基于DNA条形码的浆果及其制品真伪鉴别技术研究

发布时间：2018-09-08 10:22

【摘要】：作为一种高附加值水果,浆果在日常生活中越来越受消费者欢迎。然而,由于较短的收获期和较高的价格,浆果及其制品掺假、造假、错误标签等导致的食品质量和安全争议不断发生。目前,我国对浆果及其制品的真伪识别还处于起步阶段。对此,本文通过研究新兴的DNA条形码,建立基于Sanger测序技术的DNA条形码方法,并应用于市售浆果制品的检测,检测范围涉及完全勾兑型果汁、廉价水果替代高价浆果的产品以及标签的符合性查验。主要开展了浆果基因组DNA提取方法研究,对市场上常见的6种针对植物样品的商业试剂盒进行了比较和优化,并确定了 PCR体系中的扩增酶。结果显示:Macherey-Nagel、TIANGEN和Magen试剂盒法所得DNA质量较差,DNA严重降解和污染;Qiagen、Promega和Biotecon试剂盒法均能得到质量较好的DNA,但Promega和Biotecon试剂盒法的DNA完整性低,扩增效率低于Qiagen试剂盒法提取的DNA。最后确定Qiagen试剂盒作为浆果分子检测中基因组提取的通用性方法。在扩增效率、保真性等的比较研究中,I-5TM 2×High-Fidelity Master Mix在浆果DNA扩增中效果最好。通过对不同条形码序列扩增效率、测序成功率、单片段和组合片段的物种分辨率评价,筛选出易于标准化、可靠性高的浆果DNA条形码序列。研究结果表明:短片段rbcL(250bp)、psbA-trnH(450bp)和长片段matK(890bp)的浆果物种鉴定效果最好。其中,rbcL、psbA-trnH和matK序列的扩增成功率和测序成功率最高,均为100%。在单片段物种鉴别中,NCBI数据库比对中物种鉴定成功率为psbA-trnH= matKtrnLc/drbcLBEL1/3BEL 2/3trnLg/h,组合片段中 matK+ psbA-trnH、rbcL+psbA-trnHcL和rbcL+trnLc/d可以提高物种的识别效率,但考虑到trnLc/d的扩增效率,最终选择rbcL、psbA-trnH和watK作为浆果DNA条形码序列。进一步构建的系统发育树验证3个序列NCBI鉴定结果的准确性。另外,模拟不同加工方式对DNA断裂以致对检测结果的影响,建立了单一样品和混合样品基于Sanger测序的DNA条形码方法。结果显示,极端加工方式高温短时杀菌下短片段rbcL仍有扩增,psbA-trnH次之,matK严重降解。因此,短片段在极端处理下影响较小,基本可以用于所有浆果制品,而psbA-trnH和marK的应用范围小于rbcL序列。在模拟混合果汁的克隆测序中,鲜榨果汁、巴氏杀菌处理和高温短时杀菌处理均能检测到10%的目标浆果成分,部分甚至可以检测到1%,建立浆果单一样品直接测序和混合样品克隆测序的DNA条形码方法。采用上述建立的DNA条形码鉴别方法,对市场采集的果干、果酱、浑浊果汁以及果汁饮料共计33份样品(5种果干、12种果酱、16种果汁和果汁饮料)进行了基于rbcL和psbA-trnH的DNA条形码序列鉴定。结果显示:33份样品中,检出成分同标签相符的占48.49%,与标签不符的占45.45%,失败6.06%。研究结果表明,DNA条形码技术可以用于市售浆果制品单一成分和复杂成分的真伪鉴别,为生产的质量控制以及进出口检验检疫监控提供新的控制和检测手段。
[Abstract]:As a high value-added fruit, berry is more and more popular in daily life. However, due to the shorter harvest period and higher price, the adulteration, forgery and false labeling of berry and its products lead to food quality and safety controversy. At present, the identification of berry and its products is still in its infancy in China. In this paper, a DNA barcode method based on Angel sequencing technology was established and applied to the detection of commercial berry products. The detection range involved complete blended juice, cheaper fruit instead of high-priced berry products and the conformance test of labels. The results showed that the quality of DNA obtained by Macherey-Nagel, TIANGEN and Magen kits was poor, and the DNA was degraded and contaminated seriously; Qiagen, Promega and Biotecon kits were able to obtain better quality DNA. However, the DNA integrity of Promega and Biotecon kits was low, and the amplification efficiency was lower than that of Qiagen kits. Finally, Qiagen kits were used as a general method for genome extraction in berry molecular detection. The results showed that short fragment rbcL (250 bp), psbA-trnH (450 bp) and long fragment matK (890 bp) were the best for berry species identification. CL, psbA-trnH and matK sequences had the highest amplification and sequencing success rates of 100%. In single-fragment species identification, the NCBI database comparison showed that the success rate of species identification was psbA-trnH=matK trnLc/drbcLBEL1/3BEL 2/3trnLg/h, and the combination fragments matK+psbA-trnH, rbcL+psbA-trnH cL and rbcL+trnLc/d could improve the efficiency of species identification. However, considering the amplification efficiency of trnLc/d, rbcL, psbA-trnH and watK were selected as the barcode sequences of berry DNA. Further phylogenetic trees were constructed to verify the accuracy of the NCBI identification results of the three sequences. The results showed that short fragments of rbcL were amplified in the extreme processing mode, followed by psbA-trnH and matK, which were degraded severely. Therefore, short fragments had little effect on the extreme processing, and could be used in almost all berry products, while psbA-trnH and marK had less application scope than rbcL sequence. In cloning and sequencing, fresh juice, pasteurization and short-term high-temperature sterilization could detect 10% of the target berry components, and some could even detect 1%. A DNA barcode method for direct sequencing of single berry sample and cloning and sequencing of mixed berry samples was established. DNA barcode sequencing based on rbcL and psbA-trnH was carried out in 33 samples (5 dried fruits, 12 jams, 16 juices and juice drinks). The results showed that 48.49% of the 33 samples were identical with the labels, 45.45% were not identical with the labels, and 6.06% were unsuccessful. Shape code technology can be used to identify the authenticity and falsity of single and complex components of berry products on the market, and provide new control and detection means for quality control of production and inspection and quarantine monitoring of import and export.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：TS201.6;TP391.44

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