PCR技術在肉類成分定量分析中的應用研究進展

楊　艷,王桂姬,周廣運,任皓威,夏秀芳,劉　寧

(東北農業大學食品學院,黑龍江哈爾濱 150030)

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PCR技術在肉類成分定量分析中的應用研究進展

楊艷,王桂姬,周廣運,任皓威,夏秀芳,劉寧*

(東北農業大學食品學院,黑龍江哈爾濱 150030)

PCR技術具有特異性強、靈敏度高、可同時分析大量樣本的特點,在鑒別和定量分析食品中肉類成分上應用廣泛。本文介紹了PCR擴增產物分析、實時熒光PCR和微滴數字PCR三種應用于肉類定量的技術,通過檢出限、定量限和定量范圍等數據分析這三種方法在肉類成分定量分析中的應用效果,并對其定量的局限性進行闡述,為進一步完善PCR定量技術提供參考。

PCR,肉類成分,定量,應用

肉類摻假一直受到社會各界的廣泛關注,與肉類摻假相比,標簽欺詐問題由于其極強的隱蔽性和難以確證性,并沒有得到公眾的足夠重視。2014年和2015年,我國媒體相繼曝出“牛丸”中幾乎不含牛肉,“魚丸”中魚肉難尋的現象,而早在2009年就有學者提出,食品標簽中肉類成分含量與食品本身存在高達20%的差異[1]。為了有效規范市場秩序、保障食品安全和消費者權益,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]、質譜法[3]、免疫法[4]、蛋白質電泳[5]、核酸雜交[6]、電子鼻和電子舌[7]技術以及紅外光譜法[8]等。以DNA為研究對象的PCR(Polymerize Chain Reaction)法以較高的敏感性和特異性,得到廣泛的認可。

PCR是選擇特定的寡核苷酸雜交目標DNA,在體外合成數以百萬計的基因片段的方法。目前用于肉類鑒別的PCR方法有很多,如DNA擴增子的測序、PCR限制性酶切片段長度多態性分析、隨機擴增多態性DNA分析、PCR單鏈構象多態性分析和巢式PCR等等[9-12]。與多種多樣的PCR鑒別方法相比,用PCR對肉類進行定量的方法則相對較少。本文總結了用于肉類成分定量的三種PCR方法:PCR擴增產物分析、實時熒光PCR和微滴數字PCR,為研究者應用PCR方法定量食品中肉類成分提供參考。

1　PCR擴增產物分析

PCR擴增產物分析主要通過分析凝膠電泳條帶強度進行[13],在標準品中目標樣本質量分數0.1%～99%之間選擇8個左右的值,經過PCR擴增后,選用特定圖像分析軟件如 Kodak Digital ScienceTM和Molecular Analyst對擴增產物凝膠電泳條帶強度進行標準化,將標準化后的擴增產物凝膠強度值與樣本濃度之間建立標準曲線,對未知樣本凝膠強度進行校正,將校正值代入標準曲線,估測樣本含量。用凝膠電泳條帶強度分析來定量肉類成分的PCR方法有三種:PCR產物直接定量[14]、非競爭性對照基因定量[15]和競爭PCR[16]。

1.1PCR產物直接定量

Calvo[17]特異性擴增豬短散核序SINE(Short Interspersed Nuclear Element),用純豬DNA對豬肉質量百分數不同的標準品擴增產物條帶強度標準化,以標準化后的條帶密度作為橫坐標、豬肉質量百分數作為縱坐標建立標準曲線,在以牛肉作為主料的混合肉中檢測出1%的豬肉的添加,認為當豬肉成分在1%～75%之間線性關系良好,并且在鴨肉制品中檢測到超過標簽標識量(3%)的豬肉成分(26%)。PCR產物直接定量的優點在于,實驗設計簡單,引物要求不高,定量范圍較寬,方便操作。但是當體系中被測成分含量較低時,定量結果變異系數很高(>25%),無法做到準確定量,同時這種方法僅以豬DNA來標準化PCR擴增產物,沒有考慮到混合肉中其他肉類成分對PCR擴增的影響,定量結果參考性不強。

表1　染料法實時熒光PCR在肉類定量的應用

1.2非競爭性對照基因定量

Soares等人[18]選擇非競爭性對照基因PCR定量法,同時擴增雞和豬混合肉中線粒體DNA,以同一反應管中兩種PCR產物條帶密度之和對豬DNA擴增產物條帶密度進行標準化,分別取豬肉摻入質量百分數和相應條帶密度的對數值作為橫、縱坐標,建立標準曲線對雞肉中摻雜的豬肉進行相對定量,可以檢測到雞肉中低至0.1%的豬肉成分的添加,同時確定在豬肉添加量為0.1%～75%時,標準曲線線性關系良好(R2=0.9891),CV ≤ 7.61%;Chaumpluk[19]等以牛甲狀腺Pth特異性基因為目標基因,以真核生物12s rRNA為非競爭性對照基因,以Hoechst 33258染料標記PCR擴增產物,通過分析擴增產物條帶密度對模擬寵物食品中的牛肉成分進行定量,準確性達到90.66%。這種定量方法思路很簡單,可實現同時對混合肉中兩種成分的相對定量,節約時間和經費,但對引物要求較高,既要求擴增產物長度差異足夠大到系統可以區分,又要求兩種引物具有相似的擴增效果,這一點很難平衡。

1.3競爭PCR

競爭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)的一定比率將競爭性模板(Competitor)添加到體系中,分析PCR擴增產物凝膠電泳條帶密度與 log(RatioTarget/Competitor)的關系,建立標準曲線,對未知樣本進行定量。Wolf[20]等首次將競爭PCR應用于對豬-牛DNA混合物中對豬DNA的定量,但他們沒有制作標準曲線,對豬DNA百分含量2%和20%兩個混合體系進行擴增,通過直接觀察未知樣本凝膠電泳條帶亮度來大致判斷豬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,通過在混合DNA模板中分別摻入40%、30%、20%、10%和1%的雞DNA,建立標準曲線(R2=0.99),確定所測試的五種香腸商品中,雞肉添加量在23.87%～52.06%之間。競爭PCR是擴增產物分析中一種相對準確的定量方法,可以消除在PCR擴增過程中反應管之間、標本之間的差異,但是競爭模板的構建相對困難,并且需要多次探索以尋找合適的靶基因和競爭模板的比率,耗時較長。

2　實時熒光PCR(Real-Time PCR)

實時熒光PCR是在普通的PCR反應中添加熒光標記物,通過收集熒光信號的變化來實時監測PCR反應情況的技術,熒光信號與定量PCR反應階段的模板擴增直接相關[22]。該技術首先由Higuchi[23]等提出,在生命科學和醫學上有著廣泛的應用,在食品科學領域多用于檢測轉基因成分[24]、微生物污染[25]、肉類摻假[26]等方面。其熒光標記物主要分為兩種:DNA 嵌入熒光染料和熒光探針[27]。

2.1DNA嵌入熒光染料

在實時PCR每個循環的延伸階段,當染料結合雙鏈DNA的小溝時,能夠檢測到熒光增加,實時熒光PCR系統通過識別熒光強度的變化,繪制擴增曲線,確定Ct(Threshold Cycle,循環閾值),研究者可以知道Ct值與初始模板濃度之間的關系,對未知產物進行定量。實時PCR最常用的染料是SYBR? Green I,近年來一些研究者發表著作中提到EvaGreen和SYTO,在實時PCR中對DNA的定量時,更加穩定和靈敏[28-29],因而使用這兩種染料進行肉類鑒別和定量的論述也逐漸出現。表1總結染料法實時熒光PCR定量肉類成分的研究成果。

表2　探針法實時熒光PCR在肉類定量的應用

DNA嵌入熒光染料不用設計復雜的探針、價格相對便宜;但其能夠同時結合特異性引物、非特異性引物和引物二聚體,因此在以Ct值為判定基礎的定性和定量實驗中易出現假陽性結果和定量偏高的情況。為此,人們開發了以分析溶解曲線鑒別和半定量物種成分的方法,根據多重PCR體系中產物解鏈溫度Tm值的不同,通過擴增產物溶解曲線對物種進行鑒別和定量。María[36]等通過SYBR? Green I染色法實時熒光PCR,分析PCR產物熔解曲線,能定量檢測牛-馬混合DNA體系中1%的牛DNA和5%的馬DNA;Safdar[37]等通過建立SYBR二重實時熒光PCR反應系統,對混合肉制品(牛、羊、雞、豬)進行分析,根據實時熒光PCR產物解鏈溫度和溶解曲線峰高度,可以半定量檢測到0.003%的牛肉和0.005%的羊肉。溶解曲線分析使染色法實時熒光PCR同時鑒別和定量兩種肉類成分成為可能,但這種分析方法進行要求擴增產物Tm值有一定差異,故對特異性引物的設計要求較高。

2.2熒光探針標記

探針是附加供體或受體熒光團的寡核苷酸,常見應用于肉類鑒別和定量探針為Taqman和Taqman-MGB。Taqman探針的5′端包含一個能釋放熒光的熒光供體,3′端包含一個熒光受體,當兩種分子足夠接近時,受體分子淬滅供體分子釋放出來的熒光[38],系統即可收集到熒光信號的變化。Taqman-MGB是指結合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結合DNA雙鏈螺旋小溝的配體,這些配體能選擇性的結合AT堿基含量豐富的序列,這種特性對物種鑒別和定量有著很大的作用。與DNA嵌入熒光染料相比,探針實時熒光PCR靈敏度高,可同時進行多重PCR反應并分別進行鑒別和定量,其在肉類成分定量分析中的應用情況詳見表2。

3　微滴數字PCR

微滴數字PCR是一種絕對定量的分析方法。該方法以數字PCR為基礎,采取油包水的形式對DNA模板進行極度稀釋,將單個模板分子分配到獨立的反應容器內,通過模板特異性雜交探針來檢測PCR的終產物,若有產物生成則可觀測到熒光的產生[49]。該法最先應用于人類基因組[50]的擴增與定量,后逐漸應用于食品檢測。Cai[51]對豬-雞二元混合物(10%～90%)進行相對定量,對豬肉成分的定量偏差0.67%～17%,對雞肉成分的定量偏差2%～12.80%,與PCR擴增產物分析、實時熒光PCR兩種定量方法相比更加準確;Floren[52]等采取兩步微滴數字PCR法,通過擴增細胞核F2基因對加工肉制品中牛、馬和豬三個物種進行檢測,每個物種的檢出限低至0.001%,定量限低至0.01%。該法定量原理是進行模板分子計數,而非通過標準曲線進行標定,是相對準確的定量方法,但卻相對耗時,優化實驗步驟、降低分析時間能使這種方法在物種定量上更為廣泛的應用。

4　PCR定量方法的局限性與發展方向

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定量在實際應用中的局限性逐漸凸顯:實驗室之間、操作者之間對于實驗的實施存在一定的誤差,以實時熒光定量PCR為例,執行和解釋實時熒光定量PCR實驗缺乏統一的標準,使得一些實驗的重現性不高;由于不同種類、不同部位的動物組織DNA含量的差異相對較大,對PCR方法定量效果有一定影響;在食品的加工過程中,DNA可能發生高度的降解,使基于DNA 的定量方法失效;食品中物種成分多元化,針對每個物種逐一分析耗時耗力。

為突破這種局限性,人們嘗試從不同角度來優化PCR定量方法:一是進行誤差分析,Eugster等[53]側重于研究實驗室之間的差異,以及由此導致的定量結果的不確定性;Bustin等[54]提出了MIQE指導方針(Minimum Information for Publication of Quantitative Real-Time PCR Experiments,熒光定量PCR中至少應提供的信息),以保證研究者進行更好的實驗嘗試并提供更多可信的、不模糊的定量PCR結果。二是建立更符合肉制品實際情況的模型,進行實時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含量差異性對定量的影響,Zhang[57]等通過擴增豬特異性短散核序列SINE,Ballin等[58]通過擴增重復序列,確定以SINE為目標的擴增沒有組織差異性,定量標準差低至0.06%。四是設計新型引物和探針,提高PCR法對高度降解DNA定量的敏感性和多物種同時定量能力,Cai[59]等設計了一種新型探針,在5′端標記FAM,分別在探針的中部和3′尾部標記Zen和Iowa black FQ兩種基團,在高度降解的凝膠中,對豬和牛的檢出限低至1 pg/mL;K?ppel等[60]開發了“AllFleisch”Taqman探針,能同時對七種肉(牛肉、雞肉、豬肉、火雞肉、馬肉、山羊肉和綿羊肉)進行相對定量,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用上依然有較大的優勢。提高定量的準確性,選擇組織間含量差異小的目標基因、設計擴增產物片段較小的引物、開發靈敏的DNA標記與PCR產物分析技術、研究多物種同時定量的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，主要從事食品質量與安全方面的研究，E-mail：ningliuneau@outlook.com。

黑龍江省應用技術與開發計劃(重大項目)“調理肉制品加工關鍵技術及安全質量控制”(GA15B302)。

TS207.3

A

1002-0306(2016)17-0360-06

10.13386/j.issn1002-0306.2016.17.063