杨　艳,王桂姬,周广运,任皓威,夏秀芳,刘　宁

(东北农业大学食品学院,黑龙江哈尔滨 150030)



PCR技术在肉类成分定量分析中的应用研究进展

杨艳,王桂姬,周广运,任皓威,夏秀芳,刘宁*

(东北农业大学食品学院,黑龙江哈尔滨 150030)

PCR技术具有特异性强、灵敏度高、可同时分析大量样本的特点,在鉴别和定量分析食品中肉类成分上应用广泛。本文介绍了PCR扩增产物分析、实时荧光PCR和微滴数字PCR三种应用于肉类定量的技术,通过检出限、定量限和定量范围等数据分析这三种方法在肉类成分定量分析中的应用效果,并对其定量的局限性进行阐述,为进一步完善PCR定量技术提供参考。

PCR,肉类成分,定量,应用

肉类掺假一直受到社会各界的广泛关注,与肉类掺假相比,标签欺诈问题由于其极强的隐蔽性和难以确证性,并没有得到公众的足够重视。2014年和2015年,我国媒体相继曝出“牛丸”中几乎不含牛肉,“鱼丸”中鱼肉难寻的现象,而早在2009年就有学者提出,食品标签中肉类成分含量与食品本身存在高达20%的差异[1]。为了有效规范市场秩序、保障食品安全和消费者权益,人们提出了很多鉴别和定量肉类成分的分析方法,如色谱法[2]、质谱法[3]、免疫法[4]、蛋白质电泳[5]、核酸杂交[6]、电子鼻和电子舌[7]技术以及红外光谱法[8]等。以DNA为研究对象的PCR(Polymerize Chain Reaction)法以较高的敏感性和特异性,得到广泛的认可。

PCR是选择特定的寡核苷酸杂交目标DNA,在体外合成数以百万计的基因片段的方法。目前用于肉类鉴别的PCR方法有很多,如DNA扩增子的测序、PCR限制性酶切片段长度多态性分析、随机扩增多态性DNA分析、PCR单链构象多态性分析和巢式PCR等等[9-12]。与多种多样的PCR鉴别方法相比,用PCR对肉类进行定量的方法则相对较少。本文总结了用于肉类成分定量的三种PCR方法:PCR扩增产物分析、实时荧光PCR和微滴数字PCR,为研究者应用PCR方法定量食品中肉类成分提供参考。

1　PCR扩增产物分析

PCR扩增产物分析主要通过分析凝胶电泳条带强度进行[13],在标准品中目标样本质量分数0.1%～99%之间选择8个左右的值,经过PCR扩增后,选用特定图像分析软件如 Kodak Digital ScienceTM和Molecular Analyst对扩增产物凝胶电泳条带强度进行标准化,将标准化后的扩增产物凝胶强度值与样本浓度之间建立标准曲线,对未知样本凝胶强度进行校正,将校正值代入标准曲线,估测样本含量。用凝胶电泳条带强度分析来定量肉类成分的PCR方法有三种:PCR产物直接定量[14]、非竞争性对照基因定量[15]和竞争PCR[16]。

1.1PCR产物直接定量

Calvo[17]特异性扩增猪短散核序SINE(Short Interspersed Nuclear Element),用纯猪DNA对猪肉质量百分数不同的标准品扩增产物条带强度标准化,以标准化后的条带密度作为横坐标、猪肉质量百分数作为纵坐标建立标准曲线,在以牛肉作为主料的混合肉中检测出1%的猪肉的添加,认为当猪肉成分在1%～75%之间线性关系良好,并且在鸭肉制品中检测到超过标签标识量(3%)的猪肉成分(26%)。PCR产物直接定量的优点在于,实验设计简单,引物要求不高,定量范围较宽,方便操作。但是当体系中被测成分含量较低时,定量结果变异系数很高(>25%),无法做到准确定量,同时这种方法仅以猪DNA来标准化PCR扩增产物,没有考虑到混合肉中其他肉类成分对PCR扩增的影响,定量结果参考性不强。

表1　染料法实时荧光PCR在肉类定量的应用

1.2非竞争性对照基因定量

Soares等人[18]选择非竞争性对照基因PCR定量法,同时扩增鸡和猪混合肉中线粒体DNA,以同一反应管中两种PCR产物条带密度之和对猪DNA扩增产物条带密度进行标准化,分别取猪肉掺入质量百分数和相应条带密度的对数值作为横、纵坐标,建立标准曲线对鸡肉中掺杂的猪肉进行相对定量,可以检测到鸡肉中低至0.1%的猪肉成分的添加,同时确定在猪肉添加量为0.1%～75%时,标准曲线线性关系良好(R2=0.9891),CV ≤ 7.61%;Chaumpluk[19]等以牛甲状腺Pth特异性基因为目标基因,以真核生物12s rRNA为非竞争性对照基因,以Hoechst 33258染料标记PCR扩增产物,通过分析扩增产物条带密度对模拟宠物食品中的牛肉成分进行定量,准确性达到90.66%。这种定量方法思路很简单,可实现同时对混合肉中两种成分的相对定量,节约时间和经费,但对引物要求较高,既要求扩增产物长度差异足够大到系统可以区分,又要求两种引物具有相似的扩增效果,这一点很难平衡。

1.3竞争PCR

竞争PCR是指在PCR体系中靶基因添加量恒定的情况下,按照与靶基因(Target)的一定比率将竞争性模板(Competitor)添加到体系中,分析PCR扩增产物凝胶电泳条带密度与 log(RatioTarget/Competitor)的关系,建立标准曲线,对未知样本进行定量。Wolf[20]等首次将竞争PCR应用于对猪-牛DNA混合物中对猪DNA的定量,但他们没有制作标准曲线,对猪DNA百分含量2%和20%两个混合体系进行扩增,通过直接观察未知样本凝胶电泳条带亮度来大致判断猪DNA模板百分含量,属于一种半定量方法。Aslaminejad[21]等选择的 RatioTarget/Competitor分别为10-1、10-1.7、10-2,通过在混合DNA模板中分别掺入40%、30%、20%、10%和1%的鸡DNA,建立标准曲线(R2=0.99),确定所测试的五种香肠商品中,鸡肉添加量在23.87%～52.06%之间。竞争PCR是扩增产物分析中一种相对准确的定量方法,可以消除在PCR扩增过程中反应管之间、标本之间的差异,但是竞争模板的构建相对困难,并且需要多次探索以寻找合适的靶基因和竞争模板的比率,耗时较长。

2　实时荧光PCR(Real-Time PCR)

实时荧光PCR是在普通的PCR反应中添加荧光标记物,通过收集荧光信号的变化来实时监测PCR反应情况的技术,荧光信号与定量PCR反应阶段的模板扩增直接相关[22]。该技术首先由Higuchi[23]等提出,在生命科学和医学上有着广泛的应用,在食品科学领域多用于检测转基因成分[24]、微生物污染[25]、肉类掺假[26]等方面。其荧光标记物主要分为两种:DNA 嵌入荧光染料和荧光探针[27]。

2.1DNA嵌入荧光染料

在实时PCR每个循环的延伸阶段,当染料结合双链DNA的小沟时,能够检测到荧光增加,实时荧光PCR系统通过识别荧光强度的变化,绘制扩增曲线,确定Ct(Threshold Cycle,循环阈值),研究者可以知道Ct值与初始模板浓度之间的关系,对未知产物进行定量。实时PCR最常用的染料是SYBR® Green I,近年来一些研究者发表著作中提到EvaGreen和SYTO,在实时PCR中对DNA的定量时,更加稳定和灵敏[28-29],因而使用这两种染料进行肉类鉴别和定量的论述也逐渐出现。表1总结染料法实时荧光PCR定量肉类成分的研究成果。

表2　探针法实时荧光PCR在肉类定量的应用

DNA嵌入荧光染料不用设计复杂的探针、价格相对便宜;但其能够同时结合特异性引物、非特异性引物和引物二聚体,因此在以Ct值为判定基础的定性和定量实验中易出现假阳性结果和定量偏高的情况。为此,人们开发了以分析溶解曲线鉴别和半定量物种成分的方法,根据多重PCR体系中产物解链温度Tm值的不同,通过扩增产物溶解曲线对物种进行鉴别和定量。María[36]等通过SYBR® Green I染色法实时荧光PCR,分析PCR产物熔解曲线,能定量检测牛-马混合DNA体系中1%的牛DNA和5%的马DNA;Safdar[37]等通过建立SYBR二重实时荧光PCR反应系统,对混合肉制品(牛、羊、鸡、猪)进行分析,根据实时荧光PCR产物解链温度和溶解曲线峰高度,可以半定量检测到0.003%的牛肉和0.005%的羊肉。溶解曲线分析使染色法实时荧光PCR同时鉴别和定量两种肉类成分成为可能,但这种分析方法进行要求扩增产物Tm值有一定差异,故对特异性引物的设计要求较高。

2.2荧光探针标记

探针是附加供体或受体荧光团的寡核苷酸,常见应用于肉类鉴别和定量探针为Taqman和Taqman-MGB。Taqman探针的5′端包含一个能释放荧光的荧光供体,3′端包含一个荧光受体,当两种分子足够接近时,受体分子淬灭供体分子释放出来的荧光[38],系统即可收集到荧光信号的变化。Taqman-MGB是指结合MGB(Minor Groove Binding)配体的Taqman DNA探针[39],MGB配体是一些小分子三肽组成的非共价结合DNA双链螺旋小沟的配体,这些配体能选择性的结合AT碱基含量丰富的序列,这种特性对物种鉴别和定量有着很大的作用。与DNA嵌入荧光染料相比,探针实时荧光PCR灵敏度高,可同时进行多重PCR反应并分别进行鉴别和定量,其在肉类成分定量分析中的应用情况详见表2。

3　微滴数字PCR

微滴数字PCR是一种绝对定量的分析方法。该方法以数字PCR为基础,采取油包水的形式对DNA模板进行极度稀释,将单个模板分子分配到独立的反应容器内,通过模板特异性杂交探针来检测PCR的终产物,若有产物生成则可观测到荧光的产生[49]。该法最先应用于人类基因组[50]的扩增与定量,后逐渐应用于食品检测。Cai[51]对猪-鸡二元混合物(10%～90%)进行相对定量,对猪肉成分的定量偏差0.67%～17%,对鸡肉成分的定量偏差2%～12.80%,与PCR扩增产物分析、实时荧光PCR两种定量方法相比更加准确;Floren[52]等采取两步微滴数字PCR法,通过扩增细胞核F2基因对加工肉制品中牛、马和猪三个物种进行检测,每个物种的检出限低至0.001%,定量限低至0.01%。该法定量原理是进行模板分子计数,而非通过标准曲线进行标定,是相对准确的定量方法,但却相对耗时,优化实验步骤、降低分析时间能使这种方法在物种定量上更为广泛的应用。

4　PCR定量方法的局限性与发展方向

PCR方法是肉类鉴别和定量中较常使用的方法,但随着研究的深入,PCR定量在实际应用中的局限性逐渐凸显:实验室之间、操作者之间对于实验的实施存在一定的误差,以实时荧光定量PCR为例,执行和解释实时荧光定量PCR实验缺乏统一的标准,使得一些实验的重现性不高;由于不同种类、不同部位的动物组织DNA含量的差异相对较大,对PCR方法定量效果有一定影响;在食品的加工过程中,DNA可能发生高度的降解,使基于DNA 的定量方法失效;食品中物种成分多元化,针对每个物种逐一分析耗时耗力。

为突破这种局限性,人们尝试从不同角度来优化PCR定量方法:一是进行误差分析,Eugster等[53]侧重于研究实验室之间的差异,以及由此导致的定量结果的不确定性;Bustin等[54]提出了MIQE指导方针(Minimum Information for Publication of Quantitative Real-Time PCR Experiments,荧光定量PCR中至少应提供的信息),以保证研究者进行更好的实验尝试并提供更多可信的、不模糊的定量PCR结果。二是建立更符合肉制品实际情况的模型,进行实时PCR法肉类定量研究[55-56]。三是选择组织差异不明显的目的基因,降低组织间DNA含量差异性对定量的影响,Zhang[57]等通过扩增猪特异性短散核序列SINE,Ballin等[58]通过扩增重复序列,确定以SINE为目标的扩增没有组织差异性,定量标准差低至0.06%。四是设计新型引物和探针,提高PCR法对高度降解DNA定量的敏感性和多物种同时定量能力,Cai[59]等设计了一种新型探针,在5′端标记FAM,分别在探针的中部和3′尾部标记Zen和Iowa black FQ两种基团,在高度降解的凝胶中,对猪和牛的检出限低至1 pg/mL;Köppel等[60]开发了“AllFleisch”Taqman探针,能同时对七种肉(牛肉、鸡肉、猪肉、火鸡肉、马肉、山羊肉和绵羊肉)进行相对定量,检出限低至2%。

尽管PCR定量方法受到样本种类、加工情况等制约,但其在肉类成分鉴别和定量的应用上依然有较大的优势。提高定量的准确性,选择组织间含量差异小的目标基因、设计扩增产物片段较小的引物、开发灵敏的DNA标记与PCR产物分析技术、研究多物种同时定量的PCR方法,依然具有广阔的研究前景。

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Application progress of polymerase chain reaction in meat quantification

YANG Yan,WANG Gui-ji,ZHOU Guang-yun,REN Hao-wei,XIA Xiu-fang,LIU Ning*

(College of Food Science,Northeast Agricultural University,Harbin 150030,China)

Polymerase chain reaction(PCR)is a highly efficient,specific and sensitive technique in the field of food science. Three meat quantification methods were introduced in this paper including PCR amplicon analysis,Real-time PCR and Droplet Digital PCR. The quantification effect of these three methods were evaluated by LOD,LOQ and quantification range. The limitations were discussed when they were used as a quantitative method as well. This review aim to provide

for improving PCR technology in meat quantification.

PCR;meat;quantification;application

2016-03-01

杨艳(1988-)，女，硕士，主要从事食品质量与安全方面的研究，E-mail：yangyan2011@hotmail.com。

刘宁(1960-)，男，博士，教授，主要从事食品质量与安全方面的研究，E-mail：ningliuneau@outlook.com。

黑龙江省应用技术与开发计划(重大项目)“调理肉制品加工关键技术及安全质量控制”(GA15B302)。

TS207.3

A

1002-0306(2016)17-0360-06

10.13386/j.issn1002-0306.2016.17.063