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Retroreflection-based sandwich-type affinity sensing of isothermal amplification product for pathogen detection
- Author(s)
- 이단비
- Alternative Author(s)
- Danbi Lee
- Advisor
- 윤현철
- Department
- 일반대학원 분자과학기술학과
- Publisher
- The Graduate School, Ajou University
- Publication Year
- 2022-02
- Language
- eng
- Keyword
- Loop-mediated isothermal amplification; Molecular diagnostic; Pathogen detection; Retroreflective Janus particle; Salmonella
- Alternative Abstract
- 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.
- Fulltext
- Appears in Collections:
- Graduate School of Ajou University > Department of Molecular Science and Technology > 3. Theses(Master)
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