Melting curve analysis method

Abstract

The present invention relates to a melting curve analysis method, and provides a melting curve analysis method whereby a high-resolution melting curve result for a plurality of targets can be acquired on the basis of a real-time digital PCR device.

Description

Melting curve analysis method

[0001] The present invention relates to a melting curve analysis method, and more particularly, to a melting curve analysis method capable of obtaining high-resolution melting curve results for multiple targets based on a real-time digital PCR device.

[0002] This application claims the benefit of priority to Korean Patent Application No. 10-2024-0007506, filed on January 17, 2024, and Korean Patent Application No. 10-2025-0006092, filed on January 15, 2025, the entire contents of which are incorporated herein by reference.

[0003] Melting curve analysis began with the development of quantitative polymerase chain reaction (qPCR) using intercalating dyes (e.g., SYBR, Eva green, SYBR Gold, etc.). qPCR analysis can analyze differences in melting temperature according to the methylation level of a target gene after bisulfite treatment. However, melting curve analysis using qPCR has limitations: it has low resolution for genes with similar melting temperatures and cannot achieve absolute quantification. These shortcomings hinder the accurate analysis of low-concentration clinical samples containing a variety of methylation levels.

[0004] In melting curve analysis, for example, the melting temperature of DNA represents the "temperature at which the fluorescent signal rapidly decreases" in the context of real-time PCR using fluorescent dyes. This rapid decrease in fluorescent signal indicates that 50% of dsDNA has been converted to ssDNA, causing the DNA-binding fluorescent dye to detach from the DNA, resulting in a rapid decrease in fluorescence intensity.

[0005] Melting curve analysis is an analytical tool that must be performed to check for non-specific amplification other than the target product in real-time PCR reactions using DNA-binding fluorescent dyes.

[0006] However, as shown in Fig. 1, in the case of targets with adjacent melting temperatures, the conventional melting curve may be measured overlappingly, making it difficult to distinguish between them.

[0007] The present invention relates to a melting curve analysis method, and provides a melting curve analysis method capable of obtaining high-resolution melting curve results for multiple targets based on a real-time digital PCR device.

[0008] The technical problems to be solved by the present invention are not limited to the technical problems mentioned above, and other technical problems not mentioned can be clearly understood by a person having ordinary skill in the technical field to which the present invention belongs from the description below.

[0009] The melting curve analysis method of the present invention may use a PCR cartridge including a PCB portion including a heater; a CMOS image sensor portion laminated on an upper surface of the PCB portion; and a well array laminated on an upper surface of the CMOS image sensor portion, and a reader system that reads a signal output from the PCR cartridge and outputs real-time data.

[0010] The melting curve analysis method of the present invention is:

[0011] A sample injection step of injecting an analysis target sample containing multiple targets into a plurality of micro-wells provided in the well array;

[0012] A contact step of contacting the plurality of micro-wells into which the sample to be analyzed has been injected, to the upper surface of the CMOS image sensor unit;

[0013] A mounting step of mounting the PCR cartridge to the reader system;

[0014] A PCR step for performing PCR on the analysis target sample filled in the plurality of micro-wells by operating the heater of the PCB section in a cycle mode;

[0015] An MT measurement step of operating the heater of the PCB section in melting mode to measure the MT (melting temperature) for each of the plurality of micro-wells; and

[0016] It may include a target information calculation step for calculating target information for the sample to be analyzed based on the MT values ​​of the plurality of micro-wells.

[0017] In the sample injection step of the melting curve analysis method of the present invention, the plurality of micro-wells may be provided in the shape of a plurality of holes penetrating in the vertical direction in the well array provided as a plate having a shape perpendicular to the vertical direction.

[0018] In the sample injection step of the melting curve analysis method of the present invention, the thickness of the well array may be 10 µm to 1000 µm, and the pitch of the plurality of micro-wells may be 20 µm to 200 µm.

[0019] In the target information calculation step of the melting curve analysis method of the present invention, the type of target injected into each of the plurality of micro-wells may be determined by the MT value.

[0020] In the target information calculation step of the melting curve analysis method of the present invention, the number of micro-wells corresponding to each target type may be counted. In addition, in the target information calculation step, when displaying the counted graph by melting temperature, the distribution of melting temperature may be analyzed using a log scale. In addition, in the target information calculation step, when analyzing the data displayed by melting temperature, the distribution may be distinguished using the difference temperature (delta MT) between the melting temperature of the reference target and the melting temperature of the analysis target.

[0021] In the target information calculation step of the melting curve analysis method of the present invention, a quantitative value for each target may be calculated based on the number of micro-wells counted for each target type.

[0022] The melting curve analysis method of the present invention may further include, prior to the sample injection step, a reference sample injection step of injecting a reference sample including a reference target into a plurality of micro-wells provided in a well array of another PCR cartridge; and a reference PCR step of operating the heater of the PCR cartridge into which the reference sample has been injected in a cycle mode to perform PCR on the reference sample filled in the plurality of micro-wells.

[0023] The melting curve analysis method of the present invention may be capable of obtaining high-resolution melting curve results for multiple targets based on a real-time digital PCR device.

[0024] The melting curve analysis method of the present invention can facilitate mutual identification even for targets having adjacent melting temperatures.

[0025] Figure 1 is a graph showing a melting curve measured in a conventional manner.

[0026] Figure 2 is a conceptual diagram showing a state in which a PCR cartridge and a leader system are combined.

[0027] Figure 3 is a graph showing data on the intensity value of the fluorescence signal versus the number of cycles for each of multiple micro-wells for a reference sample.

[0028] Figure 4 is a graph showing the melting curve for each of multiple micro-wells for a reference sample.

[0029] Figure 5 is a graph showing the melting curve for each of multiple micro-wells for the sample to be analyzed.

[0030] Figure 6a is a graph showing the results of the graphs in Figure 5 grouped together.

[0031] Figure 6b is a graph showing the graph results of Figure 6a on a linear scale.

[0032] Figure 6c is a graph converted to a log scale from the graph results of Figure 6a.

[0033] Figure 7 is a diagram showing an artificial plasmid sequence according to the methylation level of the CDH13 gene, a tumor-related gene.

[0034] Figures 8a to 8e are graphs of melting curves using multiple micro-wells according to the methylation level of the CDH13 gene, a tumor-related gene.

[0035] Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings. Throughout this process, the sizes and shapes of components depicted in the drawings may be exaggerated for clarity and convenience. Furthermore, terms specifically defined in consideration of the structure and operation of the present invention may vary depending on the intentions or practices of the user or operator. Definitions of these terms should be based on the overall content of this specification.

[0036] In the description of the present invention, it should be noted that the orientation or positional relationship indicated by the terms “center,” “upper,” “lower,” “left,” “right,” “vertical,” “horizontal,” “inner,” “outer,” “one side,” “other side,” etc., is based on the orientation or positional relationship shown in the drawings, or the orientation or positional relationship in which the product of the present invention is normally placed when used, and is only for the purpose of explaining and briefly explaining the present invention, and does not suggest or imply that the indicated device or element must have a specific orientation and be configured or operated in a specific orientation, and therefore should not be understood as limiting the present invention.

[0037] Fig. 1 is a graph showing a melting curve measured by a conventional method. Fig. 2 is a conceptual diagram showing a state in which a PCR cartridge and a reader system are combined. Fig. 3 is a graph showing data on the intensity value of the fluorescence signal versus the number of cycles for each of a plurality of micro-wells for a reference sample. Fig. 4 is a graph showing a melting curve for each of a plurality of micro-wells for a reference sample. Fig. 5 is a graph showing a melting curve for each of a plurality of micro-wells for a sample to be analyzed. Figs. 6a to 6c are graphs showing the results of the graph of Fig. 5 grouped together. Fig. 7 is a diagram showing an artificial plasmid sequence according to the methylation level of the CDH13 gene, which is a tumor-associated gene. Figs. 8a to 8e are graphs of melting curves using a plurality of micro-wells according to the methylation level of the CDH13 gene, which is a tumor-associated gene.

[0038] Hereinafter, with reference to the drawings, the melting curve analysis method of the present invention will be described in detail.

[0039] As shown in Fig. 2, a PCR cartridge used in the melting curve analysis method of the present invention may include a PCB portion including a heater; a CMOS image sensor portion laminated on an upper surface of the PCB portion; and a well array laminated on an upper surface of the CMOS image sensor portion.

[0040] The PCB section includes a heater capable of applying heat to the well array. Additionally, the temperature of the well array can be measured in real time and transmitted to the reader system described below.

[0041] The CMOS image sensor unit can measure a fluorescence signal generated from the well array. The CMOS image sensor unit may be a CMOS image sensor.

[0042] The well array may be a plate made of silicon material. A plurality of microwells may be formed in the well array. The well array may contain hundreds to millions of microwells. For example, the well array may contain approximately 20,000 microwells.

[0043] The above plurality of micro-wells may be provided in the form of a plurality of holes penetrating vertically into the well array, which is provided as a plate having a shape perpendicular to the vertical direction. For example, the plurality of micro-wells may be formed through etching in a silicon substrate.

[0044] The thickness of the well array may be 10 μm to 1000 μm. For example, the thickness of the well array may be 10 μm to 700 μm. For example, the thickness of the well array may be 10 μm to 250 μm. That is, the depth of the plurality of micro-wells may be 250 μm or less.

[0045] The pitch of the plurality of micro-wells may be 20 μm to 200 μm. For example, the pitch of the plurality of micro-wells may be 50 μm to 150 μm. For example, the pitch of the plurality of micro-wells may be about 70 μm. The volume of the micro-wells may be most preferably designed so that one target enters each micro-well.

[0046] The shape of the micro-well can have various shapes such as square, pentagon, hexagon, circle, and ellipse when observed in a plane.

[0047] The bottom surface of the well array can be directly attached to the top surface of the CMOS image sensor unit or can be attached to the top surface of the CMOS image sensor unit with a fluorescent filter interposed therebetween.

[0048] The upper surface of the well array can be covered with a transparent cover after the micro-wells are filled with the sample to be analyzed.

[0049] The reader system may be a device with a removable PCR cartridge. The reader system may read signals output from the PCR cartridge and output real-time data. The reader system may be a device combining software and hardware.

[0050] As illustrated in FIG. 2, the reader system can provide a light source to the well array. Specifically, light can be irradiated from above the well array, and the fluorescence signal of the well array can be measured by the CMOS image sensor unit. Image data measured by the CMOS image sensor unit can be transmitted to the reader system through the PCB unit. The data can be processed and stored in the reader system.

[0051] The melting curve analysis method of the present invention is:

[0052] A sample injection step of injecting an analysis target sample containing multiple targets into a plurality of micro-wells provided in the well array;

[0053] A contact step of contacting the plurality of micro-wells into which the sample to be analyzed has been injected, to the upper surface of the CMOS image sensor unit;

[0054] A mounting step of mounting the PCR cartridge to the reader system;

[0055] A PCR step for performing PCR on the analysis target sample filled in the plurality of micro-wells by operating the heater of the PCB section in a cycle mode;

[0056] An MT measurement step of operating the heater of the PCB section in melting mode to measure the MT (melting temperature) for each of the plurality of micro-wells; and

[0057] It may include a target information calculation step for calculating target information for the sample to be analyzed based on the MT values ​​of the plurality of micro-wells.

[0058] In the above sample injection step, the sample to be analyzed may be transferred to the well array through a chamber provided in the PCR cartridge. The chamber may be provided in a pushable form, and after the sample to be analyzed is injected into the chamber, the chamber may be pushed to transfer the sample to the well array. The upper surface of the well array is provided with a hydrophilic coating so that the sample to be analyzed can be evenly distributed and injected throughout the multiple micro-wells.

[0059] Alternatively, a vacuum pump may be connected to the PCR cartridge to create negative pressure at one end of the well array and connect the chamber portion at the other end to rapidly deliver the sample to be analyzed throughout the well array.

[0060] In the above-described adhesion step, the well array and the CMOS image sensor unit can be completely adhered using a user's pressurized or vacuum pump. More specifically, by preventing air pockets from forming between the well array and the CMOS image sensor unit, errors in the image data measured by the CMOS image sensor unit can be prevented.

[0061] In the above mounting step, the PCB portion of the reader system and the PCR cartridge can be electrically connected. The heater, CMOS image sensor portion, etc. of the PCR cartridge can be controlled using the input device of the reader system.

[0062] The melting curve analysis method of the present invention is:

[0063] A reference sample injection step for injecting a reference sample including a reference target into a plurality of micro-wells provided in a well array of another PCR cartridge; and

[0064] It may further include a reference PCR step of operating the heater of the PCR cartridge into which the reference sample is injected in a cycle mode to perform PCR on the reference sample filled in a plurality of micro-wells.

[0065] The above-described reference sample injection step is performed in the same manner as the above-described sample injection step, and the target may be a reference sample rather than a sample to be analyzed. Prior to the reference PCR step, a PCR cartridge into which the reference sample is injected may also undergo a contact step and a mounting step. The reference sample may be one of multiple targets included in the sample to be analyzed.

[0066] In the above-mentioned reference PCR step, an absolute quantitative value can be measured for a reference target included in a reference sample. A threshold cycle (CT) value may be collected for each of a plurality of micro-wells. More specifically, heat may be repeatedly applied to the well array to perform a cycle, and data on the intensity value of a fluorescence signal with respect to the number of cycles may be acquired for each of a plurality of micro-wells. For example, Fig. 3 is a graph showing data on the intensity value of a fluorescence signal with respect to the number of cycles for each of a plurality of micro-wells. A threshold cycle (CT) value may be acquired based on the graph of Fig. 3.

[0067] By obtaining the critical cycle (CT) value for each of multiple micro-wells, an absolute quantitative value for the sample to be analyzed can be obtained as shown in Table 1 below.

[0068]

[0069] Specifically, as shown in Table 1 above, the number of copies per volume for a sample can be obtained.

[0070] In the above-mentioned standard PCR step, the cycle mode may be a mode that repeats a cycle of periodically heating and cooling the temperature of the well array using a heater of the PCB portion.

[0071] In the above MT measurement step, the melting mode may be to measure the intensity of the fluorescence signal by increasing a constant temperature at a constant time interval for each of the plurality of micro-wells. For example, the constant temperature may be 0.3°C.

[0072] As illustrated in FIG. 4, in the MT measurement step, a melting curve can be obtained for each of the multiple micro-wells into which the sample to be analyzed has been injected. The various targets contained in the sample to be analyzed may include a reference target. Positive micro-wells in the band (temperature range) corresponding to the reference target can be counted as shown in Table 2 below.

[0073]

[0074] In Table 2 above, G1 may be a reference target.

[0075] The results from the above-mentioned reference PCR step and the results from the above-mentioned reference MT measurement step can be matched. For example, if the number of positive wells in the reference target is 232 in the reference MT measurement results and the total number of valid micro-wells is 18,535, the template concentration can be calculated as 396.93 copies / ㎕ based on this.

[0076] As illustrated in FIG. 5, in the MT measurement step, for an analysis target sample including multiple targets, the heater of the PCB section is operated in melting mode to obtain MT (melting temperature) for each of the plurality of micro-wells.

[0077] In the target information generation step, the target type injected into each of the plurality of micro-wells may be determined based on the MT value. The melting curve analysis method of the present invention can assume that only one type of target is injected into each micro-well. For example, a micro-well containing two or more types of targets can be excluded as an error. As illustrated in Figures 6a to 6c, for example, the sample to be analyzed can be analyzed as containing four types of targets.

[0078] In the above target information calculation step, the number of micro-wells corresponding to each target type may be counted. In the target information calculation step, a quantitative value for each target may be calculated based on the number of micro-wells counted for each target type.

[0079] As shown in Table 2 above, the number of positive wells can be calculated for each target as a result of MT measurement. For example, the number of positive wells can be counted as 232, 554, 304, and 346 for each of the four types of targets as a result of MT measurement.

[0080] Once the number of positive wells is counted, the absolute quantitation value of each target can be predicted based on the absolute quantitation value derived from the reference sample-target relationship derived from the reference sample measurement as described above.

[0081] That is, the absolute quantitative values ​​of the four types of targets can be calculated as 396.93 copy / ㎕, 956.26 copy / ㎕, 521.14 copy / ㎕, and 593.82 copy / ㎕.

[0082] In the above target information calculation step, the distribution of melting temperature can be analyzed using the log scale when displaying the counted graph by melting temperature. The distribution of melting temperature values ​​is distributed based on a certain representative value depending on the type of target. This distribution is caused by various reasons such as the heat transfer characteristics of each micro-well and the difference in experimental conditions occurring in each well. The better the experiment is designed, the more the data will gather near the reference value. At this time, if there is a large difference between the reference value and the surrounding distribution values, it may be difficult to confirm the overall distribution of melting temperature, as shown in Fig. 6b. Therefore, using the log scale as a histogram, as shown in Fig. 6c, is highly useful in analysis using the distribution of data.

[0083] It was confirmed that melting curve analysis in multiple microwells according to the melting curve analysis method of the present invention can more accurately analyze the methylation level.

[0084] We analyzed the methylation level of CDH13, a tumor suppressor gene with functions related to cell cycle regulation, DNA repair, and apoptosis, using a model.

[0085] As shown in Fig. 7, assuming methylation at six CpG sites in the CDH13 promoter region, artificial plasmid DNA was constructed based on the assumption of bisulfate treatment according to the methylation level (unmethylated, 1-methylated, 2-methylated, 3-methylated, 4-methylated). The constructed artificial plasmid DNA was subjected to PCR amplification and melting curve analysis evaluation by designing primers that could include the methylated region, as shown in Fig. 7.

[0086] For the PCR amplification reaction, pre-denaturation at 95°C for 15 min, annealing at 95°C for 10 s, and extension at 58°C for 30 s were repeated 45 times. Melting curve analysis of the PCR amplification product was obtained between 58°C and 95°C, and the fluorescence signal was measured while increasing the temperature at a ramp rate of 0.3°C per 10 s.

[0087] In addition, in the target information calculation step, when analyzing the data displayed by melting temperature, the melting temperature of the reference target and the melting temperature of the analysis target can be distinguished using the difference temperature (delta MT) as shown in FIGS. 8a to 8e. The melting temperature value varies slightly depending on the individual experimental environment such as the experimental equipment, cartridge characteristics, experimental conditions, and experimenter, so the absolute value of the target melting temperature cannot be specified. However, within the same experiment, the difference in the melting temperature values ​​of different targets shows a reproducible value. Therefore, when conducting multiple experiments and analyzing data, realigning the data based on the melting temperature of an arbitrary reference substance rather than the absolute value of the melting temperature is a way to increase the reliability of the experiment.

[0088] Melting curve analysis was performed on plasmid DNA with six different methylation states using a PCR cartridge with multiple microwells. As shown in Figures 8a to 8e, the melting temperatures varied from 77.44°C to 78.68°C depending on the methylation level. As the methylation level increased, the proportion of cytosine (C) bases in the amplified product increased, resulting in a higher melting temperature. This can be used to analyze and differentiate the melting curves according to the methylation level of the gene.

[0089] The number of corresponding melting curves was counted from the graphs of Figures 8a to 8e, and the number of positive wells according to melting temperature was recorded and listed in Table 3. qPCR cannot perform quantitative analysis, but dPCR can perform quantitative analysis by counting the number of wells.

[0090]

[0091]

[0092] This confirmed that each methylation level can be analyzed separately. Furthermore, it was demonstrated that gene concentrations can be calculated for each methylation level, and that genes can be distinguished by each methylation level even in clinical samples containing genes with varying methylation levels.

[0093] Therefore, the melting curve analysis technique in multiple micro-wells of the present invention is useful for accurately analyzing methylated samples compared to existing techniques, and can be effectively utilized in clinical diagnostic applications.

[0094]

[0095] While the embodiments of the present invention have been described above, they are merely exemplary, and those skilled in the art will appreciate that various modifications and equivalent embodiments are possible. Therefore, the true technical protection scope of the present invention should be defined by the following claims.

Claims

1. PCB section including heater; A CMOS image sensor part laminated on the upper surface of the PCB part; and A PCR cartridge including a well array laminated on the upper surface of the CMOS image sensor portion, In a melting curve analysis method using a reader system that reads a signal output from the PCR cartridge and outputs real-time data, A sample injection step of injecting an analysis target sample containing various targets into a plurality of micro-wells provided in the well array; A step of contacting the plurality of micro-wells, into which the above analysis target sample is injected, to the upper surface of the CMOS image sensor unit; A mounting step of mounting the above PCR cartridge into the above reader system; A PCR step for performing PCR on the analysis target sample filled in the plurality of micro-wells by operating the heater of the PCB section in a cycle mode; An MT measurement step of operating the heater of the PCB section in a melting mode to measure the MT (melting temperature) for each of the plurality of micro-wells; and A melting curve analysis method comprising a target information calculation step of calculating target information for the analysis target sample based on the MT values of the plurality of micro-wells.

2. In paragraph 1, In the above sample injection step, The above plurality of micro-wells are, A melting curve analysis method, wherein a plurality of holes are formed in the shape of penetrating vertically through the well array, which is formed as a plate having a shape perpendicular to the vertical direction.

3. In paragraph 2, In the above sample injection step, The thickness of the above well array is 10 ㎛ to 1000 ㎛, A melting curve analysis method wherein the pitch of the plurality of micro-wells is 20 ㎛ to 200 ㎛.

4. In paragraph 1, In the above target information generation step, A melting curve analysis method in which the target type injected into each of the plurality of micro-wells is determined by the MT value.

5. In paragraph 4, In the above target information generation step, A melting curve analysis method that counts the number of micro-wells corresponding to each target type.

6. In paragraph 5, In the above target information generation step, A melting curve analysis method that analyzes the distribution of melting temperatures by using a log scale to display a graph counted by melting temperature.

7. In paragraph 5, In the above target information generation step, A melting curve analysis method that distinguishes data displayed by melting temperature using the temperature difference between the melting temperature of the reference target and the melting temperature of the analysis target.

8. In paragraph 5, In the above target information generation step, A melting curve analysis method for calculating quantitative values for each target based on the number of micro-wells counted for each target type.

9. In paragraph 1, Prior to the above sample injection step, A reference sample injection step for injecting a reference sample including a reference target into a plurality of micro-wells provided in a well array of another PCR cartridge; and A melting curve analysis method further comprising a reference PCR step of operating the heater of the PCR cartridge injected with the reference sample in a cycle mode to perform PCR on the reference sample filled in a plurality of micro-wells.

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