Analysis method

Abstract

The purpose of the present invention is to appropriately generate a crosstalk correction matrix by suppressing the influence of pull-up / pull-down and baseline noise on a matrix that corrects crosstalk. An analysis method according to the present invention comprises a step for creating a correction matrix for correcting spatial crosstalk observed as if fluorescence generated in a first capillary was generated in a second capillary, wherein at least one of a matrix standard serving as a reference for creating the correction matrix and a biological sample is adjusted so that the fluorescence signal intensity of the matrix standard is greater than a first reference value (see Fig. 5).

Description

Analysis method

[0001] The present invention relates to a method for analyzing a sample using electrophoresis.

[0002] A multi-capillary electrophoresis device that fills a plurality of capillaries with an electrophoresis separation medium such as an electrolyte solution, or an electrolyte solution containing a polymer gel or polymer, and performs electrophoresis analysis in parallel is widely used. In recent years, an electrophoresis method that realizes a high dynamic range and high sensitivity proposed in Non-Patent Document 1 has been desired.

[0003] There is a phenomenon in which the fluorescence of phosphors is mixed between a plurality of different capillaries. A signal generated in a certain capillary propagates as noise to an adjacent capillary and becomes a noise component. This is called spatial crosstalk. Also, since the fluorescence spectra of each phosphor electrophoresed in one capillary overlap with each other in the fluorescence wavelength components, the fluorescence of a plurality of types of phosphors may be mixed in an arbitrary wavelength band. In the present disclosure, this is called spectral crosstalk.

[0004] Patent Document 1 describes a method for reducing spatial crosstalk by calculation processing. This method cancels spatial crosstalk by previously obtaining the ratio of spatial crosstalk between different capillaries and subtracting the contribution of spatial crosstalk based on that ratio.

[0005] Patent Document 2 describes a correction method using a matrix (referred to as a spectral-spatial crosstalk correction matrix in the present disclosure) that corrects spectral crosstalk and spatial crosstalk simultaneously. The spectral-spatial crosstalk correction matrix calculates in advance the spectral crosstalk ratio and the spatial crosstalk ratio of the fluorescence signal peak, and cancels the spectral crosstalk and the spatial crosstalk by subtracting the contributions of the spectral crosstalk and the spatial crosstalk based on that ratio.

[0006] When calculating the spatial crosstalk ratio in advance, it is necessary to generate fluorescence in only one of several capillaries and measure the fluorescence propagation to the other capillaries. Furthermore, it is necessary to obtain all information regarding spatial crosstalk by sequentially performing this measurement in all capillaries.

[0007] Patent document 3 discloses a method for detecting fluorescence at different timings for each capillary. As one embodiment, the document discloses a method of injecting a phosphor contained in a single sample container into multiple different capillaries.

[0008] Patent document 4 describes a method for correcting spectral crosstalk using matrix calculations.

[0009] Non-patent document 2 reports that a correction error of about 1% occurs in spectral crosstalk. This correction error is caused by factors such as changes in experimental conditions, including temperature drift and polymer degradation, and signal saturation of the signal being corrected.

[0010] US7402817 JP 2023-101563 A JP 7340095 US9170198

[0011] Anazawa, T., et al. (2020). Highly sensitive mutation quantification by high-dynamic-range capillary-array electrophoresis (HiDy CE). Lab on a Chip, 20(6), 1083-1091.Koji, Fujii, et al. (2018). Ratios and distances of pull-up peaks observed in GlobalFiler kit data. Medicine Legal, 34, 58-63.

[0012] When generating a matrix used to correct crosstalk using matrix calculations, a sample called a matrix standard is subjected to electrophoresis, and the resulting fluorescence signal is used to generate the matrix. If the signal-to-noise ratio (S / N) of the spatial crosstalk information is low at this time, correction errors called pull-up or pull-down occur. Also, if the crosstalk appearing in the fluorescence signal of the matrix standard is small, that crosstalk may be buried in the baseline noise.

[0013] The aforementioned prior art documents do not adequately consider the effects of pull-up / pull-down and how to distinguish them from baseline noise when creating crosstalk correction matrices. Therefore, these prior art documents may have difficulty generating appropriate crosstalk correction matrices due to these effects.

[0014] This invention has been made in view of the above-mentioned problems, and aims to appropriately generate a crosstalk correction matrix by suppressing the effects of pull-up / pull-down and baseline noise on the crosstalk correction matrix.

[0015] The analytical method according to the present invention includes the step of creating a correction matrix to correct spatial crosstalk in which fluorescence generated in a first capillary is observed as if it were generated in a second capillary, and adjusting at least one of the matrix standard or biological sample so that the fluorescence signal intensity of the matrix standard used as a reference for creating the correction matrix is ​​greater than a first reference value.

[0016] According to the analysis method of the present invention, a crosstalk correction matrix can be appropriately generated by suppressing the effects of pull-up / pull-down and baseline noise on the crosstalk correction matrix. Other problems, configurations, and advantages of the present invention will become clear from the following description of embodiments.

[0017] This figure shows an overview of the configuration of the electrophoresis apparatus 100 according to Embodiment 1. This figure shows an overview of the configuration of the fluorescence detection device 115. This shows an example of the configuration of the computer 130. This figure explains the procedure by which the computer 130 performs crosstalk correction. This is a flowchart explaining the operation for capillary i in step 401. This shows a specific example of step 506. This shows a chromatogram when a matrix standard is electrophoresed without applying the present invention. This shows a chromatogram when a matrix standard is electrophoresed without applying the present invention. This shows a chromatogram when a matrix standard is electrophoresed with the present invention applied and the signal intensity brought close to the upper limit of the apparatus. This shows a chromatogram when a biological sample is electrophoresed. This shows a chromatogram when a biological sample is electrophoresed. This is the result after correction by the spectral space crosstalk correction matrix. This is the result after correction by the spectral space crosstalk correction matrix. This figure shows the flow of the analysis method according to Embodiment 2. This shows the flow up to crosstalk correction in Embodiment 3. This shows the details of step 1502. This shows a specific example of step 1606.

[0018] A preferred embodiment of the spatial crosstalk correction matrix creation method according to the present invention will be described below with reference to the attached drawings. In the following description and attached drawings, components having the same functional configuration will be denoted by the same reference numerals to avoid redundant explanations.

[0019] <Basic Principle of the Invention> When creating a crosstalk correction matrix, the fluorescence signal intensity is obtained by electrophoresis of a matrix standard, and the fluorescence signal intensity of spatial crosstalk generated in adjacent capillaries is measured. If the signal-to-noise ratio (S / N) of the fluorescence signal intensity of spatial crosstalk is insufficient, it is not possible to appropriately set the correction value for correcting spatial crosstalk. In other words, it is not possible to properly create a crosstalk correction matrix. Therefore, a method is needed to ensure a sufficient S / N ratio of the fluorescence signal intensity of spatial crosstalk in adjacent capillaries.

[0020] Through the inventors' research, it was found that by making the fluorescence signal intensity of the matrix standard greater than that of the biological sample, a sufficient signal-to-noise ratio (S / N) for spatial crosstalk fluorescence signal intensity can be ensured. In the embodiments of the present invention described below, the procedure for creating a crosstalk correction matrix using the above principle will be explained.

[0021] <Embodiment 1> [System Configuration] Figure 1 is a diagram showing an overview of an example configuration of an electrophoresis apparatus 100 according to Embodiment 1 of the present invention. As shown in Figure 1, the electrophoresis apparatus 100 has at least a measuring unit 110 and a computer 130.

[0022] (Measurement unit 110) The measurement unit 110 includes a capillary array 111, a pump unit 112, a high-voltage power supply 113, a constant temperature bath 114, a fluorescence detection device 115, a sample tray 116, and a transporter 117.

[0023] The capillary array 111 is composed of multiple capillaries 118. Each capillary 118 is hollow.

[0024] The pump unit 112 injects the electrophoretic medium M (e.g., polymer) into each capillary 118. This fills the inside of each capillary 118 with the electrophoretic medium M.

[0025] The high-voltage power supply 113 applies a high voltage to each end of the capillary 118 filled with the electrophoretic medium M.

[0026] The constant temperature bath 114 maintains a constant temperature inside the capillary 118.

[0027] The phosphor is contained in a sample container. The sample tray 116 contains multiple sample containers 119. The transporter 117 transports the sample tray 116 and inserts the tip of the capillary 118 into each sample container 119.

[0028] The phosphor has a negative charge and moves according to the electric field generated inside the capillary 118. The fluorescence detection device 115 irradiates the phosphor that has moved inside the capillary 118 with excitation light R1 at the fluorescence detection position 120, causing the phosphor to emit fluorescence. The phosphor is then discharged into the discharge container 121. The fluorescence detection device 115 detects the fluorescence R2 emitted from the phosphor. The detailed configuration of the fluorescence detection device 115 will be described later. With this configuration in the measurement unit 110, it is possible to simultaneously measure samples being electrophoresed inside multiple capillaries 118.

[0029] In Embodiment 1, a fluorescently labeled DNA fragment is assumed as the phosphor that passes through the inside of the capillary 118, but a phosphor other than a fluorescently labeled DNA fragment may be used. Also, the applied voltage may be reversed so that the phosphor passes through the fluorescence detection position 120. The number of capillaries 118 is not limited to 8, as long as there are two or more.

[0030] (Fluorescence detection device 115) Figure 2 is a diagram showing an overview of the configuration of the fluorescence detection device 115. As shown in Figure 2, the fluorescence detection device 115 includes an excitation light source 201, a shutter 202, an excitation light lens 203, an optical filter 204, a fluorescence lens 205, a diffraction grating 206, a CCD image sensor 210, and a conversion unit 220.

[0031] The excitation light source 201 emits excitation light R1. The excitation light source 201 is positioned so that the excitation light R1 is irradiated onto all capillaries 118 in the capillary array 111 that pass through the fluorescence detection position 120. The shutter 202 opens and closes repeatedly at predetermined intervals. When the shutter 202 is open, the excitation light R1 from the excitation light source 201 is irradiated onto the capillaries 118. When the shutter 202 is closed, the irradiation of the excitation light R1 to the capillaries 118 is blocked. The excitation light lens 203 focuses the excitation light R1 that has passed through the shutter 202. The excitation light R1 focused by the excitation light lens 203 is irradiated towards the fluorescence detection position 120.

[0032] The phosphors undergoing electrophoresis inside each capillary 118 are excited by excitation light R1 and emit fluorescence R2.

[0033] The optical filter 204 (for example, a color filter) cuts out light other than the fluorescent R2 emitted from the phosphor. The fluorescent lens 205 focuses the fluorescent R2 that has passed through the optical filter 204. The diffraction grating 206 spectrally separates the fluorescent R2 focused by the fluorescent lens 205 into wavelengths. The CCD image sensor 210 receives the fluorescent R2 spectrally separated by the diffraction grating 206 and outputs a charge of an intensity corresponding to the signal intensity of the fluorescent R2.

[0034] The conversion unit 220 includes a charge conversion unit 221 and a digital conversion unit 222, which is an ADC (Analog Digital Converter).

[0035] The charge conversion unit 221 converts the charge output from the CCD image sensor 210 into a voltage and outputs the converted voltage as an analog signal. The digital conversion unit 222 converts the analog signal output from the charge conversion unit 221 into a digital signal. The digital conversion unit 222 outputs the converted digital signal to the computer 130.

[0036] As the CCD image sensor 210, any of the following types can be applied: frame transfer type, full frame transfer type, interline transfer type, or frame interline transfer type. If the frame transfer type, interline transfer type, or frame interline transfer type is applied, it becomes unnecessary to provide a shutter 202. Instead of the CCD image sensor 210, a CMOS (Complementary Metal Oxide Semiconductor) image sensor may be applied for the detection of fluorescence R2.

[0037] (Computer 130) Figure 3 shows an example of the configuration of computer 130. Computer 130 includes a CPU (Central Processing Unit) 301, memory 302, storage 303, display unit 304, input unit 305, and NIF (Network Interface) 306.

[0038] The computer 130 is connected to the measurement unit 110. The computer 130 not only performs data analysis but also controls the electrophoresis apparatus 100. In addition to the output of the fluorescence detector 115, it can acquire data from the measurement unit 110, such as the temperature of the constant temperature bath 114 and the position information of the conveyor 117.

[0039] Memory 302 temporarily stores the digital signals output by the digital conversion unit 222 and stores the digital signals in storage 303. The user sets the data analysis conditions and electrophoresis apparatus control conditions through the input unit 305. The time-series raw data of signal intensity output from the electrophoresis apparatus 100 is sequentially stored in memory and displayed on the display unit 304. The computer 130 can compare the analysis results with information on the network through the network interface NIF 306. The computer 130 may be located inside the electrophoresis apparatus 100 or installed outside the electrophoresis apparatus 100.

[0040] [Operation] Figure 4 is a diagram illustrating the procedure by which the computer 130 performs crosstalk correction. Each step is performed by the computer 130. The same applies to the following flowchart. In step 401, a matrix standard, which is a reagent that serves as the basis for creating the crosstalk correction matrix, is subjected to electrophoresis in the capillary 118 to obtain crosstalk information that includes at least spatial crosstalk information. In step 402, a crosstalk correction matrix that includes at least spatial crosstalk correction is created from the acquired crosstalk information and stored in the storage 303 as the crosstalk correction matrix 403. In step 404, the biological sample is subjected to electrophoresis. In step 405, the crosstalk in the data obtained from the electrophoresis of the biological sample is corrected using the crosstalk correction matrix 403. In step 406, the crosstalk correction results are displayed.

[0041] In FIG. 4, step 404 may be performed prior to step 401. Also, the crosstalk correction matrix 403 may be only a spatial crosstalk correction matrix, or may be a matrix that simultaneously corrects spectral crosstalk and spatial crosstalk, or may be two matrices that respectively correct a spectral crosstalk correction matrix and a spatial crosstalk correction matrix.

[0042] FIG. 5 is a flowchart for explaining the operation for the i-th capillary in step 401.

[0043] Step 501: Load an assay. An assay is information that defines the operating conditions and analysis conditions for analysis. The operating conditions include the power of the laser of the detection unit, the temperature of the thermostat unit, the type of electrophoresis medium, the voltage and time applied in pre-run, the voltage and time applied in the fluorescent substance injection process, the voltage and time applied in the electrophoresis process, the laser irradiation time and sampling period at the time of data acquisition, etc. The analysis conditions include the analysis width specified by the data points and the size of the base, the threshold value of the signal height, the basecall mobility file set for reading the sequence in the case of sequencing analysis, the fragment size used for analysis in the case of fragment analysis, etc.

[0044] Step 502: Prepare for electrophoresis based on the loaded assay. The preparation for electrophoresis includes laser stabilization of the detection unit, temperature adjustment of the thermostat unit, filling of the electrophoresis medium, and pre-run. Pre-run is to apply a voltage to the filled polymer before injecting the phosphor for the purpose of stabilizing the performance of electrophoresis.

[0045] Step 503: Electrophorese the matrix standard in the i-th capillary.

[0046] Step 504: Obtain the signal intensity of the matrix standard. It may be the signal intensity of the donor peak generating spatial crosstalk or the signal intensity of the spatial crosstalk. Also, when a plurality of fluorescent dyes are included in the matrix standard, obtain the signal intensity of at least one of the plurality of fluorescent dyes.

[0047] Step 505: Determine whether the acquired signal intensity exceeds a reference value. This reference value is preferably set to be not less than the expected biological sample signal intensity. Therefore, the reference value may be calculated from the detection upper limit of the device. At this time, the reference value may be set considering variations such as injection into the capillary and the signal intensity of the matrix standard, or a range may be set to determine whether the acquired signal intensity falls within the range. When spatial crosstalk is the determination target, the reference value and range are set based on the signal intensity of the assumed spatial crosstalk from the donor peak signal intensity of the detection upper limit of the device. The reference value may be individually set for each fluorescent dye to be determined.

[0048] Step 505: Supplementary note: The fluorescence signal intensity of the matrix standard is proportional to the fluorescence signal intensity of the spatial crosstalk. Therefore, in this step, by using the fluorescence signal intensity of the matrix standard as a reference, it is possible to determine whether the fluorescence signal intensity of the spatial crosstalk is sufficient. Also, since the fluorescence signal intensity of the spatial crosstalk is generally small, measuring the fluorescence signal intensity of the matrix standard is also useful from the perspective of measurement accuracy.

[0049] Step 506: If the signal intensity of the matrix standard in Step 505 is less than the reference value, proceed to the next action. The next action can have various variations.

[0050] FIG. 6 shows a specific example of Step 506. Here, as a specific example of Step 506, an example of performing Steps 601 to 602 is shown. In Step 601, based on the result of the j-th electrophoresis, parameters such as the applied voltage, applied time, exposure time, and laser intensity of the assay are calculated so that the signal intensity of the matrix standard becomes not less than the reference value (first reference value). In Step 602, the assay is changed to the calculated parameters. Return to Step 501 and load the assay adjusted in Step 602. Based on the loaded assay, perform Steps 502 to 505. Repeat Steps 601, 602, 501 to 505 until the signal intensity of the matrix standard becomes not less than the reference value. An upper limit number of repetitions may be set. Also, when repeating, Step 502 (electrophoresis preparation) may be omitted.

[0051] The reference value in step 505 is set such that the fluorescence signal intensity of the matrix standard is at least greater than the fluorescence signal intensity of the biological sample. More preferably, the reference value in step 505 is set such that the fluorescence signal intensity of the matrix standard is as large as possible within the range below the upper limit of measurement by the electrophoresis apparatus 100. This is thought to allow the fluorescence signal intensity of spatial crosstalk to be greater than the baseline noise, as will be described later.

[0052] The above workflow is performed for all capillaries. At this time, as disclosed in Patent Document 3, the matrix standard may be injected into multiple capillaries simultaneously, or it may be injected at different timings for each capillary.

[0053] Even while the workflow shown in Figure 6 is being performed, electrophoresis of the matrix standard can be performed on different capillaries. For example, after the first electrophoresis of the matrix standard has been performed on all capillaries, step 505 may be performed on each capillary, and only the capillaries determined to be below the reference value may be subjected to step 506 (including the repetition of steps 501 to 505 from step 601 onwards).

[0054] In the above explanation, the term "capillary i" is used, but "i" is a symbol used for convenience to distinguish different capillaries. Therefore, capillary i+1 may not be spatially adjacent to capillary i.

[0055] [Effects] Figures 7 and 8 show chromatograms when the matrix standard is electrophoresed without applying the present invention. The difference between Figure 7 and Figure 8 is the maximum signal intensity. Figure 9 shows a chromatogram when the matrix standard is electrophoresed with the present invention applied, bringing the signal intensity closer to the upper limit of the instrument. Figures 10 and 11 show chromatograms when a biological sample is electrophoresed. The same sample as the matrix standard was electrophoresed as the biological sample. In Figure 11, the signal intensity is higher due to the larger sample amount compared to Figure 10. In each figure, the horizontal axis represents migration time [s], and the vertical axis represents signal intensity [RFU] after spectral crosstalk correction. The upper part of each figure shows the signal in the capillary injected with the matrix standard, i.e., the donor peak. The lower part of each figure shows the signal in a capillary adjacent to the capillary injected with the matrix standard, i.e., a capillary that has not been injected with a fluorescent dye, i.e., spatial crosstalk. The maximum signal intensity of the donor peak is largest in the order of Figure 7 < Figure 10 < Figure 8 < Figure 11 < Figure 9.

[0056] Figures 12 and 13 show the results after correction using the spectral spatial crosstalk correction matrix. Figure 12(1) is the same as the lower part of Figure 10, and Figure 13(1) is the same as the lower part of Figure 11. Figures 12(2) and 13(2) show the correction results using the spectral spatial crosstalk correction matrix created from the electrophoresis results shown in Figure 7. Figures 12(3) and 13(3) show the correction results using the spectral spatial crosstalk correction matrix created from the electrophoresis results shown in Figure 8. Figures 12(4) and 13(4) show the correction results using the spectral spatial crosstalk correction matrix created from the electrophoresis results shown in Figure 9. The above combinations are shown in the table below.

[0057]

[0058] In Figure 12(2), pull-up and pull-down resistors occurred due to correction errors (indicated by arrows). In Figures 12(3) and 12(4), spatial crosstalk was completely corrected. The reason why pull-up and pull-down resistors occurred in Figure 12(2) is that the spatial crosstalk signal in Figure 7 is buried in noise.

[0059] In Figures 13(2) and 13(3), pull-up and pull-down signals occurred due to correction errors (indicated by arrows). In Figure 13(4), spatial crosstalk was completely corrected. The reason why pull-up and pull-down signals occurred in Figure 13(2) is that the spatial crosstalk signal in Figure 7 is buried in baseline noise. The reason why pull-up and pull-down signals occurred in Figure 13(3) is that the signal-to-noise ratio of the spatial crosstalk signal in Figure 8 is insufficient. The reason why pull-up / pull-down signals occur in Figure 13(3) while no pull-up / pull-down signals occur in Figure 12(3) is presumed to be because the fluorescence signal intensity of the matrix standard in Figure 13(3) is smaller than the fluorescence signal intensity of the biological sample.

[0060] As described above, for example, if the signal intensity of the donor peak during biological sample electrophoresis is higher than the signal intensity of the donor peak during matrix preparation, the noise components contained in the matrix are amplified, and pull-up and pull-down signals occur due to correction errors.

[0061] According to Embodiment 1, the signal-to-noise ratio (S / N) of spatial crosstalk information obtained from a matrix standard can be improved. This reduces correction errors such as pull-up and pull-down that occurred due to spatial crosstalk correction matrices created from spatial crosstalk information with a low S / N ratio.

[0062] <Embodiment 1: Supplement> The above-mentioned prior art documents are supplemented below. None of the prior art documents describe raising the signal intensity of the electrophoretic matrix standard to near the upper limit of the instrument for measurement in order to create a spectral crosstalk correction matrix, as in Embodiment 1. In other words, in conventional spatial crosstalk correction, there has been no attempt to acquire the donor peak signal in calibration at a state as close as possible to the upper limit of the signal that the instrument can measure. In contrast, in Embodiment 1, the electrophoresis of the matrix standard is performed with the fluorescence signal intensity raised to near the upper limit of the signal that the electrophoresis apparatus 100 can measure (reference value in step 505). As a result, the fluorescence signal of the matrix standard's crosstalk is not buried in baseline noise, and the crosstalk correction matrix can be appropriately created.

[0063] <Embodiment 2> Embodiment 1 demonstrated the operation related to the electrophoresis of a matrix standard of a specific i-th capillary. This operation is based on the premise that the same operation will occur even if i changes. In contrast, in Embodiment 2 of the present invention, the parameters related to the signal intensity of the electrophoresis of the matrix standard are changed as i changes. The difference from Embodiment 1 is that the parameters are changed as i changes. All other points are the same as Embodiment 1. The reason for changing the parameters as i changes is as follows.

[0064] One reason is that the signal intensity of the matrix standard decreases each time it is injected from the same sample container. This decrease in signal intensity occurs because a large amount of matrix standard is injected into the capillary to acquire spatial crosstalk information. Before performing electrophoresis in capillary i+1 using the same sample container as electrophoresis in capillary i, it may be necessary to adjust the assay to compensate for the decrease in signal intensity. Since the matrix standard is used to detect crosstalk occurring in adjacent capillaries, it is usually added to one capillary at a time rather than simultaneously to multiple capillaries. Also, as explained in Embodiment 1, a large amount of matrix standard needs to be added to ensure the signal-to-noise ratio of the crosstalk. Due to these factors, the decrease in the signal intensity of the matrix standard is easily noticeable. Therefore, as in Embodiment 2, it is useful to sequentially adjust the electrophoresis parameters.

[0065] Another reason is that the electrophoresis results for capillary i+1 can be estimated from the electrophoresis results for capillary i. Generally, when preparing matrix standards, the matrix standards for multiple sample containers are adjusted in concentration and then distributed to each sample container. Therefore, the matrix standards are homogeneous across the sample containers, and if the signal intensity of the matrix standard in one sample container is low, it can be estimated that the signal intensity will also be low in the other containers. Thus, if the signal intensity of the matrix standard in capillary i is low, the parameters may be adjusted so that the signal intensity of the matrix standard in capillary i+1 before electrophoresis, when matrix standards are injected from other sample containers, becomes higher.

[0066] [Operation] Figure 14 is a diagram showing the flow of the analysis method according to Embodiment 2. Figure 14 shows an example in which the parameters are changed according to the change in i. In step 1401, capillary 1 is specified (i=1). In step 1402, electrophoresis is performed according to the workflow in Figure 5. In step 1403, it is determined whether electrophoresis of the matrix standard has been completed in all capillaries. If it has not been completed, in step 1404, the assay is modified before electrophoresis of the matrix standard in capillary i+1. For example, the applied voltage at the time of injection is increased each time a matrix standard is injected from the same sample container. In step 1405, the capillary number is advanced to the next capillary. Steps 1402 to 1405 are repeated until electrophoresis of the matrix standard is completed in all capillaries.

[0067] [Effect] In Embodiment 1, regardless of the electrophoresis result of capillary i, the first electrophoresis is performed with capillary i+1. In contrast, in Embodiment 2, by changing the electrophoresis parameters as the capillary being electrophoresed changes, it becomes possible to adjust the parameters so that the signal intensity of the matrix standard is equal to or greater than the reference value for the capillary before electrophoresis is performed.

[0068] <Embodiment 3> Embodiment 1 is based on the determination of the signal intensity of a matrix standard and the following actions based on the determination result. In contrast, Embodiment 3 of the present invention is an example that, in addition to Embodiment 1, includes the determination of the signal intensity of a biological sample and the following actions based on the determination result. It differs from Embodiment 1 in that a reference value is set for the biological sample and re-electrophoresis is performed. All other points are the same as Embodiment 1.

[0069] The reason for setting reference values ​​and re-electrophoresis in biological samples is as follows: If the signal intensity of the donor peak in the data to be corrected (biological sample) is large, the noise included in the crosstalk correction matrix will be amplified. Therefore, by making the signal intensity of the donor peak in the data to be corrected (biological sample) less than or equal to the signal intensity of the donor peak in the matrix standard, pull-up and pull-down in the correction error can be reduced. For example, in Figure 12(3), which uses the crosstalk correction matrix created from the electrophoresis data in Figure 8, no pull-up or pull-down occurs. In Figure 13(3), which applies the same crosstalk correction matrix, pull-up and pull-down occur. This is because, as the signal intensity of the donor peak in the data to be corrected (biological sample) increases, the noise component of the crosstalk is reflected more strongly.

[0070] [Operation] Figure 15 shows the flow up to crosstalk correction in Embodiment 3. Steps 401 to 403 are performed in the same manner as in Embodiment 1. In step 1501, a reference value is set for electrophoresis of the biological sample. The reference value is the signal intensity that does not emphasize the noise component of the crosstalk correction matrix. For example, it is the donor peak signal intensity of the electrophoresed matrix standard or the reference value set during matrix standard electrophoresis. A lower limit of the signal intensity of the biological sample may be set to avoid the inability to detect the target minute mutation peak due to the signal intensity of the biological sample becoming too low. In step 1502, electrophoresis of the biological sample is performed, which includes the determination of the signal intensity and the following actions based on the determination result.

[0071] Figure 16 shows the details of step 1502. Step 1601 loads the assay. Step 1602 prepares for electrophoresis based on the loaded assay. Step 1603 performs electrophoresis on the biological sample. Step 1604 obtains the signal intensity of the biological sample. Step 1605 determines whether the obtained signal intensity is below the reference value. If the signal intensity of the biological sample is above the reference value, proceed to step 1606. Various modifications are possible for the next action.

[0072] Figure 17 shows a specific example of step 1606. Here, as a specific example of step 1606, an example of performing steps 1701 to 1702 is shown. In step 1701, based on the results of the j-th electrophoresis, parameters such as the applied voltage, application time, exposure time, and laser intensity of the assay are calculated so that the signal intensity of the biological sample is less than the reference value. In step 1702, the assay is modified based on the calculated parameters. Next, proceed to step 1601 and load the assay adjusted in step 1702. Perform steps 1602 to 1605 based on the loaded assay. Repeat steps 1601 to 1605, 1701, and 1702 until the signal intensity of the biological sample is less than the reference value (second reference value). An upper limit on the number of repetitions may be set. As the reference value in step 1605, for example, the smallest of the fluorescence signal peak levels of the matrix standard can be used. This makes it possible to make the fluorescence signal intensity of the matrix standard greater than the fluorescence signal intensity of the biological sample.

[0073] [Effects] The electrophoresis apparatus 100 according to Embodiment 3 can apply crosstalk correction without emphasizing the crosstalk component by appropriately adjusting the signal intensity of the donor peak of the data to be corrected (biological sample) through the above operation. As a result, pull-up and pull-down due to correction errors can be reduced.

[0074] <Regarding Variations of the Invention> The present invention is not limited to the embodiments described above, and includes various variations. For example, the embodiments described above are described in detail to make the present invention easier to understand, and are not necessarily limited to those having all the described configurations. Furthermore, it is possible to replace a part of the configuration of one embodiment with the configuration of another embodiment, and it is also possible to add the configuration of another embodiment to the configuration of one embodiment. In addition, it is possible to add, delete, or replace parts of the configuration of each embodiment with other configurations.

[0075] In each embodiment, control lines and information lines are shown only if deemed necessary for illustrative purposes, and not all control lines and information lines are necessarily shown in the actual product. In practice, it can be assumed that almost all components are interconnected.

[0076] In the above embodiments, a step may be provided to set the reference value (second reference value) in step 1605 within a range less than or equal to the reference value (first reference value) in step 505. For example, such a step is useful in cases where the fluorescence signal intensity of the matrix standard cannot be increased, in order to make the fluorescence signal intensity of the matrix standard greater than that of the biological sample. Alternatively, such a step is also useful in cases where the fluorescence signal intensity of the biological sample is significantly greater than expected, in order to make the fluorescence signal intensity of the matrix standard greater than that of the biological sample.

[0077] 100 Electrophoresis apparatus 110 Measurement unit 111 Capillary array 112 Pump unit 113 High-voltage power supply 114 Constant temperature bath 115 Fluorescence detection device 116 Sample tray 117 Conveyor 118 Capillary 119 Sample container 120 Fluorescence detection position 121 Discharge container 130 Computer 201 Excitation light source 202 Shutter 203 Excitation light lens 204 Optical filter 205 Fluorescence lens 206 Diffraction grating 210 CCD image sensor 220 Conversion unit 221 Charge conversion unit 222 Digital conversion unit 301 CPU 302 Memory 303 Storage 304 Display unit 305 Input unit 306 NIF (Network Interface)

Claims

1. An analytical method for analyzing the characteristics of a biological sample labeled with a fluorescent dye by electrophoresis of the biological sample inside a plurality of capillaries and observing the fluorescence generated by a factor labeled with the fluorescent dye in the biological sample, comprising: acquiring crosstalk information including information relating to spatial crosstalk in which fluorescence generated in a first capillary, which is one of the plurality of capillaries, is observed as if it were generated in a second capillary, which is a different capillary from the first capillary; creating a crosstalk correction matrix, which is a matrix used in a calculation to reduce the amount of spatial crosstalk to a predetermined amount using the crosstalk information; electrophoresis of a matrix standard, which is a reagent that serves as a reference for creating the crosstalk correction matrix, inside the first capillary and acquiring the fluorescence signal intensity of the matrix standard; and adjusting at least one of the matrix standard or the biological sample so that the fluorescence signal intensity of the matrix standard is greater than a first reference value.

2. The analytical method according to claim 1, characterized in that the first reference value is set such that the fluorescence signal intensity of the matrix standard is greater than the fluorescence signal intensity of the biological sample.

3. The analytical method according to claim 1, characterized in that the first reference value is set such that the fluorescence signal intensity due to spatial crosstalk occurring in the second capillary exceeds the baseline noise.

4. The analytical method according to claim 1, characterized in that, in the step of acquiring the fluorescence signal intensity of the matrix standard, the fluorescence signal intensity in the first capillary is acquired, and in the step of adjusting the fluorescence signal intensity of the matrix standard, the fluorescence signal intensity in the first capillary is adjusted to adjust the fluorescence signal intensity in the second capillary caused by spatial crosstalk.

5. The analytical method according to claim 1, characterized in that, in the step of acquiring the signal intensity of the matrix standard, if the matrix standard contains multiple types of fluorescent dyes, the fluorescence signal intensity of at least one fluorescent dye of the matrix standard is acquired, and in the step of adjusting the fluorescence signal intensity of the matrix standard, the fluorescence signal intensity is adjusted so that the fluorescence signal intensity of at least one fluorescent dye of the matrix standard is greater than the first reference value set for each fluorescent dye.

6. The analytical method according to claim 1, characterized in that the first reference value is set to adjust the fluorescence signal intensity of the matrix standard such that at least one of the fluorescence signal intensity peaks obtained by the electrophoresis apparatus performing the electrophoresis in at least one of the first capillary or the second capillary is less than or equal to the upper limit that can be obtained by the electrophoresis apparatus and is as close as possible to the upper limit.

7. The analytical method according to claim 1, characterized in that, in the step of acquiring the fluorescence signal intensity of the matrix standard, the fluorescence signal intensity of the matrix standard is acquired for each capillary, and in the step of adjusting the fluorescence signal intensity of the matrix standard, the fluorescence signal intensity of the matrix standard is adjusted for each capillary by adjusting the electrophoresis conditions for each capillary.

8. The analytical method according to claim 1, further comprising the steps of: performing electrophoresis on the biological sample inside the first capillary to obtain the fluorescence signal intensity of the biological sample; and adjusting the fluorescence signal intensity of the biological sample so that the fluorescence signal intensity of the biological sample is less than a second reference value.

9. The analytical method according to claim 8, characterized in that the second reference value is set such that the fluorescence signal intensity of the matrix standard is greater than the fluorescence signal intensity of the biological sample.

10. The analysis method according to claim 8, further comprising the step of setting the second reference value within a range less than or equal to the first reference value.

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