Study On The Detection Of Peanut Allergen Ara H1 Gene In Food By Visual Saltatory Rolling Circle Amplification

Peanut,as a sort of popular food riched in protein,is one of the main food allergens.With the increase of peanut demand,the prevalence of peanut allergy has also risen sharply,which is hindering the development of food safety assurance.Due to the symptoms such as dermatitis,diarrhea,anaphylactic shock and even death,peanut allergy not only causes health problems,but also affects the psychology of patients seriously.The prevention of food allergy not only needs the country and the government to formulate stricter laws and regulations,but also requires more accurate testing by related agencies.Therefore,it is urgent to establish a sensitive and specific method for the rapid detection of peanut allergens.In this study,the suitable primers for Saltatory rolling circle amplification(SRCA)were designed based on the highly conserved sequence of Ara h1 gene(No.AF432231)using DNAMAN software,and selected through a large number of experiments.With the assist of dye,the visual SRCA method for detecting the peanut allergen Ara h1 gene was established and evaluated.The test results were as follows:First,the reaction system and reaction conditions were optimized based on gel electrophoresis,and the final mixture includes dNTPs 0.625 mM,MgSO4 3.00 mM,1.25× ThermoPol Reaction Buffer,0.50 ?M forward and reverse primers,3.13 ng/?L DNA template,0.60 U/?L Bst DNA polymerase(large Fragment),add sterile deionized water supplements the system to 20.00 ?L.After incubation for 55 min at a constant temperature reaction of 62?,the products was subjected to the gel electrophoresis.Then,on the basis of the above reaction system,different dyes were added to study the influence of different dyes on the color of the amplification reaction solution under different light sources and concentrations,as well as the effect on detection sensitivity and detection time.The results showed that the combination of hydroxynaphthol blue(HNB)and SRCA exhibited the best performance.The optimum addition amount of 0.8 ?L HNB was determined,with the positive sky blue and the negative violet.The sensitivity of visual SRCA detection was 6.25 × 100 fg/?L,and the result could be judged within 15 minutes.Thus,a visual HNB-SRCA method for detecting peanut allergen Ara h1 gene was successfully established.A total of 23 different species were adopted to evaluate the specificity,and 5 peanut species showed positive results,while 1 8 species of non-peanut presented negative results,which indicated that the established HNB-SRCA showed good specificity.In this study,The specie of Jihua No.4 was selected to determine the sensitivity and detection limit.After DNA extraction(62.50 ng/?L)and 10-fold serial dilution,the sensitivities were analyzed by HNB-SRCA visual method,SRCA,LAMP,and PCR detection methods respectively.The results show the sensitivity of HNB-SRCA was 6.25 ×100 fg/?L,and that of SRCA,LAMP,and PCR gel electrophoresis were 6.25 × 101 fg/?L,6.25 × 102 fg/?L,and 6.25 × 103 fg/?L,respectively.Therefore,the sensitivity of HNB-SRCA was 10-fold,100-fold,1000-fold better compared with SRCA,LAMP,PCR,respectively.The extracted genomic DNA from the binary mixture of peanut and soybean meal was detected by the HNB-SRCA visualization method.The results showed that the detection limits of HNB-SRCA,SRCA,PCR,and LAMP were 0.01%,0.05%,0.5%,and 0.05%,indicating that The detection limit of HNB-SRCA was 5-fold lower than that of SRCA and LAMP,and 50-fold lower than that of PCR.Moreover,65 actual samples were detected and compared with the detecion of the SN/T 1961.2-2007 industry standard,then,the sensitivity,accuracy and specificity of the HNB-SRCA assay were calculated to be 100.00%,95.38%,and 90.00%,respectively.In summary,the visual HNB-SRCA method established in this study can realize the on-field detection for peanut allergen Ara h1 gene rapidly under isothermal conditions.Due to the advantages of high sensitive and specific,simple operation,low cost,and high efficiency,the HNB-SRCA method is more suitable for promotion in the grassroots-testing organizations,and has a large application potential.