JP4280720B2 - Sample analysis apparatus and sample analysis method - Google Patents

Description

  The present invention relates to a DNA microarray and a method for analyzing a sample using the DNA microarray. In particular, in DNA microarray image analysis after hybridization, DNA microarray image analysis is performed by positioning the detection area defined by the analysis tool and correcting the image for the DNA microarray image read by the scanner. The present invention relates to a technique for improving the accuracy of the process and automating it.

  In recent years, gene analysis using a DNA microarray has been realized.

  In this specification, a DNA microarray or DNA chip refers to a DNA spotted in a grid shape on an array substrate made of glass or the like.

On the DNA microarray, DNA is spotted as a probe that can specifically react with a labeled DNA sample.

  When an unknown DNA sample to be analyzed is optically detectable with a luminescent or fluorescent mark and poured onto such a DNA microarray, if the DNA sample is in a complementary relationship with the DNA on the grid, it will bind and double Become a chain. When all the DNA that has not been linked to the sample is washed away, the DNA sample to be judged is lit, and this is read by a scanner, the situation of the double-stranded DNA can be observed as an image. That is, by analyzing the distribution of the marks that emit light on the DNA microarray, it is possible to analyze the presence of a desired gene, whether or not a certain gene is expressed, and how much it is expressed. In this way, by configuring a known DNA set on a DNA microarray and changing the DNA set, gene mutation, gene expression level, and the like can be detected.

  FIG. 1 shows a series of processing steps of such DNA microarray analysis.

  As shown in FIG. 1, in the hybridization step 3, an unknown DNA sample to be analyzed is dropped with a mark for light emission on a test piece 1 of a DNA microarray including a spot region 1a on which DNA is mounted. The Here, if the DNA sample is in a complementary relationship with the DNA on the grid, they are combined into a double strand. Next, in the washing step 5, when the hybridized DNA microarray 1 is washed with a predetermined washing solution, all unligated DNA is washed away. In the scanning process 7, the cleaned DNA microarray is irradiated with a laser beam having a predetermined wavelength suitable for exciting a light emission mark (for example, Cy3, Cy5, etc.) and scanned in the scanner device. Thus, the amount of luminescence of each spotted DNA (gene) is measured. The scanned image of the DNA microarray 1 is stored in the DNA microarray image file 11. In the analysis step 9, the DNA microarray image file 11 is read and a DNA microarray analysis process is executed. Specifically, in the analysis step 9, the fluorescence intensity of each spot is calculated based on the read image data, and various analyzes are executed.

  Note that the DNA microarray images read by the scanner may be sequentially input to the analysis step 9 without using the DNA microarray image file 11.

  The analysis result is recorded as the digitized data file 13 and may be displayed and output via the display device 15 or printed and output via the printing device 17 as necessary.

  FIG. 2 shows an example of a DNA microarray 1 used for DNA microarray analysis. As shown in FIG. 2, the DNA microarray 1 has a matrix in which a predetermined number of individual genes (hereinafter referred to as “spots”) of cDNA (complementary DNA) are arranged in a row direction and a column direction on a grid of the substrate 2. Blocks arranged in a shape are formed. The spots arranged in the spot region 1a on the substrate 2 respectively correspond to different DNAs whose base sequences have already been decoded, and the arrangement positions on the substrate 2 are determined in advance. .

  FIG. 3 shows an example of a template applied to the DNA microarray image file 11 in the analysis process. As shown in FIG. 3, the template is divided into a plurality of fields such as A to F, and detections arranged in a matrix of m rows and n columns (4 × 4 in FIG. 3) in each field. There are a plurality of blocks (p × 24 in FIG. 3) formed by areas (corresponding to individual spots of the DNA microarray). This block is assigned address numbers a to p and 1 to 24, for example, in both XY directions, and the position of each block can be represented as “a, 1” in each field, for example.

  In the analysis step 9 described above, the individual spots in the scanned image of the DNA microarray are applied to the detection area of the template provided by the analysis tool. The positioning operation by fitting the detection area to the image is referred to as alignment. In the analysis step 9, in order for the analysis tool to calculate the fluorescence intensity of each spot on the DNA microarray and perform an accurate analysis, the DNA microarray defined in advance for each spot on the image The alignment (positioning) process needs to be executed accurately so that the individual detection areas arranged on the substrate 2 are set correctly. (In order to correctly analyze the read image, it is necessary to apply individual detection areas corresponding to individual spots in this alignment.) In this alignment (positioning) process, misalignment is detected. In such a case, the position of the image or detection spot must be corrected so as to be correctly set in the detection area of the corresponding template.

  However, the conventional DNA microarray analysis method that has undergone the hybridization step 3 has the following problems to be solved.

    That is, the processing accuracy of the substrate 2 made of glass constituting the DNA microarray is low, the occurrence of cracks, cracks, etc., the displacement when the DNA microarray is placed on the scanner autoloader or when these are mounted on the scanner Inevitably, the image file of the DNA microarray to be analyzed is inevitably misaligned due to various causes such as the occurrence of errors, errors in machine accuracy such as autoloaders and scanners, and even minute dust adhering to the substrate 2. It will occur.

  However, the external dimensions of the substrate 2 constituting the DNA microarray are low in accuracy due to glass processing accuracy, chipping / breaking, and the like. For this reason, it has not been practically possible to use the outer shape of the substrate 2 for positioning the DNA microarray and the template provided by the analysis tool.

  Accordingly, although a large number of DNA microarrays are placed on the autoloader, and the DNA microarrays are continuously scanned by the scanner and the image data 11 is automatically accumulated, the image data 11 is automatically accumulated in the subsequent analysis step 9. Is detected by the θ deviation (rotational deviation), x-direction deviation, and y-direction deviation, and the detection area is positioned in the DNA microarray image file 11, or the detection area is positioned by an existing analyzer. It was necessary to manually determine whether a mistake was made.

  In particular, when the image misalignment is large, the existing positioning apparatus cannot automatically perform the positioning process correctly.

  Accordingly, the positioning process of the detection area to the DNA microarray image file and the determination of the success or failure of the positioning of the detection area depend on the human visual recognition and judgment, and cannot be automatically processed, and the entire bottleneck Was configured.

  In particular, when the number of images to be analyzed is large, enormous time and effort are required for the positioning processing of these detection areas and the determination of the success or failure of positioning of the detection areas. Furthermore, with the adoption of the inkjet method in the technology for forming spots on DNA microarrays, the spot diameter has become finer and the spacing between spots has become high pitch, so that accurate positioning processing by visual recognition is performed. It is even more difficult by manpower.

  The present invention has been made to solve the above technical problem, and the object of the present invention is to quantitatively and automatically position the detection area on the DNA microarray image file in the analysis of the DNA microarray. An object of the present invention is to provide a DNA microarray, a sample analysis apparatus, and a sample analysis method that can be executed with high accuracy and accuracy.

  Another object of the present invention is to provide a DNA microarray, a sample analysis device, and a sample analysis method capable of quantitatively, automatically and accurately determining whether or not a detection area is positioned successfully.

  In addition, another object of the present invention is to enable parallel analysis processing by allowing the process from the scanning process to the analysis process to obtain the desired analysis data continuously and unattended. This is to save labor and speed up analysis processing.

  In the present invention, a reference pattern used for positioning the detection area on the DNA microarray image file is provided on the substrate of the DNA microarray to be analyzed, and accurate positioning processing and position are performed using the reference pattern of the DNA microarray. A deviation correction process is executed.

  This reference pattern can be constituted by, for example, a combination of spots that always emit light and spots that never emit light.

  In the present invention, multiple stages of positioning processing may be executed in the analysis processing in units of DNA microarrays, spots, and blocks.

  According to an aspect of the present invention, a spot region in which spots that fix a substrate and a probe that can specifically react with a sample that is optically detectably labeled are formed in a matrix on the surface of the substrate. And a reference pattern region which is arranged on the substrate surface in the spot region or in the vicinity of the spot region, and which comprises a plurality of different position marks for correcting the positional deviation of the spot in the analysis of the sample. A probe-reactive chip is provided.

  According to another feature of the invention, the reference pattern region is a combination of spots that always emit light and spots that do not necessarily emit light.

  According to another aspect of the present invention, the spot region includes a plurality of blocked spot sub-regions on the substrate surface, and the reference pattern regions are respectively disposed in the spot sub-regions.

  According to another feature of the invention, the spot that always emits light in the reference pattern region is a fluorescent material that emits light at a predetermined fluorescence intensity or higher.

  According to another feature of the invention, the necessarily emitting spot in the reference pattern region is a nucleic acid adsorbing material.

  According to another feature of the invention, the spot that necessarily emits light in the reference pattern region is a housekeeping gene, a fragment of the housekeeping gene, or a part of the base sequence of the housekeeping gene or the fragment. Any one or more of the contained nucleic acids.

  According to another feature of the present invention, the reference pattern region includes a plurality of spots that do not necessarily emit light around spots that always emit light.

  According to another aspect of the present invention, the probe reactive chip further includes a reference mark disposed outside the spot area on the substrate surface for correcting a positional shift of the spot area.

  According to another feature of the invention, the reference mark is a fluorescent material that emits light at a predetermined fluorescence intensity or higher.

  According to another feature of the invention, the fiducial mark is a nucleic acid adsorbing material.

  According to another aspect of the present invention, the reference mark is any one of a housekeeping gene, a fragment of the housekeeping gene, or a nucleic acid including the housekeeping gene or the fragment in a part of a base sequence thereof. More than one.

  According to another feature of the invention, the reference mark comprises a plurality of spots that always emit light, or a combined arrangement of spots that always emit light and spots that do not necessarily emit light.

  According to another aspect of the present invention, the reference mark indicates chip-specific information including a chip type and a production lot identifier.

  According to another aspect of the present invention, the spot sub-regions are arranged with an interval of at least twice as large as each spot interval in the spot sub-regions arranged in the spot sub-region with the adjacent spot sub-regions. Is done.

  According to another aspect of the present invention, a reference pattern information storage unit that defines information of a reference pattern area formed of a plurality of different position marks for correcting a positional deviation of a spot formed on a probe reactive chip. And an image data reading unit that reads image data obtained by scanning a spot that fixes a probe that can specifically react with an optically detectably labeled sample on the probe reactive chip, Based on the information of the reference pattern area read from the reference pattern information storage unit, the image data is positioned in a predetermined detection target area, and a positional deviation between the image data and the detection target area is determined. An image data positioning unit that generates correction data to be corrected, and image data positioned by the image data positioning unit are analyzed. Accordingly, a determination unit that determines whether the positioning is successful, a correction unit that corrects a positional deviation between the image data and the detection target area based on the correction data, and an analysis of the corrected image data, There is provided a sample analysis apparatus comprising an analysis unit that outputs digitized data relating to the sample.

  According to another aspect of the present invention, the information of the reference pattern area stored in the reference pattern information storage unit is a pattern formed by a combination of spots that always emit light and spots that never emit light.

  According to another feature of the invention, the reference pattern area information stored in the reference pattern information storage unit is relative coordinates from a reference mark.

  According to another aspect of the present invention, the information of the reference pattern area stored in the reference pattern information storage unit is a relative coordinate between the reference patterns defined in advance.

  According to another aspect of the present invention, the reference pattern region information is defined for each of a plurality of blocked spot subregions on the probe reactive chip in the reference pattern information storage unit. The data positioning unit positions the image data in the spot sub-region unit.

  According to another feature of the invention, the analysis unit selectively operates in either an automatic mode or a manual mode, and the determination unit repeatedly analyzes the positioned image data a predetermined number of times. If the desired analysis result cannot be obtained, information indicating that the positioning process has failed for the chip is added to the output data.

  According to another feature of the present invention, the sample analyzer further includes a spot on the probe reactive chip for fixing a probe that can specifically react with an optically detectably labeled sample. A scanning unit that scans to obtain the image data is provided, and the scanning unit and the analysis unit execute processing in parallel.

  According to another aspect of the present invention, the image data positioning unit includes a first positioning unit that positions the spot of the image data in a detection area for each spot in the image data, and the probe reactive chip. And a second positioning unit for positioning the image data in units of a plurality of blocked spot sub-regions.

  According to another feature of the present invention, the sample analyzer further includes a third positioning unit that positions the image data in units of the entire spot area using a reference mark.

  According to another aspect of the present invention, information on a reference pattern area formed of a plurality of different position marks for correcting a spot position deviation formed on a probe reactive chip is defined in a reference pattern information storage unit. Reading image data obtained by scanning a spot on the probe-reactive chip for fixing a probe capable of specifically reacting with a sample that is optically detectably labeled; and Based on the information of the reference pattern area read from the reference pattern information storage unit, the image data is positioned in a predefined detection target area, and the positional deviation between the image data and the detection target area is corrected. Generating correction data to be determined and analyzing the positioned image data to determine whether or not the positioning is successful A step of correcting a positional deviation between the image data and the detection target area based on the correction data, and a step of analyzing the corrected image data and outputting digitized data related to the sample. A sample analysis method characterized in that it is included is provided.

  According to another aspect of the present invention, the information of the reference pattern area stored in the reference pattern information storage unit is a pattern formed by a combination of spots that always emit light and spots that never emit light.

  According to another feature of the invention, the reference pattern area information stored in the reference pattern information storage unit is relative coordinates from a reference mark.

  According to another aspect of the present invention, the information of the reference pattern area stored in the reference pattern information storage unit is a relative coordinate between the reference patterns defined in advance.

  According to another aspect of the present invention, the reference pattern region information is defined for each of a plurality of blocked spot subregions on the probe reactive chip in the reference pattern information storage unit. In the data positioning step, the image data is positioned in units of the spot sub-regions.

  According to another feature of the invention, the analyzing step selectively operates in either an automatic mode or a manual mode, and the determining step repeatedly executes the analysis of the positioned image data a predetermined number of times. If the desired analysis result cannot be obtained, information indicating that the positioning process has failed for the chip is added to the output data.

  According to another feature of the present invention, the sample analysis method further includes a step for fixing a spot on the probe reactive chip for fixing a probe that can specifically react with an optically detectably labeled sample. A scanning step of scanning to obtain the image data, wherein the scanning step and the analyzing step are performed in parallel.

  According to another aspect of the present invention, the image data positioning step includes: a first positioning step for positioning a spot of the image data in a detection area for each spot in the image data; A second positioning step of positioning the image data in units of a plurality of blocked spot sub-regions.

  According to another feature of the invention, the sample analysis method further includes a third positioning step of positioning the image data in units of the entire spot area using a reference mark.

  According to another aspect of the present invention, there is provided a program for causing a computer to execute a sample analysis process, which includes a plurality of different position marks formed on a probe-reactive chip for correcting a positional deviation of a spot. The process of defining the information of the reference pattern area in the reference pattern information storage unit, and scanning the spot on the probe reactive chip for fixing the probe that can specifically react with the optically detectable sample. Based on the process of reading the image data obtained in this manner and the information of the reference pattern area read from the reference pattern information storage unit, the image data is positioned in a predefined detection target area, and Processing for generating correction data for correcting misalignment between the image data and the detection target area, and the positioned image data Analyzing the processing to determine whether the positioning is successful, processing to correct the positional deviation between the image data and the detection target area based on the correction data, and analyzing the corrected image data, And a process of outputting digitized data relating to the sample.

  According to another aspect of the present invention, the information of the reference pattern area stored in the reference pattern information storage unit is a pattern formed by a combination of spots that always emit light and spots that never emit light.

  According to another feature of the invention, the reference pattern area information stored in the reference pattern information storage unit is relative coordinates from a reference mark.

  According to another aspect of the present invention, the information of the reference pattern area stored in the reference pattern information storage unit is a relative coordinate between the reference patterns defined in advance.

  According to another aspect of the present invention, the reference pattern region information is defined for each of a plurality of blocked spot subregions on the probe reactive chip in the reference pattern information storage unit. In the data positioning process, the image data is positioned in the spot sub-region unit.

  According to another feature of the invention, the analysis process selectively operates in either an automatic mode or a manual mode, and the determination process repeatedly executes the analysis of the positioned image data a predetermined number of times. If the desired analysis result cannot be obtained, information indicating that the positioning process has failed for the chip is added to the output data.

  According to another feature of the invention, the program further scans a spot on the probe reactive chip for immobilizing a probe that can specifically react with a sample that is optically detectably labeled. Scanning processing for obtaining the image data, and the scanning processing and the analysis processing are executed in parallel.

  According to another feature of the present invention, the image data positioning process includes: a first positioning process for positioning a spot of the image data in a detection area for each spot in the image data; A second positioning process for positioning the image data in units of a plurality of blocked spot sub-regions.

  According to another feature of the invention, the program further includes a third positioning process for positioning the image data in units of the entire spot area using a reference mark.

  According to another aspect of the present invention, there is provided a program for causing a computer to execute a sample analysis process, which includes a plurality of different position marks formed on a probe-reactive chip for correcting a positional deviation of a spot. The process of defining the information of the reference pattern area in the reference pattern information storage unit, and scanning the spot on the probe reactive chip for fixing the probe that can specifically react with the optically detectable sample. Based on the process of reading the image data obtained in this manner and the information of the reference pattern area read from the reference pattern information storage unit, the image data is positioned in a predefined detection target area, and Processing for generating correction data for correcting misalignment between the image data and the detection target area, and the positioned image data A program for determining whether the positioning is successful or not, and a process for correcting a positional deviation between the image data and the detection target area based on the correction data. The

  According to the present invention, in the analysis of the DNA microarray, the positioning process of the detection area to the DNA microarray image file and the determination process of the positioning success / failure can be performed quantitatively, automatically and with high accuracy.

  Hereinafter, a DNA microarray (DNA chip), a sample analysis apparatus, and a sample analysis method according to an embodiment of the present invention will be described in detail with reference to FIGS.

  In the present embodiment, a reference mark used for positioning a detection area on a DNA microarray image file is provided on the substrate of the DNA microarray to be analyzed, and the positioning process is performed in a block constituting the spot area of the DNA microarray. In addition, a reference pattern used for the misregistration correction process is arranged, and using this DNA microarray, the detection area positioning process and the misregistration correction process for the DNA microarray image file are executed. In the present embodiment, a multi-step positioning process is executed in the analysis process in units of DNA microarrays, spots, and blocks.

  FIG. 4 is a block diagram showing the configuration of the entire sample analysis system according to the embodiment of the present invention.

  As shown in FIG. 4, the sample analysis system according to the embodiment of the present invention includes a scanner device 71, a control personal computer 73, and an analysis personal computer 91.

  The scanner device 71 includes an autoloader mechanism for placing a plurality of DNA microarrays 1. The DNA microarray on which a DNA sample marked with a luminescent marker is dropped is excited by laser light, and the obtained image is converted into a DNA microarray. Store in the image file 11. The DNA microarray 1 is scanned at an excitation wavelength corresponding to, for example, the fluorescent dyes Cy3 and Cy5, and an image file corresponding to each excitation wavelength is obtained corresponding to one DNA microarray 1. This image file is, for example, a 16-bit grayscale Tiff format image, but may be a multi-image tiff format image, a JPEG format image, or the like.

  The control personal computer 73 may be a computer that can be connected to the scanner device 71 via, for example, a LAN cable, such as a general-purpose personal computer, and controls the scanning of the DNA microarray 1 and the image acquisition in the scanner device 71. .

  The personal computer for analysis 91 reads the DNA microarray image file 11. Alternatively, a DNA microarray image directly read from the scanner device 71 or the control personal computer 73 is input. Further, an analysis definition file group 93 that defines parameters for execution of analysis is read, the DNA microarray image is analyzed, and the digitized analysis result data is output to the digitized data file 13. The analysis personal computer 91 is installed with a program serving as an analysis tool for executing the analysis processing according to the present embodiment. The analysis program may be a program that executes the analysis processing according to the present embodiment. For example, “GenePix Pro” (TM) manufactured by InterMedical that runs on MircoSoft Windows (registered trademark) or the like is used as the basis or Can be used as part.

  The analysis program running on the personal computer 91 for analysis automatically controls the analysis of the DNA microarray image file 11. The details of the processing will be described later, but the DNA microarray image file 11 is read and analyzed. Positioning process (positioning process using the reference mark) so that the definition file group 93 is read and the reference mark on the read DNA microarray image matches the reference position on the template stored in the analysis definition file group 93 Execute. Further, the analysis program configures a plurality of spot areas on the DNA microarray using the positioning process in spot units on the DNA microarray (positioning process in spot units) and the reference pattern based on the image data and the detection area. Image data positioning processing in block units (position processing in block units) is executed. If there is a positioning error, the analysis program automatically corrects the positional deviation of the image file according to the detection area.

  If the image file to be processed within the preset time cannot be digitized, the analysis program will retry the analysis process a predetermined number of times, and notify the manual digitization if the retry exceeds the predetermined number of times. Therefore, information indicating a numerical error about the image is output to a log file. Finally, the analysis program outputs the digitized image data information to the digitized data file 13.

  Note that a plurality of analysis personal computers 91 are installed with respect to the pair of scanner devices 71 and the control personal computer 73, and these plurality of devices can analyze a large number of image files in parallel. Good. Even if the pair of scanner devices 71 and the control personal computer 73 are not provided, the DNA microarray image file 11 may be read by other means.

  Specifically, the analysis definition file group 93 includes an analysis parameter file 931 and an analysis automation information definition file 933.

  The analysis parameter file 931 includes the following information.

・ Definition of blocks and spots on DNA microarray (number of blocks in row / column direction, number of spots in row / column direction, spot diameter, etc.)
・ Definition of block and spot position information on DNA microarray ・ Definition of gene identification information such as gene name for each spot ・ Definition of attributes for each spot (for example, “not existing spot”, “good spot” (There are some attributes that change during the positioning process, such as “missing spot”)
The analysis automation information definition file 933 includes the following information.

・ Specify image file generation method (linked / not linked to scanning by scanner device)
・ Definition of image file name to be analyzed and designation of digitized data storage destination ・ Definition of fiducial marks used in positioning using the fiducial marks (number of fiducial marks, minimum fluorescence intensity, coordinates, recognition area, allowable displacement, etc.) )
・ Definition of reference patterns for positioning (alignment) (number of reference patterns, minimum fluorescence intensity, coordinates, allowable displacement, etc.)
・ Define the number of retries (or maximum waiting time) when there is no image file ・ Define the retry method at the time of positioning error (number of retries or maximum waiting time, movement order and amount of detection area, etc.)
FIG. 35 is a block diagram showing a functional configuration of the sample analyzer according to this embodiment. As shown in FIG. 35, the sample analysis apparatus according to the present embodiment is an analysis computer 91 in which the analysis program is introduced, and uses an analysis image storage unit 351, a definition file storage unit 353, and a reference mark. Positioning processing unit 355, spot unit positioning processing unit 357, block unit positioning processing unit 359, positioning determination unit 361, analysis control unit 363, analysis result output unit 365, log file 367, and analysis result A file 369 and an analysis error image file 371 are provided. The analysis image storage unit 351 stores the DNA microarray image file 11 in FIG.

  The definition file storage unit 353 stores the definition file group in FIG. 4 (analysis parameter file 931 and analysis automation information file 933 in FIG. 6 below). The definition file storage unit 353 corresponds to the reference pattern information storage unit in the claims. The positioning processing unit 355 using the reference mark includes an image data reading unit in claims. The positioning processing unit 355 using the reference marks, the spot unit positioning processing unit 357, and the block unit positioning processing unit 359 correspond to the image data positioning unit and the correction unit in the claims. The positioning determination unit 361 corresponds to the determination unit in the claims. The analysis control unit 363 and the analysis result output unit 365 correspond to the analysis unit in the claims.

  An analysis image storage unit 351, a definition file storage unit 353, a positioning processing unit 355 using a reference mark, a spot unit positioning processing unit 357, a block unit positioning processing unit 359, an analysis control unit 363, a positioning determination unit 361, Each of the analysis result processing units 365 is mounted on the analysis personal computer 91 in FIG. This analysis personal computer 91 may be one or may be configured as an analysis computer system by connecting a plurality of computers via a network.

  FIG. 5 shows an example of part of the digitized data output from the analysis personal computer 91. As shown in FIG. 5, the digitized data is obtained as, for example, fluorescence intensity data of spots detected at each of the Cy3 excitation wavelength and the Cy5 excitation wavelength. More specifically, the digitized data 13 includes the following information for each spot.

-Flag for detection spot-Block number-Row in column-Column number-Gene identification information including gene name-Center coordinates of detection spot (x, y)
・ Median, average value, standard deviation, etc. of fluorescence intensity of detection spot at Cy3 excitation wavelength ・ Median, average value, standard deviation, etc. of fluorescence intensity around detection spot (background) at Cy3 excitation wavelength ・ At Cy5 excitation wavelength Median fluorescence intensity of detection spot, average value, standard deviation, etc. Median fluorescence intensity around detection spot at Cy5 excitation wavelength (background), average value, standard deviation, etc. Various data relating to quality, etc. Next, this embodiment The procedure of the sample analysis process in the sample analysis method according to FIG.

  FIG. 6 is a flowchart showing a procedure of sample analysis processing in the sample analysis method according to the embodiment of the present invention.

  First, the analysis program is initialized and the process is started (step S1). Next, the analysis parameter file 931 and the analysis automation information file 933 are read (step S3). By reading the analysis automation information file 933, an analysis image definition file name, an analysis parameter file name, the number of image waiting retries, an image waiting time, and the like are read. From the analysis image definition file name, the image to be analyzed and the number of images are set in the analysis program.

  The processes from step S5 to step S35 are repeatedly executed until the number of images to be analyzed reaches the total number of read images (that is, end condition End) (step S5).

  Next, it is determined whether or not there is an image to be analyzed (step S7). If there is no image to be analyzed (step S7N), the number of image waiting retries is read in step S3 and stored in the analysis program. The processes from step S11 to step S15 are repeatedly executed until the set number of times is exceeded (step S11). Further, it is determined whether or not there is an image to be analyzed (step S13). If there is an image to be analyzed (step S13Y), the process proceeds to step S9, whereas if no image to be analyzed is obtained. (Step S13N) The image wait retry counter is incremented (Step S15), and if the set number of times is exceeded, the process proceeds to Step S35.

  Returning to step S7, if there is an image to be analyzed (step S7Y), an image file corresponding to the DNA microarray to be analyzed is read from the DNA microarray image file 11 (step S9). Alternatively, the image file to be analyzed may be directly input from the scanner device 71 and the control personal computer 73 side without using the DNA microarray image file 11. Subsequently, a positioning process using the reference mark (positioning process using the reference mark) is executed (step S17). After the positioning process using the reference mark in step S17, the image data is processed (step S19). If the image process is determined to be unsuccessful (step S19N), the process proceeds to step S35. On the other hand, when the image processing is determined to be successful, the image file to be analyzed after the image processing is read (step S21).

  Next, a positioning process for each spot unit on the DNA microarray (the positioning process for the spot unit) is executed (step S23), and a positioning process for the block unit on the DNA microarray (the positioning process for the block unit) is performed. It is executed (step S25). Note that either step S23 or step S25 may be executed first as long as it is after the analysis image reading process of step S21 and before the positioning determination of step S27.

  Based on the image data digitized after the positioning process using the reference marks and the spot unit positioning process, it is determined whether the positioning (alignment) has succeeded or failed (step S27). If it is determined that the positioning is successful (step S27Y), the digitized image data is output to the digitized data file 13, and the counter of the number of images to be analyzed is incremented (step S35). On the other hand, if it is determined that the positioning has failed (step S27N), it is further determined whether or not it is less than or equal to the predetermined number of movements (or movement time) of the detection area (step S31). When reaching (step S31N), the process proceeds to step S35. On the other hand, if the number of detection area movements is equal to or less than the predetermined number of detection area movements (step S31Y), detection area movement processing (correction processing) in units of DNA microarray blocks is executed (step S33), and step S23 in units of spots is performed. Return to the positioning process.

  More specifically, in the movement (correction) processing of the detection area in step S33, under the assumption that the rough positioning by the reference mark is correct, the movement amount is gradually increased from a small movement amount, for example, clockwise. Positioning between a pair of spots and a detection area is tried.

  Finally, after completing the analysis of all the images to be analyzed, the processing from step S7 to step S35 may be repeated a predetermined number of times for the image in which the analysis error has occurred (step S37).

Next, details of each positioning process in the present embodiment will be described. In the positioning process of this embodiment,
(1) First, a reference mark that emits light is placed on a DNA microarray. Similarly, on the DNA microarray, a reference pattern composed of positive control and negative control is arranged in a block on the surface of the substrate 2 (or in the vicinity of the block). As the information analysis automation information of the DNA microarray, at least information indicating the positional relationship between the reference mark and each block, and information indicating the arrangement pattern of the reference pattern and the positional relationship between each block are registered in advance and known. And

(2) After recognizing and detecting the fiducial mark on the image, the image is corrected (rotated / moved) to the position (design position) where the fiducial mark originally exists, using the position coordinates of the fiducial mark previously defined as template analysis automation information ) To roughly apply each spot on the template in advance to a detection area (positioning process using a reference mark). Thereafter, fitting of each detection area on the template to each spot is executed (positioning processing in units of spots).

(3) On the scanned image, positive control and negative control arrangement patterns (reference patterns) are detected by pattern matching using pattern information and position information about the reference patterns defined in advance in the template, The position is specified and positioning is executed in units of blocks (positioning processing in units of blocks).

  FIG. 7 is a flowchart for explaining the details of the positioning process using the reference mark in step S17 of FIG. 6 using the reference mark according to the present embodiment.

  As shown in FIG. 7, the analysis processing unit 355 using the reference mark according to the present embodiment reads the DNA microarray image file 11 (step S171), and includes a reference pattern recognition area read in advance from the analysis automation information file 933. The template is automatically recognized, and the image file is analyzed (numerically) so that the reference pattern of the read image file matches the reference pattern definition position in the reference pattern recognition area on the template (step 173). Position the fiducial mark.

  FIG. 8 shows an example of the image file read in step S171.

As shown in FIG. 8, the reference marks used for the positioning process using the reference marks according to the present embodiment are arranged on the substrate in advance around the spot area where the spots are arranged.

  The reference mark may be a mark made of a substance that emits a fluorescence signal when the DNA microarray is scanned by the scanner device 71. For example, the reference mark may be a fluorescent substance such as an autofluorescent substance, a nucleic acid adsorbing substance, or a housekeeping gene, A housekeeping gene fragment or a nucleic acid containing the housekeeping gene or a fragment thereof in a part of its base sequence can be used.

  In the present specification, an autofluorescent substance is a substance that originally has fluorescence as its own physical properties. This autofluorescent substance emits a fluorescent signal when the DNA microarray is scanned. The nucleic acid adsorbing substance does not have fluorescence as its own physical property, but may be any substance that adsorbs a fluorescently labeled nucleic acid in a sample dropped on a DNA microarray. For example, polyallylamine can be used. . Since this nucleic acid adsorbing substance adsorbs fluorescently labeled nucleic acid, it emits a fluorescent signal when the DNA microarray is scanned. A housekeeping gene is originally a gene that always has a certain expression level or more in a living body, and has no fluorescence as its own physical property. If this housekeeping gene is spotted on the DNA microarray, it will always hybridize with the fluorescently labeled nucleic acids in the sample solution, so that when the DNA microarray is scanned, a fluorescence signal is also emitted. These autofluorescent substances, nucleic acid adsorbing substances, and housekeeping genes are all spots that emit light when scanned on a DNA microarray. In this specification, these spots are referred to as positive controls. Called. On the other hand, a spot having a characteristic that does not necessarily emit light when scanned is referred to as a negative control in the present specification.

  FIG. 9 shows a state where a template including a reference mark recognition area is automatically recognized for the image of FIG. 8 read by the scanner device 71. FIG. 9 shows a state in which the definition position 10b of the reference mark in the reference mark recognition area 10a of the template and the reference mark 10c in the read image file are misaligned. The analysis program corrects the position so that the reference mark 10c in the image file coincides with the reference mark definition position 10b on the template, as shown in FIG. In FIG. 10, the reference mark recognition area 10a of the template and the definition position 10b of the reference pattern are shown for convenience, and need not be visually displayed as actual images.

  By the positioning process using the reference marks, the entire DNA microarray (that is, the entire spot area on the array) is roughly positioned with respect to the entire template.

  Returning to FIG. 7, the shift amount due to the positional deviation between the reference mark definition position 10b in the reference mark recognition area 10a of the template and the reference mark 10c in the read image file shown in FIG. 9 is calculated (step S175). ), Based on the calculated shift amount, the image is rotated and moved as shown in FIG. 10 (step S177). Finally, the corrected image file is stored in the image file 11b that stores the corrected image file. This storage destination may be the image file 11 from which the image file is read in step 171.

  Next, the details of the spot unit positioning process (step S23 in FIG. 6) in the present embodiment will be described.

  FIG. 11 is a flowchart for explaining the details of the spot-unit positioning process in step S23 of FIG. 6, which is executed for each spot on the DNA microarray according to this embodiment.

  As shown in FIG. 11, the spot-by-spot positioning processing unit 357 according to the present embodiment applies each spot image in each DNA microarray in the image file 11b after the position correction process (image processing) read in step S21. On the other hand, positioning of the template read in advance from the analysis automation information file 933 to the detection area corresponding to each spot is executed (step S231).

  FIG. 13 shows a state in which each detection area of the template is applied to the DNA microarray image after position correction read in step S21 of FIG. FIG. 13 shows a state in which each detection area 13b of the template is displaced from each spot in the read image file. In the analysis program, the spot-based positioning processing unit 357 corrects the position so that each spot image in the image file matches each detection area on the template, as shown in FIG.

  Returning to FIG. 11, the position-corrected image file shown in FIG. 14 is digitized based on the template definition information (step S233), and the digitized file is stored in the digitized data file 13 (step S233). S235).

  Next, the details of the block-unit positioning process (step S25 in FIG. 6) in the present embodiment will be described. In the block unit positioning process by the block unit positioning processing unit 359, both the positioning process using pattern matching using the pattern arrangement of the reference pattern and the positioning process using the position coordinates of the reference pattern are used. Or only one of these may be performed.

  FIG. 15 shows an example of a DNA microarray image file that is erroneously positioned by the spot-by-spot positioning process in step 23 in FIG. As shown in FIG. 15, in the spot unit positioning process, the spot unit positioning is performed for each block on the DNA microarray to test whether the block in the detection area on the template matches the spot in the block. To be executed. For this reason, for example, when dust adheres to the substrate of the DNA microarray, the detection area to which each spot is to be matched is erroneously determined, resulting in an error in block positioning. As the spot interval on the DNA microarray becomes finer, it is difficult to avoid the positioning error due to the adhering of fine dust.

  Therefore, in this embodiment, a reference pattern for adjusting the positioning in units of blocks is arranged in advance in the block on the DNA microarray, and positioning errors caused by the positioning processing in units of spots are adjusted.

  FIG. 12 shows an example of a DNA microarray used in this embodiment. As shown in FIG. 12, a plurality of block groups 1 b are arranged on the DNA microarray 1. Genes are spotted in each block, but in this embodiment, positive control that is a spot that always emits light and negative control that is a spot that always does not emit light (it is strange to shine) in each block. And place. These positive controls and negative controls are usually used as labels (markers) for judging whether or not the hybridization process has been processed without abnormality. In the present embodiment, a reference pattern composed of a combination of these positive controls and negative controls is formed. This reference pattern is used as a reference unit pattern for positioning in block units in the block unit positioning process by arranging spots for fixing genes as probes in each block formed in a matrix. This block corresponds to the spot sub-region in the claims.

  The positive control may be a mark made of a substance that emits a fluorescent signal when the DNA microarray is scanned by the scanner device 71 as in the case of the reference mark. For example, the positive control may be an autofluorescent substance, a nucleic acid adsorbing substance, or a house. A nucleic acid containing a housekeeping gene or a fragment thereof in a part of the base sequence can be used.

  Specifically, as shown by 1c in FIG. 12, a positive control is arranged in each block, and a negative control is arranged around it as shown by 1d. This reference pattern may be provided at one corner in the block, or may be provided at an arbitrary position in the block. Alternatively, if necessary, it may be provided outside the block constituting the spot area and in the vicinity thereof. The pattern arrangement of the positive control and the negative control arranged in this block and the information on the position coordinates thereof are registered in advance in the analysis automation information file 933 as one of the attribute information on the template. In the block-unit positioning process, the positions of the spots in each block can be specified by executing the determination of the fluorescence intensity and the position coordinates of these positive control and negative control.

  Another pattern of positive controls surrounded by negative controls may be placed under a reference pattern in the block, or negative on the diagonal of the block with respect to a pattern in the block・ Other patterns of positive controls surrounded by controls may be placed. By further arranging such other patterns in one block and using a plurality of reference patterns, it is possible to reduce misjudgments caused by dust or the like adhering to the DNA microarray.

  On the other hand, from the viewpoint of analyzing more genes with one DNA microarray, it is desirable that the area occupied by the reference pattern consisting of the positive control surrounded by the negative control is small.

  18 to 23 show examples of the arrangement of positive control patterns surrounded by the negative controls in the present embodiment. In FIG. 18 to FIG. 23, it is assumed that all other genes are spotted except the positive control and the negative control in the block. In addition, the determination criterion of the positioning (alignment) success in the block unit positioning processing is a threshold in which the positive control fluorescence intensity is equal to or higher than the set minimum fluorescence intensity, and the positive control coordinates are set in advance as template attributes. The following.

  In FIG. 18, a negative control 1d is arranged around one positive control 1c across the block 1b, and the required number of positive controls and negative controls is nine per block. When this pattern is used, if there is one or more dust having a minimum fluorescence intensity or more around the positive control, this is erroneously recognized as the positive control.

  In FIG. 19, the negative control 1d is arranged around two positive controls 1c separated from each other across the block 1b, and the number of necessary positive controls and negative controls is 15 per block. Become. When this pattern is used, if there are two or more dusts or the like having the minimum fluorescence intensity or more around the positive control, this is erroneously recognized as the positive control.

  In FIG. 20, the negative control 1d is arranged around one positive control 1c across the block 1b, and the negative control around the one positive control 1c is placed at a diagonal position in the block. The control 1d is arranged, and the number of necessary positive controls and negative controls is 16 per block. When this pattern is used, if there are two or more dusts or the like having the minimum fluorescence intensity or more around the positive control, this is erroneously recognized as the positive control. The pattern shown in FIG. 20 has enhanced detection of the θ shift of the block compared to the pattern of FIG.

  In FIG. 21 to FIG. 23, the pitch between the blocks 1b is arranged more than twice the pitch of the spot interval (that is, there is an interval corresponding to 1e per spot between the blocks 1b).

  In FIG. 21, the block 1b is arranged with an interval corresponding to the lowest one spot from the adjacent block 1b. A negative control 1d is arranged around one positive control 1c arranged at one of the four corners, and the number of necessary positive controls and negative controls is four per block. When this pattern is used, if there is one or more dust having a minimum fluorescence intensity or more around the positive control, this is erroneously recognized as the positive control.

  In FIG. 22, the block 1b is arranged with an interval corresponding to the lowest block and the adjacent block 1b. A negative control 1d is arranged around one positive control 1c arranged at one of the four corners, and a negative control 1d is arranged around one positive control 1c arranged at the opposite corner. The number of positive controls and negative controls required is 8 per block. When this pattern is used, if there are two or more dusts in the same arrangement as the two positive controls in the vicinity of the positive control, they are mistakenly recognized as positive controls. Will do.

  In FIG. 23, the block 1b is arranged at an interval from the adjacent block 1b and at least one spot. A negative control 1d is placed around one positive control 1c arranged at one of the four corners and another positive control spaced apart from it, and the necessary positive and negative controls are arranged. Is 8 per block. When this pattern is used, if there are two or more dusts in the same arrangement as the two positive controls in the vicinity of the positive control, they are mistakenly recognized as positive controls. Will do.

  As understood from FIGS. 21 to 23, when the interval between the blocks 1b is set to a pitch more than twice the spot interval in the block, it is necessary to surround the positive control around the block with the negative control. Disappear. Therefore, it is possible to specify the position of the block with a smaller number of positive controls and negative controls.

In any of FIGS. 18 to 23, the negative control surrounds the positive control. In the present embodiment, this arrangement is used for the following reason. That is, for example, as shown in FIG. 16, a block in which no negative control is arranged and only one positive control is arranged, a block in which two positive controls are arranged side by side, and a positive control Assume an arrangement with a block in which two controls are arranged diagonally. FIG. 17 shows a state after the block-by-block positioning process of the detection area on the template by the analysis program is executed with respect to the arrangement of FIG. In pass / fail judgment after this block unit positioning process,
(1) First, the determination of the fluorescence intensity at the spot location determined as a positive control is acceptable because DNA having a fluorescence intensity equal to or higher than the threshold exists.

(2) Next, in the determination of the position of the spot location determined as positive control, since it is greater than the allowable deviation of the position coordinates, it is rejected.

  However, since the spot interval on the DNA microarray is miniaturized, the spot interval and the allowable deviation of the position coordinates are approximated from the spot placement accuracy, the reading accuracy of the scanner device 71, the spot shape after hybridization, and the like. Have been doing. For this reason, in practice, if the allowable deviation amount of the position coordinates is set so as to be rejected, the positioning (alignment) that should be determined to pass is rejected, and accurate positioning Pass / fail judgment is not established.

  On the other hand, if the arrangement pattern according to the present embodiment surrounding the positive control with the negative control is used, the allowable deviation amount of the position coordinates can be set to be twice or more the spot interval. For this reason, significant and accurate positioning pass / fail determination can be realized. In addition, the determination of (1) and (2) is not used, and only the determination by pattern matching of positive control and negative control is performed as long as the pattern after positioning and the pattern on the template do not have the same pattern arrangement. Since pass is not judged, it is effective.

  Further, for example, when the allowable deviation amount can be set only for 3 times the pitch due to spot position accuracy, machine accuracy, etc., as shown in FIG. 34, the configuration in which the positive control is surrounded by the negative control is preferable. . If there is a DNA spot sequence that has the same fluorescence intensity by fitting the detection area to the image and it is misrecognized, even if the positive control and the negative control are determined to pass, the position coordinates are determined. Thus, it is possible to correctly determine that the positioning has failed.

  Note that the analysis processing in the present embodiment and the DNA microarray are not necessarily used in combination, and the DNA microarray is used in an analysis program other than the analysis program according to the present embodiment. Also good.

  FIG. 24 illustrates the details of the block-unit positioning process in step S25 of FIG. 6 that is executed in block units according to the sequence pattern of the positive control and negative control on the DNA microarray according to this embodiment. It is a flowchart.

  As shown in FIG. 24, the block-unit positioning processing unit 359 according to the present embodiment reads the image file 11b after the position correction processing (image processing) read in step S21 (step S251). Next, pattern matching between the positive control and negative control reference patterns defined in advance on the template and the image file is executed (step S253). Alternatively, in step S253, determination of position coordinates may be used together with pattern matching or instead of pattern matching.

  As a result of this pattern matching, an attempt is made to fit the block image to the detection area based on the calculated shift amount, and the block image is positioned (step S255). Based on the positioning in block units, the image file is digitized (step S257), and the digitized data is stored in the digitized data file 13 (step S259).

  Next, details of the positioning success / failure determination process performed by the positioning determination unit 361 according to the present embodiment will be described.

  That is, after completion of the positioning process using the reference mark, the spot unit positioning process, and the block unit positioning process, the numerical value (analysis) of each spot is executed, and the number and fluorescence of the positive control and the negative control are executed. The determination of the strength and the position coordinates is executed, thereby determining whether or not the positioning is correctly executed.

  FIG. 25 shows an example of data after the respective spots are digitized.

In FIG. 25, Pn indicates a spot of interest (for example, positive control). In indicates the fluorescence intensity of Pn. (Xn, yn) indicates real coordinates on the image of Pn. (Xn, Yn) indicates design coordinates of Pn. n is a natural number of 1 to Z.

(1) Determination method using the number of positive controls and negative controls and fluorescence intensity When all of the following conditions are satisfied, the determination of the number and fluorescence intensity is acceptable.

When the positive control for determining the number is Pn, the number is Z.

When the negative control for determining the number is Pn, the number is Z.

  When the positive control to be determined is extracted from the digitized data as Pn, the minimum positive control fluorescence intensity ≦ In is satisfied for n = 1 to Z.

When the negative control for determination is extracted from the digitized data as Pn, In ≦ maximum negative control fluorescence intensity is satisfied for n = 1 to Z.

(2) Determination method of position coordinates The determination is made by using both or one of the following determination methods.

-Determination by absolute coordinates When a spot for determining position coordinates is Pn, and n = 1 to Z, if the following condition is satisfied, the position coordinates are determined to be acceptable.

(| Xn−xn | ≦ x direction allowable deviation amount) and (| Yn−yn | ≦ y direction allowable deviation amount)
A spot for determining a determination position coordinate by relative coordinates is Pn, and P1 is a reference. If n = 1 to Z and the following condition is satisfied, the determination of position coordinates is acceptable.

(| Xn−x1 | ≦ | Xn−X1 | + x direction allowable deviation amount) and (| yn−y1 | ≦ | Yn−Y1 | + y direction allowable deviation amount)
The analysis result output unit 365 stores, in the digitized data file 13, the digitized data that has been determined to have been successfully positioned as a result of the above-described positioning judgment process (step S27 in FIG. 6). On the other hand, for an image for which the success determination is not obtained even by positioning the predetermined number of retries (step S35 in FIG. 6), the analysis result output unit 365 stores information indicating that the success determination has not been obtained in the log file. In addition to outputting to 367, an image for which success determination has not been obtained is output to the analysis error image file 371 as a miss image. This analysis error image is subjected to a positioning process by human visual recognition. The analysis control unit 363 calls the positioning processing unit 355 using the reference mark, the spot unit positioning processing unit 357, the block unit positioning processing unit 359, the positioning determination unit 361, and the analysis result output unit 365 as necessary. By executing, the entire series of analysis processing in FIG. 6 is executed.

  Next, an example of screen display in the analysis processing according to the present embodiment will be described with reference to FIGS.

  FIG. 26 shows an example of a screen display that is input from the DNA microarray image file 11 and that is executing the image recognition processing of the scanned image. As shown in the sub window 263, the position coordinates of the reference marks arranged on the left and right sides of the spot area in the scanned image are displayed, and based on the position coordinates of the reference marks, the recognition processing (image processing) of the scanned image is performed. ), A message indicating that rotational movement (θ direction correction) is being executed is displayed. A manual rotation angle calculation button 265 and a manual rotation processing button 267 are provided.

  FIG. 27 shows an example of an image display of a scanned image read from the DNA microarray image file 11. Within the block of the DNA microarray, spots of respective fluorescence intensities including a positive control and a negative control are arranged.

  FIG. 28 shows an example of a state where the template detection area registered in the analysis parameter file 931 is called for the displayed image of FIG. Each spot image in the DNA microarray image is shifted from each detection area on the template.

  FIG. 29 shows an example of a state after positioning for the image of FIG. 28 when not according to the present embodiment. When the θ-direction deviation amount, the x-direction deviation, and the y-direction deviation amount are large, correct positioning is not performed, and the detection area is incorrectly positioned. If it is not according to this embodiment, it is necessary to repeatedly perform the correction operation applied to the image while shifting the detection area one by one by visual recognition from the state of FIG.

  FIG. 30 shows an example of a state after positioning in the case where a reference pattern formed by a combination of positive control and negative control in this embodiment is not used. As the pitch between spots on the DNA microarray becomes finer, the spot position accuracy becomes closer to the spot pitch. Therefore, in the determination based on the position coordinate data of the detection area, each detection area is correctly applied to each spot. It is difficult to determine whether or not If not according to the present embodiment, each block image is enlarged and displayed from the state shown in FIG. 30, and it is confirmed whether or not the detection area is correctly applied by determining the overall balance.

  FIG. 31 shows an example of a state in which a positioning error still occurs when the reference pattern 311 by the combination of positive control and negative control is arranged only at one corner of the block in this embodiment. For example, when a large block has to be arranged on the DNA microarray, the influence of the θ-direction shift is likely to occur. If the reference pattern is arranged only at one corner on the block, the reference pattern determination and the determination based on the position coordinates ( In positioning in block units), it is determined that positioning is successful despite a positioning error. In this case, for example, if a reference pattern composed of positive control and negative control is arranged at the diagonal position 313, accurate positioning determination can be performed.

  FIG. 32 shows an example of a state in which the positioning determination is successful when the reference pattern 311 by the combination of positive control and negative control is arranged only at one corner of the block in this embodiment. A reference pattern made up of positive and negative controls is placed at one corner of the block.

  FIG. 33 is a block diagram illustrating a hardware configuration of the sample analysis apparatus and the sample analysis system according to the embodiment of the present invention.

  The sample analyzer according to the embodiment of the present invention has a configuration shown in FIG. 33, for example. That is, the sample analysis apparatus according to the embodiment of the present invention is configured by incorporating each element of sample analysis processing in one or a plurality of computer systems.

  As shown in FIG. 33, the computer system 100 includes a central processing unit 110 such as a microprocessor and many other units connected to each other via a system bus 112. The computer system includes a random access memory 114, a read-only memory 116, an I / O adapter 118 for connecting peripheral devices such as a hard disk device 120 to the system bus 112, a keyboard 124, a mouse 126, a speaker 128, and a microphone. 132, a user interface adapter 122 for connecting a user interface device such as a touch screen (not shown) to the system bus 112, a communication adapter 134 for connecting the computer system to a communication network, and a display device 138 for connecting to the system bus 112. Display adapter 136, and flexible disk 144, optical disk 146, and external disk driver 142 for driving various memory cards 148, respectively.

  Analysis programs for executing each function of the sample analysis processing according to the present embodiment are stored in various computer-readable storage media represented by the flexible disk 144, the optical disk 146, and various memory cards 148, and the like. A sample analysis program for executing each function of the sample analysis processing according to the present embodiment stored in these storage media by performing a predetermined read operation from these storage media via a disk driver is a computer system Can be installed in. By loading these programs into the random access memory 114 and executing them by the central processing unit 110, the sample analysis processing according to the present embodiment is realized. The sample analysis program may be executed on a single computer or may be executed on a plurality of computers connected to a network. When executed on a plurality of computers, modules operating on the respective computers are loaded into the respective computers by a storage medium for storing the modules.

  According to the present embodiment, the following effects can be obtained. That is, in the analysis of the DNA microarray, the positioning process of the detection area to the DNA microarray image file and the determination process of positioning success / failure can be performed quantitatively, automatically, and with high accuracy. In particular, since manual positioning processing becomes difficult as the spot interval on the DNA microarray becomes finer, the bottleneck in a series of processing of sample analysis is largely eliminated by this embodiment.

  Furthermore, by enabling unattended and continuous execution from the scanning process through the analysis process to obtaining the desired analysis data, it is possible to perform parallel analysis processing, saving labor and speeding up the analysis process. Is planned.

  In the present embodiment, the embodiment is described in which the DNA is spotted on the DNA microarray. However, the present invention is not limited to this. The DNA microarray according to the present embodiment is applicable to biomolecules that specifically bind, such as RNA and protein, and is not limited to DNA.

  It should be fully understood that the present invention includes various embodiments not described herein. Therefore, the present invention should be limited only by the matters specifying the invention according to the scope of claims reasonable from this disclosure.

It is the schematic which shows a series of processes of the sample analysis using a DNA microarray. It is a perspective view which shows an example of a DNA microarray. It is a figure which shows an example of arrangement | positioning of the detection area of the template used in a sample analysis process. It is a block diagram which shows an example of the hardware constitutions of the sample analyzer which concerns on embodiment of this invention. It is a figure which shows an example of the image data digitized by the sample analyzer which concerns on embodiment of this invention. It is a flowchart which shows the schematic process sequence of the sample analysis method which concerns on embodiment of this invention. It is a flowchart which shows the detail of the process sequence in the positioning process using the reference mark of step S17 in FIG. It is a figure which shows an example of the image image of the DNA microarray read by the sample analyzer which concerns on embodiment of this invention. FIG. 9 is a diagram illustrating a state in which a template stored in a definition file storage unit 353 overlaps the image image of FIG. 8. It is a figure which shows the state after the image of FIG. 9 is positioned by the positioning process part 355 using the reference mark which concerns on embodiment of this invention. It is a flowchart which shows the detail of the process sequence in the positioning process of the spot unit of step S23 in FIG. It is a figure which shows an example of arrangement | positioning of the spot on the DNA microarray which concerns on embodiment of this invention. It is a figure which shows an example of the image read by the positioning process part 357 of a spot unit by step S21 in FIG. It is a figure which shows an example of the image of FIG. 13 after the positioning process of the spot unit of step S23 in FIG. It is a figure which shows another example of the image of FIG. 13 after the positioning process of the spot unit of step S23 in FIG. It is a figure which shows an example of the block of the DNA microarray which has the spot area | region which has arrange | positioned positive control based on embodiment of this invention. It is a figure which shows an example of the state in which the block of FIG. 16 was positioned accidentally. It is a figure which shows an example of the reference | standard pattern arrangement | positioning which consists of positive control and negative control in the block of the DNA microarray which concerns on embodiment of this invention. It is a figure which shows another example of the reference | standard pattern arrangement | positioning which consists of positive control and negative control in the block of the DNA microarray which concerns on embodiment of this invention. It is a figure which shows another example of the reference | standard pattern arrangement | positioning which consists of positive control and negative control in the block of the DNA microarray which concerns on embodiment of this invention. It is a figure which shows another example of the reference | standard pattern arrangement | positioning which consists of positive control and negative control in the block of the DNA microarray which concerns on embodiment of this invention. It is a figure which shows another example of the reference | standard pattern arrangement | positioning which consists of positive control and negative control in the block of the DNA microarray which concerns on embodiment of this invention. It is a figure which shows another example of the reference | standard pattern arrangement | positioning which consists of positive control and negative control in the block of the DNA microarray which concerns on embodiment of this invention. It is a flowchart which shows the detail of the process sequence in the positioning process of the block unit of step S25 in FIG. It is a figure explaining the positioning determination process in embodiment of this invention. It is a figure which shows an example of the image processing screen image which the analysis control part 363 which concerns on embodiment of this invention provides. It is a figure which shows an example of the display screen image of the scan image of a DNA microarray which the analysis control part 363 which concerns on embodiment of this invention provides. It is a figure which shows an example of the display screen image of the state by which the detection area of the template was called with respect to the scan image of FIG. It is a figure which shows an example of the display screen image of the state in which incorrect positioning was performed irrespective of embodiment of this invention. It is a figure which shows another example of the display screen image of the state in which incorrect positioning was performed irrespective of embodiment of this invention. It is a figure which shows another example of the display screen image of the state in which incorrect positioning was performed irrespective of embodiment of this invention. It is a figure which shows an example of the display screen image when the positioning determination process which the analysis control part 363 which concerns on embodiment of this invention provides is successful. It is a figure which shows an example of the hardware constitutions of the control personal computer 73 and the analysis personal computer 91 which concern on embodiment of this invention. It is a figure which shows the other example of arrangement | positioning of the reference pattern on the DNA microarray which concerns on embodiment of this invention. It is a figure which shows an example of the hardware constitutions of the sample analyzer which concerns on embodiment of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 DNA microarray 1a Spot area | region 1b Block 1c Positive control 1d Negative control 11 DNA microarray image file 13 Digitized data file 71 Scanner apparatus 73 Control personal computer 91 Analysis personal computer 93 Definition file 351 Analysis image storage part 353 Definition file storage unit 355 Positioning unit 357 using reference marks Positioning unit 359 in spot units Positioning unit 361 in block units Positioning determination unit 363 Analysis control unit 365 Analysis result output unit 367 Log file 369 Analysis result file 371 Analysis error image

Claims (25)

A spot formed by blocking a plurality of spot areas on the probe reactive chip into information on a reference pattern area composed of a plurality of different position marks for correcting a positional deviation of the spot formed on the probe reactive chip. A reference pattern information storage unit defined at each position in the detection sub-region on the template corresponding to each sub-region;
An image data reading unit that reads image data obtained by scanning a spot that fixes a probe that can specifically react with a sample that is optically detectably labeled on the probe-reactive chip;
Based on the information of the reference pattern area read from the reference pattern information storage unit, the image data is positioned in a predetermined detection target area, and a positional deviation between the image data and the detection target area is determined. An image data positioning unit for generating correction data to be corrected;
A determination unit that determines pass / fail of the positioning by analyzing the image data positioned by the image data positioning unit;
Based on the correction data, a correction unit that corrects a positional deviation between the image data and the detection target area;
An analysis unit that analyzes the corrected image data and outputs digitized data related to the sample ;
The sample analysis apparatus according to claim 1, wherein the reference pattern area information is a pattern in which a plurality of spots that do not necessarily emit light are arranged around spots that always emit light . The sample analysis apparatus according to claim 1, wherein the information of the reference pattern area stored in the reference pattern information storage unit is a relative coordinate from a reference mark. The sample analysis apparatus according to claim 1, wherein the information on the reference pattern area stored in the reference pattern information storage unit is a relative coordinate between the reference patterns defined in advance. The information of the reference pattern region is
The reference pattern information storage unit is defined for each of a plurality of blocked spot sub-regions on the probe reactive chip, and the image data positioning unit positions the image data in units of the spot sub-regions. The sample analysis apparatus according to claim 1. The analysis unit selectively operates in either an automatic mode or a manual mode,
The determination unit repeatedly performs the analysis of the positioned image data a predetermined number of times, and if the desired analysis result is not obtained, adds information indicating that the positioning process has failed for the chip to the output data The sample analyzer according to claim 1, wherein: The sample analyzer further includes a scanning unit that scans a spot on the probe-reactive chip for fixing a probe that can specifically react with an optically detectably labeled sample to obtain the image data. Comprising
The sample analysis apparatus according to claim 1, wherein the scanning unit and the analysis unit execute processing in parallel. The image data positioning unit
A first positioning unit that positions a spot of the image data in a detection area for each spot in the image data and a plurality of blocked spot sub-region units on the probe reactive chip. The sample analyzing apparatus according to claim 1, further comprising: a second positioning unit that positions the second positioning unit. The sample analyzer further includes:
The sample analyzing apparatus according to claim 7 , further comprising a third positioning unit that positions the image data in units of the entire spot area using a reference mark. Information on the reference pattern area composed of a plurality of different position marks for correcting the positional deviation of the spot formed on the probe reactive chip, the spot area on the probe reactive chip in the reference pattern information storage unit Defining each position in the detection sub-region on the template corresponding to each of the spot sub-regions formed into a plurality of blocks;
Scanning image data obtained by scanning a spot on the probe-reactive chip that fixes a probe that can specifically react with an optically detectable sample; and
Based on the information of the reference pattern area read from the reference pattern information storage unit, the image data is positioned in a predetermined detection target area, and a positional deviation between the image data and the detection target area is determined. Generating correction data to be corrected; and
Determining the success or failure of the positioning by analyzing the positioned image data;
Correcting a positional deviation between the image data and the detection target area based on the correction data;
Analyzes the corrected image data, see containing and outputting a digitized data relating to the sample,
The reference pattern area information is a pattern in which a plurality of spots that do not necessarily emit light are arranged around spots that always emit light . The sample analysis method according to claim 9 , wherein the information of the reference pattern region stored in the reference pattern information storage unit is a relative coordinate from a reference mark. The sample analysis method according to claim 9 , wherein the information of the reference pattern area stored in the reference pattern information storage unit is a relative coordinate between the reference patterns defined in advance. The information of the reference pattern region is
In the reference pattern information storage unit, is defined for each of a plurality of blocked spot sub-regions on the probe reactive chip,
The sample analysis method according to claim 9 , wherein the image data positioning step positions the image data in units of the spot sub-regions. The analysis step selectively operates in either automatic mode or manual mode;
In the determination step, when the analysis of the positioned image data is repeatedly executed a predetermined number of times and a desired analysis result cannot be obtained, information indicating that the positioning process has failed for the chip is added to the output data. The sample analysis method according to claim 9 . The sample analysis method further includes:
A scanning step for obtaining the image data by scanning a spot on the probe-reactive chip for fixing a probe that can specifically react with an optically detectably labeled sample;
The sample analysis method according to claim 9 , wherein the scanning step and the analysis step are executed in parallel. The image data positioning step
A first positioning step for positioning a spot of the image data in a detection area in each spot unit in the image data; and the image data in a plurality of blocked spot sub-region units on the probe reactive chip. The sample analysis method according to claim 9 , further comprising: a second positioning step that positions the sample. The sample analysis method further includes:
The sample analysis method according to claim 15 , further comprising a third positioning step of positioning the image data in units of the entire spot area using a reference mark. A program for causing a computer to execute sample analysis processing,
The information of the reference pattern area comprising a position mark multiple heterogeneous for correcting the positional deviation of the spot formed on the probe reactive chip, the reference pattern information storage unit, a spot region on the probe reactive chip A process of defining each position in the detection sub-region on the template corresponding to each of the spot sub-regions formed into a plurality of blocks;
A process of reading image data obtained by scanning a spot that fixes a probe that can specifically react with an optically detectable sample on the probe-reactive chip; and
Based on the information of the reference pattern area read from the reference pattern information storage unit, the image data is positioned in a predetermined detection target area, and a positional deviation between the image data and the detection target area is determined. Processing for generating correction data to be corrected;
A process for determining whether the positioning is successful by analyzing the positioned image data;
Based on the correction data, a process of correcting a positional deviation between the image data and the detection target area;
Analyzes the corrected image data, see containing a process for outputting the numerical data relating to the sample,
The information of the reference pattern area is a program in which a plurality of spots that do not necessarily emit light are arranged around spots that always emit light . The program according to claim 17 , wherein the information on the reference pattern area stored in the reference pattern information storage unit is a relative coordinate from a reference mark. The program according to claim 17 , wherein the information of the reference pattern region stored in the reference pattern information storage unit is a relative coordinate between the reference patterns defined in advance. The information of the reference pattern region is
In the reference pattern information storage unit, is defined for each of a plurality of blocked spot sub-regions on the probe reactive chip,
The program according to claim 17 , wherein the image data positioning process positions the image data in units of the spot sub-regions. The analysis process selectively operates in either automatic mode or manual mode;
In the determination process, when the analysis of the positioned image data is repeatedly performed a predetermined number of times and a desired analysis result cannot be obtained, information indicating that the positioning process has failed for the chip is added to the output data. The program according to claim 17 . The above program further
A scanning process for obtaining the image data by scanning a spot on the probe-reactive chip on which a probe capable of specifically reacting with an optically detectable sample is immobilized;
The program according to claim 17 , wherein the scanning process and the analysis process are executed in parallel. Image data positioning process
A first positioning process for positioning a spot of the image data in a detection area in each spot unit in the image data; and the image data in a plurality of blocked spot sub-region units on the probe reactive chip. program according to claim 17, characterized in that it comprises a second positioning process of positioning the. The above program further
The program according to claim 23 , further comprising a third positioning process that positions the image data in units of the entire spot area using a reference mark. A program for causing a computer to execute sample analysis processing,
Information on the reference pattern area formed of a plurality of different position marks for correcting the positional deviation of the spot formed on the probe reactive chip is stored in the spot area on the probe reactive chip in the reference pattern information storage unit. A process of defining each position in the detection sub-region on the template corresponding to each of the spot sub-regions formed into a plurality of blocks;
A process of reading image data obtained by scanning a spot that fixes a probe that can specifically react with an optically detectable sample on the probe-reactive chip; and
Based on the information of the reference pattern area read from the reference pattern information storage unit, the image data is positioned in a predetermined detection target area, and a positional deviation between the image data and the detection target area is determined. Processing for generating correction data to be corrected;
A process for determining whether the positioning is successful by analyzing the positioned image data;
On the basis of the correction data, look including a process for correcting the positional deviation between the detection target area as the image data,
The information of the reference pattern area is a program in which a plurality of spots that do not necessarily emit light are arranged around spots that always emit light .

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