應用聚合酶連鎖反應技術開發加工鴕鳥肉之快速鑑定法

Abstract

目前鴕鳥肉為高價肉品,肉質近似牛肉,若以低價的假貨來欺騙消費者,對飼養鴕鳥這個新興的產業發展是一個重大傷害。本研究以鴕鳥為主要物種發展一套準確、快速及價格低廉的方法來鑑定鴕鳥肉。過去以分析蛋白質的方法來鑑定有許多的缺點,如肉品若經加工如蒸煮後即無法檢驗等。所以本試驗發展以聚合酶連鎖反應技術（polymerase chain reation；PCR）進行檢驗。此種方法須找出具有種別特異性的基因序列,設計出物種特異性的引子（primer）。依據過去的文獻,細胞粒線體的細胞色素b（cytochrome b）是電子傳遞鏈上的重要的輔酶,其結構影響到細胞的特性,故在此段基因呈現較佳的種別多樣性（diversity）。依此基因序列設計出新的鴕鳥特異性引子,用此引子進行PCR增幅可得到一段219 bp的DNA片段,經定序後在基因庫（National Center for Biotechonology Information；NCBI）比對後證實為鴕鳥的粒線體基因片段。再與其他種動物進行對照測試,確認引子的特異性,用此方法可以簡便、快速的鑑定出鴕鳥肉。無論是生肉、蒸煮過、或不同器官之材料,都不影響結果。 依同樣的方法設計出牛肉,豬肉的特異性引子並採用Herman所設計的火雞、雞特異性引子,經PCR增幅後可獲得物種專一性片段,分別為牛(384 bp)、豬(340 bp)、雞（253 bp）及火雞（242 bp）,再進行五種物種交叉測試,PCR程式中其煉合（annealing）溫度65℃時可獲得最佳的特異性,建立一組新的多物種鑑定方法。另為簡化操作的手續,再將各物種特異性引子進行以適當的比率進行混合,進行多引子聚合酶連鎖反應（multiplex PCR）試驗,結果鴕鳥及豬的種別特異性引子無交叉反應,而對其他三種物種（牛、雞、火雞）具有特異性,此種方法可對肉品的成份進行初步的檢驗。

Ostrich meat is “red meat' similar in color and taste to beef. At present ostrich meat is the higher price meat in Taiwan. Using the cheaper meat to pretend is harmful to the development of the ostrich husbandry. This research uses ostrich as the primary species to develop the fast, accurate, and inexpensive method to examine the ostrich’s processed meat. In the past, analyzing protein to examine the meat had a lot of problems, such as unable to identify after the meat has been cooked. Therefore, we developed polymerase chain reaction（PCR）to do the examination. This method has to find out the gene sequence with species diversity, to design the primers. According to the past literature, cytochrome b in cell mitochondrion（mt） is an important coenzyme on electron transfer chain, the structure of it influences the characteristic of cells. Therefore, this gene sequence has better diversity of the species. This search used this gene as gene marker to design novel specific primers for the ostrich. Partial cytochrome b gene from the ostrich meat is successfully amplified by PCR. This PCR product is sequencing, its fragment size is 219 bp. It is confirmed, by gene comparison with “National Center for Biotechnology Information” （NCBI）, this sequence is partial cytochrome b gene of ostrich. These primers are species-specific to other kind of animals. This is a fast and easy method to examine ostrich meat. The result will not be effected no matter if it’s been cooked or from different organs. Using the same method to design the species-specific primers of beef and pork, and adopt the species-specific primers for turkey and chicken designed by Herman（2001）.The five species-specific primers demostrate different bands by PCR, such as beef (384 bp), pork (340 bp), chicken (253 bp) and turkey (242 bp), and every primers are specific to other four. In this PCR program, it builds a new method to examine multi-species when the annealing temperature is adjusted to 65℃. Besides, for the purpose to simplify the operation procedure, this search mixed with two sets of the species-specific primers with the proper percentage to develop the multiplex PCR method. Only one set of primers is not cross-reaction it can simultaneously identify ostrich meat and pork. This method may carry on the preliminary examination to the ingredient of meat.