of P2 and AuNPs/GCE(10 h),hybridized time of P2 with the SPIA amplification products(90 min).Finally,the performance of this sensor was analyzed under the optimum experimental condition.First,the constructed SPIA-E method was used to detect different concentrations of Salmonella genomic DNA.There was a linear relationship between thecurrent value and the logarithm of DNA concentration.The equation was △I%=9.5941 1g Cgenomic DNA+ 57.5578,R2=0.9916.The linear response ranged from 10 fg/μL to 50 ng/μL,and the detection limit was 4.5 fg/μL.Compared with other methods,the detection limit was lower and the linear response range was wider.In addition,the specificity,repeatability and stability of the sensor were analyzed.SWV measurements were performed on 5 strains Salmonella and 7 strains different non-Salmonella strains.The method presented high specificity for Salmonella detection.The RSD of SWV measurement by 6 repeated experimental was 4%,indicating good repeatability.After 12 days of storage at 4℃,the current value was still more than 90%of the initial value,exhibiting good stability.In order to evaluate the practical application effect,the SPIA-E method and real-time fluorescence PCR(RT-PCR)were used to detect Salmonella contaminated food samples.The results of SPIA-E were highly consistent with the results of RT-PCR.The recovery rate was in the range of 93%～110%,showing that this method had broad application prospect.In summary,the SPIA method combined with electrochemical biosensor was first developed and successfully applied to the detection of Salmonella in this study,which improved detection sensitivity and expanded linear range.The SPIA-E method provides a new technique for rapid and sensitive detection of Salmonella in food,and has broad practical application prospect in food safety monitoring.