Development of real-time fluorescence-based detection and calibration of UV-Vis spectrometer using in CRISPR-Cas12a detection system

Name: Chayanit Thairat

LCSH: Kasetsart University -- Theses. M.S. (Metrology) 2022

Classification :.LCCS: QC373.S7

LCSH: Kasetsart University. -- Department of Physics -- Theses

LCSH: Spectrometer

LCSH: Fluorescence

LCSH: Rice blast disease

LCSH: Calibration

LCSH: PID controllers

This study presents a significant advancement in the field of portable molecular diagnostics with the development of a real-time fluorescence CRISPRCas12a detection system. Integrating this technology with a microcontroller provides improved performance and reliability for diagnostic analyses. The system effectively controls temperature, signal processing, and data acquisition, thereby ensuring the accurate execution of the CRISPR-Cas12a reaction and subsequent detection. Through calibration processes and comparison with LED standards, the employed DS18B20 temperature sensor and C12666MA spectrometer showed commendable accuracy, essential for precise temperature monitoring of 37°C with a low deviation ±0.16°C. A high-accuracy HR4000 spectrometer was employed as a reference point for C12666MA comparison. The comparative study resulted in a root mean square error of 5.33 nm. Uncertainty analysis revealed values between 2.02 to 2.95 nm, with a coverage factor of 2, ensuring a 95% confidence level. The findings from this metrological investigation suggest that the C12666MA spectrometer provides accurate, efficient, and reliable measurements within its operative wavelength range, confirming its robustness in spectroscopic applications. The detection system accuracy tests on positive control AvrPi9, non-target NYK56003, and non-template samples, proved successful in eliminating false-positive outcomes, and accurately distinguishing the presence and absence of the target DNA sequence. Specifically, the detection system displayed sensitivity down to 3.8 ng for the AvrPi9 PCR product comparable to that achieved by a commercial real-time PCR system, which reported a limit of detection of 1 ng, affirming its potential utility in diagnostic. Despite its current limitation of handling a single sample per reaction, the CRISPR-Cas12a detection system demonstrated impressive specificity, sensitivity, and accuracy. Moreover, this technology offers an advantage over conventional PCR and LAMP techniques in terms of simplicity, time efficiency, and a significant reduction in the occurrence of false-positives. The development of this real-time fluorescence CRISPR-Cas12a detection system serves as a promising advancement in molecular diagnostics. Its potential applications extend beyond agricultural disease diagnosis to include detection and management of various plant and human diseases. This device embodies a move towards more accessible, reliable, and rapid diagnostics, facilitating a more widespread adoption of such technologies in the future.

Kasetsart University. Office of the University Library

Address: Bangkok

Email: tdckulib@ku.ac.th

Name: Sutharat Chotikaprakhan

Role: Thesis Advisor

Name: Chatuporn Kuleung

Role: Thesis CO-Advisor

Created: 2022

Modified: 2025-07-24

Issued: 2025-07-24

วิทยานิพนธ์/Thesis

application/pdf

URL: https://www.lib.ku.ac.th/KUthesis/2565/chayanit-tha-all.pdf

CallNumber: QC373.S7 .C43

eng

DegreeName: Master of Science

Level: Master's Degree

Descipline: Metrology

Grantor: Kasetsart University

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