利用即時聚合酶連鎖反應進行乳羊之性別鑑定

本研究旨在利用即時聚合酶連鎖反應（real-time polymerase chain reaction, real-time PCR），配合SyberGreen（SYBR）與螢光標定探針（probe）進行乳羊的性別鑑定。試驗應用純化之乳羊基因組DNA與體外培養（in vitro culture）之阿爾拜因乳羊耳朵成纖維細胞做為性別鑑定之樣品。體細胞樣品先經蛋白酶K（proteinase K）處理後，利用ZFX/ZFY（ZFX/ZFY-linked zinc finger protein）引子進行PCR預先擴增（pre-amplification），獲得PCR產物後，設計nested PCR引子並應用SYBR進行real-time PCR。結果顯示，隨著起始的模板DNA增加，在較早的循環出現PCR擴增曲線，PCR產物具有高度專一之解離曲線（dissociation curve），顯示PCR產物之高度特異性。預先擴增後之初次PCR產物配合nested PCR引子及性別特異TeqMan螢光探針進行雜合反應（hybridization），因樣品產生不同螢光而判定性別。試驗結果顯示，待測之乳羊基因組DNA或少至1個體細胞，進行雜合反應後均能準確鑑定性別。本研究顯示應用real-time PCR可直接鑑定乳羊樣品之性別而不需進行電泳分析，且具較高之敏感性與重覆性，具有田間乳羊性別鑑定之應用潛力。

關鍵字

乳羊；性別鑑定；即時聚合酶連鎖反應； ZFX

並列摘要

The purpose of this study was to develop a fast method for sexing dairy goats by real-time polymerase chain reaction (PCR). DNA samples prepared from peripheral blood and in vitro cultured somatic cells were pre-amplified by traditional PCR with ZFX/ZFY primers. The resultant products were used as a template in the following real-time PCR assay, with nested primers and SYBR, and then determined with sex specific fluorogenic probes for ZFX and ZFY. Results showed that the higher the concentration of initiation template was, the earlier the amplification plot appeared. The dissociation curve of real-time PCR products showed high specificity. By using SYBR analysis and sex specific fluorogenic probes, the accuracy, efficiency, and reproducibility of sex determination for caprine genomic DNA and somatic cells were significantly improved. In addition, the time-consuming electrophoresis for traditional PCR procedure was eliminated by using fluorogenic probes for dairy goat sexing.

並列關鍵字

Dairy goat ； Sexing ； Real-time PCR ； ZFX

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