JP4458133B2 - Nucleic acid amplification equipment - Google Patents

Description

  The present invention relates to a nucleic acid amplification apparatus. More specifically, the present invention relates to a nucleic acid amplification apparatus and the like used for gene analysis such as gene expression analysis, infectious disease inspection, and SNP analysis.

  In recent years, hybridization detection techniques such as DNA chips or DNA microarrays have been put into practical use. A DNA chip is obtained by collecting and fixing a large number of 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.

  And, it is a standard technique for quantitative analysis of a small amount of nucleic acid to verify the data obtained by this microarray by performing a nucleic acid amplification reaction such as a PCR method (Polymerase Chain Reaction). As a nucleic acid amplification technique used for quantitative analysis of a small amount of nucleic acid, methods other than the PCR method are also used. Here, the real-time PCR method will be described as an example.

  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 the trace nucleic acid. In this real-time PCR method, the PCR amplification product is monitored in real time using a dedicated device or the like in which a thermal cycler and a spectrofluorometer are integrated.

This real-time PCR detection method will be described below.
First, there is an intercalator method using SYBR (registered trademark) Green I. In the intercalator method, an intercalator having a property of emitting fluorescence by binding to double-stranded DNA is used. This intercalator is bound to double-stranded DNA generated in the course of the PCR reaction, and fluorescence is emitted by irradiating it with excitation light. By detecting this fluorescence intensity, the amount of PCR amplification product produced is monitored. In this intercalator method, it is not necessary to design and synthesize a fluorescently labeled probe specific to the target, and can be easily used for measuring various targets.

  The probe method is used when it is desired to distinguish and detect sequences having similar structures, or when multiplex detection is required, such as typing of SNPs. Examples of the probe method include the TaqMan (registered trademark) probe method using, as the probe II, an oligonucleotide in which the 5 ′ end is modified with a fluorescent substance and the 3 ′ end is modified with a quencher substance.

  The TaqMan probe specifically hybridizes to the template DNA in the annealing step, but does not emit fluorescence because the quencher substance exists on the probe and is quenched even when irradiated with excitation light. However, in the extension reaction step, the TaqMan probe hybridized to the template DNA is degraded by the 5 ′ → 3 ′ exonuclease activity of Taq DNA polymerase. As a result, the fluorescent substance is released from the probe, the suppression by the quencher is released, and fluorescence is emitted. By detecting this fluorescence intensity, the amount of PCR amplification product produced can be monitored.

  The procedure for quantifying the gene expression level by real-time PCR by the above method will be described in more detail. First, PCR is performed using a standard sample with a known concentration that has been serially diluted as a template, and the number of cycles (threshold cycle; Ct value) that reaches a certain amount of amplified product is obtained. A calibration curve is created by plotting the initial DNA amount on the vertical axis with the Ct value on the horizontal axis. Based on this, a PCR reaction is performed on a sample with an unknown concentration under the same conditions to obtain a Ct value. The target DNA amount in the sample is measured from the Ct value and the calibration curve.

As technologies relating to these, Patent Literature 1 and Patent Literature 2 disclose technologies relating to temperature control and the like during an amplification reaction.
Japanese translation of PCT publication No. 2003-525617. JP 2001-136554 A.

  In a nucleic acid amplification apparatus, it is desired to analyze the amount of target nucleic acid with high accuracy. For that purpose, it is necessary to make the amplification rate of the sample (gene) constant. Accordingly, the main object of the present invention is to provide a nucleic acid amplification apparatus that can control the amplification rate of a gene with high accuracy.

First, the present invention provides a nucleic acid amplification apparatus for performing a nucleic acid amplification reaction, comprising a plurality of wells for performing the nucleic acid amplification reaction, each of the plurality of wells having a individually controllable heating unit provided for each well. A nucleic acid amplification device comprising at least optical means capable of irradiating excitation light of a specific wavelength and a fluorescence detection unit provided for each well is provided. By providing the heating unit and the fluorescence detection unit for each well, the reaction in the well can be individually controlled for each well.

  Subsequently, the present invention provides a nucleic acid amplification apparatus that uses at least the PCR method for the nucleic acid amplification reaction. In the PCR method, nucleic acid amplification is performed at a predetermined temperature cycle. However, the nucleic acid amplification device according to the present invention can control the temperature cycle performed in each well individually and with high accuracy, so that analysis with higher accuracy is possible.

  The present invention also provides a nucleic acid amplification apparatus that performs the nucleic acid amplification reaction isothermally. When used as a so-called isothermal nucleic acid amplification apparatus, the amplification rate of the nucleic acid amplification reaction in each well can be made uniform by controlling the temperature of each well. As a result, even with the isothermal amplification method, analysis with higher accuracy becomes possible.

  The present invention also provides a nucleic acid amplification device in which the heating temperature and heating time of the well are individually controlled by the heating unit corresponding to the well. By controlling the heating temperature and the heating time for each well by the heating unit, the amplification reaction in the well can be controlled with higher accuracy.

  Furthermore, the present invention provides a nucleic acid amplification device in which the heating unit is formed of a thin film transistor and performs switching control. The temperature control of each well can be performed individually using the switching of the thin film transistor.

  In addition, the present invention provides a nucleic acid amplification device in which the heating unit is formed of a heating resistor and is controlled to be switched by a thin film transistor. The temperature in each well can be individually controlled by controlling the value of the current flowing through the heating resistor using the switching of the thin film transistor.

  And this invention provides a nucleic acid amplifier provided with the Peltier device which carries out constant temperature control. By using a Peltier element, the temperature in the well can be easily controlled.

  In addition, the present invention provides a nucleic acid amplification device, wherein the optical means includes at least a light source that emits excitation light having a specific wavelength and a light guide plate that introduces the excitation light into all of the plurality of wells. Thereby, excitation light can be introduced into all the wells from the light source.

  Subsequently, the present invention provides a nucleic acid amplification device comprising a filter film that transmits light of a specific wavelength between the well and the fluorescence detection unit. By providing the filter film, the detected fluorescence can be efficiently extracted.

  The present invention also provides a nucleic acid amplification device comprising a filter film that transmits light of a specific wavelength between the well and the light guide plate. By providing the filter film, excitation light to be irradiated to each well can be efficiently extracted.

  Furthermore, the present invention provides a nucleic acid amplification device in which the light source is a light emitting diode. By using a light emitting diode as a light source, light that does not contain unnecessary ultraviolet rays, infrared rays, or the like can be easily obtained.

  The nucleic acid amplification apparatus according to the present invention can comprehensively analyze the gene expression level 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 diagram of a first embodiment of a nucleic acid amplification device according to the present invention as viewed from the side. 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 nucleic acid amplification apparatus according to the present invention. The size and layer structure of the nucleic acid amplification device 1 can be appropriately selected according to the purpose, and the configuration of the nucleic acid amplification device 1 can also be designed or changed within the scope of the object of the present invention.

  The configuration of the nucleic acid amplification device 1 will be specifically described. The nucleic acid amplification device 1 detects a reaction substrate 11, a heating unit 12 that heats the reaction substrate 11, a Peltier element 13, a light source 14, a light guide plate 15 that guides excitation light to the reaction substrate 11, and fluorescence. A fluorescence detection unit 16, a filter film 17 that transmits only light of a specific wavelength, and a measurement substrate 18 are provided.

  The reaction substrate 11 includes a plurality of wells (reaction regions) A1 and performs a predetermined reaction in the wells A1. In the present invention, the shape and the like of the well A1 are not particularly limited, and a suitable shape and capacity can be appropriately set.

  The capacity of the well A1 is not particularly limited, but is preferably a microspace, and specifically, a capacity of 1 μL or less. By using such a micro space, a small amount of reaction solution is required for the well A1, so that temperature control and the like can be performed with high accuracy and the reaction time can be shortened.

  For example, when the well A1 is 300 μm × 300 μm × 300 μm (well capacity: 27 nL), even if about 40,000 wells A1 are provided on the reaction substrate 11, a device having an area of about 6 cm square may be used. Since the device can be miniaturized in this way, the number of wells A1 equal to the number of human genes can be arranged on the reaction substrate 11 in a matrix, and comprehensive analysis can be performed more easily and easily. be able to.

  The material or the like of the reaction substrate 11 is not particularly limited as long as it is a material that can transmit the excitation light L1, and can be appropriately selected in consideration of the measurement purpose, ease of processing, and the like. For example, a low fluorescent light emitting plastic or glass can be used as the material of the reaction substrate 11.

  The reaction substrate 11 may be detachable from the heating unit 12, the fluorescence detection unit 16, the filter film 17, and the like. Or although not shown in figure, the reaction substrate 11 and the heating part 12 are made into an integral structure, and this may be made removable.

  The heating unit 12 heats each well A <b> 1 in the reaction substrate 11. This controls the temperature of the well A1. And it is desirable that the heating temperature and heating time of the well A1 can be individually controlled by the heating unit 12 corresponding to the well A1. By controlling the heating temperature and the heating time for each well A1 by the heating unit 12, the amplification reaction and the like in the well 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 the present invention, the temperature of each well can be controlled independently by providing the heating unit 12 in each well A1. Furthermore, by providing the heating unit 12 corresponding to each well A1, the heating time during the amplification reaction can be individually controlled.

  The structure or the like of the heating unit 12 is not particularly limited, but is preferably a heater that is formed of a thin film transistor (TFT) and that is switching-controlled. The temperature control of each well A1 can be performed individually using the switching function of the thin film transistor. In the temperature control, the voltage applied to the thin film transistor may be controlled to change the current value between the source and the drain, or the current between the source and the drain may be controlled as a constant current power source. In particular, it is desirable to use thin film transistors as heat sources and thin film transistors as switching elements, and to form these thin film transistors in the same circuit. As a result, the heat source and the switching control can be collectively controlled in the same circuit.

  Alternatively, the heating unit 12 may be a heater formed of a heating resistor and controlled to be switched by a thin film transistor. That is, the thin film transistor may be used only for switching. As the heating resistor, platinum (Pt), molybdenum (Mo), tantalum (Ta), tungsten (W), silicon carbide, molybdenum silicide, nickel-chromium alloy, iron-chromium-aluminum alloy, or the like can be used. In this case, the temperature can be controlled by controlling the value of the current flowing through the heating resistor.

  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.

  In the present invention, it is desirable to provide the Peltier element 13 in order to control the temperature of the well A1. The temperature of the entire well can be controlled by the Peltier element, and the temperature of each well A1 can be controlled more precisely in each heating unit 12. This makes it possible to accurately control minute temperature variations and deviations of the wells A1. By using the Peltier element 13, constant temperature control can be easily performed. As a result, highly accurate temperature control can be performed.

  Further, when performing a PCR cycle, it is necessary to control the temperature in accordance with the steps of “thermal denaturation → annealing (primer hybridization) → extension reaction”. For example, the temperature in the well A1 can be maintained in advance at the minimum temperature of the PCR cycle (for example, 55 ° C.). Then, the temperature cycle of the whole well can be controlled by the Peltier element 13, and the temperature variation and deviation of each well A 1 can be individually corrected and controlled by each heating unit 12. Thus, more accurate temperature control can be performed by performing temperature control of the whole well and temperature control of each individual well.

  In the present invention, a light source 14 or a light guide plate 15 for introducing excitation light into each well A1 can be used as an optical means capable of irradiating excitation light having a specific wavelength to all of the plurality of wells A1.

  The light source 14 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 14 and the number of light sources are not particularly limited. Although not shown, a plurality of light sources 14 may be provided so as to correspond to the respective wells A1, and each light source 14 may directly irradiate the excitation light toward the corresponding well A1. In this case, for example, since each well A1 can be directly irradiated by the light source 14, it is possible to perform uniform excitation irradiation to all the wells by taking a larger amount of excitation light and controlling the excitation light amount individually.

  The light guide plate 15 is for guiding the excitation light L <b> 1 emitted from the light source 14 to each well A <b> 1 in the reaction substrate 11. Excitation light L 1 emitted from the light source 14 is introduced into the spacer 151 inside the light guide plate 15. A reflective film 152 is provided on the light guide plate 15, and the excitation light L2 can be introduced into the reaction substrate 11 by using, for example, a dichroic mirror. Thereby, the fluorescent substance in the reaction solution in each well A1 can be excited with a uniform amount of light.

  In the present invention, it is desirable to provide a filter film 153 that transmits only the wavelength light of the excitation lights L1 and L2 at the bottom of the light guide plate 15. Thereby, the excitation light L2 can be efficiently extracted from the light emitted from the light source 14 and guided to the well A1. As the filter film 153, for example, a polarizing filter or the like can be used.

  In response to the excitation light L2 irradiated to the well A1, the fluorescence detection unit 16 detects and measures the fluorescence emitted when the fluorescent dye in the intercalated probe is excited. In the present invention, the configuration of the fluorescence detection unit 16 is not limited, and for example, a photodiode can be used.

  The fluorescence detection unit 16 is formed on the same glass substrate 15 as the heating unit 12, but may be formed on a different substrate, or the heating unit 12 and the fluorescence detection unit 16 may be stacked. Good.

  In the present invention, it is desirable to provide a filter film 17 that transmits only the wavelength light of the fluorescence L3 between each well A1 and each fluorescence detection unit 16 corresponding thereto. By providing the filter film 17 that transmits only light of a predetermined wavelength between the well A1 and the fluorescence detection unit 16, the detected fluorescence L3 can be efficiently extracted, so that a more accurate analysis can be performed. it can. For example, a polarizing filter can be used as the filter film 17.

  Thus, since the nucleic acid amplification device 1 according to the present invention has the fluorescence detection section 16, fluorescence detection can be performed in real time. In addition, by providing the fluorescence detection unit 16 individually corresponding to each well A1, individual and highly accurate detection can be performed for each well A1.

  And since the fluorescence detection in real time is possible, the detection result of the fluorescence detection part 16 can be fed back to the temperature control mechanism of the heating part 12. For example, the heating amount of the heating unit 12 can be controlled based on the reaction result (gene amplification amount) in the well A1 detected by the fluorescence detection unit 16. In this way, by feeding back the detection result of the fluorescence detection unit 16 to the heating unit 12, the gene amplification reaction of each well A1 can be controlled with higher accuracy.

  Further, the heating unit 12 and the fluorescence detection unit 16 can be provided on the measurement substrate 18. In the present invention, the position where the measurement substrate 18 is provided is not limited to the position below the heating unit 12 or the fluorescence detection unit 16 and can be provided in a suitable place. The material of the measurement substrate 18 used in the present invention is not particularly limited, and for example, a glass substrate or various resin substrates can be used.

  In the nucleic acid amplification device 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 nucleic acid amplification device 1 according to the present invention will be described.

  Primers having different base sequences designed in advance are put into each well A1. The charging method is not particularly limited, and can be, for example, a method using an inkjet or the like. Thus, the solution containing each primer is dropped into each well A1 and dried.

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

  The nucleic acid amplification device 1 according to the present invention can also be used as a real-time PCR device for performing RT-PCR reaction (Reverse Transcriptase Polymerase Chain Reaction). Since the normal PCR method uses DNA polymerase, it cannot be amplified when the analysis target is RNA. In contrast, the RT-PCR method is a method in which RNA is reverse transcribed to DNA by reverse transcriptase and then PCR is performed. According to the nucleic acid amplification device 1 of the present invention, the RT-PCR reaction is Can also be done.

  In addition, a nested primer (nested-PCR method) may be used in order to prevent amplification of a DNA fragment that is not targeted in the PCR method. And when performing PCR method in the nucleic acid amplifier 1 which concerns on this invention, it is needless to say that RT-PCR method and nested-PCR method may be used together.

  In the heat denaturation step, setting is performed by the heating unit 12 or the like so that the inside of the well A1 is 95 ° C., and the double-stranded DNA is denatured to form a single-stranded DNA. In the subsequent annealing step, the primer is set to 55 ° C. so that the primer binds to a base sequence complementary to the single-stranded DNA. In the next DNA extension step, the temperature in the well A1 is set to 72 ° C., so that 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 well A1 is amplified twice. And the temperature in each well A1 can be controlled to the optimal value of the designed primer reaction by the heating part 12 installed in each well 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 well A1 can be made constant, an accurate PCR reaction can be performed.

  SYBR Green I intercalates into ds-DNA generated 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 14 passes through the light guide plate 15 as excitation light L2 to excite the intercalated SYBR Green I to emit 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. 2 is a conceptual view of the second embodiment of the nucleic acid amplification device according to the present invention 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.

  The nucleic acid amplification device denoted by reference numeral 2 in FIG. 2 includes a reaction substrate 21, a heating unit 22 that heats the reaction substrate 21, a Peltier element 23, a light source 24, and a guide that guides excitation light to the reaction substrate 21. An optical plate 25, a fluorescence detection unit 26 that detects fluorescence, and a measurement substrate 27 are provided.

  This nucleic acid amplification device 2 is common to the first embodiment in that each well A2 includes a heating unit 22 and a fluorescence detection unit 26. However, there is a difference in that the fluorescence L3 is detected by irradiating the excitation light L2 from below the reaction substrate 21 and reflecting it in the well A2. Here, the shape of the well A2 has a curved surface portion for reflecting the fluorescence L3.

  In the nucleic acid amplification device 2, the excitation light L <b> 1 emitted from the light source 24 is irradiated to the well A <b> 2 by the light guide plate 25. In the light guide plate 25, the excitation light L <b> 1 passes through the spacer 251, and the excitation light L <b> 2 is introduced into the reaction substrate 21 by the reflective film 252 and the filter film 253. And this excitation light L2 emits fluorescence L3 by irradiating the fluorescent substance of the probe in the reaction liquid in well A2. The fluorescence L3 is reflected by the wall surface in the well A2, and is detected and measured by the fluorescence detector 26 provided below the well A2.

  Further, the temperature control is performed by the heating unit 22 provided below the well A2, and can be controlled by the Peltier element 23 or the like.

  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, in the apparatus of the present invention, the temperature can be controlled at 20 ° C. or more / second, so the time can be shortened by about 40 seconds per cycle, and the reaction time of about 25 minutes or less in the entire 30 cycles. Can be achieved.

  In the nucleic acid amplification device according to the present invention, the heating temperature and the heating time can be independently controlled and individually detected by installing a heating unit and a fluorescence detection unit for each well. Thereby, the expression level of the gene and the like can be analyzed with high accuracy in a short time.

  Furthermore, by using an apparatus in which the excitation optical system is integrated as the fluorescence detection unit, the detection system can be downsized. In addition, by making the well a micro space and reducing the reaction region, a comprehensive analysis can be efficiently performed even with a small device. As a result, the nucleic acid amplification apparatus according to the present invention enables comprehensive analysis.

  The nucleic acid amplification device according to the present invention can also be used as an isothermal nucleic acid amplification device that performs a nucleic acid amplification reaction isothermally. The term “isothermal” refers to a substantially constant temperature condition where the enzyme, primer, etc. used can function substantially. The “substantially constant temperature condition” means not only to maintain the set temperature strictly but also to an allowable temperature if the temperature change does not impair the substantial functions of the enzyme and primer to be used. It also includes holding within the range.

In the present invention, the following isothermal amplification method can be used in addition to the PCR method described above.
IVT method (In Vitro Transcription) for obtaining RNA from dsDNA as an amplification product;
TRC method (Transcription Reverse transcription Concerted amplification) which trims RNA using RNase H activity of reverse transcriptase and obtains RNA as an amplification product;
NASBA method (Nucleic Acid Sequence-Based Amplification) which obtains RNA as an amplification product from dsNDA using RNA polymerase, using dsDNA incorporating RNA promoter using reverse transcriptase or the like;
SPIA method for obtaining ssDNA from RNA as an amplification product using a primer having a chimeric structure of RNA and DNA;
LAMP method (Loop-Mediated Isothermal Amplification) that uses DNA loop formation to obtain dsDNA from DNA or RNA as an amplification product at a constant temperature;
A SMAP method (SMart Amplification Process) that uses a combination of multiple enzymes to obtain dsDNA as an amplification product from dsDNA while accurately discriminating a single base difference (SNP);
Examples include ICAN (Isothermal and Chimeric primer-initiated Amplification of Nucleic Acids), which uses a synthetic chimeric primer in which DNA and RNA are bound to obtain dsDNA from dsDNA as an amplification product.

  The isothermal amplification method that can be performed in the present invention is not limited to the above-mentioned isothermal amplification method, and it is needless to say that each of the above-described methods is not limited to the above contents. That is, the isothermal amplification method that can be performed in the present invention includes all methods for amplifying nucleic acids without performing any temperature cycle.

The isothermal amplification method does not require a temperature cycle (for example, 95 ° C. → 55 ° C. → 72 ° C.) as in the above-described PCR method, and thus has an advantage that an apparatus such as a thermal cycler is unnecessary. However, since a difference in amplification rate depending on the structure and chain length of the nucleic acid to be amplified occurs, it has been difficult to perform quantitative analysis.

  In this regard, according to the nucleic acid amplification device according to the present invention, the difference in nucleic acid amplification rate in each well can be corrected by individually controlling the reaction temperature of each well. Thereby, the reaction efficiency of the nucleic acid amplification reaction in each well can be made uniform. As a result, even when a plurality of nucleic acid amplification reactions are performed in a plurality of wells, the amount of nucleic acid amplified in each well can be quantitatively known from one calibration curve. Therefore, the nucleic acid amplification apparatus according to the present invention can quantitatively analyze the expression level.

  Furthermore, even when the appropriate reaction temperature condition is unknown when performing a predetermined nucleic acid amplification reaction, the reaction efficiency of the nucleic acid amplification reaction is fed back by feeding back the appropriate reaction temperature from the initial result of the amplification reaction. Can be made constant. First, the correlation between the amount of amplification and the temperature at that time is detected within a fixed time from the start of the amplification reaction. Then, by feeding back the result to the temperature control mechanism of the heating unit, it is possible to determine an appropriate reaction temperature condition for performing the amplification reaction.

When performing a quantitative reaction, the isothermal amplification method is difficult to apply compared to the PCR method. The main reason is that there is a large difference in amplification rate depending on the structure and chain length of the nucleic acid to be amplified. In quantitative analysis using a nucleic acid amplification reaction, it is necessary to make a comparison with a control having the same amplification rate, and such a difference in amplification rate makes quantitative analysis difficult. In particular, when quantifying multiple types of genes at the same time, it is difficult to create a calibration curve for each target gene, and it is necessary to compare those with matching amplification factors.

  On the other hand, in the nucleic acid amplification device according to the present invention, even when nucleic acid is amplified by an isothermal amplification method, an amplification reaction can be performed at an optimum temperature according to each gene or primer to be amplified. Therefore, the nucleic acid amplification rate in each well can be controlled to be constant. As a result, it is possible to perform quantitative analysis with high accuracy even with the isothermal amplification method.

  And since it reacts isothermally, the structure of the thermal cycler etc. which control a temperature cycle is unnecessary. Therefore, it can contribute more to miniaturization as a device. And it becomes possible to comprehensively analyze the quantity of many kinds of genes on the same device. Therefore, the nucleic acid amplification apparatus according to the present invention can be a high-throughput type gene analysis apparatus that can perform an isothermal amplification method in the form of a chip, a substrate, or the like provided with a microchannel.

It is the conceptual diagram which looked at 1st Embodiment of the nucleic acid amplification device concerning the present invention from the side. It is the conceptual diagram which looked at 2nd Embodiment of the nucleic acid amplifier which concerns on this invention from the side.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1, 2 Nucleic acid amplifier 11, 21 Reaction board | substrate 12,22 Heating part 13,23 Peltier element 14,24 Light source 15,25 Light guide plate 16,26 Fluorescence detection part 17 Filter film | membrane 18,27 Measurement board

Claims (11)

A nucleic acid amplification apparatus for performing a nucleic acid amplification reaction,
A plurality of wells for performing the nucleic acid amplification reaction,
An individually controllable heating section provided for each well;
Optical means capable of irradiating excitation light of a specific wavelength to all of the plurality of wells;
A fluorescence detector provided for each well;
A nucleic acid amplification apparatus comprising at least   The nucleic acid amplification apparatus according to claim 1, wherein the nucleic acid amplification reaction is performed by at least a PCR (polymerase chain reaction) method. The nucleic acid amplification apparatus according to claim 1 or 2 , wherein the nucleic acid amplification reaction is performed isothermally. The nucleic acid amplification apparatus according to any one of claims 1 to 3 , wherein a heating temperature and a heating time of the well are individually controlled by the heating unit corresponding to the well. The nucleic acid amplification device according to any one of claims 1 to 4, wherein the heating unit is formed of a thin film transistor and is switching-controlled. The nucleic acid amplification apparatus according to any one of claims 1 to 5, wherein the heating unit is formed of a heating resistor and is controlled to be switched by a thin film transistor. The nucleic acid amplification device according to any one of claims 1 to 6, further comprising a Peltier element that performs constant temperature control. The optical means includes
A light source that emits excitation light of a specific wavelength;
A light guide plate for introducing the excitation light into each well;
The nucleic acid amplification device according to any one of claims 1 to 7, further comprising: The nucleic acid amplification device according to any one of claims 1 to 8, further comprising a filter film that transmits light of a specific wavelength between the well and the fluorescence detection unit. The nucleic acid amplification device according to claim 8 or 9 , further comprising a filter film that transmits light of a specific wavelength between the well and the light guide plate. The nucleic acid amplification device according to any one of claims 8 to 10 , wherein the light source is a light emitting diode.