현장 분자 진단을 위한 랩온어칩 개발
Development of a lab-on-a-chip for point-of-care molecular diagnostics
- 주제(키워드) 도움말 분자 진단 , 랩온어칩 , bead-beating , recombinase polymerase amplification
- 발행기관 강릉원주대학교 일반대학원
- 지도교수 도움말 최석정
- 심사위원 김용주, 최석정, 박정민
- 발행년도 2018
- 학위수여년월 2018. 8
- 학위명 석사
- 학과 및 전공 도움말 일반대학원 화학과
- 실제URI http://www.dcollection.net/handler/kangnung/000000010213
- UCI I804:42001-000000010213
- 본문언어 한국어
- 저작권 강릉원주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약 도움말
체외진단 시장은 기존 치료중심의 의료 환경에서 질병의 조기진단, 예방 및 관리 의 개념으로 빠르게 변화하고 있으며 이에 따라 현장진단 시장이 확대되고 있다. 그러나 현재 개발된 현장 분자진단기기의 대부분은 전처리와 검출과정이 나눠져 있어 자동화가 어려우며 자동화된 기기는 장치가 복잡하고 가격이 비싼 단점이 있 다. 따라서 이번 연구에서는 시료의 전처리, DNA 추출 및 증폭, 검출 과정을 포함 하는 일련의 과정이 간단하게 이뤄지는 정지액체상 랩온어칩을 개발하였고 이를 통해 V. parahaemolyticus 를 검출하였다. 이 방법에서 시료의 전처리는 항체로 기능화 된 포획입자로 진행되고, DNA 추출은 포획입자의 bead-beating에 의한 lysis로 진행되기 때문에 다른 추가적인 시약이 필요하지 않아 매우 간단하다. 또 한 DNA의 증폭과 검출은 등온 증폭 방법인 recombinase polymerase amplification (RPA) 방법을 적용하여 증폭시간을 단축하고 복잡한 온도변환 장치 를 사용하지 않도록 하였다. bead-beating에 의해 추출된 DNA를 RPA에 직접 적 용할 수 있도록 lysis buffer를 최적화하였다. 각 단계가 진행되는 장치는 진동모 터가 부착된 간단한 자석 이동장치를 사용하였다. 이번 연구에서는 랩온어칩에서 DNA 추출까지 진행하고 DNA의 증폭과 검출은 microtube에서 따로 진행함으로 써 50분 이내에 Vibrio parahaemolyticus 를 최저 733 cfu까지 검출할 수 있었 다. 이후 연구에서는 포획시간과 lysis 시간의 최적화를 통해 더 짧은 시간에 검출 이 가능하도록 할 계획이며 또한 DNA 추출 단계와 증폭 및 검출 단계를 연결해 모든 과정이 하나의 칩 상에서 이뤄질 수 있도록 개선할 계획이다.
more초록/요약 도움말
The market trend of in vitro diagnostics is rapidly changing from treatment-oriented to prevention-oriented diagnosis which require rapid point-of-care (POC) molecular diagnostic systems. However, most of the current molecular diagnostic systems is not suitable for POC applications because the diagnostic process should be carried out with several different kits or instruments. Therefore, we developed a stationary liquid phase lab-on-a-chip (SLP LOC) system, in which the whole diagnostic process, including sample pretreatment, DNA extraction, amplification and detection, could be conducted. The process was simplified by using antibody-functionalized capture particles, bead- beating lysis method, and recombinase polymerase amplification (RPA) that is an isothermal amplification method. In addition, the lysis buffer was optimized for direct application of the extracted DNA to RPA amplification. The diagnostic process was carried in a simple device composed of a linear actuator and vibration motors. In this study, Vibrio parahaemolyticus was detected in the SLP LOC system except that the amplification was carried out in a separate microtube for real-time fluorescence measurement. The method enabled the bacteria with a low detection limit of 733 cfu. In future study, we will optime the time for capture and lysis and improve the system for the whole process can be carried out in the SLP LOC.
more목차 도움말
Ⅰ. 서론 ································································································································ 1
1. 체외진단(IVD, in vitro diagnostics) ····································································· 1
2. PCR 방법을 이용한 분자진단 ··················································································· 2
3. 등온증폭방법(isothermal amplification) ······························································ 3
4. Lab-on-a-chip (LOC) ······························································································ 6
5. Vibrio parahaemolyticus ····················································································· 8
6. 박테리아 세포 용해 방법 ······················································································ 10
Ⅱ. 재료 및 방법 ················································································································ 12
1. SLP LOC 시스템의 원리 ························································································· 12
2. 포획입자 제작 ················································································································ 15
1) 포획입자 제작 방법 ······························································································ 15
2) 제작한 포획입자의 성능 확인 ············································································· 16
(1) 항체 결합 확인 ··································································································· 16
(2) V. parahaemolyticus 에 대한 specificity test ········································· 17
(3) 포획 효율 확인 및 포획 시간 최적화 ···························································· 17
3. Lab-on-a-chip 제작 ·································································································· 18
4. Recombinase Polymerase Amplification ································································· 19
1) RPA primer 제작 ································································································ 19
2) RPA primer specificity test ············································································ 19
3) RPA reagent setup ···························································································· 20
5. Bead-beating lysis ········································································································ 21
1) 삼투압을 이용한 lysis 효율 확인 ······································································· 21
2) lysis buffer MgOAc 농도 최적화 ····································································· 21
3) Tween-20가 포함된 lysis buffer의 bead-beating lysis 작용확인 ·········· 22
6. DNA standard curve ······························································································· 23
7. LOC을 사용한 V. parahaemolyticus 검출 ·························································· 24
1) specificity test ····································································································· 24
2) V. parahaemolyticus 의 농도에 따른 검출 ···················································· 24
Ⅲ. 결과 및 고찰 ················································································································ 26
1. 제작한 포획입자 성능 확인 ····················································································· 26
1) 항체 결합 확인 ······································································································ 26
2) V. parahaemolyticus 에 대한 specificity test ············································· 27
3) 포획 효율 확인 및 시간 최적화 ········································································· 28
2. Recombinase Polymerase Amplification ································································· 29
1) RPA primer 작용 확인 ······················································································· 29
2) RPA primer specificity test ············································································ 30
3. Bead-beating lysis ······································································································ 31
1) 삼투압을 이용한 lysis 효율 확인 ······································································· 31
2) lysis buffer MgOAc 농도 최적화 ···································································· 32
3) Tween-20가 포함된 lysis buffer의 bead-beating lysis 작용확인 ········· 34
4. LOC를 사용한 V. prahaemolyticus 검출 ························································· 35
1) specificity test ····································································································· 35
2) V. parahaemolyticus 의 농도에 따른 검출 ···················································· 36
Ⅳ. 결론 ·································································································································· 37
