PCR技術(shù)在肉類成分定量分析中的應(yīng)用研究進(jìn)展

楊　艷,王桂姬,周廣運(yùn),任皓威,夏秀芳,劉　寧

(東北農(nóng)業(yè)大學(xué)食品學(xué)院,黑龍江哈爾濱 150030)

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PCR技術(shù)在肉類成分定量分析中的應(yīng)用研究進(jìn)展

楊艷,王桂姬,周廣運(yùn),任皓威,夏秀芳,劉寧*

(東北農(nóng)業(yè)大學(xué)食品學(xué)院,黑龍江哈爾濱 150030)

PCR技術(shù)具有特異性強(qiáng)、靈敏度高、可同時(shí)分析大量樣本的特點(diǎn),在鑒別和定量分析食品中肉類成分上應(yīng)用廣泛。本文介紹了PCR擴(kuò)增產(chǎn)物分析、實(shí)時(shí)熒光PCR和微滴數(shù)字PCR三種應(yīng)用于肉類定量的技術(shù),通過(guò)檢出限、定量限和定量范圍等數(shù)據(jù)分析這三種方法在肉類成分定量分析中的應(yīng)用效果,并對(duì)其定量的局限性進(jìn)行闡述,為進(jìn)一步完善PCR定量技術(shù)提供參考。

PCR,肉類成分,定量,應(yīng)用

肉類摻假一直受到社會(huì)各界的廣泛關(guān)注,與肉類摻假相比,標(biāo)簽欺詐問(wèn)題由于其極強(qiáng)的隱蔽性和難以確證性,并沒(méi)有得到公眾的足夠重視。2014年和2015年,我國(guó)媒體相繼曝出“牛丸”中幾乎不含牛肉,“魚(yú)丸”中魚(yú)肉難尋的現(xiàn)象,而早在2009年就有學(xué)者提出,食品標(biāo)簽中肉類成分含量與食品本身存在高達(dá)20%的差異[1]。為了有效規(guī)范市場(chǎng)秩序、保障食品安全和消費(fèi)者權(quán)益,人們提出了很多鑒別和定量肉類成分的分析方法,如色譜法[2]、質(zhì)譜法[3]、免疫法[4]、蛋白質(zhì)電泳[5]、核酸雜交[6]、電子鼻和電子舌[7]技術(shù)以及紅外光譜法[8]等。以DNA為研究對(duì)象的PCR(Polymerize Chain Reaction)法以較高的敏感性和特異性,得到廣泛的認(rèn)可。

PCR是選擇特定的寡核苷酸雜交目標(biāo)DNA,在體外合成數(shù)以百萬(wàn)計(jì)的基因片段的方法。目前用于肉類鑒別的PCR方法有很多,如DNA擴(kuò)增子的測(cè)序、PCR限制性酶切片段長(zhǎng)度多態(tài)性分析、隨機(jī)擴(kuò)增多態(tài)性DNA分析、PCR單鏈構(gòu)象多態(tài)性分析和巢式PCR等等[9-12]。與多種多樣的PCR鑒別方法相比,用PCR對(duì)肉類進(jìn)行定量的方法則相對(duì)較少。本文總結(jié)了用于肉類成分定量的三種PCR方法:PCR擴(kuò)增產(chǎn)物分析、實(shí)時(shí)熒光PCR和微滴數(shù)字PCR,為研究者應(yīng)用PCR方法定量食品中肉類成分提供參考。

1　PCR擴(kuò)增產(chǎn)物分析

PCR擴(kuò)增產(chǎn)物分析主要通過(guò)分析凝膠電泳條帶強(qiáng)度進(jìn)行[13],在標(biāo)準(zhǔn)品中目標(biāo)樣本質(zhì)量分?jǐn)?shù)0.1%～99%之間選擇8個(gè)左右的值,經(jīng)過(guò)PCR擴(kuò)增后,選用特定圖像分析軟件如 Kodak Digital ScienceTM和Molecular Analyst對(duì)擴(kuò)增產(chǎn)物凝膠電泳條帶強(qiáng)度進(jìn)行標(biāo)準(zhǔn)化,將標(biāo)準(zhǔn)化后的擴(kuò)增產(chǎn)物凝膠強(qiáng)度值與樣本濃度之間建立標(biāo)準(zhǔn)曲線,對(duì)未知樣本凝膠強(qiáng)度進(jìn)行校正,將校正值代入標(biāo)準(zhǔn)曲線,估測(cè)樣本含量。用凝膠電泳條帶強(qiáng)度分析來(lái)定量肉類成分的PCR方法有三種:PCR產(chǎn)物直接定量[14]、非競(jìng)爭(zhēng)性對(duì)照基因定量[15]和競(jìng)爭(zhēng)PCR[16]。

1.1PCR產(chǎn)物直接定量

Calvo[17]特異性擴(kuò)增豬短散核序SINE(Short Interspersed Nuclear Element),用純豬DNA對(duì)豬肉質(zhì)量百分?jǐn)?shù)不同的標(biāo)準(zhǔn)品擴(kuò)增產(chǎn)物條帶強(qiáng)度標(biāo)準(zhǔn)化,以標(biāo)準(zhǔn)化后的條帶密度作為橫坐標(biāo)、豬肉質(zhì)量百分?jǐn)?shù)作為縱坐標(biāo)建立標(biāo)準(zhǔn)曲線,在以牛肉作為主料的混合肉中檢測(cè)出1%的豬肉的添加,認(rèn)為當(dāng)豬肉成分在1%～75%之間線性關(guān)系良好,并且在鴨肉制品中檢測(cè)到超過(guò)標(biāo)簽標(biāo)識(shí)量(3%)的豬肉成分(26%)。PCR產(chǎn)物直接定量的優(yōu)點(diǎn)在于,實(shí)驗(yàn)設(shè)計(jì)簡(jiǎn)單,引物要求不高,定量范圍較寬,方便操作。但是當(dāng)體系中被測(cè)成分含量較低時(shí),定量結(jié)果變異系數(shù)很高(>25%),無(wú)法做到準(zhǔn)確定量,同時(shí)這種方法僅以豬DNA來(lái)標(biāo)準(zhǔn)化PCR擴(kuò)增產(chǎn)物,沒(méi)有考慮到混合肉中其他肉類成分對(duì)PCR擴(kuò)增的影響,定量結(jié)果參考性不強(qiáng)。

表1　染料法實(shí)時(shí)熒光PCR在肉類定量的應(yīng)用

1.2非競(jìng)爭(zhēng)性對(duì)照基因定量

Soares等人[18]選擇非競(jìng)爭(zhēng)性對(duì)照基因PCR定量法,同時(shí)擴(kuò)增雞和豬混合肉中線粒體DNA,以同一反應(yīng)管中兩種PCR產(chǎn)物條帶密度之和對(duì)豬DNA擴(kuò)增產(chǎn)物條帶密度進(jìn)行標(biāo)準(zhǔn)化,分別取豬肉摻入質(zhì)量百分?jǐn)?shù)和相應(yīng)條帶密度的對(duì)數(shù)值作為橫、縱坐標(biāo),建立標(biāo)準(zhǔn)曲線對(duì)雞肉中摻雜的豬肉進(jìn)行相對(duì)定量,可以檢測(cè)到雞肉中低至0.1%的豬肉成分的添加,同時(shí)確定在豬肉添加量為0.1%～75%時(shí),標(biāo)準(zhǔn)曲線線性關(guān)系良好(R2=0.9891),CV ≤ 7.61%;Chaumpluk[19]等以牛甲狀腺Pth特異性基因?yàn)槟繕?biāo)基因,以真核生物12s rRNA為非競(jìng)爭(zhēng)性對(duì)照基因,以Hoechst 33258染料標(biāo)記PCR擴(kuò)增產(chǎn)物,通過(guò)分析擴(kuò)增產(chǎn)物條帶密度對(duì)模擬寵物食品中的牛肉成分進(jìn)行定量,準(zhǔn)確性達(dá)到90.66%。這種定量方法思路很簡(jiǎn)單,可實(shí)現(xiàn)同時(shí)對(duì)混合肉中兩種成分的相對(duì)定量,節(jié)約時(shí)間和經(jīng)費(fèi),但對(duì)引物要求較高,既要求擴(kuò)增產(chǎn)物長(zhǎng)度差異足夠大到系統(tǒng)可以區(qū)分,又要求兩種引物具有相似的擴(kuò)增效果,這一點(diǎn)很難平衡。

1.3競(jìng)爭(zhēng)PCR

競(jìng)爭(zhēng)PCR是指在PCR體系中靶基因添加量恒定的情況下,按照與靶基因(Target)的一定比率將競(jìng)爭(zhēng)性模板(Competitor)添加到體系中,分析PCR擴(kuò)增產(chǎn)物凝膠電泳條帶密度與 log(RatioTarget/Competitor)的關(guān)系,建立標(biāo)準(zhǔn)曲線,對(duì)未知樣本進(jìn)行定量。Wolf[20]等首次將競(jìng)爭(zhēng)PCR應(yīng)用于對(duì)豬-牛DNA混合物中對(duì)豬DNA的定量,但他們沒(méi)有制作標(biāo)準(zhǔn)曲線,對(duì)豬DNA百分含量2%和20%兩個(gè)混合體系進(jìn)行擴(kuò)增,通過(guò)直接觀察未知樣本凝膠電泳條帶亮度來(lái)大致判斷豬DNA模板百分含量,屬于一種半定量方法。Aslaminejad[21]等選擇的 RatioTarget/Competitor分別為10-1、10-1.7、10-2,通過(guò)在混合DNA模板中分別摻入40%、30%、20%、10%和1%的雞DNA,建立標(biāo)準(zhǔn)曲線(R2=0.99),確定所測(cè)試的五種香腸商品中,雞肉添加量在23.87%～52.06%之間。競(jìng)爭(zhēng)PCR是擴(kuò)增產(chǎn)物分析中一種相對(duì)準(zhǔn)確的定量方法,可以消除在PCR擴(kuò)增過(guò)程中反應(yīng)管之間、標(biāo)本之間的差異,但是競(jìng)爭(zhēng)模板的構(gòu)建相對(duì)困難,并且需要多次探索以尋找合適的靶基因和競(jìng)爭(zhēng)模板的比率,耗時(shí)較長(zhǎng)。

2　實(shí)時(shí)熒光PCR(Real-Time PCR)

實(shí)時(shí)熒光PCR是在普通的PCR反應(yīng)中添加熒光標(biāo)記物,通過(guò)收集熒光信號(hào)的變化來(lái)實(shí)時(shí)監(jiān)測(cè)PCR反應(yīng)情況的技術(shù),熒光信號(hào)與定量PCR反應(yīng)階段的模板擴(kuò)增直接相關(guān)[22]。該技術(shù)首先由Higuchi[23]等提出,在生命科學(xué)和醫(yī)學(xué)上有著廣泛的應(yīng)用,在食品科學(xué)領(lǐng)域多用于檢測(cè)轉(zhuǎn)基因成分[24]、微生物污染[25]、肉類摻假[26]等方面。其熒光標(biāo)記物主要分為兩種:DNA 嵌入熒光染料和熒光探針[27]。

2.1DNA嵌入熒光染料

在實(shí)時(shí)PCR每個(gè)循環(huán)的延伸階段,當(dāng)染料結(jié)合雙鏈DNA的小溝時(shí),能夠檢測(cè)到熒光增加,實(shí)時(shí)熒光PCR系統(tǒng)通過(guò)識(shí)別熒光強(qiáng)度的變化,繪制擴(kuò)增曲線,確定Ct(Threshold Cycle,循環(huán)閾值),研究者可以知道Ct值與初始模板濃度之間的關(guān)系,對(duì)未知產(chǎn)物進(jìn)行定量。實(shí)時(shí)PCR最常用的染料是SYBR? Green I,近年來(lái)一些研究者發(fā)表著作中提到EvaGreen和SYTO,在實(shí)時(shí)PCR中對(duì)DNA的定量時(shí),更加穩(wěn)定和靈敏[28-29],因而使用這兩種染料進(jìn)行肉類鑒別和定量的論述也逐漸出現(xiàn)。表1總結(jié)染料法實(shí)時(shí)熒光PCR定量肉類成分的研究成果。

表2　探針?lè)▽?shí)時(shí)熒光PCR在肉類定量的應(yīng)用

DNA嵌入熒光染料不用設(shè)計(jì)復(fù)雜的探針、價(jià)格相對(duì)便宜;但其能夠同時(shí)結(jié)合特異性引物、非特異性引物和引物二聚體,因此在以Ct值為判定基礎(chǔ)的定性和定量實(shí)驗(yàn)中易出現(xiàn)假陽(yáng)性結(jié)果和定量偏高的情況。為此,人們開(kāi)發(fā)了以分析溶解曲線鑒別和半定量物種成分的方法,根據(jù)多重PCR體系中產(chǎn)物解鏈溫度Tm值的不同,通過(guò)擴(kuò)增產(chǎn)物溶解曲線對(duì)物種進(jìn)行鑒別和定量。María[36]等通過(guò)SYBR? Green I染色法實(shí)時(shí)熒光PCR,分析PCR產(chǎn)物熔解曲線,能定量檢測(cè)牛-馬混合DNA體系中1%的牛DNA和5%的馬DNA;Safdar[37]等通過(guò)建立SYBR二重實(shí)時(shí)熒光PCR反應(yīng)系統(tǒng),對(duì)混合肉制品(牛、羊、雞、豬)進(jìn)行分析,根據(jù)實(shí)時(shí)熒光PCR產(chǎn)物解鏈溫度和溶解曲線峰高度,可以半定量檢測(cè)到0.003%的牛肉和0.005%的羊肉。溶解曲線分析使染色法實(shí)時(shí)熒光PCR同時(shí)鑒別和定量?jī)煞N肉類成分成為可能,但這種分析方法進(jìn)行要求擴(kuò)增產(chǎn)物Tm值有一定差異,故對(duì)特異性引物的設(shè)計(jì)要求較高。

2.2熒光探針標(biāo)記

探針是附加供體或受體熒光團(tuán)的寡核苷酸,常見(jiàn)應(yīng)用于肉類鑒別和定量探針為T(mén)aqman和Taqman-MGB。Taqman探針的5′端包含一個(gè)能釋放熒光的熒光供體,3′端包含一個(gè)熒光受體,當(dāng)兩種分子足夠接近時(shí),受體分子淬滅供體分子釋放出來(lái)的熒光[38],系統(tǒng)即可收集到熒光信號(hào)的變化。Taqman-MGB是指結(jié)合MGB(Minor Groove Binding)配體的Taqman DNA探針[39],MGB配體是一些小分子三肽組成的非共價(jià)結(jié)合DNA雙鏈螺旋小溝的配體,這些配體能選擇性的結(jié)合AT堿基含量豐富的序列,這種特性對(duì)物種鑒別和定量有著很大的作用。與DNA嵌入熒光染料相比,探針實(shí)時(shí)熒光PCR靈敏度高,可同時(shí)進(jìn)行多重PCR反應(yīng)并分別進(jìn)行鑒別和定量,其在肉類成分定量分析中的應(yīng)用情況詳見(jiàn)表2。

3　微滴數(shù)字PCR

微滴數(shù)字PCR是一種絕對(duì)定量的分析方法。該方法以數(shù)字PCR為基礎(chǔ),采取油包水的形式對(duì)DNA模板進(jìn)行極度稀釋,將單個(gè)模板分子分配到獨(dú)立的反應(yīng)容器內(nèi),通過(guò)模板特異性雜交探針來(lái)檢測(cè)PCR的終產(chǎn)物,若有產(chǎn)物生成則可觀測(cè)到熒光的產(chǎn)生[49]。該法最先應(yīng)用于人類基因組[50]的擴(kuò)增與定量,后逐漸應(yīng)用于食品檢測(cè)。Cai[51]對(duì)豬-雞二元混合物(10%～90%)進(jìn)行相對(duì)定量,對(duì)豬肉成分的定量偏差0.67%～17%,對(duì)雞肉成分的定量偏差2%～12.80%,與PCR擴(kuò)增產(chǎn)物分析、實(shí)時(shí)熒光PCR兩種定量方法相比更加準(zhǔn)確;Floren[52]等采取兩步微滴數(shù)字PCR法,通過(guò)擴(kuò)增細(xì)胞核F2基因?qū)庸と庵破分信！ⅠR和豬三個(gè)物種進(jìn)行檢測(cè),每個(gè)物種的檢出限低至0.001%,定量限低至0.01%。該法定量原理是進(jìn)行模板分子計(jì)數(shù),而非通過(guò)標(biāo)準(zhǔn)曲線進(jìn)行標(biāo)定,是相對(duì)準(zhǔn)確的定量方法,但卻相對(duì)耗時(shí),優(yōu)化實(shí)驗(yàn)步驟、降低分析時(shí)間能使這種方法在物種定量上更為廣泛的應(yīng)用。

4　PCR定量方法的局限性與發(fā)展方向

PCR方法是肉類鑒別和定量中較常使用的方法,但隨著研究的深入,PCR定量在實(shí)際應(yīng)用中的局限性逐漸凸顯:實(shí)驗(yàn)室之間、操作者之間對(duì)于實(shí)驗(yàn)的實(shí)施存在一定的誤差,以實(shí)時(shí)熒光定量PCR為例,執(zhí)行和解釋實(shí)時(shí)熒光定量PCR實(shí)驗(yàn)缺乏統(tǒng)一的標(biāo)準(zhǔn),使得一些實(shí)驗(yàn)的重現(xiàn)性不高;由于不同種類、不同部位的動(dòng)物組織DNA含量的差異相對(duì)較大,對(duì)PCR方法定量效果有一定影響;在食品的加工過(guò)程中,DNA可能發(fā)生高度的降解,使基于DNA 的定量方法失效;食品中物種成分多元化,針對(duì)每個(gè)物種逐一分析耗時(shí)耗力。

為突破這種局限性,人們嘗試從不同角度來(lái)優(yōu)化PCR定量方法:一是進(jìn)行誤差分析,Eugster等[53]側(cè)重于研究實(shí)驗(yàn)室之間的差異,以及由此導(dǎo)致的定量結(jié)果的不確定性;Bustin等[54]提出了MIQE指導(dǎo)方針(Minimum Information for Publication of Quantitative Real-Time PCR Experiments,熒光定量PCR中至少應(yīng)提供的信息),以保證研究者進(jìn)行更好的實(shí)驗(yàn)嘗試并提供更多可信的、不模糊的定量PCR結(jié)果。二是建立更符合肉制品實(shí)際情況的模型,進(jìn)行實(shí)時(shí)PCR法肉類定量研究[55-56]。三是選擇組織差異不明顯的目的基因,降低組織間DNA含量差異性對(duì)定量的影響,Zhang[57]等通過(guò)擴(kuò)增豬特異性短散核序列SINE,Ballin等[58]通過(guò)擴(kuò)增重復(fù)序列,確定以SINE為目標(biāo)的擴(kuò)增沒(méi)有組織差異性,定量標(biāo)準(zhǔn)差低至0.06%。四是設(shè)計(jì)新型引物和探針,提高PCR法對(duì)高度降解DNA定量的敏感性和多物種同時(shí)定量能力,Cai[59]等設(shè)計(jì)了一種新型探針,在5′端標(biāo)記FAM,分別在探針的中部和3′尾部標(biāo)記Zen和Iowa black FQ兩種基團(tuán),在高度降解的凝膠中,對(duì)豬和牛的檢出限低至1 pg/mL;K?ppel等[60]開(kāi)發(fā)了“AllFleisch”Taqman探針,能同時(shí)對(duì)七種肉(牛肉、雞肉、豬肉、火雞肉、馬肉、山羊肉和綿羊肉)進(jìn)行相對(duì)定量,檢出限低至2%。

盡管PCR定量方法受到樣本種類、加工情況等制約,但其在肉類成分鑒別和定量的應(yīng)用上依然有較大的優(yōu)勢(shì)。提高定量的準(zhǔn)確性,選擇組織間含量差異小的目標(biāo)基因、設(shè)計(jì)擴(kuò)增產(chǎn)物片段較小的引物、開(kāi)發(fā)靈敏的DNA標(biāo)記與PCR產(chǎn)物分析技術(shù)、研究多物種同時(shí)定量的PCR方法,依然具有廣闊的研究前景。

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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

楊艷(1988-)，女，碩士，主要從事食品質(zhì)量與安全方面的研究，E-mail：yangyan2011@hotmail.com。

劉寧(1960-)，男，博士，教授，主要從事食品質(zhì)量與安全方面的研究，E-mail：ningliuneau@outlook.com。

黑龍江省應(yīng)用技術(shù)與開(kāi)發(fā)計(jì)劃(重大項(xiàng)目)“調(diào)理肉制品加工關(guān)鍵技術(shù)及安全質(zhì)量控制”(GA15B302)。

TS207.3

A

1002-0306(2016)17-0360-06

10.13386/j.issn1002-0306.2016.17.063