Retroreflection-based sandwich-type affinity sensing of isothermal amplification product for pathogen detection
- 주제(키워드) Loop-mediated isothermal amplification , Retroreflective Janus particle , Molecular diagnostic , Salmonella , Pathogen detection
- 주제(DDC) 547
- 발행기관 아주대학교
- 지도교수 윤현철
- 발행년도 2022
- 학위수여년월 2022. 2
- 학위명 석사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000031582
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Loop-mediated isothermal amplification (LAMP) is an outstanding method for molecular diagnostics due to the rapid, specific, and sensitive amplification of target genes. However, it is necessary to measure fluorescence in the quantitative analysis of LAMP products, so a sophisticated optical setup is required. This study developed a novel sensing method that can quantify target analytes with simple equipment, such as nonspectroscopic white light and a complementary metal-oxide-semiconductor (CMOS) camera. To achieve this, a retroreflective Janus particle (RJP) as a probe and specially designed loop primers, namely, fluorescein isothiocyanate (FITC)- and biotin-modified loop primers, were introduced into the LAMP system. Double-stranded amplicons possessing FITC and biotin labels at each end were generated in proportion to the quantity of the target pathogen by performing LAMP with specially designed loop primers. Using the anti-FITC antibody-modified sensing surface and streptavidin-conjugated RJP probes, amplicons could be captured in sandwich-configuration and detected under nonspectroscopic conditions composed of white light and a camera. To confirm the feasibility of the sensing system, the invA gene of Salmonella was selected as the target. It was possible to quantitatively analyze the Salmonella concentration from 0 to 106 colony-forming units (CFU), sufficiently covering the required detection range. In addition, quantitative analyses of pathogens in contaminated food sources, including milk and chicken meat, were successfully conducted with a limit of detection of 10 CFU. Taken together, we believe that the developed system will be a promising cost-effective tool for detecting foodborne pathogens.
more목차
1. Introduction 1
1.1 Standard method for detection of Salmonella 1
1.2 Isothermal nucleic acid amplification test as an important diagnostic tool 2
1.3 A novel molecular diagnostic platform using retroreflection Janus particle as a nonspectroscopic signaling probe 3
1.4 Aim of thesis 9
2. Materials and methods 10
2.1 Reagents and apparatus 10
2.2 Amplification of target gene using LAMP method 11
2.2.1 Primer design 11
2.2.2 Preparation of target genes for LAMP assay 14
2.2.3 LAMP assay 14
2.2.4 Confirmation of the presence of targeted product 15
2.3 Fabrication of the sensing channel and optical probes 18
2.3.1 Immobilization of target-specific antibody on sensing substrate 18
2.3.2 Conjugation of streptavidin on RJP 20
2.4 Analysis of S. Typhimurium using the developed system 21
2.4.1 Feasibility test of the developed system 21
2.4.2 Quantitative analysis of S. Typhimurium 23
2.4.3 Practical application of the developed system to real samples 24
3. Results and discussion 26
3.1 Principle of the RJP-based molecular diagnostic system 26
3.2 Target amplification using LAMP method 30
3.2.1 Primer design for detecting S. Typhimurium 30
3.2.2 Verification of the existence of the generated target amplicon 33
3.2.3 Optimization of LAMP reaction time 36
3.3 Confirmation of surface functionalization for target-specific analysis 40
3.3.1 Surface functionalization on sensing substrate with anti-FITC antibody 40
3.3.2 Surface functionalization on RJP with streptavidin 44
3.4 Quantitative analysis of S. Typhimurium 47
3.4.1 Verification of quantifiable potential of target amplicons 47
3.4.2 Calibration study of the RJP-based sensing system for Salmonella quantification 51
3.4.3 Application of the developed sensing system to real samples 55
4. Conclusions 60
5. References 62
