Identification of Adulterated Animal-derived Ingredients in Edible Animal Viscera Based on Capillary Gel Electrophoresis and DNA Barcoding Techniques
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摘要: 建立并优化了使用基于DNA条形码技术对可食用内脏制品中包括猪、牛、羊、鸡、鸭、鹅、兔7种常见动物源成分进行掺假鉴别的方法。用生理盐水清洗和真空冷冻干燥预处理后的内脏样品，经DNA提取扩增后，扩增产物经毛细管凝胶电泳分析系统进行确认，克隆测序结果提交本地数据库Viscera进行比对，同时筛选出适合7种动物源内脏DNA扩增的通用引物COI-A，优化DNA模板量和退火温度，验证考察了19个可食用内脏掺假模型的最低掺假比例。7种动物的5类内脏的PCR扩增效率均为100%，最佳的DNA模板量和退火温度为2 μL和53 ℃，掺假成分的最低检出比例为5%。本方法灵敏度高，可靠性好，可作为常见可食用动物内脏掺假的有效检测方法。
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关键词:
- 毛细管凝胶电泳 /
- DNA条形码 /
- 内脏 /
- 细胞色素C氧化酶亚基Ⅰ /
- 掺假
Abstract: A DNA barcoding method with cytochrome C oxidase subunit I sequence (COI) was developed to identify 7 adulterated animal-derived components (including pig, cattle, sheep, chicken, duck, goose and rabbit) in edible viscera products. Samples were cleaned with physiological saline and pretreated by vacuum freeze drying before DNA extraction and amplification. PCR products were confirmed by capillary gel electrophoresis analysis system, and the cloned sequencing results were submitted to the local database (Viscera) for comparison. The universal primer set COI-A was used for the amplification, and the amount of DNA template and annealing temperature were optimized. Meanwhile, the minimum adulteration percentage of 19 edible viscera adulteration models was validated and examined. Results showed that the 5 viscera sources from 7 animal species can be completely amplified under the above conditions, the optimal DNA template volume and annealing temperature are 2 μL and 53 ℃ respectively, and the minimum detection percentage of adulterated components was 5%. The method is sensitive and reliable, which can be used for the identification of adulterated 7 animal-derived components in the edible viscera products.-
Key words:
- capillary gel electrophoresi /
- DNA barcoding /
- viscera /
- cytochrome coxidase subunit I /
- adulteration
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图 1 不同前处理方式（A）和不同取样量（B）对鸭肠掺假模型（含10%鸡源性成分）中的掺假成分检出率的影响
Figure 1. Effect of different pretreatment methods (A) and sampling volumes (B) on the detection rate of adulterated ingredients in the duck sausage adulteration model (containing 10% chicken-derived ingredients)
注：***代表P<0.001；****代表P<0.0001。
图 2 不同DNA提取方法对可食用动物内脏DNA纯度的总体影响统计
Figure 2. Statistics on the overall effect of different DNA extraction methods on the purity of DNA from edible visceral
图 3 7种动物源的可食用内脏（肝）DNA条形码的PCR扩增电泳图
Figure 3. Electrophoresis images of DNA barcoding fragments amplified by PCR from liver of 7 anmials
注：M：Marker（15~1500 bp）；1：猪源性；2：牛源性；3：羊源性；4：鸭源性；5：鸡源性；6：鹅源性； 7：兔源性。
图 4 DNA模板量与不同退火温度对7种动物源的可食用内脏DNA条形码的PCR扩增效率的影响
Figure 4. Effect of DNA template amount and different annealing temperatures on the PCR amplification efficiency of edible visceral DNA barcodes from seven animal source
表 1 基因引物序列
Table 1. Primer sequence of genes
引物 上游引物（F-Primer 5′-3′） 下游引物（R-Primer 5′-3′） COI-A[27] TGTAAAACGACGGCCAGTTCTCAACCAACCACAARGAYATYGG CAGGAAACAGCTATGACTAGACTTCTGGGTGGCCRAARAAYCA COI-B[26] TGTAAAACGACGGCCAGTICTCAACCAACCACAAAGACATIGG CAGGAAACAGCTATGACTAGACTTCTGGGTGGCCAAAGAATCA COI-C[26] TGTAAAACGACGGCCAGTTCTCAACCAACCAIAAIGALATIGG CAGGAAACAGCTATGACTAGACTTCTGGGTGICCIAAIAAICA 
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表 2 7种动物源内脏的DNA条形码检测结果
Table 2. DNA barcoding results for 7 samples found to contain one species
序号（NO.） 内脏来源 基因相似度 物种匹配结果 1 猪 99% Sus scrofa（野猪） 2 牛 98% Bos primigenius（原始牛） 3 羊 99% Capra hircus（山羊） 4 鸭 98% Anas platyrhynchos（绿头鸭） 5 鸡 100% Gallus gallus（普通家鸡） 6 鹅 98% Anser（鹅属） 7 兔 100% Oryctolagus cuniculus（家兔）
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表 3 可食用内脏掺假模型
Table 3. Animal viscera adulteration model
模型编号 高经济价值内脏 掺假内脏 灵敏度（%） 1 牛肝 猪肝 5 2 羊肝 猪肝 5 3 兔肝 猪肝 5 4 鹅肝
鹅肝鸭肝 5 5 鸡肝 5 6 牛胃 猪胃 5 7 羊胃 猪胃 5 8 鹅胃
鹅胃鸡胃 5 9 鸭胃 5 10 牛肠 猪肠 5 11 羊肠 猪肠 5 12 鹅肠
鹅肠鸡肠 5 13 鸭肠 5 14 牛肾 猪肾 5 15 羊肾 猪肾 5 16 鹅肾
鹅肾鸡肾 5 17 鸭肾 5 18 牛肺 猪肺 5 19 羊肺 猪肺 5
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表 4 实际样品检测结果
Table 4. Actual sample test results
序号
（NO.）样品名称 DNA条形
码鉴定结果基因相似
度（%）判断
结果实时荧光定
性PCR法结果1 牛肝a 牛源性成分 98 符合 牛源性成分 2 牛肝b 牛源性成分 98 符合 牛源性成分 3 牛肝c 牛源性成分 98 符合 牛源性成分 4 羊肝a 羊源性成分 99 符合 羊源性成分 5 羊肝b 羊源性成分 99 符合 羊源性成分 6 鹅肝a 鹅源性成分 99 符合 鹅源性成分 7 鹅肝b 鹅源性成分 99 符合 鹅源性成分 8 鹅肝c 鹅源性成分 99 符合 鹅源性成分 9 鹅肝d 鸭源性成分 98 掺假 鸭源性成分 10 鹅肝e 鹅源性成分 99 符合 鹅源性成分 11 牛百叶a 牛源性成分 98 符合 牛源性成分 12 牛百叶b 牛源性成分 98 符合 牛源性成分 13 牛百叶c 牛源性成分 98 符合 牛源性成分 14 牛百叶d 猪源性成分 97 掺假 猪源性成分 15 牛百叶e 牛源性成分 98 符合 牛源性成分 16 牛百叶f 猪源性成分 97 掺假 猪源性成分 17 牛肠a 牛源性成分 98 符合 牛源性成分 18 牛肠b 牛源性成分 98 符合 牛源性成分 19 鹅肠a 鹅源性成分 99 符合 鹅源性成分 20 鹅肠b 鹅源性成分 99 符合 鹅源性成分 21 鹅肠c 鸭源性成分 99 掺假 鸭源性成分 22 鹅肠d 鹅源性成分 99 符合 鹅源性成分 23 牛肺 牛源性成分 98 符合 牛源性成分 24 羊肺a 羊源性成分 99 符合 羊源性成分 25 羊肺b 羊源性成分 99 符合 羊源性成分
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