JP2009284882A - Gene diagnosis system, and method for gene diagnosis - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a gene diagnosis system capable of accurately deciding the presence or absence of a gene even when the amount of a mixed liquid of a reagent and a specimen is changed during examination, and to provide a method for gene diagnosis. <P>SOLUTION: The gene diagnosis system includes a micro-chip having a first detecting part for mixing and reacting a specimen with a reagent, a second detecting part for mixing and reacting a positive control liquid reacting with the reagent to emit fluorescence, with the reagent, and a third detecting part for mixing and reacting a negative control liquid not reacting with the reagent, with the reagent; a fluorescence detecting means for irradiating the first detecting part, the second detecting part, and the third detecting part with excitation light, and outputting a signal corresponding to each fluorescence intensity; a reference value computing means for computing a reference value Vb for deciding the presence or absence of the gene contained in the specimen on the basis of a signal Vp and a signal Vn; and a fluorescence deciding means for comparing a signal Vs with the reference value Vb, and deciding the presence or absence of the gene. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a genetic diagnostic system and a genetic diagnostic method.

  In recent years, by making full use of micromachine technology and ultrafine processing technology, devices and means (for example, pumps, valves, flow paths, sensors, etc.) for performing conventional sample preparation, chemical analysis, chemical synthesis, etc. have been miniaturized. A system integrated on a chip has been developed (see, for example, Patent Document 1). This is also called μ-TAS (Micro Total Analysis System), microchip, bioreactor, lab-on-chip, biochip, medical examination / diagnosis field, environment Its application is expected in the field of measurement and agricultural production. In particular, as seen in genetic testing, when complicated processes, skilled procedures, and equipment operations are required, the cost, required sample volume, and time required can be reduced by using μ-TAS (microchip). Can be reduced.

  On the other hand, in a conventional genetic test, after a gene amplification is performed by reacting a mixture of a reagent and a sample, the mixture of the reagent and the sample is irradiated with excitation light, and the absolute value of the fluorescence detection value detected from the mixture is obtained. Based on this, the presence or absence of the gene was determined.

  However, in such a method, if measurement is continued until a significant difference from the fluorescence base value is obtained, it may take 30 minutes to 2 hours. Further, in order to obtain the absolute value of fluorescence detection, it is necessary to obtain a fluorescence base value, but there is a problem that the base value changes with time due to factors such as a change in intensity of the light source.

In order to solve such a problem, the measured fluorescence intensity response curve data is stored in advance as a reference amount of time from the start to the end of the increase in the fluorescence intensity and a reference amount of the fluorescence intensity after the end of the increase in the fluorescence intensity. In comparison with the above data, there has been proposed a method for determining whether or not a sample contains a gene to be determined (see, for example, Patent Document 2).
JP 2004-28589 A JP 2007-328466 A

  In the gene amplification method represented by the PCR method, it is necessary to apply a high temperature thermal cycle to the mixed solution of the reagent and the sample for a long time, but there is no particular problem when the reagent and the sample are reacted in a sealed container. However, when a reagent and a sample are reacted in a flow path having one end opened, such as μ-TAS (microchip), heating the high temperature causes the reagent and the sample to evaporate, reducing the amount of liquid, In some cases, the air inflates, and the liquid mixture of the reagent and the specimen moves from a predetermined position to change the liquid amount. As described above, when the amount of the liquid mixture of the reagent and the sample changes during the examination, the level of the fluorescence intensity detected from the liquid mixture also varies.

  However, in the method disclosed in Patent Document 2, the presence or absence of a gene is determined by comparing the measured fluorescence intensity response curve data with a predetermined reference value stored in advance. If the amount of the sample mixture changes, the correct judgment cannot be made.

  The present invention has been made in view of the above problems, and a genetic diagnostic system and a genetic diagnostic method that can accurately determine the presence or absence of a gene even if the amount of a mixture of a reagent and a sample changes during a test The purpose is to provide.

The object of the present invention can be achieved by the following constitution.
1.
In the genetic diagnosis system that determines the presence or absence of a gene from the fluorescence intensity of the mixture of the sample and reagent in the reaction by mixing the sample and reagent stored in the flow path of the microchip,
A first detection unit that mixes and reacts the specimen and the reagent; a second detection unit that mixes and reacts the positive control liquid that reacts with the reagent and emits fluorescence; and the reagent; A microchip comprising: a third detection unit that mixes and reacts a negative control solution that does not react with the reagent;
Fluorescence detection means for irradiating the first detection unit, the second detection unit, and the third detection unit with excitation light and outputting a signal corresponding to each fluorescence intensity;
Based on the signal Vp of the fluorescence detection unit according to the fluorescence intensity of the second detection unit and the signal Vn of the fluorescence detection unit according to the fluorescence intensity of the third detection unit, the gene contained in the specimen A reference value calculating means for calculating a reference value Vb for determining presence or absence;
Fluorescence determination means for comparing the signal Vs of the fluorescence detection means according to the fluorescence intensity of the first detection unit with the reference value Vb to determine the presence or absence of a gene;
A genetic diagnosis system characterized by comprising:
2.
The reference value calculation means includes
2. The gene diagnosis system according to 1, wherein the reference value Vb is calculated based on the following equation (1).
Vb = C × (Vp−Vn) + Vn (1)
However, C is a constant of 0 <C <1, Vp is a signal of the fluorescence detection means according to the fluorescence intensity of the second detection unit, Vn is the fluorescence detection according to the fluorescence intensity of the third detection unit Means signal.
3.
In a genetic diagnosis method for determining the presence or absence of a gene from the fluorescence intensity of a mixture of a sample and a reagent in a reaction by mixing a sample and a reagent,
A first fluorescence intensity obtained by mixing and reacting the specimen and the reagent; a second fluorescence intensity obtained by mixing and reacting the reagent with a positive control solution that reacts with the reagent and emits fluorescence; A fluorescence detection step for detecting a third fluorescence intensity obtained by mixing and reacting the negative control solution that does not react with the reagent and the reagent;
A reference value calculating step for calculating a reference value Vb for determining the presence or absence of a gene contained in the specimen based on the second fluorescence intensity and the third fluorescence intensity;
A fluorescence determination step of comparing the first fluorescence intensity with the reference value Vb to determine the presence or absence of a gene;
A genetic diagnosis method characterized by comprising:
4).
The reference value calculating step includes
4. The gene diagnosis method according to 3, wherein the reference value Vb is calculated based on the following equation (1).
Vb = C × (Vp−Vn) + Vn (1)
Here, C is a constant of 0 <C <1, Vp is a signal corresponding to the fluorescence intensity of the second fluorescence intensity, and Vn is a signal corresponding to the fluorescence intensity of the third fluorescence intensity.

  According to the present invention, based on the fluorescence intensity Vp obtained by mixing and reacting the positive control solution and the reagent, and the fluorescence intensity Vn obtained by mixing and reacting the negative control solution and the reagent, the gene contained in the specimen is analyzed. A reference value Vb for determining the presence or absence is calculated, and the presence or absence of the gene is determined. By doing so, it is possible to provide a genetic diagnostic system and a genetic diagnostic method that can accurately determine the presence or absence of a gene even if the amount of a mixture of a reagent and a sample changes during a test.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings.

  FIG. 1 is an external view of a genetic diagnosis system 80 according to an embodiment of the present invention.

  The genetic diagnosis system 80 includes a testing device 82 and the microchip 1. The inspection device 82 is a device that automatically detects a reaction between a sample and a reagent previously injected into the microchip 1 and displays the result on the display unit 84.

  The inspection device 82 has an insertion port 83, and the microchip 1 is inserted into the insertion port 83 and set inside the inspection device 82. The insertion port 83 is sufficiently higher than the thickness of the microchip 1 so as not to contact the insertion port 83 when the microchip 1 is inserted. Reference numeral 85 denotes a memory card slot, 86 denotes a print output port, 87 denotes an operation panel, 88 denotes an input / output terminal, and 89 denotes a power switch.

  The person in charge of inspection inserts the microchip 1 in the direction of the arrow in FIG. 1 and operates the operation panel 87 to start the inspection. Inside the inspection device 82, the reaction in the microchip 1 is automatically inspected, and when the inspection is completed, the result is displayed on the display unit 84 composed of a liquid crystal panel or the like. The inspection result can be output from the print output port 86 or stored in a memory card inserted into the memory card slot 85 by operating the operation panel 87. Further, data can be stored in the personal computer or the like from the external input / output terminal 88 using, for example, a LAN cable.

  The inspection person takes out the microchip 1 from the insertion port 83 after the inspection is completed.

  Next, an example of the microchip 1 of the present invention will be described with reference to FIG.

  FIG. 2 is an external view of the microchip 1 according to the embodiment of the present invention.

  As shown in the side view of the microchip 1 in FIG. 2A, the microchip 1 includes a groove forming substrate 108 and a covering substrate 109 that covers the groove forming substrate 108. The substrate material of the groove forming substrate 108 and the covering substrate 109 is not particularly limited, but at least the portion covering the flow path for optically detecting the reaction result needs to be made of a transparent member such as glass or resin. .

  FIG. 2B is a plan view of the microchip 1, and illustrates the grooves of the groove forming substrate 108 that can be seen through the transparent coated substrate 109. The flow path 250 is formed by covering the grooves of the groove forming substrate 108 with the covering substrate 109. The microchip 1 is provided with minute groove-like channels 250 (microchannels) and functional parts (channel elements) for performing inspections, sample processing, and the like in an appropriate manner according to the application. Has been.

  The flow path is formed on the order of micrometers, and for example, the width is several μm to several hundred μm, preferably 10 to 200 μm, and the depth is about 25 to 500 μm, preferably 25 to 250 μm.

  In the present embodiment, a microchip 1 used for a process of performing amplification and detection of a specific gene will be described as an example.

  The inlets 110a, 110b, and 110c in FIG. 2B are openings formed in the coating substrate 109 communicating with the flow path inside the microchip 1, and the driving liquid is injected from each inlet 110 into the flow path 250. Drives internal specimens and reagents. In the microchip 1 of the present embodiment, the configuration of the flow path starting from the injection port 110a and the configuration of the flow path starting from the injection port 110b are completely the same as shown in FIG. This is called a c channel. Further, each component is distinguished by adding a, b, and c.

  Reference numeral 121a denotes a specimen storage section that stores a specimen, 121b denotes a positive control liquid storage section that stores a positive control liquid that reacts with a reagent and emits fluorescence, and 121c denotes a negative control liquid storage section that stores a negative control liquid that does not react with the reagent. It is.

  The specimen storage unit 121a, the positive control liquid storage unit 121b, and the negative control liquid storage unit 121c each have a deeper groove than the other channels in order to store a predetermined amount of liquid. In the present embodiment, it is assumed that a specimen is stored in advance in the specimen storage part 121a, a positive control liquid is stored in the positive control liquid storage part 121b, and a negative control liquid is stored in the negative control liquid storage part 121c. .

  120a, 120b, and 120c are reagent storage units for storing reagents. In the present embodiment, description will be made assuming that the same reagent is stored in advance in the reagent storage unit 120a, the reagent storage unit 120b, and the reagent storage unit 120c.

  When driving liquid is injected into the flow paths 250a, 250b, and 250c from the inlets 110a, 110b, and 110c, the liquids stored in the specimen storage unit 121a, the positive control liquid storage unit 121b, and the negative control liquid storage unit 121c are pushed out. Injected into the reagent storage units 120a, 120b, and 120c downstream.

  Further downstream of the reagent storage unit 120a, the reagent storage unit 120b, and the reagent storage unit 120c, a mixing unit 130a, a mixing unit 130b, a mixing unit 130c, a mixing unit 131a, a mixing unit 131b, and a mixing unit 131c are provided. The specimen and the reagent that have flowed through the flow path 250aa are mixed by the mixing unit 130a and the mixing unit 131a. Similarly, the positive control solution and the reagent flowing through the flow channel 250ba are mixed by the mixing unit 130b and the mixing unit 131b, and the negative control solution and the reagent flowing through the flow channel 250ca are mixed by the mixing unit 130c and the mixing unit 131c.

  The sample and the reagent mixed by the mixing unit 130a and the mixing unit 131a are injected into the detection unit 111a. Similarly, the positive control solution and the reagent mixed in the mixing unit 130b and the mixing unit 131b are injected into the detection unit 111b, and the negative control solution and the reagent mixed in the mixing unit 130c and the mixing unit 131c are injected into the detection unit 111c.

  As will be described later, the temperature adjustment unit 3 is brought into close contact with the microchip 1 inside the inspection device 82, and the liquid mixture injected into each detection unit 111 is heated or absorbed to a predetermined temperature, so that the inside of the detection unit 111. The liquid is allowed to react for a predetermined time.

  The detection unit 111 is provided to optically detect the fluorescence of the injected mixed liquid, and has a groove deeper than the other flow paths to accommodate a predetermined amount of the mixed liquid.

  The liquid reservoir 141 provided downstream of each detector 111 has a shallower channel than that of the detector 111. When the detectors 111a, 111b, and 111c are filled with a predetermined amount of liquid mixture, the liquid reservoir 141 is provided. The mixed solution also flows into the portions 141a, 141b, and 141c. As will be described later, the presence / absence of the mixed liquid flowing into the liquid reservoirs 141a, 141b, and 141c is detected, and it is determined that the detection units 111a, 111b, and 111c are filled with a predetermined amount of the mixed liquid.

  The discharge ports 150a, 150b, and 150c are openings formed in the groove forming substrate 108 communicating with the flow path. The air in the flow path is pushed out from the discharge ports 150a, 150b, and 150c when the tip of the reagent or mixed solution moves.

  Note that the substrate on which the inlet 110 and the outlet 150 are formed is not particularly limited, and may be formed on either the groove forming substrate 108 or the covering substrate 109. In the description so far, the example in which the driving liquid is injected from the injection ports 110a, 110b, and 110c has been described. However, the reagent or the sample may be moved by injecting or sucking a gas such as air.

  FIG. 3 is a cross-sectional view showing an example of the internal configuration of the inspection apparatus 82 in the embodiment of the present invention.

  The inspection device 82 includes a temperature adjustment unit 3, a detection unit 22, a packing 6, a driving liquid tank 10, a tank coupling needle 13, an electromagnetic valve 26, a pipe 15, a pipe 16, a liquid detection unit 28 and the like. FIG. 3 shows a state in which the microchip 1 is in close contact with the packing 6. FIG. 3 illustrates a cross section of one of the three channels of the microchip 1 described in FIG.

  Hereinafter, an example of the internal configuration of the genetic diagnosis system 80 will be described with reference to FIG.

  The temperature adjustment unit 3 is driven by a driving member (not shown) and is movable in the vertical direction on the paper surface.

  In the initial state, the temperature adjustment unit 3 is raised from the state of FIG. 3 by the thickness of the microchip 1 by a driving member (not shown). Then, the microchip 1 can be inserted and removed in the left-right direction on the paper surface, and the person inspecting inserts the microchip 1 from the insertion port 83 until it comes into contact with a regulating member (not shown). When the microchip 1 is inserted to a predetermined position, the chip detection unit 95 using a photo interrupter or the like detects the microchip 1 and is turned on.

  Next, the temperature adjustment unit 3 and the microchip 1 are lowered by the driving member, and the microchip 1 is brought into close contact with the temperature adjustment unit 3 and the packing 6.

  The temperature adjustment unit 3 is a unit that incorporates a Peltier element, a power supply device, a temperature sensor, and the like and adjusts the surface of the microchip 1 to a predetermined temperature by generating heat or absorbing heat.

  The driving liquid tank 10 is a tank that stores the driving liquid 11 and is disposed above the microchip 1. The driving fluid amount sensor 19 is a sensor that detects the amount of the driving fluid 11 stored in the driving fluid tank 10.

  The driving liquid 11 flows to the lower pipe 15 by the weight of the driving liquid 11 through the tank coupling needle 13 provided in the driving liquid tank 10. An electromagnetic valve 26 is provided between the pipe 15 and the pipe 16, and opens and closes the flow path between the pipe 15 and the pipe 16 by giving an electric signal. The pipe 16 communicates with the inlet 110 through the packing 6.

  When the electromagnetic valve 26 is open, the driving liquid 11 can be injected from the driving liquid tank 10 into the injection port 110 due to the height difference between the driving liquid tank 10 and the microchip 1. When the electromagnetic valve 26 is closed, the injection of the driving liquid 11 into the injection port 110 can be stopped.

  In this embodiment, piping 16a, 16b, 16c is provided in each inlet 110a, 110b, 110c, respectively, and it is comprised so that liquid feeding of each channel can be controlled with three electromagnetic valves 26a, 26b, 26c. Has been.

  In the present embodiment, an example in which liquid feeding control is performed with such an inexpensive and simple configuration that does not use a pump will be described, but the present invention is not limited to such a configuration, and liquid feeding is performed using a pump. It can also be applied to control.

  If a three-way valve is used as the electromagnetic valve 26, the liquid supply of the driving liquid 11 is stopped, and then the three-way valve is connected to the atmosphere side to discharge the driving liquid 11 remaining in the pipe 16 to the outside. It is also possible to move the internal driving liquid 11 and the reagent in the reverse direction.

  The liquid detector 28 is, for example, a photo reflector, and is provided in each channel in order to optically detect the liquid flowing into the liquid reservoirs 141a, 141b, and 141c.

  The detection unit 22 includes a light emitting unit and a light receiving unit (not shown). The detection units 111a, 111b, and 111c are irradiated with light to react with the specimen, and the fluorescence emitted from the reagent is optically separated and received by the light receiving unit. Is configured to do.

  The detection unit 22 outputs an electrical signal corresponding to the fluorescence intensity detected from each detection unit 111. The detection unit 22 is a fluorescence detection means of the present invention.

  FIG. 4 is a circuit block diagram of the inspection apparatus 82 in the embodiment of the present invention.

  The control unit 99 includes a CPU 98 (central processing unit), a RAM 97 (Random Access Memory), a ROM 96 (Read Only Memory), and the like, and reads a program stored in the ROM 96 which is a nonvolatile storage unit to the RAM 97. Each part of the inspection apparatus 82 is centrally controlled according to the program.

  Hereinafter, functional blocks having the same functions as those described so far are denoted by the same reference numerals, and description thereof is omitted.

  The CPU 98 includes a liquid feeding control unit 411, a reference value calculation unit 412, and a fluorescence determination unit 413. The reference value calculation unit is the reference value calculation unit of the present invention, and the fluorescence determination unit 413 is the fluorescence determination unit of the present invention.

  The liquid supply control unit 411 controls the liquid supply to the microchip 1 by opening and closing the electromagnetic valve 26 based on the program.

  The reference value calculation unit 412 calculates a reference value Vb for determining the presence / absence of a gene contained in the sample based on the program. The reference value Vb will be described in detail later.

  The fluorescence determination unit 413 determines the presence or absence of a gene contained in the sample based on the program.

  The power supply unit 500 supplies power to each unit of the inspection apparatus 82.

  Next, a test procedure based on the genetic diagnosis method of the present invention will be described.

  FIG. 5 is a graph illustrating a procedure for determining the presence or absence of a gene based on the reference value Vb calculated in the genetic diagnosis system of the present invention. FIG. 7 is a graph for explaining a conventional procedure for determining the presence or absence of a gene, and will be described below in comparison with the procedure of the present invention shown in FIG.

  5 and 7 is a time axis, and shows a change in the signal V output from the fluorescence detection unit 22 from the start of the examination.

  5 and 7, Vs is a signal of the fluorescence detection means according to the fluorescence intensity of the detection unit 111a, Vp is a signal of the fluorescence detection means according to the fluorescence intensity of the detection unit 111b, and Vn is a detection unit. It is a signal of the fluorescence detection means according to the fluorescence intensity of 111c.

  First, FIG. 5A will be described.

  FIG. 5A shows a change due to the elapsed time of Vp, Vs, and Vn when the amount of liquid stored in the detection unit 111 does not change during the inspection.

  Since the mixture of the positive control solution and the reagent stored in the detection unit 111b reacts immediately and increases the fluorescence intensity, the signal Vp also rapidly increases its output immediately after the start of the test as shown in FIG. Saturates to level. On the other hand, since the mixed liquid of the negative control solution and the reagent stored in the detection unit 111c does not react at all and the fluorescence intensity does not change, the signal Vn is also at a certain level immediately after the start of the test as shown in FIG. is there.

  Since the reference value calculation unit 412 calculates the reference value Vb based on the following equation (1), the calculated reference value Vb indicated by the dotted line in FIG. 5A becomes a constant value after gradually increasing.

Vb = C × (Vp−Vn) + Vn (1)
However, C is a constant of 0 <C <1.

  The signal Vs corresponding to the fluorescence intensity of the mixed liquid of the specimen and the reagent stored in the detection unit 111a increases its output after a while and starts exceeding the level of the reference value Vb as shown in FIG. 5A.

  The fluorescence determination unit 413 compares the signal Vs with the reference value Vb, and determines that the sample contains a gene, that is, is positive when the signal Vs exceeds the reference value Vb. In order to prevent erroneous determination, the fluorescence determination unit 413 performs determination after time t1 when the signal Vp becomes a certain level.

  FIG. 7A shows changes in Vp, Vs, and Vn according to the elapsed time when the liquid stored in the detection unit 111 does not evaporate or move as in FIG. In the conventional genetic diagnosis method, the reference value Vc is a predetermined constant value. Conventionally, the determination is made by comparing the signal Vs with a certain reference value Vc.

  Next, a case where the liquid stored in the detection unit 111 evaporates or moves and decreases will be described with reference to FIGS. 5B and 7B.

  As shown in FIGS. 5B and 7B, the signals Vp and Vn gradually decrease with time as compared to FIGS. 5A and 7A. Further, the increase rate of the signal Vs is also smaller than that in FIGS. 5 (a) and 7 (a).

  This is because the liquid mixture stored in each of the detection unit 111a, the detection unit 111b, and the detection unit 111c decreases due to evaporation or movement. Each detection part 111 and a flow path have the same structure, and it is thought that the liquid volume of the liquid mixture stored in each detection part 111 decreases at the same rate with time. Therefore, the obtained signal Vp, signal Vn, and signal Vs also decrease at the same rate with time.

  As shown in FIG. 7B, in the conventional gene determination method, since the signal Vs does not exceed a certain reference value Vc in such a case, even if the level of the signal Vs is the same as that in FIG. It is not determined that the gene is contained, that is, positive.

  On the other hand, in the present invention, the reference value Vb calculated by the reference value calculation unit 412 based on the equation (1) decreases according to the signal Vp and the signal Vn as shown in FIG. Thus, in the present invention, even if the mixture of the specimen and the reagent decreases with time, the reference value Vb also decreases at the same rate, so that the signal Vs exceeds the reference value Vb as shown in FIG. it can.

  Thus, in the present invention, even if the liquid stored in the detection unit 111 evaporates or moves and decreases, the presence or absence of the gene can be correctly determined.

  FIG. 6 is a flowchart for explaining a test procedure performed by the genetic diagnosis system 80 in the embodiment of the present invention.

  In the flowchart, a procedure after the person in charge of inspection inserts the microchip 1 and operates the operation panel 87 to start the inspection will be described. It is assumed that the temperature adjustment unit 3 is adjusted to a predetermined temperature.

  S10: It is a step which starts liquid feeding.

  The liquid feeding control unit 411 controls the electromagnetic valve 26 to cause the pipe 15 and the pipe 16 to communicate with each other and injects the driving liquid from the inlet 110.

  S11: A step of determining whether or not the liquid has reached the liquid reservoir 141.

  The CPU 98 determines that the liquid mixture has reached the liquid reservoir 141 from the output voltage of the liquid detector 28. When the liquid mixture reaches the liquid reservoir 141, the detection unit 111 is filled with a predetermined amount of liquid mixture.

  When the liquid has not reached the liquid reservoir 141 (step S11; No), the process returns to step S11.

  When the liquid reaches the liquid reservoir 141 (step S11; Yes), the process proceeds to step S12.

  S12: It is a step which stops liquid feeding.

  The liquid feeding control unit 411 controls the electromagnetic valve 26 to close between the pipe 15 and the pipe 16 and stops the liquid feeding. The CPU 98 counts for a predetermined time by an internal timer, and reacts the mixed solution of the sample and the reagent filled in the detection unit 111 during that time to amplify the gene contained in the sample. After a predetermined time has passed, the process proceeds to the next step.

  S13: It is a step which measures a reaction result.

  The CPU 98 causes the light emitting unit of the detection unit 22 to emit light, measures the levels of the signal Vp, the signal Vn, and the signal Vs output from the detection unit 22, and stores the results in the RAM 97.

  S14: This is a step of calculating the reference value Vb.

  The reference value calculation unit 412 calculates the reference value Vb by substituting the values of the signal Vp and the signal Vn stored in the RAM 97 in step S13 into the equation (1). For the constant C, the reference value calculation unit 412 reads a value stored in advance in the RAM 97 or the ROM 96.

  S15: This is a step of determining fluorescence.

  The fluorescence determination unit 413 compares the signal Vs and the reference value Vb to determine the presence or absence of a gene.

  If Vs ≧ Vb (step S15; Vs ≧ Vb), the process proceeds to step S18.

  S18: It is a step which determines with positive.

  The fluorescence determination unit 413 determines positive, stores the result in the RAM 97, and ends the process.

  If Vs <Vb (step S15; Vs <Vb), the process proceeds to step S16.

  When the signal Vs is less than the reference value Vb, the fluorescence determination unit 413 proceeds to step S16.

  S16: This is a step of determining a timeout.

  The fluorescence determination unit 413 determines whether or not the elapsed time from the start of the inspection is a predetermined time or more.

  If it is longer than the predetermined time (step S16; Yes), the process proceeds to step S17.

  If the signal Vs does not become the reference value Vb or more even after the predetermined time has elapsed, the fluorescence determination unit 413 proceeds to step S17.

  S17: It is a step which determines with negative.

  The fluorescence determination unit 413 determines negative, stores the result in the RAM 97, and ends the process.

  If it is less than the predetermined time (step S16; No), the process returns to step S13.

  If the elapsed time from the start of the inspection is less than the predetermined time, the fluorescence determination unit 413 returns to step S13 and continues the process.

  This is the end of the description of the flowchart.

  As described above, according to the present invention, it is possible to provide a gene diagnosis system and a gene diagnosis method that can accurately determine the presence or absence of a gene even if the amount of a mixed solution of a reagent and a sample changes during a test. .

It is an external view of the genetic diagnosis system 80 in the embodiment of the present invention. 1 is an external view of a microchip 1 according to an embodiment of the present invention. It is sectional drawing which shows an example of the internal structure of the test | inspection apparatus 82 in embodiment of this invention. It is a circuit block diagram of the test | inspection apparatus 82 in embodiment of this invention. It is a graph explaining the procedure which determines the presence or absence of a gene based on the reference value Vb calculated in the genetic diagnosis system of this invention. 5 is a flowchart for explaining a test procedure performed by the genetic diagnosis system 80 in the embodiment of the present invention. It is a graph explaining the procedure which determines the presence or absence of the conventional gene.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Microchip 3 Temperature control unit 6 Packing 10 Drive liquid tank 22 Detection unit 26 Electromagnetic valve 28 Liquid detection part 80 Genetic diagnosis system 82 Inspection apparatus 83 Insertion port 84 Display part 110 Inlet 150 Outlet 411 Liquid supply control part 412 Reference value Calculation unit 413 Fluorescence determination unit

Claims (4)

In the genetic diagnosis system that determines the presence or absence of a gene from the fluorescence intensity of the mixture of the sample and reagent in the reaction by mixing the sample and reagent stored in the flow path of the microchip,
A first detection unit that mixes and reacts the specimen and the reagent; a second detection unit that mixes and reacts the positive control liquid that reacts with the reagent and emits fluorescence; and the reagent; A microchip comprising: a third detection unit that mixes and reacts a negative control solution that does not react with the reagent;
Fluorescence detection means for irradiating the first detection unit, the second detection unit, and the third detection unit with excitation light and outputting a signal corresponding to each fluorescence intensity;
Based on the signal Vp of the fluorescence detection unit according to the fluorescence intensity of the second detection unit and the signal Vn of the fluorescence detection unit according to the fluorescence intensity of the third detection unit, the gene contained in the specimen A reference value calculating means for calculating a reference value Vb for determining presence or absence;
Fluorescence determination means for comparing the signal Vs of the fluorescence detection means according to the fluorescence intensity of the first detection unit with the reference value Vb to determine the presence or absence of a gene;
A genetic diagnosis system characterized by comprising: The reference value calculation means includes
The genetic diagnosis system according to claim 1, wherein the reference value Vb is calculated based on the following equation (1).
Vb = C × (Vp−Vn) + Vn (1)
However, C is a constant of 0 <C <1, Vp is a signal of the fluorescence detection means according to the fluorescence intensity of the second detection unit, Vn is the fluorescence detection according to the fluorescence intensity of the third detection unit Means signal. In a genetic diagnosis method for determining the presence or absence of a gene from the fluorescence intensity of a mixture of a sample and a reagent in a reaction by mixing a sample and a reagent,
A first fluorescence intensity obtained by mixing and reacting the specimen and the reagent; a second fluorescence intensity obtained by mixing and reacting the reagent with a positive control solution that reacts with the reagent and emits fluorescence; A fluorescence detection step for detecting a third fluorescence intensity obtained by mixing and reacting the negative control solution that does not react with the reagent and the reagent;
A reference value calculating step for calculating a reference value Vb for determining the presence or absence of a gene contained in the specimen based on the second fluorescence intensity and the third fluorescence intensity;
A fluorescence determination step of comparing the first fluorescence intensity with the reference value Vb to determine the presence or absence of a gene;
A genetic diagnosis method characterized by comprising: The reference value calculating step includes
The genetic diagnosis method according to claim 3, wherein the reference value Vb is calculated based on the following equation (1).
Vb = C × (Vp−Vn) + Vn (1)
Where C is a constant of 0 <C <1, Vp is a signal corresponding to the fluorescence intensity of the second fluorescence intensity, and Vn is a signal corresponding to the fluorescence intensity of the third fluorescence intensity.

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