利用多目標PCR檢測基因改造木瓜

Detection of Genetically Modified Papaya with Multiplex-PCR

陳富永(F. Y. Chen) ； 蔡奇助(C. C. Tsai) ； 陳國憲(K. H. Chen) ； 王雲平(Y. P. Wang) ； 楊藹華(A. H. Yang)

《高雄區農業改良場研究彙報》 16卷4期 (2005/12) Pp. 16-27

https://doi.org/10.6958/RBKDAIS.200512.0016

摘要

木瓜產業受到木瓜輪點病毒（papaya ringspot virus，PRSV）的嚴重威脅，產量大減，中興大學植物病理系教授葉錫東博士培育的抗輪點病毒基因改造木瓜，可有效降低此病毒的威脅。但因基因改造作物是否影響生態及食用安全性之疑慮，政府單位及消費大眾均極關切基因改造木瓜的檢測。本研究利用PCR反應，進行核酸層次的檢測，以針對PRSV外鞘蛋白基因（coatprotein，CP）設計專一性之引子，可以增幅一820 bp之DNA片段，正確檢測出基因改造木瓜。為提高檢測之精準性及效率，同時開發出多目標PCR（multiplex PCR）技術，針對殖入木瓜之多個基因及DNA序列做為檢測對象，包括PRSV CP基因、NPT-II（neomycin phosphotransferase）基因、NOS（nopaline synthase）terminator序列、及CaMV（cauliflower mosaic virus）35S promoter序列等設計專一性引子，利用多組核酸引子，同時進行PCR反應，在基因改造木瓜中可分別增幅359 bp、295 bp、180 bp、及134 bp四個片段；再加上木瓜本身的木瓜酵素（papain）基因之專一性引子（增幅211 bp），做為內部對照（internal control）達到多重確認基因改造木瓜檢測精準性之效果。

關鍵字

基因改造木瓜 ； DNA檢測 ； 多目標PCR

並列摘要

Papaya (Carica papaya) is rich in vitamins and nutrition, and also an important fruit in Taiwan. Papaya ringspot virus (PRSV) is a destructive virus in papaya, and became a limit factor in papaya production. Genetically modified (GM) papaya, which has been developed by Dr. Yeh, NCHU, is a possible strategy resistant to PRSV. Exactly detection of GM papaya is an important task in papaya production. Primers specific to PRSV CP gene have been designed, which reflected 820bp fragment, exhibited good detection result in GM papaya. In order to reduce detection time and cost, and also promote the accuracy of detection, multiplex-PCR technique has been developed. Five primer pairs, which specific to PRSV CP gene, NPT II gene, papain gene, NOS terminator sequence, and CaMV 35S promoter sequence, were designed for PCR detection in one reaction. Multi-fragments patterns of 359 bp, 295 bp, 211 bp, 180 bp, and 134 bp were obtained in GM-papaya sample, but non-GM sample shown one 211 bp fragment only.

並列關鍵字

genetically modified papaya ； GMO detection ； multiplex PCR)