人類愛滋病病毒從發現至今已造成全球逾兩千五百萬人的死亡。為有效地追蹤疫情，則必須仰賴靈敏性更高的病毒檢測工具，投入例行的診斷、流行病學上的監控與病源的篩選分析。然而，現行檢測工具的弊病仍有：昂貴的價格成本、技術不夠迅速、方法不夠專門直接、檢體需求過多等因素。再者，新生兒檢體中若帶有母源性抗體或是檢體的病毒量或專一性不足，不但會干擾檢測，還會使篩選及確認感染的更加繁瑣與費工。臨床診測工具若無法兼顧精確度低和便利性，則很可能耽誤初期感染及新生兒感染的判定與診治，甚至影響到HAART病患的預後與體內病毒數的追蹤。“扣鎖探針”為一種新型態的核酸診測工具，於先前已成功的被應用於病毒RNA的診測以及準確的在人類基因體研究中診測出專一的SNP。由於諸篇研究證實了此探針兼具優異的專一性與靈敏性，因此極有潛力發展成準確度高且專門直接的診測法。在本篇研究中，我們報告人工合成的扣鎖探針應用於HIV診測的研發，利用它與保守的目標序列進行互補辨識並雜合至目標序列，此探針可被黏合催化成環型的結構，並在後續的恆溫滾式環形擴增反應(RCA)中，擴增出以100bp為倍數的梯狀診測訊號，是放大HIV感染訊息作為診測的絕佳策略。藉由比對大量的病毒株序列，我們根據坐落於LTR區域的保守序列，設計出兩個不同目標序列的探針。其中，PLP-2LTR可特別偵測出病毒的2-LTR環形產物; PLP-R則可藉偵測病毒RNA或DNA的R區域來判斷感染。經驗證，我們發現PLP-2LTR有優於PLP-R的診測靈敏度，但卻有非專一性的訊號干擾於診測中產生。因此，在應用生物素標幟探針(bio-PLP-2LTR)與磁珠來進行核酸純化後，我們成功地克服了扣鎖探針的雜訊干擾，也將病毒的診測極限突破至2個微量的病毒個體。但是目前扣鎖探針的診測仍尚具進化改良的空間。藉多重生物素的標幟的增加，可藉強化結合力而可以穩定呈現訊號，扣鎖探針也更可能有機會因此而運用於定量的檢測訊號。總體而言，本研究證實了扣鎖探針極具診測病毒感染的潛力。且扣鎖探針未來於臨床診測的運用更值得進一步的探索。

Human immunodeficiency virus (HIV) infection has resulted in more than 25 million deaths worldwide since its discovery. To effective track the disease, highly sensitive HIV detection tools are required for routine diagnosis, epidemiologic surveillance, and donor screening. However, current tools are not necessary to be cheap, rapid and static methods of accuracy, and may require a large amount of blood and/or repeated tissue biopsies. Screening and confirmation of HIV infection may also become laborious and problematic, particularly to specimens with preexisting maternal antibodies or minimized viral loads and specificity. Tools show limited precisions or convenience in practice thus may detain the detection of neonatal infection and fail to discriminate newly infections or monitor the viral loads of retroviral drug-treated adults. As molecular padlock probes (PLPs) based nucleic acid detection approach has innovative applications in localizing and signify pathogenic nucleic acids, especially in the detection of many viral RNA and human SNPs with extream specificity, it may have great potential to be developed into a static and accurate method for HIV infection. We report here the development of a synthetic PLP, combining with ligation-based recognition and isothermal rolling circle amplification (RCA) by Bst polymerase, as a strategy to signify HIV infection. By sequence alignments of HIV-1 strains from the LosAlamos Database, two PLPs were initially designed for the detection of HIV-1 conserved region in the long terminal repeat (LTR): (1) PLP-2LTR: targeting to conserved junctions of native 2LTR DNA circles; (2) PLP-R: targeting to the R regions to detect viral RNA or DNA. As these PLPs contained both ends complimentary to the targeted DNA or RNA target, which upon hybridization and ligation, they were expected to form un-nicked circles and can be amplified by Bst into 100 bp ladders. Our result indicates that PLP-2LTR was more sensitive than PLP-R for HIV detection, but non-specific amplification effects of these two PLPs were observed. After further improvement on the probe design and purification procedues, we found that the use of a biotinyl PLP-2LTR (bio-PLP-2LTR) in an implemented beads purification procedure enhanced the detection limit to 2 copies of DNA targets without confusing noises. However, the amplification signal was unstable and independent to the dosage of DNA template, presumably due to inconsistent association of the beads to the mono-labeled bio-PLP-2LTR. This preliminary study demonstrates the potential of a biotinyl PLP-2LTR for HIV-1 infection. Modifications of the bio-PLP-2LTR for clinical diagnoses are discussed and warrant further exploration.

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