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浙江大学学报（农业与生命科学版）
浙江大学学报（农业与生命科学版）  2021, Vol. 47 Issue (1): 52-59    DOI: 10.3785/j.issn.1008-9209.2020.04.291
食品科学     
基于细胞色素C氧化酶亚基Ⅰ序列的DNA微条形码技术鉴别11种生鲜肉制品掺假的研究
励炯(),吴琼,扈明洁,金朦娜,邱红钰
杭州市食品药品检验研究院，杭州 310017
Identification of adulteration in 11 fresh meat products by DNA mini-barcoding methods based on cytochrome C oxidase subunit Ⅰ (COⅠ) sequence
Jiong LI(),Qiong WU,Mingjie HU,Mengna JIN,Hongyu QIU
Hangzhou Institute for Food and Drug Control, Hangzhou 310017, China
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摘要：

建立并优化了基于细胞色素C氧化酶亚基Ⅰ（cytochrome C oxidase subunitⅠ, COⅠ）序列的DNA微条形码（DNA mini-barcoding）技术，以检测生鲜肉制品中11种常见肉类的掺假情况。样品经真空冷冻干燥处理、提取DNA后，采用通用引物进行聚合酶链式反应（polymerase chain reaction, PCR）扩增，目标条带扩增产物经切胶、回收、纯化后，进行克隆测序，并将测序结果提交到GenBank数据库（http：//blast.ncbi.nlm.nih.gov/Blast.cgi）中进行Blast比对，筛选出适合猪肉、牛肉、羊肉、鸡肉、鸭肉、鸽子肉、马肉、驴肉、鹅肉、兔肉、鼠肉等11种肉类扩增的通用引物COⅠ-A，并对PCR扩增条件进行优化，建立18个掺假模型，以对低经济价值肉类的最低掺入比例进行考察验证。结果表明：11种肉类的PCR扩增效率均为100%。在这18个掺假模型中，牛肉和羊肉中掺入6种低经济价值肉类（猪肉、鸡肉、鸭肉、马肉、驴肉、鼠肉）的最低检出比例均为5%，而在鹅肉、鸽子肉和兔肉中其最低检出比例为10%。本方法前处理简单，灵敏度高，重现性好，可作为高经济价值生鲜肉制品中掺假低经济价值肉类的有效检测方法。
关键词： DNA微条形码;  肉制品;  细胞色素C氧化酶亚基Ⅰ序列;  掺假    
Abstract:

DNA min-barcoding methods based on cytochrome C oxidase subunitⅠ (COⅠ) sequence were developed to detect adulteration in 11 fresh meat products. DNA extraction was carried out with the DNeasy blood and tissue kit after samples were treated with vacuum freeze drying. The amplification of mini-barcoding region from DNA extracts was carried out using universal primer sets. Polymerase chain reaction (PCR) products were purified with DNA purification kit and analyzed by clone sequencing. The sequencing results were queried in GenBank (http://blast.ncbi.nlm.nih.gov/Blast.cgi) using the Blast align. The primer set COⅠ-A was found to be optimal, showing 100% amplification efficiency in 11 fresh meats (including pork, beef, mutton, chicken, duck, pigeon meat, horseflesh, ass meat, goose, rabbit meat and rat meat) with optimized PCR procedure. Eighteen meat adulteration models were founded to test the minimum detected adulteration rate by low economic value meat, and the results showed that the minimum detected adulteration rates of beef and mutton were 5%, and the rates of pigeon meat, goose, and rabbit meat were 10%, when added by the six low economic value meats (including pork, chicken, duck, horseflesh, ass meat and rat meat). To sum up, the method is simple, sensitive and reproducible, which is suitable for the detection of adulteration in the 11 fresh meat products.
Key words: DNA mini-barcoding    fresh meat product    cytochrome C oxidase subunit Ⅰ sequence    adulteration
收稿日期: 2020-04-29 出版日期: 2021-03-09
CLC:  TS  
基金资助: 浙江省公益技术应用研究计划(LGN19C200001)
通讯作者: 励炯     E-mail: jokelee2@126.com
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引用本文:

励炯,吴琼,扈明洁,金朦娜,邱红钰. 基于细胞色素C氧化酶亚基Ⅰ序列的DNA微条形码技术鉴别11种生鲜肉制品掺假的研究[J]. 浙江大学学报（农业与生命科学版）, 2021, 47(1): 52-59.

Jiong LI,Qiong WU,Mingjie HU,Mengna JIN,Hongyu QIU. Identification of adulteration in 11 fresh meat products by DNA mini-barcoding methods based on cytochrome C oxidase subunit Ⅰ (COⅠ) sequence. Journal of Zhejiang University (Agriculture and Life Sciences), 2021, 47(1): 52-59.

链接本文:

http://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2020.04.291    或    http://www.zjujournals.com/agr/CN/Y2021/V47/I1/52

引物名称

Primer name

上游引物（5′→3′）

Forward primer (5′→3′)

下游引物（5′→3′）

Reverse primer (5′→3′)

COⅠ-A[27]TCCACTAATCACAARGATATTGGTACGAAAATCATAATGAAGGCATGAGC
COⅠ-B[1]GCCTCCAACTCAAAATACGCGTCAAATGGGGCTCGGT
COⅠ-C[1]GGTCAACAAATCATAAAGATATTGGACTATAAAGAAGATTATTACAAAGGC
表1  引物序列
图1  11种纯肉的DNA微条形码PCR电泳图M：DNA分子标志物；1：猪肉；2：牛肉；3：羊肉；4：鸭肉；5：鸡肉；6：马肉；7：鸽子肉；8：驴肉；9：鹅肉；10：兔肉；11：鼠肉。

序号

No.

纯肉类别

Meat species

基因相似度

Genetic

similarity/%

物种匹配结果

Species

matching result

1猪肉 Pork99野猪 Sus scrofa
2牛肉 Beef99原始牛 Bos primigenius
3羊肉 Mutton98山羊 Capra hircus
4鸭肉 Duck98绿头鸭 Anas platyrhynchos
5鸡肉 Chicken99普通家鸡 Gallus gallus
6马肉 Horseflesh99家马 Equus caballus
7

鸽子肉

Pigeon meat

99原鸽 Columba livia
8驴肉 Ass meat98驴 Equus asinus
9鹅肉 Goose98鹅属 Anser
10兔肉 Rabbit meat100家兔 Oryctolagus cuniculus
11鼠肉 Rat meat97大鼠 Rattus norvegicus
表2  11种纯肉的DNA微条形码检测结果

高经济

价值肉类

High economic

value meat

掺假肉类

Adulterating

meat

模型编号

Model No.

灵敏度

Sensitivity/%

牛肉

Beef

猪肉 PorkA15
马肉 HorsefleshA25
驴肉 Ass meatA35
鸭肉 DuckA45
鸡肉 ChickenA55
鼠肉 Rat meatA65

羊肉

Mutton

猪肉 PorkB15
马肉 HorsefleshB25
驴肉 Ass meatB35
鸭肉 DuckB45
鸡肉 ChickenB55
鼠肉 Rat meatB65

鸽子肉

Pigeon meat

鸡肉 ChickenC110
鸭肉 DuckC210

鹅肉

Goose

鸡肉 ChickenD110
鸭肉 DuckD210

兔肉

Rabbit meat

鸡肉 ChickenE110
鸭肉 DuckE210
表3  肉类掺假模型

序号

No.

样品名称

Sample name

DNA微条形码鉴定结果

DNA mini-barcoding

detecting result

基因相似度

Genetic similarity/%

判断结果

Result

1牛肉卷a Beef roll a牛肉97符合
2牛肉卷b Beef roll b牛肉97符合
3牛肉卷c Beef roll c检出猪肉成分99掺假
4牛肉卷d Beef roll d牛肉97符合
5牛排a Beef steak a牛肉97符合
6牛排b Beef steak b牛肉97符合
7牛肉丸a Beef ball a检出猪肉成分99掺假
8牛肉丸b Beef ball b牛肉97符合
9牛肉糜 Ground beef检出鸭肉成分98掺假
10牛肉条a Beef strip a牛肉97符合
11牛肉条b Beef strip b检出猪肉成分99掺假
12羊肉卷a Mutton roll a羊肉98符合
13羊肉卷b Mutton roll b羊肉98符合
14羊肉卷c Mutton roll c羊肉98符合
15羊排 Mutton steak羊肉98符合
16生羊肉串a Fresh mutton shashlik a羊肉98符合
17生羊肉串b Fresh mutton shashlik b羊肉98符合
18生羊肉串c Fresh mutton shashlik c检出鸭肉成分98掺假
19羊肉丸a Mutton ball a羊肉98符合
20羊肉丸b Mutton ball b检出猪肉成分99掺假
21鹅肉块 Goose block鹅肉97符合
22鹅肉丸Goose ball鹅肉97符合
23兔肉块a Rabbit block a兔肉100符合
24兔肉块b Rabbit block b兔肉100符合
25鸽子肉 Pigeon meat鸽子肉99符合
表4  实际样品检测结果
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