旗魚及其加工品基因定量髮技術開發之探討

Studies on quantitative gene assay for billfish and related products

吳思敏(Sz Ming Wu)

指導教授 ： 謝宥諒 ； 謝承紘

亞洲大學/健康學院/保健營養生技學系碩士班/碩士(2010年)

摘要

旗魚為台灣主要的漁獲之一，所捕獲的旗魚依其原料特性而製成不同的產品。國內現行的法規中，並無「主原料含量及品名標示相關規定」，恐業者假借其他低價魚種取代旗魚而製成假旗魚加工產品。由於所使用的基因鑑種技術，如直接定序、PCR僅能檢測假冒之旗魚加工品，卻難以檢測出摻假之旗魚產品，故急需發展旗魚加工品中旗魚肉含量之定量技術。
本研究首先對立翅旗魚 (Istiompax indica)、紅肉旗魚 (Kajikia audaxt)、黑皮旗魚(Makaira nigricans)、雨傘旗魚 (Istiophorus platypterus)、劍旗魚 (Xiphias gladius)，進行 16S rRNA 與 COI 基因序列之建立，利用 primer walking 之方式建立完整之其因資料，以所建立之資料搜尋旗魚魚種合適之基因片段，作為基因定量檢測法引子設計之參考依據。

再者，利用五種旗魚之基因資料設計一組共同且具專一性之引子對，其片段為 150 bp，進行其引子專一性及靈敏度之偵測。由結果確認此組引子是適用於作為五種旗魚之特異性引子，以此組引子進行生鮮與加工品旗魚產品之其因定量髮技術開發之用。將此組引子進行 DNA濃度靈敏度偵測其結果顯示各種檢測靈敏度為: Makaira nigricans: 10-5 ng, Kajikia audaxt: 10-5 ng, Istiophorus platypterus: 10-4 ng, Xiphias gladius: 10-3 ng and Istiompax indica: 10-5 ng。
由於市售產品之DNA 會因加熱高溫烹煮、混雜其他之魚肉等因素而有所影響，而進行加熱極限之偵測、混合魚肉、仿製市售產品之偵測。檢測結果顯示，加熱檢測極限在 100℃烹煮過後之魚肉其極限值可達 60 min ，在121℃滅菌之魚肉上其極限值可至 30 min，生鮮旗魚混合肉之檢測極限為< 0.5%之旗魚含量，在仿製產品中其針測極限為< 1%，並利用仿製之加工品作為對於市售產品偵測之標準曲線。實際採集北中南東四個地區之市售旗魚樣品:旗魚鬆 20 件 、旗魚丸 22件、其他產品:如旗魚糖、旗魚蛋捲、旗魚漿及旗魚黑輪等。經基因定量檢測原料含量結果顯示不合格之百分比為:旗魚丸: 45.45%、旗魚鬆: 5% ，有此可得知亦有不符合標示之產品。而市售之旗魚糖則未檢出，有此可得知亦有不符合標示之產品。綜合實驗結果顯示，本論文所建立之即時定量聚合酶連鎖反應之條件可適用於檢測旗魚加工品之定量分析。

關鍵字

旗魚 ； 基因定量法 ； 粒線體DNA

並列摘要

The five billfishes are the important economical fish in Taiwan. They are used in many kinds of products, including slices of meat (sashimi), fish minced products and dried fish floss. The meats and products have a relatively high market price in Taiwan. Therefore, some retail dealers misuse the other low-price fish meats instead of billfish meats in order to make a profit.
In this study we used of a specific Real-time PCR amplification of a repetitive DNA element, for the quantification by billfishes, because of its simplicity, specificity, and sensitivity.
In the beginning we amplified of cycochrome c oxidase I and 16S rRNA finished the complete of billfishes sequencing, and designed suitable primers of DNA quantitative method of Real-time PCR.
In this study, we used five billfishes assays were developed around small (amplicons 150 base pairs) region of the mitochondrial cytochrome c oxidase I (COI) gene. The assay first was tested on specificity and sensitive of primers. We defined that the primers were applicable to the five billfish, so second we used the primers detect the limit of billfish concentration, the sensitivity was as following: Makaira nigricans: 10-5 ng, Kajikia audaxt: 10-5 ng, Istiophorus platypterus: 10-4 ng, Xiphias gladius: 10-3 ng and Istiompax indica: 10-5 ng.
Third was test on different time of heated and sterilized ( 100℃ heating from 0 – 90 min, 121℃ heating from 0 – 90 min) to define the limit of five billfish. The results showed the heted of billfish meats limit detected time was > 60 min, and the sterilized detected limit time was >30 min. Forth the assay was tested on DNA extracted from raw and heat-treated mixtures of billfishes tissues in a matrix, and on DNA extracted from reference stuff samples.The mixture of billfish meats limit detected was <0.5% of billfish’s DNA concentration, and the related products of billfish limit detected was about <1%. Replicate standard curves of the threshold cycle (Ct) value (Y) vs Different percentages of billfish DNA (X) were analysed using sigama plot 10.0 software and a linear regression equation of the Ct value plotted against Different percentages of billfish’s DNA was calculated.
Furthermore, applying above the method to quantitative of billfish, we collected 44 commerical samples of billfish related products from northern region, centeral region, southern region and eastern region. It was found that qutitative of billfish in dressed fish fried marlin product were 30.47% - 100%, fish ball were 14.52% - 93.45%, surimi made by marlin was 87.21%, tempura made by marlin was 74.94%, and marlin egg roll was 71.03%.
Base on the results we concluded that Real-time PCR condition developed in our research was applicable to the quantitative analysis of detecting the marlin meat products.

並列關鍵字

billfish ； real-time PCR ； mtDNA

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