Development of cascade enzymatic reaction-based nucleic acid detection method using personal glucose meter

다국어 초록 (Multilingual Abstract)

This study presents a novel molecular diagnostic platform that integrates the structure-specific cleavage activity of flap endonuclease 1 (FEN1)-coupled cascade enzymatic reactions to detect target nucleic acids without the need for polymerase chain reaction (PCR) amplification. This system leverages AMP produced by FEN1 cleavage to trigger glucose degradation, which can be quantified using a personal glucose meter (PGM). Validation of this platform was conducted using target DNA based on the Chlamydia trachomatis (Ct) sequence, demonstrating effectiveness in detecting sequences identical to common bacterial pathogens. Utilizing this system, target nucleic acid was successfully detected with a detection limit of 4.8 pM. Additionally, the potential to detect target nucleic acids derived from Chlamydia trachomatis showed excellent selectivity compared to other STD genes and mismatches. By overcoming the limitations of PCR and fluorescence-based techniques, this platform offers a cost-effective, scalable, and user-friendly diagnostic solution with significant potential for application in resource-limited settings.

국문 초록 (Abstract)

본 논문은 flap endonuclease 1 (FEN1)의 구조 특이적 절단 활성과 연계된 연쇄 효소 반응을 통합한 새로운 분자 진단 플랫폼을 제시합니다. 이 시스템은 중합효소 연쇄 반응(PCR) 증폭 없이 표적 핵산을 검출하며, FEN1 절단으로 생성된 AMP 가 포도당 분해를 유도하여 개인용 혈당 측정기(PGM)를 통해 정량화 할 수 있습니다. 우리는 Chlamydia trachomatis (Ct) 서열을 기반으로 한 표적 DNA 를 사용하여 이 플랫폼을 검증했으며, 일반적인 세균 병원체의 서열과 동일한 서열을 검출하는 데 효과적임을 입증했습니다. 본 시스템을 이용하여 표적 핵산을 성공적으로 검출했으며, 검출 한계는 4.8 pM 이었습니다. 또한 Chlamydia trachomatis 에서 유래한 표적 핵산을 검출할 수 있는 가능성을 보였으며, 다른 성병(STD) 유전자 및 불일치 서열에 비해 뛰어난 선택성을 나타냈습니다. PCR 및 형광 기반 기술의 한계를 극복함으로써, 이 플랫폼은 비용 효율적이고 확장 가능하며 사용자 친화적인 진단 솔루션을 제공하며, 자원이 제한된 환경에서의 적용 가능성이 큽니다.

목차 (Table of Contents)

• List of Figures·············································································ⅲ
• List of Tables··············································································ⅳ
• Abstract ·····················································································ⅴ
• Ⅰ. Introduction ·············································································1
• 1. Detection of Nucleic acid ······························································1
• 2. Polymer Chain Reaction and Limitations of PCR ··································1
• 3. Personal Glucose Meter (PGM) ·······················································2
• 4. Cascade Enzyme Reaction ·····························································2
• 5. Objective of this study ··································································3
• Ⅱ. Materials and Methods ······························································4
• 1. Materials ··················································································4
• 2. FEN1 cleavage activity and verify ADP creation···································8
• 3. Buffer Selection for one-step System ················································8
• 4. Confirmation of CER results according to UP/DP ·································8
• 5. Sensitivity and selectivity ······························································9
• 6. Detection of Target DNA in 1% Human Serum ····································9
• Ⅲ. Results ················································································10
• 1. Basic Principles········································································10
• 2. FEN1 Cleavage Activity and Verification ADP Production ····················12
• 3. Buffer Selection for one-step System ··············································14
• 4. Investigation of FEN1 cleavage activity on flap structure of UP and DP·····16
• 5. Performance of system ·······························································20
• 6. Practical applicability ·································································25
• Ⅳ. Discussion············································································28
• Ⅴ. Conclusion ············································································30
• Reference··················································································31
• Abstract in Korean ·····································································39