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各界的廣泛關(guān)注,與肉類摻假相比,標(biāo)簽欺詐問題由于其極強(qiáng)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(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(kuò)增子的測序、PCR限制性酶切片段長度多態(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對肉類進(jìn)行定量的方法則相對較少。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個左右的值,經(jīng)過PCR擴(kuò)增后,選用特定圖像分析軟件如 Kodak Digital ScienceTM和Molecular Analyst對擴(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,估測樣本含量。用凝膠電泳條帶強(qiáng)度分析來定量肉類成分的PCR方法有三種:PCR產(chǎn)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(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到超過標(biāo)簽標(biāo)識量(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)計簡單,引物要求不高,定量范圍較寬,方便操作。但是當(dāng)體系中被測成分含量較低時,定量結(jié)果變異系數(shù)很高(>25%),無法做到準(zhǔn)確定量,同時這種方法僅以豬DNA來標(biāo)準(zhǔn)化PCR擴(kuò)增產(chǎn)物,沒有考慮到混合肉中其他肉類成分對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)時熒光PCR在肉類定量的應(yīng)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(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)條帶密度的對數(shù)值作為橫、縱坐標(biāo),建立標(biāo)準(zhǔn)曲線對雞肉中摻雜的豬肉進(jìn)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(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為非競爭性對照基因,以Hoechst 33258染料標(biāo)記PCR擴(kuò)增產(chǎn)物,通過分析擴(kuò)增產(chǎn)物條帶密度對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單,可實(shí)現(xiàn)同時對混合肉中兩種成分的相對定量,節(jié)約時間和經(jīng)費(fèi),但對引物要求較高,既要求擴(kuò)增產(chǎn)物長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(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,對未知樣本進(jìn)行定量。Wolf[20]等首次將競爭PCR應(yīng)用于對豬-牛DNA混合物中對豬DNA的定量,但他們沒有制作標(biāo)準(zhǔn)曲線,對豬DNA百分含量2%和20%兩個混合體系進(jìn)行擴(kuò)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(biāo)準(zhǔn)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(kuò)增產(chǎn)物分析中一種相對準(zhǔn)確的定量方法,可以消除在PCR擴(kuò)增過程中反應(yīng)管之間、標(biāo)本之間的差異,但是競爭模板的構(gòu)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(shí)時熒光PCR(Real-Time PCR)

實(shí)時熒光PCR是在普通的PCR反應(yīng)中添加熒光標(biāo)記物,通過收集熒光信號的變化來實(shí)時監(jiān)測PCR反應(yīng)情況的技術(shù),熒光信號與定量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PCR每個循環(huán)的延伸階段,當(dāng)染料結(jié)合雙鏈DNA的小溝時,能夠檢測到熒光增加,實(shí)時熒光PCR系統(tǒng)通過識別熒光強(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,對未知產(chǎn)物進(jìn)行定量。實(shí)時PCR最常用的染料是SYBR? Green I,近年來一些研究者發(fā)表著作中提到EvaGreen和SYTO,在實(shí)時PCR中對DNA的定量時,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)時熒光PCR定量肉類成分的研究成果。

表2　探針法實(shí)時熒光PCR在肉類定量的應(yīng)用

DNA嵌入熒光染料不用設(shè)計復(fù)雜的探針、價格相對便宜;但其能夠同時結(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)假陽性結(jié)果和定量偏高的情況。為此,人們開發(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,通過擴(kuò)增產(chǎn)物溶解曲線對物種進(jìn)行鑒別和定量。María[36]等通過SYBR? Green I染色法實(shí)時熒光PCR,分析PCR產(chǎn)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(shí)時熒光PCR反應(yīng)系統(tǒng),對混合肉制品(牛、羊、雞、豬)進(jìn)行分析,根據(jù)實(shí)時熒光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(jìn)行要求擴(kuò)增產(chǎn)物Tm值有一定差異,故對特異性引物的設(shè)計要求較高。

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,常見應(yīng)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(dāng)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(tǒng)即可收集到熒光信號的變化。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)時熒光PCR靈敏度高,可同時進(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)用情況詳見表2。

3　微滴數(shù)字PCR

微滴數(shù)字PCR是一種絕對定量的分析方法。該方法以數(shù)字PCR為基礎(chǔ),采取油包水的形式對DNA模板進(jìn)行極度稀釋,將單個模板分子分配到獨(dú)立的反應(yīng)容器內(nèi),通過模板特異性雜交探針來檢測PCR的終產(chǎn)物,若有產(chǎn)物生成則可觀測到熒光的產(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。Cai[51]對豬-雞二元混合物(10%～90%)進(jìn)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(kuò)增產(chǎn)物分析、實(shí)時熒光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,通過擴(kuò)增細(xì)胞核F2基因?qū)庸と庵破分信?、馬和豬三個物種進(jìn)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(jìn)行模板分子計數(shù),而非通過標(biāo)準(zhǔn)曲線進(jìn)行標(biāo)定,是相對準(zhǔn)確的定量方法,但卻相對耗時,優(yōu)化實(shí)驗(yàn)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(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、操作者之間對于實(shí)驗(yàn)的實(shí)施存在一定的誤差,以實(shí)時熒光定量PCR為例,執(zhí)行和解釋實(shí)時熒光定量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(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(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(kuò)增豬特異性短散核序列SINE,Ballin等[58]通過擴(kuò)增重復(fù)序列,確定以SINE為目標(biāo)的擴(kuò)增沒有組織差異性,定量標(biāo)準(zhǔn)差低至0.06%。四是設(shè)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(shè)計了一種新型探針,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,對豬和牛的檢出限低至1 pg/mL;K?ppel等[60]開發(fā)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(jìn)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(yīng)用上依然有較大的優(yōu)勢。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)計擴(kuò)增產(chǎn)物片段較小的引物、開發(fā)靈敏的DNA標(biāo)記與PCR產(chǎn)物分析技術(shù)、研究多物種同時定量的PCR方法,依然具有廣闊的研究前景。

[1]Ballin N Z,Vogensen F K,Karlsson H A. Species determination-Can we detect and quantify meat adulteration[J]. Meat Science,2009,83(2):165-174.

[2]Giaretta N,Giuseppe A M A D,Lippert M,et al. Myoglobin as marker in meat adulteration:a UPLC method for determining the presence of pork meat in raw beef burger[J]. Food Chemistry,2013,141(3):1814-1820.

[3]Bargen C,Dojahn J R,Waidelich D,et al. New sensitive high-performance liquid chromatography-tandem mass spectrometry method for the detection of horse and pork in halal beef[J]. Journal of Agricultural and Food Chemistry,2013,61(49):11986-11994.

[4]Liu L H,Chen F C,Dorsye J,et al. Sensitive monoclonal antibody-based sandwich elisa for the detection of porcine skeletal muscle in meat and feed products[J]. Journal of Food Science,2006,71(1):M1-M6.

[5]Montowska M,Pospiech E. Species-specific expression of various proteins in meat tissue:proteomic analysis of raw and cooked meat and meat products made from beef,pork and selected poultry species[J]. Food Chemistry,2013,136(3):1461-1469.

[6]Hunt D J,Parkes H C,Lumley I D. Identification of the species of origin of raw and cooked meat products using oligonucleotide probes[J]. Food Chemistry,1997,60(3):37-442.

[7]Tian X J,Wang J,Cui S P. Analysis of pork adulteration in minced mutton using electronic nose of metal oxide sensors[J]. Journal of Food Engineering,2013,119(4):744-749.

[8]Rohman A,Sismindari,Erwant Y,et al. Analysis of pork adulteration in beef meatball using Fourier transform infrared(FTIR)spectroscopy[J]. Meat Science,2011,88(1):91-95.

[9]Unajak S,Anyamaneeratch K,Srikulnath K,et al. Identification of species(meat and blood samples)using nested-PCR analysis of mitochondrial DNA[J]. African Journal of Biotechnology,2011,10(29):5670-5676.

[10]V Fajardo,I González,M Rojas,et al. A review of current PCR-based methodologies for the authentication of meats from game animal species[J]. Trends in Food Science and Technology,2010,21(8):408-421.

[11]Ardura A,Pola I G,Ginuino I,et al. Application of barcoding to Amazonian commercial fish labelling[J]. Food Research International,2010,43(5):1549-1552.

[12]Samart D I,H?lzel C S,Tobias J,et al. PCR-SSCP-based reconstruction of the original fungalflora of heat-processed meat products[J]. International Journal of Food Microbiology,2013,162(1):71-81.

[13]Henco K，Heibey M. Quantitative PCR:the determination of template copy numbers by temperature gradient gel electrophoresis(TGGE)[J]. Nucleic Acids Research,1990,18(22):6733-6734.

[14]Reischl U,Kochanowski B. Quantitative PCR[J]. Molecular Biotechnology,1995,3(1):55-71.

[15]Kramer F R,Tyagi S,Alland D,et al. Non-competitive co-amplification methods:US,US 6461817B1[P/OL]. https://www. researchgate.net/publication/249314529_NON-COMPETITIVE_CO-AMPLIFICATION_METHODS.

[16]Gilliland G,Perrin S,Blanchard K,et al. Analysis of cytokine mRNA and DNA:Detection and quantitation by competitive polymerase chain reaction[J]. Proceedings of the National Academy of Sciences of the United States of America,1990,87(7):2725-2729.

[17]Calvo J H,Osta R,Zaragoza P. Quantitative PCR Detection of Pork in Raw and Heated Ground Beef and Paté[J]. Agric. Food Chem,2002,50(19):5265-5267.

[18]Soares S,Amaral J S,Mafra I,et al. Quantitative detection of poultry meat adulteration with pork by a duplex PCR assay[J]. Meat Science,2010,85(3):531-536.

[19]Chaumpluk P,Chikae M,Takamura Y,et al. Novel electrochemical identification and semi quantification of bovine constituents in feedstuffs[J]. Science and Technology of Advanced Materials,2006,7(3):263-269.

[20]Wolf C,Lüthy J. Quantitative competitive(QC)PCR for quantification of porcine DNA[J]. Meat Science,2001,57(2):161-168.

[21]Aslaminejad A A,Nassiry M R,Farajollahi H,et al. Development and Use of Quantitative Competitive PCR Assay for Detection of Poultry DNA in Sausage[J]. Food Biotechnology,2010,24(3):248-257.

[22]Heid C A,Stevens J,Livak K J,et al. Real Time Quantitative PCR[J]. Genome Methods,1996,6(10):986-994.

[23]Higuchi R,Fockler C,Dollinger G,et al. Kinetic PCR analysis:real-time monitoring of DNA amplification reactions[J]. Biotechnology,1993,11(9):1026-1030.

[24]Fraiture M A,Herman P,Taverniers I,et al. Current and New Approaches in GMO Detection:Challenges and Solutions[J]. BioMed Research International,2015,Article ID 392872,22 pages.

[25]Elodie BP,Nadine B,Marc Y,et al. Development and validation of qualitative SYBR? Green Real-Time PCR for detection and discrimination of Listeria spp. and Listeria monocytogenes[J]. Applied Microbiology and Biotechnology,2012,97(9):4021-4037.

[26]Safdar M，Junejo Y. The development of a hexaplex-conventional PCR for identification of six animal and plant species in foodstuffs[J]. Food Chemistry,2016,192:745-749.

[27]Navarro E,Serrano-Heras G,Castao M J,et al. Real-time PCR detection chemistry[J]. Clinica Chimica Acta,2015,439(439):231-250.

[28]Mao F,Leung W Y，Xin X. Characterization of EvaGreen and the implication of its physicochemical properties for qPCR applications[J]. BMC Biotechnology,2007,7(20):76.

[29]Eischeid A C. SYTO dyes and EvaGreen outperform SYBR Green in real-time PCR[J]. Eischeid BMC Research Notes,2011,4(1):263.

[30]WalkerJ A,Hughes D A,Anders B A,et al. Quantitative intra-short interspersed element PCR for species-specific DNA identification[J]. Analytical Biochemistry,2003,316(2):259-269.

[31]WalkerJ A,Hughes D A,Hedges D J,et al. Quantitative PCR for DNA identification based on genome-specific interspersed repetitive elements[J]. Genomics,2004,83(3):518-527.

[32]Fajardo V,González I,Martín I,et al. Real-time PCR for detection and quantification of red deer(Cervus elaphus),fallow deer(Dama dama),and roe deer(Capreolus capreolus)in meat mixtures[J]. Meat Science,2008,79(2):289-298.

[33]Soares S,Amaral J S,Oliveira M B P P,et al. A SYBR Green real-time PCR assay to detect and quantify pork meat in processed poultry meat products[J]. Meat Science,2013,94(1):115-120.

[34]Sakaridis I,Ganopoulos I,Argiriou A,et al. A fast and accurate method for controlling the correct labeling of products containing buffalo meat using High Resolution Melting(HRM)analysis[J]. Meat Science,2013,94(1):84-88.

[35]Sakalar E,Ergün S ?,Akar E. A Simultaneous Analytical Method for Duplex Identification of Porcine and Horse in the Meat Products by EvaGreen based Real-time PCR[J]. Korean Journal of Food Science Analysis,2015,35(3):382-388.

[36]María LA,Amando G P，Antonio P. Evaluation of Post-Polymerase Chain Reaction Melting Temperature Analysis for Meat Species Identification in Mixed DNA Samples[J]. Journal of Agricultural & Food Chemistry,2006,54(21):7973-7978.

[37]Safdar M,Junejo Y. Development and validation of fast duplex real-time PCR assays based on SYBER Green florescence for detection of bovine and poultry origins in feedstuffs[J]. Food Chemistry,2015,173:660-664.

[38]Gibson U E,Heid C A,Williams P M. A novel method for real time quantitative RT-PCR[J]. Genome Res,1996,6(10):995-1001.

[39]Kutyavin I V,Afonina I A,Mills A,et al. 3’-minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures[J]. Nucleic Acids Research,2000,28(2):655-661.

[40]Martín R,Rodríguez M A,García T,et al. TaqMan real-time PCR for the detection and quantitation of pork in meat mixtures[J]. Meat Science,2005,70(1):113-120

[41]Zhang C L,Fowler M R,Scott N W,et al. A TaqMan real-time PCR system for the identification and quantification of bovine DNA in meats,milks and cheeses[J]. Food Control,2007,18(9):1149-1158.

[42]Iwobi A,Sebah D,Kraemer I,et al. A multiplex real-time PCR method for the quantification of beef and pork fractions in minced meat[J]. Food Chemistry,2015,169(1):61-66.

[43]María L A,Laura L,Amando G P,et al. Identification and quantification of species in complex DNA mixtures by real-time polymerase chain reaction[J]. Analytical Biochemistry,2005,339(1):73-82.

[44]Soichi T,Makiko H,Takeo Y,et al. A Real-Time Quantitative PCR Detection Method for Pork,Chicken,Beef,Mutton,and Horseflesh in Foods[J]. Bioscience Biotechnology & Biochemistry,2007,71(12):3131-3135.

[45]Andreo M L,Aldeguer M,Guillén I,et al. Detection and quantification of meat species by qPCR in heat-processed food containing highly fragmented DNA[J]. Food Chemistry,2012,134(1):518-523.

[46]Drummond M G,Brasil B S A F,Dalsecco L S,et al. A versatile real-time PCR method to quantify bovine contamination in buffalo products[J]. Food Control,2013,29(1):131-137.

[47]Cheng X,He W,Huang F,et al. Multiplex real-time PCR for the identification and quantification of DNA from duck,pig and chicken in Chinese blood curds[J]. Food Research International,2014,60(6):30-37.

[48]Druml B,Grandits S,Mayer W,et al. Authenticity control of game meat products-A single method to detect and quantify adulteration of fallow deer(Dama dama),red deer(Cervus elaphus)and sika deer(Cervus nippon)by real-time PCR[J]. Food Chemistry,2015,170:508-517.

[49]Hindson B J,Ness K D,Masquelier D A,et al. High-Throughput Droplet Digital PCR System for Absolute Quantitation of DNA Copy Number[J]. Analytical Chemistry,2011,83(22):8604-8610.

[50]Kline M C,Romsos E L,Duewer D L. Duewer. Evaluating Digital PCR for the Quantification of Human Genomic DNA:Accessible Amplifiable Targets[J]. Analitical Chemistry,2016,88,2132-2139.

[51]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,Article ID 810209,6 pages.

[52]Floren C,Wiedemann I,Brenig B,et al. Species identification and quantification in meat and meat products using droplet digital PCR(ddPCR)[J]. Food Chemistry,2015,173:1054-1058.

[53]Eugster A,Ruf J,Rentsch J. Quantification of beef,pork,chicken and turkey proportions in sausages:use of matrix-adapted standards and comparison of single versus multiplex PCR in an interlaboratory trial[J]. European Food Research & Technology,2009,230(1):55-61.

[54]Bustin S A,Vladimir B,Garson J A,et al. The MIQE Guidelines:Minimum Information for Publication of Quantitative Real-Time PCR Experiments[J]. Clinical Chemistry,2009,55(4):611-622.

[55]K?ppel R,Eugster A,Ruf J,et al. Quantification of Meat Proportions by Measuring DNA Contents in Raw and Boiled Sausages Using Matrix-Adapted Calibrators and Multiplex Real-Time PCR[J]. Journal of AOAC International,2012,95(2):494-499.

[56]K?ppel R,Daniels M,Felderer N.Multiplex real-time PCR for the detection and quantification of DNA from duck,goose,chicken,turkey and pork[J]. European Food Research and Technology,2013,236(6):1093-1098.

[57]Zhang C,Fang X,Qiu H,et al. A Short Interspersed Nuclear Element(SINE)-Based Real-Time PCR Approach to Detect and Quantify Porcine Component in Meat Products[J]. Journal of AOAC International,2015,98(5):1471-1473.

[58]Ballin N Z,Vogensen F K,Karlsson A H. PCR amplification of repetitive sequences as a possible approach in relative species quantification[J]. Meat Science,2012,90(2):438-443.

[59]Cai H,Gu X,Scanlan M S,el al. Real-time PCR assays for detection and quantitation of porcine and bovine DNA in gelatin mixtures and gelatin capsules[J]. Journal of Food Composition and Analysis,2012,25(1):83-87.

[60]K?ppel R,Zimmerli F,Breitenmoser A. Heptaplex realtime PCR for the identification and quantification of DNA from beef,pork,chicken,turkey,horse meat,sheep(mutton)and goat[J]. European Food Research and Technology,2009,230(1):125-133.

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)與開發(fā)計劃(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