实时荧光聚合酶链式反应技术测定肉制品中犬源性成分
Determination of Dog-Originated Ingredient in Meat Products by Real-Time PCR Technology
摘 要
根据犬线粒体脱氧核糖核酸（DNA）序列设计了特异性引物和探针，应用实时荧光聚合酶链式反应技术，检测了肉制品中犬源性成分的含量。所提取的17种犬的DNA样品扩增后均能得到典型S形扩增曲线，而所选取的14种其他动物源的DNA样品均未呈现同样的扩增曲线；同一模板所得Ct值（n=7）的相对标准偏差为1.2%。这说明所设计的条件具有较好的特异性和重复性。犬源性组分DNA的质量分数在0.000 01%~10%内与其Ct值之间呈线性关系，检测的灵敏度为0.1 pg，不同加工方法所得肉制品的扩增结果之间无显著差异。
犬源性成分
肉制品
线粒体脱氧核糖核酸
real-time fluorescence PCR technology
dog-originated ingredient
meat product
mitochondrial DNA
Abstract
According to dog's mitochondrial DNA (mtDNA) sequence, species-specific primer and probe were designed and real-time fluorescence PCR technology was applied to the determination of dog-originated ingredient in meat products. S-shaped amplification curves for the DNA samples collected from blood of 17 species of dogs were obtained while no such curve was obtained for either of the DNA samples collected from 14 other animal-originated meat; and RSD (n=7) of the values of Ct found from a same mother plate was 1.2%, which were taken as proof for good specificity and repeatability of the designed conditions of determination. Linear relationship between values of Ct and mass fractions of DNA of dog-originated ingredient was obtained in the range of 0.000 01% to 10%, with its sensitivity of 0.1 pg. No significant difference was observed among the results of various methods of processing of the meat products.
中图分类号 O65 DOI 10.11973/lhjy-hx201802017
所属栏目 工作简报
基金项目 宁夏回族自治区重点研发计划（科技惠民）项目（2016KJHM133）
收稿日期 2017/7/24
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备注岳苑(1982-),女,四川蓬溪人,高级工程师,研究方向为食品微生物检测与动物源性成分分析,yueyuan_0421@126.com
引用该论文: YUE Yuan,HE Wei,JIA Bingning,WU Ming,ZHANG Huiling. Determination of Dog-Originated Ingredient in Meat Products by Real-Time PCR Technology[J]. Physical Testing and Chemical Analysis part B:Chemical Analysis, 2018, 54(2): 204～208
岳苑,何微,贾冰凝,吴明,张慧玲. 实时荧光聚合酶链式反应技术测定肉制品中犬源性成分[J]. 理化检验－化学分册, 2018, 54(2): 204～208
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参考文献
【1】GB/T 25165-2010明胶中牛、猪源性成分的定性检测方法实时荧光PCR法[S].
【2】GB/T 21104-2007动物源性饲料中反刍动物源性成分(牛、羊、鹿)定性检测方法PCR方法[S].
【3】GB/T 21107-2007动物源性饲料中马、驴源性成分定性检测方法PCR方法[S].
【4】GB/T 21102-2007动物源性饲料中兔源性成分定性检测方法实时荧光PCR方法[S].
【5】GB/T 21105-2007动物源性饲料中狗源性成分定性检测方法PCR方法[S].
【6】GB/T 21100-2007动物源性饲料中骆驼源性成分定性检测方法PCR方法[S].
【7】RODRIÄGUEZ M A, GARCIÄA T, GONZÄLEZ I, et al. Identification of goose, mule duck, chicken, turkey, and swine in foie gras by species-specific polymerase chain reaction[J]. Agricultural and Food Chemistry, 2003,51:1524-1529.
【8】DALMASSO A, FONTANELLA E, PIATTI P, et al. A multiplex PCR assay for the identfication of animal species in feedstuffs[J]. Molecolar and Cellular Probes, 2004,18:81-87.
【9】RODRIGUEZ M A, GARCIA T, GONZALEZ I, et al. PCR identifieation of beef,sheep,goat and pork in raw and heat-treated meat mixtures[J]. Journal of Food Protection, 2004,67(1):172-177.
【10】HAUNSHI S, BASUMATARY R, GIRISH P S, et al. Identification of chicken, duck, pigeon and pig meat by species-specific markers of mitochondrial origin[J]. Meat Science, 2009,83(3):454-459.
【11】DOOLEY J J, PAINE K E, GARRETT S D, et al. Detection of meat species using TaqMan real-time PCR assays[J]. Meat Science, 2004,68:431-438.
【12】JONKER K M, TILBURG J J, HAGELE G H, et al. Species identification in meat products using real-time PCR[J]. Food Additives and Contaminants, 2008,25(5):527-533.
【13】KESMEN Z, GULLUCE A, SAHIN F, et al. Identification of meat species by TaqMan-based real-time PCR assay[J]. Meat Science, 2009,82:444-449.
【14】KESMEN Z, YETIMAN A E, SAHIN F, et al. Detection of chicken and turkey meat in meat mixtures by using real-time PCR assays[J]. Journal of Food Science, 2012,77(2):167-173.
【15】KOPPEL R, ZIMMERLI F, BREITENMOSER A. Heptaplex real-time PCR for the identification and quantification of DNA from beef,pork,chicken,turkey,horse meat,sheep and goat[J]. European Food Research and Technology, 2009,230(1):125-133.
【16】SAWYER J, WOOD C, SHANAHAN D, et al. Real-time PCR for quantitative meat species testing[J]. Food Control, 2003,14(8):579-583.
【17】王萍,乔勇升,韩芷玲.猪源性成分检测中3种DNA提取方法比较[J].生物加工过程, 2015,12(6):61-64.
【18】陈茹,林志雄,刘琳琳.应用PCR等核酸技术检验动物饲料中牛羊组织成分[J].中国兽医科技, 2001,31(9):3-8.
【19】曹际娟,卢行安,秦成,等.实时荧光PCR技术检测肉骨粉中牛羊源性成分的方法[J].生物技术通讯, 2002,13(2):158-160.
【20】杨宝华,宗卉.根据线粒体基因的特异性鉴别检测饲料中的动物源性成分[J].科学试验与研究, 2000(5):1-3.
【21】庞杰,王金玲,张莹,等.饲料中鱼源性成分定量检测的实时荧光PCR技术的建立[J].中国兽医学报, 2010(5):680-692.
【22】冯海永,韩建林.羊肉产品中若干动物源性成分的七重PCR检测技术应用研究[J].中国畜牧兽医, 2010,37(9):85-90.
【23】孙敏,高宏伟,龚方,等.利用线粒体COⅠ序列快速检测狐狸源性成分[J].畜牧与兽医, 2013,45(7):57-60.
【2】GB/T 21104-2007动物源性饲料中反刍动物源性成分(牛、羊、鹿)定性检测方法PCR方法[S].
【3】GB/T 21107-2007动物源性饲料中马、驴源性成分定性检测方法PCR方法[S].
【4】GB/T 21102-2007动物源性饲料中兔源性成分定性检测方法实时荧光PCR方法[S].
【5】GB/T 21105-2007动物源性饲料中狗源性成分定性检测方法PCR方法[S].
【6】GB/T 21100-2007动物源性饲料中骆驼源性成分定性检测方法PCR方法[S].
【7】RODRIÄGUEZ M A, GARCIÄA T, GONZÄLEZ I, et al. Identification of goose, mule duck, chicken, turkey, and swine in foie gras by species-specific polymerase chain reaction[J]. Agricultural and Food Chemistry, 2003,51:1524-1529.
【8】DALMASSO A, FONTANELLA E, PIATTI P, et al. A multiplex PCR assay for the identfication of animal species in feedstuffs[J]. Molecolar and Cellular Probes, 2004,18:81-87.
【9】RODRIGUEZ M A, GARCIA T, GONZALEZ I, et al. PCR identifieation of beef,sheep,goat and pork in raw and heat-treated meat mixtures[J]. Journal of Food Protection, 2004,67(1):172-177.
【10】HAUNSHI S, BASUMATARY R, GIRISH P S, et al. Identification of chicken, duck, pigeon and pig meat by species-specific markers of mitochondrial origin[J]. Meat Science, 2009,83(3):454-459.
【11】DOOLEY J J, PAINE K E, GARRETT S D, et al. Detection of meat species using TaqMan real-time PCR assays[J]. Meat Science, 2004,68:431-438.
【12】JONKER K M, TILBURG J J, HAGELE G H, et al. Species identification in meat products using real-time PCR[J]. Food Additives and Contaminants, 2008,25(5):527-533.
【13】KESMEN Z, GULLUCE A, SAHIN F, et al. Identification of meat species by TaqMan-based real-time PCR assay[J]. Meat Science, 2009,82:444-449.
【14】KESMEN Z, YETIMAN A E, SAHIN F, et al. Detection of chicken and turkey meat in meat mixtures by using real-time PCR assays[J]. Journal of Food Science, 2012,77(2):167-173.
【15】KOPPEL R, ZIMMERLI F, BREITENMOSER A. Heptaplex real-time PCR for the identification and quantification of DNA from beef,pork,chicken,turkey,horse meat,sheep and goat[J]. European Food Research and Technology, 2009,230(1):125-133.
【16】SAWYER J, WOOD C, SHANAHAN D, et al. Real-time PCR for quantitative meat species testing[J]. Food Control, 2003,14(8):579-583.
【17】王萍,乔勇升,韩芷玲.猪源性成分检测中3种DNA提取方法比较[J].生物加工过程, 2015,12(6):61-64.
【18】陈茹,林志雄,刘琳琳.应用PCR等核酸技术检验动物饲料中牛羊组织成分[J].中国兽医科技, 2001,31(9):3-8.
【19】曹际娟,卢行安,秦成,等.实时荧光PCR技术检测肉骨粉中牛羊源性成分的方法[J].生物技术通讯, 2002,13(2):158-160.
【20】杨宝华,宗卉.根据线粒体基因的特异性鉴别检测饲料中的动物源性成分[J].科学试验与研究, 2000(5):1-3.
【21】庞杰,王金玲,张莹,等.饲料中鱼源性成分定量检测的实时荧光PCR技术的建立[J].中国兽医学报, 2010(5):680-692.
【22】冯海永,韩建林.羊肉产品中若干动物源性成分的七重PCR检测技术应用研究[J].中国畜牧兽医, 2010,37(9):85-90.
【23】孙敏,高宏伟,龚方,等.利用线粒体COⅠ序列快速检测狐狸源性成分[J].畜牧与兽医, 2013,45(7):57-60.
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