JP2010022315A - Nucleic acid quantity detection device and nucleic acid quantity detection method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To propose a nucleic acid quantity detection device and a nucleic acid quantity detection process, capable of improving detection accuracy of proliferation reaction. <P>SOLUTION: The nucleic acid quantity detection process includes: an irradiation step in which, from each light source element matched with a plurality of containers formed as a place of proliferation reaction on a substrate, an excitation light is irradiated to a fluorescent substance contained in the containers; and a light quantity adjusting step for adjusting irradiation light quantity to each light source element so that the amount of diffuse light of excitation light, being received with a plurality light-receiving elements allotted to surroundings on the basis of optical axis of each light source element and irradiated from each light source element, becomes constant in between the substrate and each light source element. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a nucleic acid amount detection apparatus and a nucleic acid amount detection method, and is suitable for a PCR (Polymerase Chain reaction) apparatus.

  Conventionally, the present applicant has proposed a real-time PCR apparatus using a thin film transistor as a heat source for a plurality of micro-containers used as a nucleic acid growth reaction field (for example, Patent Document 1).

As a detection system for a growth reaction in this real-time PCR apparatus, a light source for irradiating excitation light of a fluorescent material put in a micro container and a light receiving element for receiving fluorescence of the fluorescent material excited by the excitation light are provided for each micro container. Is provided.
JP 2003-298068 A

  However, since each light source generally has manufacturing variations, even if the control for the light source is the same, the amount of excitation light for each micro container differs due to this variation. This difference is reflected in a signal output as a result of the growth reaction in the micro container from the light receiving element, so that the reliability of the nucleic acid amount indicated in the signal is poor.

  The present invention has been made in view of the above points, and intends to propose a nucleic acid amount detection apparatus and a nucleic acid amount detection method capable of improving the detection accuracy of a proliferation reaction.

  In order to solve such a problem, the present invention is an apparatus for detecting the amount of nucleic acid, which is associated with each of a plurality of containers formed on a substrate as a field for a proliferation reaction of nucleic acids, and emits excitation light for a fluorescent substance placed in the container. Between the plurality of light source elements to be irradiated, the substrate, and the plurality of light source elements, it is arranged around the optical axis of each light source element and receives scattered light of excitation light emitted from the light source element. A plurality of light receiving elements and a light amount adjusting unit that adjusts the amount of light applied to each light source element so that the amount of scattered light received by the plurality of light receiving elements is constant.

  The present invention also relates to a method for detecting the amount of nucleic acid, wherein each light source element associated with a plurality of containers formed on a substrate as a growth reaction field is irradiated with excitation light for a fluorescent substance placed in the container. Between the step, the substrate, and each light source element, the scattered light amount of excitation light emitted from each light source element received by a plurality of light receiving elements arranged around the optical axis of each light source element Through a light amount adjustment step of adjusting the amount of light applied to each light source element so that the light intensity becomes constant.

  In the present invention, since the light amount (scattered light amount) in the middle of the container is constant, even if there is a manufacturing variation in each light source element, the light amount difference due to the variation is canceled out. That is, the amount of excitation light reaching each container is constant regardless of variations in the light source elements. Therefore, according to the present invention, the fluorescence amount excited by the excitation light can reflect the nucleic acid amount itself in each container, and as a result, the detection accuracy of the proliferation reaction can be improved.

  Hereinafter, an embodiment to which the present invention is applied will be described in detail with reference to the drawings.

(1) Structure of real-time PCR apparatus In FIG. 1, the structure of the real-time PCR apparatus 1 according to the present embodiment is shown. This real-time PCR apparatus 1 has a structure in which a plurality of substrates 11 to 16 are arranged in layers at predetermined intervals with respect to a reaction chamber.

  The reaction substrate 11 is a substrate serving as a reference layer, and a container (hereinafter also referred to as a well) UL used as a place for nucleic acid proliferation reaction is formed on the substrate at a high density. These wells UL are given a target nucleic acid to be propagated and various substances (primers, buffers, enzymes, dNTPs, fluorescent dyes, etc.) required for the proliferation of the target nucleic acid.

  For example, when a 6 [cm] square reaction substrate 11 is used, about 40,000 wells UL having a capacity of 1 [μL] or less can be formed. Therefore, the real-time PCR apparatus 1 can handle many target nucleic acids of the same type or different types even when the reaction chamber is downsized.

  The heat generating substrate 12 is a substrate disposed as a lower layer with respect to the reaction substrate 11, and a heat source element HD is associated with each well UL on the surface of the substrate facing the reaction substrate 11. A plurality of temperature measuring elements TD are arranged around the heat source elements HD. For example, a TFT (Thin Film Transistor) or the like is used as the heat source element HD, and a pin diode or the like is used as the temperature detecting element TD.

  In this real-time PCR apparatus 1, the heat source elements HD corresponding to each well UL are individually controlled according to the temperatures sensed by the plurality of temperature measuring elements TD surrounding the heat source elements HD. Therefore, the real-time PCR apparatus 1 can determine the temperature of each well UL arranged at a high density even when the temperature conditions are different due to, for example, different target nucleic acid species given to each well UL. The temperature required for each step of the denaturation stage, annealing stage, and extension stage defined as the growth cycle can be adjusted with high accuracy.

  The heat generation auxiliary substrate 13 is a substrate disposed as a lower layer with respect to the heat generation substrate 12. The exothermic auxiliary substrate 13 maintains the reaction chamber at a set temperature by absorbing or dissipating heat in the entire reaction chamber.

  Therefore, the real-time PCR apparatus 1 can speed up the time (temperature gradient time) until the temperature of the well UL is shifted from the temperature set at the current stage to the temperature to be given at the next stage. Incidentally, for example, a Peltier element or the like is used for the heat generating auxiliary substrate 13.

  The light-emitting substrate 14 is a substrate disposed as an upper layer with respect to the reaction substrate 11. A surface of the substrate that faces the reaction substrate 11 is made to correspond to each well UL, such as an intercalator. A light source element LS for irradiating excitation light to the fluorescent material is arranged. For example, an LED (Light Emitting Diode) is used as the light source element LS.

  The excitation light transmitting substrate 15 is a substrate arranged as an intermediate layer between the reaction substrate 11 and the light emitting substrate 14, transmits the excitation light emitted from the light source element LS, and reflects light other than the excitation light. For example, a dichroic mirror is used for the excitation light transmitting substrate 15.

  The fluorescent transmission substrate 16 is a substrate disposed as an intermediate layer between the heat generation substrate 12 and the heat generation auxiliary substrate 13, and transmits fluorescence of a fluorescent material excited by excitation light and reflects light other than the fluorescence. In addition, a light receiving element LDA that receives fluorescence excited in the well is disposed on the surface facing the heat generating auxiliary substrate 13 in correspondence with each well UL. In the real-time PCR apparatus 1, the amount of target nucleic acid corresponding to the amount of fluorescence received by these light receiving elements LDA is calculated for each growth cycle.

  In the real-time PCR apparatus 1 according to this embodiment, the surface of the excitation light transmitting substrate 15 that faces the light emitting substrate 14 is surrounded by a position on the basis of the position corresponding to the optical axis of each light source element LS on the light emitting substrate 14. Thus, a light receiving element LDB that receives the scattered light of the excitation light emitted from the light source element LS is disposed.

  Further, in this real-time PCR apparatus 1, instead of immediately starting the next growth cycle at the time when one growth cycle is completed, as shown in FIG. A period for adjusting the amount of light (hereinafter also referred to as a calibration period CF) is provided.

  In each calibration period CF, the light amount of the light source element LS corresponding to each well UL is individually controlled so that the scattered light amount received by each light receiving element LDB is constant. Therefore, the real-time PCR apparatus 1 can irradiate each well UL with a certain amount of excitation light until a predetermined number of growth cycles are completed.

(2) Specific Configuration of Heat Generation Substrate Next, a specific configuration of the heat generation substrate 12 (FIG. 1) will be described with reference to FIG. The heat generating substrate 12 is made of a substrate such as glass that can transmit excitation light, and includes a plurality of pairs of heat generating circuits 30 and temperature sensing circuits 40, a scanning drive unit 50, and a temperature control unit 60.

  Each heating circuit 30 has one TFT 31, and these TFTs 31 are arranged in a matrix corresponding to each well formed on the reaction substrate 11. On the other hand, each temperature sensing circuit 40 has a plurality of pin diodes 41, and these pin diodes 41 are arranged at predetermined intervals with a certain distance around the TFT 31 of the pair of heat generating circuits 30.

  Here, specific circuit configurations of the heat generating circuit 30 and the temperature sensing circuit 40 will be described. However, since each pair of the heat generation circuit 30 and the temperature sensing circuit 40 have the same configuration, the description will be given focusing on the pair of the heat generation circuit 30 and the temperature sensing circuit 40.

  As shown in FIG. 4, the heat generating circuit 30 includes a TFT 31 that functions as a heat source element, transistors T1, T2, and T3 that function as switching elements, and a capacitor 32 that functions as a heat control element.

  On the other hand, the temperature sensing circuit 40 includes a plurality of pin diodes 41 that function as temperature sensing elements, transistors T4 and T5 that function as switching elements, and a voltmeter 42.

  A pulse signal PS1 is supplied from the first scan driver 50A (FIG. 3) to the gates of the transistors T1, T2, and T3 in the heating circuit 30, and a pulse signal is supplied to the gates of the transistors T3 and T4 in the temperature sensing circuit 40. A pulse signal PS2 having an inverted relationship with PS1 is supplied from the second scan driver 50B (FIG. 3).

  For example, when the pulse signal PS1 is in the “High” state (when the heat generation circuit 30 is in the driving state), the pulse signal PS2 is in the “Low” state (the temperature sensing circuit 40 is in the non-driving state). In this state, the transistors T1 and T2 in the heat generating circuit 30 are in the on state, and the transistor T3 in the heat generating circuit 30 and the transistors T4 and T5 in the temperature sensing circuit 40 are in the off state.

  In this state, the temperature control unit 60 (FIG. 3) is given to the heat generating circuit 30 a signal (hereinafter, also referred to as a heating control signal) Isig corresponding to the temperature to be heated. In the heat generation circuit 30, since the drain and gate of the TFT 31 are short-circuited by the transistor T2, the heat generation control signal Isig flows to the TFT 31 through the transistor T1.

  As a result, a voltage between the gate and the source corresponding to the heat generation control signal Isig is generated in the TFT 31, and this voltage is held in the capacitor 32 as a heat source element to be generated in the TFT 31.

  When the TFT 31 is an enhancement type transistor (that is, threshold Vth> 0), the TFT 31 operates in a saturation region. Therefore, when the gate-source voltage in the TFT 31 is Vgs, the voltage Vgs and the heat generation control signal Isig In the meantime,

The relationship is established. Incidentally, in this equation (1), “μ” is the carrier mobility, “Cox” is the gate capacity per unit area, “W” is the channel width, and “L” is the channel length. is there.

  On the other hand, when the pulse signal PS1 is in the “Low” state (when the heat generation circuit 30 is in the non-driven state), the pulse signal PS2 is in the “High” state (the temperature sensing circuit 40 is in the non-driven state). ). In the heat generation circuit 30 in this state, the transistors T1 and T2 are in the off state and the transistor T3 is in the on state, so that a current flows from the power supply voltage VDD toward the source end GND in the TFT 31.

  The voltage supplied from the power supply voltage VDD to the TFT 31 is set higher than the voltage at which the TFT 31 operates in the saturation region. That is, the power supply voltage VDD for the TFT 31 is set sufficiently high and the on-resistance of the transistor T3 is set sufficiently low so that the TFT 31 operates in the saturation region.

  Therefore, the current Idrv flowing through the TFT 31 does not depend on the drain-source voltage Vds, and

Given by.

  In general, in TFT, each parameter appearing on the right side of the equations (1) and (2) varies, but in this heating circuit 30, since the TFT 31 operates in the saturation region, the current does not depend on the value of the parameter. Idrv and the heat generation control signal Isig coincide with each other.

  Therefore, the amount of heat generated in the TFT 31 is an accurate value determined by the product of the heat generation control signal Isig supplied from the temperature control unit 60 (FIG. 3) and the power supply voltage VDD, regardless of variations in characteristics of the TFT 31.

  Further, the heat generation control signal Isig can change the current value accurately in the temperature controller 60 (FIG. 3). Therefore, the heat generating circuit 30 can accurately vary the temperature of the well through the TFT 31 by controlling the current in the temperature control unit 60.

  On the other hand, in the temperature sensing circuit 40, when the pulse signal PS1 is in the “Low” state (the heat generating circuit 30 is not driven) and the pulse signal PS2 is in the “High” state (the temperature sensing circuit 40 is driven), A side temperature control signal Idet is supplied to the temperature control unit 60 (FIG. 3).

  In this case, in the temperature sensing circuit 40, the side temperature control signal Idet flows to the plurality of pin diodes 41 connected in series via the transistor T4. As a result, a voltage is applied to these pin diodes 41, and the voltage is measured by the voltmeter 42.

  The measured value in the voltmeter 32 is taken in as a parameter indicating the average temperature around the TFT 31 by the temperature control unit 60 (FIG. 3), and the current value of the heat generation control signal Isig in the temperature control unit 60 (FIG. 3). Used to determine

  In this way, each pair of the heat generating circuit 30 and the temperature sensing circuit 40 operate alternately under the control of the temperature control unit 60.

  The scanning drive unit 50 (FIG. 3) sends, to each heat generating circuit 30, an in-phase or different-phase pulse signal having a driving period at predetermined intervals from the first scanning driving unit 50A, while A pulse signal having a reversed relationship with the pulse signal sent to the pair of heat generating circuits 30 is sent from the second scanning drive unit 50B to the sensing circuit 40.

  In this way, the scanning drive unit 50 sets each heating circuit 30 and temperature sensing circuit 40 in pairs, with the driving of the heating circuit 30 and the driving of the temperature sensing circuit 40 as units (hereinafter also referred to as driving cycle). It is made to circulate and drive.

  The temperature control unit 60 is configured as a computer including a CPU (Central Processing Unit), a ROM (Read Only Memory), and a RAM (Random Access Memory) as a work memory of the CPU.

  The temperature controller 60 initially sets a temperature that is increased or decreased according to the type of the target nucleic acid from the temperature required for the denaturation step (95 ° C.) as the temperature to be given to the well (hereinafter also referred to as well temperature).

  The temperature control unit 60 measures the transition timing to the denaturation stage, annealing stage, and extension stage in the growth cycle, and when the transition timing is reached, the well temperature is set to the temperature required for the stage to be transferred (95 The temperature is changed from 0 ° C., 55 ° C. or 72 ° C. to a temperature increased or decreased depending on the species of the target nucleic acid.

  The temperature control unit 60 generates a current value signal corresponding to the temperature to be heated (hereinafter also referred to as a heating control signal) for each drive cycle with reference to the well temperature.

  That is, the temperature control unit 60 gives, for example, a side temperature control signal Idet1 of 100 [μA] and a side temperature control signal Idet2 of 10 [μA] to the temperature sensing circuit 40 to be driven. The temperature in the heat generating circuit 30 paired with the temperature sensing circuit 40 is obtained from the measured value of the voltmeter 42 (FIG. 4).

  Specifically, the difference between the voltage value when the side temperature control signal Idet1 is given and the voltage value when the side temperature control signal Idet2 is given becomes the measurement value of the voltmeter 42 (FIG. 4). If the measured value is ΔV,

Ask for. In this equation (3), “5.0072” is a Boltzmann constant.

  Further, when the temperature control unit 60 uses the temperature sensing circuit 40 to be driven to obtain the temperature in the pair of heat generating circuits 30, the temperature control unit 60 is proportional to the temperature difference between the obtained temperature (measured temperature) and the well temperature. The current value to be determined is determined as the current value of the heat generation control signal Isig, and the heat generation control signal Isig having the determined current value is generated.

  The temperature control unit 60 sends the heat generation control signal Isig to the temperature sensing circuit 40 when the pair of heat generation circuits 30 are driven.

  In this manner, the temperature control unit 60 performs each driving cycle according to the temperature difference between the temperature measured by the temperature sensing circuit 40 paired with the heat generating circuit 30 and the well temperature until a predetermined number of proliferation cycles are passed. A heating control signal to be sent to the heating circuit 30 is generated.

  Therefore, the temperature control unit 60 makes the temperature gradient smoothly (constantly) change as compared with the case where the heating control signal having a fixed current value corresponding to the well temperature is continuously applied until the well temperature is reached. As a result, deterioration of the target nucleic acid or the like due to a rapid temperature change can be avoided.

(3) Specific Configuration of Light Source Substrate Next, a specific configuration of the light emitting substrate 14 (FIG. 1) will be described with reference to FIG. The light emitting substrate 14 includes a plurality of light emitting circuits 70, a scan driving unit 80, and an adjusting unit 90.

  Each light emitting circuit 70 has one LED 71, and these LEDs 71 are arranged in a matrix corresponding to each well formed on the reaction substrate 11. Here, a specific circuit configuration in the light emitting circuit 70 will be described. However, since each light emitting circuit 70 has the same configuration, the description will be made with attention paid to one light emitting circuit 70.

  As shown in FIG. 6, the light emitting circuit 70 includes an LED 71 that functions as a light source element LS, transistors T10 and T11 that function as switching elements, and a capacitor 72 that functions as a light emission amount control element.

  The pulse signal PS is supplied from the scan driver 80 (FIG. 5) to the gate of the transistor T10 in the light emitting circuit 70. For example, when the pulse signal PS is in the “High” state (driving period), the transistor T10 in the light emitting circuit 70 is turned on.

  In this state, the light emitting circuit 70 is supplied with a signal Isg (hereinafter also referred to as a light emission control signal) Isg corresponding to the light emission amount to be irradiated from the adjusting unit 90 (FIG. 5), and the transistor T10 is connected to the light emitting circuit 70. Flowing through.

  As a result, a voltage corresponding to the light emission control signal Isg is generated between the source of the transistor T10 and the gate of the transistor T11, and this voltage is held in the capacitor 72 provided between the source and gate.

  As a result, an amount of current corresponding to the voltage held in the capacitor 72 is applied to the LED 71 from the power supply voltage VDD, and the LED 71 is irradiated with excitation light having a light emission amount corresponding to the amount of current. Become.

  The holding voltage for the capacitor 72 is determined according to the value of the light emission control signal Isg, and this value is varied by the adjusting unit 90 (FIG. 5). Therefore, the light emitting circuit 70 can irradiate the LED 71 with the excitation light for each well UL for each LED 71 in accordance with the current control in the adjustment unit 90.

  The scanning drive unit 80 (FIG. 5) applies a pulse signal whose drive period is the calibration period CF (FIG. 2) to each light emitting circuit 70, and drives the light emitting circuit 70 to circulate simultaneously or in a predetermined order. ing.

  The adjustment unit 90 is configured as a computer including a CPU, a ROM, and a RAM as a work memory of the CPU.

  Based on the program stored in the ROM, the adjustment unit 90 adjusts the amount of light for each LED 71 at every calibration period CF (FIG. 2) from the current multiplication cycle to the next multiplication cycle. The amount of current to be supplied to the corresponding light emitting circuit 70 is adjusted.

  In addition, the adjustment unit 90 is configured to determine the difference between the scattered light amount received by each light receiving element LDB in the current calibration period CF and the scattered light amount received by each light receiving element LDB in the previous calibration period CF. The target nucleic acid amount calculated in the previous growth cycle is individually adjusted for each well UL.

  Specific adjustment processing in the adjustment unit 90 is performed according to a flowchart shown in FIG. 7, for example.

  That is, the adjustment unit 90 starts this adjustment processing procedure when an amplification reaction start operation is performed. In step SP1, the adjustment unit 90 generates a light emission control signal Isig having a current value set as an initial value. To send.

  Then, the adjustment unit 90 proceeds to the next step SP2, determines whether or not the time specified for the extension stage in the growth cycle has elapsed, and if it determines that the time has elapsed, In step SP3, it is determined whether or not the specified number of cycles has been completed.

  Here, when the prescribed number of cycles has not been completed, the adjustment unit 90 recognizes that the period has shifted to the calibration period CF, proceeds to the next step SP4, and is scattered light received by each light receiving element LDB. Is stored as data in the internal memory.

  In step SP5, the adjustment unit 90 sets the current value of the light emission control signal Isig to be given to each light emitting circuit 30 so that the amount of scattered light acquired from each light receiving element LDB is constant.

  Then, the adjustment unit 90 proceeds to the next step SP6, generates the light emission control signal Isig set for each light emitting circuit 30 in step SP4, and sends it to the corresponding light emitting circuit 70.

  Thereafter, the adjustment unit 90 proceeds to step SP7, and when the shift to the calibration period CF is the second time or later, the scattering of each light receiving element LDB acquired in step SP3 within the current calibration period CF. The amount of light and the amount of scattered light of each light receiving element LDB acquired in step SP3 during the previous calibration period CF are compared between the corresponding light receiving elements LDB.

  Here, when there is even one light receiving element LDB having a difference in the amount of scattered light between the current calibration period CF and the previous calibration period CF, this is the case with the LED 71 corresponding to the light receiving element LDB. It means that there is a difference in the amount of light.

  In this case, the adjustment unit 90 proceeds to step SP8, and for the well UL targeted for irradiation by the light receiving element LDB having a difference in the amount of scattered light between the current calibration period CF and the previous calibration period CF, the previous proliferation cycle At this time, data indicating the amount of target nucleic acid calculated according to the amount of fluorescence received by the light receiving element LDA is acquired.

  Then, the adjustment unit 90 adds or subtracts an amount proportional to the difference in the amount of scattered light between the current calibration period CF and the previous calibration period CF with respect to the target nucleic acid amount in this data, so that After correcting the amount of deviation due to a difference in the amount of light, the process returns to step SP2. The “amount proportional to the difference” may be added with a proportionality constant corresponding to the species of the target nucleic acid. In this way, a target nucleic acid amount closer to the true value can be obtained.

  On the other hand, when there is no light receiving element LDB having a difference in the amount of scattered light between the current calibration period CF and the previous calibration period CF, the adjustment unit 90 returns to step SP2 without passing through step SP8.

  In this way, in step SP3, the adjustment unit 90 adjusts the light amount for each LED 71 within the calibration period CF until the next growth cycle until the specified number of cycles has passed. The target nucleic acid amount calculated in the cycle is appropriately corrected.

(4) Operation and Effect In the above configuration, in the real-time PCR device 1, the light receiving element LDB that receives the scattered light of the excitation light emitted from the LEDs 31 (light source elements LS) respectively associated with the plurality of wells UL (see FIG. 1) is provided between the well UL and the LED 31 (light source element LS). These light receiving elements LDB are arranged around the optical axis of the corresponding LED 31 (light source element LS) (FIG. 1).

  The adjustment unit 90 (FIG. 6) in the real-time PCR apparatus 1 adjusts the amount of light emitted from each LED 31 so that the amount of scattered light received by these light receiving elements LDB is constant.

  In this real-time PCR device 1, since the light amount (scattered light amount) in the middle of the well UL is constant, even if there is a manufacturing variation in each LED 31, the light amount difference due to the variation is canceled out. become. That is, the amount of excitation light reaching each well UL is constant regardless of variations in the LEDs 31.

  Therefore, in this real-time PCR device 1, the amount of fluorescence excited by the excitation light can reflect the amount of nucleic acid itself in each well UL, and as a result, the detection accuracy of the proliferation reaction can be improved. .

  In the case of this embodiment, the adjustment unit 90 performs such adjustment every time the proliferation cycle ends (for each calibration period CF) without performing only the first adjustment (FIG. 2).

  Therefore, in this real-time PCR apparatus 1, the light amount (scattered light amount) in the middle of the well UL can be made constant according to the state of each LED 31 before the start of the next growth cycle. That is, the excitation light amount reaching each well UL can be made constant not only in the manufacturing variation in each LED 31 but also in the variation of the LED 31 over time. As a result, the real-time PCR apparatus 1 can further improve the detection accuracy of the proliferation reaction.

  In the real-time PCR apparatus 1, each LED 31 is arranged in a sealed space where the temperature changes drastically. Therefore, it is particularly useful that the amount of excitation light reaching each well UL can be made constant even if the LED 31 varies with time.

  By the way, when there is a difference between the amount of scattered light received by the light receiving element LDB in the current calibration period CF and the amount of scattered light received by the light receiving element LDB in the previous calibration period CF (the LED 31 varies over time). In the present calibration period CF, the amount of excitation light reaching the well UL is constant. For this reason, the amount of fluorescence excited by excitation light in the next growth cycle reflects the amount of nucleic acid itself in each well UL.

  However, in the relationship between the amount of fluorescence obtained in the next growth cycle and the amount of fluorescence in the previous growth cycle, a difference with time in the LED 31 remains. In the case of this embodiment, when there is a difference between the current scattered light amount and the previous scattered light amount, the adjustment unit 90 calculates the nucleic acid amount in the well UL associated with the LED 31 that emits the scattered light amount having the difference. Adjust according to.

  Therefore, in this real-time PCR device 1, even if the LED 31 changes over time, the conversion of the amount of nucleic acid can be made uniform, and as a result, the detection accuracy of the proliferation reaction can be further improved.

  Further, in the case of this embodiment, the light receiving element LDB (FIG. 1) is opposed to the LED 31 in the excitation light transmitting substrate 15 disposed between the plurality of wells UL and the LED 31 (light source element LS). Arranged on the surface.

  Therefore, in this real-time PCR device 1, light other than the excitation light is blocked in the excitation light transmission substrate 15 as long as the light amount (scattering light amount) in the stage up to the surface of the excitation light transmission substrate 15 is constant. Mixing of noise light from the excitation light transmitting substrate 15 to the well UL can be prevented in advance. As a result, in the real-time PCR apparatus 1, the amount of excitation light reaching each well UL can be made constant.

  According to the above configuration, the real-time PCR apparatus 1 that can improve the detection accuracy of the proliferation reaction can be realized by making the excitation light amount reaching each well UL constant regardless of the variation of the LEDs 31.

(5) Other Embodiments In the above-described embodiments, the circuit configuration shown in FIG. However, the circuit configuration is not limited to this embodiment, and various circuit configurations can be applied.

  Moreover, although the above-described embodiment PCR method is applied, other growth reaction methods can be applied. For example, IVT (In Vitro Transcription) method for obtaining RNA from dsDNA, TRC (Transcription Reverse transcription Concerted amplification) method for obtaining RNA by trimming RNA using RNase activity of reverse transcriptase, RNA using RNA polymerase NASBA (Nucleic Acid Sequence-Based Amplification) method for obtaining RNA from dsDNA with a promoter incorporated therein, SPIA method for obtaining ssDNA from RNA using a primer comprising a chimeric structure of RNA and DNA, and DNA loop formation, LAMP (Loop-Mediated Isothermal Amplification) method to obtain dsDNA from RNA at constant temperature, SMAP (SMart Amplification) to obtain dsDNA while identifying single nucleotide polymorphism (SNP (Single Nucleotide Polymorphism)) by combining multiple enzymes Process), DNA and RNA Is like ICAN method (Isothermal and Chimeric primer-initiated Amplification) method to obtain the dsDNA using chimeric primers.

  In the above-described embodiment, a so-called transmission type apparatus is applied as the real-time PCR apparatus 1. However, instead of this, for example, a so-called reflective real-time PCR device 100 may be applied as shown in FIG.

  In the reaction substrate 111 in the real-time PCR apparatus 100, a plurality of wells UL whose bottom surfaces are formed in an R shape are formed, and light receiving elements LDA are arranged on one surface corresponding to the wells UL. Excitation light emitted from each light source element LS is guided to a corresponding well UL formed on the reaction substrate 111 via the excitation light transmitting substrate 15. In each well UL, the fluorescence of the fluorescent material excited by the excitation light is reflected by the wall surface of the well UL and enters the light receiving element LDA on one surface of the reaction substrate 111.

  Even when such a reflection-type real-time PCR apparatus 100 is applied, the same effects as those of the above-described embodiment can be obtained.

  Note that the components of the real-time PCR apparatus 1 or 100 are not limited to those shown in the drawings, and can be appropriately changed in design.

  The present invention can be used in the bioindustry such as gene analysis, gene diagnosis, or creation of a medicine.

It is sectional drawing which shows schematic structure of a real-time PCR apparatus. It is an approximate line figure used for explanation of a calibration period. It is the schematic which shows the structure of a heat generating board. It is a figure which shows the structure of a heat generating circuit and a temperature sensing circuit. It is the schematic which shows the structure of a light emitting substrate. It is a figure which shows the structure of a light emitting circuit. It is a flowchart which shows an adjustment process procedure. It is sectional drawing which shows schematic structure of the real-time PCR apparatus by other embodiment.

Explanation of symbols

  DESCRIPTION OF SYMBOLS 1,100 ... Real-time PCR apparatus 11, 111 ... Reaction substrate, 12 ... Heat generation substrate, 13 ... Heat generation auxiliary substrate, 14 ... Light emission substrate, 15 ... Excitation light transmission substrate, 16 ... Fluorescence transmission substrate , 30... Heating circuit, 40... Temperature sensing circuit, 50, 80... Scanning drive unit, 60... Temperature control unit, 70. Part, T10, T11... Transistor, VDD.

Claims (6)

A plurality of light source elements that are respectively associated with a plurality of containers formed on a substrate as a field for a proliferation reaction of nucleic acid, and that irradiates excitation light with respect to a fluorescent substance placed in the container;
Between the substrate and the plurality of light source elements, a plurality of light receiving elements arranged around the optical axis of each light source element and receiving scattered light of excitation light emitted from the light source element;
A nucleic acid amount detection apparatus comprising: a light amount adjustment unit that adjusts an irradiation light amount for each light source element so that a scattered light amount received by the plurality of light receiving elements is constant. The light amount adjustment unit is
The nucleic acid amount detection device according to claim 1, wherein the amount of light applied to each light source element is adjusted for each period provided between cycles of the proliferation reaction. A reflector disposed between the substrate and the plurality of light source elements in parallel with the substrate and reflecting light other than the excitation light;
The plurality of light receiving elements are:
The nucleic acid amount detection device according to claim 1, wherein the nucleic acid amount detection device is disposed on a surface of the reflection plate facing the plurality of light source elements. For each of the plurality of light receiving elements, the amount of scattered light in the current period is compared with the amount of scattered light in the previous period, and the reaction results in the plurality of containers in the previous growth reaction are shown. The reaction result adjusting unit according to claim 2, further comprising: a reaction result adjusting unit that adjusts a value indicating a reaction result in a container associated with a light source element that irradiates a scattered light amount having the difference among the values according to the difference. Nucleic acid amount detection device. The reaction result adjustment unit is
The nucleic acid amount detection apparatus according to claim 4, wherein a value proportional to the difference is added to or subtracted from a value indicating a reaction result in a container associated with a light source element that irradiates the scattered light amount having the difference. Irradiation step of irradiating excitation light to the fluorescent substance placed in the container from each light source element respectively associated with a plurality of containers formed on the substrate as a field of proliferation reaction;
A scattered light amount of excitation light emitted from each light source element received by a plurality of light receiving elements arranged around the optical axis of each light source element between the substrate and each light source element. And a light amount adjustment step for adjusting the amount of light applied to each of the light source elements so as to be constant.

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