基于微滴数字PCR技术检测致敏原芒果成分

Detection of allergen mango components by using droplet digital PCR technology

[目的]建立微滴数字PCR技术检测致敏原芒果成分的方法。[方法]选取叶绿体trnH-psbA基因作为致敏原芒果成分检测的靶基因,设计特异性引物探针,对微滴数字PCR方法反应条件进行优化,对方法的灵敏度和稳定性进行验证,并应用于10批次食品样品中致敏原芒果成分检测。[结果]试验建立的微滴PCR方法特异性较好,不与芒果以外10种其他水果交叉反应,在上、下游引物和探针浓度分别为200,200,100 nmol/L,退火温度为52℃的最适微滴PCR反应条件下,方法的核酸水平检测灵敏度为5 pg/μL,芒果质量含量检测灵敏度为5 mg/kg,方法变异系数<25%,重复性好,应用于10批次食品样品检测结果与食品标签一致。[结论]该方法具有快速、特异性、灵敏度高的优点,适用于致敏原芒果成分的检测。

[Objective]A droplet digital PCR method for detect allergen mango components was established in this study.[Methods]Firstly,the chloroplast trnH-psbAgene was selected as the target gene for detect allergen mango components.Secondly,specific primers and probe were designed.Finally,the reaction conditions of droplet digital PCR was optimized to verify the sensitivity and stability of the method.[Results]This method had been applied to detect allergen mango components in 10 batches of food samples.The results of establishment of droplet digital PCR method was showed good specificity and not cross-react with 10 kinds of other fruits except mango.Under the optimal ddPCR reaction conditions,which were to have the concentration of forward and reverse primers and probe set as 200,200 and 100 nmol/L,respectively,and to have annealing temperature set as 52℃,the detection sensitivity of nucleic acid level reached 5 pg/μL,and the detection sensitivity of mango content reached 5 mg/kg,the coefficient of variation of the method was less than 25%.The test results of 10 batches of food samples applied with the method were consistent with the food labels.[Conclusion]This method has the advantages of being fast,specific,and highly sensitive,making it suitable for the detection of allergenic mango components.