基于微滴数字PCR阿胶定量检测方法的建立
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摘要
为对阿胶及相关产品进行精确定量和纯度鉴定,本研究建立了一种基于微滴数字PCR(dd PCR)的阿胶定量检测方法。结果显示,在一定范围内阿胶质量与DNA含量以及DNA含量与DNA绝对拷贝数间均呈线性关系。以DNA含量作为换算值,确定出阿胶质量M阿胶(mg)与DNA绝对拷贝数C(copies/μL)的线性关系为M阿胶=0.13C+3.05。最终利用dd PCR检测样品中目的基因的绝对拷贝数来确定样品中的阿胶含量。准确性验证实验显示实测值与真实值基本保持一致,实测偏差最大仅为4.0%,这表明所建立的方法准确可靠。利用该方法对市场上不同品牌的8个阿胶和2个阿胶速溶粉进行检测,结果显示8个阿胶中,3款阿胶较纯,2款阿胶的阿胶含量较低,最低的仅为23.3%。而2款阿胶速溶粉的阿胶含量分别为44.8%和39.8%。以上表明该方法能有效对市售的阿胶产品进行定量检测和纯度鉴定。本实验建立的基于dd PCR的阿胶定量检测方法准确、高效、稳定,有较大的应用价值和应用前景,可有效预防阿胶掺假现象的发生。
A quantitative detection method based on droplet digital PCR( dd PCR) technique was established to determine the purity of donkey-hide gelatin and its subsidiary products.The results showed that in a given range,the correlationship between raw gelatin weight and DNA concentration and between DNA concentration and target DNA copy number were both close to linear.Using DNA concentration as intermediate,the linear formula between raw gelatin weight M( mg) and target DNA copy number C( copies/μL),M = 0.13 C + 3.05,was calculated out.Thus,gelatin quantification was carried out in the samples by the formula between raw gelatin weight and target DNA copy number,which was calculated by dd PCR. Accuracy test showed that measured value was basically the same as the real value and the maximum deviation was just 4%,which indicates that the method was accurate and reliable.Using this method,8 gelatins and 2 gelatin instant powder in the market were tested.Results showed that,in 8 gelatins,3 were relatively pure and 2 of them had low gelatin content and the lowest was just 23.3%. The content of gelatin in 2 gelatin instant powder was 44.8% and 39.8%,respectively.This showed that this method could be used effectively in quantitative detection and purity identification for donkey-hide gelatin and its subsidiary products. The quantitative detection method based on dd PCR in this paper was accurate,efficient and stable.It had great application value and application prospect,which can effectively prevent the occurrence of gelatin adulteration.
A quantitative detection method based on droplet digital PCR( dd PCR) technique was established to determine the purity of donkey-hide gelatin and its subsidiary products.The results showed that in a given range,the correlationship between raw gelatin weight and DNA concentration and between DNA concentration and target DNA copy number were both close to linear.Using DNA concentration as intermediate,the linear formula between raw gelatin weight M( mg) and target DNA copy number C( copies/μL),M = 0.13 C + 3.05,was calculated out.Thus,gelatin quantification was carried out in the samples by the formula between raw gelatin weight and target DNA copy number,which was calculated by dd PCR. Accuracy test showed that measured value was basically the same as the real value and the maximum deviation was just 4%,which indicates that the method was accurate and reliable.Using this method,8 gelatins and 2 gelatin instant powder in the market were tested.Results showed that,in 8 gelatins,3 were relatively pure and 2 of them had low gelatin content and the lowest was just 23.3%. The content of gelatin in 2 gelatin instant powder was 44.8% and 39.8%,respectively.This showed that this method could be used effectively in quantitative detection and purity identification for donkey-hide gelatin and its subsidiary products. The quantitative detection method based on dd PCR in this paper was accurate,efficient and stable.It had great application value and application prospect,which can effectively prevent the occurrence of gelatin adulteration.
引文
[1]吴海燕,孙佳明,张辉.阿胶的研究进展[J].吉林中医药,2016,36(1):57-60.
[2]Liu M,Tan H,Zhang X,et al.Hematopoietic effects and mechanisms of Fufange’jiaojiang on radiotherapy and chemotherapy-induced myelosuppressed mice[J].Journal of Ethnopharmacology,2014,152(3):575-584.
[3]果敏,马洪宇,沈继朵,等.复方阿胶浆对H22肝癌荷瘤小鼠5-FU化疗的增效减毒作用[J].中国实验方剂学杂志,2012,18(20):216-219.
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[6]Cai L,Feng J,Regenstein J,et al.Confectionery gels:Effects of low calorie sweeteners on the rheological properties and microstructure of fish gelatin[J].Food Hydrocolloids,2017,67:157-165.
[7]Yilmaz M T,Kesmen Z,Baykal B,et al.A novel method to differentiate bovine and porcine gelatins in food products:Nano UPLC-ESI-Q-TOF-MS(E)based data independent acquisition technique to detect marker peptides in gelatin[J].Food Chemistry,2013,141(3):2450.
[8]Flaudrops C,Armstrong N,Raoult D,et al.Determination of the animal origin of meat and gelatin by MALDI-TOF-MS[J].Journal of Food Composition&Analysis,2015,41:104-112.
[9]Hashim D M,Man Y B C,Norakasha R,et al.Potential use of Fourier transform infrared spectroscopy for differentiation of bovine and porcine gelatins[J].Food Chemistry,2010,118(3):856-860.
[10]Cebi N,Durak M Z,Toker O S,et al.An evaluation of Fourier transforms infrared spectroscopy method for the classification and discrimination of bovine,porcine and fish gelatins[J].Food Chemistry,2016,190:1109-1115.
[11]Venien A,Levieux D.Differentiation of bovine from porcine gelatines using polyclonal anti-peptide antibodies in indirect and competitive indirect ELISA[J].Journal of Pharmaceutical&Biomedical Analysis,2005,39(3-4):418-424.
[12]古今,刘萍,胡景华.三种动物胶的SDS不连续聚丙烯酰胺电泳法鉴别[J].北京中医杂志,2003,22(1):33-34.
[13]Azira T N,Man Y B C,Hafidz R,et al.Use of principal component analysis for differentiation of gelatine sources based on polypeptide molecular weights[J].Food Chemistry,2014,151(20):286-292.
[14]Mutalib S A,Muin N M,Abdullah A,et al.Sensitivity of polymerase chain reaction(PCR)-southern hybridization and conventional PCR analysis for Halal authentication of gelatin capsules[J].LWT-Food Science and Technology,2015,63(1):714-719.
[15]罗晖明,肖炳燚,聂平,等.阿胶原料驴源性成分的DNA分子鉴别方法[J].药物分析杂志,2017,37(2):202-211.
[16]Hindson C,Chevillet J,Briggs H,et al.Absolute quantification by droplet digital PCR versus analog real-time PCR[J].Nature Methods,2013,10(10):1003-1005.
[17]Sanders R,Huggett J,Bushell C,et al.Evaluation of digital PCR for absolute DNA quantification[J].Analytical Chemistry,2011,83(17):6474-6484.
[18]Pinheiro L,Coleman V,Hindson C,et al.Evaluation of a droplet digital polymerase chain reaction format for DNA copy number quantification[J].Analytical Chemistry,2012,84(2):1003-1011.
[19]Maker M.Digital PCR hits its stride[J].Nature Methods,2012,9(6):541-544.
[20]Cai Y,Li X,Lv R,et al.Quantitative analysis of pork and chicken products by droplet digital PCR[J].Biomed Research International,2014,2014:810209.
[21]Ren J,Deng T,Huang W,et al.A digital PCR method for identifying and quantifying adulteration of meat species in raw and processed food[J].Plos One,2017,12(3):e0173567.
[22]苗丽,张秀平,陈静,等.肉制品中羊源性成分微滴数字PCR法定量检测方法的研究[J].食品工业科技,2016,37(4):73-76.
[23]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.
[24]中华人民共和国出入境检验检疫行业标准.SN/T 3730.4-2013.食品及饲料中常见畜类品种的鉴定方法第4部分:驴成分检测实时荧光PCR法[S].2013.
[2]Liu M,Tan H,Zhang X,et al.Hematopoietic effects and mechanisms of Fufange’jiaojiang on radiotherapy and chemotherapy-induced myelosuppressed mice[J].Journal of Ethnopharmacology,2014,152(3):575-584.
[3]果敏,马洪宇,沈继朵,等.复方阿胶浆对H22肝癌荷瘤小鼠5-FU化疗的增效减毒作用[J].中国实验方剂学杂志,2012,18(20):216-219.
[4]高景会,王蕊,范锋.阿胶现代研究进展[J].中国药事,2011,25(4):396-401.
[5]Karim AA,Bhat R.Fish gelatin:Properties,challenges,and prospects as an alternative to mammalian gelatins[J].Food Hydrocolloids,2009,23(3):563-576.
[6]Cai L,Feng J,Regenstein J,et al.Confectionery gels:Effects of low calorie sweeteners on the rheological properties and microstructure of fish gelatin[J].Food Hydrocolloids,2017,67:157-165.
[7]Yilmaz M T,Kesmen Z,Baykal B,et al.A novel method to differentiate bovine and porcine gelatins in food products:Nano UPLC-ESI-Q-TOF-MS(E)based data independent acquisition technique to detect marker peptides in gelatin[J].Food Chemistry,2013,141(3):2450.
[8]Flaudrops C,Armstrong N,Raoult D,et al.Determination of the animal origin of meat and gelatin by MALDI-TOF-MS[J].Journal of Food Composition&Analysis,2015,41:104-112.
[9]Hashim D M,Man Y B C,Norakasha R,et al.Potential use of Fourier transform infrared spectroscopy for differentiation of bovine and porcine gelatins[J].Food Chemistry,2010,118(3):856-860.
[10]Cebi N,Durak M Z,Toker O S,et al.An evaluation of Fourier transforms infrared spectroscopy method for the classification and discrimination of bovine,porcine and fish gelatins[J].Food Chemistry,2016,190:1109-1115.
[11]Venien A,Levieux D.Differentiation of bovine from porcine gelatines using polyclonal anti-peptide antibodies in indirect and competitive indirect ELISA[J].Journal of Pharmaceutical&Biomedical Analysis,2005,39(3-4):418-424.
[12]古今,刘萍,胡景华.三种动物胶的SDS不连续聚丙烯酰胺电泳法鉴别[J].北京中医杂志,2003,22(1):33-34.
[13]Azira T N,Man Y B C,Hafidz R,et al.Use of principal component analysis for differentiation of gelatine sources based on polypeptide molecular weights[J].Food Chemistry,2014,151(20):286-292.
[14]Mutalib S A,Muin N M,Abdullah A,et al.Sensitivity of polymerase chain reaction(PCR)-southern hybridization and conventional PCR analysis for Halal authentication of gelatin capsules[J].LWT-Food Science and Technology,2015,63(1):714-719.
[15]罗晖明,肖炳燚,聂平,等.阿胶原料驴源性成分的DNA分子鉴别方法[J].药物分析杂志,2017,37(2):202-211.
[16]Hindson C,Chevillet J,Briggs H,et al.Absolute quantification by droplet digital PCR versus analog real-time PCR[J].Nature Methods,2013,10(10):1003-1005.
[17]Sanders R,Huggett J,Bushell C,et al.Evaluation of digital PCR for absolute DNA quantification[J].Analytical Chemistry,2011,83(17):6474-6484.
[18]Pinheiro L,Coleman V,Hindson C,et al.Evaluation of a droplet digital polymerase chain reaction format for DNA copy number quantification[J].Analytical Chemistry,2012,84(2):1003-1011.
[19]Maker M.Digital PCR hits its stride[J].Nature Methods,2012,9(6):541-544.
[20]Cai Y,Li X,Lv R,et al.Quantitative analysis of pork and chicken products by droplet digital PCR[J].Biomed Research International,2014,2014:810209.
[21]Ren J,Deng T,Huang W,et al.A digital PCR method for identifying and quantifying adulteration of meat species in raw and processed food[J].Plos One,2017,12(3):e0173567.
[22]苗丽,张秀平,陈静,等.肉制品中羊源性成分微滴数字PCR法定量检测方法的研究[J].食品工业科技,2016,37(4):73-76.
[23]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.
[24]中华人民共和国出入境检验检疫行业标准.SN/T 3730.4-2013.食品及饲料中常见畜类品种的鉴定方法第4部分:驴成分检测实时荧光PCR法[S].2013.