微滴式数字PCR定量检测杏仁露中杏仁、花生源性成分
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摘要
为定量分析市售杏仁露中杏仁源性成分和掺假花生源性成分的含量,本研究基于微滴式数字聚合酶链式反应(droplet digital polymerase chain reaction,ddPCR)技术建立准确的检测方法。从杏仁、花生的植物组织提取核酸后,建立植物源性成分质量(M)与DNA拷贝数(C)之间的线性关系。ddPCR研究结果显示,在一定范围内杏仁、花生的质量和DNA含量、DNA含量和DNA拷贝数均呈显著的线性关系,以DNA含量为中间值换算得出公式:M_(杏仁)=0.13C+1.24,M花生=0.081C-0.63。通过已知混合比例的两物种掺假模型验证该公式的准确性和适用性,并对市售的12个杏仁露样品进行检测。研究表明,该方法准确可靠,能达到精准定量。本研究建立的ddPCR方法可有效甄别杏仁露中的花生源性成分为无意沾染或有意掺杂,在杏仁露中杏仁和花生含量的定量检测方面具有较大应用潜力,为植物蛋白饮料的市场监管提供技术支持。
An accurate method to quantitatively analyze the contents of apricot kernel-derived and peanut-derived components in apricot kernel protein drink products was established based on droplet digital polymerase chain reaction(ddPCR) in this study. We established a linear relationship between plant material mass(M) and DNA copy number(C) after the extraction of nucleic acids from both apricot kernels and peanuts. According to the results of ddPCR, we found that the relationships between the mass of each plant material and DNA concentration and between DNA concentration and DNA copy number were both significantly linear within a certain range. The DNA concentration was utilized as an intermediate value to establish the following formulae: M_(apricot kernel) = 0.13 C + 1.24, Mpeanut = 0.081 C-0.63. The accuracy and applicability of this method were tested and verified using known mixtures of apricot kernel and peanut and 12 commercial apricot protein drink products. This method enabled accurate and reliable quantitative analysis, and effective identification of whether the adulteration in apricot kernel protein drink products is inadvertent or intentional. Hence ddPCR has strong potential in quantifying apricot kernel and peanut in apricot kernel protein drink products and provides technical support for market supervision of plant protein drink products.
An accurate method to quantitatively analyze the contents of apricot kernel-derived and peanut-derived components in apricot kernel protein drink products was established based on droplet digital polymerase chain reaction(ddPCR) in this study. We established a linear relationship between plant material mass(M) and DNA copy number(C) after the extraction of nucleic acids from both apricot kernels and peanuts. According to the results of ddPCR, we found that the relationships between the mass of each plant material and DNA concentration and between DNA concentration and DNA copy number were both significantly linear within a certain range. The DNA concentration was utilized as an intermediate value to establish the following formulae: M_(apricot kernel) = 0.13 C + 1.24, Mpeanut = 0.081 C-0.63. The accuracy and applicability of this method were tested and verified using known mixtures of apricot kernel and peanut and 12 commercial apricot protein drink products. This method enabled accurate and reliable quantitative analysis, and effective identification of whether the adulteration in apricot kernel protein drink products is inadvertent or intentional. Hence ddPCR has strong potential in quantifying apricot kernel and peanut in apricot kernel protein drink products and provides technical support for market supervision of plant protein drink products.
引文
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[19]ZHU P Y,WANG C G,HUANG K L,et al.A novel pretreatmentfree duplex chamber digital PCR detection system for the absolute quantitation of GMO samples[J].International Journal of Molecular Sciences,2016,17(3):402.DOI:10.3390/ijms17030402.
[20]DALMIRA F,MELINA P,JOSéBENIGNO V T,et al.Development,optimization,and evaluation of a duplexdroplet digital PCR assay to quantify the T-nos/hmg copy number ratio in genetically modified maize[J].Analytical Chemistry,2015,88(1):812-819.DOI:10.1021/acs.analchem.5b03238.
[21]STRAIN M C,LADA S M,LUONG T,et al.Highly precise measurement of HIV DNA by droplet digital PCR[J].PLoS ONE,2013,8(4):e55943.DOI:10.1371/journal.pone.0055943.
[22]BLAYA J,LLORET E,SANTíSIMA-TRINIDAD A B,et al.Molecular methods(digital PCR and real-time PCR)for the quantification of low copy DNA of Phytophthora nicotianae in environmental samples[J].Pest Management Science,2016,72(4):747.DOI:10.1002/ps.4048.
[23]周巍,李月华,孙勇,等.微滴式数字PCR技术定量检测发酵乳中金黄色葡萄球菌[J].食品科学,2017,38(16):287-291.DOI:10.7506/spkx1002-6630-201716046.
[24]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.
[25]CAI Y C,LI X,LV R,et al.Quantitative analysis of pork and chicken products by droplet digital PCR[J].Biomed Research International,2014,2014:810209.DOI:10.1155/2014/810209.
[26]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.
[27]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.
[28]陈晨,张岩,李永波,等.微滴式数字PCR对肉制品中羊肉和猪肉定量分析[J].现代食品科技,2018,34(1):221-226;194.DOI:10.13982/j.mfst.1673-9078.2018.1.034.
[29]国家认证认可监督管理委员会.食品中过敏原成分检测方法第2部分:实时荧光PCR法检测花生成分:SN/T 1961.2-2007[S].北京:中国标准出版社,2007.
[30]国家认证认可监督管理委员会.出口食品过敏原成分检测第9部分:实时荧光PCR方法检测杏仁成分:SN/T 1961.9-2013[S].北京:中国标准出版社,2013.
[2]KOPPEL R,JURG R,JURG R.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(6):151-155.DOI:10.1007/s00217-010-1371-y.
[3]KANE D E,HELLBERG R S.Identification of species in ground meat products sold on the U.S.commercial market using DNA-based methods[J].Food Control,2016,59:158-163.DOI:10.1016/j.foodcont.2015.05.020.
[4]CALVO J H,OSTA R,ZARAGOZA P.Quantitative PCR detection of pork in raw and heated ground beef and paté[J].Journal of Agricultural and Food Chemistry,2002,50(19):5265-2567.DOI:10.1021/jf0201576.
[5]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.DOI:10.1016/j.meatsci.2012.02.031.
[6]HINDSON B J,NESS K D,MASQUELIER D A,et al.Highthroughput droplet digital PCR system for absolute quantitation of DNA copy number[J].Analytical Chemistry,2011,83(22):8604-8610.DOI:10.1021/ac202028g.
[7]HINDSON C M,CHEVILLET J R,BRIGGS H A,et al.Absolute quantification by droplet digital PCR versus analog real-time PCR[J].Nature Methods,2013,10(10):1003-1005.DOI:10.1038/nmeth.2633.
[8]PINHEIRO L B,COLEMAN V A,HINDSON C M,et al.Evaluation of a droplet digital polymerase chain reaction format for DNA copy number quantification[J].Analytical Chemistry,2012,84(2):1003-1011.DOI:10.1021/ac202578x.
[9]POHL G,SHIH I M.Principle and applications of digital PCR[J].Expert Review of Molecular Diagnostics,2004,4(1):41-47.DOI:10.1586/14737159.4.1.41.
[10]SYKES P J,NEOH S H,BRISCO M J,et al.Quantitation of targets for PCR by use of limiting dilution[J].Biotechniques,1992,13(3):444-449.
[11]刘津,刘二龙,谢力,等.数字聚合酶链式反应技术在食品安全检测领域的研究应用进展[J].食品科学,2016,37(17):275-280.DOI:10.7506/spkx1002-6630-201617046.
[12]ZHANG Y,TANG E T,DU Z.Detection of MET gene copy number in cancer samples using the droplet digital PCR method[J].PLoSONE,2016,11(1):e0146784.DOI:10.1371/journal.pone.0146784.
[13]BHARUTHRAM A,PAXIMADIS M,PICTON A C,et al.Comparison of a quantitative real-time PCR assay and droplet digital PCR for copy number analysis of the CCL4L genes[J].Infection Genetics&Evolution,2014,25(7):28-35.DOI:10.1016/j.meegid.2014.03.028.
[14]BHAT S,POLANOWSKI A M,DOUBLE M C,et al.The effect of input DNA copy number on genotype call and characterising SNPmarkers in the humpback whale genome using a nanofluidic array[J].PLoS ONE,2012,7(6):e39181.DOI:10.1371/journal.pone.0039181.
[15]LO Y M,LUN F M,CHAN K C,et al.Digital PCR for the molecular detection of fetal chromosomal aneuploidy[J].Proceedings of the National Academy of Sciences of the United States of America,2007,104(32):13116-13121.DOI:10.1073/pnas.0705765104.
[16]BARRETT A N,MCDONNELL T C,CHAN K C,et al.Digital PCRanalysis of maternal plasma for noninvasive detection of sickle cell anemia[J].Clinical Chemistry,2012,58(6):1026-1032.DOI:10.1373/clinchem.2011.178939.
[17]ORHANT L,ANSELEM O,FRADIN M,et al.Droplet digital PCRcombined with minisequencing,a new approach to analyze fetal DNA from maternal blood:application to the non-invasive prenatal diagnosis of achondroplasia[J].Prenatal Diagnosis,2016,36(5):397-406.DOI:10.1002/pd.4790.
[18]MORISSET D,?TEBIH D,MILAVEC M,et al.Quantitative analysis of food and feed samples with droplet digital PCR[J].PLoS ONE,2013,8(5):e62583.DOI:10.1371/Journal.pone.0062583.
[19]ZHU P Y,WANG C G,HUANG K L,et al.A novel pretreatmentfree duplex chamber digital PCR detection system for the absolute quantitation of GMO samples[J].International Journal of Molecular Sciences,2016,17(3):402.DOI:10.3390/ijms17030402.
[20]DALMIRA F,MELINA P,JOSéBENIGNO V T,et al.Development,optimization,and evaluation of a duplexdroplet digital PCR assay to quantify the T-nos/hmg copy number ratio in genetically modified maize[J].Analytical Chemistry,2015,88(1):812-819.DOI:10.1021/acs.analchem.5b03238.
[21]STRAIN M C,LADA S M,LUONG T,et al.Highly precise measurement of HIV DNA by droplet digital PCR[J].PLoS ONE,2013,8(4):e55943.DOI:10.1371/journal.pone.0055943.
[22]BLAYA J,LLORET E,SANTíSIMA-TRINIDAD A B,et al.Molecular methods(digital PCR and real-time PCR)for the quantification of low copy DNA of Phytophthora nicotianae in environmental samples[J].Pest Management Science,2016,72(4):747.DOI:10.1002/ps.4048.
[23]周巍,李月华,孙勇,等.微滴式数字PCR技术定量检测发酵乳中金黄色葡萄球菌[J].食品科学,2017,38(16):287-291.DOI:10.7506/spkx1002-6630-201716046.
[24]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.
[25]CAI Y C,LI X,LV R,et al.Quantitative analysis of pork and chicken products by droplet digital PCR[J].Biomed Research International,2014,2014:810209.DOI:10.1155/2014/810209.
[26]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.
[27]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.
[28]陈晨,张岩,李永波,等.微滴式数字PCR对肉制品中羊肉和猪肉定量分析[J].现代食品科技,2018,34(1):221-226;194.DOI:10.13982/j.mfst.1673-9078.2018.1.034.
[29]国家认证认可监督管理委员会.食品中过敏原成分检测方法第2部分:实时荧光PCR法检测花生成分:SN/T 1961.2-2007[S].北京:中国标准出版社,2007.
[30]国家认证认可监督管理委员会.出口食品过敏原成分检测第9部分:实时荧光PCR方法检测杏仁成分:SN/T 1961.9-2013[S].北京:中国标准出版社,2013.