WO2024072111A1 - Procédé d'analyse pcr en temps réel numérique - Google Patents

Procédé d'analyse pcr en temps réel numérique Download PDF

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Publication number
WO2024072111A1
WO2024072111A1 PCT/KR2023/015053 KR2023015053W WO2024072111A1 WO 2024072111 A1 WO2024072111 A1 WO 2024072111A1 KR 2023015053 W KR2023015053 W KR 2023015053W WO 2024072111 A1 WO2024072111 A1 WO 2024072111A1
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sample
time pcr
fractions
analysis method
digital real
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PCT/KR2023/015053
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English (en)
Korean (ko)
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송현우
김재헌
엄재원
변재오
이도영
최경학
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(주)옵토레인
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Priority claimed from KR1020230130511A external-priority patent/KR20240046076A/ko
Publication of WO2024072111A1 publication Critical patent/WO2024072111A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • 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
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • 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
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • 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
    • G01N21/84—Systems specially adapted for particular applications
    • G—PHYSICS
    • G06—COMPUTING; CALCULATING OR COUNTING
    • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
    • G06T7/00—Image analysis
    • G06T7/90—Determination of colour characteristics
    • G—PHYSICS
    • G06—COMPUTING; CALCULATING OR COUNTING
    • G06V—IMAGE OR VIDEO RECOGNITION OR UNDERSTANDING
    • G06V10/00—Arrangements for image or video recognition or understanding
    • G06V10/10—Image acquisition

Definitions

  • the present invention relates to a digital real-time PCR analysis method, which allows digital real-time PCR analysis of high or low concentration samples beyond existing measurement limits, that is, samples with a wide concentration range.
  • Gene amplification technology is an essential process in molecular diagnosis and is a technology that repeatedly copies and amplifies a specific base sequence of trace amounts of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in a sample.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • PCR polymerase chain reaction
  • digital PCR is a new PCR technology that detects and quantifies nucleic acids.
  • the goal is to generate thousands to millions of nanoliter to picoliter size fractions and determine the presence or absence of nucleic acids in each fraction.
  • the number of nucleic acids can be calculated. Therefore, there is no need to rely on a standard curve using standard substances for quantification, and it is an absolute quantification method that can be quantified with just one PCR. It has high measurement precision and enables analysis of complex mixtures and linear quantitative detection of the results.
  • ddPCR droplet digital PCR
  • a droplet generator to form droplets of a sample, a special device called a droplet generator must be used, and the formed droplets must be transferred to a 96-well PCR plate and then proceed with the PCR reaction.
  • the droplets are easily broken and some of the sample is lost.
  • 5 to 6 other equipment are required, and skilled personnel are required for analysis, and it has the disadvantage of being difficult to measure real-time curves.
  • the end-point method has a limited dynamic range that can be measured in one test, which is determined by the number of fractions and the volume of the fractions. For example, if there are 1 nanoliter per fraction and the number of fractions is 20,000, the dynamic range that can detect a target is from 1 to a minimum of 100,000 and a maximum of 300,000. If 20 microliters of a sample contains 1 million high-concentration targets, it cannot be quantified in a single test.
  • the present invention relates to a digital real-time PCR analysis method, and is intended to provide a digital real-time PCR analysis method capable of performing digital real-time PCR analysis on high or low concentration samples that exceed existing measurement limits, that is, samples with a wide concentration range.
  • the digital real-time PCR analysis method of the present invention includes a microfluidic chamber in which a liquid sample to be analyzed is stored; a well array attached to the bottom of the microfluidic chamber and receiving the sample to be analyzed from the microfluidic chamber; And using a digital real-time PCR cartridge including a CMOS photosensor array located on the bottom of the well array and capturing in real time a reaction image of the sample to be analyzed filled in a plurality of fractions provided in the well array, the present invention
  • the digital real-time PCR analysis method is,
  • It may include a final calculation step (S50) of calculating the target concentration in the sample to be analyzed based on the plurality of target numbers.
  • the digital real-time PCR analysis method of the present invention can provide a digital real-time PCR analysis method capable of performing digital real-time PCR analysis on high- or low-concentration samples that exceed existing measurement limits, that is, samples with a wide concentration range.
  • the digital real-time PCR analysis method of the present invention can implement digital PCR that provides real-time graphs for individual fractions, thereby eliminating false positive or false negative errors.
  • the digital real-time PCR analysis method of the present invention can achieve the same effect as repeatedly evaluating digital real-time PCR tens of thousands of times with one cartridge, and calculates the number of targets present in each unit compartment based on the values measured in each unit compartment. By doing so, the initial concentration in the sample can be calculated.
  • Figure 1 is a perspective view schematically explaining a cartridge for digital real-time PCR according to an embodiment.
  • Figure 2 is an exploded perspective view of the cartridge for digital real-time PCR shown in Figure 1.
  • FIG. 3 is a perspective view schematically illustrating an example of the well array shape shown in FIG. 2.
  • Figures 4a and 4b are diagrams showing digital real-time PCR results.
  • FIG. 5A is a front perspective view for schematically explaining the microfluidic chamber shown in FIG. 2
  • FIG. 5B is a rear perspective view for schematically explaining the microfluidic chamber shown in FIG. 2
  • FIG. 5C is a front perspective view for schematically explaining the microfluidic chamber shown in FIG. 2. This is an exploded perspective view of the microfluidic chamber.
  • Figure 6 is a cross-sectional view schematically explaining the PCR module shown in Figure 2.
  • Figures 7 to 21 are cross-sectional views showing the schematic use of a cartridge for digital real-time PCR.
  • Figure 22 is a block diagram showing the digital real-time PCR analysis method of the present invention.
  • the digital real-time PCR analysis method of the present invention includes a microfluidic chamber in which a liquid sample to be analyzed is stored; a well array attached to the bottom of the microfluidic chamber and receiving the sample to be analyzed from the microfluidic chamber; And a digital real-time PCR cartridge including a CMOS photosensor array located on the bottom of the well array and capturing in real time a reaction image of the sample to be analyzed filled in a plurality of fractions provided in the well array may be used.
  • It may include a final calculation step (S50) of calculating the target concentration in the sample to be analyzed based on the plurality of target numbers.
  • the microfluidic chamber may be made of polydimethylsiloxane (PDMS) material.
  • the well array may be completely brought into close contact with the CMOS photosensor array.
  • the raw data acquisition step (S20) of the digital real-time PCR analysis method of the present invention is performed after confirming that the sample to be analyzed is filled in all of the plurality of fractions, and in the raw data acquisition step (S20), the plurality of fractions
  • the image for may be acquired as the raw data.
  • the raw data may be collected at least once per cycle.
  • a fluorescence intensity function with fluorescence intensity as a dependent variable and time or cycle number as an independent variable is used for each of the plurality of fractions from the raw data.
  • the Ct value may be the time or cycle number value at which the second derivative of the fluorescence intensity function is maximum.
  • each of the plurality of target numbers for each of the plurality of fractions may be calculated using Equation 1 above.
  • N i is the number of targets in the ith fraction
  • Ct i is the Ct value of the ith fraction
  • Ct ref is determined by at least one of the number of amplicons required to measure Ct and the fluorescence efficiency of the probe,
  • a is a constant determined by PCR efficiency.
  • the Ct ref may be calculated using Equation 2 below.
  • Ct 0 may be a Ct value obtained from a standard sample.
  • the concentration of the standard sample in the digital real-time PCR analysis method of the present invention may be 10 7 copies/rxn to 10 8 copies/rxn.
  • the concentration Con of the sample to be analyzed may be calculated using Equation 3 below.
  • n is the total number of the plurality of fractions.
  • the negative or positive nature of the sample to be analyzed may be determined based on the Con value.
  • the terms “center”, “top”, “bottom”, “left”, “right”, “vertical”, “horizontal”, “inside”, “outside”, “one side” The orientation or positional relationship indicated by “, “other side”, etc. is based on the orientation or positional relationship shown in the drawings or the orientation or positional relationship normally placed when using the product of the present invention, and is for the purpose of explanation and brief explanation of the present invention. However, it does not suggest or imply that the displayed device or element must necessarily be constructed or operated in a specific orientation and should not be construed as limiting the present invention.
  • first, second, etc. may be used to describe various components, but the components should not be limited by the terms. The above terms are used only for the purpose of distinguishing one component from another. For example, a first component may be referred to as a second component without departing from the scope of the present invention, and similarly, the second component may also be referred to as a first component. Singular expressions include plural expressions unless the context clearly dictates otherwise.
  • the present invention is a structure for mounting samples, sample volumes or reaction volumes in a certain arrangement on a substrate, and more specifically, relates to mounting samples in a certain arrangement of respective reaction positions in the substrate.
  • devices, devices, systems, and methods are provided for loading samples into articles used to detect targets in large quantities of small volume samples.
  • targets may be any suitable biological target, but may also include DNA sequences (including cell-free DNA), RNA sequences, genes, oligonucleotides, molecules, proteins, biomarkers, and cells (e.g., circulating tumors). cells), or other suitable target biomolecules.
  • these biological components may be used in applications such as prenatal diagnosis, multiplexed PCR, virus detection, and quantitative normalization, genotyping, sequence verification, mutation detection, genetic biology, rare allele detection, and copy number change detection. It may also be used in connection with various PCR, qPCR, and/or dPCR methods and systems.
  • PCR quantitative polymerase chain reaction
  • Suitable PCR methods include, for example, digital PCR, allele-specific PCR, asymmetric PCR, ligationmediated PCR, multiplex PCR, nested PCR, qPCR, casting PCR, genome walking, bridge ( bridge) including but not limited to PCR.
  • reaction locations include, but are not limited to, for example, through holes, sample holding areas, wells, indentations, spots, cavities, and reaction chambers. It doesn't work.
  • dPCR digital PCR
  • solutions containing relatively small numbers of target polynucleotides or nucleic acid sequences may be further divided into a larger number of smaller samples, whereby each sample typically contains one molecule of the target nucleotide sequence. It may or may not contain the target nucleotide sequence.
  • samples are thermally cycled in a PCR protocol, procedure, or experiment, samples containing the target nucleotide sequence are amplified and produce a positive detection signal, while samples that do not contain any target nucleotide sequence are not amplified and are detected. Does not generate signal.
  • Poisson statistics the number of target nucleotide sequences in the original solution may be correlated with the number of samples producing a positive detection signal.
  • a simple and cost-effective method can be used to sample tens or hundreds of thousands of samples, i.e., samples with volumes of a few nanoliters, 1 or approximately 1 nanoliter, or less than 1 nanoliter, respectively.
  • the initial sample solution can be divided. Because the number of target nucleotide sequences can be very small, the entire contents of the initial solution may also be important in those environments where multiple reaction sites are taken into account.
  • Embodiments described herein address these and other dPCR design constraints by distributing the initial sample solution to a plurality of reaction sites in a manner that takes into account all, or all of the essentials, of the sample solution.
  • the array of reaction volumes of the liquid sample may be a substrate with a plurality of reaction sites.
  • Reaction locations may be, but are not limited to, through holes, wells, recesses, points, cavities, reaction chambers, or any structures capable of holding a sample according to various embodiments described herein. It doesn't work.
  • the through holes or wells may be tapered in diameter.
  • reaction volumes can be further increased by reducing the reaction volume of the liquid sample so that more reactions can be performed within a given area.
  • an array of reaction sites consisting of 300 ⁇ m diameter through through holes in the substrate may contain a reaction volume of approximately 30 nL.
  • each reaction volume may be, for example, 100 pL of liquid sample.
  • Reaction volumes according to various embodiments described herein may range from about 1 pL to 30 nL of liquid sample.
  • the dynamic range may be increased by using more than one dilution of the liquid sample.
  • a well array attached to the bottom of the microfluidic chamber and receiving the sample to be analyzed from the microfluidic chamber;
  • a cartridge for digital real-time PCR may be used, which is located on the bottom of the well array and includes a CMOS photosensor array that captures in real time a reaction image of the sample to be analyzed filled in a plurality of fractions provided in the well array.
  • Figure 1 is a perspective view schematically illustrating a cartridge for digital real-time PCR according to an embodiment.
  • Figure 2 is an exploded perspective view of the cartridge for digital real-time PCR shown in Figure 1.
  • the cartridge for digital real-time PCR is a cartridge-type test module and includes an upper case 110, a bottom case 120, a microfluidic chamber 130, and a well array 140. , CMOS photosensor array 150 and PCB 160.
  • the cartridge for digital real-time PCR may be detachably coupled to the reader system of the digital real-time PCR equipment.
  • the microfluidic chamber 130, well array 140, CMOS image sensor 150, and PCB 160 may define a PCR module.
  • the upper case 110 includes a donut-shaped cover body including an upper hole formed in a central area and a window member disposed in the upper hole.
  • the upper case 110 is fastened to the bottom case 120.
  • the upper case 110 and the bottom case 120 are shown as having a flat cylindrical shape, but various shapes are possible.
  • the window member may include a transparent material.
  • the window member may include PDMS material.
  • the bottom case 120 accommodates the microfluidic chamber 130, the well array 140, the CMOS photosensor array 150, and the PCB 160, and is fastened to the upper case 110.
  • the bottom case 120 and the upper case 110 may be fastened using a hook method.
  • the microfluidic chamber 130 includes a membrane switch protruding upward and an inlet formed on one side of the membrane switch for injection of a liquid sample.
  • the lower edge area of the microfluidic chamber 130 contacts the edge area of the PCB 160.
  • a receding space is formed in the lower central area of the microfluidic chamber 130 to accommodate the CMOS photosensor array 150 and the well array 140 mounted on the PCB 160.
  • the microfluidic chamber 130 may be made of a material such as PDMS.
  • the microfluidic chamber 130 has flexibility, transparency, PCR compatibility, and low autofluorescence, and can be injection molded.
  • the well array 140 is attached to the lower surface of the microfluidic chamber 130 and disposed on the CMOS photosensor array 150.
  • the well array 140 may be made of an etched silicon material.
  • the well array 140 includes a plurality of micro wells (approximately 1,000 to 100,000) that serve as PCR reaction sites.
  • FIG. 3 is a perspective view schematically illustrating an example of the well array shape shown in FIG. 2.
  • the shape of the micro well is shown in Figure 3 as being circular, various shapes such as hexagon, square, etc. are possible.
  • the shape, size, and volume of the microwell can be adjusted.
  • the micro-well has a shape with upper and lower portions penetrating.
  • the micro-well may have a thickness of less than 700 ⁇ m and a pitch of less than 150 ⁇ m.
  • the microwell may have various shapes such as hexagonal shape, circular shape, etc.
  • Well array 140 may be treated with a hydrophilic coating.
  • Well array 140 may be attached to microfluidic chamber 130 by plasma or thermal or UV curing adhesive.
  • the CMOS photosensor array 150 is disposed below the well array 140, and photographs PCR reaction products in a plurality of micro-wells of the well array 140 in real time. Specifically, the CMOS photosensor array 150 is arranged to correspond to the substrate hole formed in the PCB 160 to receive the light emitted from the well array 140 to produce the PCR reaction product performed in the PCR device as shown in FIG. 4A. As shown, measure. The measured PCR reaction product can be analyzed graphically, etc., as shown in FIG. 4B.
  • Figures 4a and 4b are diagrams showing digital real-time PCR results.
  • Figure 4a is a photograph showing PCR positive/negative results in a well array
  • Figure 4b is a graph that measures fluorescence in each micro-well in real time to determine whether the well is positive or negative.
  • the upper surface of the CMOS photosensor array 150 may be coated with a thin layer of a material such as PDMS to form the bottom of the micro-well after sealing.
  • the PCB 160 accommodates the CMOS image sensor 150.
  • a vent 152 for vacuum processing may be formed in the PCB 160 so that the liquid sample introduced through the inlet is quickly provided to the well array 140.
  • the vent 152 is connected to the space between the CMOS photosensor array 150 and the well array 140.
  • the cartridge for digital real-time PCR may further include a heater 170 for thermal circulation.
  • thermal cycling uses a thermal cycler, isothermal amplification, thermal convention, infrared mediated thermal cycling, or helicase dependent amplification. It may also include steps to:
  • the chip may be integrated with an embedded heating element.
  • the chip may be integrated with semiconductors. Detection of a target according to various embodiments may be performed singly or in combination, for example, fluorescence detection, positive or negative ion detection, acidity (pH) detection, voltage detection, or current detection, but is not limited thereto.
  • FIG. 5A is a front perspective view for schematically explaining the microfluidic chamber 130 shown in FIG. 2
  • FIG. 5B is a rear perspective view for schematically explaining the microfluidic chamber 130 shown in FIG. 2
  • FIG. 5C. is an exploded perspective view of the microfluidic chamber 130 shown in FIG. 2.
  • the microfluidic chamber 130 includes a square-shaped base member 132 and a square-shaped top member 134 disposed on the base member 132.
  • the base member 132 and the top member 134 are shown as physically separated, but may be formed as one piece.
  • the base member 132 includes a square-shaped first flat part 132a, a square-shaped support hole 132b formed in the center area of the first flat part 132a, and a corner area of the first flat part 132a. It includes a circular flat hole 132c. Guide protrusions protruding from the central area may be formed in the support hole 132b to define a square sawtooth shape.
  • the top member 134 includes a square-shaped second flat part 134a, a dish-shaped membrane switch 134b, and a perupe-shaped inlet part 134c.
  • the second flat portion 134a is in close contact with the upper surface of the first flat portion 132a.
  • the membrane switch 134b is disposed in the central area of the second flat portion 134a and protrudes upward.
  • the lower area of the membrane switch 134b corresponds to the support hole 132b of the base member 132. Accordingly, a receding space is formed at the bottom of the microfluidic chamber 130.
  • the CMOS image sensor 150 and the well array 140 can be accommodated in the retreated space.
  • the inlet portion 134c has a fence shape and protrudes upward on one side of the membrane switch 134b to block the liquid sample from flowing out to the outside.
  • An inlet 134d is formed in the central area of the inlet portion 134c in the vertical direction of the base member 132.
  • a micro channel 134e is formed in an area connecting the inlet 134d of the inlet portion 134c and the bottom edge area of the membrane switch 134b.
  • the liquid sample injected into the inlet 134d of the inlet portion 134c reaches the bottom edge area of the membrane switch 134b through the micro flow path 134e.
  • the top member 134 may further include a grip portion 134f protruding upward from the observer's viewpoint on the other side of the membrane switch 134b.
  • the grip portion 134f is disposed to face the inlet portion 134c with respect to the membrane switch 134b.
  • the height of the grip portion 134f may be higher than the height of the inlet portion 134c.
  • the height of the inlet portion 134c and the height of the membrane switch 134b are the same.
  • the inlet 134d and the micro channel 134e are connected to each other.
  • the liquid sample falls into the space formed below the membrane switch 134b through the micro flow path 134e.
  • each of the plurality of micro wells of the well array 140 exposed through the micro flow path 134e may be completely filled with the liquid sample.
  • Figure 6 is a cross-sectional view schematically explaining the PCR module shown in Figure 2.
  • Figure 7 is an exploded cross-sectional view for schematically explaining the PCR module shown in Figure 6.
  • the PCR module includes a microfluidic chamber 130, a well array 140, a CMOS photosensor array 150, and a PCB 150.
  • a receding space is formed in the bottom area of the microfluidic chamber 130 to accommodate the CMOS photosensor array 150 and the well array 140 disposed on the PCB 150. Since the microfluidic chamber 130 has been described in FIGS. 5A to 5C, its description is omitted.
  • the well array 140 is attached to the lower surface of the microfluidic chamber 130.
  • the well array 140 is disposed on the CMOS photosensor array 150 and is inserted into a substrate hole formed in the PCB 150.
  • the well array 140 includes a plurality of micro wells 142.
  • the shape, size, number, etc. of the micro wells 142 may vary.
  • Each of the plurality of micro wells 142 may contain an analysis sample such as a powder sample or a liquid sample.
  • the analysis sample is a specific component for analyzing biological substances.
  • the analysis sample refers to a component for quantitative or qualitative analysis of a specific biological material, such as protein, DNA, RNA, etc., and refers to primers, probes, antibodies, aptamers, DNA or RNA polymerase, etc., especially in real time. It refers to ingredients needed to perform polymerase chain reaction, constant temperature enzyme reaction, or LCR (Ligase Chain Reaction).
  • the CMOS photosensor array 150 is disposed below the well array 140 and captures images of PCR reaction products performed in the plurality of micro wells 142 of the well array 140 in real time.
  • the CMOS photosensor array 150 is inserted and placed into a substrate hole formed in the PCB 150.
  • the CMOS photosensor array 150 receives the emitted light and photographs the PCR reaction product performed in the PCR device in real time. That is, the CMOS photosensor array 150 detects fluorescence (emission light) generated from a plurality of probes by excitation light. Detection of the above-mentioned fluorescence may be performed by a time separation method or a wavelength separation method.
  • the fluorescent sensor array or a single sensor constituting the array detects the emission light passing through the emission filter and sets the time constant of the detected emission light. Obtain and detect fluorescence.
  • the fluorescent sensor array or a single sensor constituting the array detects the emission light passing through the emission filter and specifies the spectrum of the detected emission light. Fluorescence is detected through analysis.
  • the PCB 150 is disposed below the microfluidic chamber 130 and accommodates the mounted CMOS photosensor array 150.
  • the PCB 150 is placed in contact with the bottom edge area of the microfluidic chamber 130.
  • a vent hole 152 is formed in a portion of the PCB 150 that is in contact with the bottom edge area of the microfluidic chamber 130.
  • the vent 152 is connected to the space between the CMOS photosensor array 150 and the well array 140.
  • the inlet 134d When a liquid sample is introduced into the inlet 134d, air is pumped through a vacuum device (not shown) connected to the vent 152. According to the pumping of air, the liquid sample introduced into the inlet 134d passes through the micro channel 134e formed in the microfluidic chamber 130 to a partial region of the well array 140, an upper region of the partial region, and the partial region. It is provided in the lower area of .
  • a sticker 180 forming the bottom of the micro channel 134e formed in the microfluidic chamber 130 may be further included. By attaching the sticker 180, the liquid sample flowing into the micro channel 134e can be provided intact to the well array 140 without flowing out to other areas.
  • the thickness of the sticker 180 may be the same as the thickness of the well array 140.
  • the PCR module may further include a light provider (not shown) arranged to irradiate an excitation light toward the probe accommodated in each of the plurality of micro wells 142 of the well array 140.
  • the light providing unit may include a light source that emits light, such as a light emitting diode (LED) light source, a laser light source, or the like. Light emitted from the light source passes through or reflects the micro well 142 of the well array 140, and in this case, the CMOS photosensor array 150 can detect the optical signal generated by nucleic acid amplification.
  • LED light emitting diode
  • the PCR module may further include an emission filter (not shown) that serves to select light having a predetermined wavelength.
  • the emission filter may be disposed on the CMOS photosensor array 150.
  • Figures 8 to 16 are cross-sectional views showing the process of injecting a liquid sample into the well array 140.
  • an empty PCR module is placed in a flat position.
  • a liquid sample is pipetted into the inlet 134d of the microfluidic chamber 130.
  • the amount of liquid sample pipetted into the inlet 134d is preferably an amount that can fill the space between the well array 140 and the microfluidic chamber 130 and the plurality of microwells 142 of the well array 140.
  • the liquid sample pipetted into the inlet 134d cannot flow into the micro channel 134e due to air resistance present in the micro channel 134e (shown in FIG. 3B).
  • liquid sample pipetted into the inlet 134e is provided to the well array 140 along the micro flow path 134e.
  • the liquid sample provided to the well array 140 reaches the CMOS photosensor array 150 through the micro well 142 adjacent to the end of the micro channel 134e.
  • Liquid samples can be provided more quickly to a plurality of microwells through the pumping of such a vacuum device. Additionally, air that may exist in the corners or edge areas of the well array can be more easily removed through pumping of the vacuum device.
  • the vacuum device connected to the vent 152 stops operating. As the negative pressure caused by the vacuum device is eliminated, the liquid sample that has reached the lower space of the well array 140 is gradually pulled up to the upper space of the well array 140 by capillary force.
  • the liquid sample is filled in the space between the well array 140 and the microfluidic chamber 130 and the plurality of micro wells 142 of the well array 140.
  • CMOS photosensor array 150 when the window member disposed in the center area of the upper case 110 is pressed according to pressure from the user or pressure from machinery, a plurality of micro wells 142 ) The well array 140, each of which has a filled liquid sample, is pressed against the CMOS photosensor array 150.
  • It may include a final calculation step (S50) of calculating the target concentration in the sample to be analyzed based on the plurality of target numbers.
  • the microfluidic chamber may be made of polydimethylsiloxane (PDMS) material.
  • the plurality of fractions may correspond to the plurality of micro wells 142 described above. That is, one fraction may be one microwell.
  • the sample to be analyzed may be the liquid sample described above.
  • the sample injection step (S10) the sample to be analyzed may be injected into a plurality of fractions through the above-described processes. At this time, it can be confirmed that the injection of the sample to be analyzed into the plurality of fractions has been completed through the Emptywell algorithm. Images obtained after it is confirmed that the sample to be analyzed have arrived can be used as raw data.
  • the raw data acquisition step (S20) is performed after confirming that all of the plurality of fractions are filled with the sample to be analyzed, and in the raw data acquisition step (S20), images for the plurality of fractions are converted into the raw data. It may be obtained as.
  • the raw data may be collected at least once per cycle.
  • a cycle is a temperature-controlled time period provided to repeat the denaturation, annealing, and elongation steps.
  • the area between fractions can be distinguished through a contour algorithm.
  • the wall between microwells can be distinguished through the contour algorithm.
  • the fluorescence intensity value can be calculated by extracting the color variable of the image area for each of the plurality of fractions.
  • Fluorescence intensity values can be obtained as time series (cycle number) data. That is, the fluorescence intensity value for each of the plurality of fractions can be obtained as real-time data (every cycle period).
  • a color variable may include at least one of brightness, saturation, hue, contrast, and color code.
  • a fractional image can be processed into a complementary color image, and a contrast value can be assigned to each pixel to extract it as a color variable.
  • the saturation value of each pixel can be extracted as a color variable.
  • the brightness value, saturation value, color value, and contrast value can be extracted from each pixel, and a linear function value with independent weights for each variable can be extracted as a color variable.
  • the target number may be the copy number of the target gene in the sample to be analyzed.
  • Each of the plurality of target numbers for each of the plurality of fractions may be calculated using Equation 1 above.
  • N i is the number of targets in the ith fraction.
  • Ct i is the Ct value of the ith fraction.
  • Ct ref is determined by at least one of the number of amplicons required to measure Ct and the fluorescence efficiency of the probe.
  • a is a constant determined by PCR efficiency.
  • PCR efficiency may indicate the rate at which a target gene is amplified by a PCR reaction. For example, 100% PCR efficiency means that the target is amplified 2-fold for each PCR reaction cycle, and 90% PCR efficiency means that the target is amplified 1.8-fold for each PCR reaction cycle.
  • the relationship between a and PCR efficiency Ep may be equivalent to Equation 4 below.
  • PCR efficiency can be set to 90% to 110%, so a can be -3.103 to -3.587.
  • Equation 1 above may be derived from Equation 5 below.
  • Equation 5 may be the value obtained in the Ct extraction step (S30).
  • the digital real-time PCR analysis method of the present invention obtains the value of Ct ref through a standard sample, making it possible to analyze even ultra-high concentration samples that cannot be analyzed conventionally.
  • the Ct ref may be calculated using Equation 2 below.
  • Vunit is the volume of one fraction. For example, it may mean the volume of one microwell, and may actually be the average volume of a plurality of microwells.
  • Ct 0 may be a Ct value obtained from a standard sample.
  • Ct 0 is the Ct value obtained by PCR amplification when the initial target number in all of the plurality of fractions is 1.
  • the Ct value may be obtained by PCR amplification when the number of targets is 1 in a volume of 20,000
  • Ct0 is a value obtained by measuring a standard sample of known concentration after serial dilution.
  • the concentration of the standard sample may be 10 7 copies/rxn to 10 8 copies/rxn.
  • the standard sample may be an international standard material or a self-produced standard material.
  • standard samples may be WHO biological reference materials (WHO international standards and reference materials).
  • the concentration Con of the sample to be analyzed may be calculated using Equation 3 below.
  • n is the total number of the plurality of fractions.
  • the final calculation step (S50) it may be determined whether the sample to be analyzed is negative or positive based on the Con value.
  • the digital real-time PCR analysis method of the present invention can provide a digital real-time PCR analysis method capable of performing digital real-time PCR analysis on high- or low-concentration samples that exceed existing measurement limits, that is, samples with a wide concentration range.
  • the digital real-time PCR analysis method of the present invention can implement digital PCR that provides real-time graphs for individual fractions, thereby eliminating false positive or false negative errors.
  • the digital real-time PCR analysis method of the present invention can achieve the same effect as repeatedly evaluating digital real-time PCR tens of thousands of times with one cartridge, and calculates the number of targets present in each unit compartment based on the values measured in each unit compartment. By doing so, the initial concentration in the sample can be calculated.

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Abstract

La présente invention concerne un procédé d'analyse PCR en temps réel numérique et vise à fournir un procédé PCR en temps réel numérique permettant une analyse PCR en temps réel numérique pour des échantillons sur une large plage de concentration, comprenant à la fois des échantillons à haute et basse concentration qui dépassent les limites de mesure de procédés classiques.
PCT/KR2023/015053 2022-09-30 2023-09-27 Procédé d'analyse pcr en temps réel numérique WO2024072111A1 (fr)

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Citations (4)

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Publication number Priority date Publication date Assignee Title
KR20190096614A (ko) * 2018-02-09 2019-08-20 단국대학교 천안캠퍼스 산학협력단 등온 증폭의 핵산 농도 실시간 모니터링 장치
KR20190120947A (ko) * 2018-04-17 2019-10-25 (주)로고스바이오시스템스 핵산증폭반응산물을 실시간으로 검출하는 장치
KR102185443B1 (ko) * 2018-04-25 2020-12-01 (주)옵토레인 디지털 실시간 pcr용 카트리지
KR20210094385A (ko) * 2020-01-21 2021-07-29 (주)옵토레인 푸시타입 pcr용 카트리지

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KR20190096614A (ko) * 2018-02-09 2019-08-20 단국대학교 천안캠퍼스 산학협력단 등온 증폭의 핵산 농도 실시간 모니터링 장치
KR20190120947A (ko) * 2018-04-17 2019-10-25 (주)로고스바이오시스템스 핵산증폭반응산물을 실시간으로 검출하는 장치
KR102185443B1 (ko) * 2018-04-25 2020-12-01 (주)옵토레인 디지털 실시간 pcr용 카트리지
KR20210094385A (ko) * 2020-01-21 2021-07-29 (주)옵토레인 푸시타입 pcr용 카트리지

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