微滴式数字聚合酶链式反应精准定量检测羊肉中掺杂猪肉
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摘要
以羊和猪的单拷贝持家基因DNA复制蛋白A1为靶基因设计合成了适用于微滴式数字聚合酶链式反应的特异性引物和探针,通过理论推导获得了单位质量两种肉基因拷贝数之比的固定值,并进行了验证,据此将样品中羊肉和猪肉的拷贝数转换为相对质量分数,从而建立了羊肉中掺杂猪肉的精准定量检测方法。该方法可以很好地应用于羊肉中掺杂猪肉的含量检测,猪肉的最低定量限为1%,在5%~80%范围内绝对误差小于±1.30%,相对误差小于±10%,定量结果准确、重复性高,可以为肉类掺假的监管工作提供有力的技术参考。
The adulteration of meat products happened frequently in recent years, and authentication of meat products is necessary to protect consumers from an inferior product with a false description. Although at present, there are numerous qualitative methods for meat species identification, fewer quantitative detection methods have been reported. Herein, a droplet digital PCR method for the quantitative determination of pork incorporated in mutton products was developed. The single copy house-keeping gene encoding replication protein A1(PRA1) was chosen as the target to design species-specific primers and probes for mutton and pig, respetively. Each assay was proved specific to the target species, respectively. The ratio constants between copy numbers and unit mass of pork and mutton were obtained by theoretical analysis and then verified by experiments. Consequently, the relative mass fractions of pork and mutton in the sample were measured based on the DNA copy numbers. The limit of quantitation(LOQ) of the method was confirmed to be 1%. The results showed that the absolute error was less than ±1.3%, and the relative error was less than ±10% in the range of pork proportion from 5% to 80%. The method developed in this paper was successfully applied to quantitate pork content incorporated into commercial mutton products and it may be useful for food administration laboratories to carry out meat species quantification in raw and processed foods.
The adulteration of meat products happened frequently in recent years, and authentication of meat products is necessary to protect consumers from an inferior product with a false description. Although at present, there are numerous qualitative methods for meat species identification, fewer quantitative detection methods have been reported. Herein, a droplet digital PCR method for the quantitative determination of pork incorporated in mutton products was developed. The single copy house-keeping gene encoding replication protein A1(PRA1) was chosen as the target to design species-specific primers and probes for mutton and pig, respetively. Each assay was proved specific to the target species, respectively. The ratio constants between copy numbers and unit mass of pork and mutton were obtained by theoretical analysis and then verified by experiments. Consequently, the relative mass fractions of pork and mutton in the sample were measured based on the DNA copy numbers. The limit of quantitation(LOQ) of the method was confirmed to be 1%. The results showed that the absolute error was less than ±1.3%, and the relative error was less than ±10% in the range of pork proportion from 5% to 80%. The method developed in this paper was successfully applied to quantitate pork content incorporated into commercial mutton products and it may be useful for food administration laboratories to carry out meat species quantification in raw and processed foods.
引文
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[19]苗丽,张秀平,陈静,等.微滴数字PCR法对肉制品中牛源和猪源成分的定量分析[J].食品科学,2016,37(8):187-191.DOI:10.7506/spkx1002-6630-201608033.
[20]BALLIN N Z,VOGENSEN F K,KARLSSON A H.Species determination-can we detect and quantify meat adulteration?[J].Meat Science,2009,83(2):165-174.DOI:10.1016/j.meatsci.2009.06.003.
[21]WANG Wei,HAN Jianxun,WU Yajun,et al.Simultaneous detection of eight food allergens using optical thin-film biosensor chips[J].Journal of Agricultural and Food Chemistry,2011,59(13):6889-6894.DOI:10.1021/jf200933b.
[22]Codex Committee on Methods of Analysis and Sampling.Guidelines on performance criteria and validation of methods for detection identification and quantification of specific DNA sequences and specific proteins in foods:CAC/GL740-2010[S].Rome:Codex Alimentariusliment,2010.
[23]K?PPEL R,RUF J,RENTSCH J.Multiplex real-time PCR for the detection and quantification of DNA from beef,pork,horse and sheep[J].European Food Research and Technology,2011,232(1):151-155.DOI:10.1007/s00217-010-1371-y.
[24]RODRIGUEZ M A,GARCIA T,GONZALEZ I,et al.Identification of goose,mule duck,chicken,turkey,and swine in foiegras by speciesspecific polymerase chain reaction[J].Journal of Agricultural and Food Chemistry,2003,51(6):1524-1529.DOI:10.1021/jf025784+.
[2]李志强,王济民.我国畜产品消费及消费市场前景分析[J].中国农村经济,2000(7):46-51.
[3]金萍,丁洪流,李培,等.2013年苏州地区肉及其制品掺假情况调查[J].中国食品卫生杂志,2014,26(2):168-172.
[4]NAKYINSIGE K,MAN Y B C,SAZILI A Q.Halal authenticity issues in meat and meat products[J].Meat Science,2012,91(3):207-214.DOI:10.1016/j.meatsci.2012.02.015.
[5]SENTANDREU Má,SENTANDREU E.Authenticity of meat products:tools against fraud[J].Food Research International,2014,60:19-29.DOI:10.1016/j.foodres.2014.03.030.
[6]Europe:In official journal of the European communities.Method of analysis for the determination of constituents of animal origin for the official control of feed:EC152-2009[S].Europe,2009.
[7]LIU L,CHEN F C,DORSEY J L,et al.Sensitive monoclonal antibody-based sandwich ELISA for the detection of porcine skeletal muscle in meat and feed products[J].Journal of Food Science,2006,71(1):M1-M6.DOI:10.1111/j.1365-2621.2006.tb12393.x.
[8]MONTOWSKA M,POSPIECH E.Species-specific expression of various proteins in meat tissue:proteomic analysis of raw and cooked meat and meat products made from beef,pork and selected poultry species[J].Food Chemistry,2013,136(3):1461-1469.DOI:10.1016/j.foodchem.2012.09.072.
[9]CHOU C C,LIN S P,LEE K M,et al.Fast differentiation of meats from fifteen animal species by liquid chromatography with electrochemical detection using copper nanoparticle plated electrodes[J].Journal of Chromatography B,2007,846(1):230-239.DOI:10.1016/j.jchromb.2006.09.006.
[10]AMARAL J S,SANTOS C G,MELO V S,et al.Authentication of a traditional game meat sausage(Alheira)by species-specific PCR assays to detect hare,rabbit,red deer,pork and cow meats[J].Food Research International,2014,60:140-145.DOI:10.1016/j.foodres.2013.11.003.
[11]IWOBI A,SEBAH D,KRAEMER I,et al.A multiplex real-time PCR method for the quantification of beef and pork fractions in minced meat[J].Food Chemistry,2015,169(169):305-313.DOI:10.1016/j.foodchem.2014.07.139.
[12]LO?PEZ-ANDREO M,ALDEGUER M,GUILLéN I,et al.Detection and quantification of meat species by q PCR in heat-processed food containing highly fragmented DNA[J].Food Chemistry,2012,134(1):518-523.DOI:10.1016/j.foodchem.2012.02.111.
[13]GUO G,HUSS M,TONG G Q,et al.Resolution of cell fate decisions revealed by single-cell gene expression analysis from zygote to blastocyst[J].Developmental Cell,2010,18(4):675-685.DOI:10.1016/j.devcel.2010.02.012.
[14]DREO T,PIRC M,RAM?AK?,et al.Optimising droplet digital PCR analysis approaches for detection and quantification of bacteria:a case study of fire blight and potato brown rot[J].Analytical and Bioanalytical Chemistry,2014,406(26):6513-6528.DOI:10.1007/s00216-014-8084-1.
[15]MORISSET D,?TEBIH D,MILAVEC M,et al.Quantitative analysis of food and feed samples with droplet digital PCR[J].PLo S ONE,2013,8(5):e62583.DOI:10.1371/journal.pone.0062583.
[16]FLOREN C,WIEDEMANN I,BRENIG B,et al.Species identification and quantification in meat and meat products using droplet digital PCR(dd PCR)[J].Food Chemistry,2015,173(173):1054-1058.DOI:10.1016/j.foodchem.2014.10.138.
[17]RENéK,THOMAS B,ANNA F,et al.Droplet digital PCR versus multiplex real?time PCR method for the detection and quantification of DNA from the four transgenic soy traits MON87769,MON87708,MON87705 and FG72,and lectin[J].European Food Research Technology,2015,241(4):521-527.DOI:10.1007/s00217-015-2481-3.
[18]CAI Y,XIANG L,RONG L,et al.Quantitative analysis of pork and chicken products by droplet digital PCR[J].Biomed Research International,2014,2014(2014):810209.DOI:10.1155/2014/810209.
[19]苗丽,张秀平,陈静,等.微滴数字PCR法对肉制品中牛源和猪源成分的定量分析[J].食品科学,2016,37(8):187-191.DOI:10.7506/spkx1002-6630-201608033.
[20]BALLIN N Z,VOGENSEN F K,KARLSSON A H.Species determination-can we detect and quantify meat adulteration?[J].Meat Science,2009,83(2):165-174.DOI:10.1016/j.meatsci.2009.06.003.
[21]WANG Wei,HAN Jianxun,WU Yajun,et al.Simultaneous detection of eight food allergens using optical thin-film biosensor chips[J].Journal of Agricultural and Food Chemistry,2011,59(13):6889-6894.DOI:10.1021/jf200933b.
[22]Codex Committee on Methods of Analysis and Sampling.Guidelines on performance criteria and validation of methods for detection identification and quantification of specific DNA sequences and specific proteins in foods:CAC/GL740-2010[S].Rome:Codex Alimentariusliment,2010.
[23]K?PPEL R,RUF J,RENTSCH J.Multiplex real-time PCR for the detection and quantification of DNA from beef,pork,horse and sheep[J].European Food Research and Technology,2011,232(1):151-155.DOI:10.1007/s00217-010-1371-y.
[24]RODRIGUEZ M A,GARCIA T,GONZALEZ I,et al.Identification of goose,mule duck,chicken,turkey,and swine in foiegras by speciesspecific polymerase chain reaction[J].Journal of Agricultural and Food Chemistry,2003,51(6):1524-1529.DOI:10.1021/jf025784+.