肉制品中牛源性成分Taqman荧光定量PCR检测法的建立
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摘要
本实验根据牛线粒体差异序列设计特异性PCR引物和Taqman探针,并基于16S r DNA内参基因设计通用引物及Taqman探针,绘制并通过标准曲线确定样品中总DNA浓度以及牛源性成分DNA浓度,采用相对定量法测定肉制品中牛源性成分的质量分数。并通过特异性、灵敏度实验,及混合肉样回收率及市售肉制品检测,对本方法进行验证。结果表明,方法特异性强,可对牛肉DNA进行特异性扩增。最低检出限为10 pg/μL,具有较高的灵敏度。并根据回收实验可知,平均回收率为98.62%。可以为肉制品中牛肉含量的测定提供技术参考。
In this experiment,specific PCR primers and Taqman probes were designed based on the differential mitochondrial DNA sequences of bovine mitochondria,and based on the 16 S r DNA internal reference gene,universal primers and Taqman probes were designed. The total DNA concentration and DNA concentration of bovine source samples were determined by standard curve.Relative quantification method was used to determine the quality fraction of bovine source in meat products.The method was validated by specificity,sensitivity experiments,mixed meat recovery,and commercial meat testing.The results show that that method is specific and can amplify specific DNA of beef. The minimum observable limit with high sensitivity is10 pg/μL. According to the recovery test,the average recovery was 98.62%,which can provide technical reference for the determination of beef content in meat products.
In this experiment,specific PCR primers and Taqman probes were designed based on the differential mitochondrial DNA sequences of bovine mitochondria,and based on the 16 S r DNA internal reference gene,universal primers and Taqman probes were designed. The total DNA concentration and DNA concentration of bovine source samples were determined by standard curve.Relative quantification method was used to determine the quality fraction of bovine source in meat products.The method was validated by specificity,sensitivity experiments,mixed meat recovery,and commercial meat testing.The results show that that method is specific and can amplify specific DNA of beef. The minimum observable limit with high sensitivity is10 pg/μL. According to the recovery test,the average recovery was 98.62%,which can provide technical reference for the determination of beef content in meat products.
引文
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[13]吴迪,郭青.Taq Man实时荧光定量PCR法检测紫河车掺伪的研究[J].中药材,2017,40(1):38-41.
[14]陈默颖.超微量核酸蛋白测定仪在核酸和蛋白质浓度测定方面的应用[C]//全国光谱仪器与分析监测学术研讨会,2013.
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[16]董志雪,唐蜻,程超华,等.悬铃叶苎麻基因组DNA的六种提取方法比较[J].中国麻业科学,2015(5):233-238.
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[20]高丹丹,曹郁生,王迎华.PCR技术在食品动物源性检测中的应用[J].食品研究与开发,2007,28(1):141-144.
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[2]李宗梦,赵良娟,赵宏,等.肉及肉制品动物源性成分鉴别技术研究进展[J].食品研究与开发,2014(18):122-127.
[3]钟文涛,王芳妹,李白玉,等.四重PCR反应体系的建立及在牛肉掺假检测中的应用[J].现代生物医学进展,2016,16(18):3574-3577.
[4]冼钰茵,易敏英,张璜,等.环介导等温扩增技术快速检测肉及肉制品中的牛源性成分[J].食品工业科技,2016,37(7):278-282.
[5]郭燕华,王德莲,王强,等.重组酶介导等温扩增技术快速检测牛肉及牛肉制品中的牛源性成分[J].食品安全质量检测学报,2017,8(5):1745-1749.
[6]苗丽,张秀平,陈静,等.微滴数字PCR法对肉制品中牛源和猪源成分的定量分析[J].食品科学,2016,37(8):187-191.
[7]汪永信,安虹,程坚,等.双重实时荧光PCR法检测食品和饲料中的鸡源性成分[J].生物技术通报,2012(1):134-138.
[8]赵新,刘娜,陈锐,等.应用通用引物量化检测鲜肉及其制品中羊源性成分[J].食品研究与开发,2016,37(16):179-184.
[9]K9ppel R,Ruf J,Zimmerli F,et al.Multiplex real-time PCR for the detection and quantification of DNA from beef,pork,chicken and turkey[J].European Food Research and Technology,2008,227(4):1199-1203.
[10]刘娜,赵新,陈锐,等.动物肌肉组织DNA的提取方法及实时荧光定量PCR检测[J].食品工业科技,2016,37(18):74-80.
[11]杨彤晖,孟镇,钟其顶,等.猪肉类罐头食品中总DNA提取方法的比较[J].食品与发酵工业,2015,41(4):62-67.
[12]王阿娜,裴瑾,刘薇,等.桑叶片总RNA提取方法的比较研究[J].中成药,2012,34(7):1377-1380.
[13]吴迪,郭青.Taq Man实时荧光定量PCR法检测紫河车掺伪的研究[J].中药材,2017,40(1):38-41.
[14]陈默颖.超微量核酸蛋白测定仪在核酸和蛋白质浓度测定方面的应用[C]//全国光谱仪器与分析监测学术研讨会,2013.
[15]赵新,王永,兰青阔,等.荧光定量PCR方法鉴别肉制品中羊源性成分[J].食品工业科技,2015,36(1):299-302.
[16]董志雪,唐蜻,程超华,等.悬铃叶苎麻基因组DNA的六种提取方法比较[J].中国麻业科学,2015(5):233-238.
[17]Druml B,Kaltenbrunner M,Hochegger R,et al.A novel reference real-time PCR assay for the relative quantification of(game)meat species in raw and heat-processed food[J].Food Control,2016,70:392-400.
[18]Calvo J H,Zaragoza P,Osta R.Random Amplified Polymorphic DNA Fingerprints for Identification of Species in Poultry Paté[J].Poultry Science,2001,80(4):522.
[19]胡谦,陈颖,倪凯,等.肉制品异源基因检测技术研究进展[J/OL].食品科学:1-10(2017-10-13).http://kns.cnk.net/kcms/detail/11.2206.TS 20171013.1056.082.html.
[20]高丹丹,曹郁生,王迎华.PCR技术在食品动物源性检测中的应用[J].食品研究与开发,2007,28(1):141-144.
[21]Listed N.Proceedings of the National Academy of Sciences,USA.[J].Current Biology Cb,1996,6(6):628.
[22]Ebbehoj K F,Thomsen P D.Differentiation of closely related species by DNA hybridization.[J].Meat Science,1991,30(4):359-66.