應用聚合酶連鎖反應技術開發鑑定鴕鳥肉成分之方法

對肉製品而言，鑑定物種來源、檢測成分有無攙雜或非法添加其他肉品是重要課題。本研究目的在以聚合酶連鎖反應技術(polymerase chain reaction; PCR)發展一套準確快速且成本經濟的方法來鑑定鴕鳥肉。以具有種別特異性的細胞粒線體之細胞色素b(cytochrome b)設計出對鴕鳥組織具物種特異性之引子對(primers)。用此引子對進行PCR增幅可得到－大小為219bp的DNA產物，經定序比對後證實為鴕鳥的粒線體基因片段；並同時利用其他常見食用動物組織進行引子的特異性測試確認成功。此方法不但可以簡便、快速的鑑定出鴕鳥肉，且無論是生肉、蒸煮加工過、或不同器官之鴕鳥組織都不影響檢測結果。進一步依同樣方法設計出可供鑑定牛肉(384bp)、豬肉(340bp)之特異性引子對並引用他人已發表之火雞、雞特異性引子對，找出合適於多物種鑑定PCR程式中的關鍵煉合溫度65℃，據此可以同一溫度程式及反應液濃度進行多物種鑑定。在發展多引子聚合酶連鎖反應方面，將所設計的鴕鳥及豬的特異性引子對於2: 1比例混合時，具有可同時鑑定出是否存在混合樣本的能力。

關鍵字

鴕鳥肉；聚合酶連鎖反應技術；細胞色素b ；特種鑑定

並列摘要

Identification of meat species and examination of combining species of meat are essential for meat products. In this study, a rapid, accurate, and inexpensive PCR assay was developed for identifying ostrich tissue. The cytochrome b gene of cell mitochondria was used to design ostrich species-specific primers. By employing this set of primers, a size 219-bp of amplicon was amplified and then proved to be part of the ostrich cytochrome b gene by blasting. In addition, this set of primers was demonstrated to have high species-specificity. The results of this PCR assay were not altered regardless of the condition of meat sample. Raw meat, processed meat, and even different organs produced same results. Furthermore, our own designed cattle-(384bp) and pig-(340bp) species-specific primers as well as previously published turkey and chicken primers were employed to develop an all-purpose PCR program in which the annealing temp was applied as 65℃. To simplify the procedure of examining multi-species samples, a multiplex PCR assay was also developed in which the ratio between ostrich and pig primers was 2: 1. This multiplex PCR was proven to be able to identify mixed ostrich and pig samples.

並列關鍵字

ostrich meat ； PCR ； cytochrome b ； animal species identification

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應用聚合酶連鎖反應技術開發鑑定鴕鳥肉成分之方法

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