WO2020067742A1 - Dispositif de détection de fluorescence d'amplification en chaîne par polymérase, en temps réel - Google Patents

Dispositif de détection de fluorescence d'amplification en chaîne par polymérase, en temps réel Download PDF

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Publication number
WO2020067742A1
WO2020067742A1 PCT/KR2019/012544 KR2019012544W WO2020067742A1 WO 2020067742 A1 WO2020067742 A1 WO 2020067742A1 KR 2019012544 W KR2019012544 W KR 2019012544W WO 2020067742 A1 WO2020067742 A1 WO 2020067742A1
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Prior art keywords
fluorescence detection
filter
real
polymerase chain
chain reaction
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PCT/KR2019/012544
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English (en)
Korean (ko)
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김종대
황지수
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주식회사 바이오메듀스
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Publication of WO2020067742A1 publication Critical patent/WO2020067742A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N2021/6463—Optics
    • G01N2021/6467—Axial flow and illumination
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N2021/6463—Optics
    • G01N2021/6471—Special filters, filter wheel

Definitions

  • the present disclosure relates to a real-time polymerase chain reaction fluorescence detection device, and more particularly, to a real-time polymerase chain reaction fluorescence detection device capable of miniaturizing the fluorescence detection device and reducing manufacturing cost.
  • the PCR method consists of three steps. After undergoing a heat denaturation process that separates two strands of DNA using heat, the temperature is lowered so that the primer is annealed to the end of the sequence to be amplified, and heat is slightly raised again to synthesize DNA.
  • the polymerization reaction (polymerization or extension) proceeds.
  • the heat denaturation process is a process in which hydrogen bonds of complementary bases of two strands of DNA are dropped to one strand by using heat at 95 ° C, and the binding reaction is complementary to a strand of DNA at about 55 to 65 ° C. It is the process of binding to the base sequence.
  • the polymerization reaction is followed by attaching the starter to one strand of DNA (template DNA) and then synthesizing the complementary base of the template DNA using DNA polymerase to extend the two strands of DNA.
  • the illumination unit may be arranged such that excitation light is irradiated on the PCR chip.
  • the optical lens and image sensor are camera modules for a smartphone, and the image sensor may be a complementary metal-oxide-semiconductor (CMOS) image sensor or a charge-coupled device (CCD) image sensor.
  • CMOS complementary metal-oxide-semiconductor
  • CCD charge-coupled device
  • the PCR chip includes: a housing, a black matt PCB substrate disposed over the housing, and a temperature sensor disposed under the black matt PCB substrate, a double-sided tape including a reaction chamber, and a temperature sensor disposed under the black matt PCB substrate, and It includes a cover film disposed on the double-sided tape, the reagent for fluorescence detection can be accommodated in the reaction chamber.
  • FIG. 3 is a view showing a fluorescence detection unit according to an embodiment of the present invention.
  • FIG. 5 is a block diagram showing a fluorescence detector according to an embodiment of the present invention.
  • FIG. 6 is a diagram of a 4-channel laten filter according to an embodiment of the present disclosure.
  • FIG. 7 is a view showing a real-time polymerase chain reaction fluorescence detection device according to an embodiment of the present invention.
  • FIG. 8 is a view showing the configuration of a PCR chip according to an embodiment of the present invention.
  • FIG. 9 is a view showing a cross-sectional view of the PCR chip shown in FIG. 8.
  • FIG. 10 is a view showing a real picture of a PCR chip according to an embodiment of the present invention.
  • FIG. 11 is a photograph of the real-time polymerase chain reaction fluorescence detection device of FIG. 7.
  • FIG. 12 is a view showing the results of fluorescence detection in real-time PCR using the real-time polymerase chain reaction fluorescence detection device of FIG.
  • FIG. 13 is a view showing the results of the same fluorescence detection using Roche's LightCycler 480 to confirm the fluorescence amplification performance of the real-time polymerase chain reaction fluorescence detection device (FIG. 11) of the present invention.
  • the real-time polymerase chain reaction fluorescence detection device 100 is within the illumination unit 110 configured to irradiate the PCR chip 140 with light, an excitation filter 120 attached to one end of the illumination unit, and the PCR chip 140 It may be configured to include a fluorescent detection unit 130 configured to detect a fluorescent material.
  • the lighting unit 110 may include various lights such as LEDs, lasers, and fibers connected to the LEDs, and may include at least one light source. In addition, for fluorescence detection, the illumination unit 110 may be arranged such that light is side illuminated.
  • the excitation filter 120 attached to one end of the lighting unit 110 is filtered to pass only a specific wavelength region (excitation wavelength region) of light emitted from a light source installed inside the lighting unit 110 so that the excitation light is transmitted to the PCR chip 140. It can be configured to investigate.
  • the fluorescent material in the PCR chip 140 may absorb excitation light provided from the illumination unit 110 and emit light (emission light) in a longer wavelength region (emission wavelength region).
  • the fluorescence detector 130 may be configured to detect emitted light emitted by the fluorescent material in the PCR chip 140. To this end, the fluorescence detector 130 may include an image sensor 132, an emission filter 134, and an optical lens 136.
  • the emission filter 134 may be configured to block excitation light and pass only the emission light emitted by the fluorescent material.
  • the excitation filter 120 may be a Ratten filter, and may be composed of a single channel or multiple channels.
  • the excitation filter 120 may be configured as a multi-channel laten filter, in which case, the illumination unit 110 is provided with the multi-channel excitation filter 120 ) May include a plurality of light sources.
  • the excitation filter 120 may be composed of a 2-channel or 4-channel laten filter.
  • the excitation filter 120 may be an interference filter, but is not limited thereto, and may be configured using various filters.
  • the emission filter 134 may be disposed on one side of the optical lens 136. That is, the optical lens 136 may be disposed between the emission filter 134 and the image sensor 132.
  • the emission filter 134 may be composed of a single channel or a single channel. When configured as a single channel, an interference filter or a laten filter can be used, and when configured as a multi-channel, a laten filter can be used.
  • an emission filter 134 may be disposed between the optical lens 136 and the image sensor 132.
  • the emission filter 134 may be composed of a single channel or multiple channels.
  • an interference filter or a laten filter can be used, and when configured as a multi-channel, a laten filter can be used.
  • the emitted light passing through the emission filter 134 may be transmitted to the image sensor 132.
  • the image sensor 132 may be configured to convert light into an electrical signal, and may be, for example, a Complementary Metal-Oxide-Semiconductor (CMOS) image sensor, a charge-coupled device (CCD) image sensor, and the like. No, any image sensor can be used. Since the emission filter 134 passes only the emitted light, the image sensor 132 may convert the emitted light into an electrical signal to detect a fluorescent material.
  • CMOS Complementary Metal-Oxide-Semiconductor
  • CCD charge-coupled device
  • the real-time polymerase chain reaction fluorescence detection device 200 is in a state where the control unit 210 is connected to the fluorescence detection unit 130 and the PCR chip 140.
  • the control unit 210 may be a terminal equipped with a computational processing device such as a laptop or desktop.
  • the temperature sensor 220 may measure the temperature of the PCR chip 140 and provide it to the control unit 210.
  • the control unit 210 may control the temperature of the PCR chip 140 by controlling the heater 230 and the fan 240 of the PCR chip 140 using PWM (Pulse Width Modulation) and FET (Field Effect Transistor). . Specifically, the controller 210 may calculate the PWM based on the Proportional-Integral-Derivative (PID) control mechanism based on the temperature value provided from the temperature sensor 220.
  • PWM Proportional-Integral-Derivative
  • the control unit 210 may provide not only temperature control, but also a GUI environment related to PCR protocol execution.
  • the lighting unit 110 may irradiate the excitation light diagonally toward the PCR chip 140, and it is also possible to illuminate using several lighting components in front of the PCR chip 140.
  • the image sensor 132 of the fluorescent detector 130 may detect the emitted light that has passed through the optical lens 136 and the emission filter 134, amplify the optical signal, and output the amplified signal to the control unit 210.
  • the control unit 210 may receive and store an optical signal transmitted by the image sensor 132.
  • the emission filter 320 is shown in FIG. 3 as a two-channel filter, the present invention is not limited thereto, and may be composed of two or more multi-channel filters.
  • the fluorescence detection unit 300 is illustrated in FIG. 3 as being capable of separating the image sensor 310 and the lens mount 330, an integrated camera module may be used.
  • the emission filter 320 may be fixed in front of the optical lens 340. In particular, when a short channel laten filter is used, such a configuration can be used.
  • a fluorescence detection unit can be formed in a small volume without using a filter wheel even when detecting fluorescence of several channels.
  • a camera module for a smartphone may be used to configure the fluorescence detector, and the image sensor 310 may be a CMOS image sensor, a CCD image sensor, or the like.
  • the emission filter 320 can be disposed between the image sensor 310 and the optical lens 340.
  • the emission filter 320 may be configured to block excitation light from light passing through the optical lens 340 and transmit only the emission light.
  • the emitted light passing through the emission filter 320 is converted into an electrical signal by the image sensor 310, so that a fluorescent material can be detected.
  • the fluorescence detector 500 includes an image sensor 510, a first laten filter 520, a second laten filter 530, a lens mount 540 and an optical lens 550.
  • the optical lens 550 is a lens for condensing emitted light, and the lens mount 540 may serve to fix the optical lens 550.
  • the first laten filter 520 and the second laten filter 530 may be configured to transmit light in wavelengths of different regions.
  • the first laten filter 520 and the second laten filter 530 may be disposed between the image sensor 510 and the optical lens 550. In another embodiment, the first laten filter 520 and the second laten filter 530 may be disposed at one end of the optical lens 550.
  • the image sensor 510 may acquire a fluorescence image corresponding to the pass band of each laten filter. Although two laten filters are shown in FIG. 5, the present invention is not limited thereto, and various filters such as an interference filter may be used.
  • the four channel laten filter 600 may include first to fourth laten filters 610, 620, 630, and 640 having different pass bands.
  • the light transmitted through the 4-channel laten filter 600 is converted into an electrical signal by an image sensor, and fluorescence is detected.
  • the first to fourth laten filters 610, 620, 630, and 640 may use a laten filter having a pass band corresponding to an image for a plurality of wavelength bands to be acquired.
  • a laten filter having a pass band corresponding to an image for a plurality of wavelength bands to be acquired For example, when it is desired to detect emitted light in the wavelength bands of a, b, c and d, four types of laten filters having pass bands of a, b, c and d can be used. According to such a configuration, it is possible to perform fluorescence detection of several channels without using a bulky filter wheel. In addition, since a thin laten filter with a thickness of about 100 ⁇ m is used, it is also possible to configure a filter with 4 or more channels. Since the filter wheel is not used, the configuration of the fluorescence detector is simplified and it is possible to downsize the entire system. Although four laten filters are shown in FIG. 6, the present invention is not limited thereto, and various filters such as an interference filter
  • Real-time polymerase chain reaction fluorescence detection device 700 includes an illumination unit 710, excitation filter 720, chip connector 730, PCR chip 732, camera module holder 740, camera module 750 and emission filter 760.
  • the camera module 750 may be a smartphone camera module, and the camera module holder 740 serves to fix the camera module 750.
  • the lighting unit 710 may include LED, optical fiber lighting, laser lighting, and the like.
  • An excitation filter 720 is attached to one end of the illumination unit 710 so that the excitation light is irradiated to the PCR chip 732 at an angle of 35 degrees.
  • a chip connector 730 may be connected to drive the PCR chip 732, and a fan (not shown) may be disposed near the PCR chip 732 to control the temperature of the PCR chip 732.
  • the excitation light is shown to be irradiated to the PCR chip 732 at an angle of 35 degrees, but is not limited thereto, and the illumination unit 710 is disposed so that the excitation light is side-illuminated to the PCR chip 732.
  • the emission filter 760 may be fixed to the front end of the camera module 750, and the camera module 750 may be disposed at a distance of 43 mm from the chip connector 730.
  • the emission filter 760 may be a multi-channel or short-channel laten filter, or may be an edge filter.
  • the light emitted from the illumination unit 710 passes through the excitation filter 720 and the excitation light is irradiated to the PCR chip 732.
  • the fluorescent material in the PCR chip 732 absorbs excitation light and emits emitted light.
  • the emission filter 760 transmits the emitted light emitted by the fluorescent material, and the camera module 750 converts the emitted light into an electrical signal to perform fluorescence detection.
  • a blue LED of 9600mcd is used as the illumination unit 710, an interference filter is used as the excitation filter 720, and an interference filter is used as the emission filter 760,
  • the entire system can be placed in the dark and fluorescence detection can be performed in real time.
  • real-time PCR can be performed according to the PCR protocol. Specifically, PCR was performed according to the procedures of Pre-incubation, Pre-Heating, Denaturation, and Annealing, respectively, for 2 minutes at 50 ° C, 10 minutes at 95 ° C, 15 seconds at 95 ° C, and 1 minute at 60 ° C for a total of 40 Cycle can be performed, and fluorescence detection can be performed in the section of 60 °C.
  • the reagent used for PCR is DNA (Chlamydia Trachomatis) 1ng / 5.4 ⁇ l, Master mix 18 ⁇ l, Primer mix (primer F, primer R, Probe) 10pM / 9 ⁇ l, distilled water 3.6 ⁇ l (total 36 ⁇ l) and experiment You can proceed.
  • 1 ng / ⁇ L concentration of CT (Chlamydia Trachomatis) DNA, Roche's Master mix, Primer mix, distilled water may be used as a reagent used in the fluorescence detection process.
  • the PCR process can be performed for 50 minutes at 50 ° C for a pre-incubation process for 2 minutes, a 95 ° C denaturation process for 30 seconds, and a 58 ° C annealing process for 50 seconds.
  • the PCR chip 800 is a heater pattern for transferring heat in the cover film 810, the first double-sided tape 820, the box tape 830, and the PCR chip 800 to accommodate a small amount of sample and reaction reagent.
  • the engraved printed circuit board (PCB) 840, the second double-sided tape 850, and the housing 860 may be included.
  • the cover film 810 is 200 ⁇ m
  • the first double-sided tape 820 is 400 ⁇ m
  • the box tape 830 is 50 ⁇ m
  • the PCB 840 is 200 ⁇ m
  • the second double-sided tape 850 is 200 ⁇ m. It may be configured to a thickness of ⁇ m.
  • the heater pattern for heating to the black matt PCB may be printed on the PCB 840 in a white silk legend, or may be coated with silver, gold, tin, or the like.
  • a box tape 830 may be attached to prevent DNA from being adsorbed on the PCB 840.
  • a channel serving as a micro-fluidic channel may be cut on the first double-sided tape 820 and used as a reaction chamber.
  • the cover film 810 it may be composed of a polycarbonate film.
  • the PCR chip 800 may include a sample inlet 910 having a passage through which samples and reagents can be introduced.
  • the sample inlet 910 may be formed in the housing 860.
  • Samples and reagents may be accommodated between the PCB 840 / box tape 830 and the cover film 810.
  • samples and reagents may be accommodated in a micro-fluidic channel or reaction chamber formed in the first double-sided tape 820.
  • a temperature sensor 920 for detecting the temperature of the sample and reagent may be disposed under the PCB 840.
  • the temperature of the heater pattern formed on the PCB 840 may be controlled based on the temperature measured by the temperature sensor 920.
  • the temperature sensor 920 is illustrated as being disposed under the PCB 840, but is not limited thereto, and may be disposed at any position capable of detecting the temperature of the sample and reagent.
  • the PCR chip 1000 may be connected to a chip connector, and the control unit may control heating through the heater pattern of the PCR chip 1000 and cooling through the pen based on the temperature measured by the temperature sensor in the PCR chip 1000. have. By controlling the heating and cooling of the PCR chip 1000, it is possible to proceed with the process of DNA denaturation, primer binding, and DNA synthesis in the PCR chip 1000.
  • FIG. 11 is a photograph of the real-time polymerase chain reaction fluorescence detection device 700 of FIG. 7.
  • a smartphone camera module is used, and a fluorescence detection unit is constructed by fixing an emission filter in front of the lens of the smartphone camera module. Since the fluorescence detection device using the smart phone camera module can only detect the intensity of brightness when detecting fluorescence with a photodiode when compared with a photodiode, singularities or problems that occur during the actual experimental process that cannot be measured with a photodiode Can be monitored for quick troubleshooting.
  • the camera module used in the smart phone is inexpensive and the use of the module is universal, the parts can be easily obtained and there is no difficulty in manufacturing.
  • the smartphone camera module is a compact and high-definition module, it is advantageous in terms of price and performance. Therefore, it is possible to downsize the entire system. In addition, it is possible to perform quantitative analysis by distribution of fluorescence rather than analyzing the average value of brightness.
  • FIG. 12 is a view showing the results of fluorescence detection in real-time PCR using the real-time polymerase chain reaction fluorescence detection device of FIG.
  • 1 ng / ⁇ L concentration of CT (Chlamydia Trachomatis) DNA was used.
  • FIG. 13 is a view showing the results of the same fluorescence detection using Roche's LightCycler 480 to confirm the fluorescence amplification performance of the real-time polymerase chain reaction fluorescence detection device (FIG. 11) of the present invention.
  • CT Ribonuclear Tumor

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  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Physics & Mathematics (AREA)
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Abstract

L'invention concerne un dispositif de détection de fluorescence d'amplification en chaîne par polymérase, en temps réel. Le dispositif selon l'invention comprend un illuminateur destiné à irradier de la lumière, au moins un filtre d'excitation fixé sur une extrémité de l'illuminateur pour être traversé par une lumière d'excitation, ainsi qu'un détecteur de fluorescence destiné à détecter une substance fluorescente dans une puce PCR. Le détecteur de fluorescence peut comprendre une lentille optique, au moins un filtre d'émission destiné à être traversé par la lumière émise par la substance fluorescente qui a absorbé la lumière d'excitation, ainsi qu'un capteur d'image destiné à convertir la lumière en un signal électrique.
PCT/KR2019/012544 2018-09-28 2019-09-26 Dispositif de détection de fluorescence d'amplification en chaîne par polymérase, en temps réel WO2020067742A1 (fr)

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KR10-2018-0116500 2018-09-28
KR1020180116500A KR102261902B1 (ko) 2018-09-28 2018-09-28 실시간 중합효소 연쇄반응 형광 검출 장치

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CN113624726A (zh) * 2020-05-07 2021-11-09 纬创资通股份有限公司 检测装置、检测方法及荧光实时定量聚合酶连锁反应系统
CN114015550A (zh) * 2021-10-29 2022-02-08 广州国睿科学仪器有限公司 一种荧光定量pcr光学检测装置
CN114480111A (zh) * 2022-02-15 2022-05-13 深圳阿斯克医疗有限公司 一种实时荧光定量pcr仪
WO2023178710A1 (fr) * 2022-03-23 2023-09-28 无锡百泰克生物技术有限公司 Instrument portable de pcr quantitative fluorescente ultrarapide en temps réel

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CN113862144B (zh) * 2021-11-05 2023-11-17 中元汇吉生物技术股份有限公司 全自动荧光定量pcr分析仪

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WO2023178710A1 (fr) * 2022-03-23 2023-09-28 无锡百泰克生物技术有限公司 Instrument portable de pcr quantitative fluorescente ultrarapide en temps réel

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