- 學位論文
以多測試線之側向流免疫層析法對基因HLA–A*3101單核苷酸多態性變異之快速偵測
Rapid Detection of Single Nucleotide Polymorphism (SNP) Variation of Gene HLA–A*3101 Using Multiple-line Membrane-based Lateral-flow Immunoassay.
摘要
卡馬西平是抗癲癇藥物之一，當HLA–A*3101基因存在單核甘酸多態性（SNP）時，會造成皮膚的不良反應，因此醫生在開立處方籤前，若能先檢測病患是否存在單核苷酸多態性（SNP），便能降低用藥的造成傷害的風險。因此本研究以辨認HLA–A*3101基因是否存在單核甘酸多態性（SNP）為研究方向規劃。 本研究利用前端引子與模版的鹼基對相互不互補的理念，辨識HLA–A*3101是否存在單核苷酸多態性（SNP），再以側向流體免疫層析試紙測試法（LFIA）顯示。實驗設計九個前端引子（編號F1～F9），將其末端包含SNP位點，利用與模版鹼基對不互補的設計，以聚合酶鏈鎖反應（PCR）先行確認出F6號前端引子，與F8號前端引子為可辨認HLA–A*3101的單核苷酸多態性（SNP）。將F6號前端引子修飾FITC與F8號前端引子修飾Biotin，使用相同後端引子修飾Digoxigenin後，再對HLA–A*3101模版進行側向流體免疫層析試紙測試法（LFIA）的一系列檢測。實驗結果表明F6號與F8號引子的PCR產物，可於側向流體免疫層析試紙辨別HLA–A*3101有無存在單核甘酸多態性。雖然檢測另一個基因型會有微弱的訊號，但不影響整體判斷。兩採用之引子對於其他基因具有良好的專一性，以肉眼檢測極限濃度為0.1ng∕μL，inter-assay與intra-assay的變異係數在1.72﹪～15.86﹪區間，具有良好的一致性。 本研究不侷限於任何地點及環境，可為應用於臨床治療､家用看護及偏郊醫療，為醫療檢驗帶來更多的發展。
並列摘要
Carbamazepine is one of regular antiepileptic medicine. When a patient’s HLA-A*3101 gene has a variation of single nucleotide polymorphism (SNP), that condition could cause the patient serious adverse skin reactions if he takes that medicine. The clinic doctors thus could detect if patients have that specific SNP variation before signing a prescription to reduce the risk of wrong medication. Therefore, the research direction of this study is to identify whether HLA-A*3101 gene has SNP. In this study, we report the idea using mismatched base pairs between a forward primer and its template to recognize whether or not there is a SNP in the template. The lateral flow immunochromatographic assay(LFIA) is applied to visually report the result. In the experimental design, nine forward primers (F1~F9) are designed to include their terminal base is mismatched. Polymerase chain reaction (PCR) was used to confirm that F6 and F8 forward primers perfectly recognized SNP on its corresponding template, gene HLA-A*3101. After individually modifying ligands FITC on F6, biotin on F8 forward primers, and digoxigenin on the reverse primer, the HLA–A*3101 PCR product was successfully detected on LFIA strips. Both forward primers are able to identify regular gene HLA-A*3101 from its allele with a detection limit of 0.1 ng/μL by naked eyes. The detection coefficients of variation of inter assay and intra assay were in the range of 1.72 to 15.86, which indicates a good consistency among tests. The results obtained from this study can apply on many care points. They can further apply on clinic treatment, household care, and suburban medication. It opens many opportunities for medical diagnoses.