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The polymerase chain reaction is an important technique in biological research. However, it is time consuming and has a number of disadvantages. Therefore, real-time PCR technology that can be used in real-time monitoring has emerged, and many studies are being conducted regarding its use. Real-time PCR requires many optical components and imaging devices such as expensive, high-performance cameras. Therefore, its cost and assembly process are limitations to its use. Currently, due to the development of smart camera devices, small, inexpensive cameras and various lenses are being developed. In this paper, we present a Compact Camera Fluorescence Detector for use in parallel-light lens-based real-time PCR devices. The proposed system has a simple optical structure, the system cost can be reduced, and the size can be miniaturized. This system only incorporates Fresnel lenses without additional optics in order for the same field of view to be achieved for 25 tubes. In the center of the Fresnel lens, one LED and a complementary metal-oxide semiconductor camera were placed in directions that were as similar as possible. In addition, to achieve the accurate analysis of the results, image processing was used to correct them. As a result of an experiment using a reference fluorescent substance and double-distilled water, it was confirmed that stable fluorescence detection was possible.

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RESUMEN

The polymerase chain reaction is an important technique in biological research because it tests for diseases with a small amount of DNA. However, this process is time consuming and can lead to sample contamination. Recently, real-time PCR techniques have emerged which make it possible to monitor the amplification process for each cycle in real time. Existing camera-based systems that measure fluorescence after DNA amplification simultaneously process fluorescence excitation and emission for dozens of tubes. Therefore, there is a limit to the size, cost, and assembly of the optical element. In recent years, imaging devices for high-performance, open platforms have benefitted from significant innovations. In this paper, we propose a fluorescence detector for real-time PCR devices using an open platform camera. This system can reduce the cost, and can be miniaturized. To simplify the optical system, four low-cost, compact cameras were used. In addition, the field of view of the entire tube was minimized by dividing it into quadrants. An effective image processing method was used to compensate for the reduction in the signal-to-noise ratio. Using a reference fluorescence material, it was confirmed that the proposed system enables stable fluorescence detection according to the amount of DNA.

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BACKGROUND: In general, the image analysis of nucleic acid for detecting DNA is dependent on the gel documentation system. These experiments may deal with harmful staining agents and are time consuming. To address these issues, real-time polymerase chain reaction (PCR) devices have been developed. The advantages of real-time PCR are its capabilities for real-time diagnosis, improved sensitivity, and digitization of measurement results. However, real-time PCR equipment is still too bulky and expensive for use in small hospitals and laboratories. METHODS: This paper describes an evaluation-independent real-time PCR system that differs from conventional systems in that it uses a side-illumination optical detection system and a temperature adjustment coefficient for DNA detection. The overall configuration of the evaluation-independent system includes the PCR chip and system hardware and software. The use of the side-illumination method for detection enables the system size to be reduced compared to systems using a typical illumination method. Furthermore, the results of a PCR test are strongly affected by the reaction temperature. Thus, extremely precise control of the temperature of the reaction is needed to obtain accurate results and good reliability. We derived a temperature compensation coefficient that allows us to compensate for the differences between the measured temperature of the negative temperature coefficient (NTC) thermistor sensor and the real temperature of the thermocouple. RESULTS: Applying the temperature compensation coefficient parameter using the NTC thermistor and using the side-illumination method resulted in an increase in the initial sensor value. The occurrence of the DNA section amplification decreased to 22 cycles from 24 cycles. CONCLUSIONS: The proposed system showed comparable performance to that of an existing real-time PCR, even with the use of simpler and smaller optical devices.

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Many image acquisition methods of nucleic acids still depend on UV illumination, especially after the electrophoresis when determining the size of the target DNA. Therefore the quality of the UV illuminator in the gel documentation system, and the comparison of the fluorescence detected are crucial. This paper presents a fluorescence standard reference plate using quantum dots compared to the conventional method where an agarose gel containing ethidium bromide is loaded with standard samples. The fluorescence standard reference plate consists of chambers filled with commercially available quantum dots such as phosphor dots. The chamber is made by thermally attaching nylon and polyester, the former on the inside and the latter on the outside, for increased stability. The images of the proposed reference plate were captured more than 2 months in regular intervals. The intensity analysis of the images shows that the proposed reference plate delivers stable fluorescence over a long term period. The proposed reference plate can be utilized to compare the performance of various UV illuminators, or to set a standard fluorescence point for certain analyses.

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RESUMEN

Many image acquisition methods of nucleic acids still depend on UV illumination, especially after the electrophoresis when determining the size of the target DNA. Therefore the quality of the UV illuminator in the gel documentation system, and the comparison of the fluorescence detected are crucial. This paper presents a fluorescence standard reference plate using quantum dots compared to the conventional method where an agarose gel containing ethidium bromide is loaded with standard samples. The fluorescence standard reference plate consists of chambers filled with commercially available quantum dots such as phosphor dots. The chamber is made by thermally attaching nylon and polyester, the former on the inside and the latter on the outside, for increased stability. The images of the proposed reference plate were captured more than 2 months in regular intervals. The intensity analysis of the images shows that the proposed reference plate delivers stable fluorescence over a long term period. The proposed reference plate can be utilized to compare the performance of various UV illuminators, or to set a standard fluorescence point for certain analyses.