基于核酸分子學(xué)方法的肉類成分鑒別技術(shù)研究進(jìn)展

王金斌，李 文*，白 藍(lán)，劉 華，蔣 瑋，吳 瀟，王榮談，唐雪明,*

（1.上海市農(nóng)業(yè)科學(xué)院，上海 201106；2.上海海洋大學(xué)食品學(xué)院，上海 200090；3.上海市農(nóng)業(yè)遺傳育種重點(diǎn)實(shí)驗(yàn)室，上海 201106；4.上海瑞豐農(nóng)業(yè)科技有限公司，上海 201106）

基于核酸分子學(xué)方法的肉類成分鑒別技術(shù)研究進(jìn)展

王金斌1,2,3，李 文1,3,*，白 藍(lán)1,3，劉 華1,3，蔣 瑋1,3，吳 瀟1,3，王榮談4，唐雪明1,2,3,*

（1.上海市農(nóng)業(yè)科學(xué)院，上海 201106；2.上海海洋大學(xué)食品學(xué)院，上海 200090；3.上海市農(nóng)業(yè)遺傳育種重點(diǎn)實(shí)驗(yàn)室，上海 201106；4.上海瑞豐農(nóng)業(yè)科技有限公司，上海 201106）

近年來，肉類摻假問題頻繁發(fā)生?；诤怂岬姆肿由飳W(xué)肉類成分鑒別技術(shù)已成為研究熱點(diǎn)，其具有靈敏度高、特異性強(qiáng)、檢測時(shí)間短以及成本低的優(yōu)點(diǎn)。本文綜述了基于核酸分子學(xué)的肉類成分種屬鑒別技術(shù)在肉類摻假檢驗(yàn)中的應(yīng)用，著重于量化各種方法的檢測限，并重點(diǎn)對實(shí)時(shí)熒光定量聚合酶鏈?zhǔn)椒磻?yīng)（polymerase chain reaction，PCR）和數(shù)字PCR技術(shù)在動(dòng)物成分鑒別定量分析的研究現(xiàn)狀與前景做介紹。探討不同來源的靶基因（核DNA和線粒體DNA）在動(dòng)物成分鑒別中，定性和定量檢測靈敏度與特異性的區(qū)別。

摻假；肉類制品；數(shù)字PCR；物種測定

王金斌, 李文, 白藍(lán), 等. 基于核酸分子學(xué)方法的肉類成分鑒別技術(shù)研究進(jìn)展[J]. 食品科學(xué), 2017, 38(11): 318-327. DOI:10.7506/spkx1002-6630-201711049. http://www.spkx.net.cn

WANG Jinbin, LI Wen, BAI Lan, et al. A review of current DNA-based methodologies for meat authentication[J]. Food Science, 2017, 38(11): 318-327. (in Chinese with English abstract) DOI:10.7506/spkx1002-6630-201711049. http://www.spkx.net.cn

民以食為天，食以安為先，吃放心健康綠色的肉類制品是食品質(zhì)量安全的一個(gè)重要方面。食品摻假是一個(gè)老生常談的話題，我國古代就有“掛羊頭賣狗肉”的說法。為確保食品成分的真實(shí)性，質(zhì)檢“十二五”規(guī)劃綱要明確指出，需重點(diǎn)加強(qiáng)開展食品摻假鑒別技術(shù)研究。國內(nèi)市場近期曝光的多起肉類摻假事件引發(fā)了公眾對食品安全的擔(dān)憂。即使是在擁有世界上最嚴(yán)格食品安全制度的歐洲，2013年亦出現(xiàn)“掛牛頭賣馬肉”的造假現(xiàn)象，值得人們深思。

在肉類摻假形式層出不窮的情勢下，對動(dòng)物源性成分鑒別技術(shù)的研究逐步成為食品安全領(lǐng)域的研究熱點(diǎn)。目前，使用的動(dòng)物物種鑒別分析方法主要基于蛋白質(zhì)和DNA分析。其中，蛋白質(zhì)技術(shù)包括免疫[1]、色譜[2]和光譜[3]。但基于蛋白質(zhì)的檢測技術(shù)有一定的局限性，當(dāng)材料進(jìn)行熱處理時(shí)，多數(shù)蛋白質(zhì)會發(fā)生變性，不能滿足檢測的需要。而以核酸為基礎(chǔ)的分析方法可以克服這些困難，因?yàn)镈NA存在于所有生物的所有組織，且DNA比蛋白質(zhì)的耐熱性強(qiáng)，高溫處理過的食品中仍能提取出片段化的DNA。另外，DNA比蛋白質(zhì)具有更豐富的種間多態(tài)性，有利于品種鑒定[4]。目前，以檢測DNA為基礎(chǔ)的方法主要有：常規(guī)聚合酶鏈?zhǔn)椒磻?yīng)（polymerase chain reaction，PCR）-凝膠電泳法、多重PCR-凝膠電泳法、PCR-隨機(jī)擴(kuò)增多態(tài)性DNA（random amplified polymorphic DNA，RAPD）分析、PCR-限制性內(nèi)切酶片段長度多態(tài)性（restriction fragment length polymorphism，RFLP）分析、DNA條形碼（DNA barcoding）、熒光定量PCR、微滴數(shù)字（droplet digital，dd）PCR方法等。本文將分別綜述基于核酸檢測的食品中肉類成分鑒別定性和定量技術(shù)的現(xiàn)狀與問題。

1 基于核酸分子學(xué)方法的動(dòng)物成分鑒別

1.1 常規(guī)PCR-凝膠電泳法

DNA片段經(jīng)擴(kuò)增后，通過瓊脂糖凝膠電泳進(jìn)行片段大小的檢測，這是基于核酸分子學(xué)肉類成分鑒別方法中應(yīng)用廣泛且操作最簡單的技術(shù)。其基本的實(shí)驗(yàn)思路為：根據(jù)不同物種細(xì)胞核或線粒體基因組序列中的特征位點(diǎn)設(shè)計(jì)物種特異性引物，利用PCR反應(yīng)實(shí)現(xiàn)食品中目標(biāo)基因片段的指數(shù)級擴(kuò)增，繼而通過電泳鑒別食品中可能的物種來源。目前利用常規(guī)PCR-凝膠電泳法對肉類成分檢測的方法研究匯總見表1。

自1998年Tartaglia等[5]首次報(bào)道基于普通PCR方法檢測飼料中的牛、羊源性成分至今，大量相關(guān)研究報(bào)道了應(yīng)用PCR-凝膠電泳對食品中多種肉類成分的鑒別方法，檢測限大部分在0.1%以下。其中，對未加工（生鮮肉）和加工過的（腌制或者熱加工）肉類產(chǎn)品進(jìn)行對比測試，雖然DNA高度受損，但兩者PCR特異性引物的檢測靈敏度差別不大。這是因?yàn)槲锓N特異性引物的PCR技術(shù)的靶基因是短的DNA片段，具有簡單性、特異性和高靈敏度的優(yōu)點(diǎn)；主要的缺點(diǎn)是只有依賴針對靶序列的準(zhǔn)確數(shù)據(jù)才能設(shè)計(jì)出相應(yīng)的特異性引物。

表1 常規(guī)PCR-凝膠電泳法的匯總Table 1 Reported PCR-gel electrophoresis methods

1.2 多重PCR-凝膠電泳法

多重PCR是設(shè)計(jì)多種普通PCR的引物，加入同一PCR反應(yīng)體系里，同時(shí)擴(kuò)增出多個(gè)核酸片段，以同時(shí)檢測多種不同動(dòng)物源成分的一項(xiàng)具有發(fā)展前景的技術(shù)[34]。相比于單一物種的PCR系統(tǒng)，多重PCR技術(shù)具有節(jié)省成本，提高分析速率、效率和可靠性的優(yōu)點(diǎn)。表2匯總了運(yùn)用多重PCR方法檢測動(dòng)物成分的相關(guān)研究。

表2 多重PCR-凝膠電泳法的匯總表Table 2 Reported multiplex PCR-gel electrophoresis methods

Matsunaga等[42]首次進(jìn)行了多重PCR的研究，通過分析Cty b研究了牛、豬、山羊、雞、綿羊、馬肉的六重PCR，并得出了檢測限是25 ng DNA。近幾年，大量研究實(shí)現(xiàn)了在同一PCR體系里加入多對引物同時(shí)檢測牛、豬、驢、山羊、綿羊、禽類等多種肉類成分的多重PCR鑒定。例如，Kitpipit等[47]用豬、雞、馬、牛、鴕鳥和山羊肉進(jìn)行了六重PCR的研究，其分析目標(biāo)是Cyt b、COI和12S rRNA基因，檢測靈敏度可達(dá)到到12 500線粒體拷貝（相當(dāng)于7 fg）。

1.3 PCR-RAPD法和PCR-RFLP法

食品中DNA成分復(fù)雜，加之PCR技術(shù)的高靈敏度，使得應(yīng)用PCR擴(kuò)增方法來鑒定近緣物種時(shí)具有因非特異性擴(kuò)增而產(chǎn)生假陽性結(jié)果的缺點(diǎn)。因此，以PCR為基礎(chǔ)，應(yīng)用改良的引物設(shè)計(jì)策略或其他驗(yàn)證手段的鑒定方法為肉類定性鑒別提供了新方向。其中，PCR-RPLF與PCR-RAPD是兩種主要策略。表3總結(jié)了部分至今為止有關(guān)RFLP法和RAPD法在肉類檢測中的研究。

表3 PCR-RFLP和PCR-RAPD方法的匯總Table 3 Reported PCR-RFLP and PCR-RAPD methods

PCR-RPLF法是將通用引物擴(kuò)增后的PCR產(chǎn)物進(jìn)行限制性內(nèi)切酶酶切，然后通過凝膠電泳進(jìn)行觀察，作為單一物種的特有模式進(jìn)行定性分析，可進(jìn)行親緣性較近的物種間的鑒別。RFLP分析技術(shù)用于區(qū)分動(dòng)物成分，依賴于其特定的限制性酶切位點(diǎn)識別的差異。然而，PCR-RPLF的缺點(diǎn)是容易受到目標(biāo)基因序列中酶切位點(diǎn)隨機(jī)突變的影響，易產(chǎn)生不確定的檢測結(jié)果，且RFLP方法絕大多數(shù)都是定性檢測純動(dòng)物組織，因?yàn)榛旌衔锟赡墚a(chǎn)生復(fù)雜的結(jié)果導(dǎo)致無法解釋。

PCR-RAPD利用任意PCR短的引物擴(kuò)增產(chǎn)生一系列的產(chǎn)物，得到PCR產(chǎn)物的指紋圖譜，根據(jù)指紋圖間的差別區(qū)分不同的種屬。當(dāng)參考材料可用的很少或DNA序列信息未知時(shí)，RAPD技術(shù)是非常強(qiáng)大的。但PCR-RAPD的重復(fù)性較差且受到食品中其他DNA成分的嚴(yán)重干擾，存在不易標(biāo)準(zhǔn)化與廣泛應(yīng)用的缺點(diǎn)。

1.4 DNA條形碼

在生物物種鑒定領(lǐng)域，DNA條形碼技術(shù)是發(fā)展最為迅速的一種新技術(shù)，最早由Hebert等[57]于2003年提出并用于物種鑒別和分類。目前DNA條形碼技術(shù)的研究主要集中在魚類[58-59]、動(dòng)物[60]、植物[61]的成分鑒定。

表4 DNA條形碼技術(shù)的匯總Table 4 Reported DNA Barcoding methods

DNA條形碼技術(shù)最初用于生物分類和物種鑒別，后發(fā)展到應(yīng)用于動(dòng)物肉類鑒定中并得到廣泛研究，是一種簡單、快速、可靠、有效的分子鑒定技術(shù)。大多數(shù)動(dòng)物物種的DNA條形碼是線粒體上一段約650 bp編碼基因COI，研究者將其作為動(dòng)物鑒定的條形碼標(biāo)準(zhǔn)片段[57]。表4總結(jié)了部分至今為止有關(guān)DNA條形碼法在肉類檢測中的研究。COI基因作為公認(rèn)的DNA條形碼被認(rèn)為能夠很好地對動(dòng)物進(jìn)行分類鑒定[74]。COI基因在雙鏈環(huán)狀閉合的核外線粒體基因組上，保證了其相對完整性和熱穩(wěn)定性，在深加工的肉類食品中有相對足夠量的DNA被用于PCR擴(kuò)增，確保足夠高的PCR產(chǎn)物含量。COI序列還具有足夠變異性、易擴(kuò)增、片段自身在物種種內(nèi)具有特異性和種間多樣性等特點(diǎn)。然而，DNA條形碼技術(shù)也存在局限性，首先，數(shù)據(jù)庫中關(guān)于家禽家畜的DNA條形碼序列很少，缺乏大量數(shù)據(jù)作為支撐[74]。其次，只適用于含有單一成分的鑒別，不能鑒別同一產(chǎn)品中的幾種成分[75]。

雖然DNA條形碼技術(shù)在動(dòng)物成分鑒別中存在局限性，但DNA條形碼技術(shù)能避免形態(tài)學(xué)分類的缺陷，對鑒定者的經(jīng)驗(yàn)和專業(yè)知識背景要求較低，使科研和檢疫檢驗(yàn)工作更加高效，加速了物種鑒別進(jìn)程。隨著動(dòng)物物種DNA條形碼數(shù)據(jù)庫的建立和不斷完善，該技術(shù)將在肉類食品鑒別中發(fā)揮更完善的作用，為我國肉類食品安全檢測和可追溯體系的建立提供參考。

1.5 實(shí)時(shí)熒光定量PCR技術(shù)

實(shí)時(shí)熒光定量PCR技術(shù)是利用熒光分子提供的熒光強(qiáng)度與PCR產(chǎn)物的豐度之間的相關(guān)性來實(shí)現(xiàn)實(shí)時(shí)數(shù)據(jù)采集[76]。相比于在末期進(jìn)行分析的凝膠瓊脂糖或聚丙烯酰胺電泳技術(shù)，實(shí)時(shí)熒光定量PCR方法自然更加精確靈敏。在復(fù)雜的混合而成的產(chǎn)品中，即使只有微量的不同物種成分，也能分析出來，因此被認(rèn)為是在肉類鑒定中最有前途的分子工具之一[77]。研究人員利用實(shí)時(shí)熒光定量PCR技術(shù)對肉類成分定性定量檢測進(jìn)行了大量的研究，如表5匯總。

表5 實(shí)時(shí)熒光定量PCR法的匯總Table 5 Reported real-time PCR methods

續(xù)表5

Lahiff等[78]在2002年首次利用熒光定量PCR（TaqMan探針法），通過分析線粒體tRNA ATP6和ATP8對熱加工后牛肉成分檢測進(jìn)行了研究，檢測限為0.001%。近年來肉類成分實(shí)時(shí)熒光PCR檢測技術(shù)有了長足發(fā)展，但相關(guān)方法的完善與推廣仍面臨著挑戰(zhàn)。肉類成分的定量分析通過已知樣品中相同的目標(biāo)基因（參照）繪制的濃度校準(zhǔn)曲線與已知成分含量（m/m）進(jìn)行對比分析建立。然而，在量化動(dòng)物摻假時(shí)，動(dòng)物組織組成、樣品處理和肉類加工程度是不被研究者知道的，因此很難確定市場樣品肉類含量（m/m）與測量方法的相關(guān)性。首先，實(shí)時(shí)定量PCR技術(shù)的準(zhǔn)確度及定量性潛能受DNA產(chǎn)量的影響，比如由于加工處理的原因及同一樣品不同組織中的DNA種類和生產(chǎn)細(xì)胞的數(shù)量存在有差異會影響DNA降解的程度。如經(jīng)過粗加工和高度加工的肉類產(chǎn)品的定量結(jié)果相差約為10 倍[77]。其次，大多數(shù)動(dòng)物細(xì)胞含有許多拷貝的線粒體DNA，并且在不同組織中的線粒體DNA拷貝數(shù)是不同的，導(dǎo)致未知樣品中基于線粒體DNA序列測量結(jié)果和肉類含量（m/m）的相關(guān)性不準(zhǔn)確。雖然該技術(shù)具有值得肯定的定量潛力，但很難實(shí)現(xiàn)肉類成分組織組成和加工過程盡可能與建立方法的條件保持一致，對商業(yè)肉制品中的目標(biāo)物種進(jìn)行定量測定僅僅只是可用，大部分研究主要還是利用實(shí)時(shí)熒光定量PCR的靈敏性對動(dòng)物成分進(jìn)行定性及半定量的判斷。而且，由于使用了特殊的熒光探針，通常成本也相對較高。

可靠的熒光定量PCR檢測方法必須考慮到DNA降解程度，才能推算出食品中原料肉的用量[137]。MY基因和GH基因被開發(fā)作為定量動(dòng)物成分的校正參照標(biāo)準(zhǔn)。由于這些校正參照基因在不同哺乳動(dòng)物及禽類組織中的表達(dá)水平相當(dāng)，因此將對校正參照基因的定量結(jié)果與生肉組織中的定量結(jié)果相比，即可計(jì)算出基因組DNA的降解程度，從而進(jìn)一步通過降解程度校正物種特異性擴(kuò)增的定量結(jié)果。

1.6 ddPCR技術(shù)

ddPCR技術(shù)作為一種全新的準(zhǔn)確定量核酸檢測方法，通過把反應(yīng)體系均分到大量反應(yīng)單元中獨(dú)立地進(jìn)行PCR，并根據(jù)泊松分布和陽性比例來計(jì)算核酸數(shù)量。與傳統(tǒng)PCR、定量PCR相比，其結(jié)果的精確度、準(zhǔn)確性和靈敏度更佳。定量結(jié)果不再依賴于Ct值，直接給出靶序列的起始濃度，實(shí)現(xiàn)真正意義上的絕對定量。2014年Cai Yicun等[138]首次采用數(shù)字PCR技術(shù)，以DNA含量為中間值計(jì)算出DNA拷貝數(shù)與生鮮肉質(zhì)量之間的線性關(guān)系，對肉制品中豬肉和雞肉進(jìn)行成分鑒定及含量的分析。王珊等[139]建立一種定量檢測羊肉制品中羊源和豬源性成分的ddPCR方法，并將該方法與SN/T 2051—2008《食品、化妝品和飼料中牛羊豬源性成分檢測方法 實(shí)時(shí)PCR法》中實(shí)時(shí)熒光定量PCR方法做對比，來檢測3 份羊肉制品中的羊源和豬源性成分，得出結(jié)論：在肉種成分真?zhèn)舞b定上，ddPCR方法較實(shí)時(shí)熒光PCR方法更科學(xué)、準(zhǔn)確。苗麗等[140]基于數(shù)字PCR技術(shù)，建立了定量檢測肉及肉制品中牛肉和豬肉質(zhì)量的方法。方法利用在一定范圍內(nèi)生鮮肉質(zhì)量與DNA含量、DNA含量與DNA拷貝數(shù)之間均呈現(xiàn)明顯的線性關(guān)系，以DNA含量為中間值計(jì)算出DNA拷貝數(shù)（C）與生鮮肉質(zhì)量（M）之間的換算公式M牛= 0.062C－0.943、M豬=0.045C－1.72。對已知目標(biāo)肉種含量的混合肉樣進(jìn)行檢測，結(jié)果表明測量值和真實(shí)值基本一致，且不受外源物種的干擾。2015年Floren等[141]研究了基于ddPCR技術(shù)利用線粒體Cyt b基因151（馬）、146（牛）、147 bp（豬）和染色體凝血因子Ⅱ基因（F2）95（馬）、96 bp（牛）和97 bp（豬）作為標(biāo)記基因鑒定和定量肉和肉制品。研究得出線粒體Cyt b基因作為標(biāo)記基因進(jìn)行物種量化是不合適的，利用染色體凝血因子Ⅱ基因（F2）作為標(biāo)記基因，通過兩步ddPCR，能實(shí)現(xiàn)牛、馬、豬的精確量化，定量限（limit of quantity，LOQ）和檢測限（limit of detection，LOD）分別在0.01%和0.001%。

ddPCR是一個(gè)擁有巨大潛力的新興技術(shù)，具有高靈敏度、高精確度、高耐受性和絕對定量的優(yōu)點(diǎn)，可以對諸如肉制品這種復(fù)雜樣品中物種特異性靶基因?qū)崿F(xiàn)更靈敏、更準(zhǔn)確的檢測，并且很容易將現(xiàn)成的實(shí)時(shí)熒光定量PCR檢測體系進(jìn)行直接轉(zhuǎn)化，甚至無需優(yōu)化。ddPCR技術(shù)將會在動(dòng)物成分特別是精加工肉類產(chǎn)品的定量檢測上進(jìn)一步發(fā)展與完善，應(yīng)用范圍也會大大擴(kuò)展。

2 基于核酸分子學(xué)方法的動(dòng)物成分定量方法的優(yōu)缺點(diǎn)

表6 動(dòng)物成分物種鑒別定性、定量方法存在的優(yōu)缺點(diǎn)Table 6 Advantages and disadvantages of qualitative and quantitative PCR-based methods for species determination

基于核酸分子學(xué)水平的動(dòng)物成分定性檢測技術(shù)中一個(gè)重要的步驟是選擇合適的靶基因。用于動(dòng)物成分鑒別不同靶基因所存在的優(yōu)缺點(diǎn)見表6。線粒體基因、染色體DNA和重復(fù)序列作為靶基因被廣泛地用于識別野生和馴養(yǎng)肉類物種。如來源于線粒體的靶基因位點(diǎn)主要為線粒體Cty b、12S和16S核糖體RNA亞基、和D-loop等，這些都是動(dòng)物源性成分檢測最常用的標(biāo)記基因。此外，核基因組特異性標(biāo)記基因（生長激素基因、肌動(dòng)蛋白基因、和黑毛發(fā)受體1基因）和重復(fù)序列（SINE、LINE）也可以有效鑒別肉類物種。在產(chǎn)品中檢測到未標(biāo)識的動(dòng)物成分是相對簡單的（摻假或不摻假），主要面臨的挑戰(zhàn)是定量的問題。在目標(biāo)基因選擇方面，線粒體基因組編碼序列雖然是肉類定性鑒別的首選，但不同動(dòng)物組織中線粒體數(shù)量區(qū)別較大，在動(dòng)物組織種類未知的情況下以之作為定量檢測的目標(biāo)基因，則可能導(dǎo)致定量結(jié)果的偏差，而現(xiàn)有的部分研究卻未考慮這一因素；細(xì)胞基因組中的重復(fù)序列不存在組織差異，但重復(fù)序列間的高同源性加大了特異性引物與探針設(shè)計(jì)的難度；細(xì)胞基因組中單拷貝的編碼序列在食品加工過程中降解嚴(yán)重，導(dǎo)致檢測靈敏度過低。

3 結(jié) 語

基于核酸DNA肉類成分定性分析的靶序列可來源于線粒體或基因組，包括單拷貝和重復(fù)序列，標(biāo)記基因DNA序列的選擇對檢測方法的檢測限有很大的影響。定量的分析應(yīng)根據(jù)實(shí)時(shí)PCR或ddPCR分析，PCR擴(kuò)增產(chǎn)物的序列必須來源于基因組，其中單拷貝和重復(fù)序列都可以使用。用線粒體DNA進(jìn)行定量分析是不可能的，因?yàn)樵谖粗獦悠分忻總€(gè)組織細(xì)胞的線粒體拷貝數(shù)是不確定的。

食品中肉類成分的種屬鑒定技術(shù)是打擊肉制品摻假、維護(hù)市場秩序的有效保障。如今，隨著實(shí)時(shí)熒光定量PCR和ddPCR技術(shù)的飛速發(fā)展為肉類成分檢測開辟了新的途徑，使得食品中肉類成分的定量分析與溯源成為可能。在定量檢測中，通過對反應(yīng)體系的精巧設(shè)計(jì)而提升方法的準(zhǔn)確性與實(shí)用價(jià)值將成為相關(guān)技術(shù)的發(fā)展方向與趨勢。

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A Review of Current DNA-Based Methodologies for Meat Authentication

WANG Jinbin1,2,3, LI Wen1,3,*, BAI Lan1,3, LIU Hua1,3, JIANG Wei1,3, WU Xiao1,3, WANG Rongtan4, TANG Xueming1,2,3,*
(1. Shanghai Academy of Agricultural Sciences, Shanghai 201106, China; 2. College of Food Science and Technology, Shanghai Ocean University, Shanghai 200090, China; 3. Key Laboratory of Agricultural Genetics and Breeding, Shanghai 201106, China; 4. Shanghai Ruifeng Agricultural Sci-Tech Company Ltd., Shanghai 201106, China)

In recent years, the problem of meat adulteration has occurred frequently. Full attention has been paid to DNA-based methodologies for meat species identi?cation because of their high sensitivity and speci?city, as well as rapid processing time and low cost. This article presents an overview of the commonly used DNA-based methodologies to verify the authenticity of meat and meat products with focus on their detection limits. Moreover, this review highlights the current applications and future prospects of real-time fluorescence quantitative polymerase chain reaction (PCR) and digital PCR in the identification of animal origin ingredients. Finally, target genes from different sources (nuclear DNA and mitochondrial DNA) are compared in terms of their characteristics and their in?uence on the sensitivity and speci?city of species identi?cation and quanti?cation.

adulteration; meat products; digital PCR; species determination

10.7506/spkx1002-6630-201711049

TS207.3

A

1002-6630（2017）11-0318-10引文格式：

2016-05-12

上海市閔行區(qū)產(chǎn)學(xué)研合作計(jì)劃項(xiàng)目（2016MH256）；上海市農(nóng)委青年人才成長計(jì)劃項(xiàng)目（滬農(nóng)青字（2014）第1-20號）作者簡介：王金斌（1982—），男，助理研究員，博士研究生，研究方向?yàn)槭称钒踩c檢測技術(shù)。E-mail：wangjinbin2013@126.com

*通信作者：李文（1982—），女，助理研究員，碩士，研究方向?yàn)楣δ苁称芳夹g(shù)研發(fā)和利用。E-mail：liwen@saas.sh.cn唐雪明（1970—），男，研究員，博士，研究方向?yàn)樯锛夹g(shù)。E-mail：xueming70@foxmail.com