核桃乳DNA提取方法优化与DNA条形码鉴定

Optimization of DNA extraction method and DNA barcoding identification of walnut milk

随着乳饮料行业快速发展,掺杂使假现象层出不穷,对核桃乳真实成分的准确鉴定变的尤为重要。核桃乳属深加工食品,DNA破坏严重,DNA提取是开展核桃乳DNA条形码鉴定的首要环节。为优化核桃乳DNA提取方法,并基于psbA-trnH基因DNA条形码建立核桃乳的掺假造假鉴定方法。以10种不同品牌的核桃乳为样品,采用3种方法(静置抽提、异丙醇沉淀、抽真空冻干)进行预处理,再用2种CTAB裂解沉淀方法和3种试剂盒方法(康为新型植物基因组DNA提取试剂盒、爱思进植物基因组DNA提取试剂盒和天根深加工食品DNA提取试剂盒)提取核桃乳DNA,并在此基础上创新尝试了CTAB与爱思进试剂盒结合方法。以提取的市售核桃乳DNA为模板,利用自行设计的特异性基因psbA-trnH-wal鉴定样品中是否含有核桃成分,确定造假情况;选择常见植物候选基因psbA-trnH鉴定样品中是否含有其他成分,确定掺假情况。结果表明,抽真空冻干预处理方法优于其他2种预处理方式。CTAB与爱思进试剂盒结合方法能提取到纯度好、得率高、扩增能力强的核桃乳DNA,是最佳的核桃乳DNA提取方法。扩增及比对结果显示,1款核桃乳样品中含有花生,属于掺假产品。抽真空冻干预处理、CTAB与爱思进试剂盒结合为优化后的核桃乳DNA提取方法,psbA-trnH-wal基因和psbA-trnH基因结合能够对核桃乳及其掺假造假品进行快速准确的鉴定。

With the rapid development of milk beverage industry,adulteration of milk beverage was common,thus it is particularly important to accurately identify the true ingredients of walnut milk.Walnut milk is a deep-processed food with severe DNA damage.DNA extraction is the first step in DNA barcode identification of walnut milk.The purpose of this experiment was to optimize the DNA extraction method of walnut milk,and to establish an adulteration and fraud identification method based on psbA-trnH gene DNA barcoding.Taking 10 different brands of walnut milk as samples,three methods(static extraction,isopropanol precipitation and vacuum lyophilization)were used for pretreatment.Walnut milk DNA was extracted using two CTAB lysate precipitation methods and three kit methods(Kangwei Nuclean Plant Genomic DNA Kit,AxYGen AxyPrep Multisource Genomic DNA Miniprep Kit,and TianGen Deep Processing Food DNA Extraction Kit).Furthermore,CTAB and AxYGen kit was combined for DNA extraction.Using the extracted commercial walnut milk DNA as a template,the self-designed specific gene psbA-trnH-wal was used to identify whether the sample contained walnut components,and the fraud situation was determined.The common plant candidate gene psbA-trnH was selected to identify whether the sample contained other components,to determine adulteration.Vacuum freezing method was superior to the other two pretreatment methods.Walnut milk extracted DNA using CTAB and AxYGen kit combination method was in good purity,high yield and strong amplification ability,thus it was the best method for walnut milk DNA extraction.Amplification and comparison results showed that one walnut milk sample contained peanuts,which belonged to adulterated products.The vacuum freezing method and the combination of CTAB and AxYGen kit combination method were the optimized methods for DNA extraction from walnut milk.The combination of psbA-trnH-wal gene and psbA-trnH gene could quickly and accurately identify walnut milk and its adulterated fakes.