Enhancing the Fluorescence and Cycle Threshold of qPCR Devices Through Excitation Time Point Adjustment

Abstract

Quantitative polymerase chain reaction (qPCR) has been widely employed for the positive or negative detection of bacteria or viruses, particularly SARS-CoV-2. Fluorescence signal and cycle threshold information is critical for the positive and negative detection of target test samples in qPCR systems. To determine viral concentration, the fluorescence intensity of each cycle must be recorded using a qPCR system. In general, the time points of fluorescence excitation and excitation light intensity affect fluorescence intensity. Thus, this study proposed an effective excitation method for enhancing fluorescence intensity. Several parameters, including excitation light intensity, the excitation time point, and the reaction time of the reagent at each temperature stage, were modified in assessing fluorescence performance and determining suitable parameters for fluorescence excitation in a qPCR system. Fluorescence intensity resulted in the most optimal fluorescence performance; specifically, excitation was triggered by using a 30 mA current, and the excitation light was activated when the temperature decreased to 60 °C. Total reaction time was 1 s, and the concentrated fluorescence value and suitable cycle threshold value were obtained. Overall, high efficiency, low fluorescence decay, and high light stability were observed. The present findings demonstrate that controlling the time point of excitation light can enhance the fluorescence efficiency and performance of qPCR systems, with relevant benefits in medical diagnostics and rapid viral detection, among other applications.