台灣常見消費肉品之晶片快速檢鑑技術之開發

Development of A Multiplex Biochip System for Quick Detection and Identification of Different Meat Commonly Consumed in Taiwan

賴品娟(PIN-JYUAN LAI)

指導教授 ： 張清安

朝陽科技大學/理工學院/應用化學系生化科技碩博士班/碩士(2017年)

摘要

近年來台灣屢次發生重大食品安全問題，引起消費者恐慌及對食品之安全疑慮。另外衛生部門也查獲廠商以不合格或低價肉品混充販售之情事，甚至在素食食材發現混雜動物肉品或者違法販售狗肉之案例。這些案例不僅可能造成消費者健康問題，有些甚至引發宗教信仰或道德上的爭議。因此開發可快速檢測鑑別食用肉品之技術，遂成為把關肉品真偽之關鍵。本研究依消費普遍性之差異，將台灣常見肉品分為一般消費肉品與特殊肉品兩大類別，分別建立其檢測與鑑別(檢鑑)系統，前者包括牛(Bos taurus)、羊(Ovis aries)、雞(Gallus gallus)及豬(Sus scrofa)。後者則為消費頻率較低之鴕鳥(Struthio camelus)、鹿(Cervus elaphus)及兔子(Oryctolagus cuniculus)。另外也將狗(Canis lupus familiaris)肉列入特殊肉品進行檢鑑系統開發。本研究終極目標為開發出能夠快速檢鑑肉品種類之多目標型PCR增幅及生物晶片雜合技術。以有別於傳統技術需以專一性引子或抗體進行多次單目標檢測，而致耗費時間與成本。本研究以各標的動物之cytochrome b基因設計增幅用專一性引子進行PCR增幅，選出最適引子後再加以整合成多目標型增幅系統，後續再針對各物種之增幅產物序列設計專一性互補探針，將其固定於塑膠晶片上進行雜合反應以捕捉被增幅之PCR產物。由於我們事先將增幅引子以biotin標定，所以可利用標定有鹼性磷酸酶( alkaline phosphatase, AP )的鏈親和素( streptavidin )去追蹤帶有生物素( biotin )的增幅產物，最後可以根據不同探針位置之AP酵素反應之呈色結果，鑑別肉品之種類。本研究已成功建立上述二組肉品之快速檢測與鑑別系統，可以就標註不明或有混雜疑慮之肉品，在6小時內完成正確之肉品種類判別，研究中也證實此二檢鑑系統具有可信賴之重複性與再現性，且晶片雜合反應之偵測極限明顯高出電泳分析2-8倍以上。實際應用於市面上不同來源肉品之檢鑑時，確認可以快速且明確判別單一或混雜之肉品與種類。部分案例證實本檢鑑系統之晶片雜合反應可以偵測到以傳統PCR電泳分析無法測出之微量夾雜肉品種類。

關鍵字

肉品 ； 多目標增幅 ； 生物晶片

並列摘要

Food safety issues have been occurring repeatedly in Taiwan in recent years, which caused consumers being panic and always doubted the safety of various foods selling in the market. For example, Health Department of the government had seized some stores selling unqualified or low-cost mixed meat; or cases such as vegetarian food products were detected intentionally mixed with animal meat and some even worse cases of selling illegal dog meat. These cases might not only cause the health problems to the consumers but also lead to the issues of violating the religious beliefs and moral insistence. Therefore, developing feasible technologies for fast detecting and identifying qualified foods has become the key to certify the genuineness of meat products. This research is aiming to develop a system for quick detection and identification of different animal meats. Actually, two separate systems for different animal meats will be targeted based on their commonness and popularity in the markets. The first system is for ordinary meats including beef (Bas Taurus), lamb (Ovis aries), chicken (Gallus gallus) and pork (Sus scrofa) and the second one is for special meats including ostrich (Struthio camelus), deer (Cervus elaphus), and rabbit (Oryctolagus cuniculus). We also included the dog (Canis lupus familiaris) meat as one of the targets in the second special meat checking system. The technique we are developing is the multiplex polymerase chain reaction (PCR) amplification along with the biochip hybridization technology which are different from the traditional methodologies such as the uniplex PCR or ELISA that always aims at single target during detection which apparently more time consuming and costly in labor. The mitochondria cytochrome b genes from the targeted animals were used as templates to design the amplification specific PCR primers. After choosing the best primer for each animal, they were integrated into a multiplex amplification system. Subsequently, the sequences of PCR products from different animals were used to design specific oligonucleotide probes and implanted onto the surface of plastic biochips, which could be used to hybridize and capture the PCR amplicons from various meat targets. Since the primers were pre-labelled with biotin at their 5’ end, each PCR product once amplified will be biotin-labelled and they will be detected by the further use of streptavidin, a bacterial compound that has remarkably high affinity to biotin. Since the streptavidin was labelled with alkaline phosphatase (AP), its reaction with biotin-labelled PCR amplicon could be visualized by reacting with its enzyme substrate. It is therefore, the PCR amplicons from different animal meats will be identified by the coloration of the probe diagram on the biochip and the corresponding meat types can thus be confirmed. In this research, we have successfully developed two multiplex detection and identification systems; one for four ordinary animal meats and another for four special ones. The whole detection process can be completed within six hour time. We have also confirmed the two systems are repeatable and reproducible in different laboratory situation. The detection limits for both systems were also justified and confirmed that biochip hybridization has at least 2-8 times higher sensitivity than the conventional PCR electrophoresis analyses. When both systems were applied in checking meats from diversified market sources, correct ingredient and purity of each meat sale could be quickly identified and confirmed. We even came across some cases that minor contamination of different meats could be readily detected by these newly developed systems.

並列關鍵字

Meat ； Multiplex PCR ； Biochip

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