JP6609954B2 - DNA chip image spot effectiveness determination apparatus, DNA chip image spot effectiveness determination method, and DNA chip image spot effectiveness determination program - Google Patents

Description

  The present invention relates to a technique for identifying a non-test object using a DNA chip. In particular, in a DNA chip on which a probe is immobilized, a labeled test object binds to the probe and is obtained by photographing the DNA chip. The present invention relates to a DNA chip image spot validity determination device, a DNA chip image spot validity determination method, and a DNA chip image spot validity determination program.

In recent years, testing using a DNA chip has been performed in testing of microorganisms such as mold and food poisoning bacteria in food testing, environmental testing, clinical testing, livestock hygiene, and genetic testing. In an inspection using such a DNA chip, in general, first, a part of DNA contained in a test sample is amplified and fluorescently labeled by PCR (polymerase chain reaction) or the like, and the obtained amplification product is used as a DNA chip. The test object is identified by binding to the probe immobilized on and detecting the label of the bound amplification product.
This detection of the label is performed by photographing a DNA chip in which the amplification product is bound to the probe, and detecting a spot in the obtained DNA chip image. That is, the spot of the DNA chip image appears on the image corresponding to the label of the amplification product.

Therefore, in such an inspection using a DNA chip, it is important that spots appear accurately and satisfactorily in the DNA chip image.
However, conventionally, when dust is present on the DNA chip, there is a problem that dust may be erroneously detected as a spot.
In addition, when spots are not originally formed due to problems in the manufacture of the DNA chip, or due to touching the chip surface during the inspection work using the DNA chip, there is a quasi-unevenness or shape abnormality. There is a problem that a spot is generated and this is erroneously detected as a spot.

As a technique related to determination of the presence or absence of a spot, for example, a DNA microarray fluorescence image analysis method described in Patent Document 1 can be cited.
In this analysis method, a template having a detection area is applied to a spot of a fluorescent image, the luminance is measured while jumping the frame n pixels at a time in the detection area, and each of the upper and lower n pixels from the frame where the luminance is detected in each detection area, The presence / absence of a spot is determined by measuring the luminance of a frame in a block of left and right n pixels.

  In addition, in the microarray analysis method described in Patent Document 2, a microarray substrate having a plurality of spots is imaged, a luminance distribution curve for each spot is calculated from the obtained digital image data, and a peak coordinate is detected. The presence / absence of a spot is determined by determining the positional relationship of the spot from the coordinates of the peak of each luminance distribution curve and measuring the luminance of the determined spot.

  Furthermore, in the determination method described in Patent Document 3, a spot position is determined in image data, a pixel group corresponding to the spot is extracted, a median value of detection intensity in the extracted pixel group, and an upper predetermined ratio from the pixel group And / or the ratio or difference of the detection intensity median value in the group excluding the lower-predetermined proportion of pixels, and the reliability of the spot is determined based on the calculated ratio or difference and a predetermined reference value. Judgment is made.

JP 2009-204414 A JP 2009-68996 A JP 2013-186007 A JP 2008-256428 A

However, although these analysis methods can improve spot detection accuracy, it is effective that the dust is erroneously detected as a spot when the DNA chip has dust near the center of the position where the spot is formed. It was not something that could be excluded.
In addition, when a spot having unevenness or shape abnormality is generated due to a problem in manufacturing a DNA chip or in an inspection operation, the erroneous detection cannot be effectively eliminated.

Here, in Patent Document 4, the position of a block having a plurality of spots is not affected by noise distributed in the microarray image, for example, by binarizing the microarray image, without information such as the reference spot position. Techniques for automatic detection are disclosed.
However, the technique described in Patent Document 4 also cannot prevent erroneous detection of dust as a spot or erroneous detection due to a spot with unevenness or shape abnormality.

  The present invention has been considered in view of the above circumstances, and can prevent erroneous detection based on dust or a pseudo spot in a DNA chip image, and can improve the accuracy of inspection using the DNA chip. An object of the present invention is to provide an image spot effectiveness determining apparatus, a DNA chip image spot effectiveness determining method, and a DNA chip image spot effectiveness determining program.

  In order to achieve the above object, the DNA chip image spot validity determination device according to the present invention is a DNA chip having a probe immobilized thereon, wherein a labeled test object is bound to the probe and the DNA chip A DNA chip image spot validity determination device obtained by photographing a plurality of threshold values distributed over the entire luminance of the DNA chip image as threshold values used for binarization processing of the image, A multistage binarization processing unit that creates a plurality of binarized images from a DNA chip image, a spot shape and position detection unit that detects the shape and position of a spot in the binarized image, and a spot shape and position detection unit The configuration includes a spot shape and position determination unit that determines the quality of the spot shape and position detected by.

  In addition, the DNA chip image spot validity determination method of the present invention is obtained by photographing the DNA chip in a state where a labeled test object is bound to the probe in the DNA chip on which the probe is immobilized. A DNA chip image spot validity determination method, wherein a plurality of threshold values distributed over the entire luminance of the DNA chip image are used as threshold values used in the binarization processing of the image, and a plurality of threshold values are used. The method includes a step of creating a binarized image, a step of detecting the shape and position of a spot in the binarized image, and a step of determining pass / fail of the detected shape and position of the spot.

  In addition, the DNA chip image spot validity determination program according to the present invention is obtained by photographing the DNA chip in a state where a labeled test object is bound to the probe on the DNA chip on which the probe is immobilized. A DNA chip image spot effectiveness determination program, wherein the computer inputs the DNA chip image, and a plurality of threshold values used for binarization processing of the image are distributed over the entire luminance of the DNA chip image. Using a threshold value to create a plurality of binarized images from the DNA chip image, to detect the shape and position of the spot in the binarized image, and to determine the quality of the detected spot shape and position; It is meant to be executed.

  According to the present invention, erroneous detection based on dust and pseudo spots in a DNA chip image can be prevented, and the accuracy of inspection using a DNA chip can be improved.

It is a block diagram which shows the structure of the spot effectiveness determination apparatus of the DNA chip image which concerns on embodiment of this invention. It is explanatory drawing which shows the multistep binarization process by the spot effectiveness determination apparatus of the DNA chip image which concerns on embodiment of this invention. It is explanatory drawing which shows the analysis area | region for calculating the brightness nonuniformity and S / N ratio value by the spot effectiveness determination apparatus of the DNA chip image which concerns on embodiment of this invention. It is a flowchart which shows the process process by the spot effectiveness determination apparatus of the DNA chip image which concerns on embodiment of this invention. It is a flowchart which shows the process process of the reference | standard spot detection by the spot effectiveness determination apparatus of the DNA chip image which concerns on embodiment of this invention. It is a flowchart which shows the process process of the normal spot detection by the spot effectiveness determination apparatus of the DNA chip image which concerns on embodiment of this invention. It is a figure which shows the spot shape and the position determination result by the spot effectiveness determination apparatus of the DNA chip image which concerns on embodiment of this invention. It is a figure which shows the nonuniformity of a brightness | luminance by the spot effectiveness determination apparatus of the DNA chip image which concerns on embodiment of this invention, a S / N ratio determination result, and a comprehensive determination result.

  Hereinafter, preferred embodiments of the DNA chip image spot effectiveness determining apparatus, DNA chip image spot effectiveness determining method, and DNA chip image spot effectiveness determining program of the present invention will be described.

The DNA chip image spot validity determination device according to the present embodiment is a DNA chip obtained by photographing a DNA chip in a state where a labeled test object is bound to the probe in the DNA chip on which the probe is immobilized. An image spot validity determination device that uses a plurality of threshold values distributed over the entire luminance of a DNA chip image as threshold values used in the binarization processing of the image, and a plurality of binarized images from the DNA chip image. A multi-stage binarization processing unit for creating a spot shape, a position detection unit for detecting the shape and position of a spot in a binarized image, and the quality of the spot shape and position detected by the spot shape and position detection unit And a spot shape and position determination unit.
In addition, the DNA chip image spot validity determination device according to the present embodiment includes a brightness unevenness detection unit that detects unevenness in brightness at a spot detected by the spot shape and position detection unit, and a brightness detected by the brightness unevenness detection unit. It is also preferable to further include a luminance unevenness determination unit that determines whether the unevenness is good or bad.
Further, the DNA chip image spot validity determination device of the present embodiment includes an S / N ratio calculation unit that calculates an S / N ratio value at a spot detected by a spot shape and position detection unit, and an S / N ratio calculation. It is also preferable to further include an S / N ratio determination unit that determines the quality of the S / N ratio value calculated by the unit.

The DNA chip image spot validity determination method according to the present embodiment is a DNA chip obtained by photographing a DNA chip in a state where a labeled test object is bound to the probe in a DNA chip on which a probe is immobilized. A method for determining the validity of an image spot, wherein a plurality of binarized images are obtained from a DNA chip image by using a plurality of threshold values distributed throughout the luminance of the DNA chip image as threshold values used for binarization processing of the image. , A step of detecting the shape and position of the spot in the binarized image, and a step of determining pass / fail of the shape and position of the detected spot.
In addition, the spot validity determination method of the DNA chip image of the present embodiment further includes a step of detecting luminance unevenness in the detected spot and a step of determining whether the detected luminance unevenness is good or bad. It is also preferable to do.

The DNA chip image spot validity determination program according to the present embodiment is a DNA chip obtained by photographing a DNA chip in a state where a labeled test object is bound to the probe in the DNA chip on which the probe is immobilized. An image spot validity determination program that allows a computer to input a DNA chip image and uses a plurality of threshold values distributed throughout the luminance of the DNA chip image as threshold values used for binarization processing of the image, A plurality of binarized images are created from a DNA chip image, the shape and position of a spot in the binarized image are detected, and the quality of the detected spot shape and position is determined. .
The DNA chip image spot validity determination program according to the present embodiment preferably causes the computer to detect luminance unevenness in the detected spot and determine whether the detected luminance unevenness is good or bad.

  As a premise of the present embodiment, as described above, a probe is immobilized (spotted) on a DNA chip that is a subject of a DNA chip image, and a spot made of the probe is formed on the DNA chip. Then, a DNA chip image used in the present embodiment can be obtained by photographing the DNA chip in a state where a test object labeled with fluorescence or the like is bound (hybridized) to the probe. .

When the DNA to be inspected is included in the inspection sample, spots indicated by fluorescence or the like appear in the DNA chip image corresponding to the position of the spot where the probe is immobilized.
As described above, the term “spot” generally has two meanings, “spot formed on a DNA chip” and “spot appearing on a DNA chip image”. In this embodiment, When simply referred to as “spot”, it is used to mean “spot appearing in a DNA chip image”.

  Further, as a premise of the present embodiment, on the DNA chip, there are a spot (reference spot) serving as a reference for estimating the position of each spot and a normal spot (normal spot) for detecting the inspection object. It is fixed. In the DNA chip image, a reference spot and a normal spot corresponding to each of them appear.

For example, a total of 64 spots of 8 vertically and 8 horizontally are formed in a matrix on a DNA chip, the spots at both ends at the bottom are used as reference spots, and the remaining spots are used as normal spots. In the chip image, a reference spot and a normal spot corresponding to these can appear.
In the present embodiment, when simply referred to as “spot”, it is used to mean including both a reference spot and a normal spot.

  Next, the DNA chip image spot effectiveness determination apparatus of the present embodiment will be described with reference to FIGS. 1 to 3. However, the DNA chip image spot effectiveness determination apparatus of the present invention is limited to this. The present invention is not intended to be modified in any way as long as it has the features of the present invention described above and exhibits the same effects. FIG. 1 is a block diagram showing a configuration of a DNA chip image spot effectiveness determination apparatus according to the present embodiment. FIG. 2 is an explanatory diagram showing multi-level binarization processing, and FIG. 3 is an explanatory diagram showing an analysis region for calculating luminance unevenness and an S / N ratio value.

  As shown in FIG. 1, a DNA chip image spot validity determination device 10 according to this embodiment includes a DNA chip image input unit 101, a multistage binarization processing unit 102, a spot shape and position detection unit 103, a spot shape. And position determination unit 104, detection error processing unit 105, luminance unevenness detection unit 106, luminance unevenness determination unit 107, S / N ratio calculation unit 108, S / N ratio determination unit 109, normal spot position estimation unit 110, and determination result An output unit 111 is provided.

  The DNA chip image spot validity determination device 10 can be configured by using various computers such as a personal computer, a workstation, a server, a smartphone, and a tablet computer, and each of the above configurations can be configured by a CPU and a memory in the computer. it can. Moreover, it can also be comprised as a dedicated counting device provided with such each structure.

  The DNA chip image input unit 101 inputs a DNA chip image obtained by photographing the DNA chip image to the spot validity determination device 10 for the DNA chip image. The input DNA chip image can be stored in a hard disk, a memory, or the like in the spot validity determination device 10 for the DNA chip image.

The multistage binarization processing unit 102 can perform binarization processing on the input DNA chip image to create a plurality of binarized images.
That is, the binarization processing in the present embodiment is characterized in that a plurality of binarized images can be created using a plurality of threshold values distributed over the entire luminance of the DNA chip image.

  Specifically, the multi-level binarization processing unit 102 uses the minimum and maximum values of the luminance of the DNA chip image, for example, as shown in FIG. A plurality of threshold values can be comprehensively set such as intervals (N steps, N = positive integer), and a plurality of binarized images can be created using each threshold value. The minimum and maximum luminance values may be those previously stored in the DNA chip image spot validity determination device 10 and determined based on the DNA chip image by the multistage binarization processing unit 102. May be used.

The spot shape and position detection unit 103 detects the shape and position of the spot in the binarized image created by the multistage binarization processing unit 102.
Further, the spot shape and position detection unit 103 can detect the circularity and size of the spot as the spot shape, and can detect the center position of the spot as the spot position.

As a method for detecting the circularity, size, and center position of the spot, for example, a binarized image can be labeled.
Specifically, in a binarized image, raster scanning is performed on a certain range where spot shape and position detection processing is performed, and eight continuous portions in the vertical, horizontal, and diagonal directions are made the same label. By performing a labeling process, spots can be detected based on the pixels indicated by the same label, and the number of pixels can be acquired as the area S (size). In addition, the circularity can be obtained by the equation of 4πS / L ^{2 where} the length connecting the outer pixels with a line is the peripheral length L. Further, an average value of all coordinates of the pixels indicated by the same label can be calculated and obtained as the center position.

The fixed range in which the spot shape and position detection processing is performed can be stored in advance in the DNA chip image spot validity determination device 10 as a fixed range for searching for the reference spot.
As for the normal spot, a spot estimated by the normal spot position estimation unit 110 described later and stored in the spot validity determination device 10 of the DNA chip image can be used as a certain range for searching for the normal spot.

The spot shape and position determination unit 104 determines whether or not the circularity, size, and center position of the spot obtained by the spot shape and position detection unit 103 are within a setting range that is determined to be good. To do.
Specifically, for example, when the circularity is 0.6 or more, the size is 50 pixels or more and 300 pixels or less, and the radius is within 10 pixels with respect to the position where the center position is estimated, it can be determined to be good. . In such a case, it can be determined that the spot shape is not abnormal and the position is good.
However, the present embodiment is not limited to these numerical values, and the determination criteria can be changed as appropriate. In particular, in this specific example, pixels are used as units. However, the size and the center position vary greatly depending on the number of pixels of the camera used for photographing and the lens magnification. Therefore, these numerical values are shown for reference only.

The reason for determining the center position of the spot is that there is a large deviation between a certain range stored in the spot validity determination device 10 of the DNA chip image as a range for searching for the spot and the actual spot position in the DNA chip image. This is because it may occur.
The main cause of such misalignment is when the probe is spotted on the DNA chip in the DNA chip manufacturing process, the accuracy of the spot position depends on the mechanical accuracy of the manufacturing equipment and the surface tension of the chemicals containing the probe. There is to be.
According to the present embodiment, it is possible to determine the effectiveness of a spot with high accuracy by determining the center position of the spot.

  In the detection of the reference spot, when the spot shape and position determination unit 104 determines that the spot shape or position is not good, the detection error processing unit 105 causes the determination result output unit 111 to output an error and perform processing. Can be terminated.

The brightness unevenness detection unit 106 detects unevenness in brightness at the spot detected by the spot shape and position detection unit 103.
At this time, the luminance unevenness detection unit 106 detects the luminance unevenness of the spot in the DNA chip image input by the DNA chip image input unit 101.
Further, as shown in FIG. 3, the luminance unevenness detection unit 106 sets a range smaller than the spot size as the luminance analysis range 4, and detects luminance unevenness in the analysis range. Thus, by setting the analysis range to a range smaller than the spot size, it is possible to set the highest brightness portion in the spot as a detection target and obtain a stable detection result.

The following two can be used as indicators for detecting unevenness in luminance.
First, as the first luminance unevenness index, a fluctuation value (CV value), that is, a value obtained by dividing the standard deviation of luminance at a spot by the average value of luminance can be used.
Further, as the second luminance unevenness index, a value calculated based on the following equation (1) by acquiring the minimum value, the maximum value, and the average value from the luminance at the spot can be used.
(Maximum luminance value−minimum luminance value) / average luminance value (1)

The brightness unevenness determination unit 107 determines whether the brightness unevenness detected by the brightness unevenness detection unit 106 is good or bad.
Specifically, for example, when the variation value is 20 or less, it can be determined that there is no unevenness and is good. More preferably, it can be determined that the fluctuation value is 15 or less, more preferably 10 or less.
Further, for example, when the second luminance unevenness index is 0.5 or less, it can be determined that there is no unevenness and is good. More preferably, it can be determined that the second luminance unevenness index is good when it is 0.3 or less.

The S / N ratio calculation unit 108 calculates the S / N ratio value at the spot detected by the spot shape and position detection unit 103.
At this time, the S / N ratio calculation unit 108 calculates the S / N ratio value of the spot in the DNA chip image input by the DNA chip image input unit 101.
Specifically, the S / N ratio calculation unit 108 acquires the median value of the luminance at the spot, acquires the median value of the luminance around the spot as the background value, and calculates S based on the following equation (2). / N ratio value can be calculated.
(Median luminance-background value) / background value ... Equation (2)
For example, as shown in FIG. 3, the background value can be acquired for the outer four corners of the spot in the rectangle surrounding the spot.

The S / N ratio determination unit 109 determines whether the S / N ratio value calculated by the S / N ratio calculation unit 108 is good or bad.
Specifically, for example, when the S / N ratio value is 3 or more, it can be determined to be good. However, since the threshold value of the S / N ratio value varies depending on the type of DNA chip, it may be a value that can be determined that the luminance is sufficiently detected for each type of DNA chip, and is limited to the threshold value. It goes without saying that it is not done.

The normal spot position estimation unit 110 estimates the position of the normal spot in the DNA chip image based on the spot shape and the position of the reference spot detected by the position detection unit 103.
Although the method is not particularly limited, for example, it can be performed by calculating the coordinates (grid) of the position where the normal spot is supposed to be arranged based on the center position of the detected reference spot.

The determination result output unit 111 outputs at least the determination result by the spot shape and position determination unit 104.
The determination result output unit 111 can also output the determination result by the luminance unevenness determination unit 107 and / or the determination result by the S / N ratio determination unit 109.
Further, the determination result output unit 111 can output a comprehensive determination result for determining the effectiveness of the spot based on all the determination results.

The determination result output unit 111 can also output an error result determined by the detection error processing unit 105 as a reference spot detection error.
Although the specific output method of the determination result is not particularly limited, for example, it can be output to a display device such as a display connected to the DNA chip image spot validity determination device 10.

  Next, a processing procedure in the DNA chip image spot validity determination device 10 will be described with reference to FIGS. FIG. 4 is a flowchart showing the processing steps by the DNA chip image spot validity determination device of the present embodiment. FIG. 5 is a flowchart showing the processing steps for reference spot detection, and FIG. 6 is a flowchart showing the processing steps for normal spot detection. The DNA chip image spot validity determination program of this embodiment causes an information processing apparatus such as a computer to execute the following processing.

As shown in FIG. 4, first, the DNA chip image input unit 101 inputs a DNA chip image to the DNA chip image spot validity determination device 10 (step 10). The input DNA chip image is stored in the DNA chip image spot validity determination device 10.
Next, a reference spot detection process is executed (step 20), then a normal spot detection process is executed (step 30). Finally, the determination result output unit 111 outputs a determination result of the spot validity. (Step 40).

Next, the reference spot detection process (step 20) will be described in detail with reference to FIG.
First, the multistage binarization processing unit 102 executes binarization processing on the DNA chip image to create a binarized image (step 200).

At this time, the multistage binarization processing unit 102 uses, for example, the minimum and maximum luminance values stored in advance in the spot validity determination device 10 for the DNA chip image, and the luminance between the minimum value and the maximum value. The threshold value used in the binarization process can be set so as to be in increments of N. Further, the minimum value and the maximum value of the luminance may be determined by the multistage binarization processing unit 102 based on the DNA chip image.
Then, the multistage binarization processing unit 102 first creates the first binarized image based on the DNA chip image using the first threshold.

Next, the spot shape and position detection unit 103 compares the spot shape with respect to a certain range stored in advance in the spot validity determination device 10 of the DNA chip image as a reference spot range to be searched first among the reference spots. And the position detection process is executed (step 201).
For example, in the first binarization process shown in FIG. 2, spot shape and position detection processing is performed for a certain range 2-1 for searching for one reference spot 2 a in the DNA chip image 1 of the threshold value b 1. Executed.

Next, the circularity, size, and center position of the detected reference spot by the spot shape and position determination unit 104 are stored in advance in the DNA chip image spot validity determination device 10 as a good range. Is determined (step 202).
If any of the circularity, size, and center position of the reference spot is not within the setting range (NO in step 202), the spot shape and position of all the binarized images corresponding to all the threshold values are set. It is confirmed whether or not the detection process has been executed (step 203).

If there is still a threshold that is not used for binarization processing (NO in step 203), a binarized image is created using the next threshold, and spot shape and position detection processing is executed. If any of the circularity, size, and center position of the detected reference spot is not within the set range, the same processing is repeated (steps 200 to 202).
If spot shape and position detection processing has been executed for all binarized images corresponding to all threshold values (YES in step 203), error processing is executed by the detection error processing unit 105. (Step 204).

On the other hand, if the circularity, size, and center position of the reference spot are within the set range (YES in step 202), it is determined that the shape or position of the reference spot is good, and then the luminance unevenness detection unit 106 Then, processing for detecting unevenness in luminance of the reference spot in the DNA chip image is executed (step 205).
Therefore, in this case, even when a threshold value that is not used for the binarization process still exists, the generation of the binarized image using the next threshold value, the detection of the spot shape and position, and the determination process Is not done. That is, when it is determined that the shape or position of the reference spot is good in one binarized image, the detection and luminance related to the reference spot are detected without performing binarization processing on the reference spot thereafter. Judgment processing is performed.

Next, the brightness unevenness determination unit 107 determines whether or not the brightness unevenness is within a set range stored in advance in the DNA chip image spot validity determination device 10 as a good range (step 206). .
If the luminance unevenness is not within the set range (NO in step 206), error processing is executed by the detection error processing unit 105 (step 204).

On the other hand, if the luminance unevenness is within the set range (YES in step 206), the S / N ratio calculation unit 108 executes a process of calculating the S / N ratio value of the reference spot in the DNA chip image. (Step 207).
Next, the S / N ratio determination unit 109 determines whether or not the S / N ratio value is within a set range stored in advance in the DNA chip image spot effectiveness determination device 10 as a good range. (Step 208).
If the S / N ratio value is not within the set range (NO in step 208), error processing is executed by the detection error processing unit 105 (step 204).

On the other hand, if the S / N ratio value is within the set range (YES in step 208), it is confirmed whether or not searching has been performed for all reference spots (step 209).
If the reference spot to be searched still remains (NO in step 209), the process after the creation of the binarized image is executed again for the next reference spot (steps 200 to 209).

  In this case, the binarized images created by the binarized image creation process that has already been performed are stored in the spot validity determination device 10 for the DNA chip image, thereby omitting the creation process. be able to. In addition, when step 100 is executed for the first time, binarized images can be created for all threshold values. The same applies to normal spot detection described later, and a binarized image created in reference spot detection can also be used in normal spot detection.

  If the search is completed for all the reference spots (YES in step 209), the normal spot detection process (step 30) is then executed.

Next, the normal spot detection process (step 30) will be described in detail with reference to FIG.
First, the normal spot position estimation unit 110 estimates the position (coordinates) of the normal spot in the DNA chip image based on the spot shape and the position of the reference spot detected by the position detection unit 103 (step 300). The estimated position of the normal spot is stored in the spot validity determination device 10 for the DNA chip image, and the following processing is performed for each normal spot.

First, the multistage binarization processing unit 102 performs binarization processing on the DNA chip image to create a binarized image (step 301).
As in the case of the reference spot detection process, the multistage binarization processing unit 102 first creates an initial binarized image based on the DNA chip image using the initial threshold value.

Next, the spot shape and position detection unit 103 compares the spot shape and the position with respect to a certain range stored in the spot validity determination device 10 of the DNA chip image as the normal spot range to be searched first among the normal spots. A position detection process is executed (step 302).
For example, in the first binarization process shown in FIG. 2, spot shape and position detection processing is executed for a certain range 3-1 for searching for a normal spot 3 a in the DNA chip image 1 of the threshold value b 1. The

Next, the circularity, size, and center position of the detected normal spot by the spot shape and position determination unit 104 are stored in advance in the DNA chip image spot validity determination device 10 as a good range. Is determined (step 303).
If any of the circularity, size, and center position of the normal spot is not within the setting range (NO in step 303), the spot shape and position of all the binarized images corresponding to all the threshold values are set. It is confirmed whether or not the detection process has been executed (step 304).

If there is still a threshold that is not used in the binarization process (NO in step 304), a binarized image is created using the next threshold, and the spot shape and position detection process is executed. If any of the circularity, size, and center position of the detected normal spot is not within the set range, the same processing is repeated (steps 301 to 303).
If spot shape and position detection processing has been executed for all binarized images corresponding to all threshold values (YES in step 304), it is determined that the shape or position of the normal spot is not good. Next, luminance unevenness detection processing is performed (step 305).

  Since the reference spot should always be detected, if it cannot be detected, error processing is performed. However, the normal spot includes the inspection object to be detected corresponding to the normal spot in the inspection sample. If it is not detected, it is normal that it is not detected. Therefore, even if it is determined that the shape or position of the normal spot is not good, error processing is not executed.

On the other hand, if the circularity, size, and center position of the normal spot are within the set range (YES in step 303), it is determined that the shape or position of the normal spot is good, and then the luminance unevenness detection unit 106 Then, processing for detecting unevenness in luminance of the normal spot in the DNA chip image is executed (step 305).
Therefore, in this case, even when a threshold value that is not used for the binarization process still exists, the generation of the binarized image using the next threshold value, the detection of the spot shape and position, and the determination process Is not done. That is, when it is determined that the shape or position of the normal spot is good in one binarized image, the detection and luminance related to the normal spot are detected without performing the binarization process for the normal spot thereafter. Judgment processing is performed.

  Next, the brightness unevenness determination unit 107 determines whether or not the brightness unevenness is within the set range stored in advance in the DNA chip image spot validity determination device 10 as a good range (step 306). . As described above, unlike the case of the reference spot, the error processing is not performed for the normal spot even if it is determined that the luminance unevenness is not good.

Next, the S / N ratio calculation unit 108 executes a process of calculating the S / N ratio value of the normal spot in the DNA chip image (step 307).
Then, the S / N ratio determination unit 109 determines whether or not the S / N ratio value is within a set range stored in advance in the DNA chip image spot effectiveness determination device 10 as a good range ( Step 308). In this case, as described above, unlike the case of the reference spot, the error processing is not performed for the normal spot even if it is determined that the S / N ratio value is not good.

Then, it is confirmed whether or not the search has been performed for all the normal spots (step 309), and when the normal spots to be searched still remain (NO in step 309), a binarized image is created for the next normal spot. The subsequent processing is executed again (steps 301 to 309).
When the search is completed for all the reference spots (YES in step 309), the determination result output unit 111 executes determination result output processing (step 40).

The judgment results to be output include, for each of all the reference spots and normal spots, whether the spot shape and position are good, whether the luminance unevenness is good, and the S / N ratio value is good. Information on whether or not there is can be included. And a spot where all of these are good can include information that it is an effective spot as a comprehensive determination result, and a spot determined that any of these is not good is a comprehensive determination result , Information that is not a valid spot can be included.
The determination result can be output to the display of the DNA chip image spot validity determination device 10 by the determination result output unit 111, or output as a form from a printer.

Note that the processing flow described in the present embodiment is not limited to this, and can be appropriately changed within a range in which similar results are obtained.
For example, in the flowcharts shown in FIGS. 5 and 6, the determination process is performed immediately after the various detection processes, but the determination process may be collectively executed before the determination result output process.
Specifically, it is possible to determine the luminance unevenness and the S / N ratio value after both the detection of the luminance unevenness and the detection of the S / N ratio value are performed first. In addition, binarized images of all thresholds are first created by multi-level binarization processing, spot shapes and positions are detected, and the determination is performed together with the determination of uneven brightness and S / N ratio value. It is also possible to do so.

  In addition to performing processing for each spot, after detecting and determining the spot shape and position for all spots, detection and determination of luminance unevenness is performed for all spots, and then the S / N ratio value is detected. It is also possible to perform the determination for all spots.

As described above, according to the spot validity determination device for DNA chip image, the spot validity determination method for DNA chip image, and the spot validity determination program for DNA chip image according to the present embodiment, the brightness of the DNA chip image is improved. A binarized image can be created by setting a plurality of threshold values in an exhaustive manner, and these can be used to determine whether or not the spot shape and position are good.
Therefore, it is possible to detect and determine the spot shape and position with high accuracy without considering a specific optimum threshold.
Further, since it is possible to determine whether the brightness unevenness of the spot and the S / N ratio value are good, it is possible to further improve the accuracy of determining the effectiveness of the spot.

  Using the DNA chip image spot effectiveness determination apparatus, DNA chip image spot effectiveness determination method, and DNA chip image spot effectiveness determination program of the above-described embodiment, the effectiveness of the spot in the DNA chip image is determined. The experiment was conducted.

Specifically, as shown in FIGS. 7 and 8, two reference spots (Examples 1 and 2) and seven normal spots (Examples 3 and 4 and Comparative Examples 1 to 5) are shown in FIG. The processing of the flow shown in FIG. 6 was executed to determine the effectiveness of each spot.
The spots of Examples 1 to 4 are all normal in shape and position of the spot, uneven brightness, and S / N ratio value.
Further, the spots of Comparative Examples 1 and 5 are deformed, the spot of Comparative Example 2 is shifted in the center position, and the spot of Comparative Example 3 has uneven brightness. A spot has a small S / N ratio value.

As a result, the spots of Examples 1 to 4 were judged to be effective as a comprehensive judgment because of good circularity, size, center position, luminance unevenness, and S / N ratio value. .
On the other hand, in Comparative Example 1, it was determined that all the results other than the S / N ratio value were not good, and it was determined that it was not effective as a comprehensive determination.
In Comparative Example 2, it was determined that the center position and the luminance unevenness were not good, and it was determined that it was not effective as a comprehensive determination.

In Comparative Example 3, it was determined that the luminance unevenness was not good, and it was determined that it was not effective as a comprehensive determination.
Further, in Comparative Example 4, it was determined that the circularity, size, center position, and S / N ratio value were not good, and it was determined that it was not effective as a comprehensive determination.
Further, in Comparative Example 5, it was determined that the circularity, size, and center position were not good, and it was determined that it was not effective as a comprehensive determination.

  As described above, according to the DNA chip image spot validity determination device, the DNA chip image spot validity determination method, and the DNA chip image spot validity determination program according to the above-described embodiment, the determination of the spot validity is performed. It was confirmed that it can be performed with high accuracy.

The present invention is not limited to the above-described embodiments and examples, and it goes without saying that various modifications can be made within the scope of the present invention.
For example, the components shown in the block diagram of FIG. 1 can be combined, or divided into more detailed functions. Further, in order to apply the present invention to a DNA chip that does not have a reference spot, the reference spot detection process is omitted from FIGS. It is possible to make appropriate changes such as storing and executing.

  INDUSTRIAL APPLICABILITY The present invention can be suitably used in fields such as inspection of microorganisms such as mold and food poisoning bacteria in food inspection, environmental inspection, clinical test, livestock hygiene and the like, and genetic inspection.

1 DNA chip image 2 (2-1, 2-2,...) Reference spot search range 2a Reference spot 3 (3-1, 3-2,...) Normal spot search range 3a Normal spot 4 Luminance Analysis range 5 Background analysis range 10 DNA chip image spot validity determination device 101 DNA chip image input unit 102 Multi-stage binarization processing unit 103 Spot shape and position detection unit 104 Spot shape and position determination unit 105 Detection error processing unit 106 brightness unevenness detection unit 107 brightness unevenness determination unit 108 S / N ratio calculation unit 109 S / N ratio determination unit 110 normal spot position estimation unit 111 determination result output unit

Claims (15)

In a DNA chip on which a probe is immobilized, a spot validity determination device for a DNA chip image obtained by photographing the DNA chip in a state where a labeled test object is bound to the probe,
A multi-stage binarization processing unit that creates a plurality of binarized images from the DNA chip image using a plurality of threshold values distributed over the entire luminance of the DNA chip image as thresholds used for the binarization processing of the image When,
A spot shape and position detection unit for detecting the shape and position of the spot in the binarized image;
A spot shape and position determination unit for determining the quality of the spot shape and position detected by the spot shape and position detection unit, and a DNA chip image spot validity determination device. A luminance unevenness detection unit for detecting unevenness in luminance in the spot detected by the spot shape and position detection unit;
The DNA chip image spot validity determination device according to claim 1, further comprising: a luminance unevenness determination unit that determines whether or not the luminance unevenness detected by the luminance unevenness detection unit is good. The multistage binarization processing unit uses a plurality of threshold values comprehensively set between the minimum value and the maximum value based on the minimum value and the maximum value of the luminance of the DNA chip image, A plurality of binarized images are created. The DNA chip image spot validity determination device according to claim 1 or 2.   4. The DNA chip image spot validity determination device according to claim 3, wherein the plurality of threshold values are set at equal intervals between the minimum value and the maximum value. The spot shape and position detection unit detects the circularity and size of the spot as the spot shape, and detects the center position of the spot as the spot position. The spot effectiveness determining apparatus for DNA chip images according to 1. The brightness unevenness detection unit, as spot brightness unevenness, obtains a value obtained by dividing the standard deviation of brightness at the spot by the average value of brightness, or the minimum value, maximum value, and average value from the brightness at the spot, The spot validity determination device for DNA chip images according to claim 2, wherein the calculation is based on the following equation (1).
(Maximum luminance value−minimum luminance value) / average luminance value (1) An S / N ratio calculation unit for calculating an S / N ratio value at a spot detected by the spot shape and position detection unit;
The DNA according to any one of claims 1 to 6, further comprising an S / N ratio determination unit that determines the quality of the S / N ratio value calculated by the S / N ratio calculation unit. Chip image spot effectiveness determination device. The S / N ratio calculation unit acquires a median value of brightness at the spot, acquires a median value of brightness around the spot as a background value, and calculates an S / N ratio value based on the following equation (2). The spot effectiveness determination device for DNA chip images according to claim 7, wherein the spot effectiveness determination device calculates.
(Median brightness-background value) / background value ... Equation (2) The spot shape and position detection unit detects the spot shape and position with respect to a certain range corresponding to the position of the reference spot serving as a reference for estimating the position of each spot in the DNA chip image, A spot detection device for DNA chip images according to any one of claims 1 to 8, wherein a reference spot is detected. A spot position estimation unit that estimates the position of a spot other than the reference spot in the DNA chip image based on the detected position of the reference spot;
The spot shape and position detection unit detects a spot by detecting the shape and position of the spot with respect to a certain range corresponding to the spot position estimated by the spot position estimation unit. The DNA chip image spot effectiveness determination device according to claim 9.   The DNA chip image spot validity determination device according to any one of claims 1 to 10, further comprising a determination result output unit that outputs at least a determination result by the spot shape and position determination unit. In a DNA chip on which a probe is immobilized, a method for determining the spot effectiveness of a DNA chip image obtained by photographing the DNA chip in a state in which a labeled test object is bound to the probe,
Creating a plurality of binarized images from the DNA chip image using a plurality of threshold values distributed throughout the luminance of the DNA chip image as threshold values used for the binarization processing of the image;
Detecting a spot shape and position in the binarized image;
Determining the quality of the detected spot shape and position, and a method for determining the effectiveness of a DNA chip image spot.   13. The determination of spot effectiveness of a DNA chip image according to claim 12, further comprising: detecting luminance unevenness in the detected spot; and determining whether the detected luminance unevenness is good or bad. Method. In a DNA chip on which a probe is immobilized, a spot validity determination program for a DNA chip image obtained by photographing the DNA chip in a state where a labeled test object is bound to the probe,
On the computer,
Inputting the DNA chip image;
Using a plurality of threshold values distributed throughout the luminance of the DNA chip image as a threshold value used for the binarization processing of the image, a plurality of binarized images are created from the DNA chip image,
Detecting the shape and position of the spot in the binarized image;
A DNA chip image spot validity determination program for determining whether or not the shape and position of a detected spot are good.   15. The program for determining the effectiveness of a spot on a DNA chip image according to claim 14, further comprising: causing a computer to detect luminance unevenness in the detected spot and determining whether the detected luminance unevenness is good or bad.