開發可正確且同時診斷多種病原菌的檢測平台將有助於感染性疾病的治療。傳統以培養及生化測試為主的診斷方式受限於需要較長的鑑定時間及人為操作誤差；而以PCR為主的分子診斷技術缺點為效率不彰及診斷的準確度。扣鎖探針 (padlock probe)為一新型核酸檢測工具，其為一條長約100個鹼基的單股寡聚核苷，在其5’和3’端各別約有20個鹼基序列用來辨認與其互補的特定標的核醣核酸分子，聯結這兩端的中間序列則常被設計成有可被檢測的功能。其標的核醣核酸分子與扣鎖探針兩端互補的鹼基序列是連續的。因此，當單股寡核苷酸探針的兩端與之互補雜交後，原來呈直線的探針會彎曲成環形，在接合酶(ligase)的催化下，可將探針的兩個端點接合，使得這扣鎖探針扣成環形。然而，如果直線型的扣鎖探針沒有與其標的核醣核酸分子正確的互補雜交，接合酶也無法將探針接合成環形。因此，環型扣鎖探針的形成反應了其標的核醣核酸分子的存在。經過訊號放大後，可利用探針上的標籤(Tag)序列與一個含有多組標籤序列的DNA microarray做雜交檢測，藉此區分病原菌。為了改善現有的診斷技術，我們希望藉由結合扣鎖探針與DNA array技術開發一套檢測平台，並以此平台檢測造成泌尿道感染之病原菌。首先，我們設計出一組扣鎖探針，針對台灣地區常見的尿道感染致病菌 (Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Proteus mirabilis, and Gram-positive bacteria)，接著調整出最佳的檢測條件。由實驗結果可知，探針的純度、使用濃度及不同類型的接合作用(ligation)皆會影響探針的靈敏性及特異性。調整好檢測條件後，我們測試各扣鎖探針對其標的病原的特異性與靈敏性，最低可偵測到2 pg genomic DNA及104~105菌數。我們以扣鎖探針檢測了41株臨床分離菌株。另外，我們成功藉由結合扣鎖探針與DNA array同時正確地偵測多種病原菌。在進行臨床檢驗前，我們先藉由與標準訊號比對的方式來估算菌量。最後，我們利用所開發的平台直接檢測臨床病人檢體並與傳統培養為主的資料進行比對，來測試此平台的可用性。希望這套檢測平台在未來能確實有效應用在臨床檢驗上，使感染性疾病診斷系統更加完善。

Development of molecular approaches for pathogen detection is the key to improve treatment of infectious diseases. A new class of oligonucleotide probes, called padlock probes, has been developed for detecting pathogen-specific nucleotide sequences. A padlock probe comprises two target-complementary end-sequences which can recognize adjacent sequences on the target DNA (or RNA). When hybridizing to the correct target sequences, the ends of the probe get into adjacent and can be jointed by enzymatic ligation to form a circularized probe. Therefore, the production of the circular forms of the padlock probes indicates the existence of their target sequences. Then, the signals of the circularized probes can be amplified by PCR with primers recognizing the sequences in the padlock probes. In addition, each of the padlock probes contains a unique 20-bp sequence which can serve as a name tag. By combing DNA array analysis with the name tags, the existence of the circular forms of multiple padlock probes can be detected. Our objective is to develop a diagnostic tool utilizing padlock probes and DNA array analyses to simultaneously detect multiple bacterial pathogens causing urinary tract infections (UTI). We have constructed 5 padlock probes specifically targeting the five most common UTI bacterial pathogens (Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Proteus mirabilis, and Gram-positive bacteria), respectively. The optimized working conditions for the padlock probes to detect targets have been determined. Sensitivity and specificity of each padlock probes were also assessed. The lowest target DNA concentrations and the minimal bacterial numbers can be detected by the probes were around 2 pg and 104~105, respectively. So far, the probes have been used to successfully detect 41 bacterial isolates using PCR to check the existence of their circularized forms. We have also successfully detected multiple pathogens at the same time by combining padlock probe techniques and dot-blotting assays. Finally, we evaluate whether this diagnostic system is applicable in detecting bacterial UTI pathogens in clinical urine specimens.

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