KIT COMPRISING A POLYNUCLEOTIDE PROBE FOR DETECTING A TARGET NUCLEIC ACID

專利類型

發明

專利國別 (專利申請國家)

美國

專利申請案號

14/538,965

專利證號

US 9,902,997 B2

專利獲證名稱

KIT COMPRISING A POLYNUCLEOTIDE PROBE FOR DETECTING A TARGET NUCLEIC ACID

專利所屬機關 (申請機關)

國立臺灣大學

獲證日期

2018/02/27

技術說明

此體外診斷光學感測套組乃利用CdSe/ZnS核/殼量子點上修飾雙功能DNA探針；此功能型DNA探針由二段DNA序列組成。相對遠離量子點為與目標miR互補之辨識序列探針，尾端攜有一淬滅劑(Quencher, BHQ2)；而緊鄰量子點的DNA片段為訊號引子。在目標miR之存在下，DNA探針與目標miR進行互補配對後，可被溶液中存在的雙股核酸酶(Duplex specific nuclease, DSN)辨識，已配對之DNA:RNA雙股中只有探針DNA被剪切分解；尾端淬滅劑因而遠離量子點表面，使量子點之螢光訊號回復。同一時間，目標miR從原本雙股DNA:RNA中釋放至溶液中，並可再重覆與其它探針DNA進行互補配對，使得量子點螢光再度回復。本發明藉由目標miR的循環利用，引發相關反應並造成螢光訊號回復的原理，有效測量miR。此外，DNA探針被剪切後，造成鄰近量子點的DNA訊號引子片段裸露，而在有端粒酶(Tolemerase)之反應溶液下進行序列延長；又因延長之序列結構含有數個重覆GGG鹼基片段，可堆疊排列形成三級立體結構並具催化能力，引發受質溶液產生化學冷光，並隨目標miR濃度增加呈現線性增長，偵測極限下探。該套組有高度選擇性，只專一性辨識目標miR，對於測量其他序列相似度高之同源家族miR及其他miR並無交叉反應(Cross-reactivity)現象。若將此套組實際應用測量血液樣本時，發現可成功鑑別健康男性及攝護腺癌確診病人，顯示其對於臨床診斷攝護腺癌有良好應用前景。 rostate cancer (PC) is a frequently diagnosed cancer-related cause of death in men worldwide. A blood-based in vitro diagnostic test allows for fast and convenient prognosis, diagnosis, and better treatment options. Because the current prostate-specific antigen screening usually gives false-positive results, it is a crucial prerequisite for such diagnostics to target specifically prostate cancer-associated biomarkers. Circulating miRNAs in blood have been revealed as cancer-specific and stable biomarkers for identification of various cancers. We designed and developed two optical genosensors toward the detection of the prostate cancer-associated miRNA, for screening prostate cancer. We took advantage of probe-DNA modified QDs along with nucleases to create the target-triggered, nuclease-induced recycling of miR for the amplified regeneration of fluorescence signal of QDs, resulting in the “turn-on” fluorescent genosensor.

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