利用实时荧光定量PCR法检测食品中鹌鹑源性成分
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摘要
为建立基于TaqMan实时荧光聚合酶链式反应(polymerase chain reaction,PCR)技术在食品中的鹌鹑源性成分的定量检测方法,根据现行检验检疫标准(SN/T 2727-2010)合成引物及探针。首先对13种不同动物鲜肉组织的DNA进行鹌鹑源性成分特异性检测,然后对鹌鹑源性DNA模板原液进行梯度稀释,检测方法灵敏度,最后在加工制品中检测方法的适用性。研究结果表明:建立的方法特异性强,除鹌鹑肉外,牛、羊、猪、马、驴、狗、兔子、鸡、鸭、鸽子、火鸡、鱼12种动物鲜肉组织均无特异性扩增;方法的灵敏度较高,鹌鹑组分DNA的检出限可达10 fg/mL,灵敏度可达0.001%;方法的适用性较广,可以用于加工制品中鹌鹑源性成分的检测。
In order to establish a quantitative detection method of quail origin in foods based on Taq Man realtime polymerase chain reaction(PCR).In this study,we synthesized primers and probes according to the industry standard(SN/T 2727-2010)for quarantine inspection.The primers and probe's specificity was tested by DNA templates extracted from raw meat from quail and 12 other animal species.Gradient dilutions of the quail-derived DNA template were used to evaluate the sensitivity.The applicability of the PCR assay for food products was also investigated.The results showed that this PCR assay was highly specific and could specifically amplify DNA from quail rather than cattle,sheep,pig,horse,donkey,dog,rabbit,chicken,duck,pigeon,turkey and fish.It was sensitive.The limit of detection for quail DNA was 10 fg/mL and the sensitivity was 0.001 %.Wide applicability,can be used for the test of processed meat products.
In order to establish a quantitative detection method of quail origin in foods based on Taq Man realtime polymerase chain reaction(PCR).In this study,we synthesized primers and probes according to the industry standard(SN/T 2727-2010)for quarantine inspection.The primers and probe's specificity was tested by DNA templates extracted from raw meat from quail and 12 other animal species.Gradient dilutions of the quail-derived DNA template were used to evaluate the sensitivity.The applicability of the PCR assay for food products was also investigated.The results showed that this PCR assay was highly specific and could specifically amplify DNA from quail rather than cattle,sheep,pig,horse,donkey,dog,rabbit,chicken,duck,pigeon,turkey and fish.It was sensitive.The limit of detection for quail DNA was 10 fg/mL and the sensitivity was 0.001 %.Wide applicability,can be used for the test of processed meat products.
引文
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[19]马凌艳.速食鹌鹑蛋加工工艺的优化研究[D].咸阳:西北农林科技大学,2017:1
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[23]Zhang C L,Chen D F,Mccormac A C,et al.Use of the GFP reporter as a vital marker for Agrobacterium-mediated transformation of sugar beet(Beta vulgaris L.)[J].Molecular Biotechnology,2001,17(2):109
[24]Murray M G,Thompson W F.Rapid isolation of high molecular weight plant DNA[J].Nucleic Acids Research,1980,8(19):4321-4325
[25]张慧霞,宗卉,吴建平,等.应用SYBR GreenⅠ实时荧光PCR溶解曲线检测鹌鹑源性成分[J].中国畜牧杂志,2010,46(17):70-73
[26]Von H C,Honikel K O,Unglaub W,et al.Determination of an appropriate heat treatment of animal waste using the ELISA technique:results of a validation study[J].Meat Science,2000,54(1):1-7
[27]付理文,张宇,依含,等.Taq Man多重实时荧光PCR同步定量检测6种动物源性成分方法的建立[J].中国生物工程杂志,2017,37(9):48-59
[28]张驰,邱皓璞,张筠.荧光定量PCR检测肉制品中鸭源性成分[J].食品科学,2013,34(18):154-157
[29]卞如如,范阳阳,刘艳艳,等.一种驴和马及狐狸源性成分快速检测方法的研究[J].中国畜牧杂志,2017,53(1):100-104
[2]Mamani-Linares L W,Gallo C,Alomar D.Identification of cattle,llama and horse meat by near infrared reflectance or transflectance spectroscopy[J].Meat Science,2012,90(2):378-385
[3]冯静,刘琳,黄孟军,等.基于飞行时间质谱的快速肉类溯源检测方法[C].第四届国际食品安全高峰论坛论文集,2011:200-204
[4]Bendixen E.The use of proteomics in meat science[J].Meat science,2005,71(1):138-149
[5]Rencova E,Necidova I.Identification by ELISA of poultry,horse,kangaroo and rat muscle specific proteins in heat processed products[J].Veterinarni Medicina,2000,45(12):353-356
[6]Ali M E,Hashim U,Mustafa S,et al.Analysis of pork adulteration in commercial meatballs targeting porcine-specific mitochondrial cytochrome b gene by Taq Man probe real-time polymerase chain reaction[J].Meat Science,2012,91(4):454-459
[7]Haider N,Nabulsi I,Al-Safadi B.Identification of meat species by PCR-RFLP of the mitochondrial COI gene[J].Meat Science,2012,90(2):490-493
[8]Mane B G,Mendiratta S K,Tiwari A K.Beef specific polymerase chain reaction assay for authentication of meat and meat products[J].Food Control,2012,28(2):246-249
[9]Sakaridis I,Ganopoulos I,Argiriou A,et al.A fast and accurate method for controlling the correct labeling of products containing buffalo meat using high resolution melting(HRM)analysis[J].Meat Science,2013,94(1):84-88
[10]Soares S,Amaral J S,Oliveira M B,et al.A SYBR green real-time PCR assay to detect and quantify pork meat in processed poultry meat products[J].Meat Science,2013,94(1):115-120
[11]Zhang C.Semi-nested multiplex PCR enhanced method sensitivity of species detection in further-processed meats[J].Food Control,2013,31(2):326-330
[12]Raamsdonk L W D V,Holst C V,Baeten V,et al.New developments in the detection and identification of processed animal proteins in feeds[J].Animal Feed Science&Technology,2007,133(1):63-83
[13]国家质量监督检验检疫总局.SN/T 2978-2011动物源性产品中鸡源性成分:PCR检测方法[S].北京:中国标准出版社,2011:2
[14]Brodmann P D,Moor D.Sensitive and semi-quantitative TaqMan 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(1):599-607
[15]Zhang C L,Scott N W,Lawson G,et al.A Taq Man real-time PCRsystem for the identication and quantication of bovine DNA in meats,milks and cheeses[J].Food Control,2007,18(9):1149-1158
[16]Imaizumi K,Akutsu T,Miyasaka S,et al.Development of species identification tests targeting the 16S ribosomal RNA coding region in mitochondrial DNA[J].International Journal of Legal Medicine,2007,121(3):184-191
[17]Murugaiah C,Noor Z M,Mastakim M,et al.Meat species identification and Halal authentication analysis using mitochondrial DNA[J].Meat science,2009,83(1):57-61
[18]姜文灿,岳素文,江洪,等.Taq Man探针法实时荧光定量PCR的应用和研究进展[J].临床检验杂志,2015,4(1):797-805
[19]马凌艳.速食鹌鹑蛋加工工艺的优化研究[D].咸阳:西北农林科技大学,2017:1
[20]钱伯钦.动物人参鹌鹑、鹌鹑蛋[J].肉品卫生,2001(3):35
[21]付晶,白秀娟.鹌鹑的研究进展[J].饲料博览·技术版,2009(7):36-37
[22]国家质量监督检验检疫总局.SN/T 2727-2010饲料中禽源性成分检测方法:实时荧光PCR方法[S].北京:中国标准出版社,2010:4
[23]Zhang C L,Chen D F,Mccormac A C,et al.Use of the GFP reporter as a vital marker for Agrobacterium-mediated transformation of sugar beet(Beta vulgaris L.)[J].Molecular Biotechnology,2001,17(2):109
[24]Murray M G,Thompson W F.Rapid isolation of high molecular weight plant DNA[J].Nucleic Acids Research,1980,8(19):4321-4325
[25]张慧霞,宗卉,吴建平,等.应用SYBR GreenⅠ实时荧光PCR溶解曲线检测鹌鹑源性成分[J].中国畜牧杂志,2010,46(17):70-73
[26]Von H C,Honikel K O,Unglaub W,et al.Determination of an appropriate heat treatment of animal waste using the ELISA technique:results of a validation study[J].Meat Science,2000,54(1):1-7
[27]付理文,张宇,依含,等.Taq Man多重实时荧光PCR同步定量检测6种动物源性成分方法的建立[J].中国生物工程杂志,2017,37(9):48-59
[28]张驰,邱皓璞,张筠.荧光定量PCR检测肉制品中鸭源性成分[J].食品科学,2013,34(18):154-157
[29]卞如如,范阳阳,刘艳艳,等.一种驴和马及狐狸源性成分快速检测方法的研究[J].中国畜牧杂志,2017,53(1):100-104