Nucleic acid analyzer, nucleic acid analysis method, and program

Abstract

To provide a nucleic acid analysis device that reduces an artifact component.SOLUTION: A nucleic acid analysis device is provided which includes a distribution unit, a sign part, an information acquisition unit, and an information processing unit. The information processing unit includes an information extraction unit, an artifact component determination unit, a correction unit, and a specification unit. The artifact component determination unit determines an artifact component from information corresponding to a portion other than a plurality of extracted individual separation sections. The correction unit corrects information corresponding to the plurality of extracted individual separation sections by using the artifact component.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a nucleic acid analysis apparatus, a nucleic acid analysis method, and a program.

Background Art

[0002] Jennifer Doudna et al. of the University of California demonstrated that different strains of human papillomavirus (HPV) in human samples can be accurately detected by distinguishing them from each other using Cas12a (Non-Patent Document 1). The complex composed of Cas12a and crRNA specifically recognizes and binds to the target DNA sequence, and Cas12a cleaves the bound target DNA. At this time, when a reporter molecule in which a fluorescent substance and a quencher are linked by a single-stranded DNA is added to the reaction system, Cas12a cleaves the single-stranded DNA of the reporter molecule by a trans-cleavage reaction. As a result, the fluorescent substance and the quencher are separated, and fluorescence occurs. That is, when the target DNA is present in the sample, the fluorescence derived from the fluorescent substance of the reporter molecule is generated by activating the trans-cleavage reaction of Cas12a, so that the target DNA can be detected based on the fluorescence.

Prior Art Documents

Non-Patent Documents

[0003]

Non-Patent Document 1

Non-Patent Document 2

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] However, as shown in Non-Patent Document 2, when reaction reagents are isolated in a minute area and fluorescence images are acquired using a fluorescence microscope or the like, it is known that signals other than the signal (artifacts) can occur in the fluorescence image due to various factors. Particularly problematic are those caused by uneven brightness of incident light (shading) and the influence of light from adjacent wells (optical crosstalk). These artifacts significantly reduce the accuracy of fluorescence detection, and their removal or reduction is desirable.

[0006] Several methods have been proposed to reduce artifacts. Regarding shading, Patent Document 1 discloses a method in which artifact components (shading components) caused by brightness unevenness of incident light are acquired in advance using a diffuser plate or the like, and the captured image is corrected based on this. Regarding optical crosstalk, Non-Patent Document 2 discloses a method in which optical crosstalk from adjacent wells is calculated for each pattern, and the captured image is corrected based on this. However, acquiring shading components in advance in order to correct shading is a complicated process. Furthermore, the shading components may differ in the captured image, in which case effective correction is difficult. In addition, regarding optical crosstalk, the process of calculating artifact components (optical crosstalk components) caused by the influence of light from adjacent wells is complex, and in reality, it is necessary to consider optical crosstalk components caused by the influence of light from all wells, making it difficult to completely eliminate optical crosstalk. Therefore, the present invention aims to provide a nucleic acid analyzer that reduces artifacts without complicating the process. [Means for solving the problem]

[0007] Embodiments of the present invention are: A nucleic acid analyzer comprising a distribution unit, a labeling unit, an information acquisition unit, and an information processing unit, The information processing unit includes an information extraction unit, an artifact component determination unit, a correction unit, and a specification unit. The distribution unit distributes the sample containing nucleic acids and reagents into a plurality of individual separation compartments, forming an assembly that includes the plurality of individual separation compartments and the portion other than the plurality of individual separation compartments. The labeling unit makes a change in the individual isolation compartment to which the sample containing the target nucleic acid is distributed, so that the individual isolation compartment to which the sample containing the target nucleic acid is distributed can be distinguished from the individual isolation compartment to which the sample not containing the target nucleic acid is distributed. The information acquisition unit acquires information corresponding to the aggregate, The information extraction unit extracts from the acquired information corresponding to the aggregate information information corresponding to the plurality of individual separation sections and information corresponding to the parts other than the plurality of individual separation sections, respectively. The artifact component determination unit determines artifact components from information corresponding to the parts other than the extracted plurality of individual separation sections. The correction unit corrects the information corresponding to the extracted plurality of individual separated sections using the artifact component. The identifying unit identifies individual isolation sections in which the magnitude of the change exceeds a predetermined threshold, based on the corrected information corresponding to the plurality of individual isolation sections. The present invention provides a nucleic acid analyzer characterized by the following features.

[0008] Furthermore, embodiments of the present invention are A nucleic acid analysis method comprising a distribution step, a labeling step, an information acquisition step, an information extraction step, an artifact component determination step, a correction step, and a identification step, In the distribution step, the sample containing nucleic acids and reagents are distributed into a plurality of individual separation compartments to form an assembly including the plurality of individual separation compartments and the portion other than the plurality of individual separation compartments. In the labeling step, a change is made to the individual isolation compartment to which the sample containing the target nucleic acid is distributed, so that it is possible to distinguish between the individual isolation compartment to which the sample containing the target nucleic acid is distributed in the specific step, In the information acquisition step, information corresponding to the aggregate is acquired, In the information extraction step, information corresponding to the plurality of individual separation compartments and information corresponding to portions other than the plurality of individual separation compartments are respectively extracted from the information corresponding to the aggregate obtained In the artifact component determination step, an artifact component is determined from the information corresponding to the portions other than the plurality of extracted individual separation compartments, In the correction step, the information corresponding to the plurality of extracted individual separation compartments is corrected using the artifact component, In the specifying step, based on the information corresponding to the plurality of corrected individual separation compartments, an individual separation compartment in which the magnitude of the change exceeds a predetermined threshold is specified. There is provided a nucleic acid analysis method characterized by the above.

[0009] Also, an embodiment of the present invention provides a program for causing a computer to execute the nucleic acid analysis method.

Advantages of the Invention

[0010] According to the present invention, by determining an artifact component from information corresponding to portions other than a plurality of individual independent separation compartments and correcting the information corresponding to the plurality of individual separation compartments using the artifact component, analysis of nucleic acids from which artifacts have been removed or reduced becomes possible.

Brief Description of the Drawings

[0011] [Figure 1] Configuration diagram of a nucleic acid analyzer [Figure 2] Cross-sectional view of a well plate [Figure 3] Flowchart showing the flow of a nucleic acid analysis method [Figure 4] Flowchart for correcting an artifact component by estimation [Figure 5] Conceptual diagram of a fluorescence image of a well plate [Figure 6] Flowchart for correcting an artifact component by calculation [Figure 7] Diagram showing the effect using this embodiment

Mode for Carrying Out the Invention

[0012] Hereinafter, embodiments of the present invention will be illustratively described in detail with reference to the drawings. However, the components described in this embodiment are merely examples, and the technical scope of the present invention is determined by the scope of the claims and is not limited by the following individual embodiments.

[0013] Embodiments of the Present Invention FIG. 1 is a functional block diagram of a nucleic acid analyzer 100 according to an embodiment of the present invention. The nucleic acid analyzer 100 includes a dispensing unit 101, a labeling unit 102, an information acquisition unit 103, and an information processing unit 104.

[0014] The dispensing unit 101 dispenses a sample and a reagent containing nucleic acid into a plurality of individual separation compartments, and forms an aggregate including the plurality of individual separation compartments and a portion other than the plurality of individual separation compartments.

[0015] The plurality of individual separation compartments can be wells containing a sample and a reagent containing the dispensed nucleic acid, and the aggregate can be a well plate including the wells. In this case, the portion other than the plurality of individual separation compartments includes a part of the region other than the wells of the well plate.

[0016] Alternatively, the plurality of individual separation compartments can be droplets containing a sample and a reagent containing the dispensed nucleic acid, and the aggregate can include the droplets and a dispersion medium. In this case, the portion other than the plurality of individual separation compartments can include a part of the dispersion medium.

[0017] Preferred examples of the volume of the individual separation compartments can include 0.1 fL or more and 1000 fL or less, and further 0.5 fL or more and 400 fL or less.

[0018] The sample may contain nucleic acids, including DNA and RNA, and can be any biological material, extract from living organisms, blood, blood-derived materials, food, food-derived materials, natural products, natural product-derived materials, culture medium-derived materials, etc. The reagent may contain, for example, an effector protein, a crRNA that binds to the target nucleic acid, and a reporter molecule.

[0019] The labeling unit 102 causes a change in the individual isolation compartment to which the sample containing the target nucleic acid is distributed, so that the individual isolation compartment to which the sample containing the target nucleic acid is distributed can be distinguished from the individual isolation compartment to which the sample without the target nucleic acid is distributed. The change can be the generation of fluorescence, in which case the information acquisition unit acquires the information corresponding to the aggregate, which includes the fluorescence image of the aggregate. For example, in the labeling unit 102, the binding of crRNA to the target nucleic acid activates the effector protein, and the activated effector protein modifies the reporter molecule to generate fluorescence. The target nucleic acid can be any nucleic acid having any sequence, for example, a nucleic acid that can be applied to the diagnosis of disease states or constitutional diagnosis, and such disease states include cancer, autoimmune diseases, and infectious diseases, and infectious diseases include, for example, infectious diseases caused by DNA viruses or RNA viruses.

[0020] The information acquisition unit 103 acquires information corresponding to the aggregate. For example, it can detect fluorescence. The information processing unit 104 identifies individual separation sections whose magnitude of change exceeds a predetermined threshold, based on the information corresponding to the aggregate.

[0021] The information processing unit 104 includes an information extraction unit 105, an artifact component determination unit 106, a correction unit 107, and a specification unit 108. These will be described later.

[0022] The information processing unit 104 may have a display unit 109, which displays the results calculated by the information processing unit 104 on a monitor or the like. The information processing unit 104 may also have a storage unit 110. The information processing unit 104 can have the functions of a computer. For example, the information processing unit 104 may be integrated with a desktop PC (Personal Computer), laptop PC, tablet PC, smartphone, etc. The information processing unit 104 may further have a function to control the operation of the distribution unit 101, indicator unit 102, and information acquisition unit 103 according to a predetermined program. To realize the functions of a computer that performs calculations and storage, the information processing unit 104 may be equipped with a CPU (Central Processing Unit), RAM (Random Access Memory), ROM (Read Only Memory), and HDD (Hard Disk Drive). The information processing unit 104 may also be equipped with a communication interface (I / F), a display device, and an input device.

[0023] The nucleic acid analyzer according to an embodiment of the present invention can be used for nucleic acid analysis using CHRISPR-Cas technology. In this case, by distributing a sample containing the target nucleic acid into individual independent isolation compartments, the sample appears to be concentrated, making it possible to detect the target nucleic acid without an amplification step and to shorten the time it takes for the fluorescence signal to saturate. Furthermore, by making the volume of each individual independent isolation compartment sufficiently small, it is possible to set the amount of target nucleic acid contained in each compartment to one molecule or less, and by counting the number of compartments from which a fluorescence signal is obtained, it is possible to calculate the concentration of the target nucleic acid in the sample. In this case, the multiple individual isolation compartments can be droplets containing the sample containing the distributed nucleic acid and reagents, and a preferred example of a droplet is a water-in-oil emulsion (W / O emulsion). Alternatively, the multiple individual isolation compartments can be wells containing the sample containing the distributed nucleic acid and reagents, and as wells, for example, wells in a well plate with the configuration shown in Figure 2 can be used. In Figure 2, 200 is a well plate, 201 is a lower substrate, 202 is an upper substrate, 203 is a partition, 204 is a well, and 205 is a space.

[0024] Figure 3 is a flowchart showing the flow of nucleic acid analysis according to an embodiment of the present invention. A specific example of nucleic acid analysis will be explained in detail using Figure 3.

[0025] Steps S301 and S302 are performed by the distribution unit 101. The reaction solution consisting of the sample and the detection reagent is delivered from the inlet to the space 205 of the well plate 200, which has an inlet (not shown) and an outlet (not shown) that are open.

[0026] In step S302, the reaction solution is filled into the wells 204. One method for filling the reaction solution is to leave the well plate 200 under reduced pressure and degas the space 205. Specifically, it is preferable to leave the well plate 200 in a depressurized desiccator at 0.1 atmospheres for a predetermined time. By degassing, the air in the wells 204 is removed, and the reaction solution can be efficiently filled into the wells 204. The degassing time is not particularly limited and can be set arbitrarily. The method of filling the reaction solution is not limited to the degassing method.

[0027] Steps S303 and S304 are performed by the labeling unit 102. In step S303, a hydrophobic solvent is introduced into the space 205 and sealed. That is, the reaction solution present in the space 205 above the well 204 is replaced with a hydrophobic solvent. Examples of hydrophobic solvents include fluorinated oils, saturated aliphatic hydrocarbons, unsaturated aliphatic hydrocarbons, aromatic hydrocarbons, and silicone oils. Examples of fluorinated oils include Fluorinert, Asahi Clean AE-3000 (manufactured by AGC), and Fomblin (manufactured by Solvay). Examples of saturated aliphatic hydrocarbons include Isopar (manufactured by ExxonMobil) and mineral oil. In step S304, the labeling unit 102 and the fluorescence generation unit 103 incubate the well plate 200 filled with the reaction solution in an incubator at 37°C. However, the reaction temperature can be set arbitrarily and is not limited to 37°C. This incubation process allows the CHRISPR-Cas trans-cleavage reaction to proceed, generating fluorescence derived from the fluorescent substance present in the reporter molecule.

[0028] Step S305 is performed by the information acquisition unit 103. In step S305, the pre-set incubation is completed, and bright-field images and fluorescence images are acquired for each well 204. Any device that can acquire bright-field and fluorescence images can be used by the information acquisition unit 103, but here a fluorescence microscope is used. Bright-field images are taken for well detection, but if a reporter is placed in the reaction solution and all wells are made to glow, fluorescence images taken under imaging conditions that match the fluorescence wavelength of the reporter can also be used. Here, it is best to select a reporter that has a fluorescence wavelength sufficiently far from the fluorescence wavelength of the reporter that CHRISPR-Cas cleaves and fluoresces.

[0029] Step S306 is performed by the information extraction unit 105. In step S306, the information extraction unit 105 uses the bright-field image to create a mask image for each well (well: 1, non-well: 0). That is, it creates a default mask based on the shape of the wells, and the created default mask is sent to the storage unit 110 and shared with the correction unit 107, etc. The following methods can be considered for creating the mask image. For example, since the wells are arranged regularly, a template of the well mask can be created in advance, and the translation direction and angle can be adjusted using template matching, etc. Alternatively, edge detection such as a Zobel filter can be used to emphasize the outer perimeter of the wells, and circle detection can be performed using the Hough transform, etc. Furthermore, detection may be performed using deep learning.

[0030] Step S307 is performed by the artifact component determination unit 106 and the correction unit 107 to correct the artifact components of the fluorescence image. A specific example of the flow for correcting artifact components will be explained in detail with reference to Figure 4.

[0031] [Determination of artifact components] In step S401, the fluorescence intensity of the out-of-well region 502 is sampled along with its coordinate information from the mask image created in step S306 and the fluorescence image 500 shown in Figure 5. The sampling method is to sample the fluorescence intensity of the fluorescence image at the coordinates where the pixel value of the mask image is 0.

[0032] In step S402, the correction unit 107 can estimate the artifact component using the brightness and coordinate information sampled in step S401, using methods such as two-dimensional surface approximation. Estimating the artifact component improves the accuracy of the analysis. For estimation, for example, basis function approximation such as radial basis function approximation, or polynomial approximation can be used. To avoid overestimating or underestimating the artifact component in the well region, it is preferable to use a linear function as the basis function. Also, shading components and optical crosstalk components are low-frequency components that have a certain degree of spatial spread. Taking this into consideration, it is preferable to estimate a smooth surface by including a regularization term when approximating. Alternatively, surface approximation can be performed without regularization, and the approximated surface artifact component can be blurred by applying a low-pass filter such as a Gaussian filter. Surface approximation has the challenge of increasing computational cost and memory usage as the number of sampling points increases. In particular, memory is often limited, so it is possible to reduce the number of sampling points to reduce memory usage. To achieve accurate surface approximation even with a reduced number of sampling points, it is preferable to distribute the sampled coordinates across the entire image. For example, by dividing the image into small ROIs (Regions of Interest) 503 separated by dotted lines as shown in Figure 5, and sampling multiple points from the well-external region 502 of each small ROI, it is possible to sample without bias across the entire fluorescence image 500.

[0033] In step S403, the correction unit 107 corrects the artifact component by subtracting or dividing the artifact component created in step S402 from the fluorescence image.

[0034] In step S308, the identification unit 108 calculates the fluorescence intensity of each well using the fluorescence image corrected for artifact components in step S307, and identifies well 204 having a fluorescence intensity exceeding a predetermined threshold (positive determination). The threshold may be automatically determined from the fluorescence intensity of each well using the Otsu method or the like, or it may be determined from the variation in fluorescence intensity after measuring well plates with a DNA concentration of 0.

[0035] In step S309, after the trans-cleavage reaction during incubation in S304, the concentration of the target nucleic acid is calculated from the number of wells that fluoresce. If the sample contains a large number of target nucleic acids, a single droplet may contain two or more molecules of the target nucleic acid. Therefore, the number of target nucleic acid molecules and the number of wells that fluoresce may not match. For the reasons above, it is preferable to calculate the concentration of the target nucleic acid by a calculation that takes into account the Poisson distribution. In the Poisson distribution, when the average number of molecules per droplet is λ, the proportion of droplets that fluoresce P(k) can be expressed by the following equation 1. P(k)=(λk / k!)e-λ (k=0, 1, 2,...) Equation 1

[0036] From the number of droplets exhibiting fluorescence, P(k) can be determined, and λ can be calculated. Therefore, using Equation 1, the concentration of the target nucleic acid can be calculated from the number of droplets in which fluorescence was detected out of all droplets.

[0037] By using this embodiment, artifact components occurring in fluorescence images can be appropriately removed, fluorescence intensity can be corrected, and fluorescence detection accuracy can be improved. In this embodiment, a well plate was used for the explanation, but the individual independent separation compartments may be droplets. By replacing the wells with droplets, artifact components can be corrected in the same manner as in this embodiment, and analytical accuracy can be improved.

[0038] [Determination of artifact components through calculation] The above describes the case where artifact components are determined by estimation. However, when artifact components are determined by estimation, there is a problem in that the computational effort and memory increase significantly by increasing the number of points for sampling luminance information. By determining artifact components by calculation, it is possible to suppress the increase in memory, computational effort, and memory consumption. This embodiment will be explained in detail with reference to Figure 6. Note that the steps are the same as those for determining artifact components, except that the estimation of artifact components is changed to calculation.

[0039] In step S601, the artifact component determination unit 106 creates a binned image from the mask image created in step S306 and the fluorescence image 500 shown in Figure 5. The binning method involves replacing the small ROI 503 with statistical values ​​such as the mean, and excluding the pixel information of the mask region during averaging. In other words, for example, if the fluorescence image is divided into 64x64 small ROI 503, a binned image of size 64x64 will be created. The binned image created in this way is an image that reflects artifact components other than signal components, such as background components, shading components, and optical crosstalk.

[0040] In step S602, the binning image created in step S601 is enlarged to the same image size as the fluorescence image. That is, the image of the parts other than the individually separated sections is made into a 64x64 binning image and used as the artifact component. For the enlargement process, bilinear, bicubic, Lanczos, etc., can be used. Also, the shading component and optical crosstalk component are low-frequency components that have a certain degree of spatial spread. Considering this, the bicubic or Lanczos method, which uses surrounding information, is preferable. Alternatively, a low-pass filter such as a Gaussian filter may be applied to blur the image to prevent overestimation or underestimation of the artifact component due to abrupt changes. The enlarged image is called the artifact component image.

[0041] In step S603, the artifact component is corrected by subtracting or dividing the artifact component image created in step S602 from the fluorescence image.

[0042] The process of determining the positive / negative state and calculating the density from the artifact-corrected fluorescence image, after artifact component correction, is the same as in the embodiment described above.

[0043] In this way, by creating a regular image like a binned image, it becomes possible to create an artifact component image using interpolation processing with relatively low computational cost.

[0044] To appropriately correct optical crosstalk, which is relatively steep compared to shading, it is preferable to determine the artifact component by estimation. However, to save memory and computational costs due to hardware constraints, it is preferable to determine the artifact component by calculation.

[0045] Examples An example of determining the artifact components by estimation is explained using Figure 7. Figure 7(b) is the fluorescence image before correcting the artifact components. Figure 7(a) is the artifact components estimated in this embodiment from Figure 7(b). Figure 7(c) is the fluorescence image after artifact correction, obtained by subtracting Figure 7(a) from Figure 7(b). Figures 7(d) and 7(e) show the profiles of the dotted line portion at the top of the fluorescence images before and after correction. In Figure 7(d), the baseline of the profile is non-uniform, whereas in Figure 7(e), the baseline is corrected and uniformly becomes 0. From this, it can be seen that the shading components that were present in the image have been appropriately corrected. Furthermore, the optical crosstalk from adjacent wells is also reduced by the local brightness change pattern in Figure 7(a). By appropriately correcting artifact components such as baselines and optical crosstalk, the accuracy of negative / positive determination is improved, leading to improved analysis accuracy.

[0046] Table 1 below shows an example of measuring DNA concentration (relative value) using fluorescence analysis with beads. With correction, the values ​​are clearly different for each DNA concentration, and the detection limit is below a DNA concentration of 0.001. However, without correction, the values ​​for DNA0 and DNA0.001 are almost the same, and the detection limit is between a DNA concentration of 0.01 and 0.001. This result is due to the improvement in the signal-to-noise ratio and thus the accuracy of nucleic acid analysis, as shading was removed by the correction.

[0047] [Table 1]

[0048] Although specific nucleic acid analyzers have been described above, the present invention is not limited to them. [Explanation of symbols]

[0049] 100: Nucleic acid analyzer 101:Distribution section 102: Sign section 103: Information acquisition department 104: Information Processing Section 105: Information extraction section 106: Artifact component determination unit 107: Correction section 108: Specific part 109:Display section 110: Storage section 200: Well Plate 201: Lower board 202: Upper board 203: Bulkhead 204: Well 205: Space 500: Fluorescence image of a well plate 501: Well area 502: Outer well region 503: Small ROI

Claims

1. A nucleic acid analyzer comprising a distribution unit, a labeling unit, an information acquisition unit, and an information processing unit, The information processing unit includes an information extraction unit, an artifact component determination unit, a correction unit, and a specification unit. The distribution unit distributes the sample containing nucleic acids and reagents into a plurality of individual separation compartments, and an aggregate is formed between the plurality of individual separation compartments and the portion other than the plurality of individual separation compartments. The labeling unit makes a change in the individual isolation compartment to which the sample containing the target nucleic acid is distributed, so that the individual isolation compartment to which the sample containing the target nucleic acid is distributed can be distinguished from the individual isolation compartment to which the sample not containing the target nucleic acid is distributed. The information acquisition unit acquires an image of the aggregate including artifact components, The information extraction unit extracts from the acquired image the regions corresponding to the plurality of individual separation sections and the regions corresponding to the parts other than the plurality of individual separation sections, The artifact component determination unit determines the artifact component in the image of the region corresponding to the portion other than the extracted plurality of individual separation sections. The correction unit corrects the images corresponding to the extracted plurality of individual separated sections using the determined artifact components. The identifying unit identifies individual isolation compartments in which the magnitude of the change occurring in the individual isolation compartment where the sample containing the target nucleic acid is distributed exceeds a predetermined threshold, based on the corrected image. A nucleic acid analyzer characterized by the following features.

2. The assembly is A substrate, and a plurality of individual separation compartments arranged on the substrate, in which the sample containing the nucleic acid and the reagents are distributed, The nucleic acid analyzer according to claim 1, comprising a portion of the substrate other than the plurality of individual isolation compartments, which consists of a region where the plurality of individual isolation compartments are not present.

3. The aforementioned change is the generation of fluorescence. The nucleic acid analyzer according to claim 1 or 2, wherein the information acquired by the information acquisition unit, corresponding to the aggregate, includes a fluorescence image of the aggregate.

4. The nucleic acid analyzer according to any one of claims 1 to 3, wherein the image of the aggregate, including artifact components, further includes a bright-field image of the aggregate.

5. The nucleic acid analyzer according to any one of claims 1 to 4, characterized in that the information extraction unit uses a default mask based on the shapes of the plurality of individual separation compartments in the aggregate.

6. The aforementioned plurality of individual isolation compartments are wells containing a sample and reagents containing distributed nucleic acids. The nucleic acid analyzer according to any one of claims 1 to 5, characterized in that the aggregate is a well plate containing the wells.

7. The nucleic acid analyzer according to claim 6, characterized in that the portion other than the plurality of individual isolation compartments includes a part of the region of the well plate other than the wells.

8. The nucleic acid analyzer according to any one of claims 1 and 3 to 5, characterized in that the plurality of individual separation compartments are droplets containing a sample and reagents containing distributed nucleic acids, and the aggregate contains the droplets and a dispersion medium.

9. The nucleic acid analyzer according to claim 8, characterized in that the portion other than the plurality of individual separation compartments includes a part of the dispersion medium.

10. The nucleic acid analyzer according to any one of claims 1 to 9, characterized in that the artifact component determination unit determines the artifact component by estimation using basis functions.

11. The nucleic acid analyzer according to claim 10, characterized in that the basis function is a linear function.

12. The nucleic acid analyzer according to any one of claims 1 to 9, characterized in that the artifact component determination unit determines the artifact component by estimation using a regularization term.

13. The nucleic acid analyzer according to any one of claims 1 to 9, characterized in that the artifact component determination unit determines the artifact component by estimation using a polynomial approximation.

14. The nucleic acid analyzer according to any one of claims 1 to 9, characterized in that the artifact component determination unit determines the artifact component by calculation using binning.

15. The aforementioned information and artifact components each include coordinate information and the brightness of the coordinates, The correction unit, The brightness of the artifact component is subtracted from the brightness of the region corresponding to the plurality of individual separated sections, or, The nucleic acid analyzer according to any one of claims 3 to 14, characterized in that the brightness of the region corresponding to the plurality of individual isolation compartments is divided by the brightness of the artifact component.

16. The reagent comprises an effector protein, a crRNA that binds to the target nucleic acid, and a reporter molecule. The crRNA binds to the target nucleic acid and activates the effector protein. The labeling portion is characterized by generating fluorescence by modifying the reporter molecule with the activated effector protein. A nucleic acid analyzer according to any one of claims 3 to 15.

17. A nucleic acid analysis method comprising a distribution step, a labeling step, an information acquisition step, an information extraction step, an artifact component determination step, a correction step, and a identification step, In the distribution step, the sample containing nucleic acids and reagents are distributed into a plurality of individual separation compartments, and an assembly is formed that includes the plurality of individual separation compartments and the portion other than the plurality of individual separation compartments. In the labeling step, in order to distinguish between individual isolation compartments into which a sample containing the target nucleic acid was distributed in the specific step and individual isolation compartments into which a sample not containing the target nucleic acid was distributed, The sample containing the target nucleic acid causes a change in the individual isolation compartments to which it is distributed. In the information acquisition step, an image of the aggregate including artifact components is acquired. In the information extraction step, regions corresponding to the plurality of individual separation sections and regions corresponding to the parts other than the plurality of individual separation sections are extracted from the acquired image, In the artifact component determination step, the artifact component in the image of the region corresponding to the part other than the extracted plurality of individual separation sections is determined. In the correction step, the determined artifact components are used to correct the images corresponding to the extracted plurality of individual separated sections. In the aforementioned specific step, based on the corrected image, an individual isolation compartment in which the magnitude of the change occurring exceeds a predetermined threshold is identified, in which the sample containing the target nucleic acid is distributed. A nucleic acid analysis method characterized by the following features.

18. A program for causing a computer to perform the nucleic acid analysis method described in claim 17.

Images