【摘要】：作為一種高附加值水果,漿果在日常生活中越來(lái)越受消費(fèi)者歡迎。然而,由于較短的收獲期和較高的價(jià)格,漿果及其制品摻假、造假、錯(cuò)誤標(biāo)簽等導(dǎo)致的食品質(zhì)量和安全爭(zhēng)議不斷發(fā)生。目前,我國(guó)對(duì)漿果及其制品的真?zhèn)巫R(shí)別還處于起步階段。對(duì)此,本文通過(guò)研究新興的DNA條形碼,建立基于Sanger測(cè)序技術(shù)的DNA條形碼方法,并應(yīng)用于市售漿果制品的檢測(cè),檢測(cè)范圍涉及完全勾兌型果汁、廉價(jià)水果替代高價(jià)漿果的產(chǎn)品以及標(biāo)簽的符合性查驗(yàn)。主要開(kāi)展了漿果基因組DNA提取方法研究,對(duì)市場(chǎng)上常見(jiàn)的6種針對(duì)植物樣品的商業(yè)試劑盒進(jìn)行了比較和優(yōu)化,并確定了 PCR體系中的擴(kuò)增酶。結(jié)果顯示:Macherey-Nagel、TIANGEN和Magen試劑盒法所得DNA質(zhì)量較差,DNA嚴(yán)重降解和污染;Qiagen、Promega和Biotecon試劑盒法均能得到質(zhì)量較好的DNA,但Promega和Biotecon試劑盒法的DNA完整性低,擴(kuò)增效率低于Qiagen試劑盒法提取的DNA。最后確定Qiagen試劑盒作為漿果分子檢測(cè)中基因組提取的通用性方法。在擴(kuò)增效率、保真性等的比較研究中,I-5TM 2×High-Fidelity Master Mix在漿果DNA擴(kuò)增中效果最好。通過(guò)對(duì)不同條形碼序列擴(kuò)增效率、測(cè)序成功率、單片段和組合片段的物種分辨率評(píng)價(jià),篩選出易于標(biāo)準(zhǔn)化、可靠性高的漿果DNA條形碼序列。研究結(jié)果表明:短片段rbcL(250bp)、psbA-trnH(450bp)和長(zhǎng)片段matK(890bp)的漿果物種鑒定效果最好。其中,rbcL、psbA-trnH和matK序列的擴(kuò)增成功率和測(cè)序成功率最高,均為100%。在單片段物種鑒別中,NCBI數(shù)據(jù)庫(kù)比對(duì)中物種鑒定成功率為psbA-trnH= matKtrnLc/drbcLBEL1/3BEL 2/3trnLg/h,組合片段中 matK+ psbA-trnH、rbcL+psbA-trnHcL和rbcL+trnLc/d可以提高物種的識(shí)別效率,但考慮到trnLc/d的擴(kuò)增效率,最終選擇rbcL、psbA-trnH和watK作為漿果DNA條形碼序列。進(jìn)一步構(gòu)建的系統(tǒng)發(fā)育樹(shù)驗(yàn)證3個(gè)序列NCBI鑒定結(jié)果的準(zhǔn)確性。另外,模擬不同加工方式對(duì)DNA斷裂以致對(duì)檢測(cè)結(jié)果的影響,建立了單一樣品和混合樣品基于Sanger測(cè)序的DNA條形碼方法。結(jié)果顯示,極端加工方式高溫短時(shí)殺菌下短片段rbcL仍有擴(kuò)增,psbA-trnH次之,matK嚴(yán)重降解。因此,短片段在極端處理下影響較小,基本可以用于所有漿果制品,而psbA-trnH和marK的應(yīng)用范圍小于rbcL序列。在模擬混合果汁的克隆測(cè)序中,鮮榨果汁、巴氏殺菌處理和高溫短時(shí)殺菌處理均能檢測(cè)到10%的目標(biāo)漿果成分,部分甚至可以檢測(cè)到1%,建立漿果單一樣品直接測(cè)序和混合樣品克隆測(cè)序的DNA條形碼方法。采用上述建立的DNA條形碼鑒別方法,對(duì)市場(chǎng)采集的果干、果醬、渾濁果汁以及果汁飲料共計(jì)33份樣品(5種果干、12種果醬、16種果汁和果汁飲料)進(jìn)行了基于rbcL和psbA-trnH的DNA條形碼序列鑒定。結(jié)果顯示:33份樣品中,檢出成分同標(biāo)簽相符的占48.49%,與標(biāo)簽不符的占45.45%,失敗6.06%。研究結(jié)果表明,DNA條形碼技術(shù)可以用于市售漿果制品單一成分和復(fù)雜成分的真?zhèn)舞b別,為生產(chǎn)的質(zhì)量控制以及進(jìn)出口檢驗(yàn)檢疫監(jiān)控提供新的控制和檢測(cè)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.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：TS201.6;TP391.44

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