Detection device and detection method

Abstract

To prevent detection errors in a melting temperature for each user.SOLUTION: A detection device 200 includes a CPU 211 and a memory 212. The CPU 211 sets a boundary point at which a melting curve is divided into a front region and a rear region, sets a start point and an end point, calculates an approximate straight line by the least squares method, and repeats setting processing of the approximate straight line. The CPU 211 sets a first straight line in the front region, sets a second straight line in the rear region, and detects, as a melting temperature, a temperature of a nucleic acid corresponding to an intersection between a melting curve and a line connecting intermediate values of the absorbance of the nucleic acid at which temperatures of the nucleic acid are the same on the first straight line and the second straight line.SELECTED DRAWING: Figure 7

Description

[Technical field]

[0001] The present disclosure relates to a detection device and a detection method for detecting the melting temperature of a nucleic acid. [Background technology]

[0002] The melting temperature (hereinafter also referred to as Tm value) is an index showing the stability of nucleic acids such as DNA (deoxyribonucleic acid). Nucleic acids have a double-stranded structure. When the temperature of a solution in which the nucleic acid is dissolved is increased, the double-stranded structure of the nucleic acid gradually separates, and in a high-temperature solution, the double-stranded structure is completely separated and becomes a single-stranded structure. When the nucleic acid changes from a double-stranded structure to a single-stranded structure, the absorbance of ultraviolet light at a wavelength of about 260 μm increases significantly. Because nucleic acids have such characteristics, when a graph of a melting curve is created with temperature on the horizontal axis and absorbance on the vertical axis, a region where the graph changes suddenly appears. The Tm value is the temperature at which the proportions of single-stranded structure and double-stranded structure in such a region become equal.

[0003] Japanese Patent Laid-Open Publication No. 2003-121396 (Patent Document 1) discloses an apparatus capable of calculating the absorbance of DNA with high accuracy, which makes it possible to create a graph of a melting curve with high accuracy. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2003-121396 A Summary of the Invention [Problem to be solved by the invention]

[0005] The Tm value can be determined, for example, by the following method (hereinafter also referred to as the "midline method"). In the midline method, a start point and an end point are set in the front region (region of double-stranded structure) and the rear region (region of single-stranded structure) of a graph of the nucleic acid, respectively, and a straight line is drawn connecting the two points. The midline method is a method in which the Tm value is determined as the temperature of the nucleic acid at the intersection of the melting curve and a line connecting the median values ​​of absorbance of the nucleic acid at which the temperature of the nucleic acid is the same temperature from the two straight lines. JP 2003-121396 A (Patent Document 1) does not disclose a detailed method for detecting the Tm value.

[0006] When the midline method is used to calculate the Tm value, the start and end points are set arbitrarily by the user. Therefore, even when the same data is used, the start and end points of the Tm value vary from user to user, which may result in errors in detection.

[0007] The present disclosure has been made to solve such problems, and has as its object to provide a detection device and a detection method that are capable of eliminating detection errors in the melting temperature (Tm value) for each user. [Means for solving the problem]

[0008] The detection device according to the present disclosure relates to a detection device that detects the melting temperature of a nucleic acid using data indicating the relationship between the temperature of the nucleic acid measured by an analysis device and the absorbance of the nucleic acid corresponding to ultraviolet light. The detection device includes a control device and a storage device that stores data from the analysis device. The data is represented by a melting curve in which the absorbance of the nucleic acid increases with an increase in the temperature of the nucleic acid when the data is plotted with the temperature of the nucleic acid on the horizontal axis and the absorbance of the nucleic acid on the vertical axis. The control device sets a boundary point that divides the data stored in the storage device into a front region and a rear region according to the temperature of the nucleic acid when the data stored in the storage device is expressed as a melting curve, sets the minimum temperature as a first start point and the maximum temperature as a first end point for first data defined by a predetermined number of data that continuously changes according to the temperature of the nucleic acid among the data in the front region, calculates an approximation line by a least squares method using data in a range between the first start point and the first end point, moves the range of the first data in the direction of the boundary point until the first end point reaches the boundary point, and repeats the process of setting an approximation line using each first data after the movement, sets the maximum temperature as a second start point and the minimum temperature as a second end point for second data defined by a predetermined number of data that continuously changes according to the temperature of the nucleic acid among the data in the rear region, calculates an approximation line by a least squares method using data in the range between the second start point and the second end point, a first line is set such that K1×W1+1 / r is maximum, where K1 is a first constant preset based on the plot interval between adjacent data in the first data, W1 is the temperature difference from the first start point to the first end point of the first data, and r is the slope of the approximation line calculated from the first data; a second line is set such that K2×W2+1 / r is maximum, where K2 is a second constant preset based on the plot interval between adjacent data in the second data, W2 is the temperature difference from the second start point to the second end point of the second data, and r is the slope of the approximation line calculated from the second data; and a temperature of the nucleic acid corresponding to an intersection of the melting curve and a line connecting the intermediate values ​​of absorbance of the nucleic acid at which the nucleic acid has the same temperature on the first line and the second line is detected as the melting temperature.

[0009] The detection method according to the present disclosure relates to a method for detecting the melting temperature of a nucleic acid using data showing the relationship between the temperature of the nucleic acid measured by an analysis device and the absorbance of the nucleic acid in response to ultraviolet light. The data is represented by a melting curve in which the absorbance of the nucleic acid increases with an increase in the temperature of the nucleic acid when the data is plotted with the temperature of the nucleic acid on the horizontal axis and the absorbance of the nucleic acid on the vertical axis. The method includes the steps of: storing data from the analysis device; setting a boundary point that divides the stored data into a front region and a rear region according to the temperature of the nucleic acid when the data is expressed as a melting curve; setting a start point and an end point on the melting curve in order from the point where the nucleic acid temperature is low for a first number of data in the front region that changes continuously according to the temperature of the nucleic acid; calculating an approximation line by a least squares method using the first number of data in the range between the start point and the end point; repeating the setting process of the approximation line determined from the first number of data while moving the start point and the end point one by one in the direction of the boundary point until the end point reaches the boundary point; setting a start point and an end point on the melting curve in order from the point where the nucleic acid temperature is high for a second number of data in the rear region that changes continuously according to the temperature of the nucleic acid; calculating an approximation line by a least squares method using the second number of data in the range between the start point and the end point; the width from the start point to the end point of the second number of data is W2, and the slope of the approximation line calculated from the second number of data is r; where K1 is a first constant preset based on the plot interval of adjacent first number of data on the melting curve, W1 is a width from the start point to the end point of the first number of data, and r is a gradient of the approximation line calculated from the first number of data, the steps of setting a first line that maximizes K1×W1+1 / r are as follows: the second constant preset based on the plot interval of adjacent second number of data on the melting curve is K2, W2 is a width from the start point to the end point of the second number of data, and r is a gradient of the approximation line calculated from the second number of data; and detecting, as the melting temperature, the temperature of the nucleic acid corresponding to the intersection of the melting curve and a line connecting the median values ​​of absorbance of the nucleic acid at which the nucleic acid has the same temperature on the first line and the second line.

[0010] According to the detection device and detection method of the present disclosure, the melting temperature is uniquely determined, so that detection errors in the melting temperature (Tm value) for each user can be eliminated. [Brief description of the drawings]

[0011] [Figure 1] FIG. 2 is a diagram showing the configuration of a main part of an analysis device and a detection device. [Diagram 2] FIG. 1 shows a graph of an ideal melting curve. [Diagram 3] FIG. 1 is a graph showing a melting curve according to an embodiment. [Figure 4] FIG. 13 is a graph showing a melting curve when a data range is set. [Diagram 5] FIG. 13 is a graph showing melting curves when the data range is shifted. [Figure 6] FIG. 1 shows a graph of melting curves with reduced data range. [Figure 7] 1 is a flowchart showing a process for detecting a Tm value. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The present embodiment will now be described in detail with reference to the drawings. In the drawings, the same or corresponding parts are designated by the same reference characters and, in principle, descriptions thereof will not be repeated.

[0013] An analysis device 100 to which a detection device 200 according to an embodiment is applied will be described with reference to Fig. 1. Fig. 1 is a diagram showing the configuration of the analysis device 100 and the main part of the detection device 200.

[0014] The analytical device 100 includes, as its main components, a photometric unit 1 of a so-called double-beam type ultraviolet-visible spectrophotometer, a first control device 110 that performs various calculation processes and control processes, and a sample unit 30 equipped with a multicell 32. A predetermined control program is installed in the first control device 110, and various processes described below are performed by executing this control program. A detection device 200 is connected to the analytical device 100.

[0015] The first control device 110 includes a CPU (Central Processing Unit) 111 and a memory 112. The CPU 111 executes a program stored in the memory 112 to control the operation of the photometry unit 1 and each part in the sample unit 30. The operation unit 24 connected to the first control device 110 is, for example, a keyboard, a pointing device, etc., and is used to set various parameters related to the measurement and to instruct various measurements and processes. The display unit 25 connected to the first control device 110 displays auxiliary information for operation, measurement results, etc. on a screen.

[0016] In the photometry section 1, light emitted from a light source 2 is incident on a spectroscope 3, where monochromatic light having a desired wavelength is extracted. As for the wavelength, ultraviolet light in the range of about 230 to 280 μm is often used. The monochromatic light is sent to a sector mirror 5 by a reflector 4, and is split by the sector mirror 5 into two beams, a sample-side beam S and a control-side beam R. A light shielding portion is provided in the sector mirror 5, so that a light-shielding period occurs alternately with a period during which the sample-side beam S and the control-side beam R are generated.

[0017] The sample-side light beam S is irradiated via a reflector 6 onto one of the cells of the multi-cells 32 provided in the sample unit 30. The light that has passed through one of the cells of the multi-cells 32 is sent to the light-receiving surface of the photodetector 11 via reflectors 8 and 10. On the other hand, the control-side light beam R is irradiated via a reflector 7 onto an aperture plate 31 in the sample unit 30. By passing through the aperture plate 31, the light beam diameter is made uniform with that of the sample side. The light that has passed through the aperture plate 31 is also sent to the light-receiving surface of the photodetector 11 via a reflector 9. Note that a dummy cell may be disposed in place of the aperture plate 31.

[0018] An output signal from the photodetector 11 is input to the detection device 200 via an interface section (I / F) 12 including a sample-and-hold circuit, an analog-digital converter, etc. The detection device 200 includes a second control device 210. The second control device 210 includes a CPU (Central Processing Unit) 211 and a memory 212. The CPU 211 executes various types of arithmetic processing by executing programs stored in the memory 212.

[0019] The CPU 211 executes various calculation processes for calculating absorbance using, for example, data from the analysis device 100 input to the detection device 200 and stored in the memory 212. The CPU 211 creates a melting curve, which is a graph showing the relationship between the temperature and absorbance of the nucleic acid, using the absorbance that is the calculation result. The CPU 211 detects the Tm value based on the graph of the melting curve. A specific method for detecting the Tm value will be described later.

[0020] In the sample unit 30, the multi-cell 32 is a plurality of small quartz cells with a capacity of about 10 to 100 μl arranged in a line, and the whole is configured to perform a linear reciprocating motion in a direction substantially perpendicular to the sample-side light beam S by a slide drive unit 37. This allows the sample-side light beam S to be selectively irradiated onto any of the cells. The multi-cell 32 is held in a constant temperature block 33. The constant temperature block 33 is designed to be quickly heated or cooled by a heating / cooling unit 34 using a Peltier element or the like. The constant temperature block 33 is provided with one or more temperature sensors 36. The temperature detected by the temperature sensor 36 is provided to a temperature control unit 35. The temperature control unit 35 receives a temperature control target value Tc from the CPU 111 and controls the power supplied to the heating / cooling unit 34 so that the detected temperature becomes the control target value Tc, that is, so that the difference between the detected temperature and the control target value Tc becomes zero. In the analytical device 100, by appropriately setting the control target value Tc, it is possible to measure the absorbance of a sample at any temperature within a predetermined temperature range (usually within the capacity range of the heating / cooling unit 34).

[0021] Figure 2 shows a graph of an ideal melting curve. Although noise occurs in an actual graph depending on the analysis conditions, sample state, etc., Figure 2 shows a graph of an ideal melting curve without noise. The horizontal axis of Figure 2 shows the temperature of the solution in which the nucleic acid is dissolved, and the vertical axis shows the absorbance of ultraviolet light with a wavelength of about 260 μm.

[0022] In FIG. 2, the region where the temperature is low and the rate of change in absorbance is low is called the pre-transition region. The pre-transition region is a region where the double-stranded structure gradually separates by increasing the temperature of the solution in which the nucleic acid is dissolved. In FIG. 2, the region where the temperature is high and the rate of change in absorbance is low is called the post-transition region. The post-transition region is a region where most of the nucleic acid becomes a single-stranded structure by increasing the temperature of the solution in which the nucleic acid is dissolved. In FIG. 2, the region between the pre-transition region and the post-transition region, where the rate of change in absorbance is high, is called the transition region. The transition region is a region where the double-stranded structure transitions to a single-stranded structure, and where the absorbance increases rapidly. The temperature at which the proportion of the single-stranded structure and the double-stranded structure in the transition region becomes equal is the melting temperature (Tm value).

[0023] Here, detection of melting temperature (Tm value) by a conventional method will be described. A user arbitrarily sets a start point and an end point in a portion where the slope of the graph in the pre-transition region is stable. Similarly, a user arbitrarily sets a start point and an end point in a portion where the slope of the graph in the post-transition region is stable. In calculating the melting temperature (Tm value), a line (midline) is obtained that connects the median values ​​of the absorbance of the nucleic acid at which the temperature of the nucleic acid is the same on a straight line connecting the start point and the end point in the pre-transition region and a straight line connecting the start point and the end point in the post-transition region. The melting temperature (Tm value) is detected as the temperature of the nucleic acid at the intersection of this midline and the graph of the melting curve.

[0024] Here, in the conventional method of detecting melting temperature (Tm value), the user arbitrarily sets the start point and end point. Therefore, even when the same data is used, the start point and end point of the Tm value may vary from user to user, resulting in detection errors. In addition, noise may occur in the actual graph depending on the analysis conditions, the state of the sample, etc. Therefore, when such a graph is used, there is a possibility that the error in the detection of the Tm value may become large. According to the detection device 200 and the detection method using the detection device 200 of the present disclosure, the error in the detection of the Tm value for each user can be eliminated. A specific method of detecting the Tm value will be described below.

[0025] FIG. 3 is a diagram showing a graph of a melting curve according to an embodiment. As shown in FIG. 3, the actual graph is a graph in which noise is superimposed on the ideal graph of FIG. 2. The process shown below will be described as a process executed by the CPU 211 in the second control device 210 of the detection device 200. The CPU 211 determines a boundary point in the graph of the melting curve. The boundary point is a point on the graph that is set in a region corresponding to the transition region, and is set, for example, at the median value of the absorbance data. The region before the boundary point is the front region, and the region after the boundary point is the rear region.

[0026] FIG. 4 is a diagram showing a graph of a melting curve when a data range ΔT is set. The CPU 211 sets the data range ΔT with the lowest temperature as the start point and the highest temperature as the end point for the first data defined by a predetermined number of data in the range of the previous region. The data range ΔT is a temperature range that can be arbitrarily set by the user. The data range ΔT may be set automatically by the CPU 211. Setting the data range ΔT also means setting the number of data points in the range including the start point and the end point. The CPU 211 calculates an approximate straight line by the least squares method using data in the range including the two points at both ends of the data range ΔT. The least squares method is a method of finding the most likely relational expression by minimizing the sum of the squares of the errors in processing measured values ​​that include errors.

[0027] In the detection device 200 of this embodiment, the set data range ΔT is moved and reduced, and the process of setting an approximate line is repeated for the moved and reduced data. In the detection device 200 of this embodiment, the most appropriate approximate line from among the multiple approximate lines obtained is set in each of the front and rear regions. This makes it possible to set an appropriate approximate line compared to the case where the data range ΔT is simply set only once to obtain an approximate line. Below, the case of moving the data range ΔT and the case of reducing the data range ΔT will be explained using graphs.

[0028] FIG. 5 is a diagram showing a graph of a melting curve when the data range ΔT is moved. The CPU 211 moves the data range ΔT in the range of the previous region in the direction of the boundary point until the end point reaches the boundary point. The CPU 211 calculates an approximate line by the least squares method using the data of the moved data range ΔT as the calculation target. The CPU 211 repeats the calculation process of the approximate line until the end point reaches the boundary point. FIG. 5 shows a situation when the initial position is moved by multiple points. Note that the initial position of the start point may be the point on the graph with the lowest temperature, or may be a point moved several points toward the boundary point. The approximate line on the graph in the previous region has a characteristic that the slope of the approximate line is greater when the temperature is high than when the temperature is low.

[0029] FIG. 6 is a diagram showing a graph of a melting curve when the data range ΔT is reduced. After executing a process of moving the end point of the data in the data range ΔT to the boundary point and setting an approximate straight line, the CPU 211 similarly executes a process of setting an approximate straight line in the data range ΔT×α, which is obtained by multiplying the data in the data range ΔT as shown in FIG. 6 by a predetermined ratio α and reducing the number of data points. α is a value that can be arbitrarily set by the user. For example, the user may reduce the value in increments of 0.1 (10%) within a range from 0.9 (90%) to 0.2 (20%). Note that if the value obtained by multiplying ΔT by α is not an integer, the number of data points may be set within the range of integer values ​​by rounding down the decimal point.

[0030] Next, the Tm value detection process will be specifically described. Fig. 7 is a flowchart showing the Tm value detection process. The Tm value detection process is performed by CPU 211 executing various programs stored in memory 212. In the following, each step in the flowchart will simply be represented as "S".

[0031] In the process of detecting the Tm value, the CPU 211 acquires information on the sampling points (S11). Specifically, the CPU 211 calculates the absorbance using data input from the analysis device 100 to the memory 212 of the detection device 200, and creates data showing the relationship between the temperature of the nucleic acid and the calculated absorbance. This data is expressed as a melting curve in which the absorbance of the nucleic acid increases with an increase in the temperature of the nucleic acid when the data is plotted with the temperature of the nucleic acid on the horizontal axis and the absorbance of the nucleic acid on the vertical axis. The CPU 211 acquires data on the melting curve created from a plurality of points as information on the sampling points.

[0032] Next, CPU211 sets a boundary point that is the boundary between the front region and the rear region as shown in Fig. 3 (S12). CPU211 sets a point obtained as the median value of the absorbance data as the boundary point. Next, CPU211 sets temperature range data of a data range ΔT in the range of the front region as shown in Fig. 4 (S13). The process of S13 also sets the minimum temperature of the data range ΔT in the front region as the start point and the maximum temperature as the end point, and sets the number of data points in the data range ΔT.

[0033] Next, the CPU 211 performs linear fitting by the least squares method using the data between ΔT as shown in Fig. 4 (S14). The CPU 211 repeats the process of setting an approximated line using each piece of data after moving while shifting the data range one point at a time in the direction of the boundary point until the end point reaches the boundary point in S14. The CPU 211 repeats the process of S14 in a state where the process of setting the approximated line is not completed, such as when the process of S15 described later is NO, or when the process of S18 is NO.

[0034] Next, the CPU 211 judges whether the end point of the data range ΔT has reached the boundary point (S15). If the CPU 211 judges that the end point of the data range ΔT has not reached the boundary point (NO in S15), it executes the process of S14. If the CPU 211 judges that the end point of the data range ΔT has reached the boundary point (YES in S15), it reduces the data range ΔT by 10% (S16). The process of S16 reduces the width of the data range ΔT and also reduces the number of data points. The reduction rate can be set arbitrarily by the user. If the value obtained by multiplying the data range ΔT by the reduction rate is not an integer, the decimal point is rounded down and the number of data points can be set within the range of integer values.

[0035] Next, the CPU 211 judges whether the data range ΔT has decreased to 20% or less (S17). 20% is a lower limit value that can be arbitrarily set by the user. However, if the number of data points is reduced too much, the influence of data errors becomes large, which hinders accurate detection of the Tm value, so it is desirable to set the reduction rate to about 20%. If the CPU 211 judges that the data range ΔT has not decreased to 20% or less (NO in S17), it judges whether the number of sampling points has decreased to less than two in S18. The number of sampling points being less than two (single sampling point) means that the setting process of linear fitting is not executed. Note that even if the data range ΔT has decreased to 20% or less, it is not usually expected that the number of sampling points will be less than two, but the process of S18 is provided as an error process in case some error occurs.

[0036] If the CPU 211 determines that the number of sampling points is not less than two (NO in S18), it repeats the process of S14. If the CPU 211 determines that the data range ΔT has decreased to 20% or less in S17 (YES in S17) or if the CPU 211 determines that the number of sampling points is less than two in S18 (YES in S18), it proceeds to the process of S19. The CPU 211 determines in S19 whether the setting process of the straight line fitting in the rear region has been completed.

[0037] When the CPU 211 determines that the setting process of the linear fitting in the rear region is not completed (NO in S19), it inverts the data of the rear region with the boundary point as the center (S20). Here, in the rear region, the maximum temperature of the data range ΔT is set as the start point and the minimum temperature as the end point, but in S20, the data is inverted in order to use the processing method of the front region for the processing of the rear region. The processing of S20 is a processing of inverting the plot position of the data of the rear region so as to rotate the melting curve point-symmetrically with the boundary point as the base. In other words, in the processing of S20, a processing of changing the data is executed so as to invert the data of each sampling point in the rear region corresponding to the temperature of the nucleic acid and the absorbance of the nucleic acid with the boundary point as the base.

[0038] Next, the CPU 211 sets data of the temperature range of the data range ΔT in the range of the subsequent region (S21) as executed in S13 of Fig. 7, and proceeds to the process of S14. The process of S21 also sets the maximum temperature of the data range ΔT in the subsequent region as the start point and the minimum temperature as the end point, and sets the number of data points of the data range ΔT. Note that in S21, since the data was inverted in S20, in the actual process, the start point is the minimum temperature of the data range ΔT, and the end point is the maximum temperature of the data range ΔT.

[0039] By performing a process of inverting the data of the rear region based on the boundary point in S20, the CPU 211 can standardize the processes of S14 to S18 and reduce the processing load. Note that, without performing the process of S20, the maximum temperature in the data range ΔT in the rear region may be set as the start point and the minimum temperature as the end point, and the data range ΔT may be moved until the end point reaches the boundary point, and linear fitting may be performed using each data after the movement.

[0040] When the CPU 211 determines that the setting process of the straight line fitting in the rear region is completed (YES in S19), it ends the calculation process of the straight line fitting (S22). Next, the CPU 211 sets the straight line that is the maximum value in the front region and the rear region from the relational expression between the gradient of the obtained approximate straight line and the weighting as the fitting line (S23).

[0041] The process of S23 will be specifically described. When a simulation is performed with the plot interval (temperature difference) between adjacent data in the melting curve as W, the weighting factor K as a constant that increases in proportion to the change in W, and C as a constant previously set in memory 212, the relationship K=W×C is established. In this way, the weighting factor K changes based on the plot interval of the data. The weighting factor K1 is a first constant previously set based on the plot interval of the data in the data range ΔT of the front region, and the weighting factor K2 is a second constant previously set based on the plot interval of the data in the data range ΔT of the rear region.

[0042] In the process of S23, if the temperature difference from the start point to the end in the front region is W1 and the slope of the approximation line obtained in S14 is r, the approximation line where A1 is maximum in A1=K1×W1+1 / r is set as the first line as the fitting line in the front region. In the process of S23, if the temperature difference from the start point to the end in the rear region is W2 and the slope of the approximation line obtained in S14 is r, the approximation line where A2 is maximum in A2=K2×W2+1 / r is set as the fitting line in the rear region as the second line.

[0043] Next, CPU 211 draws the fitting line set in the processing of S23 between the front and rear regions, detects the temperature of the nucleic acid at the intersection of the melting curve and the fitting line using the midline method as the Tm value (melting temperature), outputs it to display unit 25 (S24), and ends the processing.

[0044] Here, a specific example of actual measurement of nucleic acid will be described. Reagent M13-25mer is used as nucleic acid, the concentration of the reagent is 20 μM, the optical path length is 1 mm, and the measurement temperature is 40 to 95° C. When the measurement is performed by the analysis device 100 under such conditions, the data range ΔT of the front region can be set to a range of about 30° C., and the data range ΔT of the rear region can be set to a range of about 15° C. In the reagent M13-25mer, the rear region is narrower than the front region, so the data range ΔT of the rear region is smaller than the data range ΔT of the front region. In this way, the data range ΔT changes depending on various conditions. In the detection device 200 of this embodiment, regardless of how the data range ΔT is set, the Tm value can be uniquely detected by the process of FIG. 7.

[0045] 7, in the detection device 200, the CPU 211 sets fitting lines for the anterior region and posterior region one by one from a plurality of approximation straight lines by the least squares method. The CPU 211 detects the temperature of the nucleic acid at the intersection of the melting curve and the fitting line by the median method as the Tm value (melting temperature) from the first straight line which is the fitting line in the anterior region and the second straight line which is the fitting line in the posterior region, and outputs it to the display unit 25. In this way, in the detection device 200, the fitting lines are automatically determined one by one in the anterior region and the posterior region, so that it is possible to eliminate detection errors of the melting temperature (Tm value) for each user.

[0046] [Aspects] It will be appreciated by those skilled in the art that the exemplary embodiments described above are examples of the following aspects.

[0047] (Item 1) A detection device according to one embodiment detects the melting temperature of a nucleic acid using data indicating the relationship between the temperature of the nucleic acid measured by an analysis device and the nucleic acid's absorbance in response to ultraviolet light. The detection device includes a control device and a storage device that stores data from the analysis device. The data is represented by a melting curve in which the absorbance of the nucleic acid increases with an increase in the temperature of the nucleic acid when the data is plotted with the temperature of the nucleic acid on the horizontal axis and the absorbance of the nucleic acid on the vertical axis. The control device sets a boundary point that divides the data stored in the storage device into a front region and a rear region according to the temperature of the nucleic acid when the data stored in the storage device is expressed as a melting curve, sets the minimum temperature as a first start point and the maximum temperature as a first end point for first data defined by a predetermined number of data that continuously changes according to the temperature of the nucleic acid among the data in the front region, calculates an approximation line by a least squares method using data in a range between the first start point and the first end point, moves the range of the first data in the direction of the boundary point until the first end point reaches the boundary point, and repeats the process of setting an approximation line using each first data after the movement, sets the maximum temperature as a second start point and the minimum temperature as a second end point for second data defined by a predetermined number of data that continuously changes according to the temperature of the nucleic acid among the data in the rear region, calculates an approximation line by a least squares method using data in the range between the second start point and the second end point, a first line is set such that K1×W1+1 / r is maximum, where K1 is a first constant preset based on the plot interval between adjacent data in the first data, W1 is the temperature difference from the first start point to the first end point of the first data, and r is the slope of the approximation line calculated from the first data; a second line is set such that K2×W2+1 / r is maximum, where K2 is a second constant preset based on the plot interval between adjacent data in the second data, W2 is the temperature difference from the second start point to the second end point of the second data, and r is the slope of the approximation line calculated from the second data; and a temperature of the nucleic acid corresponding to an intersection of the melting curve and a line connecting the intermediate values ​​of absorbance of the nucleic acid at which the nucleic acid has the same temperature on the first line and the second line is detected as the melting temperature.

[0048] According to the detection device described in paragraph 1, the melting temperature is uniquely detected, so that detection errors of the melting temperature for each user can be eliminated.

[0049] (Clause 2) In the detection device described in paragraph 1, the control device reduces the number of data points of the first data by a predetermined number in the front region, changes W1 according to the reduced number of data points, and repeats the process of setting the first line, reduces the number of data points of the second data by a predetermined number in the rear region, changes W2 according to the reduced number of data points, and repeats the process of setting the second line, and detects, as the melting temperature, the temperature of the nucleic acid corresponding to the intersection of the melting curve and a line connecting the midpoints of the absorbance of the nucleic acid where the temperature of the nucleic acid is the same in the first line and the second line.

[0050] According to the detection device described in paragraph 2, the first line and the second line are set after the number of pieces of data is reduced by a predetermined number at a time, so that detection errors in the melting temperature can be eliminated.

[0051] (Clause 3) In the detection device described in paragraph 1 or 2, the control device terminates the setting process of the first line when the number of data items in the first data reaches a predetermined lower limit, and terminates the setting process of the second line when the number of data items in the second data reaches a predetermined lower limit.

[0052] According to the detection device described in paragraph 3, since the lower limit value is determined, it is possible to avoid using data in an unnecessary range that would increase detection errors.

[0053] (4) In the detection device described in any one of paragraphs 1 to 3, the control device inverts the plot position of the data in the rear region so as to rotate the dissolution curve point-symmetrically based on the boundary point, and after executing the setting process of the second straight line, inverts the plot position of the data of the set second straight line again based on the boundary point.

[0054] According to the detection device described in paragraph 4, the rear region can be processed in the same manner as the front region, so that the processing load can be reduced.

[0055] (Item 5) A detection method according to one embodiment relates to a method for detecting the melting temperature of a nucleic acid using data showing the relationship between the temperature of the nucleic acid measured by an analysis device and the nucleic acid's absorbance in response to ultraviolet light. The data is represented by a melting curve in which the absorbance of the nucleic acid increases with an increase in the temperature of the nucleic acid when the data is plotted with the nucleic acid temperature on the horizontal axis and the nucleic acid absorbance on the vertical axis. The method includes the steps of: storing data from the analysis device; setting a boundary point that divides the stored data into a front region and a rear region according to the temperature of the nucleic acid when the data is expressed as a melting curve; setting a start point and an end point on the melting curve in order from the point where the nucleic acid temperature is low for a first number of data in the front region that changes continuously according to the temperature of the nucleic acid; calculating an approximation line by a least squares method using the first number of data in the range between the start point and the end point; repeating the setting process of the approximation line determined from the first number of data while moving the start point and the end point one by one in the direction of the boundary point until the end point reaches the boundary point; setting a start point and an end point on the melting curve in order from the point where the nucleic acid temperature is high for a second number of data in the rear region that changes continuously according to the temperature of the nucleic acid; calculating an approximation line by a least squares method using the second number of data in the range between the start point and the end point; the width from the start point to the end point of the second number of data is W2, and the slope of the approximation line calculated from the second number of data is r; where K1 is a first constant preset based on the plot interval of adjacent first number of data on the melting curve, W1 is a width from the start point to the end point of the first number of data, and r is a gradient of the approximation line calculated from the first number of data, the steps of setting a first line that maximizes K1×W1+1 / r are as follows: the second constant preset based on the plot interval of adjacent second number of data on the melting curve is K2, W2 is a width from the start point to the end point of the second number of data, and r is a gradient of the approximation line calculated from the second number of data; and detecting, as the melting temperature, the temperature of the nucleic acid corresponding to the intersection of the melting curve and a line connecting the median values ​​of absorbance of the nucleic acid at which the nucleic acid has the same temperature on the first line and the second line.

[0056] According to the detection method described in item 5, the melting temperature is uniquely detected, so that detection errors of the melting temperature for each user can be eliminated.

[0057] (Clause 6) The detection method described in clause 5 executes the steps of reducing the width of a first number of data by a predetermined percentage in the front region, changing W1 according to the reduced width, and repeating the process of setting a first line, reducing the width of a second number of data by a predetermined percentage in the rear region, changing W2 according to the reduced width, and repeating the process of setting a second line, and detecting, as the melting temperature, the temperature of the nucleic acid corresponding to the intersection of the melting curve and a line connecting the midpoints of the absorbance of the nucleic acid where the temperature of the nucleic acid is the same on the first line and the second line.

[0058] According to the detection method described in item 6, the first straight line and the second straight line are set after the width is reduced by a predetermined percentage, so that detection errors in the melting temperature can be eliminated.

[0059] (Clause 7) The detection method described in clause 5 or 6 includes a step of terminating the process of setting a first straight line when the number of plots of the first number of data reaches a predetermined lower limit, and a step of terminating the process of setting a second straight line when the number of plots of the second number of data reaches a predetermined lower limit.

[0060] According to the detection method described in paragraph 7, since a lower limit value is determined, it is possible to avoid using data in an unnecessary range that would increase detection errors.

[0061] (Item 8) The detection method described in any one of Items 5 to 7 includes a step of inverting the plot position of the data in the posterior region so as to rotate the dissolution curve point-symmetrically with respect to the boundary point, executing a process for setting a second straight line, and then again inverting the plot position of the data of the set second straight line with respect to the boundary point.

[0062] According to the detection method described in item 8, the rear region can be processed in the same manner as the front region, so that the processing load can be reduced.

[0063] The embodiments disclosed herein should be considered to be illustrative and not restrictive in all respects. The scope of the present disclosure is defined by the claims, not by the description of the embodiments described above, and is intended to include all modifications within the meaning and scope of the claims. [Explanation of symbols]

[0064] 1 photometry unit, 2 light source, 3 spectroscope, 4, 6, 7, 8, 9 reflector, 5 sector mirror, 11 photodetector, 24 operation unit, 25 display unit, 30 sample unit, 31 aperture plate, 32 multicell, 33 constant temperature block, 34 hot / cold unit, 35 temperature control unit, 36 temperature sensor, 37 slide drive unit, 100 analysis device, 110 first control device, 111, 211 CPU, 112, 212 memory, 200 detection device, 210 second control device.

Claims

1. A detection device that detects the melting temperature of a nucleic acid using data indicating the relationship between the temperature of the nucleic acid measured by an analysis device and the absorbance of the nucleic acid corresponding to ultraviolet light, a control device; a storage device that stores the data from the analysis device; the data is represented by a melting curve in which the absorbance of the nucleic acid increases with an increase in the temperature of the nucleic acid when the data is plotted with the temperature of the nucleic acid on the horizontal axis and the absorbance of the nucleic acid on the vertical axis; The control device When the data stored in the storage device is expressed as the melting curve, a boundary point is set that divides the data into a front region and a rear region according to the temperature of the nucleic acid; Among the data in the preceding region, the first data is defined by a predetermined number of data that continuously change depending on the temperature of the nucleic acid, and the lowest temperature is set as a first start point and the highest temperature is set as a first end point; calculating an approximate straight line by a least squares method using data in the range between the first start point and the first end point; repeating a process of setting an approximate straight line using each of the first data after the movement while moving the range of the first data in a direction toward the boundary point until the first end point reaches the boundary point; Among the data in the subsequent region, second data is defined by a predetermined number of data that continuously change depending on the temperature of the nucleic acid, and the highest temperature is set as a second start point and the lowest temperature is set as a second end point; calculating an approximate straight line by a least squares method using data in the range between the second start point and the second end point; repeating a process of setting an approximate straight line using each second data item after the movement while moving the range of the second data item in a direction toward the boundary point until the second end point reaches the boundary point; a first straight line that maximizes K1×W1+1 / r, where K1 is a first constant that is preset based on the plot interval between adjacent data in the first data, W1 is a temperature difference from the first start point to the first end point of the first data, and r is a gradient of an approximation straight line calculated from the first data; a second straight line that maximizes K2 × W2 + 1 / r, where K2 is a second constant that is preset based on the plot interval between adjacent data in the second data, W2 is a temperature difference from the second start point to the second end point of the second data, and r is a gradient of an approximation straight line calculated from the second data; A detection device that detects, as the melting temperature, the temperature of the nucleic acid corresponding to the intersection of the melting curve and a line connecting the median values ​​of absorbance of the nucleic acid where the temperature of the nucleic acid is the same on the first straight line and the second straight line.

2. The control device the number of data points of the first data is reduced by a predetermined number in the previous region, W1 is changed in accordance with the reduced number of data points, and the process of setting the first straight line is repeated; the number of data points in the second data set is reduced by a predetermined number in the rear area, W2 is changed in accordance with the reduced number of data points, and the process of setting the second straight line is repeated; The detection device according to claim 1, wherein the melting temperature is detected as the temperature of the nucleic acid corresponding to the intersection of the melting curve and a line connecting the median values ​​of absorbance of the nucleic acid where the temperature of the nucleic acid is the same on the first straight line and the second straight line.

3. The control device When the number of data of the first data reaches a predetermined lower limit value, the setting process of the first straight line is terminated. The detection device according to claim 2 , wherein the process of setting the second line is terminated when the number of data in the second data reaches a predetermined lower limit value.

4. 4. The detection device according to claim 1, wherein the control device inverts the plot position of the data of the posterior region so as to rotate the melting curve point-symmetrically based on the boundary point, and after executing the setting process of the second straight line, inverts the plot position of the data of the set second straight line again based on the boundary point.

5. A detection method for detecting the melting temperature of a nucleic acid using data indicating a relationship between the temperature of the nucleic acid measured by an analyzer and the absorbance of the nucleic acid corresponding to ultraviolet light, comprising: the data is represented by a melting curve in which the absorbance of the nucleic acid increases with an increase in the temperature of the nucleic acid when the data is plotted with the temperature of the nucleic acid on the horizontal axis and the absorbance of the nucleic acid on the vertical axis; The detection method includes: a step of setting a boundary point that divides the stored data into a front region and a rear region according to the temperature of the nucleic acid when the data is represented as the melting curve; setting the lowest temperature as a first start point and the highest temperature as a first end point for first data defined by a predetermined number of data that continuously change depending on the temperature of the nucleic acid among the data in the previous region; calculating an approximate straight line by a least squares method using data in the range between the first start point and the first end point; a step of repeating a process of setting an approximate line using each of the first data after the movement while moving the range of the first data in a direction toward the boundary point until the first end point reaches the boundary point; setting the highest temperature as a second start point and the lowest temperature as a second end point for second data defined by a predetermined number of data that continuously change depending on the temperature of the nucleic acid among the data in the subsequent region; calculating an approximate straight line by a least squares method using data in the range between the second start point and the second end point; a step of repeating a process of setting an approximate line using each second data item after the movement while moving the range of the second data item in a direction toward the boundary point until the second end point reaches the boundary point; a step of setting a first straight line that maximizes K1×W1+1 / r, where K1 is a first constant that is preset based on the plot interval between adjacent data in the first data, W1 is a temperature difference from the first start point to the first end point of the first data, and r is a gradient of an approximate straight line calculated from the first data; a step of setting a second straight line that maximizes K2×W2+1 / r, where K2 is a second constant that is preset based on the plot interval between adjacent data in the second data, W2 is a temperature difference from the second start point to the second end point of the second data, and r is a gradient of an approximate straight line calculated from the second data; and detecting, as the melting temperature, the temperature of the nucleic acid corresponding to the intersection of the melting curve with a line connecting the median values ​​of absorbance of the nucleic acid where the temperature of the nucleic acid is the same on the first straight line and the second straight line.

6. The detection method includes: a step of reducing the number of data points of the first data in the previous region by a predetermined number, changing W1 in accordance with the reduced number of data points, and repeating the process of setting the first straight line; a step of reducing the number of data points of the second data in the rear region by a predetermined number, changing W2 in accordance with the reduced number of data points, and repeating the process of setting the second straight line; and detecting, as the melting temperature, the temperature of the nucleic acid corresponding to the intersection of the melting curve with a line connecting the median values ​​of absorbance of the nucleic acid at which the temperature of the nucleic acid is the same on the first straight line and the second straight line.

7. The detection method includes: a step of terminating the setting process of the first straight line when the number of data of the first data reaches a predetermined lower limit value; The detection method according to claim 6 , further comprising the step of terminating the process of setting the second line when the number of data in the second data reaches a predetermined lower limit value.

8. The detection method according to any one of claims 5 to 7, further comprising the steps of: inverting the plot position of the data of the posterior region so as to rotate the melting curve point-symmetrically with respect to the boundary point; executing the setting process of the second straight line; and then again inverting the plot position of the data of the set second straight line with respect to the boundary point.

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