牦牛肉产品的分子追溯研究
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摘要
为了建立牦牛、黄牛(包括瘤牛和普通牛)和水牛肉在线粒体和核基因水平上快速、准确的鉴定方法,用于牦牛肉产品的来源追溯和物种成分鉴别,以牦牛、黄牛、水牛肉和来源于10个不同厂家的牦牛肉产品作为研究材料,尝试并系统地比较了异硫氰酸胍法、酚-氯仿抽提法和试剂盒提取法提取DNA效果进行了比较分析,并采用Cyt b基因和三个特异性微卫星位点进行检测,主要获得以下研究结果：
1.通过定量分析、凝胶电泳和PCR片段大小梯度扩增等手段对利用异硫氰酸胍法、酚-氯仿抽提法和试剂盒提取法提取的基因组DNA的数量和质量进行了比较。研究认为异硫氰酸胍法操作简便、快速、廉价,能从牦牛肉产品中提取到高质量的基因组DNA。
2.在Cyt b基因(472 bp)区域发现牦牛、黄牛和水牛各自的特异性酶切位点：牦牛的酶切位点为GACGCNNNN/,酶切片段分别为220 bp和252 bp；普通牛的酶切位点为GGGCC/C,酶切片段分别为173 bp和299 bp；水牛的酶切位点为CCANNNN/NTGG,酶切片段分别为69 bp和403 bp,且没有交叉酶切现象,可用于混合牛肉产品的牛种来源的鉴别。
3.通过对10个厂家的30袋牦牛肉产品的135份样品进行Cyt b基因片段的测序检测,发现其中有68条牦牛序列,仅占50.4%；另外有51条黄牛序列和16条水牛序列,占到了49.6%。说明黄牛肉、水牛肉掺杂到牦牛肉产品中的现象十分严重。
4.通过研究发现,对于存在基因渗透的畜种,结合3个特异性微卫星位点检测的结果才能得到可靠的结论。
In order to develop a comprehensive, rapid and accurate method that can be used to identify yak, taurine and zebu cattle as well as buffalo meats using both mtDNA and nuclear genomic information. this study explored three different extraction protocols of phenol-chloroform extraction, guanidine thiocyanate method and commercial DNA extraction Kit, separately. this experiment extracted DNAs from the fresh meats with known species origins of yak, cattle and buffloes as well as highly processed yak meat products from 10 manufacturers for molecular tests using sequences of Cyt b genes as well as three species specific microsatellite DNA markers for yak and cattle. The following results were obtained:
1. The quality and quantity of the extracted DNAs were assessed using three methods of quantitative analysis through spectrophotometer, argarose gel electrophoresis and PCR amplifications for different sizes of mtDNA and nuclear genomic gene fragments.we suggest that guanidine thiocyanate method is a simple, rapid, cheap and sufficient protocol that can be used for routine DNA extraction from the yak meat products for their further molecular traceability tests.
2. Within a fragment of 472 bp long Cyt b gene amplified using the universal primers, the species specific recognition sites were identified for yak as GACGCNNNN/with two fragments of 220 bp and 252 bp, for cattle as GGGCC/C with two fragments of 173 bp and 299 bp and for buffaloes as CCANNNN/NTGG with also two fragments in 69 bp and 403 bp. There was no cross-species reaction, therefore this protocol can be used for the identification of species origins in the mixed meat products.
3. A total of 135 specimens from 30 packages of yak meat products marketed by the 10 manufacturers were sampled for sequencing the informative Cyt b gene fragment. The results showed that there were only 68 yak sequences, taking up 50.4%, while the remaining half of them (49.6%) were of cattle (51) and buffaloe (16) sequences. These results suggest that the yak meat products have been seriously contaminated by other sources of meats, which certainly affect badly the healthy market development of the yak meat industry.
4. Combining the data from the three species specific microsatellite DNA markers for yak and cattle, the accuccy of the molecular traceability of the highly processed yak meats can be enhanced and quaranteed.
1.通过定量分析、凝胶电泳和PCR片段大小梯度扩增等手段对利用异硫氰酸胍法、酚-氯仿抽提法和试剂盒提取法提取的基因组DNA的数量和质量进行了比较。研究认为异硫氰酸胍法操作简便、快速、廉价,能从牦牛肉产品中提取到高质量的基因组DNA。
2.在Cyt b基因(472 bp)区域发现牦牛、黄牛和水牛各自的特异性酶切位点：牦牛的酶切位点为GACGCNNNN/,酶切片段分别为220 bp和252 bp；普通牛的酶切位点为GGGCC/C,酶切片段分别为173 bp和299 bp；水牛的酶切位点为CCANNNN/NTGG,酶切片段分别为69 bp和403 bp,且没有交叉酶切现象,可用于混合牛肉产品的牛种来源的鉴别。
3.通过对10个厂家的30袋牦牛肉产品的135份样品进行Cyt b基因片段的测序检测,发现其中有68条牦牛序列,仅占50.4%；另外有51条黄牛序列和16条水牛序列,占到了49.6%。说明黄牛肉、水牛肉掺杂到牦牛肉产品中的现象十分严重。
4.通过研究发现,对于存在基因渗透的畜种,结合3个特异性微卫星位点检测的结果才能得到可靠的结论。
In order to develop a comprehensive, rapid and accurate method that can be used to identify yak, taurine and zebu cattle as well as buffalo meats using both mtDNA and nuclear genomic information. this study explored three different extraction protocols of phenol-chloroform extraction, guanidine thiocyanate method and commercial DNA extraction Kit, separately. this experiment extracted DNAs from the fresh meats with known species origins of yak, cattle and buffloes as well as highly processed yak meat products from 10 manufacturers for molecular tests using sequences of Cyt b genes as well as three species specific microsatellite DNA markers for yak and cattle. The following results were obtained:
1. The quality and quantity of the extracted DNAs were assessed using three methods of quantitative analysis through spectrophotometer, argarose gel electrophoresis and PCR amplifications for different sizes of mtDNA and nuclear genomic gene fragments.we suggest that guanidine thiocyanate method is a simple, rapid, cheap and sufficient protocol that can be used for routine DNA extraction from the yak meat products for their further molecular traceability tests.
2. Within a fragment of 472 bp long Cyt b gene amplified using the universal primers, the species specific recognition sites were identified for yak as GACGCNNNN/with two fragments of 220 bp and 252 bp, for cattle as GGGCC/C with two fragments of 173 bp and 299 bp and for buffaloes as CCANNNN/NTGG with also two fragments in 69 bp and 403 bp. There was no cross-species reaction, therefore this protocol can be used for the identification of species origins in the mixed meat products.
3. A total of 135 specimens from 30 packages of yak meat products marketed by the 10 manufacturers were sampled for sequencing the informative Cyt b gene fragment. The results showed that there were only 68 yak sequences, taking up 50.4%, while the remaining half of them (49.6%) were of cattle (51) and buffaloe (16) sequences. These results suggest that the yak meat products have been seriously contaminated by other sources of meats, which certainly affect badly the healthy market development of the yak meat industry.
4. Combining the data from the three species specific microsatellite DNA markers for yak and cattle, the accuccy of the molecular traceability of the highly processed yak meats can be enhanced and quaranteed.
引文
[1]《中国牦牛学》编写委员会.中国牦牛学[M].成都：四川科学技术出版社,1989,1-35.
[2]王喜群,王明金.牦牛肉营养成分分析与研究[J].肉类研究.2006,5：24-26.
[3]牛小莹,郭淑珍,赵君,等.甘南牦牛肉营养成分含量研究分析[J].畜牧兽医杂志,2009,28(7)：101-103.
[4]食品安全与食源性疾病控制研究进展[J].安徽预防医学杂志,2002,8(6)：384-387.
[5]潘良文,陈家华.食品和饲料中动物源成分的检测方法的进展[J].检验检疫科学,2002,12(2)：45-47.
[6]马小珍,陈学群,周围.浅谈饲料与队伍源性食品安全[J].饲料研究,2004,11：8-10.
[7]Hofmann K, Bauer F.Proof of proper heating at meat and bone meal[J]. Digital Object Identifier,1996,76(10):1037-1039.
[8]Momcilovic D, Rasooly A. Detection and analysis of animal materials in food and feed[J]. Food Protection,2000,63:1602-1609.
[9]Siklenka P, Fotta M, Korenekova B, et al. Detection of specific bovine proteins in heat-processed meat product using rabbit antiserum. Bulletin of the Veterinary Institute in Pulawy,2004,48:277-281.
[10]Berger R. G, Mageau R P,Schwab B, et al. Detection of poultry and pork in cooked and canned meat by enzyme-linked immunosorbent assays[J]. Journal of Association of Official Analytical chemists,1988,71:406-409.
[11]Hofmann K, Fisher K, Miller E, et al. Experiments to demonstrate the effectiveness of heat treatments applied tocanned meatsand meat and bone meals. Fleischwirschaft,1996,76: 920-923.
[12]Janssen F W, Buntjer J B, Lenster J A,et al. Species identification in meat by using PCR-gennerated satellite DNA probes[J]. Industy Microbiol. Biotechnol.1988,21:115-120.
[13]Hsieh Y. H, Sheu S C,Bridgman R C. Production and partial characterization of monoclonal antibodies specific to cooked mammalian meats. Journal of Food Protect.1998,61(4): 476-481.
[14]Quinteoro J, Sotelo C G, RehBein H, et al. Use of mtDNA direct polymerase chain reaction (PCR) sequencing and PCR-Restriction Fragment Length Polymorphism methodologies in species identification of canned tuna[J]. Journal of Agricultural and Food Chemistry,1998, 46:1662-1669.
[15]Sotelo C,Gallardo J M, Perez-Martin R. I. Fish species identification in seafood products[J]. Trends Food Science Technology.1993,4,395-401.
[16]Montowska M, Pospiech E. Species identification of meat by electrophoretic methods. ACTA Scientiarum Polonorum-Technologia Alimentaria,2007,6(1):5-16..
[17]Ilhak O I, Arslan A. Identification of meat species by polymerasechain reaction (PCR) technique[J]. Turkish Journal of Veterinary and Animal Sciences,2007,31(3):159-163.
[18]Necidova L, Rencova E, Svoboda I. A simple method for the detection of species specific muscle proteins in heat-processed products[J]. Medicine Czech,2002,47(5):143-147
[19]Chikuni K, Ozutsumi K, Koishikawa T, et al. Species identification of cooked meats by DNA hybridization assay[J]. Meat Science,1990,27:119-128.
[20]EtienneM, Etienne, M, Jerome, M,et al. A standardized method of identification of raw andheat-processed fish by urea isoelectric focusing:A collaborative study [J]. Electrophoresis,1999,20:1923-1933.
[21]Ebbehoj E F, Thomsen P D. Differentiation of closely related species by DNA hybridization[J]. Meat Science,1991,31:359-366
[22]Buntijer J. B.,et al. J. Sci. Food Agriculture.1999,79:53-57.
[23]Buntijer J.B.,et al. Z.Lebensm. Unters Forsch.1995,201:577-582.
[24]Jeffrey L.R, infante,et al. Applied species specific PCR and mutiplex PCR assay for the product of mitochondrial DNA[J]. Agiric. Food Chem.1996,44:2460-2467.
[25]Buntijer J. B.,et al. J.Ind. M icrobiol. Biotechnol.1998,21:121-127.
[26]Bellagamba F, Moretti V.M,Comincini S. Identification of species in animal feedstuffs by polymerase chain reaction -restriction fragment length polymorphism analysis of mithocondrial DNA[J]. agriculture. Food Chem.2001,49:3775-3781.
[27]Wolf C, Rentseh J, Hubner, P. PCR-RFLP analysis of mitochondrial DNA:a reliable method for species identification[J]. Journal of Agricultural and Food Chemistry,1999,47,1350-1355.
[28]Meyer,R,Hoefelin,C,Luethy,J,&Candrian,U. Polymerase chain reaction-restriction fragment length Polymorphism analysis:a simple method for species identification in food[J].Journal of AOAC international,1995,78(6),1542-1551.
[29]Partis L, Croan D, Guo, Z, et al. Evaluation of a DNA fingerprinting Method for deter mining the species origin of meats[J]. Meat Science,2000,54,369-376.
[30]Avise J C,Lansm an R A. Polymorphism of mitochondrial DNA in population of high animals [A].In:NeiM, Koehn R K. Evolution of genes and proteins[C]. Sinauser Associates Inc.Sunderland,1983.
[31]曹黛丽,陈超,宋平,等.一种简易的猪肉和牛肉分子生物学鉴别方法研究[J].中国农学通报.2009,25(18)：110-112
[32]Zuckerkandl and L. Pauling. Evolutionary divergence and convergence in proteins. In V. Bryson and H. J. Vogel,editors,Evolving Genes and Proteins,Academic Press,New York,1965: 97-166.
[33]H. Jianlin, Ochieng J.W, Rege J.E.O. Low level of cattle introgression in yak populations from Bhutanand China:Evidences from Y-specific microsatellites and mitochondrial DNA markers[C]. Proceedings of the 3th international cogress on yak production center asian highlands.2002; 190-196
[34]Qi X B, Han J L, Wang G, et al. Assessment of cattle genetic introgression into domestic yak populations using mitochondrial and microsatellite DNA markers[J]. Animalgenetics,2009, doi:10.1111/j.1365-2052.2009.01989.x.
[35]Yu Y, Nie L, He Z Q, et al. Mitochondrial DNA variation in cattle of South China:origin and introgression. Animal Genetics [J],1999,30:245-250.
[36]Takeda K, Sotoh M, Neopane S P, et al. Mitochondrial DNA analysis of Nepalese domestic dwarf cattle Lulu[J]. Animal Science Journal,2004,75:103-110.
[37]Bailey J F, Healy B, Han J L,et al. Gengtic variation of mitochondrial DNA within domestic yak populations[C]. Proceedings of the third international cogress on yak, in Lhase, china,international livestock Research Institute(ILRL),2002:181-189.
[38]Chikuni K, Tabata T, Kosugiyama,et al. Polymerase chain reaction assay for detection of sheep and goat meats[J].Meat Science,1994,37,337-345
[39]Unseld M, Beyermann B, Brandt P. Identification of the species origin of highly processed meat Products by mitochondrial DNA sequences[J]. PCR methods and applications, 1995,4(4),241-243.
[40]Wolf C, Rentseh J, Hubner, P. PCR-RFLP analysis of mitochondrial DNA:a reliable method for species identification[J]. Journal of Agricultural and Food Chemistry,1999,47,1350-1355.
[41]Meyer,R,Hoefelin,C,Luethy,J,&Candrian,U. Polymerase chain reaction-restriction fragment length Polymorphism analysis:a simple method for species identification in food[J].Journal of AOAC international,1995,78(6),1542-1551.
[42]Partis L, Croan D, Guo, Z, et al. Evaluation of a DNA fingerprinting Method for deter mining the species origin of meats[J]. Meat Science,2000,54,369-376.
[43]胡强,郑玉才,金素钰等.用通用引物扩增细胞色素b基因进行牦牛肉的鉴定[J].食品科学,2007,28(11)：319-321.
[44]Tartaglia M, Saulle E, Pestalozza S, et al. Detection of bovine mitochondrial DNA in ruminant feeds:a molecular approch to test the presence of bovine derived materials[J]. Food Protect,1998,61:513-518
[45]Myers M J, Friedman S L, Farrell D E,et al. Validation of a polymerase chain reaction method for the detection of rendered bovine-derived materials in feedstuffs[J]. Food Protect, 2001,64(4),564-566.
[46]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.
[47]杨宝华,宗卉,林庆燕,等.用分子生物学方法鉴别检测动物源性饲料中的牛羊源性成分[J].中国畜牧杂志.2002,38(1)：3-5.
[48]宗卉,曾少灵,林庆燕,等.饲料中马、驴源性成分的分子生物学检测技[J].中国草食动物,2005,25(5)：3-6.
[49]陈颖,吴亚君,徐宝梁,等.食品及饲料中马属动物源性成分的PCR检测研究[J].中国生物工程杂志.2004,24(5)：78-83.
[50]陈颖,吴亚君,徐宝梁,等.进出口动物源性产品中牛羊成分[J].食品工业科技.2004,25(8)：144-146.
[51]邵碧英,陈文炳,郑腾,等.动物产品中牛、羊源性成分多重PCR检测方法的建立[J].畜牧与兽医.2004,36(3)：7-9.
[52]陈冬,柏凡,周明亮,等.基于线粒体12S rRNA基因鉴别混合牛肉及制品的牛种来源[J].遗传,2008,30(8)：1008-1014.
[53]Daiytt C, Marchi M D, Cassandro M. Genetic traceability of livestock products[J]. Meat Science,2007,77:437-449.
[54]高琳,徐幸莲,周光宏,等.PCR技术用于食品中原料肉物种鉴别的研究进展[J].肉类研究,2006,92(10)：19-20.
[55]管峰,杨利国,艾君涛,等.STR基因座在奶牛个体识别中的应用[J].中国农学通报,2005,21(2)：4-6.
[56]潘良文,陈家华,丁燕,等.进口肉骨粉中牛成分检测研究[J].生物技术通报,2001,(5)：23-26.
[57]周丽,李飞,魏刚.动物DNA提取方法概述[J].畜牧与兽医,2008,40(3)：66-68.
[58]邵碧英,陈文炳,郑腾,等.动物产品中牛、羊源性成分多重PCR检测方法的建立[J].畜牧与兽医,2004,36(3)：7-9.
[59]邵碧英,杨婕,张体银.动物产品的DNA提取方法[J].畜牧与兽医,2005,37(9)：47-49
[60]Aslan O, Hamll R M, Sweeney T, et al. Integrity of nuclear genomic DNA in cooked meat: implications for food traceability[J].Journal of Animal Science,2009,87:57-61.
[61]Pascol A, Prado M, Calo P, et al. Detection of bovine DNA in raw and heat-processed foodstuffs, commercial foods and specific risk materials by a novel specific polymerase chain reaction method[J].European Food Research and Technology,2005,220:444-450.
[62]Arslanr A, Lhak O I, Calicioglu M. Effect of method of cooking on identification of heat processed beef using polymerase chain reaction (PCR) technique[J]. Meat Science, 2006,72:326-330.
[63]潘和平,杨具田,阎萍,等.白牦牛肉干的研制[J].食品与发酵工程,2005,31(2)：147-148
[64]薛志成.肉干的加工技术[J].肉类工艺,2005,291(7)：6-7.
[65]韩玲.新型牦牛肉干加工工艺[J].甘肃农业大学学报,2002,37(4)：456-460.
[66]Samnrook J, Fritsh E F, Manttatis T.分子克隆实验指南(3版)[M].北京：科学出版 社,2002：463-471.
[67]张维铭.现代分子生物学实验手册[M].北京：科学出版社,2003：83-89
[68]裴杰,阎萍,姬国红,等.高原牦牛乳铁蛋白素基因克隆及序列分析[J].中国畜牧兽医,2008,35(4)：37-41.
[69]Hassanin A, Douzsery E J P. Evolutionary affinities of the enigmatic saola (Pseudoryx nghetinhensis) in the context of the molecular phylogeny of Bovidae[J]. Proceedings of the Royal Society B:Biological Sciences,1999,266:893-900.
[70]李齐发,李艳华,赵兴波等.牛亚科MHC DRB3基因exon2的序列变异分析[J].农业生物技术学报,2005,13(4)：441-446.
[71]Cardill E J, Womack J E. Detection of polymorphisms in bovine toll-like receptors 3,7,8, and 9[J].Genomics,2007,89(6):745-755
[72]高丹丹,陈燕,王迎华等.动物肉制品基因组DNA的提取和纯化[J].食品科技,2007,8：42-44.
[73]Hird H, Chisolm J, Sanchez A, et al. Effect of heat and pressure processing on DNA fragmentation and implications for the detection of meat using a real-time polymerase chain reaction[J].Food Additives and Contaminants,2006,23(7):645-650.
[74]Matsunage 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 Science,1999,51:143-148.
[75]Brown W M, George M J, Wilson A C, et al. Rapid evolution of animal mitochondrial DNA[J]. Proc Nat Academe Science USA,1979,76:1967-1971.
[76]Meyer A, Kocher T D, Basabwaki P, et al. Monophyletic origin of Victoria cichlid fish suggested by mitochondrial DNA sequence[J]. Nature,1990,347:550-553.
[77]Fajardo V, Gonzalez I, Martin I,et al. Real-time PCR for detection and quantification of red deer(Cervus elaphus), fallow deer(Dama dama), and roe deer(Capreolus capreolus) in meat mixtures. Meat Science,2007,80(2):718-726.
[78]Verma S K, Singh L. Novel universal primers establish identity of an enormous number of animal species for forensic application[J]. MolEcol Notes,2003(3):28-31
[79]冯冬梅,韩建林,罗玉柱,等.牦牛和黄牛三个微卫星基因座上的种间特异性等位基因[J].甘肃农业大学学报,2010
[80]Girish P S, Anjaneyulu A S R, Viswas K N, et al. Sequence analysis of mitochondrial 12S rRNA gene can identify meat species. Meat Science,2004,66(3):551-556.
[81]Qi X B. Genetic diversity, differentiation and relationship of domestic yak populations: microsatellite and mitochondrial DNA study [D]. Lanzhou University, Lanzhou.2004
[2]王喜群,王明金.牦牛肉营养成分分析与研究[J].肉类研究.2006,5：24-26.
[3]牛小莹,郭淑珍,赵君,等.甘南牦牛肉营养成分含量研究分析[J].畜牧兽医杂志,2009,28(7)：101-103.
[4]食品安全与食源性疾病控制研究进展[J].安徽预防医学杂志,2002,8(6)：384-387.
[5]潘良文,陈家华.食品和饲料中动物源成分的检测方法的进展[J].检验检疫科学,2002,12(2)：45-47.
[6]马小珍,陈学群,周围.浅谈饲料与队伍源性食品安全[J].饲料研究,2004,11：8-10.
[7]Hofmann K, Bauer F.Proof of proper heating at meat and bone meal[J]. Digital Object Identifier,1996,76(10):1037-1039.
[8]Momcilovic D, Rasooly A. Detection and analysis of animal materials in food and feed[J]. Food Protection,2000,63:1602-1609.
[9]Siklenka P, Fotta M, Korenekova B, et al. Detection of specific bovine proteins in heat-processed meat product using rabbit antiserum. Bulletin of the Veterinary Institute in Pulawy,2004,48:277-281.
[10]Berger R. G, Mageau R P,Schwab B, et al. Detection of poultry and pork in cooked and canned meat by enzyme-linked immunosorbent assays[J]. Journal of Association of Official Analytical chemists,1988,71:406-409.
[11]Hofmann K, Fisher K, Miller E, et al. Experiments to demonstrate the effectiveness of heat treatments applied tocanned meatsand meat and bone meals. Fleischwirschaft,1996,76: 920-923.
[12]Janssen F W, Buntjer J B, Lenster J A,et al. Species identification in meat by using PCR-gennerated satellite DNA probes[J]. Industy Microbiol. Biotechnol.1988,21:115-120.
[13]Hsieh Y. H, Sheu S C,Bridgman R C. Production and partial characterization of monoclonal antibodies specific to cooked mammalian meats. Journal of Food Protect.1998,61(4): 476-481.
[14]Quinteoro J, Sotelo C G, RehBein H, et al. Use of mtDNA direct polymerase chain reaction (PCR) sequencing and PCR-Restriction Fragment Length Polymorphism methodologies in species identification of canned tuna[J]. Journal of Agricultural and Food Chemistry,1998, 46:1662-1669.
[15]Sotelo C,Gallardo J M, Perez-Martin R. I. Fish species identification in seafood products[J]. Trends Food Science Technology.1993,4,395-401.
[16]Montowska M, Pospiech E. Species identification of meat by electrophoretic methods. ACTA Scientiarum Polonorum-Technologia Alimentaria,2007,6(1):5-16..
[17]Ilhak O I, Arslan A. Identification of meat species by polymerasechain reaction (PCR) technique[J]. Turkish Journal of Veterinary and Animal Sciences,2007,31(3):159-163.
[18]Necidova L, Rencova E, Svoboda I. A simple method for the detection of species specific muscle proteins in heat-processed products[J]. Medicine Czech,2002,47(5):143-147
[19]Chikuni K, Ozutsumi K, Koishikawa T, et al. Species identification of cooked meats by DNA hybridization assay[J]. Meat Science,1990,27:119-128.
[20]EtienneM, Etienne, M, Jerome, M,et al. A standardized method of identification of raw andheat-processed fish by urea isoelectric focusing:A collaborative study [J]. Electrophoresis,1999,20:1923-1933.
[21]Ebbehoj E F, Thomsen P D. Differentiation of closely related species by DNA hybridization[J]. Meat Science,1991,31:359-366
[22]Buntijer J. B.,et al. J. Sci. Food Agriculture.1999,79:53-57.
[23]Buntijer J.B.,et al. Z.Lebensm. Unters Forsch.1995,201:577-582.
[24]Jeffrey L.R, infante,et al. Applied species specific PCR and mutiplex PCR assay for the product of mitochondrial DNA[J]. Agiric. Food Chem.1996,44:2460-2467.
[25]Buntijer J. B.,et al. J.Ind. M icrobiol. Biotechnol.1998,21:121-127.
[26]Bellagamba F, Moretti V.M,Comincini S. Identification of species in animal feedstuffs by polymerase chain reaction -restriction fragment length polymorphism analysis of mithocondrial DNA[J]. agriculture. Food Chem.2001,49:3775-3781.
[27]Wolf C, Rentseh J, Hubner, P. PCR-RFLP analysis of mitochondrial DNA:a reliable method for species identification[J]. Journal of Agricultural and Food Chemistry,1999,47,1350-1355.
[28]Meyer,R,Hoefelin,C,Luethy,J,&Candrian,U. Polymerase chain reaction-restriction fragment length Polymorphism analysis:a simple method for species identification in food[J].Journal of AOAC international,1995,78(6),1542-1551.
[29]Partis L, Croan D, Guo, Z, et al. Evaluation of a DNA fingerprinting Method for deter mining the species origin of meats[J]. Meat Science,2000,54,369-376.
[30]Avise J C,Lansm an R A. Polymorphism of mitochondrial DNA in population of high animals [A].In:NeiM, Koehn R K. Evolution of genes and proteins[C]. Sinauser Associates Inc.Sunderland,1983.
[31]曹黛丽,陈超,宋平,等.一种简易的猪肉和牛肉分子生物学鉴别方法研究[J].中国农学通报.2009,25(18)：110-112
[32]Zuckerkandl and L. Pauling. Evolutionary divergence and convergence in proteins. In V. Bryson and H. J. Vogel,editors,Evolving Genes and Proteins,Academic Press,New York,1965: 97-166.
[33]H. Jianlin, Ochieng J.W, Rege J.E.O. Low level of cattle introgression in yak populations from Bhutanand China:Evidences from Y-specific microsatellites and mitochondrial DNA markers[C]. Proceedings of the 3th international cogress on yak production center asian highlands.2002; 190-196
[34]Qi X B, Han J L, Wang G, et al. Assessment of cattle genetic introgression into domestic yak populations using mitochondrial and microsatellite DNA markers[J]. Animalgenetics,2009, doi:10.1111/j.1365-2052.2009.01989.x.
[35]Yu Y, Nie L, He Z Q, et al. Mitochondrial DNA variation in cattle of South China:origin and introgression. Animal Genetics [J],1999,30:245-250.
[36]Takeda K, Sotoh M, Neopane S P, et al. Mitochondrial DNA analysis of Nepalese domestic dwarf cattle Lulu[J]. Animal Science Journal,2004,75:103-110.
[37]Bailey J F, Healy B, Han J L,et al. Gengtic variation of mitochondrial DNA within domestic yak populations[C]. Proceedings of the third international cogress on yak, in Lhase, china,international livestock Research Institute(ILRL),2002:181-189.
[38]Chikuni K, Tabata T, Kosugiyama,et al. Polymerase chain reaction assay for detection of sheep and goat meats[J].Meat Science,1994,37,337-345
[39]Unseld M, Beyermann B, Brandt P. Identification of the species origin of highly processed meat Products by mitochondrial DNA sequences[J]. PCR methods and applications, 1995,4(4),241-243.
[40]Wolf C, Rentseh J, Hubner, P. PCR-RFLP analysis of mitochondrial DNA:a reliable method for species identification[J]. Journal of Agricultural and Food Chemistry,1999,47,1350-1355.
[41]Meyer,R,Hoefelin,C,Luethy,J,&Candrian,U. Polymerase chain reaction-restriction fragment length Polymorphism analysis:a simple method for species identification in food[J].Journal of AOAC international,1995,78(6),1542-1551.
[42]Partis L, Croan D, Guo, Z, et al. Evaluation of a DNA fingerprinting Method for deter mining the species origin of meats[J]. Meat Science,2000,54,369-376.
[43]胡强,郑玉才,金素钰等.用通用引物扩增细胞色素b基因进行牦牛肉的鉴定[J].食品科学,2007,28(11)：319-321.
[44]Tartaglia M, Saulle E, Pestalozza S, et al. Detection of bovine mitochondrial DNA in ruminant feeds:a molecular approch to test the presence of bovine derived materials[J]. Food Protect,1998,61:513-518
[45]Myers M J, Friedman S L, Farrell D E,et al. Validation of a polymerase chain reaction method for the detection of rendered bovine-derived materials in feedstuffs[J]. Food Protect, 2001,64(4),564-566.
[46]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.
[47]杨宝华,宗卉,林庆燕,等.用分子生物学方法鉴别检测动物源性饲料中的牛羊源性成分[J].中国畜牧杂志.2002,38(1)：3-5.
[48]宗卉,曾少灵,林庆燕,等.饲料中马、驴源性成分的分子生物学检测技[J].中国草食动物,2005,25(5)：3-6.
[49]陈颖,吴亚君,徐宝梁,等.食品及饲料中马属动物源性成分的PCR检测研究[J].中国生物工程杂志.2004,24(5)：78-83.
[50]陈颖,吴亚君,徐宝梁,等.进出口动物源性产品中牛羊成分[J].食品工业科技.2004,25(8)：144-146.
[51]邵碧英,陈文炳,郑腾,等.动物产品中牛、羊源性成分多重PCR检测方法的建立[J].畜牧与兽医.2004,36(3)：7-9.
[52]陈冬,柏凡,周明亮,等.基于线粒体12S rRNA基因鉴别混合牛肉及制品的牛种来源[J].遗传,2008,30(8)：1008-1014.
[53]Daiytt C, Marchi M D, Cassandro M. Genetic traceability of livestock products[J]. Meat Science,2007,77:437-449.
[54]高琳,徐幸莲,周光宏,等.PCR技术用于食品中原料肉物种鉴别的研究进展[J].肉类研究,2006,92(10)：19-20.
[55]管峰,杨利国,艾君涛,等.STR基因座在奶牛个体识别中的应用[J].中国农学通报,2005,21(2)：4-6.
[56]潘良文,陈家华,丁燕,等.进口肉骨粉中牛成分检测研究[J].生物技术通报,2001,(5)：23-26.
[57]周丽,李飞,魏刚.动物DNA提取方法概述[J].畜牧与兽医,2008,40(3)：66-68.
[58]邵碧英,陈文炳,郑腾,等.动物产品中牛、羊源性成分多重PCR检测方法的建立[J].畜牧与兽医,2004,36(3)：7-9.
[59]邵碧英,杨婕,张体银.动物产品的DNA提取方法[J].畜牧与兽医,2005,37(9)：47-49
[60]Aslan O, Hamll R M, Sweeney T, et al. Integrity of nuclear genomic DNA in cooked meat: implications for food traceability[J].Journal of Animal Science,2009,87:57-61.
[61]Pascol A, Prado M, Calo P, et al. Detection of bovine DNA in raw and heat-processed foodstuffs, commercial foods and specific risk materials by a novel specific polymerase chain reaction method[J].European Food Research and Technology,2005,220:444-450.
[62]Arslanr A, Lhak O I, Calicioglu M. Effect of method of cooking on identification of heat processed beef using polymerase chain reaction (PCR) technique[J]. Meat Science, 2006,72:326-330.
[63]潘和平,杨具田,阎萍,等.白牦牛肉干的研制[J].食品与发酵工程,2005,31(2)：147-148
[64]薛志成.肉干的加工技术[J].肉类工艺,2005,291(7)：6-7.
[65]韩玲.新型牦牛肉干加工工艺[J].甘肃农业大学学报,2002,37(4)：456-460.
[66]Samnrook J, Fritsh E F, Manttatis T.分子克隆实验指南(3版)[M].北京：科学出版 社,2002：463-471.
[67]张维铭.现代分子生物学实验手册[M].北京：科学出版社,2003：83-89
[68]裴杰,阎萍,姬国红,等.高原牦牛乳铁蛋白素基因克隆及序列分析[J].中国畜牧兽医,2008,35(4)：37-41.
[69]Hassanin A, Douzsery E J P. Evolutionary affinities of the enigmatic saola (Pseudoryx nghetinhensis) in the context of the molecular phylogeny of Bovidae[J]. Proceedings of the Royal Society B:Biological Sciences,1999,266:893-900.
[70]李齐发,李艳华,赵兴波等.牛亚科MHC DRB3基因exon2的序列变异分析[J].农业生物技术学报,2005,13(4)：441-446.
[71]Cardill E J, Womack J E. Detection of polymorphisms in bovine toll-like receptors 3,7,8, and 9[J].Genomics,2007,89(6):745-755
[72]高丹丹,陈燕,王迎华等.动物肉制品基因组DNA的提取和纯化[J].食品科技,2007,8：42-44.
[73]Hird H, Chisolm J, Sanchez A, et al. Effect of heat and pressure processing on DNA fragmentation and implications for the detection of meat using a real-time polymerase chain reaction[J].Food Additives and Contaminants,2006,23(7):645-650.
[74]Matsunage 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 Science,1999,51:143-148.
[75]Brown W M, George M J, Wilson A C, et al. Rapid evolution of animal mitochondrial DNA[J]. Proc Nat Academe Science USA,1979,76:1967-1971.
[76]Meyer A, Kocher T D, Basabwaki P, et al. Monophyletic origin of Victoria cichlid fish suggested by mitochondrial DNA sequence[J]. Nature,1990,347:550-553.
[77]Fajardo V, Gonzalez I, Martin I,et al. Real-time PCR for detection and quantification of red deer(Cervus elaphus), fallow deer(Dama dama), and roe deer(Capreolus capreolus) in meat mixtures. Meat Science,2007,80(2):718-726.
[78]Verma S K, Singh L. Novel universal primers establish identity of an enormous number of animal species for forensic application[J]. MolEcol Notes,2003(3):28-31
[79]冯冬梅,韩建林,罗玉柱,等.牦牛和黄牛三个微卫星基因座上的种间特异性等位基因[J].甘肃农业大学学报,2010
[80]Girish P S, Anjaneyulu A S R, Viswas K N, et al. Sequence analysis of mitochondrial 12S rRNA gene can identify meat species. Meat Science,2004,66(3):551-556.
[81]Qi X B. Genetic diversity, differentiation and relationship of domestic yak populations: microsatellite and mitochondrial DNA study [D]. Lanzhou University, Lanzhou.2004