JP6756142B2 - Computer program, judgment method and judgment device - Google Patents

Description

The present invention relates to a computer program, a determination method, and a determination device for determining a test result using a test piece.

Conventionally, a test piece for determining drug resistance of Mycobacterium tuberculosis has been known (Patent Document 1). A plurality of types of probes are fixed to such a test piece. This test piece and the tubercle bacillus gene (hereinafter referred to as "target gene") contained in the sample are amplified and reacted (hybridized) by the PCR method, and the presence or absence of the reaction is colored in the test piece. The drug resistance of Mycobacterium tuberculosis is determined from the coloring pattern.

There is known a system or the like in which an inspection result is determined by analyzing image data obtained by photographing a test piece (Patent Document 2). In the technique disclosed in Patent Document 2, a test paper unit is photographed by a portable device, and the image data is transmitted to a remote computing device. Then, the image data of the test strip unit is analyzed by the test strip analysis system of the remote calculation device, and the analysis result is transmitted to the portable device.

Japanese Unexamined Patent Publication No. 2011-087465 Japanese Unexamined Patent Publication No. 2012-202089

In the method of analyzing the coloring pattern of the test piece as in the above-mentioned examination of the drug resistance of Mycobacterium tuberculosis, if the coloring pattern of the test piece can be determined by the arithmetic unit, the determination work becomes simple and the determination result is obtained. Stabilize. However, the color due to coloring changes in the image data in which the test piece is photographed depending on the variation in the amplification of the target gene by the PCR method, the variation in the color development in the color development reaction, the imaging environment of the test piece, and the like. Even if such variations occur, it is desirable to obtain stable determination results.

The present invention has been made in view of the above-mentioned circumstances, and an object of the present invention is to obtain a stable determination result even if the coloring of the test piece varies in the image data due to amplification, color development, imaging environment, etc. in the inspection. It is an object of the present invention to provide a computer program, a determination method, and a determination device capable of the present invention.

(1) In the computer program according to the present invention, a plurality of detectors that develop color depending on the presence or absence of reaction with the substance to be inspected are fixed, and the image data of the test piece on which the position marker is marked is stored in the storage unit. Based on the image data storage step to be stored and the position of the position marker along the reference direction in the image data, at least the plurality of detectors can refer to the detector position information stored in advance in the storage unit. The difference between the position specifying step for obtaining each existing existing region, the maximum value of the brightness value in each existing region of the image data, and the minimum value of the brightness value in each non-existing region adjacent to each existing region. A difference value acquisition step for acquiring each difference value in association with each detector, and each of the above-mentioned ones higher than the first cutoff value set as a default among a plurality of cutoff values stored in advance in the storage unit. A second cutoff from a plurality of cutoff values stored in advance in the storage unit based on the representative value acquisition step of extracting the difference value and acquiring the representative value of each of the extracted difference values and the representative value. A cutoff value selection step for selecting a value, and a reaction determination step for determining a positive reaction of the plurality of detectors based on the difference value and the second cutoff value and displaying the positive reaction on the display unit are included. ..

According to the above configuration, the second cutoff value for determining the positive reaction of the plurality of detectors is determined according to the difference value between the luminance values of the present region and the non-existent region of the detector, and thus is amplified. Even if the coloring of the test piece in the image data varies depending on the color development, the shooting environment, etc., a stable determination result can be obtained.

(2) Preferably, a plurality of cutoff values and a threshold range are stored in advance in the storage unit in association with each other. In the cutoff value selection step, the cutoff value corresponding to the threshold range including the representative value is selected as the second cutoff value.

According to the above configuration, the second cutoff value is easily selected based on the representative value.

(3) Preferably, in the representative value acquisition step, the average value of the difference values higher than the first cutoff value is set as the representative value.

(4) Preferably, in the reaction determination step, the display unit is provided on the condition that the difference between each difference value and the second cutoff value is within a predetermined range preset in the storage unit. The determination of the positive reaction of the plurality of detectors in the above is suspicious.

According to the above configuration, the user can know the certainty of determining the positive reaction of the detector.

(5) Preferably, the detector is a plurality of types of probes that react with the amplified target gene. In the test piece, the position where the plurality of detectors are present is colored by the reaction between the labeling substance of the primer used for the amplification of the target gene and the coloring reagent.

(6) Preferably, the type of the gene included in the test target is determined based on the determination result of the positive reaction of the plurality of detectors and the determination pattern information stored in advance in the storage unit, and the above display is performed. The gene determination step to be displayed in the section is further included.

According to the above configuration, the user can know the type of gene included in the test target.

(7) Preferably, the test piece has a color control to which the labeling substance is fixed. In the position specifying step, the area where the color control exists is obtained by referring to the detector position information stored in advance in the storage unit based on the position of the position marker along the reference direction in the image data. On the condition that the brightness value in the region is lower than the first cutoff value, the display unit is displayed to indicate that the color development reaction is poor.

According to the above configuration, the user can know whether or not the color development reaction is poor.

(8) Preferably, the test piece has an amplification control in which a probe that reacts with a control gene that is simultaneously amplified in the amplification of the target gene is fixed. In the position specifying step, the region where the amplification control exists is obtained by referring to the detector position information stored in advance in the storage unit based on the position of the position marker along the reference direction in the image data. On the condition that the brightness value in the region is lower than the first cutoff value, the display unit is displayed to indicate that the amplification reaction is poor.

According to the above configuration, the user can know whether or not the amplification reaction is defective.

(9) Preferably, in the representative value acquisition step, it is displayed on the display unit that there is no positive reaction of the plurality of detectors, provided that each of the difference values higher than the first cutoff value does not exist. ..

According to the above configuration, the user can easily know that there is no positive reaction for all the detectors.

(10) Preferably, in the position specifying step, the plurality of detector position information stored in advance in the storage unit is referred to based on the position of the position marker along the reference direction in the image data. The detectors are divided into a plurality of detector groups, and the difference acquisition step, the representative value acquisition step, the cutoff value selection step, and the reaction determination step are executed for each detector group.

According to the above configuration, the user can know the determination result of the positive reaction of the plurality of detector groups for the test piece having the plurality of detector groups.

(11) The determination method according to the present invention is a determination method for determining a positive reaction of a plurality of detectors using a computer. A plurality of detectors that develop color depending on the presence or absence of reaction with the substance to be inspected are fixed, and an image data storage step for storing the image data of the test piece on which the position marker is written in the storage unit and the above image data. With reference to the detector position information stored in advance in the storage unit based on the position of the position marker along the reference direction in the above, the position specifying step of obtaining each existing area where at least the plurality of detectors are present. , Each difference value, which is the difference between the maximum value of the brightness value in each existing region of the image data and the minimum value of the brightness value in each non-existing region adjacent to each existing region, is associated with each detector. Of the difference value acquisition step to be acquired and the plurality of cutoff values stored in advance in the storage unit, each of the above difference values higher than the first cutoff value set as the default is extracted and extracted. A representative value acquisition step for acquiring a representative value of a difference value, a cutoff value selection step for selecting a second cutoff value from a plurality of cutoff values stored in advance in the storage unit based on the representative value, and a cutoff value selection step. A reaction determination step for determining a positive reaction of the plurality of detectors based on each of the difference values and the second cutoff value is included.

(12) The determination device according to the present invention is a determination device including an image pickup device for acquiring image data, a storage unit for storing data, a display unit for displaying an image, and a calculation unit. In the calculation unit, a plurality of detectors that develop color according to the presence or absence of reaction with the substance to be inspected are fixed, and an imaging step of acquiring image data of a test piece marked with a position marker by the imaging device. Based on the image data storage step of storing the image data in the storage unit and the position of the position marker along the reference direction in the image data, the detector position information stored in advance in the storage unit is referred to. In the position specifying step for obtaining each existing region in which at least the plurality of detectors exist, the maximum value of the brightness value in each existing region of the image data, and each non-existing region adjacent to each existing region. The difference value acquisition step for acquiring each difference value, which is the difference from the minimum brightness value, in association with each detector, and the plurality of cutoff values stored in advance in the storage unit are set as defaults. A representative value acquisition step of extracting each of the above difference values higher than the first cutoff value and acquiring the representative value of each of the extracted said difference values, and a plurality of stored in advance in the above storage unit based on the above representative values. Based on the cutoff value selection step of selecting the second cutoff value from the cutoff value of the above, each of the above difference values, and the above second cutoff value, the positive reaction of the plurality of detectors is determined and the display unit is described. The reaction determination step displayed in is executed.

According to the present invention, the second cutoff value used for determining the positive reaction is selected by the luminance value of the region where the detector exists, so that the test piece is colored in the image data depending on the amplification, color development, shooting environment, and the like. Even if there are variations, stable judgment results can be obtained.

FIG. 1 is a schematic view of a test piece 10 according to an embodiment of the present invention. FIG. 2 is a plan view showing the appearance of the mobile terminal 50. FIG. 3 is a block diagram showing an electrical configuration of the mobile terminal 50. FIG. 4 is a diagram showing a cutoff value selection table. FIG. 5 is a flowchart showing the flow of the main process. FIG. 6 is a flowchart showing the flow of the test piece type determination process. FIG. 7 is a flowchart showing the flow of the Δ value calculation process. FIG. 8 is a flowchart showing the flow of the gene type determination process. FIG. 9 is a diagram showing an example of determining a positive reaction. FIG. 10 is a schematic diagram showing a display example of the gene type determination result on the mobile terminal 50.

Hereinafter, preferred embodiments of the present invention will be described. Needless to say, the present embodiment is only one embodiment of the present invention, and the embodiments can be changed without changing the gist of the present invention.

[Test pieces]
Mycobacterium tuberculosis has a wild type and a mutant type mutated from the wild type. There are multiple types of variants that have drug resistance to different drugs. Therefore, in order to select a drug effective against a specific M. tuberculosis bacterium, it is necessary to specify the gene type of the M. tuberculosis bacterium. The test piece 10 is used in a test for identifying the gene type of a drug resistance gene of Mycobacterium tuberculosis (hereinafter referred to as "target gene"). The target gene is an example of a substance to be tested.

As shown in FIG. 1, the test piece 10 has an elongated strip shape extending in the reference direction 11. A plurality of marker lines 12 are marked on one surface of the test piece. The marker line 12 is elongated in a direction orthogonal to the reference direction 11. The marker line 12 is, for example, a test piece 10 colored in blue. A plurality of marker lines 12 (4 in the test piece 10 shown in FIG. 1) are marked with a predetermined interval in the reference direction 11 of the test piece 10. The region of the test piece 10 sandwiched by the pair of marker lines 12 is used as a region for testing a specific target gene. The number of marker lines 12 and the arrangement interval in the reference direction 11 (hereinafter, referred to as “arrangement pattern of marker lines 12”) are different for each type of test piece 10.

In the test piece 10, a plurality of probes 13 of one type or different types are fixed between the marker lines 12 in the reference direction 11. The probe 13 is an oligonucleotide that can hybridize with the target gene. The probe 13 of a specific type is elongated in a direction orthogonal to the reference direction 11. The probes 13 of different types are arranged at a predetermined distance in the reference direction 11 of the test piece 10. A probe group 14 is composed of a plurality of probes 13 arranged between a pair of adjacent marker lines 12. In the test piece 10 shown in FIG. 1, there are three probe groups 14. Each probe 13 of one probe group 14 hybridizes to a different region of the target gene. The probe group 14 is an example of a detector group.

In the present embodiment, a plurality of probes 13 arranged between a pair of adjacent marker lines 12 constitute one probe group 14, but they are not necessarily arranged between a pair of adjacent marker lines 12. It is not necessary that the same probe group 14 is configured only by the plurality of probes 13. For example, probes belonging to another probe group 14 may be arranged between a pair of adjacent marker lines 12. Further, only a part of the plurality of probes 13 belonging to one probe group 14 is arranged between the pair of adjacent marker lines 12, and the other part is other than between the pair of marker lines 12. It may be arranged in another position.

In order to determine the gene type of the target gene, the target gene is hybridized to the probe 13 immobilized on the test piece 10. Specifically, first, the target gene is extracted from the sample and amplified by, for example, the PCR method. The amplified target gene hybridizes with the probe 13 immobilized on the test piece 10.

The primers used in the PCR method are, for example, modified with biotin. Therefore, the amplified target gene is biotin-modified. After the target gene is hybridized with the probe 13 of the test piece 10, the test piece 10 is reacted with a solution containing streptavidin labeled with alkaline phosphatase, and then NBT / BCIP (Nitro-Blue Tetrazolium /) as a coloring reagent is used. By 5-Bromo-4-Chloro-3-Indolyl-Phosphat), the probe 13 to which the target gene is hybridized is colored. In the test piece 10, the gene type included in the test target is determined by the color development patterns of the plurality of probes 13 between the marker lines 12.

Further, in the PCR method, a control gene different from the target gene is added and amplified by a biotin-modified primer together with the target gene.

[Color control 15]
As shown in FIG. 1, a color control 15 is fixed to the test piece 10. The color control 15 is for determining whether or not color development is normally performed. As the color control 15, for example, a probe labeled with biotin modification is fixed to the test piece 10.

[Amplification control probe 16]
As shown in FIG. 1, an amplification control probe 16 (hereinafter, referred to as “AC probe”) is fixed to the test piece 10. The AC probe 16 is a probe for determining whether or not the amplification of the target gene has been normally performed by determining whether or not the amplification of the control gene has been normally performed in the PCR method. As the AC probe 16, a probe that hybridizes with the control gene is immobilized on the test piece 10.

[Mobile terminal 50]
The mobile terminal 50 is, for example, a smartphone, and as shown in FIGS. 2 and 3, the display 53, the input interface 54 (hereinafter, referred to as “input I / F”), the camera 55, and the CPU ( (Abbreviation of Central Processing Unit) 61, a memory 62, and a communication bus 63 are mainly provided. The mobile terminal 50 is an example of a determination device and a computer, a camera 55 is an example of an image pickup device, a display 53 is an example of a display unit, a CPU 61 is an example of a calculation unit, and a memory 62 is an example of a calculation unit. This is an example of a storage unit.

As the display 53, for example, a liquid crystal display (abbreviation of Liquid Crystal Display), an organic EL display (abbreviation of Organic Electro-Luminence Display), and the like are adopted.

The input I / F 54 is a user interface that accepts input operations by the user. Specifically, the input I / F 54 is a film-like touch sensor superimposed on the display 53. That is, the display 53 is configured as a touch panel display. Further, the input I / F 54 may be a button included in the mobile terminal 50. When the input I / F is operated by the user, the input I / F 54 outputs various operation signals associated with the operation of the input I / F 54 to the CPU 61.

A CMOS (Complementary Metal Oxide Semiconductor) image sensor is used in the camera 55. With the camera 55 activated, the image captured by the camera 55 is displayed on the display 53 in real time. Further, the image data of the still image captured by the camera 55 is stored in the memory 62 described later as color image data. A CCD (Charge Coupled Device) image sensor may be used for the camera 55.

The CPU 61 controls the overall operation of the mobile terminal 50. The CPU 61 acquires various programs described later from the memory 62 and executes them based on various information output from the input I / F 54.

The memory 62 has a program storage area 62A and a data storage area 62B. The OS (abbreviation of Operating System) 64 and the application program 65 are stored in the program storage area 62A. OS64 is, for example, Android (registered trademark of Google inc.) OS, iOS (registered trademark of Cisco Systems, Inc.), Windows Phone (registered trademark of Microsoft Corporation) Operating System, and the like.

The program stored in the program storage area 62A is executed by the CPU 61. The application program 65 may be a single program or a collection of a plurality of programs. Data necessary for executing the application program 65 is stored in the data storage area 62B.

The memory 62 includes, for example, RAM (abbreviation of Random Access Memory), ROM (abbreviation of Read Only Memory), EEPROM (abbreviation of Electrically Erasable Programmable Read-Only Memory, abbreviation of Read-Only Memory), HDD (Hard Disk). It is composed of (abbreviation of Drive), etc., or a combination thereof.

The data storage area 62B of the memory 62 stores marker line pattern information, probe position information, cutoff value selection table, determination pattern information, CC flag, and AC flag. The probe position information is an example of the detector position information.

The marker line pattern information is information in which the arrangement pattern of the marker line 12 on the test piece 10, the type of the test piece 10, and the number of probe groups 14 on the test piece 10 are associated with each other. The position of the marker line 12 is, for example, a position along the reference direction 11. In the marker line pattern information, as the arrangement pattern of the marker lines 12, the positions of the plurality of marker lines 12 fixed to the test piece 10 are along the reference direction 11 from the marker line 12 located on the leftmost side as a reference. The distance ratio is memorized.

The probe position information is information for specifying a region (an example of an existing region, hereinafter referred to as “existing region”) in which the probe 13 is fixed in the test piece 10. The probe position information is stored for each type of the test piece 10. The probe position information is, for example, for each probe 13 in the reference direction 11 based on the position of each marker line 12 in the reference direction 11 for the test piece 10 of the target type (for example, the test piece 10 shown in FIG. 1). Information on the range or position occupied by the existing area of. The range of the probe 13 is represented, for example, in units of pixels in the image data.

As shown in FIG. 4, a plurality of cutoff values are stored in the cutoff value selection table. The cutoff value is a threshold value for determining a positive reaction of the probe 13. The cutoff value is provided corresponding to each probe 13 and forms a cutoff value unit. One of the plurality of cutoff value units is set as the first cutoff value. The cutoff value unit set as the first cutoff value may be changed by the user. Although not shown, the cutoff value selection table stores the cutoff value of the color control 15 and the cutoff value of the AC probe 16, and stores the first cutoff value of the color control 15 and the AC probe 16. The first cutoff value of is set. In the cutoff value selection table, a single cutoff value may be set for each probe 13 instead of the cutoff value unit. Further, the cutoff value selection table may be composed of a single table or a plurality of tables.

The threshold range is stored in the cutoff value selection table. Each threshold range is associated with each cutoff value unit. The threshold range is the range of the positive Δ average value described later. The positive Δ average value is an example of a representative value.

The determination pattern information is information for determining the gene type by the test piece 10. Specifically, the determination pattern information is information in which the arrangement pattern of the probe 13 determined to be positive and the probe 13 determined to be negative and the gene type are associated with each other in each type of test piece 10. .. Judgment pattern information is provided for each type of test piece 10 for all gene types that can be determined by the test piece 10 of that type.

The CC flag is a flag indicating whether the color control 15 is negative or positive. In the CC flag, for example, 0 is stored when the color control 15 is determined to be negative, and 1 is stored when the color control 15 is determined to be positive.

The AC flag is a flag indicating whether the amplification control probe 16 is negative or positive. In the AC flag, for example, 0 is stored when the amplification control probe 16 is determined to be negative, and 1 is stored when the amplification control probe 16 is determined to be positive.

[Main processing]
The main process shown in FIG. 5 is executed by the CPU 61 when the user activates the application program 65 for determining the gene type of Mycobacterium tuberculosis on the mobile terminal 50.

In the main process, the CPU 61 transmits a start signal to the camera 55 to start the camera 55 (step S11). As a result, as shown in FIG. 2, the image captured by the camera 55 is displayed on the display 53.

After transmitting the start signal to the camera 55, the CPU 61 determines whether or not the press of the end button 23 (see FIG. 2) is accepted (step S12). The end button 23 is one of the input I / F 54, and is, for example, a button included in the mobile terminal 50. When the CPU 61 determines that the press of the end button 23 has been accepted (step S12: Yes), the CPU 61 ends the main process.

In a state where the camera 55 is activated, as shown in FIG. 2, the display 53 displays a red line 21 (hereinafter, referred to as a “reference line”) that crosses the display 53. The user can view the positional relationship between the test piece 10 and the camera 55 so that the reference line 21 displayed on the display 53 is along the reference direction 11 of the test piece 10 and the entire test piece 10 can be seen in the display 53. To adjust. Then, after the adjustment, the image pickup button 22 (see FIG. 2) is pressed. The image pickup button 22 is one of the inputs I / F 54. The image pickup button 22 may be a button displayed on the display 53, or may be a button included in the mobile terminal 50.

When the CPU 61 determines that the press of the end button 23 is not accepted (step S12: No), the CPU 61 determines whether or not the image pickup button 22 has been pressed (step S13). When the CPU 61 determines that the image pickup button 22 is not pressed (step S13: No), the CPU 61 re-executes the process of step S12.

When the CPU 61 determines that the imaging button 22 has been pressed (step S13: Yes), the CPU 61 transmits an imaging instruction to the camera 55. When the camera 55 receives the imaging instruction, it executes imaging and transmits the captured image data to the CPU 61. Along with this, the CPU 61 receives the image data from the camera 55 (step S14). Then, the CPU 61 stores the received image data in the memory 62 (step S15). The process of step S14 is an example of an imaging step, and the process of step S15 is an example of an image data storage step.

Then, the CPU 61 executes the test piece type determination process (step S16). The test piece type determination process is a process for determining the type of the test piece 10 described above. Details of the test piece type determination process will be described later.

Next, the CPU 61 executes the gene type determination process (step S17). The gene type determination process is a process for determining the gene type of the target gene based on the color development state of each probe 13 of the test piece 10. The details of the gene type determination process for the CPU 61 will be described later. After the gene type determination process is completed, the main process is completed.

[Test piece type judgment processing]
The details of the test piece type determination process executed in step S16 of the main process shown in FIG. 5 are shown in FIG.

In the test piece type determination process, the CPU 61 reads the image data of the test piece 10 captured by the camera 55 from the memory 62, and specifies the position of the blue pixel in each pixel along the reference line 21 as the position of the marker line 12. (Step S21). For example, the position of the marker line 12 in the image data is specified for each pixel, and the position of the marker line 12 is the position of a group of blue pixels indicating one marker line 12 along the reference line 21. Let (range) be the position (range) of the marker line 12.

The CPU 61 acquires the interval ratio of the plurality of marker lines 12 fixed to the test piece 10 along the reference direction 11 by using the position of the marker line 12 specified in step S21 (step S22). This interval ratio is calculated as the ratio of the distances at which each marker line 12 is separated from the reference marker line 12 in the reference direction 11 with reference to the marker line 12 located on the leftmost side of the plurality of marker lines 12. ..

The CPU 61 reads the marker line pattern information from the memory 62, compares it with the acquired marker line interval ratio, and acquires the type of the test piece 10 corresponding to the arrangement pattern of the matching marker lines 12 (step S23). The arrangement pattern of the marker lines 12 does not necessarily have to match all the interval ratios, and may be determined from the fact that the interval ratios of a specific ratio among the plurality of marker lines 12 match. Then, the CPU 61 stores an identifier indicating the type of the imaged test piece 10 in the memory 62 (step S24).

[Delta value calculation process]
The Δ value calculation process shown in FIG. 7 is a process of calculating the Δ values for all the probes 13 of the imaged test piece 10. The Δ value calculation process is an example of the difference value acquisition step.

In the Δ value calculation process, the CPU 61 reads the type of the imaged test piece 10 from the memory 62, and reads the probe position information corresponding to the read type from the memory 62 (step S51).

Then, the CPU 61 reads the pixel on the reference line 21 of the test piece 10 in the image data from the memory 62, acquires the pixel value (gradation) of each read pixel, and calculates the luminance value for each pixel (step S52). ). The brightness value is calculated from the pixel value using a predetermined formula.

Next, the CPU 61 compares the probe position information with the pixels on the reference line 21, and for the pixels on the reference line 21, the pixels located in the region where the probe 13 exists, the probe 13 and the marker line 12 are fixed. The pixel located in the non-existing region is specified (step S53). The process of step S53 is an example of the position specifying step. The non-existing region may be specified as a predetermined range or position between the existing regions of each probe 13 specified based on the probe position information, or the brightness value between the existing regions of each probe 13 may be specified. It may be specified based on the position where the lowest value exists. Further, among the brightness values of the pixels on the reference line 21, the position where the pixel less than the threshold value exists may be specified as the non-existing region.

Then, the CPU 61 selects one from the existing region of the probe 13 for which the Δ value has not been calculated (step S54). For example, the probe 13 located on the leftmost side is selected from the plurality of target probes 13.

Then, the CPU 61 acquires the highest value among the luminance values of the pixels belonging to the selected existing region (step S55). The highest value may be the highest value of one of the brightness values of the pixels belonging to the selected existing region, or may be the average value of a plurality of high values selected in order from the highest value. ..

Further, the CPU 61 acquires the lowest value among the brightness values of the pixels belonging to the non-existing regions located on both sides of the reference direction 11 of the selected existing region (step S56). The lowest value may be the lowest value of one of the brightness values of the pixels belonging to the selected non-existing region, or a plurality of low values are selected in order from the lowest value, and the average value of these multiple low values. You may. It should be noted that the height of the brightness value is a relative concept, and in the present embodiment, the brightness value is lower as it is not colored, and the brightness value is higher as it is colored.

Then, the CPU 61 stores the value obtained by subtracting the minimum value of the brightness value from the maximum value of the acquired brightness value in the memory 62 as the Δ value of the probe 13 corresponding to the selected existing area (step S57).

Next, the CPU 61 determines whether or not there is an existing region for which the Δ value has not been calculated (step S58). Then, when there is an existing area for which the Δ value has not been calculated (step S58: Yes), the CPU 61 re-executes the process of step S54. For example, it is determined whether or not there is a probe 13 to the right of the already selected probe 13, and if there is a probe 13 to the right, the process of step S54 is executed for the probe 13. When there is no existing area for which the Δ value has not been calculated (step S58: No), the CPU 61 ends the Δ value calculation process.

[Gene type determination processing]
Details of the gene type determination process performed in step S17 of the main process shown in FIG. 5 are shown in FIG.

In the gene type determination process, the CPU 61 executes the above-mentioned Δ value calculation process (step S31). The Δ value calculated in the Δ value calculation process is stored in the memory 62. The Δ value is an example of the difference value.

Next, the CPU 61 reads the first cutoff value and the Δ value of the color control 15 from the memory 62, and determines whether or not the Δ value of the color control 15 is equal to or greater than the first cutoff value (step S32). .. When the CPU 61 determines that the Δ value of the color control 15 is equal to or greater than the first cutoff value (step S32: Yes), the CPU 61 stores “1” as information indicating a positive result in the CC flag stored in the memory 62. (Step S33).

Then, the CPU 61 reads the first cutoff value and the Δ value of the AC probe 16 from the memory 62, and determines whether or not the Δ value of the AC probe 16 is equal to or greater than the first cutoff value (step S34). When the CPU 61 determines that the Δ value of the AC probe 16 is equal to or greater than the first cutoff value (step S34: Yes), the CPU 61 stores “1” as information indicating a positive result in the AC flag stored in the memory 62. (Step S35).

On the other hand, when the CPU 61 determines in step S34 that the Δ value of the AC probe 16 is smaller than the first cutoff value (step S34: No), the information indicating negative to the AC flag stored in the memory 62 is ". 0 ”is stored (step S36).

After the processing of step S35 or step S36, the CPU 61 reads out the type of the test piece 10 stored in the memory 62, and the number of probe groups 14 corresponding to the test piece 10 (for example, three probe groups) from the marker line pattern information. Hereafter, they are referred to as group A, group B, and group C, respectively.) Then, the CPU 61 selects one probe group (for example, group A) based on the number of probe groups 14 (step S37).

The CPU 61 is included in the first cutoff value unit (hereinafter, simply referred to as “first cutoff value”) corresponding to the group A selected as the first cutoff value from the memory 62, and the group A. Read out the Δ values of all the probes 13. Then, the CPU 61 compares the first cutoff value with the Δ value of each probe 13 and determines whether or not there is one or more probes 13 having a Δ value larger than the first cutoff value (step). S38). At this time, the CPU 61 stores the Δ values of all the probes 13 having a Δ value larger than the first cutoff value in the memory 62.

When the CPU 61 determines that there is one or more probes 13 having a Δ value larger than the first cutoff value (step S38: Yes), the CPU 61 uses each Δ value stored in the memory 62 in step S38 to perform the first step. An average value of Δ values larger than the cutoff value is calculated and stored in the memory 62 as a positive Δ average value (step S39). The process of step S39 is an example of the representative value acquisition step.

For example, as shown in FIG. 9, it is assumed that the Δ values of the five probes 13 are “18”, “13”, “5”, “5”, and “7”, respectively. For example, if the first cutoff value corresponding to each probe 13 is "10", "10", "8", "12", "4", the Δ value larger than the first cutoff value is "18". , "13", "7". The average value of these three Δ values is calculated, and “12.67” is obtained as the positive Δ average value.

Then, the CPU 61 reads the cutoff value stored in the memory 62 in association with the threshold range including the positive Δ average value in the cutoff value selection table shown in FIG. 4 from the memory 62, and reads the second cutoff value. (Step S40). As shown in FIG. 4, if the positive Δ average value is “12.67”, the second cutoff value corresponding to each probe 13 is “5”, “5”, “4”, “6”, "2" is acquired. The process of step S40 is an example of the cutoff value selection step.

Then, the CPU 61 compares the second cutoff value with the Δ values of all the probes 13 included in the A group, determines a positive reaction for each probe 13, and stores the determination result in the memory 62 (step S41). ). Specifically, as a determination of a positive reaction, the CPU 61 determines that the probe 13 having a Δ value of the second cutoff value or more is positive, and the probe 13 having a Δ value smaller than the second cutoff value is negative. At this time, if the Δ value is included in a predetermined range of the second cutoff value or more (for example, a value obtained by multiplying the second cutoff value by 1.3), it is determined as suspicious positive. Further, when the Δ value is included in a predetermined range less than the second cutoff value (for example, a value obtained by multiplying the second cutoff value by 0.7), it is determined as suspicious negative. Here, the above-mentioned predetermined range is stored in the memory 62 in advance as a value for determining a suspicious positive and a suspicious negative. This value may be changeable by the user. The process of step S41 is an example of the reaction determination step.

For example, in the example shown in FIG. 9, the above Δ value “2” is used by using the second cutoff values “5”, “5”, “4”, “6”, and “2” corresponding to each probe 13. When a positive reaction is determined for "18", "13", "5", "5", and "7", the Δ values "18", "13", and "7" of probe numbers 1, 2, and 5 are positive. Is determined. Of these, the Δ value “5” of probe number 3 is determined to be suspicious positive, and the Δ value “5” of probe number 4 is determined to be suspicious negative.

Next, the CPU 61 determines whether or not there is still a probe group 14 for which a positive reaction has not been determined (step S42). When the CPU 61 determines that there is still a probe group 14 (for example, B group, C group) for which a positive reaction has not been determined (step S42: Yes), the CPU 61 returns to step S37 and returns to the probe group for which no positive reaction has been determined. 14 is selected to execute the process from step S38.

On the other hand, when the CPU 61 determines in step S38 that there is no probe 13 having a Δ value larger than the first cutoff value (step S38: No), the memory 62 indicates that all the probes 13 in the group A are negative. (Step S43), and the process of step S42 is executed.

When the CPU 61 determines that there are no more probe groups 14 that have not determined a positive reaction (step S42: No), the CPU 61 determines the gene type based on the determination result of the probe group 14 that has determined a positive reaction (step S44). ). Specifically, the CPU 61 reads the determination pattern information and the determination result of each probe group 14 stored in the memory 62 in step S41 from the memory 62. Then, the CPU 61 compares a plurality of determination patterns corresponding to the test piece 10 included in the determination pattern information with the determination results of all the probes 13 and selects a matching determination pattern.

Further, when the CPU 61 determines in step S32 that the Δ value of the color control 15 is smaller than the first cutoff value (step S32: No), the information indicating negative to the CC flag stored in the memory 62 is ". 0 ”is stored (step S45). When it is determined that the Δ value of the color control 15 is smaller than the first cutoff value, the color of the target gene is not normally developed. Therefore, the gene type determination in step S44 is not performed.

After the treatment of step S44 or the treatment of step S45, the CPU 61 is negative or positive for the gene type of the matching determination pattern, each probe 13, the color control 15, and the AC probe 16 as shown in FIG. In addition, whether or not it is a suspicious negative or a suspicious positive is displayed on the display 53 as a determination result (step S46). The processing of steps S44 and S46 is an example of the gene determination step.

In the display example of the determination result shown in FIG. 10, for example, "katG mutant type" determined as the gene type is displayed in the gene type display column 31. For the probe 13, probe group type 32, probe number 33, Δ value 34, negative / positive type 35, and suspicious negative / suspicious positive type 36 are displayed for each probe group 14. In the probe group type 32 (display column of "group" in FIG. 10), the type of the probe group to which the probe 13 belongs, for example, "A" is displayed. In the probe number 33 (display column of "probe" in FIG. 10), the probe numbers, for example, the numbers assigned to each probe 13 in order from the left of the test piece 10 are displayed. In the Δ value 34 (display column of “Δ value” in FIG. 10), the Δ value calculated for each probe 13 is displayed. In the negative / positive type 35 (display column of "+/-" in FIG. 10), the determination result determined for each probe 13 is "-" in the case of negative and "+" in the case of positive. Is displayed. As the suspicious negative / suspicious positive type 36 (display column of "Reliability" in FIG. 10), "R" is displayed in the case of suspicious negative or suspicious positive, and nothing is displayed in the case of not suspicious negative or suspicious positive.

The negative / positive type 35 and the suspicious negative / suspicious positive type 36 may be indicated by the background color in the display of the Δ value 34. For example, the background color in the display of the Δ value 34 may be red if it is negative, orange if it is suspiciously negative, blue if it is positive, or green if it is suspiciously positive.

[Action and effect of this embodiment]
As described above, the second cutoff value for determining the positive reaction of the plurality of probes 13 is determined according to the Δ value of the probes 13. The Δ value is a difference value between the maximum value of the brightness value in the existing region and the minimum value of the brightness value in the non-existing region of the probe 13. Then, the second cutoff value is determined according to the average value (positive Δ average value) of the Δ values in all the probes 13 whose Δ values are higher than the first cutoff value. Therefore, the positive reaction is stably determined according to the characteristics of the target gene such as easy amplification, the reaction state of the positive reaction of the probe 13, the imaging environment of the test piece 10, and the like.

There are variations in the amplification of the target gene by the PCR method and the hybridization between the target gene and the probe 13. The cutoff value is set corresponding to each probe 13 of the plurality of probes 13 according to the variation in hybridization between the target gene and the probe 13. In addition, the cutoff value is reselected from the first cutoff value to the second cutoff value according to the variation in the amplification of the target gene. Thereby, the positive reaction of each probe 13 can be determined by the amplification of the target gene by the PCR method and the appropriate cutoff value corresponding to the variation in the hybridization between the target gene and the probe 13.

The CPU 61 displays on the display 53 that there is no positive reaction of the plurality of probes 13 on condition that the probes 13 having each Δ value higher than the first cutoff value do not exist. Therefore, the user can easily know that there is no positive reaction for all the probes 13.

The CPU 61 has a suspicious positive or suspicious negative in addition to the positive or negative determination result for the probe 13 in which the difference between each Δ value and the second cutoff value is within a predetermined range preset in the memory 62. Is displayed on the display 53. Therefore, the user can know that the determination of the positive reaction of the probe 13 is suspicious positive or suspicious negative.

The CPU 61 determines the gene type included in the target gene based on the determination result of the positive reaction of the plurality of probes 13 and the determination pattern information stored in advance in the memory 62, and displays the gene type on the display 53. Therefore, the user can know the gene type of the target gene.

The test piece 10 has a color control 15. Therefore, the user can know whether the color development reaction is normal or poor.

The test piece 10 has an amplification control probe 16. Therefore, the user can know whether the amplification reaction is normal or bad.

Since the CPU 61 determines a positive reaction for the plurality of probe groups 14, the user can know the determination result of the positive reaction of the plurality of probe groups 14 for the test piece 10 having the plurality of probe groups 14.

[Modification example]
In the above-described embodiment, the Δ value is calculated using the brightness value of the pixel corresponding to each probe 13. However, the Δ value may be calculated using the brightness value instead of the brightness value. Further, the Δ value may be calculated using both the brightness value and the brightness value.

Further, in the above-described embodiment, the Δ value or the like is obtained using only the pixels located on the reference line 21, but the Δ value or the like is obtained at a position away from the reference line 21 in the direction orthogonal to the reference line 21 along the test piece 10. The Δ value or the like may be calculated using a plurality of existing pixels.

Further, the substance to be inspected using the test piece 10 does not necessarily have to be the drug resistance gene of Mycobacterium tuberculosis. The test piece 10 includes, for example, HPV (human papilloma) test, HLA (human leukocyte antigen) test, MRSA (methicillin-resistant staphylococcus) detection, Staphylococcus (staphylococcus) group detection, Helicobacter pylori (Helicobacter pylori) detection, Detection of Clostridium diffile (Helicobacter pylori), E.I. It may be used for genetic testing such as detection of E. coli.

Further, in the above-described embodiment, the mobile terminal 50 is used as the determination device, but another information processing device such as a PC (personal computer) or a dedicated determination device may be used. Further, the determination device does not necessarily have to be provided with an image pickup device (camera 55). The image pickup device may be provided separately from the determination device, and image data may be transmitted from the image pickup device to the determination device by using a communication means such as Bluetooth (registered trademark of Bluetooth SIG) or infrared communication. The communication means may be a wired connection or a wireless connection. The communication means may be one that directly communicates with the image pickup apparatus and the determination apparatus, or may be an indirect communication.

10 ... Test piece 11 ... Reference direction 12 ... Marker line (position marker)
13 ... Probe (detector)
14 ... Probe group (detector group)
15 ... Color control probe 16 ... Amplification control probe 50 ... Mobile terminal (judgment device, computer)
53 ... Display (display unit)
55 ... Camera (imaging device)
61 ... CPU (calculation unit)
62 ... Memory (storage unit)

Claims (12)

A plurality of detectors that develop color depending on the presence or absence of reaction with the substance to be inspected are fixed, and an image data storage step of storing the image data of the test piece on which the position marker is marked is stored in the storage unit.
Based on the position of the position marker along the reference direction in the image data, the position for obtaining each existing area where at least the plurality of detectors are present by referring to the detector position information stored in advance in the storage unit. With specific steps
Each difference value, which is the difference between the maximum value of the luminance value in each of the existing regions of the image data and the minimum value of the luminance value in the non-existing region of the detector adjacent to each existing region, is given to each detector. The difference value acquisition step to be acquired in association with
A representative that extracts each of the above-mentioned difference values higher than the first cut-off value set as the default from the plurality of cut-off values stored in advance in the above-mentioned storage unit, and acquires the representative value of each of the extracted said-mentioned difference values. Value acquisition step and
A cutoff value selection step of selecting a second cutoff value from a plurality of cutoff values stored in advance in the storage unit based on the representative value, and
A reaction determination step of determining whether or not the plurality of detectors are positive based on the respective difference values and the second cutoff value, and displaying whether or not the plurality of detectors are positive on the display unit. A computer program that causes a computer to run . A plurality of cutoff values and a threshold range are stored in advance in the above storage unit in association with each other.
The computer program according to claim 1, wherein in the cutoff value selection step, a cutoff value corresponding to a threshold range including the representative value is selected as the second cutoff value. The computer program according to claim 1 or 2, wherein in the representative value acquisition step, the average value of each difference value higher than the first cutoff value is set as the representative value. In the reaction determination step, the plurality of detectors in the display unit are provided on the condition that the difference between the difference value and the second cutoff value is within a predetermined range preset in the storage unit. The computer program according to any one of claims 1 to 3, which indicates that the determination as to whether or not is positive is suspicious. The detector is a plurality of types of probes that react with the amplified target gene.
The test piece according to any one of claims 1 to 4, wherein the position where the plurality of detectors are present is colored by the reaction between the labeling substance of the primer used for amplifying the target gene and the coloring reagent. Computer program. Based on the determination result of whether or not the plurality of detectors are positive and the determination pattern information stored in advance in the storage unit, the type of the gene included in the test target is determined, and the determined gene is determined. The computer program according to claim 5, further comprising a gene determination step of displaying the type of the above on the display unit. The test piece has a color control to which the labeling substance is fixed.
In the position specifying step, the region where the color control exists is obtained by referring to the detector position information stored in advance in the storage unit based on the position of the position marker along the reference direction in the image data. The computer program according to claim 6, wherein the display unit displays an indication that the color development reaction is poor, provided that the luminance value in the region is lower than the first cutoff value. The test piece has an amplification control in which a probe that reacts with a control gene that is simultaneously amplified in the amplification of the target gene is fixed.
In the position specifying step, the region where the amplification control exists is obtained by referring to the detector position information stored in advance in the storage unit based on the position of the position marker along the reference direction in the image data. The computer program according to claim 6 or 7, wherein the display unit displays an indication that the amplification reaction is defective, provided that the luminance value in the region is lower than the first cutoff value. Any of claims 1 to 8 for displaying on the display unit that the plurality of detectors are not positive, provided that each of the difference values higher than the first cutoff value does not exist in the representative value acquisition step. The computer program described in Crab. In the position specifying step, a plurality of the plurality of detectors are detected by referring to the detector position information stored in advance in the storage unit based on the position of the position marker along the reference direction in the image data. The method according to any one of claims 1 to 9, wherein the detection body group is divided into body groups, and the difference value acquisition step, the representative value acquisition step, the cutoff value selection step, and the reaction determination step are executed for each detection body group. Computer program. It is a determination method for determining whether or not a plurality of detectors are positive using a computer.
A plurality of detectors that develop color depending on the presence or absence of reaction with the substance to be inspected are fixed, and an image data storage step of storing the image data of the test piece on which the position marker is marked is stored in the storage unit.
Based on the position of the position marker along the reference direction in the image data, the position for obtaining each existing area where at least the plurality of detectors are present by referring to the detector position information stored in advance in the storage unit. With specific steps
Each difference value, which is the difference between the maximum value of the luminance value in each of the existing regions of the image data and the minimum value of the luminance value in the non-existing region of the detector adjacent to each existing region, is given to each detector. The difference value acquisition step to be acquired in association with
A representative that extracts each of the above-mentioned difference values higher than the first cut-off value set as the default from the plurality of cut-off values stored in advance in the above-mentioned storage unit, and acquires the representative value of each of the extracted said-mentioned difference values. Value acquisition step and
A cutoff value selection step of selecting a second cutoff value from a plurality of cutoff values stored in advance in the storage unit based on the representative value, and
A determination method including a reaction determination step for determining whether or not the plurality of detectors are positive based on each of the difference values and the second cutoff value. A determination device including an imaging device for acquiring image data, a storage unit for storing data, a display unit for displaying an image, and a calculation unit.
The above calculation unit
A plurality of detectors that develop color depending on the presence or absence of reaction with the substance to be inspected are fixed, and an imaging step of acquiring image data of a test piece marked with a position marker by the above imaging device, and
An image data storage step for storing the image data in the storage unit, and
Based on the position of the position marker along the reference direction in the image data, the position for obtaining each existing area where at least the plurality of detectors are present by referring to the detector position information stored in advance in the storage unit. With specific steps
Each difference value, which is the difference between the maximum value of the luminance value in each of the existing regions of the image data and the minimum value of the luminance value in the non-existing region of the detector adjacent to each existing region, is given to each detector. The difference value acquisition step to be acquired in association with
A representative that extracts each of the above difference values higher than the first cutoff value set as the default from the plurality of cutoff values stored in advance in the storage unit, and acquires the representative value of each of the extracted difference values. Value acquisition step and
A cutoff value selection step of selecting a second cutoff value from a plurality of cutoff values stored in advance in the storage unit based on the representative value, and
A reaction determination step of determining whether or not the plurality of detectors are positive based on the respective difference values and the second cutoff value, and displaying whether or not the plurality of detectors are positive on the display unit. And, a judgment device that executes.

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