JP5909420B2 - Genetic testing device, genetic testing reagent, and genetic testing method - Google Patents

Description

  The present invention relates to a genetic test apparatus, a genetic test reagent, and a genetic test method for detecting the presence or absence of a gene by fluorescence detection using a reagent.

  Whether biological samples collected from human bodies, animals and plants (hereinafter abbreviated as specimens) are infected with genetic diseases (hereinafter abbreviated as genetic test items) such as bacteria, viruses and cancers A genetic test device that has the function of judging from the fluorescence intensity by extracting the nucleic acid contained in the sample, specifically amplifying the base sequence of the gene corresponding to the genetic test item, and fluorescently labeling the corresponding gene About.

  A genetic testing device is a testing device that tests one gene item in one reaction container, and includes a sample, a reagent necessary for amplification and testing of the base sequence of one corresponding gene, and a fluorescently labeled probe for labeling the corresponding gene. It includes a step of dispensing into one reaction vessel.

  On the other hand, when testing multiple genetic test items at once, it is necessary to dispense samples into multiple containers, and to amplify and test the nucleic acid sequences of genetic test items for each dispensed reaction container. .

  To simplify this process, use a genetic tester that can identify amplified and fluorescently labeled nucleic acid sequences in a reaction container even when multiple nucleic acid sequences are amplified and fluorescently labeled in a single reaction container. It is necessary.

  When it is desired to amplify and fluorescently label a plurality of base sequences in one reaction vessel, for example, multiplex is known as an identifiable device. In Patent Document 1, amplification of a plurality of base sequences is identified as a multiplex using fluorescent labels of different colors.

Special table 2007-525184 gazette

  However, the types of genes that can be amplified at one time in one reaction vessel are limited by the number of colors of fluorescent labels. When the number of genes that can be tested at one time in a single reaction container is small, it is necessary to dispense reaction containers and to install many reaction containers.

  An object of the present invention is to provide a genetic testing device, a genetic testing reagent, and a genetic testing method capable of identifying a plurality of nucleic acid sequences using a single color fluorescent label when a plurality of genes are amplified. .

    The reagent used in the present invention includes a fluorescent mixed probe, and the fluorescent mixed probe emits the same color fluorescence with the same color excitation light, and a plurality of fluorescent probes are mixed, and each fluorescent probe is a specific gene. Each fluorescent probe is characterized by having a different plateau emission value of the fluorescence intensity at the time of genetic testing.

    The genetic test apparatus of the present invention has an advantage that a plurality of nucleic acid sequences can be identified using a single color fluorescent label.

The figure which shows the combination and plateau luminescence value of a some genetic test item. Type 1 of gene amplification section and detection section of nucleic acid analyzer. Type 2 of the gene amplification section and detection section of the nucleic acid analyzer. Type 3 of gene amplification part and detection part of nucleic acid analyzer. Excitation light source type 1 for nucleic acid analyzers. Excitation light source type 2 for nucleic acid analyzers. Nucleic acid analyzer excitation light source type 3. 1 is a schematic diagram of the overall configuration of a nucleic acid analyzer. Flow of creating a fluorescent mixed probe in which common fluorescent probes of the same color are mixed. Calibration flow of a fluorescent mixed probe in which fluorescent probes of the same color are mixed. Measurement flow of a fluorescent mixed probe in which fluorescent probes of the same color are mixed. Display screen of fluorescent mixed probe information. A test result screen in which multiple genes are tested using a fluorescent probe mixed with fluorescent probes of the same color. Display screen of fluorescence blend probe information. The figure which shows a plateau luminescence value.

  The gene testing apparatus of the present invention amplifies the gene corresponding to the gene item to be tested contained in the reaction container, and when the gene in the reaction container is amplified, the fluorescence intensity of the fluorescent label contained in the reaction container increases. It is a type of genetic testing device that uses the properties to determine whether a specimen contains a gene for a genetic test item based on the presence or absence of fluorescence or emission intensity, or to quantitatively evaluate the gene concentration. This is a genetic test apparatus of a type in which the fluorescence intensity is increased, and is a type of genetic test apparatus having a feature that when the gene is sufficiently increased, the fluorescence intensity does not increase any more and reaches a saturated state.

  The gene testing apparatus of the present invention includes a step of dispensing a sample, a reagent necessary for amplification and testing of a base sequence of one corresponding gene, and a fluorescently labeled probe for labeling the corresponding gene into one reaction container.

  The gene testing apparatus includes a step of bringing the solution contained in the reaction vessel into an appropriate high temperature state and an appropriate low temperature state with respect to the reaction vessel, and further including a step of repeating this step, thereby It is desirable to include a step of PCR amplification having a mechanism for amplifying. However, it is not necessary to add a PCR amplification step if it is a genetic testing apparatus including a step for amplifying genes.

The genetic test apparatus of the present invention preferably includes a real-time PCR function for measuring fluorescence intensity during amplification of a gene in a reaction vessel.
In the case of a genetic testing device having a real-time PCR function, the fluorescence intensity in the reaction vessel increases as shown in FIG. 15 each time the corresponding gene is amplified. However, if gene amplification is sufficiently performed, the fluorescence intensity does not increase any more as shown in FIG. 15, and the fluorescence intensity becomes saturated. (In the present invention, the fluorescence intensity at which the fluorescence intensity is saturated is called a plateau emission value.)
Hereinafter, means for solving the problems of the present invention will be described.

The plateau luminescence value has characteristics that do not depend on the amplified gene concentration.
The first means of the present invention utilizes this feature, when a plurality of fluorescently labeled probes that each emit fluorescence of the same color, amplify the gene of the specimen in the reaction container, and test with a plurality of fluorescently labeled probes The ratio of the plateau luminescence values of the respective fluorescently labeled probes (hereinafter abbreviated as the plateau luminescence value ratio) so that which fluorescently labeled probe has reached the plateau luminescence value can be identified from the fluorescence intensity reaching the plateau luminescence value. However, it is to use a fluorescence mixing lobe which mixes so that it may become a special ratio with a genetic test device. This is because, for example, fluorescently labeled probes a, b, and c of the same color are prepared, and fluorescent mixed probes mixed so that the plateau emission ratio of fluorescently labeled probes a, b, and c is 4: 3: 2. It is to be used in a genetic testing device.

  The second means of the present invention uses a calibration sample containing a known gene for the fluorescent mixed probe, and calibrates the plateau luminescence value of all combinations of genetic test items that can be tested with the fluorescent mixed probe. Perform Here, the combination of genetic test items is, for example, when the fluorescence mixed probe can test the genetic test items of gene A, gene B, and gene C, the total combination of genetic test items is (1) gene A, (2 (7) Gene B, (3) Gene C, (4) Gene A and B, (5) Gene A and C, (6) Gene B and C, (7) Gene A, B and C. This means includes a function capable of storing calibration information including a calibration result of a plateau luminescence value for each combination of gene items in a genetic test apparatus. In addition, it is preferable that the means compares the plateau luminescence values for each gene item so that the ratio of the plateau luminescence values can be registered in the calibration information.

  The third means of the present invention is to dispense a specimen, a reagent necessary for amplification / testing of a plurality of corresponding genes, and a fluorescent mixed probe into one reaction container, perform gene amplification, and then obtain a plateau luminescence obtained. By comparing the value with the calibration information, searching for the plateau luminescence value closest to the plateau luminescence value registered in the calibration information, and extracting the combination of genetic test items that reach that plateau luminescence value, A means for identifying a gene contained in the specimen is used.

  The first effect of the present invention is that, even when a gene amplification / inspection is performed using a reagent containing a fluorescent mixed probe in which a plurality of fluorescently labeled probes of the same color are mixed, a plateau emission value of the fluorescent intensity This has the effect of identifying whether the gene was present.

  The second effect is that even when a plurality of genes are amplified in one reaction container, the number of genes that can be amplified at one time is not limited by the number of colors of the fluorescent label.

  The third effect is that, when handling multiple genetic item tests with multiple specimens, the process of dispensing each genetic item into the reaction container can be reduced, and even when there are few reaction container installation locations, the test can be performed at once. By doing so, the inspection time can be shortened.

  Next, the apparatus configuration of the present invention will be described.

  As shown in FIG. 8, the overall configuration of the genetic test apparatus of the present invention is as follows. (1) A user operation unit having an input screen 109 for inputting test information by the user and an output screen 110 for checking the test result and calibration result by the user 180, (2) a data analysis unit 111 that analyzes fluorescence intensity data from the photodetector, (3) a probe information registration unit 112 that registers probe and reagent information and calibration information, and (4) a used reaction container and A sample disposal unit 100 for discarding a waste liquid containing a sample, a detection unit 101 for detecting fluorescence of a fluorescent label contained in the reaction vessel, a gene amplification unit 102 for amplifying a gene contained in the sample in the reaction vessel, and a sample in the reaction vessel And a sample / probe / reagent adjustment unit 103 for dispensing fluorescent probes and reagents necessary for gene amplification / testing, and a sample. A specimen placement unit 104 to be stored, a probe / reagent placement / storage unit 105 for storing fluorescent mixed probes and reagents necessary for gene amplification / testing, and a specimen to the specimen / probe / reagent adjustment unit 103 using a robot arm. Sample transport section Y-axis 107 to be moved, fluorescence mixed probe and reagent necessary for gene testing / amplification are transported to the sample / probe / reagent adjustment section 103 using a robot arm. Probe / reagent / reaction container transport section The X-axis 108, the reaction vessel installation unit 113 for installing the reaction vessel, and the like are included in the analysis unit 181. These operations are managed by a computer such as the PC unit 106.

  However, the sample disposal unit 100, the sample / probe / reagent adjustment unit 103, the sample installation unit 104, the probe / reagent installation / storage unit 105, the sample transport unit Y-axis 107, the probe / reagent / reaction container of the analysis unit 181 When the functions performed by the transport unit X-axis 108 and the reaction vessel installation unit 113 can be replaced manually, the analysis unit 181 can be configured by the detection unit 101 and the gene amplification unit 102 alone.

  FIG. 2 shows a first form of the gene amplification unit 102 and the detection unit 101 of the genetic test apparatus necessary for the present invention.

  The gene amplification unit 102 has a plurality of temperature control units 4 for gene amplification in the carousel 5, and a reaction vessel 3 whose temperature is controlled by each temperature control unit 4 can be installed. It is assumed that each reaction vessel 3 can be installed at a position away from the rotation shaft 7 by a certain distance. The carousel 5 is rotatable in the direction of reference numeral 2.

  It is desirable that the temperature control unit 4 of the gene amplification unit 102 be installed so that the temperature of each reaction container 3 is evenly controlled.

  The temperature control unit 4 of the gene amplification unit 102 is installed for each reaction vessel 3. Here, the temperature control unit 4 is provided for each reaction vessel 3, but the temperature control unit 4 is not limited thereto, and the temperature control of the plurality of reaction vessels 3 may be performed by one temperature control unit 4.

  The detector of the genetic test apparatus is the first photodetector 1 that can recognize the fluorescence intensity in an analog or digital manner. The first photodetector 1 is preferably a photodetector such as a photomultiplier (PMT). The first photodetector 1 is installed so that the light receiving surface of the detector is parallel to an angle at which the fluorescence emitted from the reaction vessel 3 can be detected most efficiently, for example, one surface of the reaction vessel 3. Is desirable.

  The first photodetector 1 has a distance from the reaction vessel 3 to the first photodetector 1 when the carousel 5 rotates and the reaction vessel 3 reaches the position detected by the first photodetector 1. It is desirable to install them so as to be equal to all the reaction vessels 3.

  As shown in FIG. 5, the optical axis of the first photodetector indicates the direction from the reaction vessel 3 toward the detector, and the first fluorescence generated by the excitation light of the first excitation light source 6 is shown. It is assumed that the optical axis 14 (hereinafter abbreviated as the first fluorescent optical axis 14).

  The optical axis 12 of the excitation light emitted from the first excitation light source 6 of the genetic testing device (hereinafter abbreviated as the optical axis 12 of the first excitation light) is the first fluorescence light as shown in FIG. Excitation light is irradiated from an optical axis direction different from the axis 14, and the first excitation light source 6 is installed at a position below the carousel 5 by placing the reaction vessel 3 between the rotation axis 7 and the first photodetector 1. When the reaction vessel 3 reaches the position where the carousel 5 rotates and the first photodetector 1 detects, all the reaction vessels 3 are installed so that they can receive the same amount of excitation light. It is desirable.

  The optical axis 12 of the first excitation light of the genetic testing device can be irradiated with the excitation light from the same optical axis direction as the optical axis 14 of the first fluorescence. In this case, it is necessary to add a mechanism for distinguishing excitation light from fluorescence by using optical components such as a half mirror, a total reflection mirror, a dichroic mirror, a low pass filter, and a band pass filter in combination.

  FIG. 3 shows a second configuration of the gene amplification unit 102 and the detection unit 101 of the genetic test apparatus necessary for the present invention.

  The second form of the genetic test apparatus is a form in which a plurality of first photodetectors are installed around the carousel 5. As shown in FIG. 3, the second mobile phone of the diffusion inspection apparatus has the first photodetector 1 and the second photodetector 20 around the carousel 5. Here, the number of photodetectors is two, that is, the first photodetector 1 and the second photodetector 20, but more detectors can be installed.

  It is assumed that the first photodetector 1 and the second photodetector 20 of the second form of the genetic test apparatus detect fluorescence of different wavelengths by the detectors.

  As shown in FIG. 6, the excitation light source of the second form of the genetic testing apparatus is assumed to be the first excitation light source 6 and the excitation light 11 that emit excitation light of different wavelengths, and the installation position is the rotation axis 7 and The first excitation light source 6 is installed at a position where the reaction vessel 3 can be irradiated from the lower part of the carousel 5 between the first photodetectors 1, and the reaction vessel 3 is interposed between the rotary shaft 7 and the second photodetector 20. 2nd excitation light source 11 shall be installed in the position which can irradiate from the lower part of carousel 5. FIG. The optical axis 13 of the excitation light emitted from the second excitation light source 11 irradiates excitation light from an optical axis direction different from the second fluorescence optical axis 15 as shown in FIG. The installation position of the light source 11 is a position where the reaction vessel 3 can be irradiated from the lower part of the carousel 5 between the rotating shaft 7 and the second photodetector 20. The carousel 5 rotates and the second photodetector 20 detects it. It is desirable that all reaction containers 3 be installed so that the same amount of excitation light can be received when the reaction containers 3 reach the position where the reaction is performed.

  When the reaction vessel 3 reaches the position detected by the first photodetector 1 when the carousel 5 rotates, or the reaction vessel 3 reaches the position detected by the second photodetector 20 when the carousel 5 rotates. At this time, it is desirable to install all the reaction vessels 3 so as to receive the same amount of excitation light.

  FIG. 4 (front view) shows a third form of the gene amplification unit 102 and the detection unit 101 of the genetic test apparatus necessary for the present invention.

  The gene amplification unit 102 includes a plurality of temperature control units 4 for gene amplification in an incubator 10 that does not rotate, and a reaction vessel 3 that is temperature-controlled by each temperature control unit 4 can be installed. Each reaction container 3 can be installed at equal intervals.

  It is desirable that the temperature control unit 4 of the gene amplification unit 102 be installed so that the temperature of each reaction container 3 is evenly controlled.

  The temperature control unit 4 of the gene amplification unit 102 performs temperature control of the plurality of reaction vessels 3 with one temperature control unit 4. Here, the temperature control of the plurality of reaction vessels 3 is performed by one temperature control unit 4, but the temperature control unit 4 can be installed for each reaction vessel 3.

  Fig. 4 (side view) shows the detector of the genetic testing device.

  The detector is capable of recognizing the fluorescence intensity in an analog or digital manner, and has a function of spatially recognizing the position of each reaction container 3 and a function of identifying the light intensity of each reaction container 3. The light detector 40 is required. Here, the third photodetector 40 is, for example, a CCD camera. The third photodetector 40 has an angle at which the fluorescence emitted from the reaction vessel 3 can be detected most efficiently, for example, one surface of the reaction vessel 3 is parallel to the light receiving surface of the third photodetector 40. It is desirable to install as follows.

  In the third photodetector 40, the distance 8 from the reaction vessel 3 to the third photodetector 40 (hereinafter abbreviated as the distance 8 to the photodetector) is different for each reaction vessel 3. The distance 8 to the third light detector 40 has a unique distance for each reaction container 3, and even if time passes after installing the genetic testing device, It is assumed that the distance 8 between the reaction vessel 3 and the third photodetector 40 does not change.

  Further, unlike the first embodiment, the optical axis 12 of the first excitation light in the third form of the genetic test apparatus is directed to the same optical axis direction as the first fluorescent light axis 14 as shown in FIG. The first excitation light source 6 is installed in the lower part of the incubator. However, the distance 9 from each reaction vessel 3 to the first excitation light source 6 (hereinafter, abbreviated as the distance 9 to the excitation light source) has a unique distance for each reaction vessel 3, and the genetic test apparatus described above. It is assumed that the distance 9 between each reaction vessel 3 and the excitation light source does not change even if time elapses after the installation location of the above and the genetic testing device are installed.

  Next, an implementation procedure, which is a characteristic part of the present invention, will be described.

  In the above gene testing apparatus, the test procedure for identifying a plurality of nucleic acid sequences using a fluorescent mixed probe of the same color fluorescent label is as follows: (1) a fluorescent mixed probe mixed with a fluorescent labeled fluorescent probe emitting the same color of fluorescence (2) Calibration of a fluorescent mixed probe, (3) A procedure for measuring a plurality of gene items.

  (1) Preparation of a fluorescent mixed probe in which fluorescent probes of the same color that emit fluorescence are mixed so that the plateau emission value of each fluorescent probe has a special ratio as shown in FIG. To mix. Specifically, first, the user or the probe creator selects a plurality of fluorescent probes having different target genes from each other with respect to the fluorescent probes emitting the same color fluorescence with the same color excitation light, step S1, the selected fluorescent probe Step S2 for selecting the plateau emission value ratio of the first, and then when all the selected fluorescent probes have reached the plateau emission value, step S3 for determining the upper limit value of the fluorescence emission value, and setting the upper limit value of the fluorescence emission value as the emission value. Step S4, which is defined as the maximum value of the ratio, and then, based on the fact that the maximum value of the emission value ratio is defined as the upper limit value of the fluorescence emission value, the fluorescence emission value indicated by the plateau emission value ratio of each fluorescent probe is determined. Step S5 for determining, and then, using a sample in which a gene that reacts with each fluorescent probe is sufficiently amplified, reacting each fluorescent probe with the sample to measure the fluorescence emission value. In step S6, the amount or concentration of the fluorescent probe is adjusted until the determined fluorescent emission value of each fluorescent probe is reached. Next, as a mixing operation of the fluorescent probes, the emission ratio of the adjusted fluorescent probes does not change. It is desirable to create a fluorescent probe according to the creation flow of step S7 of mixing.

  However, the creation flow in FIG. 9 is an example, and if the plateau emission values of the fluorescent probes included in the fluorescent mixed probe are finally mixed at a special ratio, the method and procedure for creating the fluorescent mixed probe are not questioned. Make it not exist.

  For example, in the case of fluorescent probes A, B, and C that react specifically with three types of genes A, B, and C and have fluorescent labels of the same color, the plateau emission values of fluorescent probes A, B, and C are special. Fluorescent probes are mixed so that the ratio is 4: 3: 2.

(2) The calibration of the fluorescent mixed probe is performed by, for example, calibrating the relationship between the amplified gene item and the emission value of the fluorescent mixed probe as in the calibration flow shown in FIG. Register in the information registration unit 112.
Specifically, first, a user or a probe creator selects a fluorescent mixed probe to be calibrated in step S8, and then the apparatus reads information on the selected fluorescent mixed probe and uses excitation light and light detection. Step S9 for determining the vessel, and then the user prepares at least a combination of genetic test items that can be tested with the selected fluorescent mixed probe for samples (calibration samples) that contain the known genes (calibration samples). Step S10, next, the apparatus dispenses the calibration sample, the fluorescent mixed probe, and the reagent necessary for the analysis into one reaction container. Next, the apparatus amplifies the gene of the corresponding reaction container. Step S12, and then the apparatus irradiates the corresponding reaction vessel with excitation light and detects the fluorescence emission value from the detector. 13. Next, the apparatus calibrates the fluorescent mixed probe according to the calibration flow of step S14 in which the relationship between the gene item included in the calibration sample and the fluorescent emission value of the fluorescent mixed probe is registered as calibration information. Is desirable.

  However, the calibration flow in FIG. 10 is an example. If the difference between the plateau luminescence value of each fluorescent probe at the time of preparing the fluorescent mixed probe and the plateau luminescence value of each probe when actually used in the apparatus can be corrected, calibration is performed. It doesn't matter how.

  The calibration information includes the relationship between the amplified gene item and the plateau luminescence value of the fluorescent mixed probe. As shown in FIG. 1, the information that can identify the gene type contained in the specimen from the plateau luminescent value of the fluorescent mixed probe. It is.

  In addition, the calibration information preferably includes a result of calculating a ratio between plateau luminescence values (plateau luminescence value ratio) for each amplified gene item.

  (3) For measurement of a plurality of gene items, for example, in a measurement flow as shown in FIG. 11, a plurality of corresponding enzyme reagents necessary for gene amplification, a corresponding fluorescent mixed probe, and a sample are put in one reaction container. After dispensing, gene amplification is performed until the fluorescence value of the fluorescence mixed probe reaches a plateau. Specifically, first, the user selects a plurality of gene items to be examined from the operation screen.

  Step S15, next, the user selects a fluorescent mixed probe capable of inspecting the gene item to be inspected from the operation screen. Step S16. Next, the apparatus reads the information of the fluorescent mixed probe and uses the excitation light and the detector. Step S17, and then the apparatus dispenses and adjusts all the reagents, fluorescent mixed probes, and specimens necessary for the test into the reaction container, and then the apparatus selects the gene in the corresponding reaction container. Amplifying step S19, and then the apparatus irradiates the corresponding reaction vessel with excitation light and detects the fluorescence emission value from the detector. Next, the apparatus detects the gene item and plateau of the registered fluorescent mixed probe. The gene item corresponding to the acquired fluorescence emission value is identified from the relationship of the emission value, and the measurement process in step S21 for determining the gene contained in the specimen of the reaction container Perform measurements in the over.

  At this time, it is assumed that the time for amplifying the gene is predetermined. The timing for measuring the fluorescence value is either the method of measuring multiple times during gene amplification, such as real-time PCR, or the method of measuring the fluorescence value after performing gene amplification in a time that clearly reaches the plateau luminescence value. Be good.

  Here, when acquiring a fluorescence value, the reaction vessel is irradiated with excitation light from an excitation light source, and fluorescence is acquired from a photodetector. The fluorescence acquired by the photodetector is converted as a plateau emission value by the data analysis unit of the computer.

  Here, if there is an error in the plateau luminescence value of the specimen, the amplified gene item and the plateau luminescence value of the fluorescence mixed probe are obtained from the calibration information of the corresponding fluorescence mixed probe registered in the probe information registration unit 112. The relationship is read, the plateau luminescence value detected from the first photodetector 1, the closest plateau luminescence value registered as calibration information and the corresponding plateau luminescence value ratio are searched, and they are included in the sample in the reaction container. Identify the relevant gene.

  Next, the effects of the present invention will be described.

  When three kinds of fluorescent probes with the same color are mixed in the above procedure, seven combinations of genetic items can be examined in one reaction vessel at a time as shown in FIG.

  When the fluorescently labeled probes A, B, and C of the same color are mixed so that the ratio of the plateau emission values of the fluorescent values is 4: 3: 2, as in the above procedure, The combination of the plateau luminescence value ratio and the gene contained in the specimen is as follows.

  Here, when the sample contains gene A and gene A is amplified, the fluorescent label a is present, when the sample contains gene B and gene B is amplified, the fluorescent label b, and the sample contains gene C. Thus, when gene C is amplified, fluorescent label c emits light specifically. (Here, the reaction container contains all reagents and specimens such as primers and enzymes necessary for gene amplification and testing in addition to the fluorescent mixed probe in which the fluorescently labeled probes A, B, and C are mixed. (This is omitted.)

  When the presence of genes A, B, and C contained in a sample in one reaction container is examined using a fluorescent mixed probe that is a mixture of fluorescently labeled probes A, B, and C, the plateau emission ratio of the photodetector Can be identified as gene A, if the plateau luminescence value ratio is 3, gene B can be identified, and if the plateau luminescence value ratio is 2, gene C can be identified.

Here, the plateau luminescence value ratio is 9 when the fluorescent labels a, b, and c all emit plateau light, excluding the background light of the photodetector, and 4 when the plateau light is emitted only by the fluorescent label a. It is expressed as
The fluorescent mixed probe can identify a gene even when a biological sample in one reaction vessel contains a plurality of genes. For example, when this mixed reagent is used, genes A and B are included if the plateau luminescence value ratio is 7, and A if the plateau luminescence value ratio is 6, and B if the plateau luminescence value ratio is 5. Can be identified as contained in the reaction vessel (Figure 1).

The first effect of the present invention is that when a fluorescently labeled probe of the same color is used for a sample that is conventionally desired to test a plurality of genetic test items, it cannot be tested at once in one reaction container. There was a problem that a step of dispensing the sample into the reaction container by the number of the plurality of genetic test items had to be added, but the fluorescently labeled probe of the same color was used by the first means of the present invention. Even in this case, the effect of testing a plurality of genetic test items at a time in one reaction container can be expected.
According to the first effect, even when a fluorescently labeled probe of the same color is used, there is a third effect that can eliminate the step of dispensing the sample into the reaction container by the number of genetic test items. I can expect.

  The amount of excitation light irradiated to each fluorescent probe mixed in the fluorescent mixed probe is a short time and a weak excitation light intensity that does not easily cause a phenomenon that the fluorescent intensity is weakened (bleaching) when the fluorescent label is continuously excited. Moreover, it is desirable that the excitation light intensity emits a certain fluorescence intensity that can be distinguished from the background light when fluorescence is acquired by the photodetector.

  The amount of each fluorescent probe mixed in the fluorescent mixed probe reaches the plateau luminescence value when each fluorescent probe reacts with the corresponding gene and the plateau luminescent value of the fluorescent mixed probe reaches the plateau luminescent value. It is necessary to adjust the amount so that it can be distinguished depending on the type of the material.

  It is necessary to adjust the amount of the fluorescent mixed probe and the amount of excitation light so that the detection limit of the photodetector is not exceeded when all the fluorescent probes mixed in the fluorescent mixed probe reach the plateau emission value. .

  The excitation light source needs to have the same excitation light intensity at the time of specimen measurement and at the time of calibration.

  When performing gene amplification of a sample in the gene amplification unit 102, it is necessary that the enzyme reagent or the fluorescently labeled probe necessary for gene amplification has a gene amplification mechanism that is difficult to deactivate. Specifically, when a gene is amplified in the PCR amplification step, it is necessary to have a mechanism that does not reach the temperature at which the fluorescently labeled probe and reagent are deactivated when high and low temperatures are repeated.

  As for the registered calibration information, it is desirable that the fluorescence mixture probe ID 1001, the inspection item 1002, the probe emission value information 1003, and the like can be displayed as in the fluorescence mixture probe information screen 1000 of FIG. It is desirable that the probe emission value information can display the inspection item, the mixed fluorescent probe ID, the excitation light wavelength to be used, the fluorescence wavelength, the plateau emission value of the fluorescent probe alone, the plateau emission value ratio, and the like.

  When a genetic test is performed using a fluorescent mixed probe in which fluorescent probes of the same color are labeled, it is desirable that the test result can be displayed on the output screen as in the test result screen 1004 of FIG. . It is desirable that the inspection result can display the inspection reference number 1005, the reaction container ID 1006, the fluorescent mixed probe ID 1007 used, and the inspection result 1008. In the inspection result, it is desirable to be able to display the inspection item, determination, fluorescent mixed probe ID used, measured luminescence value, and measured luminescence value ratio.

  The genetic test apparatus can also use a plurality of fluorescent mixed probes using different fluorescent labels for one reaction vessel in the fluorescent mixed probe. As shown in Fig. 3 and Fig. 6, multiple excitation light sources with different excitation light and the first photodetector 1 are installed, and only one type of fluorescent-labeled fluorescent probe corresponding to each excitation light source is dispensed into the reaction vessel. It is desirable that it is possible.

  The above-mentioned genetic test device is a fluorescent mixed probe in which fluorescent probes of the same color are mixed in a special ratio. The fluorescent mixed probes of different colors are mixed in advance, and information is registered in the device as a fluorescent blend probe. It shall be possible to keep.

  In addition, calibration is performed using a calibration sample so that the type of gene contained in the sample can be identified from the plateau emission value of the fluorescent mixed probe, and the calibration information obtained by calibration is used as the probe information of the device. It is desirable to be able to register with the registration unit 112.

  Calibration information and fluorescence blend probe information registered in the probe information registration unit 112 include inspection items, mixed fluorescence probe IDs, excitation light wavelength, fluorescence wavelength, plateau emission value, plateau emission value ratio, and position number of the detector to be detected. It is desirable to register.

  Specifically, it is desirable to display the fluorescence blend probe information such as the fluorescence blend probe ID 1010, the inspection item 1002, and the blend probe emission value information 1011 as in the fluorescence blend probe information screen 1009 in FIG. Blend probe luminescence value information can display inspection items, mixed fluorescent probe ID, excitation light wavelength to be used, fluorescence wavelength, plateau luminescence value of single fluorescent probe, plateau luminescence value ratio, detector position to be detected, etc. It is desirable to do.

  In the case of a genetic testing apparatus in which a plurality of excitation light sources of different excitation light and the first photodetector 1 are installed as described above, light is emitted at the wavelength of the excitation light source of the fluorescent mixed probe registered in the probe information registration unit 112 of the apparatus Only the fluorescence value is analyzed by the data analysis unit.

  Detector that detects fluorescence values stronger than the background when gene amplification and fluorescence values are measured by dispensing the above fluorescent blend probe, enzyme reagents necessary for gene amplification and testing, and specimens into a single reaction vessel From the position, the corresponding fluorescent mixed probe is identified based on the registered calibration information, and the corresponding fluorescent probe is identified from the fluorescence value, and it is determined which gene is included in the specimen.

  At this time, when the fluorescence mixed probe is excited with a plurality of excitation light wavelengths and the fluorescence has a plurality of wavelengths, it is desirable to cut the fluorescence into one type of wavelength with an optical filter or the like.

  The second effect of the present invention is that the number of genes that can be amplified at one time is not limited by the number of colors of the fluorescent label even when a plurality of genes are amplified in one reaction vessel.

  For example, if it is possible to identify, for example, 3 genes, the number of genes that can be amplified at one time in one reaction vessel can be increased by using fluorescent labels of different colors. The number of genes that can be generated is limited by the number of colors of the fluorescent label, such as the reproducibility of the genetic test apparatus and the resolution of the photodetector.

  The third effect of the present invention is that when handling a plurality of genetic item tests with a plurality of specimens, it is possible to reduce the process of dispensing each gene item into a reaction container, even when the number of reaction container installation locations is small, Inspection time can be shortened by inspecting at a time.

  In the fluorescent mixed probe and the fluorescent blend probe, it is also possible to use a fluorescent probe that specifically labels a plurality of genes as the fluorescent probe to be mixed and blended. It is desirable that these fluorescent mixed probes can be registered in the probe information registration unit 112. This is because when a fluorescent probe that specifically labels multiple genes reaches a plateau luminescence value, instead of identifying the corresponding gene, the corresponding multiple genes are regarded as one gene group, and the gene group is used for screening purposes. Is used to determine whether any of the samples is included in the specimen.

  Further, in the fluorescent mixed probe, each fluorescent probe is mixed so that the ratio of the plateau emission values becomes a special ratio. Therefore, the number of fluorescent probes that emit light at each plateau emission value is usually one. However, the number of fluorescent probes that emit light at each plateau emission value can be plural. In this case, fluorescent probes that emit light at each plateau emission value are grouped into one group, and one fluorescent probe group labels a plurality of corresponding genes as one group. For example, if genes A, D, and E are set as one gene group A, fluorescent probes a, d, and e that are fluorescently labeled specifically for gene group A are set as one fluorescent probe group a. At this time, assuming that the fluorescent probes that react with gene B and gene C are fluorescent probe b and fluorescent probe c, the plateau emission values of fluorescent probe group a, fluorescent probe b, and fluorescent probe c are 4: 3: 2. Mix. At this time, the fluorescent probes a, d, e included in the fluorescent probe group a are desirably mixed so that the plateau emission value ratio is 1: 1: 1. Therefore, if the gene d is contained in the specimen, the plateau luminescence value ratio shows a luminescence value of 4/3, and even if the gene e is contained, the plateau luminescence value ratio shows a luminescence value of 4/3. Furthermore, if the sample contains genes d and e, the plateau emission ratio is 8/3, and if the sample contains genes a, d and e, the plateau emission ratio Indicates a light emission value of 4. It is desirable to be able to register the information of the fluorescent mixed probe including these fluorescent probe groups and the calibration information in the probe information registration unit 112 of the apparatus.

  The genetic testing device uses a fluorescent probe that specifically labels the plurality of genes or a fluorescent mixed probe including a fluorescent probe group, and when fluorescence is acquired by a detector, a plurality of fluorescence is detected from a plateau emission value of the fluorescence. It is possible to identify the corresponding gene group of the fluorescent probe or fluorescent probe group that specifically labels the gene of, and to express that the sample contains any of the genes included in the gene group To. This function does not identify a gene, but is used for the purpose of screening the gene.

  It is desirable that the gene testing apparatus is equipped with a real-time PCR method, and creates a standard for judging that the increase in fluorescence value due to gene amplification has reached a plateau luminescence value. Specifically, using the property that the fluorescence intensity increases each time the gene is amplified, the fluorescence intensity is first-order differential, second-order derivative, third-order derivative according to the PCR cycle or time, and the fluorescence intensity increase amount, or When either the fluorescence intensity increase degree or the differential value of the fluorescence intensity increase degree is monitored by a computer and the monitoring data reaches a certain value, for example, when the fluorescence intensity increase degree becomes 0, the plateau emission value has been reached. It is desirable to add a mechanism for determining

1 First photodetector
3 Reaction vessel
4 Temperature controller
5 Carousel
6 First excitation light source
7 Rotating axis
8 Distance from reaction vessel to third photodetector
9 Distance from reaction vessel to first excitation light source
10 Incubator
11 Second excitation light source
12 Optical axis of the excitation light emitted from the first excitation light source (optical axis of the first excitation light)
13 Optical axis of excitation light emitted from the second excitation light source
14 Optical axis of the first fluorescence generated by the excitation light of the first excitation light source
15 Optical axis of the second fluorescence generated by the excitation light of the second excitation light
20 Second photodetector
40 Third photo detector
100 specimen disposal department
101 detector
102 Gene amplification section
103 Sample / probe / reagent adjustment unit
104 Sample setting section
105 Probe / Reagent Installation / Storage Section
106 PC
107 Sample transport Y-axis
108 Probe / Reagent / Reaction Container Transport Unit X-Axis
109 Input screen
110 Output screen
111 Data analysis section
112 Probe information registration section
113 Reaction vessel installation
180 User control
181 Analysis Department
1000 Fluorescence mixed probe information screen
1001 fluorescent mixed probe ID
1002 Inspection items
1003 Probe emission information
1004 Inspection result screen
1005 Inspection reference number
1006 Reaction vessel ID
1007 Fluorescent mixed probe ID used
1008 Test result
1009 Fluorescence blend probe information screen
1010 Fluorescent blend probe ID
1011 Blend probe emission information

Claims (9)

A reagent for detecting fluorescence by amplifying a gene ;
A reagent container containing the reagent;
A rack for the reagent container;
A genetic test apparatus having a fluorescence detection unit comprising an excitation light source and a photodetector,
The reagent contained in the reagent container is for determining whether a given gene is present,
The reagent includes a fluorescent mixed probe,
The fluorescent mixed probe emits the same color fluorescence with the same color excitation light, a plurality of fluorescent probes are mixed,
Each fluorescent probe is a probe that emits fluorescence corresponding to a specific gene,
A genetic test apparatus characterized in that each fluorescent probe has a different plateau emission value of fluorescence intensity at the time of genetic test. In claim 1,
There are three or more fluorescent probes,
At least any two fluorescent probes are probes that emit fluorescence corresponding to the same gene. In claim 1,
Any one of the fluorescent probes is a probe that emits fluorescence corresponding to a plurality of genes. In claim 1,
The reagent includes three or more fluorescent mixed probes,
Among them, each fluorescent probe included in at least one fluorescent mixed probe is different from each fluorescent probe included in another fluorescent mixed probe in a combination of excitation light and fluorescent color. In claim 1,
A plurality of fluorescence detection units, the light wavelength used in the excitation light source of at least one of the fluorescence detection units, and the light wavelength detectable by the photodetector are the light wavelengths used in the excitation light sources of other fluorescence detection units, And the genetic test | inspection apparatus different from the light wavelength which can be detected with a photodetector. In claim 1,
Gene items that can be tested with reagents, mixed fluorescent probe IDs contained in the reagents, plateau luminescence values of each fluorescent probe of the fluorescent mixed probe ID, relationship between the types of genes contained in the specimen and the luminescence values of the detected fluorescence, A genetic test apparatus comprising a storage unit in which is stored. In claim 6,
A genetic test apparatus characterized in that the storage unit registers a relationship with at least one of an excitation wavelength and a fluorescence wavelength with a fluorescence mixed probe. A gene testing reagent used for genetic testing to detect fluorescence by amplifying a gene,
Including a fluorescent mixed probe,
The fluorescent mixed probe emits the same color fluorescence with the same color excitation light, a plurality of fluorescent probes are mixed,
Each fluorescent probe is a probe that emits fluorescence corresponding to a specific gene,
A reagent for genetic testing, wherein each fluorescent probe has a different plateau emission value of fluorescence intensity at the time of genetic testing. A genetic test method for detecting fluorescence by amplifying a gene,
Whether or not a given gene is present is determined using a reagent,
The reagent includes a fluorescent mixed probe,
The fluorescent mixed probe emits the same color fluorescence with the same color excitation light, a plurality of fluorescent probes are mixed,
Each fluorescent probe is a probe that emits fluorescence corresponding to a specific gene,
A genetic testing method, wherein each fluorescent probe has a different plateau emission value of fluorescence intensity at the time of genetic testing.