多重微滴式数字PCR定量检测市售核桃乳中核桃、大豆源性成分

Duplex Digital Droplet PCR for the Determination of Walnut-Derived and Soybean-Derived Ingredients in Walnut Protein Drink

目的:为市售核桃乳中核桃源及主要掺杂物种大豆两种源性成分建立准确、快速定量检测方法。方法:从植物组织(核桃、大豆)或是植物蛋白饮料(核桃乳饮料)提取核酸后,主要采用多重微滴式数字聚合酶链式反应(polymerase chain reaction,PCR)技术,测算单位质量核桃和大豆的目的基因拷贝数比值,建立基因拷贝数与质量之间的关系,进而利用该比例关系换算出植物蛋白饮料中核桃和大豆的投料量,以实现对植物蛋白饮料中植物源性成分的定量检测。结果:基于多重微滴式数字PCR定量检测市售产品中大豆和核桃源性组分的方法,引物探针特异性好,目标物种间无交叉反应;灵敏度高,核桃中掺杂大豆的质量检测限为0.5%,相对误差为5.6%。将单一源性5种不同质量下靶基因拷贝数之比与5种不同比例混合源性靶基因拷贝数之比通过重复3次检测,综合比较并拟合后得到单位质量下拷贝数(C_(大豆)/C_(核桃)=1.771 1)的换算比例值。在从商品超市中抽取的11份不同品牌的核桃乳中(仅含核桃一种植物源成分),多重微滴式数字PCR方法检测显示:11份样品均检出核桃源性成分,6份样品检出大豆源性成分,其中4份样品中大豆与核桃质量之比高于10%,判断存在掺杂使假;2份样品大豆与核桃质量之比低于0.2%,极低的检出量推断为工艺沾染。结论:采用多重微滴式数字PCR准确、快速的定量方法可作为鉴别核桃乳中掺杂使假问题的有效手段。

Objective:The aim of this study was to establish a new method for the rapid and accurate quantification of walnut-derived ingredients and adulterated ingredients,mainly derived from soybean,in commercial walnut beverage using droplet digital polymerase chain reaction(ddPCR).Methods:This method was based on the ratio between the numbers of target gene copies per unit mass of walnut and soybean,which could represent the relationship between gene copy number and the mass of plant materials.The soybean lectin gene and the walnut Jugr2gene were chosen as the target genes according to the commercial inspection standard SN/T1961-2013.The specificity of the assay was evaluated by testing DNA from six different species using the species-specific primers and probes.The results showed that there was no crossreaction among the target species and high sensitivity was observed.The ddPCR assay showed a limit of detection(LOD)for added soybean-derived ingredients in walnut protein drink was0.5%with a relative error of5.6%.The ratios of target gene copy numbers between soybean and walnut in pure samples of five different masses and between the two species in five mixtures with different proportions were determined three times,and the experimental data were fitted to a linear equation to calculate the ratio between gene copy numbers per unit mass soybean and walnut(Csoybean/Cwalnut ratio=1.7711).Furthermore,we applied this method to test11commercial brands of walnut beverage,and it turned out that walnut-derived ingredients were detected in all these samples and6of them were found to also contain soybean-derived ingredients,out of which4were adulterated with more than10%soybean and the others contained as low as0.2%soybean,which may be unintentionally incorporated during the production process.Conclusion:The ddPCR assay can provide a rapid and accurate quantitative method for the identification of adulterated walnut beverage.