应用微滴式数字聚合酶链式反应定量检测牛肉制品中的猪源性成分
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摘要
参照GB/T 25165—2010《明胶中牛、羊、猪源性成分的定性检测方法实时荧光聚合酶链式反应法》,以牛的生长激素(growth hormone,GH)基因和猪的朊蛋白(prion protein,PRNP)基因2种核基因为靶基因合成特异性引物和探针。理论推导单位质量牛肉的GH基因与猪肉PRNP基因拷贝数之比为一固定值,通过微滴式数字聚合酶链式反应(droplet digital polymerase chain reaction,ddPCR)方法对该固定值进行检测和验证,并以该固定值对牛肉/猪肉人工混合样本和市售牛肉制品中的猪肉成分进行定量检测。结果表明:该方法具有良好的准确性及可重复性;牛肉中掺杂猪肉质量分数在5%～99%范围内时,检测结果绝对误差小于1.28%,变异系数小于6.5%,猪肉成分的回收率为99.09%～102.80%;20份牛肉制品中,4份检出猪肉成分,其中3份的相对含量为29.19%～98.15%,1份为0.12%(低于本方法检出限5%)。因此,dd PCR方法可以较好地应用于牛肉制品中掺杂猪肉成分的定量检测。
According to the national standard GB/T 25165-2010, specific primers and probes targeting bovine growth hormone gene(GH) and porcine prion protein gene(PRNP), respectively were synthesized. The ratio of bovine GH gene copy number per unit mass of beef to porcine PRNP gene per unit mass of pork was derived to be a constant value theoretically, which was analyzed and verified by droplet digital polymerase chain reaction(ddPCR). This value was used to quantitatively detect added pork components in beef samples and pork components in adulterated commercial beef products. The results showed that ddPCR was highly accurate and repeatable. When beef added with 5%–99% was detected by ddPCR, the absolute error was less than 1.28%, the coefficient of variation less than 6.5%, and the recovery of pork 99.09%–102.80%. Pork components were detected in 4 out of 20 beef products; the content of pork was 29.19%–98.15% in three of the four and 0.12%(lower than the limit of detection of dd PCR, 5%) in the remaining one. The above results demonstrated that the developed ddPCR method is highly applicable for the quantitative determination of pork components in adulterated commercial beef products.
According to the national standard GB/T 25165-2010, specific primers and probes targeting bovine growth hormone gene(GH) and porcine prion protein gene(PRNP), respectively were synthesized. The ratio of bovine GH gene copy number per unit mass of beef to porcine PRNP gene per unit mass of pork was derived to be a constant value theoretically, which was analyzed and verified by droplet digital polymerase chain reaction(ddPCR). This value was used to quantitatively detect added pork components in beef samples and pork components in adulterated commercial beef products. The results showed that ddPCR was highly accurate and repeatable. When beef added with 5%–99% was detected by ddPCR, the absolute error was less than 1.28%, the coefficient of variation less than 6.5%, and the recovery of pork 99.09%–102.80%. Pork components were detected in 4 out of 20 beef products; the content of pork was 29.19%–98.15% in three of the four and 0.12%(lower than the limit of detection of dd PCR, 5%) in the remaining one. The above results demonstrated that the developed ddPCR method is highly applicable for the quantitative determination of pork components in adulterated commercial beef products.
引文
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[19]杨德平,刘维薇.数字PCR技术在临床诊断中的应用进展[J].临床检验杂志,2016,34(10):785-787.DOI:10.13602/j.cnki.jcls.2016.10.21.
[20]王珊,李志娟,苗丽.微滴式数字PCR与实时荧光PCR检测羊肉制品中羊源和猪源性成分方法的比较[J].肉类工业,2015(7):38-41.DOI:10.3969/j.issn.1008-5467.2015.07.012.
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[25]杨华,汪小福,肖英平,等.牛肉及其制品中掺入鸡肉、鸭肉和猪肉的多重数字PCR快速检测方法研究[J].浙江农业学报,2017,29(6):994-1000.DOI:10.3969/j.issn.1004-1524.2017.06.20.
[26]王建昌,王金凤,陈瑞春,等.鸭肉冒充牛羊肉的分子生物学检测[J].肉类研究,2012,26(6):20-23.
[27]尚柯,段庆梓,张玉,等.多重PCR法用于畜肉源性鉴定的研究[J].食品工业科技,2015,36(1):299-303.
[28]沙才华,杨素,廖秀云,等.多重PCR法用于畜肉源性鉴定的研究[J].食品工业科技,2013,34(8):83-85.
[29]宗卉,范万红,温燕辉,等.应用多重P C R同时检测牛、羊源性成分[J].中国草食动物,2006,26(6):21-23.DOI:10.3969/j.issn.2095-3887.2006.06.008.
[30]中国检验检疫科学研究院.明胶中牛、羊、猪源性成分的定性检测方法实时荧光PCR法:GB/T 25165-2010[S].北京:中国标准出版社,2010.
[31]联合国粮食及农业组织,世界卫生组织.检测、鉴定和量化食品中特定基因序列和蛋白质的执行标准和方法确认准则:CAC/GL 74-2010[S].罗马:食品法典委员会,2010.
[32]中国商业联合会商业标准中心,双汇实业集团有限责任公司,中国肉类食品综合研究中心,等.火腿肠:GB/T 20712-2006[S].北京:中国标准出版社,2006.
[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,2011,2(9):340-347.
[3]张弛,邱郝璞,张筠.实时荧光定量P C R检测肉制品中鸭源性成分[J].食品科学,2013,34(18):154-157.DOI:10.3969/j.issn.1007-5038.2016.11.010.
[4]ZUKAL E,KORMENDY L.On calculation of‘meat content’according to the quantitative ingredient declarations(QUID)[J].Journal of Food Engineering,2007,78(2):614-621.DOI:10.1016/j.jfoodeng.2005.10.033.
[5]任君安,黄文胜,葛毅强,等.肉制品真伪鉴别技术研究进展[J].食品科学,2016,37(1):247-257.DOI:10.7506/spkx1002-6630-201601043.
[6]蒋士强.《食品安全法》的启迪:对风险评估和标准体系的反思(修订稿)[C]//BCEIA2009分析仪器应用技术报告会论文集.北京:中国分析测试协会,2009:1-9.
[7]佚名.国务院严打肉类产品掺假售假等违法行为[J].当代畜牧,2013(15):4.
[8]WASIN’SKI B,OSEK J.New methods of meat species identification and detection of meat adulterations[J].Medycyna Weterynaryjna,2013,69(6):348-352.
[9]BARAI B K,NAYAK R R,SINGHAL R S,et al.Approaches to the detection of meat adulteration[J].Trends in Food Science and Technology,1992,3:69-72.DOI:10.1016/0924-2244(92)90133-H.
[10]薛超波,王萍亚,李素芳,等.基于PCR多物种鉴定技术及其在肉类鉴定中的应用[J].食品安全质量检测学报,2016,7(2):561-566.
[11]PERESTAM A T,FUJISAKI K K,NAVA O,et al.Comparison of realtime PCR and ELISA-based methods for the detection of beef and pork in processed meat products[J].Food Control,2017,71:346-352.DOI:10.1016/j.foodcont.2016.07.017.
[12]MANE B G,MENDIRATTA S K,RAUT A A,et al.PCR-RFLPassay for identification of species origin of meat and meat products[J].Journal of Meat Science and Technology,2014,2(2):31-36.
[13]王宽,刘树柏,张晓梅,等.肉类掺假检测技术研究进展[J].食品安全质量检测学报,2014,5(9):2634-2639.
[14]蔡一村.利用实时荧光PCR和数字PCR技术对肉类制品进行定性和定量检测研究[D].北京:中国科学院大学,2015:52-54.
[15]张明,王东妍,杨文奇,等.牛羊肉中猪源性成分检测能力验证研究[J].食品安全质量检测学报,2013,4(5):1605-1610.
[16]董莲华,张玲,姜君,等.大肠杆菌O157:H7微滴数字PCR定量方法的建立[J].分析化学,2015(3):319-324.
[17]潘广,章桂明,黄新,等.应用双重数字PCR对转基因玉米成分进行定量方法研究[J].植物检疫,2016,30(3):65-71.
[18]胡伟,陈荣华,张晨,等.微滴式数字PCR技术用于生物样品种属鉴定和绝对定量[J].法医学杂志,2014,30(5):342-345.
[19]杨德平,刘维薇.数字PCR技术在临床诊断中的应用进展[J].临床检验杂志,2016,34(10):785-787.DOI:10.13602/j.cnki.jcls.2016.10.21.
[20]王珊,李志娟,苗丽.微滴式数字PCR与实时荧光PCR检测羊肉制品中羊源和猪源性成分方法的比较[J].肉类工业,2015(7):38-41.DOI:10.3969/j.issn.1008-5467.2015.07.012.
[21]REN Junan,DENG Tingting,HUANG Wensheng,et al.A digital PCRmethod for identifying and quantifying adulteration of meat species in raw and processed food[J].PLoS One,2017,12(3):e0173567.DOI:10.1371/journal.pone.0173567.
[22]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.
[23]苗丽,张秀平,陈静,等.肉制品中羊源性成分微滴数字P C R法定量检测方法的研究[J].食品工业科技,2016,37(4):73-76.DOI:10.13386/j.issn1002-0306.2016.04.005.
[24]任君安,邓婷婷,黄文胜,等.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[J].食品科学,2017,38(2):311-316.DOI:10.7506/spkx1002-6630-201702049.
[25]杨华,汪小福,肖英平,等.牛肉及其制品中掺入鸡肉、鸭肉和猪肉的多重数字PCR快速检测方法研究[J].浙江农业学报,2017,29(6):994-1000.DOI:10.3969/j.issn.1004-1524.2017.06.20.
[26]王建昌,王金凤,陈瑞春,等.鸭肉冒充牛羊肉的分子生物学检测[J].肉类研究,2012,26(6):20-23.
[27]尚柯,段庆梓,张玉,等.多重PCR法用于畜肉源性鉴定的研究[J].食品工业科技,2015,36(1):299-303.
[28]沙才华,杨素,廖秀云,等.多重PCR法用于畜肉源性鉴定的研究[J].食品工业科技,2013,34(8):83-85.
[29]宗卉,范万红,温燕辉,等.应用多重P C R同时检测牛、羊源性成分[J].中国草食动物,2006,26(6):21-23.DOI:10.3969/j.issn.2095-3887.2006.06.008.
[30]中国检验检疫科学研究院.明胶中牛、羊、猪源性成分的定性检测方法实时荧光PCR法:GB/T 25165-2010[S].北京:中国标准出版社,2010.
[31]联合国粮食及农业组织,世界卫生组织.检测、鉴定和量化食品中特定基因序列和蛋白质的执行标准和方法确认准则:CAC/GL 74-2010[S].罗马:食品法典委员会,2010.
[32]中国商业联合会商业标准中心,双汇实业集团有限责任公司,中国肉类食品综合研究中心,等.火腿肠:GB/T 20712-2006[S].北京:中国标准出版社,2006.