使用磁珠分離及鏈置換增擴放大法開發子宮內膜樣腺癌偵檢策略

Abstract

子宮內膜癌是目前世界上女性第四常見的癌症，其中子宮內膜樣腺癌佔了病例之九成，其發病率更在近年來大幅上升，因此早期診斷子宮內膜樣腺癌的分析平台有其迫切需求。據先前研究指出，microRNA-205之表現水平與子宮內膜樣腺癌具有高度相關性，可作為子宮內膜樣腺癌之生物標誌分子。本篇研究首先藉由鏈結合素及生物素之間的親和作用，將核酸探針修飾到磁珠上，再以強力磁鐵進行磁吸分離並將目標核酸microRNA-205進行分離純化。接著加入核酸聚合酶及核酸限制酶，於磁珠上進行鏈置換增擴放大反應以產生核酸產物。反應後再次磁吸分離並蒐集產物之上清液後，加入與產物序列互補之產物核酸探針，並形成富含腺苷酸及胸苷酸之雙股核酸分子，產物探針之利用可做為第二道檢測專一性之檢查點。而本篇研究著重於磁珠對於鏈置換增擴放大反應的使用策略，在確立酵素反應之最佳化反應時間為六十分鐘後，核酸聚合酶與核酸限制酶可於修飾於磁珠上的探針附近持續進行反應，並發現可對於產物之產率產生影響：於相同反應時間內，相較於未使用磁珠的組別，使用磁珠的組別之產率明顯較高，並且保持在無目標核酸出現時的良好反應專一性，不會有偽陽性之結果發生。而修飾於磁珠上的探針選擇性，與未修飾於磁珠上的探針相同，對於非目標序列之真實存在microRNA序列具有良好的選擇性。經本篇研究確立：磁珠之使用，於使用鏈置換增擴放大反應作為放大訊號的方式，具有良好的產率增加之效果；磁吸分離之方式亦可簡化子宮內膜樣腺癌之偵檢流程，於疾病檢測上更為便利。

Endometrioid Endometrial Carcinoma (EEC) is the fourth most common cancer among women in the world, and the incidence of EEC has increased sharply in recent years. Therefore, it is urgent to develop an EEC diagnostic platform for early detection of EEC. MicroRNA-205 was previously reported as a potential biomarker for EEC, which motivated us to develop an ultrasensitive sensing platform to further confirm its correlation to EEC. Strand displacement amplification (SDA) reaction is an isothermal technique to amplify nucleic acid target, relying on a polymerase and a restriction enzyme. We herein utilized the concept of SDA, and designed a new isothermal amplification method for the detection of EEC-related miR-205. We first conjugated DNA probe on magnetic beads via streptavidin-biotin interaction, it was followed by the enrichment of miR-205 present in the sample through magnetic separation. DNA polymerase (3' → 5' exo-) and nicking enzyme were added afterwards to induce the synthesis of the product DNA. After SDA reaction time optimization as 60 min, we found that applying magnetic beads to SDA amplification reaction can increase the yield of product DNA, compared with the magnetic bead- free group. In specificity, probes conjugated on magnetic beads can distinguish microRNA-205 from others non-target microRNA. Magnetic separation can also simplify the process of diagnosis of Endometrioid Endometrial Carcinoma