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dc.contributor.advisor이혁진-
dc.contributor.author최보람-
dc.creator최보람-
dc.date.accessioned2018-03-06T16:30:20Z-
dc.date.available2018-03-06T16:30:20Z-
dc.date.issued2018-
dc.identifier.otherOAK-000000147677-
dc.identifier.urihttp://dcollection.ewha.ac.kr/common/orgView/000000147677en_US
dc.identifier.urihttps://dspace.ewha.ac.kr/handle/2015.oak/240256-
dc.description.abstractFood contamination with bacteria can cause various food poisoning and the number of patients is increasing every year worldwide. Conventional approaches for bacterial detection have utilized clonogenic assays, a method of counting bacterial colonies on a plate. This test requires a skilled healthcare personnel to perform a laboratory work and it takes 2-7 days to complete multiple procedures and analysis. Meanwhile, immunological tests and molecular detection techniques such as PCR have developed to improve the sensitivity and accuracy of detection. However, these methods are still difficult to be applied in clinics, since they require well equipped-laboratory for detection analysis. To overcome these problems, we have developed an advanced diagnostic platform based on isothermal rolling circle amplification (RCA) to visually identify the presence of bacteria pathogens. With the combination of DhITACT-magnetic beads (DhITACT-MBs) and capillary tube based testing, it is appropriate to use this platform as a versatile molecular diagnosis test for point of care (POC) applications in clinical field.;박테리아에 의한 음식물 오염은 다양한 식중독을 유발할 수 있으며 전세계 환자 수는 매년 증가하고 있습니다. 박테리아 검출을 위한 종래의 접근법은 플레이트 상에 세균성 콜로니를 계수하는 방법 인 클로로젠 분석법을 이용 하였다. 이 검사는 숙련 된 의료 인력이 연구실에서 수행해야 하며, 여러 절차와 분석을 완료하는 데 2-7 일이 소요됩니다. 한편, 면역 학적 검사 및 PCR과 같은 분자 검출 기술은 검출의 민감도와 정확도를 향상시키기 위해 개발되었습니다. 그러나 이러한 방법은 탐지 분석을 위해 잘 갖추어 진 검사실이 필요하기 때문에 클리닉에서 적용하기가 여전히 어렵습니다. 이러한 문제를 극복하고, 박테리아 병원균의 존재를 시각적으로 확인하기 위해 등온 롤링 서클 증폭 (RCA)을 기반으로 한 고급 진단 플랫폼을 개발했습니다. DhITACT-magnetic beads (Dhitact-MBs)와 모세 혈관 기반 테스트가 결합된 진단 플랫폼은 임상 분야의 POC (point of care) 적용하여 다기능 분자 진단 테스트로 사용하는 것이 적절합니다.-
dc.description.tableofcontents1. Introduction 1 2. Materials and Methods 4 2.1. Materials 4 2.2. Sequence of DNA probes, target RNA and immobilized primers used in capillary-based diagnostic platforms 4 2.3. Design of dumbbell DNA probe for detection of target gene 5 2.4. DNA ligation formed by accessing of target gene 5 2.5. Improvement of ligation efficiency by Splint R ligase 6 2.6. Hybridization of dumbbell DNA probe and 5-biotinylated primer 6 2.7. Immobilization of 5 biotinylated DNA hybrid on the surface of avidin-bead 6 2.8. Generation of amplification DNA products on the bead surface based on DhITACT system 7 2.9. Observation of bead behavior change through particle 7 2.10. Optical analysis using fluorescence microscopy 8 2.11. DhITACT-MBs -based capillary device 8 2.12. Total RNA extraction from bacterial cell using magnetic bead 8 2.13. Limit of detection (LOD) test using capillary-based device with all in one process 9 3. Results 10 3.1. PAGE gel analysis to determine the location of the DNA probe in the presence of the bacterial pathogen 10 3.2. The improvement of the ligation formation by using Splint R ligase as compared to T4 DNA ligase 11 3.3. Comparison of ligation efficiency according to the length of the bacterial pathogen 12 3.4. Detection sensitivity of bacterial genes at various concentrations 13 3.5. Confirmation of rapid and easy immobilization of primer-DNA probe on the bead surface 14 3.6. Detection of target bacterial pathogen using DhITACT-bead system 15 3.7. Fluorescent imaging-based assay 16 3.8. Photograph images of the bead aggregation assay 17 3.9. Observation of beads aggregation in capillary-based device 18 3.10. Volume difference of beads inside the capillary by amplified DNA product 19 3.11. Approach for a capillary based-diagnostic platform with all in on process 20 3.12. Capillary-based detection test with all in one process using RNA samples extracted from cultured bacterial cells 21 3.13. Limit of detection (LOD) test of capillary-based device using real sample 22 4. Discussion 23 5. Conclusion 25 6. Reference 26 7. List of research article published 28 Abstract in Korean 29-
dc.formatapplication/pdf-
dc.format.extent839631 bytes-
dc.languageeng-
dc.publisher이화여자대학교 대학원-
dc.subject.ddc600-
dc.titlePoint-of-care testing (POCT) application-
dc.typeMaster's Thesis-
dc.title.subtitleVisual detection of bacterial genes based on rolling circle amplification (RCA)-
dc.format.pagevii, 29 p.-
dc.identifier.thesisdegreeMaster-
dc.identifier.major대학원 제약산업학과-
dc.date.awarded2018.2-
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