台灣常見消費肉品之晶片快速檢鑑技術之開發

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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 , Masters　　Advisor：張清安　　

繁體中文

肉品；多目標增幅；生物晶片； Meat ； Multiplex PCR ； Biochip

Abstract │ Reference (70) │ Altmetrics

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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.

Reference ( 70 ) 〈TOP〉

黃尚仁，可同時檢測三種病毒之香蕉生物晶片系統之開發，碩士論文，朝陽科技大學生化科技研究所，台中(2012)。
連結：
黃莉真，可同時檢測多種百香果病毒之多目標型增幅及晶片檢測系統，碩士論文，朝陽科技大學生化科技研究所，台中(2014)。
連結：
Adrian, L., Shanan S, Tobe., “An overview to the investigative approach to species testing in wildlife forensic science” Investigative Genetics 2: 2-10(2011).
連結：
Ali, M. E., Hashim, U., Mustafa, S., Che Man, Y. B., Dhahi, Th. S., Kashif, M., Kamal Uddin, Md., Abd Hamid a, S. B. “Analysis of pork adulteration in commercial meatballs targeting porcine-specific mitochondrial cytochrome b gene by TaqMan probe real-time polymerase chain reaction” Meat Science. 91:454-459 (2012).
連結：
Asensio, L., González, I., García, T., Martín, R., “Determination of food authenticity by enzyme-linked immunosorbent assay (ELISA)”. Food Control 19: 1–8. (2008).
連結：

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