Study On Quantitative Detection For Plant-derived Ingredients In Food By Digital PCR

Huge consumer market of plant-derived processed food has attracted various Economically Motivated Adulteration(EMA)for illegal profit,such as making lotus seed-paste moon cakes with kidney bean as raw material,adding peanut in sesame paste,and so on.The main reason for such adulteration includes insufficient supply of raw materials,high production costs,difficult processing and adventitious contamination caused by sharing production lines.These production condition not only disturbs the order of the market economy,but also seriously damages the rights and interests of consumers.In recent years,digital PCR method has been gradually developed with its strong specificity,good stability and high sensitivity,which could provide a new method for quantitative detection.In this study,droplet digital PCR(dd PCR)and chip digital PCR(cd PCR)were used to establish qualitative detection with high sensitivity and specificity for lotus seed,kidney bean,sesame and peanut.Quantitative detection for mass ratio of kidney bean in lotus paste and mass ratio of peanut in sesame paste was explored by duplex digital PCR,and applied to simulated samples,commercial lotus seed paste and sesame paste samples,which could provide technical support for identifying the authenticity of paste products and combating adulteration.(1)Primers and probes of lotus seed,kidney bean,sesame and peanut were designed based on their genome sequence.Both q PCR and digital PCR were used to verify the specificity of the primers and probes.Result showed that these primers and probes have excellent speciesspecificity and amplification efficiency.Four plant genomic DNA extraction kits were compared and TIANGEN was verified to be the best one for its DNA extraction efficiency and quality.(2)Qualitative detection of lotus seed and kidney bean,sesame and peanut were firstly established on duplex dd PCR and duplex cd PCR platform.Theoretical values and measured values of DNA copy concentration for lotus seed,kidney bean,sesame and peanut were in good consistency.Limit of detection for DNA copy concentration on dd PCR was 0.90 copies/?L for lotus seed-original DNA,1.03 copies/?L for kidney bean-original DNA,1.07 copies/?L for sesame-original DNA,and 1.07 copies/?L for peanut-original DNA,respectively.Detection sensitivity could reach 0.01%.Results of prepackaged commercial lotus seed paste and sesame paste samples showed that there is the presence of adulteration.Besides,establishment of highsensitivity detection method for peanut allergen has practical significance for protecting susceptible consumers.(3)A novel idea of mass ratio quantification was adopted in this study.For the first time,duplex dd PCR and duplex cd PCR were used to establish quantitative detection for lotus seed and kidney bean,sesame and peanut.Limit of quantification of DNA copy concentration on dd PCR was 5.07 copies/?L for lotus seed-original DNA,4.97 copies/?L for kidney beanoriginal DNA,4.57 copies/?L for sesame-original DNA,and 5.70 copies/?L for peanut-original DNA,respectively.(4)Formulas of “mass ratio-DNA copy concentration ratio” were established for lotus seed and kidney bean,sesame and peanut,and linearity of formula with mass ratio of kidney bean or peanut was performed good from 5% to 80%.Relationships between mass and DNA copy concentration for lotus seed,kidney bean,sesame and peanut were performed good linearity in the range of 10-100 mg.(5)Measured values of quantitative detection for simulated lotus seed paste with kidney bean at mass ratio of more than 5%,and simulated sesame paste with peanut at mass ratio of more than 5%,were close to theretical value,and recovery rate was between 80% and 120%.Thus,duplex digital PCR quantitative detection method established in this study could be effectively applied to quantitative detection of commercial samples to identify adulteration.(6)Detected results on dd PCR and cd PCR were basically with no significant difference.In summary,this study established a qualitative and mass ratio quantitative detection method based on duplex digital PCR,which could achieve qualitative and quantitative detection of kidney bean component in the lotus seed paste,peanut component in the sesame paste,providing strong technical support for the authenticity and quality supervision of plant-derived food.