Subject：Species identification and quantitation of meat using real-time polymerase chain reaction

• Main Authors: Yueh-EChen, 陳月娥
• Other Authors: Mi-Li Wu
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/53116477764930644640

碩士 === 國立屏東科技大學 === 食品科學系 === 94 === Taiwan has joined the World Trade Organization (WTO). Base on the premise of trend of trade liberalization between nations, the predicted amount of imported raw meat will be increased significantly. Therefore it’s necessary to exam the purity of imported flesh raw meat, its products and domestic meat products. We don’t have official standard quantitative methods for identifying meat products in Taiwan now. The aim of the research was to develop the quantitative and qualitative methods by using the real-time Polymerase Chain Reaction (real-time PCR) that could identify the domestic fresh raw meat, poultry and meat products effectively, conveniently and rapidly. We used the traditional PCR and real-time PCR to identify six kinds of meat, including pork, beef, lamb, chicken, turkey and ostrich. Mixing proper proportion of primer pairs to have PCR, and then we could get species-specific DNA fragments. The primer pairs for pork and beef were designed according to the specific DNA sequences of growth hormones genome. For sheep, was designed according to the DNA sequence of satellite I and for chicken, turkey and ostrich were designed according to the DNA sequence of mitochondrion. The PCR primers of the six kinds of meat could produce bands of DNA sequences with different molecular weight. The species-specific sequences were 108bp (pork), 130bp (beef), 374bp (lamb), 234bp (chicken), 216bp (turkey) and 247bp (ostrich). In traditional PCR the test sensitivity of six kinds of meat samples was as following: 5ng for pork and beef; 1ng for sheep; 0.1ng for chicken, turkey and ostrich meat. We used the real-time PCR (SYBER GREEN Ⅰ), the sensitivity was as following: 1ng for pork and beef and 0.1ng for lamb, chicken, turkey and ostrich meat. Using traditional PCR and real-time PCR to test samples treated with 80℃, 100℃ and 120℃ heat separately, the results were shown that the traditional PCR could not be used for detecting the pork and beef after treated with 120℃ heat but the real-time PCR method still worked. We used the real-time PCR, species-specific primer pairs and TaqMan probe to quantify the meat samples. The results were as following: for the mixture of beef and pork samples, the detecting limit of pork was 0.1%; for the mixture of lamb and pork samples, the detecting limit of pork was 0.1%; for the mixture of chicken and ostrich samples, the detecting limit of chicken was 0.1%; for the mixture of ostrich and turkey samples, the detecting limit of turkey was 0.1%. Base on the results we concluded that the real-time PCR condition developed in our research was applicable to the qualitative quantitative/ analysis of detecting fake meat or meat mixture. Keywords: Polymerase Chain Reaction（PCR）, Meat species identification, real-time Polymerase Chain Reaction（real-time PCR）、qualitative /quantitative analysis