食品中动物源性成分的多重PCR快速检测
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摘要
目的利用多重PCR技术对市售的多种食品进行检测,确定动物源性成分的种类,进行掺假及真伪鉴别。方法对鸡、猪、牛、兔、绵羊、山羊、马、鹿8种动物源性产品进行DNA提取,根据不同动物线粒体DNA设计特异性引物,根据脊椎动物细胞色素b基因mt DNA序列设计通用引物作为内源参照,对8种动物源性产品的DNA进行多重PCR扩增,同时对多种成分进行鉴定。结果琼脂糖凝胶电泳表明,此方法能同时扩增出8种动物的特异性条带,检测灵敏度达到0.01%。结论所建立的鉴定多种动物源性成分的新方法,操作简便、快速准确,可为各检验机构对食品进行检测提供方法指导。
Objective To detect variety of food products on the market by multiple PCR technology, identify the types of animal species, and make a identification of adulteration and authenticity. Methods DNA was extracted from 8 kinds of animal-derived products including chicken, pig, bovine, rabbit, sheep, goat, horse and deer, and specific primers were designed according to the mitochondrial DNA of different kinds of animals. According to the mt DNA sequence of cytochrome b gene in vertebrate, a universal primer was designed for endogenous reference. Multiple PCR amplification was performed on the DNA of 8 kinds of animal-derived products and multiple components were identified. Results Agarose gel electrophoresis showed that the method could simultaneously expand the specific bands of 8 kinds of animals, and the detection sensitivity reached 0.01%. Conclusion The method established for identification of animal-derived species is simple, rapid and accurate, which can provide methodological instruction for the inspection agencies.
Objective To detect variety of food products on the market by multiple PCR technology, identify the types of animal species, and make a identification of adulteration and authenticity. Methods DNA was extracted from 8 kinds of animal-derived products including chicken, pig, bovine, rabbit, sheep, goat, horse and deer, and specific primers were designed according to the mitochondrial DNA of different kinds of animals. According to the mt DNA sequence of cytochrome b gene in vertebrate, a universal primer was designed for endogenous reference. Multiple PCR amplification was performed on the DNA of 8 kinds of animal-derived products and multiple components were identified. Results Agarose gel electrophoresis showed that the method could simultaneously expand the specific bands of 8 kinds of animals, and the detection sensitivity reached 0.01%. Conclusion The method established for identification of animal-derived species is simple, rapid and accurate, which can provide methodological instruction for the inspection agencies.
引文
[1]何金兴,崔一笑,赵晓磊.肉类掺假检测技术研究进展[J].河南工业大学学报(自然科学版),2015,36(1):106-112.He JX,Cui YX,Zhao XL.Advances in meat adulteration detection technology[J].J Henan Univ Technol,2015,36(1):106-112.
[2]刘畅,张奇,王思颖,等.驴肉及其制品真伪鉴别技术研究进展[J].食品安全质量检测学报,2017,8(7):2657-2663.Liu C,Zhang Q,Wang SY,et al.Research progress on authenticity identification of donkey meat and its products[J].J Food Saf Qual,2017,8(7):2657-2663.
[3]Li S,Wang JH,Gao JX,et al.Detection of cattle and sheep-derived materials in feed products of ruminant feed and animal-derived feed in Shaanxi province[J].Prog Vet Med,2007,28(4):34-38.
[4]Luo JQ,Wang JQ,Bu DP,et al.Development and application of a PCR detection of bovine,sheep,pig and chicken derived materials in feedstuff[J].Sci Agric Sin,2008,41(7):2112-2119.
[5]Ilhak O I,Arslan A.Identification of meat species by polymerase chain reaction(PCR)technique[J].Turk J of Vet Anim Sci,2007,31(3):159-163.
[6]侯东军,韩合敬,郝智慧,等.三重荧光PCR法鉴定食品中牛、猪、鸭3种动物源性成分[J].畜牧与兽医,2016,48(4):45-48.Hou DJ,Han HJ,Hao ZH,et al.Triple fluorescence PCR assay was used to identify three animal-derived components of cattle,pigs and ducks in food[J].Anim Hus Vet Med,2016,48(4):45-48.
[7]杜巍.定量PCR技术在食源性致病微生物检测中的应用[J].食品科学,2006,27(4):260-263.Du W.Quantitative PCR technology in the detection of foodborne pathogenic microorganisms[J].Food Sci,2006,27(4):260-263.
[8]程欣.应用实时荧光PCR方法鉴别食品中的鸭源性成分[D].南京:南京农业大学,2015.Cheng X.Real-time fluorescence PCR method was used to identify duckderived ingredients in food[D].Nanjing:Nanjing Agricultural University,2015.
[9]Fajardo V,gonzalez I,lopez-calleja I,et al.Identification of meats from red deer(Cervus elaphus),fallow deer(Dama dama),and roe deer(Capreolus capreolus)using polymerase chain reaction targeting specific sequences from the mitochondrial 12S r RNA gene[J].Meat Sci,2007,76(2):234-240.
[10]何玮玲.应用多重PCR及实时荧光PCR方法鉴别食品中肉类成分[D].南京:南京农业大学,2013.He WL.Multiple PCR and real-time fluorescence PCR were used to identify meat components in food[D].Nanjing:Nanjing Agricultural University,2013.
[11]黄宇锋,罗海英,罗东辉,等.PCR方法快速鉴别食品中肉的种类[J].现代食品科技,2013,29(7):1725-1729.Huang YF,Luo HY,Luo DH,et al.PCR method for rapid identification of meat species in food[J].Mod Food Technol,2013,29(7):1725-1729.
[12]高菲.利用DNA-Barcoding技术鉴别动物源性成分[D].雅安:四川农业大学,2014.Gao F.DNA-Barcoding technology was used to identify animal origin components[D].Ya'an:Sichuan Agricultural University,2014.
[13]王冬亮.畜禽类加工肉制品种属鉴定的研究[D].贵阳:贵州大学,2016.Wang DL.Research on species identification of meat products processed by livestock and poultry[D].Guiyang:Guizhou University,2016.
[14]凌胜男,吴亚君,韩建勋,等.DNA条形码技术在深加工动物制品源性成分鉴定中的应用研究进展[J].肉类研究,2017,31(1):48-54.Ling SN,Wu YJ,Han JX,et al.Advances in the application of DNA barcode technology in the identification of the source components of processed animal products[J].Meat Res,2017,31(1):48-54.
[15]Ballin NZ,Vogensen FK,Karlsson AH.Species determination can we detect and quantify meat adulteration[J].Meat Sci,2009,83(2):165-174.
[16]Ghovvati S,Nassiri MR,Mirhoseini SZ,et al.Fraud identification in industrial meat products by multiplex PCR assay[J].Food Control,2009,20(8):696-699.
[17]Soares S,Amaral J S,Mafra I,et al.Quantitative detection of poultry meat adulteration with pork by a duplex PCR assay[J].Meat Sci,2010,85(3):531-536.
[18]吕二盼,张伟,周正.动物源性食品中各种动物源性肉及肉制品鉴别检验的研究[D].保定:河北农业大学,2012.Lv EP,Zhang W,Zhou Z.Identification and testing of various animal-derived meat and meat products in animal-derived food[D].Baoding:Hebei Agricultural University,2012.
[19]宗卉,范万红,温燕辉.应用多重PCR同时检测牛、羊源性成分[J].中国草食动物,2006,26(6):21-23.Zong H,Fan WH,Wen YH.Multiplex PCR was used to detect both cattle and sheep[J].Chin Herbiv,2006,26(6):21-23.
[20]曾少灵,秦智锋,阮周曦,等.多重实时荧光PCR检测牛、山羊和绵羊源性成分[J].生物工程学报,2009,25(1):139-146.Zeng SL,Qin ZF,Ruan ZX,et al.Multiple real-time fluorescence PCR detection of cattle,goats and sheep[J].Chin J Biotechnol,2009,25(1):139-146.
[21]巩元芳,李祥龙,刘铮铸.我国主要地方绵羊品种mt DNA细胞色素b基因PCR-RFLP研究[J].畜牧兽医学报,2005,36(7):649-653.Gong YF,Li XL,Liu ZZ.Studies on the cytochrome b gene pcr-rflp of mt DNA in major local sheep breeds in China[J].Anim Husb Vet Med,2005,36(7):649-653.
[22]Matsunaga T,Chikuni K,Tanabe R,et al.A quick and simple method for the identification of meat species and meat products by PCR assay[J].Meat Sci,1999,51(2):143-148.
[23]田晨曦,周巍,王爽,等.基于DNA条形码技术常见肉类掺假鉴别技术研究[J].现代食品科技,2016,32(8):295-301.Tian C X,Zhou W,Wang S,et al.Research on common meat adulteration identification technology based on DNA barcode technology[J].Mod Food Technol,2016,32(8):295-301.
[24]Arslan A,Ilhak OI,Calicioglu M.Effect of method of cooking on identification of heat processed beef using polymerase chain reaction(PCR)technique[J].Meat Sci,2006,72(2):326-330.
[25]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].生物技术,2010,37(9):85-90.Feng HY,Han JL.Research on application of seven-fold PCR detection technology for several animal-derived components in mutton products[J].Biotech,2010,37(9):85-90.
[2]刘畅,张奇,王思颖,等.驴肉及其制品真伪鉴别技术研究进展[J].食品安全质量检测学报,2017,8(7):2657-2663.Liu C,Zhang Q,Wang SY,et al.Research progress on authenticity identification of donkey meat and its products[J].J Food Saf Qual,2017,8(7):2657-2663.
[3]Li S,Wang JH,Gao JX,et al.Detection of cattle and sheep-derived materials in feed products of ruminant feed and animal-derived feed in Shaanxi province[J].Prog Vet Med,2007,28(4):34-38.
[4]Luo JQ,Wang JQ,Bu DP,et al.Development and application of a PCR detection of bovine,sheep,pig and chicken derived materials in feedstuff[J].Sci Agric Sin,2008,41(7):2112-2119.
[5]Ilhak O I,Arslan A.Identification of meat species by polymerase chain reaction(PCR)technique[J].Turk J of Vet Anim Sci,2007,31(3):159-163.
[6]侯东军,韩合敬,郝智慧,等.三重荧光PCR法鉴定食品中牛、猪、鸭3种动物源性成分[J].畜牧与兽医,2016,48(4):45-48.Hou DJ,Han HJ,Hao ZH,et al.Triple fluorescence PCR assay was used to identify three animal-derived components of cattle,pigs and ducks in food[J].Anim Hus Vet Med,2016,48(4):45-48.
[7]杜巍.定量PCR技术在食源性致病微生物检测中的应用[J].食品科学,2006,27(4):260-263.Du W.Quantitative PCR technology in the detection of foodborne pathogenic microorganisms[J].Food Sci,2006,27(4):260-263.
[8]程欣.应用实时荧光PCR方法鉴别食品中的鸭源性成分[D].南京:南京农业大学,2015.Cheng X.Real-time fluorescence PCR method was used to identify duckderived ingredients in food[D].Nanjing:Nanjing Agricultural University,2015.
[9]Fajardo V,gonzalez I,lopez-calleja I,et al.Identification of meats from red deer(Cervus elaphus),fallow deer(Dama dama),and roe deer(Capreolus capreolus)using polymerase chain reaction targeting specific sequences from the mitochondrial 12S r RNA gene[J].Meat Sci,2007,76(2):234-240.
[10]何玮玲.应用多重PCR及实时荧光PCR方法鉴别食品中肉类成分[D].南京:南京农业大学,2013.He WL.Multiple PCR and real-time fluorescence PCR were used to identify meat components in food[D].Nanjing:Nanjing Agricultural University,2013.
[11]黄宇锋,罗海英,罗东辉,等.PCR方法快速鉴别食品中肉的种类[J].现代食品科技,2013,29(7):1725-1729.Huang YF,Luo HY,Luo DH,et al.PCR method for rapid identification of meat species in food[J].Mod Food Technol,2013,29(7):1725-1729.
[12]高菲.利用DNA-Barcoding技术鉴别动物源性成分[D].雅安:四川农业大学,2014.Gao F.DNA-Barcoding technology was used to identify animal origin components[D].Ya'an:Sichuan Agricultural University,2014.
[13]王冬亮.畜禽类加工肉制品种属鉴定的研究[D].贵阳:贵州大学,2016.Wang DL.Research on species identification of meat products processed by livestock and poultry[D].Guiyang:Guizhou University,2016.
[14]凌胜男,吴亚君,韩建勋,等.DNA条形码技术在深加工动物制品源性成分鉴定中的应用研究进展[J].肉类研究,2017,31(1):48-54.Ling SN,Wu YJ,Han JX,et al.Advances in the application of DNA barcode technology in the identification of the source components of processed animal products[J].Meat Res,2017,31(1):48-54.
[15]Ballin NZ,Vogensen FK,Karlsson AH.Species determination can we detect and quantify meat adulteration[J].Meat Sci,2009,83(2):165-174.
[16]Ghovvati S,Nassiri MR,Mirhoseini SZ,et al.Fraud identification in industrial meat products by multiplex PCR assay[J].Food Control,2009,20(8):696-699.
[17]Soares S,Amaral J S,Mafra I,et al.Quantitative detection of poultry meat adulteration with pork by a duplex PCR assay[J].Meat Sci,2010,85(3):531-536.
[18]吕二盼,张伟,周正.动物源性食品中各种动物源性肉及肉制品鉴别检验的研究[D].保定:河北农业大学,2012.Lv EP,Zhang W,Zhou Z.Identification and testing of various animal-derived meat and meat products in animal-derived food[D].Baoding:Hebei Agricultural University,2012.
[19]宗卉,范万红,温燕辉.应用多重PCR同时检测牛、羊源性成分[J].中国草食动物,2006,26(6):21-23.Zong H,Fan WH,Wen YH.Multiplex PCR was used to detect both cattle and sheep[J].Chin Herbiv,2006,26(6):21-23.
[20]曾少灵,秦智锋,阮周曦,等.多重实时荧光PCR检测牛、山羊和绵羊源性成分[J].生物工程学报,2009,25(1):139-146.Zeng SL,Qin ZF,Ruan ZX,et al.Multiple real-time fluorescence PCR detection of cattle,goats and sheep[J].Chin J Biotechnol,2009,25(1):139-146.
[21]巩元芳,李祥龙,刘铮铸.我国主要地方绵羊品种mt DNA细胞色素b基因PCR-RFLP研究[J].畜牧兽医学报,2005,36(7):649-653.Gong YF,Li XL,Liu ZZ.Studies on the cytochrome b gene pcr-rflp of mt DNA in major local sheep breeds in China[J].Anim Husb Vet Med,2005,36(7):649-653.
[22]Matsunaga T,Chikuni K,Tanabe R,et al.A quick and simple method for the identification of meat species and meat products by PCR assay[J].Meat Sci,1999,51(2):143-148.
[23]田晨曦,周巍,王爽,等.基于DNA条形码技术常见肉类掺假鉴别技术研究[J].现代食品科技,2016,32(8):295-301.Tian C X,Zhou W,Wang S,et al.Research on common meat adulteration identification technology based on DNA barcode technology[J].Mod Food Technol,2016,32(8):295-301.
[24]Arslan A,Ilhak OI,Calicioglu M.Effect of method of cooking on identification of heat processed beef using polymerase chain reaction(PCR)technique[J].Meat Sci,2006,72(2):326-330.
[25]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].生物技术,2010,37(9):85-90.Feng HY,Han JL.Research on application of seven-fold PCR detection technology for several animal-derived components in mutton products[J].Biotech,2010,37(9):85-90.