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Optimization of capillary gel electrophoresis for DNA fragment analysis and its application to surveillance of meat contents in canine canned food
|關鍵字:||capillary gel electrophoresis;毛細管膠體電泳;canine canned food;meat species;犬罐頭;肉品種別||出版社:||獸醫學系暨研究所||引用:||Aebersold R, Mann M. Mass spectrometry-based proteomics. Nature 422: 198-207, 2003. Allmann M, Candrian U, Höfelein C, Lüthy J. Polymerase chain reaction (PCR): a possible alternative to immunochemical methods assuring safety and quality of food Detection of wheat contamination in non-wheat food products. Zeitschrift für Lebensmitteluntersuchung und-Forschung A 196: 248-251, 1993. Anderson S, Bankier AT, Barrell BG, De Bruijn MHL, Coulson AR, Drouin J, Eperon IC, Nierlich DP, Roe BA, Sanger F. Sequence and organization of the human mitochondrial genome. Nature 290: 457-65, 1981. Aristoy MC, Toldra F. Histidine dipeptides HPLC-based test for the detection of mammalian origin proteins in feeds for ruminants. Meat Sci 67: 211-217, 2004. Asensio L, González I, García T, Martín R. Determination of food authenticity by enzyme-linked immunosorbent assay (ELISA). 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隨著民眾逐漸重視動物用食品之安全及品質，動物用食品中之成分檢測就顯得格外重要，其中肉品成分之誠實標示對於健康、宗教及商業公平性皆具顯著意義。然而此一動物用食品安全領域卻少見重視及研究。此外，各種分析方法之適用性也需要檢視。本研究針對蛋白質與DNA兩大類分子，選用酵素免疫分析（enzyme-linked immunosorbent assay，ELISA）與聚合酶鏈鎖反應合併限制片段多形性（polymerase chain reaction-restriction fragment length polymorphism，PCR-RFLP）方法，分析市售犬用罐頭之肉品標示種別。為增進PCR-RFLP分析圖譜的解析度與偵測極限，本研究除以平板電泳（slab gel electrophoresis，SGE）搭配紫外光（ultraviolet，UV）偵測PCR-RFLP產物外，更以毛細管膠體電泳（capillary gel electrophoresis，CGE）搭配雷射激發螢光（laser-induced fluorescence，LIF）進一步偵測。由於CGE對於不同大小的DNA片段有其合適的分析條件，故本實驗亦對肉品DNA切割後之產物（100 ~ 400 bp）進行方法之最佳化。PCR-RFLP結果顯示牛與豬肉之DNA經AluI與MboI切割之後片段大小具有良好的種別特異性；而羊肉與禽肉之片段則因與部分物種相似故以ELISA方法區別度較佳。此外，部分以SGE檢測不出牛或豬肉成分者，可以CGE測得該成分波峰，顯示CGE-LIF比SGE-UV具有更低的偵測極限及敏感度。本研究發現11個不同品牌的罐頭多數可測出未標示之豬或羊肉成分，而部分則測不出所標示之雞肉成分，顯示標籤之正確性仍有改善空間。研究結果同時顯示各分析方法所產生的結果並不完全一致，代表單一方法的準確性亦有進一步討論的必要。本實驗額外以PCR-RFLP搭配SGE-UV檢測市售潔牙骨是否含有肉品來源，在6個不同品牌標示不含肉品成分之潔牙骨產品中，有1個產品可檢出肉品反應。簡言之，藉由這些樣品檢測，研究發現多數犬用食品之肉品成分並未完全符合其標籤內容，建議廠商在產品標示上應做出更精確的肉品成分與添加比例說明。合併多重分析方法，可以使檢測的準確度提高，因此，現行以單一方法檢測的方式值得進一步檢討。未來，若能擴大更多品牌、產地及生產批號之樣品檢驗，以增進採樣的統計意義，將能更進一步了解伴侶動物食品之肉品標示的正確性。
With the increasing awareness of animal food safety and quality, species identification of meat content in animal food products has gaining importance for health, economic and legal reasons. To obtain reliable and accurate information regarding meat contents in canine canned food, several analytical techniques have been developed for the identification of meat species in mixed samples, including protein-based and DNA-based methods. We used both enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods for the initial screening of canine canned food, and coupled with slab gel electrophoresis (SGE), and capillary gel electrophoresis (CGE) with laser-induced fluorescence (LIF) detector for confirmation of PCR-RFLP products. CGE method for the analysis of PCR-RFLP products sized between 100 to 400 bp was optimized. The results suggested that while the specificity for beef and pork was better using PCR-RFLP, ELISA was better for differientiation of sheep/goat and avian species. CGE was shown to have improved resolution and sensitivity than SGE revealing more meat species in the same sample. The results of canned food surveillance indicated that none of the 11 tested canned food meat species totally matched the label. On the other hand, one out of six canine vegan bone treats was found with meat content. In conclusion, it is better to use multiple analytical methods to improve assay spectrum although there is a significant discrepancy between the labeled canned food meat species and the tested results. Manufactures are advised to be more accurate in stating their products labels. Sample numbers should be increased for better biostatistic interpretation and accurate surveillance of meat contents in companion animals.
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