以側向流免疫層析法對基因序列HLA-A3101單核苷酸多態性變異之快速偵測

本研究利用鹼基不互補的概念，對具有單核苷酸多態性(SNP)的HLA-A3101 DNA進行辨識，並使用側向流薄膜試條對，具有特殊修飾的PCR產物進行判讀。藉由鹼基不互補概念，設計14個前端引子來分辨其SNP，得到引子11與引子14能夠辨識HLA-A3101的SNP。將前端引子分別修飾biotin與FITC，後端引子皆修飾digoxigenin之後，對具有SNP的HLA-A3101雙股DNA進行一系列偵測。在實驗結果裡，利用引子11所得到的未純化PCR產物之肉眼檢測極限為0.001µl(0.553ng)；引子11對其他控制基因的專一性測試下，雖然對其他控制基因，有微弱的測試線畫痕，但是並不影響肉眼的判讀，表示所設計的前後引子有良好的專一性。兩種再現性測試，intra-assay與inter-assay得到結果，變異係數為4.94%~13.1%，皆小於15%，代表所使用的側向流薄膜試條具有良好的一致性。本研究證實已成功的設計出可辨識SNP的引子，並成功利用側向流薄膜試條進行SNP判讀，未來若能進一步應用於臨床實驗，可在有限的儀器下，避免錯誤的藥物治療。

關鍵字

側向流；基因序列；單核苷酸多態性；快速偵測

並列摘要

In this study, HLA-A3101 DNA with a single nucleotide polymorphism (SNP) was identified by base-mismatched primers. The lateral-flow strips were used to detect the DNA’s PCR products. Fourteen forward primers, denoted from #1 to #14, were designed in different base mismatching scenarios around the SNP spot. As a result, the forward primers #11 and #14 were recognized to be able to identify HLA-A3101 from its allele gene. After being labeled with biotin or FITC ligands on the forward primer and digoxigenin on the reverse primer, a series of detections of HLA-A3101 SNP on the membrane-based lateral-flow strips was conducted. In the experimental results, the unpurified PCR product using primer #11 received a detection limit of 0.001 μl (0.553 ng) by naked eyes. Primer #11 also had a good specificity from other negative control genes. In two tests, intra-assay and inter-assay, the strips showed a good data reproducibility in variation coefficients of 4.94%~13.1%. This study can be further applied on clinic diagnosis with limited instrument support to avoid medication treatment from wrong drug prescription.