JP2008283870A - Real-time pcr device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a real-time PCR device which enables comprehensive analyses and can detect fluorescent light obtained by reaction, with high accuracy. <P>SOLUTION: This real time PCR device 1 for detecting a gene expression quantity comprises at least a plurality of reaction zones A1; heating portions 16, disposed for the reaction zones A1 and used for heating the reaction zones with heat sources equipped with temperature-controlling mechanisms, respectively; an optical means of irradiating all of the reaction zones with exciting lights L1, L2 having specific wavelengths; and fluorescent light-detecting portions 15, having fluorescent light detection mechanisms for converting the quantities of received fluorescent lights L3 into electrical signals and detect the fluorescent lights L3. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a real-time PCR apparatus. More specifically, the present invention relates to a real-time PCR apparatus that analyzes gene expression and the like.

  In recent years, hybridization detection techniques such as DNA chips and DNA microarrays have been put into practical use. A DNA chip is obtained by collecting and fixing various and various DNA probes on a substrate surface. By using this DNA chip to detect hybridization on the surface of the DNA chip substrate, gene expression in cells / tissues can be comprehensively analyzed.

  In addition, verification of data obtained by the microarray using a PCR method (polymerase chain reaction) is a standard technique for quantitative analysis of a small amount of nucleic acid.

  In the real-time PCR method, DNA and the like can be amplified several hundred thousand times by continuously performing an amplification cycle of “thermal denaturation → annealing with a primer → polymerase extension reaction”. In this method, the PCR amplification product thus obtained is monitored in real time to quantitatively analyze each trace amount of acid.

  In the real-time PCR method, the PCR amplification product can be monitored in real time using a dedicated device or the like in which a thermal cycler and a spectrofluorometer are integrated. As such an apparatus, there is a real-time PCR apparatus.

  A real-time PCR device is a reaction processing device that can detect fluorescence signals in real time by irradiating excitation light when an amplification reaction proceeds in a sample. It can be used as a detector for observing genomic DNA used for research.

  For example, in a PCR method for amplifying DNA, when a fluorescent dye used for characterizing double-stranded DNA is labeled, changes in the fluorescent dye can be observed by heating the double-stranded DNA. There are several detection methods for real-time PCR.

  For example, when only the target of interest can be amplified with a highly specific primer, an intercalator method using SYBR (registered trademark) GREEN I is used.

  An intercalator that emits fluorescence by binding to double-stranded DNA binds to double-stranded DNA synthesized by a PCR reaction and emits fluorescence when irradiated with excitation light. By detecting this fluorescence intensity, the amount of amplification product generated can be monitored. There is no need to design and synthesize fluorescently labeled probes specific to the target, and it can be easily used for measuring various targets.

  In addition, the probe method is used when it is necessary to distinguish and detect similar sequences or when multiplex detection is required, such as typing of SNPs. One of them is the TaqMan (registered trademark) probe method in which an oligonucleotide whose 5 ′ end is modified with a fluorescent substance and whose 3 ′ end is modified with a quencher substance is used as a probe.

  The TaqMan probe specifically hybridizes to the template DNA in the annealing step. However, since a quencher is present on the probe, fluorescence emission is suppressed even when irradiated with excitation light. In the extension reaction step, when the TawMan probe hybridized to the template is decomposed by the 5 ′ → 3 ′ exonuclease activity of Taq DNA polymerase, the fluorescent dye is released from the probe, and the suppression by the quencher is released and fluorescence is released. Emits light. By measuring the fluorescence intensity, the amount of amplification product generated can be monitored.

  The principle of quantifying gene expression level by real-time PCR by such a method will be described below. First, PCR is carried out using serially diluted standard samples with known concentrations as templates. Then, the number of cycles (threshold cycle; Ct value) reaching a certain amount of amplification product is obtained. A calibration curve is created by plotting the Ct value on the horizontal axis and the initial DNA amount on the vertical axis.

  For a sample of unknown concentration, a Ct value is obtained by performing a PCR reaction under the same conditions. From this value and the calibration curve described above, the target DNA amount in the sample can be measured.

As techniques related to these, Patent Documents 1 and 2 disclose techniques related to temperature control during amplification reaction.
JP2003-298068A. Japanese Patent Application Laid-Open No. 2004-025426.

  Such a PCR apparatus has a feature that it is excellent in quantitativeness in detecting the gene expression level. However, there are the following problems. First, the number of samples that can be analyzed at one time is small, and exhaustive analysis is not possible. In addition, in the case of excitation light irradiation or fluorescence detection, a reflection type optical system structure using a reflecting mirror is used. In such a case, the detected fluorescence becomes weak, so a signal amplifier etc. Must be used separately. For this reason, there is a problem that the structure of the detector becomes complicated or expensive.

  Thus, the main object of the present invention is to provide a real-time PCR apparatus that can perform comprehensive analysis and can detect fluorescence obtained by the reaction with high accuracy.

The present invention is a real-time PCR device for detecting gene expression level, a plurality of reaction regions, a heating unit that is provided for each reaction region and heats the reaction region with a heat source having a temperature control mechanism, An optical means capable of irradiating excitation light of a specific wavelength to all of the plurality of reaction regions, and a fluorescence detection unit provided for each of the reaction regions and having a fluorescence detection mechanism for detecting the amount of received fluorescence by converting it into an electrical signal And at least a real-time PCR apparatus.
By providing a fluorescence detection unit for each reaction region and providing a fluorescence detection mechanism that detects the amount of fluorescence converted into an electrical signal, fluorescence can be detected with high accuracy and in real time. Then, highly accurate analysis can be performed by individually performing temperature control and detection control for each reaction region.
In the present invention, the fluorescence detection unit is provided with means for processing the electrical signal detected by the fluorescence detection mechanism based on the correlation between the electrical signal stored in advance and the gene expression level. A PCR device is provided. By performing an arithmetic process on the electrical signal detected by the fluorescence detection mechanism based on the correlation, highly accurate fluorescence detection can be performed for each reaction region.
Subsequently, the present invention provides a real-time PCR apparatus in which the control medium of the temperature control mechanism and the detection medium of the fluorescence detection mechanism are provided in the same pixel. By providing the control medium of the temperature control mechanism and the detection medium of the fluorescence detection mechanism in the same pixel, it is possible to perform highly accurate reaction control and fluorescence detection corresponding to each reaction region and to reduce the size of the device. It can be made easier.
The present invention also provides a real-time PCR apparatus using an EL element (Electro Luminescence) as a fluorescence detection mechanism of the light receiving unit. By using an EL element for a fluorescence detection mechanism, it is possible to respond quickly to the electrical signal, so that more accurate fluorescence detection can be performed.
Furthermore, the present invention provides a real-time PCR apparatus using a temperature control thin film transistor (TFT) as a control medium of the temperature control mechanism and using a fluorescence detection thin film transistor as a detection medium of the fluorescence detection mechanism.
The present invention also provides a real-time PCR apparatus using a temperature control EL element as a control medium of the temperature control mechanism and using a fluorescence detection thin film transistor as a detection medium of the fluorescence detection mechanism.

  According to the real-time PCR apparatus according to the present invention, the present invention enables comprehensive analysis and can detect fluorescence obtained by the reaction with high accuracy.

  Embodiments of the method according to the present invention will be described below with reference to the accompanying drawings. It should be noted that the embodiments and the like shown in the drawings illustrate preferred embodiments of the present invention, and the present invention is not construed narrowly.

  FIG. 1 is a conceptual view of a first embodiment of a real-time PCR apparatus according to the present invention as viewed from the side. FIG. 2 is a conceptual diagram of an example in which the control medium of the temperature control mechanism and the detection medium of the fluorescence detection mechanism are formed in the same circuit in the same embodiment. FIG. 3 is an example of a circuit diagram in which the control medium of the temperature control mechanism and the detection medium of the fluorescence detection mechanism are formed in the same circuit in the same embodiment. In the drawings used below, for the convenience of explanation, the configuration of the apparatus is shown in a simplified manner.

  Reference numeral 1 in FIG. 1 indicates a real-time PCR apparatus according to the present invention. The size and layer structure of the real-time PCR device 1 can be selected as appropriate according to the purpose, and the configuration of the real-time PCR device 1 can be designed or changed within the scope of the object of the present invention.

  The real-time PCR apparatus 1 includes a well substrate 11 having a plurality of reaction regions A1, a light source 12, and an excitation light scanning plate 13 that guides excitation lights L1 and L2 emitted from the light source 12. A filter 14, a fluorescence detection unit 15 that detects fluorescence L 3, and a heating unit 16 that heats the reaction region A 1 are provided on the measurement substrate 17.

  In the real-time PCR apparatus 1, the excitation light L1 emitted from the light source 12 passes through the excitation light scanning plate 13 and is irradiated to each reaction region A1 as the excitation light L2. Then, the fluorescence L3 emitted from the reaction region A1 is detected and measured by the fluorescence detector 15. The fluorescence detection unit 15 includes at least a fluorescence detection mechanism that detects the fluorescence L3 by converting it into an electrical signal.

  By providing the fluorescence detection unit 15 for each reaction region A1 and including a fluorescence detection mechanism that detects the amount of fluorescence L3 converted into an electrical signal, the fluorescence L3 in each reaction region A1 can be individually and accurately detected in real time. Can be analyzed.

  As will be described later, in the present invention, the detection medium of the fluorescence detection mechanism of the fluorescence detection unit 15 and the medium of the temperature control mechanism of the heating unit 16 can be provided in the same pixel. Thereby, miniaturization as a device becomes possible. Hereinafter, each component of the real-time PCR apparatus 1 will be described in detail.

  The well substrate 11 includes a plurality of reaction regions (wells) A1 in the substrate. A predetermined reaction is performed in the reaction region A1. Further, it is formed of a low fluorescent light emitting plastic material or glass, and for example, a number comparable to the number of human genes can be arranged in a matrix.

  In the present invention, the reaction region (well) for the PCR reaction is preferably a microspace. For example, if the wells are 300 μm × 300 μm × 300 μm (capacity of about 30 nL) and about 40,000 wells are arranged, a device having an area of about 6 cm square is obtained.

  Here, the shape of each reaction region A1 is not particularly limited, and is not limited to the rectangular parallelepiped, and may be any shape as long as the reaction solution can be held. A suitable shape can be appropriately selected in consideration of an optical path for irradiating the excitation lights L1 and L2, an optical path for detecting the fluorescence L3, and the like. For example, in the real-time PCR device 2, the reaction region A1 has a curved surface portion in order to reflect the fluorescence L3 in the reaction region A1.

  Moreover, in order to suppress a decrease in detection sensitivity due to the influence of light scattering and the influence of external light, the reaction region A1 is preferably coated with a light shielding material (for example, diamond-like carbon).

  In the present invention, as an optical means capable of irradiating the plurality of reaction regions A1 with excitation light L1 and L2 having a specific wavelength, the light source 12 and excitation light scanning for introducing the excitation light L1 and L2 into each reaction region A1. A plate 13 or the like can be used.

  The light source 12 is not particularly limited as long as it emits light of a specific wavelength, and it is preferable to use a white or monochromatic light emitting diode (LED). By using a light emitting diode, light that does not contain unnecessary ultraviolet rays and infrared rays can be easily obtained.

  In the present invention, the installation location of the light source 12 and the number of light sources are not particularly limited. Although not shown, a plurality of light sources 12 may be provided so as to correspond to each reaction region A1, and each light source 12 may directly irradiate excitation light toward each corresponding reaction region A1.

  In this case, since each reaction region A1 can be directly irradiated with the light source 12, more light can be obtained from the excitation lights L1 and L2, and uniform excitation irradiation can be performed on all the reaction regions A1. Furthermore, it is possible to individually control the excitation wavelengths and the light amounts of the excitation light L1 and L2 irradiated to each reaction region A1. In order to individually control the excitation wavelength, for example, a plurality of light emitting elements having different wavelengths can be used as the light source 12.

  The excitation light scanning plate 13 guides the excitation light L1 emitted from the light source 12 to each reaction region A1 in the well substrate 11. Excitation light L 1 emitted from the light source 12 is introduced into the spacer 151 inside the excitation light scanning plate 13. A reflection film 131 is provided on the bottom of the excitation light scanning plate 13 so that the excitation light L <b> 2 can be introduced into the well substrate 11. Thereby, the fluorescent substance in the reaction solution in each reaction region A1 can be excited with a uniform amount of light. The material of the reflective film 131 is not particularly limited, but it is preferable to use a dichroic mirror.

  In the present invention, it is desirable to provide a filter 133 that transmits only the wavelength light of the excitation light L1 and L2 above the excitation light scanning plate 13. Thereby, the excitation light L2 can be efficiently extracted from the light emitted from the light source 12 and guided to the reaction region A1. As this filter 133, a polarizing filter etc. can be used, for example.

  The excitation light L2 irradiated to the reaction region A1 emits fluorescence L3 when irradiated to the fluorescent substance of the probe in the reaction solution in the reaction region A1. The fluorescence L3 is reflected by the wall surface in the reaction region A1, and is detected and measured by the fluorescence detection unit 15 provided below the reaction region A1.

  In the present invention, the filter 14 can also be disposed between the reaction region A1 and the fluorescence detection unit 15 so that light of a specific wavelength (fluorescence L3 or the like) can be extracted. The filter 14 only needs to be able to extract light of a specific wavelength, and the material thereof is not limited. For example, a dichroic mirror can be used.

  The fluorescence detection unit 15 detects and measures the fluorescence emitted by the excitation of the fluorescent dye in the intercalated probe in response to the excitation light L2 irradiated to the reaction region A1.

  And in this invention, the fluorescence detection part 15 is equipped with the fluorescence detection mechanism which converts and detects the light quantity of the received fluorescence L3 to an electrical signal. With this fluorescence detection mechanism, the received light quantity of the fluorescence L3 can be converted into an electrical signal, and the light quantity of the fluorescence L3 can be measured and detected with high accuracy based on this electrical signal. Further, by continuing to measure this electrical signal, the amount of fluorescence L3 can be detected in real time, so that the gene expression level can be measured in real time.

  In addition, the type of electrical signal detected by the present invention is not particularly limited, and may be a current value or a voltage value, for example. Furthermore, in the present invention, the detection medium of the fluorescence detection mechanism of the fluorescence detection unit 15 is not particularly limited, but it is desirable to use a photosensor such as a photodiode, and more preferably an EL element, a thin film transistor, or the like. desirable.

  Since the ratio between the amount of received light and the amount of current generated is stable, the photodiode can be suitably used for the real-time PCR device 1 as a highly accurate light receiving element. Furthermore, by using an EL element, the response speed is high, and the current generation phenomenon due to fluorescence reception can be immediately detected, so that the gene expression level can be analyzed with high accuracy in real time.

  Furthermore, in the present invention, it is desirable to provide means for performing an arithmetic processing on the electrical signal detected by the fluorescence detection mechanism of the fluorescence detection unit 15 based on the correlation between the gene expression level stored in advance and the electrical signal.

  As the arithmetic processing means, for example, a correspondence table (corresponding to the correlation between the physical quantity and the gene amplification signal) by converting the amount of fluorescence received by the fluorescence detection mechanism into an electrical signal (current value, voltage value, etc.) Table) and the like. Thus, the correlation between the electrical signal in each reaction region A1 and the corresponding gene amplification signal is obtained in advance, and the electrical signal amount obtained at the time of actual measurement is calculated based on this correlation information. Thus, it is possible to perform a highly accurate analysis in accordance with each reaction region A1.

  The means for performing the arithmetic processing based on the correlation between the electrical signal stored in advance and the gene expression level is not particularly limited. For example, the inside of the real-time PCR apparatus 1 (for example, the fluorescence detection unit 15 or the heating unit 16). Etc.), and a method of recording the correlation on the recording medium can be used.

  And in the real-time PCR apparatus 1, the heating part 16 is provided in each reaction area | region A1, respectively. The heating unit 16 includes a temperature control mechanism, whereby the temperature of the reaction region A1 can be controlled by the heating unit 16. Thereby, for example, when performing a PCR cycle, more accurate temperature control can be performed for the steps of thermal denaturation → annealing → extension reaction.

  Furthermore, according to the present invention, the heating time of the reaction region A1 can be individually controlled by the heating unit 16 corresponding to the reaction region A1. By controlling the heating temperature and the heating time for each reaction region A1 by the heating unit 16, the amplification reaction and the like in the reaction region A1 can be controlled with higher accuracy.

  Although temperature control by a thermal cycler or the like used in the conventional real-time PCR method may be added with a gradient mechanism, there is a problem that temperature control of each sample cannot be performed individually. On the other hand, in this invention, each reaction area | region A1 can be temperature-controlled separately by providing the heating part 16 in each reaction area | region A1.

  Furthermore, in the heating unit 16, each reaction region A1 can be temperature-controlled with higher accuracy by heating each reaction region A1 using a heat source having a temperature control mechanism. Examples of such a temperature control mechanism include means for controlling the temperature of the heat source using a switching element or the like, and it is preferable to use a thin film transistor as a control medium of the temperature control mechanism.

  That is, the temperature control for the heat source corresponding to each reaction region A1 can be individually performed using the switching function of the thin film transistor or the like. The type of the thin film transistor used in the present invention is not particularly limited, and for example, a type such as polysilicon or α-silicon can be used as appropriate.

  Further, a heating resistor can be used as a heat source used in the heating unit 16, for example, platinum (Pt), molybdenum (Mo), tantalum (Ta), tungsten (W), silicon carbide, molybdenum silicide, nickel- A chromium alloy, an iron-chromium-aluminum alloy, or the like can be used.

  By controlling electric signals such as a current value and a voltage value flowing through the heat source, temperature control with high accuracy becomes possible. For example, the voltage applied to the thin film transistor is controlled to make the current value between the source (S) and the drain (D) variable, or the current between the source (S) and the drain (D) is controlled as a constant current power source. Thus, the temperature control of the heat source can be performed.

  The fluorescence detection unit 15 is formed on the same substrate as the heating unit 16, and the fluorescence detection mechanism and control medium of the fluorescence detection unit 15 and the control medium of the temperature control mechanism of the heating unit are formed in the same pixel. ing. About the form formed in the same pixel, the case where the fluorescence detection part 15 and the heating part 16 are laminated | stacked may be sufficient as well as the state currently formed planarly. Thus, by providing the detection medium of the fluorescence detection mechanism of the fluorescence detection unit 15 and the control medium of the temperature control mechanism of the heating unit 16 in the same pixel, it is possible to reduce the size of the device.

  That is, in the present invention, it is desirable to provide the control medium of the temperature control mechanism of the heating unit 16 in the same pixel circuit as the detection medium of the fluorescence detection unit 15. With such a structure, temperature control and fluorescence detection can be performed in the same pixel layer. As a result, it is possible to omit the necessity of performing complicated steps in device fabrication, so that the device can be miniaturized, and real-time detection can be performed more simply and easily.

  FIG. 2 is a conceptual diagram of an example in which the control medium of the temperature control mechanism and the detection medium of the fluorescence detection mechanism are formed in the same circuit in the real-time PCR apparatus 1 according to the present invention. That is, the temperature control medium T that is a control medium of the temperature control mechanism and the fluorescence detection medium L that is a detection medium of the fluorescence detection mechanism are formed in the same pixel. The detected electric signal is fed back to the comparison circuit, whereby the heating is controlled. In FIG. 2, the temperature of the heat source H is controlled by the temperature control medium T, and the fluorescence detection medium L is provided in the same pixel.

  Thus, by providing the thermal control medium T and the fluorescence detection medium L in the same pixel, thermal control and fluorescence detection can be performed in the same pixel layer, and a highly accurate reaction corresponding to each reaction region A1. Control and fluorescence detection can be performed, and a complicated structure such as a laminated structure is not required for device fabrication, and the device can be further reduced in size.

  The pixel composed of the temperature control medium T and the fluorescence detection medium L corresponds to the reaction region A1, and the pixel is arranged in a matrix along the gate line (for example, the X direction) and the data line (for example, the Y direction). It is also possible to arrange a plurality of shapes. That is, the pixels corresponding to each reaction region A1 (see FIG. 1) can be arranged in a matrix on the measurement substrate 17 (see FIG. 1).

  Further, the temperature control medium T and the fluorescence detection medium L can be stored in the same pixel as a two-stage structure with a protective layer removed.

  FIG. 3 is an example of a circuit diagram in which the control medium of the temperature control mechanism and the detection medium of the fluorescence detection mechanism are formed in the same circuit in the same embodiment. That is, it is an example of a circuit diagram in which the temperature control medium T and the fluorescence detection medium L are formed in the same circuit. Thereby, in each pixel, the temperature control and the fluorescence detection can be directly detected and collectively controlled in the vicinity of the reaction part.

  In the present invention, it is desirable to use a temperature control thin film transistor as a control medium of the temperature control mechanism and to use a fluorescence detection thin film transistor as a detection medium of the fluorescence detection mechanism. For each reaction region A1, by providing the thin film transistors for temperature control and fluorescence detection in the same pixel, it is possible to realize fluorescence detection and the same configuration control simultaneously with thermal control closely placed in the well. There is no need to provide a separate detection means such as a detection substrate separately from the detection means. By having the detection means on the same configuration, detection accuracy can be improved by detecting reaction detection more closely and directly. Furthermore, by configuring the thermal control wiring and the fluorescence detection wiring matrix configuration as the same pixel, the detection signal is detected in real time more than a device that is currently required as a separate means as a detection means, and further miniaturization is realized. Can make it possible.

  Alternatively, in the present invention, it is desirable to use a temperature control thin film transistor as the control medium of the temperature control mechanism and use a fluorescence detection EL element as the detection medium of the fluorescence detection mechanism. As a result, it is possible to realize the same configuration control of the fluorescence detection simultaneously with the thermal control that is closely installed in the well, thereby eliminating the need for providing the reaction detection unit separately, for example, another configuration unit such as a detection substrate. By having the detection means on the same configuration, detection accuracy can be improved by detecting reaction detection more closely and directly. Furthermore, by configuring the thermal control wiring and the fluorescence detection wiring matrix configuration as the same pixel, it is possible to detect detection signals in real time more than devices currently required as separate means as detection means, and further miniaturization. Realization can be possible.

  Thus, by providing the pixel circuit of the EL element L dedicated to light reception in the pixel circuit of the thin film transistor element T which is the thermal control medium, thermal control and fluorescence detection can be performed in the same pixel layer, and real-time detection is performed. Can be performed with high accuracy. In addition, a complicated structure such as a laminated structure is not necessary for device creation, and the device can be further reduced in size.

  In the present invention, it is desirable to provide the Peltier element 18 in order to control the temperature of the reaction region A1. In the PCR cycle, it is necessary to control the temperature in accordance with the steps of “thermal denaturation → annealing (primer hybridization) → extension reaction”, but by providing the Peltier element 18, constant temperature control is easier, Accurate temperature control can be performed. For example, the temperature in the reaction region A1 can be maintained in advance at the lowest temperature of the PCR cycle (for example, 55 ° C.).

  In the real-time PCR apparatus 1 according to the present invention, a commonly used PCR method can be performed. Specifically, (1) target DNA to be amplified, (2) at least two oligonucleotide primers that specifically bind to the target DNA, (3) buffer, (4) enzyme, (5) dATP, By using a deoxyribonucleotide triphosphate such as dCTP, dGTP, dTTP, etc., the cycle of “thermal denaturation → annealing (primer hybridization) → extension reaction” is repeated to amplify the target DNA to a desired amount. Can do so.

  Hereinafter, an example of a measurement procedure using the real-time PCR apparatus 1 according to the present invention will be described.

  Primers having different gene sequences designed in advance are introduced into each reaction region A1. The charging method is not particularly limited, and can be, for example, a method using an inkjet or the like. In this manner, a solution containing each primer is dropped into each reaction region A1 and dried.

  Subsequently, the total RNA extracted from the specimen is transcribed into cDNA by the reverse transcription method and put into each reaction region A1. In addition to this, deoxynucleotide triphosphate (dNTP), intercalator (“SYBR (registered trademark) GREEN I”), an enzyme DNA polymerase necessary for DNA extension amplification reaction, etc., which are raw materials of each base necessary for amplification .

  In the heat denaturation step, the reaction zone A1 is set by the heating unit 16 or the like so as to be 95 ° C., and the double-stranded DNA is denatured to form a single-stranded DNA. In the subsequent annealing step, the primer is bound to the single-stranded DNA-complementary base sequence by setting the reaction region A1 to be 55 ° C. In the next DNA extension step, the reaction region A1 is set to be 72 ° C., and the primer is used as a starting point for DNA synthesis to advance the polymerase reaction to extend the cDNA.

  For each temperature cycle of “95 ° C. (thermal denaturation) → 55 ° C. (primer hybridization) → 72 ° C. (DNA extension)”, the cDNA in each reaction region A1 is amplified by a double amount. And the temperature in each reaction area | region A1 can be controlled to the optimal value of the designed primer reaction by the heating part 16 each installed in each reaction area | region A1. In addition, since the primer hybridization time and the polymerase reaction time can be controlled, generation of unnecessary reaction byproducts can also be controlled. As a result, since the amplification rate of the gene (cDNA) in each reaction region A1 can be made constant, a highly accurate PCR reaction can be performed.

  SYBR GREEN I intercalates into ds-DNA produced during the DNA replication reaction. This SYBR GREEN I is a substance that intercalates into ds-DNA and then emits fluorescence when irradiated with excitation light L2 (excitation light wavelength: 497 nm, emission wavelength: 520 nm).

  Thereby, at the time of DNA replication by the DNA polymerase, the light L1 from the light source 12 is excited as the excitation light L2 via the excitation light scanning plate 13 to excite the intercalated SYBR GREEN I to emit the fluorescence L3. The light emission amount of the fluorescence L3 is measured by the fluorescence detection unit 11 for each temperature cycle and quantified. Then, based on the correlation between the number of temperature cycles and the amount of luminescence corresponding thereto, the initial cDNA amount can be obtained as the gene expression amount.

  FIG. 4 is a conceptual view of the second embodiment of the real-time PCR device according to the present invention as viewed from the side. Hereinafter, the difference from the first embodiment will be mainly described, and the description of the common parts will be omitted.

  This real-time PCR device 2 is common to the first embodiment in that it includes a fluorescence detection unit 25 and a heating unit 26 for each reaction region (well) A2. However, the difference is that, for example, the fluorescence L3 transmitted through the reaction region A2 is detected by irradiating the excitation light L2 from above the well substrate 21.

  In the real-time PCR device 2, the excitation light L <b> 1 emitted from the light source 22 is guided to the reaction region A <b> 2 by the excitation light scanning plate 23. In the excitation light scanning plate 23, the excitation light L 1 passes through the spacer 231, and the excitation light L 2 is introduced into the well substrate 21 by the reflective film 232 and the filter 233.

  And this excitation light L2 emits fluorescence L3 by irradiating the fluorescent substance etc. of the probe in the reaction liquid in reaction region A2. This fluorescence L3 is detected and measured by a fluorescence detection unit 25 provided below the reaction region A2.

  Further, the temperature control is performed by the heating unit 26 provided below the reaction region A1, and can be controlled at a constant temperature by the Peltier element 28 or the like. Further, the fluorescence detection unit 25 and the heating unit 26 are provided on the measurement substrate 27.

  According to the present invention, gene expression levels can be comprehensively analyzed in a short time by performing individual temperature control and individual fluorescence detection.

  In a general real-time PCR apparatus, a reaction time of 25 to 30 minutes is required to perform about 30 cycles consisting of “thermal denaturation → annealing → extension reaction”. At that time, the temperature is controlled at about 2 ° C./second. On the other hand, the real-time PCR apparatus according to the present invention can control the temperature at 20 ° C. or more / second, so that the time can be shortened by about 40 seconds per cycle, and the total of 30 cycles is about 25 minutes or less. Reaction time can be achieved.

  In addition, the annealing time and extension reaction time can be controlled with high accuracy according to the design of the primer, so that the amplification rate can be adjusted to a constant magnification (for example, 2 times, etc.), so that the detection accuracy of the gene expression level can be adjusted. Can be improved.

It is the conceptual diagram which looked at 1st Embodiment of the real-time PCR apparatus which concerns on this invention from the side. It is a conceptual diagram of an example in which the control medium of the temperature control mechanism and the detection medium of the fluorescence detection mechanism are formed in the same circuit in the real-time PCR apparatus 1 according to the present invention. FIG. 3 is an example of a circuit diagram in which a control medium of a temperature control mechanism and a detection medium of a fluorescence detection mechanism are formed in the same circuit in the same embodiment. It is the conceptual diagram which looked at 2nd Embodiment of the real-time PCR apparatus which concerns on this invention side.

Explanation of symbols

1, 2 Real-time PCR device 11, 21 Well substrate 12, 22 Light source 13, 23 Excitation light scanning plate 14, 24 Filter 15, 25 Fluorescence detection unit 16, 26 Heating unit 17, 27 Measurement substrate 18, 28 Peltier element

Claims (6)

A real-time PCR device for detecting gene expression level,
Multiple reaction zones;
A heating unit that is provided for each reaction region and heats the reaction region by a heat source having a temperature control mechanism;
Optical means capable of irradiating excitation light of a specific wavelength to all of the plurality of reaction regions;
A fluorescence detection unit that is provided for each reaction region and includes a fluorescence detection mechanism that detects the amount of received fluorescence by converting it into an electrical signal;
A real-time PCR apparatus comprising at least   The said fluorescence detection part is provided with a means to process the electrical signal detected by the said fluorescence detection mechanism based on the correlation of the electrical signal and gene expression level which were stored beforehand. The real-time PCR apparatus described.   The real-time PCR apparatus according to claim 1, wherein the control medium of the temperature control mechanism and the detection medium of the fluorescence detection mechanism are provided in the same pixel.   2. The real-time PCR apparatus according to claim 1, wherein an EL element is used for the fluorescence detection mechanism of the light receiving unit. The control medium of the temperature control mechanism is a temperature control thin film transistor,
The real-time PCR apparatus according to claim 3, wherein the detection medium of the fluorescence detection mechanism is a thin film transistor for fluorescence detection. The control medium of the temperature control mechanism is a temperature control EL element,
The real-time PCR apparatus according to claim 3, wherein the detection medium of the fluorescence detection mechanism is a thin film transistor for fluorescence detection.

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