검색 상세

Non-spectroscopic multiplex molecular diagnostic platform based on loop-mediated isothermal amplification (LAMP) with specially designed primers

  • 진양원 (Jin Yangwon, 아주대학교 일반대학원)

초록/요약

Herein, we developed a novel multiplex molecular diagnostic platform based on the integration of loop-mediated isothermal amplif ication (LAMP) and retroreflective signaling. LAMP is a striking method that allows for rapid and specific gene amplif ication at a constant temperature, simplifying the need for equipment, such as a thermocycler. However, it sti l l faces challenges when it comes to post-amplif ication analysis. This is due to the intricate optical setup it demands, involving a complex arrangement of optical components. To minimize the se requirements, we util ized retroreflective signaling which can induce signals from non-spectral l ight source. This approach with LAMP eliminates the need for sophisticated equipment and makes the molecular diagnostics accessible. In this study, we introduced specially designed primer sets to generate double - labeled amplicons. These possess small molecule antigen and biotin at each end, facil itating their detection in the same manner as the sandwich-type assay. Multiplex sensing with avidin-conjugated RJPs (A-RJPs) could be available in this system by applying different pairs of small molecule antigens and capture molecule while maintaining biotin. We introduced FITC and Texas Red as small molecule antigens for the invA of Salmonella and tetracycline resistance gene ( tet), respectively. In addition, for the validation of the chip uti l ized in the assay, a control channel was integrated into the multi-sensing chip. Therefore, it allows the simultaneous detection of multiple targets and validation of the results using only o ne type of optical probe. Based on the developed platform, a highly sensitive and selective quantif ication of antibiotic-resistant Salmonella was successfully performed with a detection l imit of 1 colony-forming units (CFU).

more

목차

1. Introduction 1
1.1 LAMP as an important molecular diagnostic tool 1
1.2 Retroref lect ive Janus particle as a non-spectroscopic optical sensing probe 2
1.3 Multiplex molecular diagnostic platform by integrating LAMP and retroreflective signaling 4
1.4 Aim of thesis 9
2. Materials and methods 11
2.1 Reagents and apparatus 11
2.2 Development of a new lamp method for forming double labeled amplicon 12
2.2.1 Introducing a new developed LAMP method 12
2.2.2 Primer design 16
2.2.3 Preparation of target genes for LAMP assay 18
2.2.4 LAMP assay 18
2.3 Fabrication of the sensing surface and optical probes 20
2.3.1 Modif icat ion of target-specif ic capture molecule on sensing substrate 20
2.3.2 Modification of Avidin on RJPs 23
2.4 Analysis of antibiotic-resistant Salmonella using the developed multiplex system 25
2.4.1 Quantitative analysis of antibiotic-resistant Salmonella 25
2.4.2 Quantitative analysis of specific amplicon from double-labeled amplicon mixture 27
3. Results and discussion 29
3.1 Optimization of the developed LAMP method 29
3.2 Confirmation of surface modification for target specific analysis 32
3.2.1 Verification of sensing surface 32
3.2.2 Verification of optical signaling probe 35
3.3 Optimization of double-labeled amplicon concentration and RJP reaction time for maximize the sensitivity 37
3.4 Quantitative analysis of antibiotic-resistant Salmonella 41
3.4.1 Calibration study of the developed platform for antibiotic-resistant Salmonella quantif ication 41
3.4.2 Calibration study of the developed platform using mixture of double labeled amplicon 46
4. Conclusions 49
5. References 51

more
반출 Meta View 목록