基于TaqMan PCR鉴定猪源性成分的引物和探针研发
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摘要
为了研发具有自主知识产权的猪源性成分检测的引物和探针,比对猪、牛、羊、马、鸡、鸭、兔、鹅等8种动物的线粒体基因组,筛选并设计猪特异性的引物和探针组合进行荧光定量PCR。研究结果表明,所研发的猪源性成分检测的引物和探针仅对猪相关DNA模板有典型扩增曲线,对其他动物来源的DNA模板无扩增,具有高度的猪源性特异性。灵敏度实验表明,利用此引物和探针的检测方法具有高度的灵敏度,可达1pg级。肉制品中猪源性定量检测实验说明此方法具有较好的定量检测能力。此引物和探针组合适用于猪源性食品和农畜产品的检验检测。
The objective of this study was to design the primer and probe with intellectual property which can be used to detect porcine-derived materials by means of TaqMan PCR. The results showed that the technique based on the primers and probe was specific for species identification and quantification,and was able to detect 1 pg template.This biotechnological method based on TaqMan PCR was appropriate to identify porcine-derived materials in farm products and foods.
The objective of this study was to design the primer and probe with intellectual property which can be used to detect porcine-derived materials by means of TaqMan PCR. The results showed that the technique based on the primers and probe was specific for species identification and quantification,and was able to detect 1 pg template.This biotechnological method based on TaqMan PCR was appropriate to identify porcine-derived materials in farm products and foods.
引文
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[15]徐琼,张奕南,顾文佳,等.Taq Man实时荧光PCR法定量检测生肉中猪源性成分的建立[J].食品科技,2016,41(2):309-313.XU Q,ZHANG Y N,GU W J,et al.Establishment of quantitative Taq Man real-time PCR targeting the swine-derived in raw meat[J].Food Science and Technology,2016,41(2):309-313.(in Chinese with English abstract)
[16]王晶晶,傅春玲.Taqman荧光PCR技术在地沟油猪源性成分检测中的应用初探[J].大家健康(学术版),2014,8(3):5,9.WANG J J,FU C L.Application of Taqman real-time PCRin the identification of porcine ingredient in illegal cooking oil[J].For all Health,2014,8(3):5,9.(in Chinese)
[2]李家鹏,乔晓玲,田寒友,等.食品和饲料中动物源性成分检测技术研究进展[J].食品科学,2011,32(9):340-347.LI J P,QIAO X L,TIAN H Y,et al.Research progress of identification techniques for animal derived materials in food and feed[J].Food Science,2011,32(9):340-347.(in Chinese with English abstract)
[3]石盼盼,李旭,吴昊,等.肉及肉制品中动物源性成分核酸检测方法研究进展[J].食品研究与开发,2016,37(10):211-214.SHI P P,LI X,WU H,et al.Research progress in nucleic acid test for animal-derived material in meat and meat product[J].Food Research and Development,2016,37(10):211-214.(in Chinese with English abstract)
[4]LAHIFF S,GLENNON M,BRIEN L O,et al.Species-specific PCR for the identification of ovine,porcine and chicken species in meat and bone meal(MBM)[J].Molecular and Cellular Probes,2001,15(1):27-35.
[5]NATONEK-WI S/NIEWSKA M,KRZY S/CIN P,PIESTRZY N/SKA-KAJTOCH A.The species identification of bovine,porcine,ovine and chicken components in animal meals,feeds and their ingredients,based on COX I analysis and ribosomal DNA sequences[J].Food Control,2013,34(1):69-78.
[6]WANG Q,ZHANG X,ZHANG H Y,et al.Identification of12 animal species meat by T-RFLP on the 12S r RNA gene[J].Meat Science,2010,85(2):265-269.
[7]李秀婷,孙宝国,吕跃钢,等.动物源性食品中药物残留的快速检测技术研究进展[J].食品科学,2009,30(19):346-350.LI X T,SUN B G,LYU Y G,et al.Progress in rapid detection techniques for drug residues in animal-origin foods[J].Food Science,2009,30(19):346-350.(in Chinese with English abstract)
[8]LWDVAN R,CVON H,BAETEN V,et al.New developments in the detection and identification of processed animal proteins in feeds[J].Animal Feed Science and Technology,2007,133(1/2):63-83.
[9]WALKER J A,HUGHES D A,ANDERS B A,et al.Quantitative intra-short interspersed element PCR for species-specific DNA identification[J].Analytical Biochemistry,2003,316(2):259-269.
[10]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[J].Meat Science,2008,79(2):289-298.
[11]ZHANG C L,FOWLER M R,SCOTT N W,et al.ATaq Man real-time PCR system for the identification and quantification of bovine DNA in meats,milks and cheeses[J].Food Control,2007,18(9):1149-1158.
[12]PEGELS N,GONZALEZ I,LOPEZ-CALLEJA I,et al.E-valuation of a Taq Man real-time PCR assay for detection of chicken,turkey,duck,and goose material in highly processed industrial feed samples[J].Poultry Science,2012,91(7):1709-1719.
[13]于雷,姜艳彬,王海,等.荧光定量PCR法的优化及其在牛羊源性成分检测中的应用研究[J].中国畜牧杂志,2009,45(24):51-54.YU L,JIANG Y B,WANG H,et al.Optimization and application of real-time PCR in the identification of bovine and ovine materials[J].Chinese Journal of Animal Science,2009,45(24):51-54.(in Chinese)
[14]李鑫,孙甸甸,张慧娟,等.一种食品中猪源性成分的快速检测技术[J].今日畜牧兽医,2018,34(2):1-3.LI X,SUN D D,ZHANG H J,et al.A method for rapid detection of pig ingredient in meat products[J].Today Animal Husbandry and Veterinary Medicine,2018,34(2):1-3.(in Chinese with English abstract)
[15]徐琼,张奕南,顾文佳,等.Taq Man实时荧光PCR法定量检测生肉中猪源性成分的建立[J].食品科技,2016,41(2):309-313.XU Q,ZHANG Y N,GU W J,et al.Establishment of quantitative Taq Man real-time PCR targeting the swine-derived in raw meat[J].Food Science and Technology,2016,41(2):309-313.(in Chinese with English abstract)
[16]王晶晶,傅春玲.Taqman荧光PCR技术在地沟油猪源性成分检测中的应用初探[J].大家健康(学术版),2014,8(3):5,9.WANG J J,FU C L.Application of Taqman real-time PCRin the identification of porcine ingredient in illegal cooking oil[J].For all Health,2014,8(3):5,9.(in Chinese)