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Extraction method for fluorescence information from droplet digital PCR images

Guangxue Jingmi Gongcheng/Optics and Precision Engineering 2022年第7期30卷 821-829页

作者： Li, Shanshan Wang, Zichao Yu, Chengzhuang Wei, Chunyang Li, Junwei School of Mechanical Engineering Hebei University of Technology Tianjin300401 China Hebei Key Laboratory of Robotic Sensing and Human-Robot Interactions Tianjin300130 China State Key Laboratory of Reliability and Intelligence of Electrical Equipment Tianjin300132 China Institute of Biophysics Hebei University of Technology Tianjin300401 China

Droplet digital polymerase chain reaction (ddPCR) is one of the main methods used in nucleic acid detection. Counting the droplets in ddPCR experiments can be difficult if the number of droplets is too high or low, the fluorescence intensity is uneven, or the droplets are too closely arranged. We propose an image analysis method in which the image information is collected via the gray traversal method and droplets are classified and counted by differential analysis. Noise is removed from the image using the convolution algorithm, and the gray contrast of the image is enhanced by gray distribution equalization. The image is then converted to binary form by the gray traversal method with the geometric conditions taken as screening conditions, and the number of droplets is counted by differential analysis. Experiments with human gDNA (genomic DNA) as a detection sample, with approximately 20 000 droplets, showed an average detection accuracy of 99.36 % as compared to those conducted using commercial instrument algorithms or similar, with an average improvement of 2.24 % and 2.53 %, respectively, in the detection accuracy. The developed method can therefore be considered to provide reliable results and is suitable for use in ddPCR experiments. © 2022, Science Press. All right reserved.

关键词： Drops

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Hybrid Nucleic Acid Detection Approach for Ion Concentration Polarization Enabled Polymerase Chain Reaction with Reduced Cycle Numbers

SSRN

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SSRN 2024年

作者： Yu, Chengzhuang Li, Junwei Meng, Jiyu Qiu, Xinchen Bu, Tianxi Dai, Shijie Li, Shanshan Hebei Key Laboratory of Robotic Sensing and Human-robot Interactions School of Mechanical Engineering Hebei University of Technology Tianjin300132 China Institute of Biophysics School of Science Hebei University of Technology Tianjin300401 China College of Mechanical Engineering Tianjin University of Science and Technology Tianjin300222 China Arizona College of Technology Hebei University of Technology Tianjin300132 China School of Energy and Environmental Engineering Hebei University of Technology Tianjin300132 China Engineering Research Center of the Ministry of Education for Intelligent Rehabilitation Equipment and Detection Technologies Hebei University of Technology Tianjin300132 China

Polymerase chain reaction (PCR) plays important roles in the detection and quantification of nucleic acid targets. To cut-off the detection time, and also to reduce any potential by-products during the thermal cycles, it is required to keep the cycle numbers as few as possible. Here, in this work, a hybrid PCR microfluidic device is proposed to perform a PCR test with only 25 thermal cycles, which are much lower than 35 or 40 cycles in standard operations. With a novel hybrid design, the target nucleic acid molecules were amplified by 25 thermal cycles to avoid any potential by-products. And then the amplified nucleic acid molecules were actively enriched by ion concentration polarization (ICP) effects to increase the local concentration significantly. Owe to the hybrid function of "amplification and active concentration", the PCR- ICP device enables the nucleic acid detection more credible and allows to take a faster PCR test with less thermal cyclings. As a demonstration, meat adulteration experiments were taken to show the sensitivity and lower limit of detection of the hybrid device, verifying the performance of the proposed method. © 2024, The Authors. All rights reserved.

关键词： Polymerase chain reaction

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A Droplet-Based Multivolume Microfluidic Device for Digital Polymerase Chain Reaction

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SSRN 2022年

作者： Wei, Chunyang Yu, Chengzhuang Li, Shanshan Meng, Jiyu Li, Tiejun Cheng, Jingmeng Li, Junwei Hebei Key Laboratory of Robotic Sensing and Human-robot Interactions School of Mechanical Engineering Hebei University of Technology Tianjin300132 China State Key Laboratory of Reliability and Intelligence of Electrical Equipment Hebei University of Technology Tianjin300130 China Institute of Biophysics School of Health Sciences and Biomedical Engineering Hebei University of Technology Tianjin300401 China Department of Computer Science and Electrical Engineering Hebei University of Technology Langfang065000 China

Digital PCR plays an essential role in detecting and quantifying nucleic acid targets, while there are still challenges, including reduction of cost, simplification of operations, and system miniaturization. This work proposes a droplet-based multivolume microfluidic device for easy-to-operate and integrated droplet digital PCR (ddPCR). With an elaborate " asymmetrical droplet-splitting" microstructure, we have successfully prepared a small droplet population ( n = 1.5k, Φ = 59.1 ± 2.2 μm) and a large droplet population ( n = 1.5k, Φ = 106.3 ± 3.9 μm) in the same chip for realizing multivolume design, by which the ddPCR chip achieves a dynamic range of over 10^4 for DNA molecule quantification. The chip integrates droplet generation, droplet array self-assembly, droplet dPCR, and fluorescence detection on a single device. The quantitative capability of the ddPCR chip was evaluated by measuring template DNA at concentrations from 20 to 12500 copies/μL. Benefiting from the characteristics of wide dynamic range, easy operation, and miniaturization, our ddPCR device is expected to be used in the applications involving DNA molecule quantification, including diagnosis of genetic disease, single-cell gene expression, and point of care field. © 2022, The Authors. All rights reserved.

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