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中國精品科技期刊2020

微滴式數字PCR定量檢測歐洲甜櫻桃中摻假蘋果成分的研究

陳佳 秦麗 劉浩 楊帛 張凱江 趙冬梅 郭金穎 張媛媛

陳佳,秦麗,劉浩,等. 微滴式數字PCR定量檢測歐洲甜櫻桃中摻假蘋果成分的研究[J]. 食品工業科技,2022,43(19):349?355. doi:  10.13386/j.issn1002-0306.2022010002
引用本文: 陳佳,秦麗,劉浩,等. 微滴式數字PCR定量檢測歐洲甜櫻桃中摻假蘋果成分的研究[J]. 食品工業科技,2022,43(19):349?355. doi:  10.13386/j.issn1002-0306.2022010002
CHEN Jia, QIN Li, LIU Hao, et al. Quantitative Detection of Adulterated Apple Components in European Sweet Cherries by Droplet Digital PCR[J]. Science and Technology of Food Industry, 2022, 43(19): 349?355. (in Chinese with English abstract). doi:  10.13386/j.issn1002-0306.2022010002
Citation: CHEN Jia, QIN Li, LIU Hao, et al. Quantitative Detection of Adulterated Apple Components in European Sweet Cherries by Droplet Digital PCR[J]. Science and Technology of Food Industry, 2022, 43(19): 349?355. (in Chinese with English abstract). doi:  10.13386/j.issn1002-0306.2022010002

微滴式數字PCR定量檢測歐洲甜櫻桃中摻假蘋果成分的研究

doi: 10.13386/j.issn1002-0306.2022010002
基金項目: 河北省市場監管局科技計劃項目(2022ZD36);石家莊學院博士科研啟動基金(21BS004)。
詳細信息
    作者簡介:

    陳佳(1982?),女,博士,講師,研究方向:食品營養與安全,E-mail:chenjia0311@aliyun.com

    秦麗(1981?),女,碩士,高級工程師,研究方向:食品安全,E-mail:88426413@qq.com

    通訊作者:

    張媛媛(1983?),女,博士,副教授,研究方向:食品微生物、植物活性物提取,E-mail:zhyy701@163.com

  • 中圖分類號: TS251.7

Quantitative Detection of Adulterated Apple Components in European Sweet Cherries by Droplet Digital PCR

  • 摘要: 目的:基于微滴式數字PCR技術(Droplet Digital PCR,ddPCR)建立歐洲甜櫻桃中摻假蘋果成分定量檢測的方法。方法:通過篩選歐洲甜櫻桃、蘋果成分特異性引物探針,建立ddPCR定量檢測方法,構建質量和DNA含量的擬合曲線及DNA含量與拷貝數之間的擬合曲線,以DNA含量作為中間值,獲得質量和擴增拷貝數的計算公式。其中線性擬合曲線R2均在0.99以上,且建立了相應的摻假模型以驗證方法的準確性。結果:歐洲甜櫻桃和蘋果的質量和擴增DNA拷貝數的計算公式:M=0.0833C?3.8420、M=0.4084C?1.5747。在歐洲甜櫻桃與蘋果的摻假模型中相對誤差最大為?19.17%,符合統計學要求。結論:建立歐洲甜櫻桃制品快速檢測方法為打擊歐洲甜櫻桃摻雜使假提供了強有力手段,保障了消費者的權益,對促進小漿果及其制品行業健康發展具有重要意義。
  • 圖  1  歐洲甜櫻桃引物特異性篩選

    Figure  1.  Screening of cherry specific primers

    注:A01、A02為杏;A03、A04為蘋果;A05、A06為桃;A07、A08為梨;A09、A10為歐洲甜櫻桃;A11、A12為葡萄;B1、B2為樹莓;B3、B4為小櫻桃;B5、B6為蔓越莓;B07、B08為黑加侖;B09、B10為ddH2O。

    圖  2  蘋果引物特異性篩選

    Figure  2.  Screening of apple specific primers

    注:C02、C03為杏;C04、C05為蘋果;C06、C07為桃;C08、C09為梨;C10、C11為歐洲甜櫻桃;C12、D01為葡萄;D02、D03為樹莓;D04、D05為小櫻桃;D06、D07為蔓越莓;D08、D09為黑加侖;C01、D10為ddH2O。

    圖  3  歐洲甜櫻桃質量和DNA濃度的關系

    Figure  3.  Relationship between mass and DNA concentration of cherry

    圖  4  蘋果質量和DNA濃度的關系

    Figure  4.  Relationship between mass and DNA concentration of apple

    圖  5  梯度DNA含量條件下歐洲甜櫻桃(a)、蘋果(b)的拷貝數圖譜

    Figure  5.  Copies number map of cherry (a) and apple (b) under gradient DNA content conditions

    注:A、B為歐洲甜櫻桃;A01、A02、A03為5 ng/μL;A04、A05、A06為10 ng/μL;A07、A08、A09為20 ng/μL;A10、A11、A12為40 ng/μL;B01、B02、B03為60 ng/μL;B04、B05、B06為80 ng/μL;B07、B08、B09為100 ng/μL;B10、B11、B12為ddH2O。E、F為蘋果;E01、E02、E03為5 ng/μL;E04、E05、E06為10 ng/μL;E07、E08、E09為20 ng/μL;E10、E11、E12為40 ng/μL;F01、F02、F03為60 ng/μL;F07為陽性;F05、F08、F09、F10為ddH2O。

    圖  6  歐洲甜櫻桃DNA濃度和DNA拷貝數之間的關系

    Figure  6.  Linear relationship between DNA copy number and nucleic acid concentration of cherry

    圖  7  蘋果DNA濃度和DNA拷貝數之間的關系

    Figure  7.  Linear relationship between DNA copy number and nucleic acid concentration of apple

    表  1  漿果實驗所需引物和探針

    Table  1.   Primers and probes required for berry experiments

    引物、探針
    名稱
    序列
    (5′→3′)
    來源篩選
    結果
    歐洲甜櫻桃-FCCAGACAGGCAAGAGCGATTEF
    165590.1
    歐洲甜櫻桃-RGGAGACACGTGGGTCTGATAGG
    歐洲甜櫻桃-ProbeTCCGCCGATGCGTG
    蘋果-FGGAATATGAACGAAAGAGCGFM
    887031.1
    蘋果-RATCCGTTGCCGAGAGTCGTT
    蘋果-ProbeTGCGTCGTCGTCTTCGATAA
    下載: 導出CSV

    表  2  已知比例歐洲甜櫻桃與蘋果摻假模型櫻桃分析結果

    Table  2.   Analysis results of cherries with known proportion of cherries and apple adulteration model


    實際質量
    (mg)
    拷貝數
    (copies/μL)
    平均值
    (copies/μL)
    變異系
    數(%)
    測得質
    量(mg)
    相對誤
    差(%)
    15103100101101.331.514.60?8.02
    210147151149149.001.348.57?14.30
    315196189190191.671.9812.12?19.17
    420267274278273.002.0418.90?5.51
    525340344339341.000.7824.56?1.75
    630389400394394.331.4029.01?3.31
    735495503482493.332.1537.256.44
    840533504499512.003.5938.81?2.98
    945579577587581.000.9144.56?0.99
    下載: 導出CSV

    表  3  已知比例歐洲甜櫻桃與蘋果摻假模型蘋果分析結果

    Table  3.   Analysis results of apple with known proportion of cherries and apple adulteration model


    實際質量
    (mg)
    拷貝數
    (copies/μL)
    平均值
    (copies/μL)
    變異系
    數(%)
    測得質
    量(mg)
    相對誤
    差(%)
    145121.3120.1118.9120.11.0047.475.50
    240104.2103.5108.8105.52.7341.513.78
    33591.691.185.289.33.9934.90?0.30
    43075.273.97574.70.9428.93?3.56
    52560.858.962.760.83.1223.26?6.98
    62048.14948.248.41.0218.19?9.04
    71537.639.237.838.22.2814.03?6.49
    81027.426.827.127.11.119.49?5.07
    9515.81615.915.90.634.92?1.62
    下載: 導出CSV

    表  4  市售樣品分析結果

    Table  4.   Commercial sample analysis results

    樣品檢測項目拷貝數(copies/μL)平均值
    (copies/μL)
    質量比
    (%)
    123
    Y-1歐洲甜櫻桃00000
    蘋果6.710.28.88.5715.37
    Y-2歐洲甜櫻桃00000
    蘋果00000
    Y-3歐洲甜櫻桃00000
    蘋果4.55.34.74.833.17
    Y-4歐洲甜櫻桃00000
    蘋果5.65.14.75.134.15
    下載: 導出CSV
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  • 收稿日期:  2022-01-04
  • 網絡出版日期:  2022-08-19
  • 刊出日期:  2022-09-23

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