双重实时荧光PCR定量检测动物产品中牛源性成分
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摘要
旨在建立一种定量检测动物产品中牛源性成分含量的双重实时荧光PCR方法。以牛卫星IV DNA为检测靶基因,以肌肉生长抑制素基因作为内对照合成引物探针,采用基于TaqMan的双重实时荧光PCR技术对已知牛肉添加比例的冻干混合肉粉标准品进行检测并建立线性模型,并应用模拟样品和送检样品进行了验证检测。结果显示,该方法可检出牛源成分含量为0.1%的冻干肉粉,不与其他种动物组织发生交叉反应,重复性试验的相对标准偏差小于5%,方法的扩增效率为93.6%。该方法适用于肉品中牛源性成分的定量检测,可用于测定市场上动物产品中的牛源性成分含量。
The objective of this work is to develop a duplex real-time quantitative PCR method for detecting the amount of bovine derived material in animal product. Using bovine satellite IV DNA as detection target gene and myostatin gene as an internal control to synthesize primerprobe,the standard freeze-dried meat mixture powder with known beef proportion was detected by duplex real-time PCR based on TaqMan,and the linear model was established. Then the model was verified by detecting mocked and delivered samples. The method detected 0.1%bovine material in freeze-dried meat powder,and no cross reaction with the tissues of other animal species occurred. The relative standarddeviation of repeatability test was < 5%,and the amplification efficiency of the method was 93.6%. Conclusively,the established method issuitable for quantitative detection of bovine derived material in meat products,and can be used to determine the content of bovine derived material in animal products on the market.
The objective of this work is to develop a duplex real-time quantitative PCR method for detecting the amount of bovine derived material in animal product. Using bovine satellite IV DNA as detection target gene and myostatin gene as an internal control to synthesize primerprobe,the standard freeze-dried meat mixture powder with known beef proportion was detected by duplex real-time PCR based on TaqMan,and the linear model was established. Then the model was verified by detecting mocked and delivered samples. The method detected 0.1%bovine material in freeze-dried meat powder,and no cross reaction with the tissues of other animal species occurred. The relative standarddeviation of repeatability test was < 5%,and the amplification efficiency of the method was 93.6%. Conclusively,the established method issuitable for quantitative detection of bovine derived material in meat products,and can be used to determine the content of bovine derived material in animal products on the market.
引文
[1]Jonker KM, Tilburg JJ, Hagele GH, et al. Species identification in meat products using real-time PCR[J]. Food Additives and Contaminants, 2008, 25:527-533.
[2]Ren YF, Li X, Liu YM, et al. A novel quantitative real-time PCR method for identification and quantification of mammalian and poultry species in foods[J]. Food Control, 2017, 76:42-51.
[3]Brodmann PD, Moor D. Sensitive and semi-quantitative TaqManTM real-time polymerase chain reaction systems for the detection of beef(Bos taurus)and the detection of the family Mammalia in food and feed[J]. Meat Science, 2003, 65:599-607.
[4]GB/T 21103-2007.动物源性饲料中哺乳动物源性成分定性检测方法实时荧光PCR方法[S].中国国家标准. 2017.
[5]Ines L, Jutta Z, Almuth S, et al. Development and design of a‘readyto-use’ reaction plate for a PCR-based simultaneous detection of animal species used in foods[J]. Int J Food Sci Technol, 2007, 42(1):9-17.
[6]Koppel R, Daniels M, Felderer N, et al. Multiplex real-time PCR for the detection and quantification of DNA from duck, goose, chicken,Turkey and pork[J]. Eur Food Res Technol, 2013, 2:1093-1098.
[7]Koppel R, Zimmerli F, Breitenmoser A. Heptaplex real-time PCR for the detection and quantification of DNA from beef, pork, chicken,Turkey, horse meat, sheep(mutton)and goat[J]. Eur Food Res Technol, 2009 230(1):125-133.
[8]Ballin NZ, Vogensen FK, Karlsson AH. Species determination. Can we detect and quantify meat adulteration?[J]. Meat Science,2009, 83:165-174.
[9]林露,严维凌,朱祖琪,等.实时荧光PCR技术快速检测混合肉制品中牛肉含量[J].中国食品学报, 2016(3):152-159.
[10]陈志宣,龚国利,钱云开.阿胶DNA提取方法优化[J].中国实验方剂学杂志, 2014, 20(21):13-16.
[2]Ren YF, Li X, Liu YM, et al. A novel quantitative real-time PCR method for identification and quantification of mammalian and poultry species in foods[J]. Food Control, 2017, 76:42-51.
[3]Brodmann PD, Moor D. Sensitive and semi-quantitative TaqManTM real-time polymerase chain reaction systems for the detection of beef(Bos taurus)and the detection of the family Mammalia in food and feed[J]. Meat Science, 2003, 65:599-607.
[4]GB/T 21103-2007.动物源性饲料中哺乳动物源性成分定性检测方法实时荧光PCR方法[S].中国国家标准. 2017.
[5]Ines L, Jutta Z, Almuth S, et al. Development and design of a‘readyto-use’ reaction plate for a PCR-based simultaneous detection of animal species used in foods[J]. Int J Food Sci Technol, 2007, 42(1):9-17.
[6]Koppel R, Daniels M, Felderer N, et al. Multiplex real-time PCR for the detection and quantification of DNA from duck, goose, chicken,Turkey and pork[J]. Eur Food Res Technol, 2013, 2:1093-1098.
[7]Koppel R, Zimmerli F, Breitenmoser A. Heptaplex real-time PCR for the detection and quantification of DNA from beef, pork, chicken,Turkey, horse meat, sheep(mutton)and goat[J]. Eur Food Res Technol, 2009 230(1):125-133.
[8]Ballin NZ, Vogensen FK, Karlsson AH. Species determination. Can we detect and quantify meat adulteration?[J]. Meat Science,2009, 83:165-174.
[9]林露,严维凌,朱祖琪,等.实时荧光PCR技术快速检测混合肉制品中牛肉含量[J].中国食品学报, 2016(3):152-159.
[10]陈志宣,龚国利,钱云开.阿胶DNA提取方法优化[J].中国实验方剂学杂志, 2014, 20(21):13-16.