A polymerase chain reaction device capable of detecting more than one fluorescent signal in real time

Abstract

The present invention relates to a polymerase chain reaction device capable of detecting more than one fluorescent signal in real time. The device performs polymerase chain reaction by controlling the temperature range of the reagent container storage space, and detects the generated fluorescent signal by adding a specific probe, a fluorescent substance, a light source, and a spectrometer. At the same time, the device is also preloaded with an algorithm for real-time analysis and quantification of fluorescent signals.

Description

Technical Field

[0001] The present invention relates to a polymerase chain reaction device. More specifically, the present invention relates to a polymerase chain reaction device capable of instantly quantifying more than one fluorescent signal. Background Art

[0002] Polymerase Chain Reaction (PCR) is a technology that rapidly amplifies DNA signals. Its principle and main operation steps are as follows: (a) Denaturation: using high temperature (90-95°C) to dissociate double-stranded DNA into single-stranded DNA, which is then used as a template for replication; (b) Primer annealing: when the temperature is lowered to an appropriate level, the primer will attach to the correct target gene location; (c) Primer extension: when the reaction temperature is adjusted to 72°C, DNA polymerase will attach deoxyribonucleotide triphosphates (dNTPs) to the primers one by one to synthesize another new DNA fragment.

[0003] The nucleic acid signal is amplified by repeating the three steps of denaturation-primer binding-primer extension. Each time the three steps are repeated, the number of target genes can be doubled. If the three steps are repeated for a total of 40 cycles, the number of target genes can be amplified by nearly 10 9 PCR can obtain a large number of target gene fragments in vitro. Therefore, as one of the molecular diagnostic techniques currently used in a large number of clinical diagnosis, it can be applied to projects including diagnosis of genetic diseases, diagnosis of pathogens, diagnosis and prognosis evaluation of tumors and cancers, as well as basic research. Therefore, it is also a technology currently used in a large number of clinical diagnosis.

[0004] In recent years, in response to the needs of technological development, real-time polymerase chain reaction (RT-PCR), also known as quantitative real-time polymerase chain reaction (Q-PCR) (hereinafter referred to as real-time PCR) has been developed. Although both real-time PCR and traditional PCR use thermal cycling steps to amplify the trace amount of DNA of the target gene to achieve the purpose of amplification, the difference between the two is that real-time PCR adds non-specific fluorescent substances or specific fluorescent probes to the PCR reaction. After each PCR amplification cycle, the target gene DNA is amplified and a fluorescent signal is generated. The fluorescent signal of the generated product is detected and recorded. After the PCR reaction is completed, the cycle number and the fluorescent signal are plotted to obtain a reaction curve graph, which fully presents the product generation situation of each cycle in the PCR reaction. Then, the built-in program analysis is used to achieve real-time quantitative results.

[0005] The currently commonly used non-specific fluorescent dye is SYBR Green 1, which releases fluorescence after binding to the minor groove of DNA. Therefore, during the PCR process, the fluorescence intensity can be measured at the end of the primer extension step of each cycle to know how many PCR products are produced in each cycle. However, since SYBR Green I binds to all double-stranded DNA, it is impossible to distinguish between specific products and non-specific products. Therefore, its specificity for products is poor, and sometimes false positive test results will occur.

[0006] The currently commonly used specific fluorescent probe is TaqMan probe, which is an artificially synthesized oligonucleotide that is specific to the target gene sequence and is labeled with different fluorescent substances at both ends of the oligonucleotide. The 5' end fluorescence is called the reporter and the 3' end fluorescence is called the quencher. If the specific probe is in a free state, the interaction between the reporter and the quencher will shield each other's fluorescence, so no fluorescence will be generated; but when the PCR product is generated, the specific probe is hydrolyzed, and the quencher loses the effect of shielding the quencher, so the fluorescence of the reporter can be detected. Since the specific probe is the only oligonucleotide that is specific to the target gene, it will not bind to other non-specific products. Currently, the fluorescent dyes commonly used in combination with TaqManprobe include FAM, VIC, HEX, ROX, CY3, CY5, CY5.5, JOE, TET, Texas Red, TAMRO, NED, Quasar705, Alexa488, Alexa546, Alexa594, Alexa633, Alexa643, and Alexa680.

[0007] Each of these fluorescent dyes has its own optimal absorption wavelength range and scattering wavelength range, but the optimal absorption wavelength ranges and scattering wavelengths of the above fluorescent dyes also overlap. Therefore, if more than two fluorescent dyes are needed to quantify two or more different target genes in the same sample in the same test tube at the same time, in practice, fluorescent dyes that are less likely to produce a high degree of signal overlap (crosstalk) are selected for combination use, and the acquired data are analyzed in a traditional data method to finally obtain a distinguishable fluorescent signal to assist in interpretation; another method is to test only one target gene of the sample in one test tube at a time, but at the same time, several test tubes are used to detect different target genes of the same sample, and finally the test results of the several detection targets of the sample are combined to avoid the situation where the data cannot be analyzed due to the overlap of fluorescent signals. The advantage of the former is that no matter how many different target genes are tested, they can be conducted in the same test tube, which will not cause waste of consumables. The disadvantage is that because there are multiple sources of fluorescent signals, signal overlap will occur, resulting in errors in data analysis, or it is impossible to effectively identify which target gene the overlapping signal part belongs to, which can easily lead to misjudgment of experimental results; the latter can effectively solve the problem of signal overlap because only one target gene is tested in one test tube at a time, and then multiple test results are combined for judgment. The accuracy of signal interpretation is also much higher than the former, but it will cause waste of consumables and require more samples. In practice, if there is a need to quantify multiple target genes in the same sample, the latter method is mostly used.

[0008] Currently, the devices commonly used in laboratories for real-time PCR mostly use temperature-controlled metal as a heater. By taking advantage of its rapid heating and cooling characteristics, the device performs repeated heating and cooling operations to achieve the reaction temperature of the three steps of denaturation, primer binding, and primer extension. The heat is transferred to the reagents and reactants (which contain fragments of the target gene) in the test tube by heating the reagent container made of plastic, so as to achieve the effect of amplifying the target gene signal and detecting the fluorescent signal. However, such a machine that uses temperature-controlled metal to repeatedly heat and cool is generally large in size. In order to achieve effective temperature control, the entire temperature control system must have a larger volume and heat capacity ratio. In addition, according to the current design of the machine, most of the time is spent waiting for the temperature-controlled metal to heat up or cool down to the reaction temperature. For example, the number of cycles required for a general test is about 30-35 times, and the reaction time required by the traditional machine is about two to three hours, which makes it difficult to reduce the reaction time. Therefore, it cannot be used in tests that require results in a very short time.

[0009] In order to improve the problems of traditional devices, some researchers have developed real-time PCR applied to microfluidic chip technology. The advantage of this technology is that the microfluidic chip can reduce the volume of reagents or reactants and the overall heat capacity ratio, thereby reducing the reaction time and reagent consumption. However, since this technology still requires repeated heating and cooling in three different temperature ranges, there is still the problem of too long heating and cooling time.

[0010] Another developed real-time PCR microfluidic chip eliminates the setting of repeated heating and cooling of the heater, and instead uses a specially designed driving force to pressurize the reactants and reagents in the flow channel, so that the reactants and reagents repeatedly flow through three different temperature ranges in the specially processed and designed flow channel, thereby achieving the effect of amplifying the target gene and detecting the signal. Although the use of this technology for real-time PCR can eliminate the time consumption caused by heating and cooling, the system of this technology needs to include a complex pressurization system and a liquid drive system, and the liquid drive system is closely related to the volume and viscosity of the liquid, which causes difficulties in system and instrument manufacturing and regulation, and thus indirectly limits the development of this technology.

[0011] Researchers have also developed another technology that uses thermal convection circulation to perform real-time PCR to solve the problems of high heat capacity ratio and high time consumption of traditional machines. This technology uses two heat sources at different temperatures to heat the upper and lower ends of a closed test tube containing reagents and reactants. The temperature difference between the upper and lower ends drives the reagents and reactants in the test tube to flow through different temperature ranges to perform real-time PCR reactions. Although this technology overcomes the time loss caused by repeated heating and cooling of the heater, and does not require external pressure to drive the liquid flow circulation in the test tube, since the heater is mostly block metal, the volume of the machine cannot be reduced because the heat capacity ratio cannot be reduced, and its complex temperature control mechanism and metal heating system also make the manufacturing cost high.

[0012] If multiple target genes or markers are to be quantified for the same sample, the aforementioned problems with fluorescent probe combinations and equipment will become more prominent. That is, in order to obtain a more reliable fluorescent signal value, in practice, only one target gene is tested in one test tube at a time, and then multiple test results are combined for judgment. This will result in the need for multiple test tubes for each test sample. If the device used is the machine commonly used in the aforementioned laboratory, or a microfluidic chip that repeatedly heats and cools in different temperature ranges, in addition to the problem of long test time, there is also the problem of waste of consumables and the need for a large amount of sample. Even if the test is performed on a microfluidic chip with a special flow channel design or a device that uses thermal cycling for real-time PCR testing, although the problem of long test time can be eliminated, the problem of waste of consumables and the need for a large amount of sample cannot be eliminated.

[0013] In view of the above problems, the present invention discloses a polymerase chain reaction device which can solve the problems of the aforementioned machine being too large in size, time wasting due to repeated temperature rise and fall, and can also solve the problems of waste of consumables and a large amount of sample required. Summary of the invention

[0014] The present invention relates to a polymerase chain reaction device capable of detecting more than one fluorescent signal in real time. The device comprises a reagent container accommodating space, the reagent container accommodating space is composed of a heat-resistant material, the surface layer of the heat-resistant material may comprise a heat-resistant insulating material or a conductive film coated with a conductive material (conductive material) or both; and the reagent container accommodating space may have different structural designs according to the different external profiles of the reagent container and its different temperature control conditions, and other conductive accessories such as circuit boards or conductive metal accessories may also be added to facilitate operation. The heat-resistant material may be metal or non-metal; if the heat-resistant material is metal, it may be aluminum sheet, copper sheet, or other heat-resistant metal; if the heat-resistant material is non-metal, it may be glass, plastic, or ceramic; if the heat-resistant material is metal, a layer of heat-resistant insulating material such as aluminum oxide, Teflon, and polyimide (polyimide) will be coated on the metal first.

[0015] Regardless of whether the heat-resistant material is metal or not, whether a conductive film is to be coated on its surface to provide a specific resistance value depends on the design of the heating mechanism of the reagent container accommodating space. If the heating mechanism design only includes a conductive material of a metal material and no other accessories can provide heating, the device disclosed in the present invention will coat a conductive film on the surface of the heat-resistant material. Since the conductive film has a certain resistance value, it can start heating after receiving the current from the conductive material; on the contrary, if the heating mechanism design directly includes an electric heating device, such as a circuit board with a soldered electric heating element, once the power is turned on, the reagent container accommodating space can start heating and heating, then there is no need to coat the heat-resistant material with a conductive film. The conductive material of the present invention can be tin oxide, indium oxide, zinc oxide, indium tin oxide, chromium, titanium, tantalum or copper, and the electric heating element of the present invention can be a resistor or circuit wiring (PCB layout). Through the design of the heating mechanism disclosed in the present invention, the volume of the heating mechanism can be greatly reduced, and compared with the traditional PCR machine, it has more volume advantages and derivability.

[0016] At least one temperature sensor is provided at the part of the reagent container accommodating space that contacts the reagent container. The placement position and number of the temperature sensor can be determined based on the position where the temperature change is relatively easy to detect when the reagent container is actually placed in the reagent container accommodating space without affecting the reaction. The temperature sensor is used to monitor and report the temperature at the location.

[0017] The present invention also includes a power supply, a heat sink, and a processor. The power supply is used to supply power required for the temperature rise and fall of the reagent container storage space and the operation of the entire device, the heat sink is used to cool the system, and the processor is preloaded with a program to quantify and analyze more than one fluorescent signal using an algorithm, and the algorithm can be an algorithm that sets parameters based on the least squares method.

[0018] In addition to analyzing the fluorescent signal, the aforementioned processor can also be used to control the power supply to start providing power to the reagent container accommodating space to heat up, start the heat dissipation device, and the time point of the system light source switch. The power supply is connected to the reagent container accommodating space. More precisely, the connector of the power supply can be connected to the conductive film on the surface of the reagent container accommodating space, or to other electric heating devices in the reagent container accommodating space. When the power supply starts to supply power, the reagent container accommodating space begins to heat up to a predetermined temperature. At this time, the temperature sensor set in the reagent container accommodating space will start to detect the temperature and report it to the processor. When the temperature sensor detects that the temperature of the reagent container accommodating space exceeds the maximum temperature preset by the system, the heat dissipation device will be started within a predetermined time to start cooling to the low temperature range preset by the system. The installation or position of the heat dissipation device relative to the reagent container accommodating space is not specific, as long as the device temperature can be quickly and effectively reduced. In the temperature setting of the present invention, the temperature range that allows the reagent container accommodating space to heat up is 85°C to 130°C, and the temperature range that allows the reagent container accommodating space to be low is 50°C to 75°C.

[0019] In a preferred embodiment of the present invention, the heat dissipation device may be a fan, and a thermoelectric cooler (TE cooler) or more than one fan may be used in combination with the heat dissipation device to achieve a rapid and effective temperature reduction effect, and the heat-resistant material constituting the reagent container accommodating space may also include a plurality of heat dissipation holes to accelerate the heat dissipation effect.

[0020] The device disclosed in the present invention is equipped with at least one light source, a spectrometer, a light illumination lens, and a light detection lens. The light source is used to excite fluorescent dyes or fluorescent probes to produce detectable fluorescence. The device disclosed in the present invention can also use a plurality of light sources with different excitation light wavelengths in combination. The light emitted by the light source can be injected into the aforementioned reagent container through the light excitation lens at a specific angle, and excite the fluorescent substance to generate a fluorescent signal. The generated fluorescent signal passes through the fluorescent signal emission port through the light detection lens and the spectrometer to send the signal to the processor for analysis. The light sources applicable to the present invention include LED lamps, laser lamps, or other light sources whose wavelengths match the wavelengths of fluorescent dyes or fluorescent probes, and they can also be used in combination; and the light excitation lenses and light detection lenses applicable to the present invention can be biconvex lenses, plano-convex lenses, double lenses, aspheric lenses, achromatic lenses, aberration-free lenses, Fresnel lenses, plano-concave lenses, biconcave lenses, positive / negative meniscus lenses, axicons, gradient refractive index lenses, microlens arrays, cylindrical lenses, diffractive optical elements, and holographic optical elements, and can also be a combination of the above various lenses, elements, and arrays. The processor can control the time when the light source is turned on and off, and can also control whether the spectrometer and the light source should start detecting fluorescent signals at the same time.

[0021] The device disclosed in the present invention does not have a particularly limited arrangement of the light source, spectrometer, light excitation lens, light detection lens and reagent container accommodating space. Basically, it is only necessary to confirm that the light source and its light emission path and the light excitation lens are located on one side of the reagent container accommodating space, and the light emitted by the light source can pass through the light excitation lens to guide into the reagent container to effectively excite the fluorescent dye or fluorescent probe in the reagent; another part, when the fluorescent signal is generated, can pass through the light detection lens to transmit the fluorescent signal to the spectrometer, and then transmit it to the processor to analyze the relevant signal. In a preferred embodiment of the present invention, the light source and the light excitation lens are located vertically below the reagent container accommodating space, and the light detection lens and the spectrometer are arranged at a 90-degree angle to the aforementioned light source and light excitation lens. In a preferred embodiment disclosed in the present invention, the reagent container accommodating space will be provided with a fluorescent signal emission port at the position of the fluorescent emission path to facilitate the implementation of the device disclosed in the present invention.

[0022] In the device disclosed in the present invention, the fluorescence signal range that can be detected by the spectrometer is between 340nm and 850nm. The emission spectrum (Emission Wavelength) of the fluorescent dye or fluorescent probe that can be used in the present invention must also be between 340nm and 850nm, including but not limited to the following substances: FAM, VIC, HEX, ROX, CY3, CY5, CY5.5, JOE, TET, SYBR, Texas Red, TAMRO, NED, Quasar705, Alexa488, Alexa545, Alexa594, Alexa633, Alexa643, Alexa680. If there are other fluorescent dyes whose emission spectrum falls between 340nm and 850nm, they can also be used in the present invention.

[0023] The device disclosed in the present invention can detect multiple target genes or markers in the same reagent container, that is, add more than one fluorescent dye or fluorescent probe to the same reagent container for real-time PCR, collect the fluorescent signal of each reaction, and analyze the fluorescent signal through the algorithm preloaded by the processor to achieve the qualitative and quantitative effects of multiple target genes or markers. If more than one fluorescent dye or fluorescent probe is added, the fluorescent signal will produce a signal overlap. In order to effectively and instantly detect and identify more than two fluorescent signals in the same reagent container, first, for the fluorescent dye or fluorescent probe to be used, its standard fluorescent spectrum is input into the algorithm preloaded in the processor, and the standard spectrum of the excitation light source used by the device is also input into the processor. The two are used as the reference standard values ​​of the algorithm, and then the measured original fluorescent signal and the standard spectrum are compared through the algorithm operation to obtain its fluorescent signal value. For example, if six different target genes or markers are expected to be detected in the same reagent container, six different fluorescent dyes or fluorescent probes need to be used. Therefore, the standard spectra of the six fluorescent dyes or fluorescent probes must first be input into the algorithm preloaded in the processor, and the standard spectrum of the excitation light source must also be input into the algorithm preloaded in the processor before starting real-time PCR. After the program is completed, the processor will first obtain the measured spectrum raw data, which is the total cumulative fluorescence signal of the six fluorescent dyes or fluorescent probes. Then, after deducting the background value, the algorithm is used to calculate the respective proportions of the six fluorescent signals in the fluorescence spectrum for each PCR cycle. Through calculation, the real-time fluorescence signal value of each fluorescent dye or fluorescent probe can be separated and obtained, and then the qualitative or quantitative results of the six different target genes or markers can be obtained through conversion.

[0024] When the device disclosed in the present invention is in operation, the power supply is first activated, the conductive material immediately starts to be energized, and the reagent container storage space begins to heat up. The reagent container with reactants and reagents added is placed in the reagent container storage space, and the reactants and reagents begin to undergo denaturation, primer annealing, and primer extension by rapidly and repeatedly heating and cooling the reagent container storage space. At this time, the processor activates the light source switch, and the emitted excitation light source is injected into the reagent container through the light excitation lens and excites the fluorescent material therein to emit fluorescence. The generated fluorescence passes through the light detection lens and is received by the spectrometer as a signal, and the signal is then transmitted to the processor for signal analysis. Finally, qualitative or quantitative analysis can be performed on the target gene or marker to be detected in the reagent container.

[0025] The device disclosed in the present invention utilizes a conductive film or electric heating element, combined with a small volume heating mechanism to provide a rapid temperature rise and fall function, effectively shortening the PCR reaction time. At the same time, the built-in algorithm can also be used to analyze the fluorescence value of more than one fluorescence signal in real time, achieving the function of qualitative and quantitative analysis of the target gene in a short time. In order to achieve the above-mentioned purpose, a preferred embodiment is provided according to the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a schematic diagram of the parts configuration of a preferred embodiment of the present invention.

[0027] Figure 2-1 and Figure 2-2 The reagent container accommodating space, heat dissipation holes,

[0028] And the temperature sensor diagram.

[0029] Figure 2-3 FIG. 1 is a top view of a reagent container accommodating space according to a preferred embodiment of the present invention.

[0030] Figure 2-4 A side view of a reagent container accommodating space according to a preferred embodiment of the present invention.

[0031] Figure 3 FIG. 4 is a diagram showing an original fluorescent signal according to a preferred embodiment of the present invention.

[0032] Figure 4 FIG. 4 is a diagram showing the fluorescent signal analysis results according to a preferred embodiment of the present invention.

[0033] Figure 5-1 FIG. 4 is a schematic diagram of a reagent container accommodating space according to another preferred embodiment of the present invention.

[0034] Figure 5-2FIG. 1 is a top view of a reagent container accommodating space according to another preferred embodiment of the present invention.

[0035] Figure 6-1 FIG. 4 is a schematic diagram of a reagent container accommodating space according to another preferred embodiment of the present invention.

[0036] Figure 6-2 FIG. 4 is a schematic diagram of a reagent container accommodating space according to another preferred embodiment of the present invention. DETAILED DESCRIPTION

[0037] The structure and function of a preferred embodiment of the present invention are described in detail below with reference to the accompanying drawings. In addition, in this specification, the positions of the mechanisms or their parts are described with "front", "back", "left", "right", "up", "down", etc., corresponding to the spatial relationship when the user operates the preferred embodiment.

[0038] Please refer to Figure 1, the configuration of the components of a device (1) according to a preferred embodiment of the present invention is shown in the figure. A device (1) according to a preferred embodiment of the present invention comprises a reagent container accommodating space (10), a reagent container (20), a first lens (30), a light source (40), a second lens (50), a spectrometer (60), a fan (70), a thermoelectric cooler (80), a processor (90), and a power supply (100). The reagent container accommodating space (10) is used to accommodate the reagent container (20) and provide a field for heating the reagent and the reactant, and the power supply (100) is directly connected to the reagent container accommodating space (10), while the fan (70) and the thermoelectric cooler (80) are used to cool the device (1).

[0039] Please refer to Figures 2-1 to 2-4, a reagent container storage space (10) of a preferred embodiment of the present invention is shown in the figure. It includes a first substrate (101) having a first concave surface (1011), a second substrate (102) having a second concave surface (1021), a first metal sheet (103), and a second metal sheet (104). The first substrate (101) has a heat dissipation hole (105) and a second heat dissipation hole (107) for heat dissipation, and the first substrate (101) also has a first fluorescent signal outlet (106) and a first temperature sensor (108). The first temperature sensor (108) is used to detect the temperature of the reagent container storage space (10) and report the temperature to the processor (90). The second substrate (102) only has a first heat dissipation hole (105) and a second heat dissipation hole (107).

[0040] The first substrate (101) and the second substrate (102) are arranged in parallel to each other so that the first concave surface (1011) and the second concave surface (1021) are coupled to each other in a vertical horizontal plane manner to form a space for placing the reagent container (20), and the outer wall of the reagent container (20) is respectively connected to the first concave surface (1011) and the second concave surface (1021). The shapes of the first concave surface (1011) and the second concave surface (1021) are not particularly limited, and basically only need to be consistent with the reagent container (20). In this embodiment, because the reagent container (20) is a test tube, the first concave surface (1011) and the second concave surface (1021) are coupled to form a space for placing the reagent container (20) and the shape of the outer wall of the test tube. The first metal sheet (103) and the second metal sheet (104) are clamped by the first substrate (101) and the second substrate (102), and are approximately parallel to the first substrate (101) and the second substrate (102), and the first metal sheet (103) and the second metal sheet (104) are respectively located on both sides of the first substrate (101) and the second substrate (102). The first metal sheet (103) and the second metal sheet (104) are respectively connected to the power supply (100).

[0041] In the present embodiment, the base material of the first substrate (101) and the second substrate (102) is aluminum. The base material is firstly treated by anodizing to coat a layer of aluminum oxide (Aluminum Oxide), and then a conductive film is coated thereon. The material of the conductive film in the present embodiment can be tin oxide, indium oxide, zinc oxide, indium tin oxide, chromium, titanium, tantalum or copper. As for the material of the first metal sheet (103) and the second metal sheet (104), in the present embodiment, they are copper.

[0042] Please refer to Figure 1. In this embodiment, the light source (40) is a laser diode, which can be used to excite fluorescent dyes or fluorescent probes to produce detectable fluorescent signals. If there are other needs, the light source (40) can also be changed to a laser light group or LED. The first lens (30) is a telecentric lens, which is used to guide the light emitted by the light source (40) to the reagent container (20). In this embodiment, the reagent container (20) has been placed in the reagent container accommodating space (10), and the light source (40) and the first lens (30) are located below the reagent container (20), and are arranged vertically with the reagent container (20), and the first lens (30) is located between the light source (40) and the reagent container (20). Such an arrangement ensures that the excitation light emitted by the light source (40) is guided by the first lens (30) to the bottom of the reagent container (20) to excite the fluorescent dye or fluorescent probe in the reagent container (20).

[0043] Please refer to FIG. 1. In this embodiment, the second lens (50) is a conjugate focal lens, which is used to transmit the generated fluorescent signal to the spectrometer (60), and the spectrometer (60) transmits it to the processor (90) for signal analysis. The second lens (50) and the spectrometer (60) are located on the side of the reagent container accommodating space (10) closer to the first substrate (101), and the second lens (50), the spectrometer (60) and the first fluorescent signal emission port (106) should be arranged as horizontally as possible, so that the fluorescent signal excited in the reagent container (20) can pass through the first fluorescent signal emission port (106) and the second lens (50) in a straight line and be detected by the spectrometer (60). In this embodiment, the second lens (50) and the spectrometer (60) are located on the right side of the reagent container accommodating space (10), and the arrangement of the second lens (50) and the spectrometer (60) is approximately perpendicular to the arrangement of the first lens (30) and the light source (40).

[0044] Please refer to Figure 1. In this embodiment, the fan (70) is a heat dissipation device, which is used in combination with the thermoelectric cooler (80). Its position is not specific. In this embodiment, the fan (70) is located on the side of the reagent container storage space (10) closer to the second substrate (102), and the thermoelectric cooler (80) is located above it. When the device (1) starts to cool down, the first heat dissipation hole (105) and the second heat dissipation hole (107) will cooperate to dissipate heat. This embodiment includes a processor (90) preloaded with an algorithm using the least squares method for fluorescent signal analysis. In this embodiment, the standard spectrum of the pre-used fluorescent dye or fluorescent probe and the wavelength of the light source (40) must be pre-input for subsequent data analysis.

[0045] The processor (90) also receives a signal from the first temperature sensor (108). When the temperature of the reagent container storage space (10) is higher than the system set temperature range, the fan (70) and the thermoelectric cooler (80) are activated to start cooling the reagent container storage space (10); and when the temperature of the reagent container storage space (10) is higher than the system set temperature range, the power supply (100) starts to heat the device (1). In this embodiment, in order to coordinate the temperature rise speed control, the processor (90) can further control the first metal sheet (103) and the second metal sheet (104) to be powered on and heated at the same time, or only heat the first metal sheet (103) or the second metal sheet (104).

[0046] The present embodiment also includes a power supply (100) for providing the power required by the entire device (1). When the power supply (100) is activated, current is transmitted to the first metal sheet (103) and the second metal sheet (104). Since the first metal sheet (103) and the second metal sheet (104) are made of copper, they can conduct current to the contacting parts of the first substrate (101) and the second substrate (102). Since the surface of the first substrate (101) and the second substrate (102) is coated with a conductive film, which has a certain resistance value, the reagent container accommodating space (10) begins to heat up to a temperature range of 95°C to 100°C. This temperature range is the heating temperature range set in the present embodiment. In the present embodiment, the processor (90) sets the reagent container accommodating space (10) to maintain this heating temperature range for about 6 seconds to 15 seconds to provide real-time PCR reaction in the reagent container (20). Then the processor (90) starts the fan (70) and the thermoelectric cooler (80) to start cooling the reagent container storage space (10) to a temperature range of 60°C to 62°C, which is the low temperature range set in this embodiment. In this embodiment, the processor (90) sets the reagent container storage space (10) to maintain this low temperature range for about 1 second to 5 seconds to provide the real-time PCR reaction in the reagent container (20). In order to provide an effective and sufficient reaction time and temperature, the processor (90) also controls that during the reaction, the temperature of the reagent container storage space (10) will be repeatedly cycled in the heating temperature range and the low temperature temperature range until the reaction is completed. When the temperature of the reagent container storage space (10) rises and falls in this stable manner, the reactants and reagents in the reagent container (20) can then reach the three required temperature ranges for real-time PCR.

[0047] When the real-time PCR reaction begins, the reagents and reactants at the contact portion between the reagent container (20) and the reagent containing space (10) begin to be heated first. When the reactants and reagents closer to the contact portion are heated to 95°C, the reactants and reagents at that portion begin to undergo a denaturation step. The temperature cycle of the reagent container containing space (10) is controlled to reach a temperature for primer annealing and primer extension.

[0048] At this time, the processor (90) activates the light source (40) switch, and the emitted excitation light enters the reagent container (20) through the first lens (30) and excites the fluorescent material therein to emit fluorescence. The generated fluorescence passes through the second lens (50) and is received by the spectrometer (60) as an original fluorescence signal. The original fluorescence signal is then transmitted to the processor (90). Through the least squares algorithm preloaded in the processor (90), after deducting the background light signal value, the data is verified and analyzed with the pre-input fluorescent dye or fluorescent probe standard spectrum. Finally, the proportion value of each fluorescent dye or fluorescent probe can be obtained. Through conversion, qualitative or quantitative analysis can be performed on the target gene or marker to be detected in the reagent container (20).

[0049] Through the above-mentioned setting of this embodiment, if four fluorescent probes of different concentrations are added to four reagent containers (20), and each reagent container (20) contains four fluorescent probes of the same concentration of FAM, VIC, Alexa594, and Alexa647, through the implementation of this device (1), the mixed raw fluorescent data in the four reagent containers (20) can be obtained as follows: Figure 3 Then, through the built-in least squares method calculation algorithm and analysis, the concentration analysis of each fluorescent probe in the four reagent containers (20) can be obtained. Figure 4 .

[0050] Please refer to Figure 5-1 and Figure 5-2 In another preferred embodiment, the device (1) can also use another reagent container accommodating space (210) to replace the above-mentioned reagent container accommodating space (10), and the rest of the device settings are the same as the previous embodiment. The reagent container accommodating space (210) includes a third substrate (201), a fourth substrate (202), and an insulating sheet (204). The third substrate (2101) has a temperature sensor (205), and the temperature sensor (2105) is used to detect the temperature of the reagent container accommodating space (210) and report the temperature to the processor (90). The third substrate (201) and the fourth substrate (202) are the same heat-resistant material, which is only bent at a specific angle and arranged in a symmetrical manner. The bending part constitutes the reagent container accommodating part (203). There is no special restriction on the shape of the reagent container accommodating part (203), basically as long as it matches the reagent container (220). In this embodiment, since the reagent container (220) is a test tube, the reagent container receiving portion (2103) is the shape of the outer wall of the reagent container (220). An insulating sheet (204) is included between the third substrate (201) and the fourth substrate (202), and the third substrate (201) and the fourth substrate (202) are respectively connected to the power supply (100).

[0051] In this embodiment, the base material of the third substrate (201) and the fourth substrate (202) is aluminum, which is firstly treated by anodizing to be coated with a layer of aluminum oxide, and then a conductive film is coated thereon. The conductive film of this embodiment can be made of tin oxide, indium oxide, zinc oxide, indium tin oxide, chromium, titanium, tantalum or copper. The power supply (100) is respectively connected to the third substrate (201) and the fourth substrate (202) to be powered. The third substrate (201) and the fourth substrate (202) are respectively positive and negative electrodes, and the insulating sheet (204) is used to ensure that the positive and negative electrodes do not touch each other.

[0052] Please refer to Figure 6-1 and Figure 6-2 In another preferred embodiment, the device (1) can also use another reagent container accommodating space (310) to replace the above-mentioned reagent container accommodating space (10), and the remaining parts and configurations of the device (1) are the same as those of the above-mentioned disclosed embodiment. The reagent container accommodating space (310) includes a fifth substrate (301) having a third concave surface (3011), a sixth substrate (102) having a fourth concave surface (3021), a first circuit board (303), and a second circuit board (304). The fifth substrate (301) has a third heat dissipation hole (305) and a fourth heat dissipation hole (307) for heat dissipation, and the fifth substrate (301) also has a second fluorescent signal outlet (306) and a second temperature sensor (308). The second temperature sensor (308) is used to detect the temperature of the reagent container accommodating space (310) and report the temperature to the processor (90). The sixth substrate (302) only has a third heat dissipation hole (305) and a fourth heat dissipation hole (307).

[0053] The fifth substrate (301) and the sixth substrate (302) are arranged in parallel to each other so that the third concave surface (3011) and the fourth concave surface (3021) are coupled to each other in a vertical horizontal plane manner to form a space for placing the reagent container (20), and the outer wall of the reagent container (20) is respectively connected to the third concave surface (3011) and the fourth concave surface (3021). The shapes of the third concave surface (3011) and the fourth concave surface (3021) are not particularly limited, and basically only need to be consistent with the reagent container (20). In this embodiment, because the reagent container (20) is a test tube, the third concave surface (3011) and the fourth concave surface (3021) are coupled to form a space for placing the reagent container (20) and the shape of the outer wall of the test tube. The first circuit board (303) and the second circuit board (304) are clamped by the fifth substrate (301) and the sixth substrate (302), and are approximately parallel to the fifth substrate (301) and the sixth substrate (302), and the first circuit board (303) and the second circuit board (304) are respectively located on both sides of the fifth substrate (301) and the sixth substrate (302). The first circuit board (303) and the second circuit board (304) are respectively welded with resistors (309) on both sides of the front and back sides of the first circuit board (303) and the second circuit board (304), and are connected to the fifth substrate (301) and the sixth substrate (302). The first circuit board (303) and the second circuit board (304) are also respectively connected to the power supply (100). In this embodiment, the base material of the fifth substrate (301) and the sixth substrate (302) is aluminum, and the base material is anodized and plated with a layer of aluminum oxide.

[0054] When the power supply (100) is activated, the current is transmitted to the first circuit board (303) and the second circuit board (304). At this time, the resistor (309) starts to heat up after being energized, and drives the fifth substrate (301) and the sixth substrate (302) to start heating up accordingly. Therefore, when the reagent container storage space (310) starts to heat up to the temperature range of 95°C to 100°C, the real-time PCR reaction begins. The second temperature sensor (308) detects the temperature of the reagent container storage space (310) and reports it to the processor. When the temperature exceeds the temperature range set by the system, the third heat dissipation hole (305) and the fourth heat dissipation hole (307) will assist the fan (70) and the thermoelectric cooler (80) to cool down. After the real-time PCR reaction generates a fluorescent signal, the fluorescent signal will be emitted through the fluorescent signal emission port (306) and detected by the second lens (50) and the spectrometer (60).

[0055] Explanation of symbols

[0056] 1 Device 10 Reagent container storage space

[0057] 101 first substrate 102 second substrate

[0058] 1011 first concave surface 1021 second concave surface

[0059] 103 first metal sheet 104 second metal sheet

[0060] 105 first heat dissipation hole 106 first fluorescent signal outlet

[0061] 107 Second heat dissipation hole 108 First temperature sensor

[0062] 20 reagent container 30 first lens

[0063] 40 light source 50 second lens

[0064] 60 Spectrometer 70 Fan

[0065] 80 Thermoelectric Cooler 90 Processor

[0066] 100 Power supply 210 Reagent container storage space

[0067] 201 third substrate 202 fourth substrate

[0068] 203 Reagent container storage 204 Insulation sheet

[0069] 205 Temperature sensor 220 Reagent container

[0070] 301 fifth base plate 302 sixth base plate

[0071] 3011 third concave surface 3021 fourth concave surface

[0072] 303 first circuit board 304 second circuit board

[0073] 305 Third heat dissipation hole 306 Fluorescent signal outlet

[0074] 307 fourth heat dissipation hole 308 second temperature sensor

[0075] 309 resistor 310 reagent container storage space

Claims

1. A polymerase chain reaction device capable of real-time detection and quantification of more than one fluorescent signal, the device comprising: a reagent container loaded with reactants and reagents, wherein the reagents comprise more than one fluorescent probe or fluorescent dye; The reagent container accommodating space is provided on a horizontal plane and is used to place the reagent container, wherein the reagent container accommodating space is composed of a heat-resistant material; wherein the reagent container accommodating space is composed of the following methods: The invention comprises a first substrate having a first concave surface, a second substrate having a second concave surface, a first conductive accessory and a second conductive accessory, wherein the first conductive accessory and the second conductive accessory are metal sheets, are clamped by the first substrate and the second substrate and are approximately parallel to the first substrate and the second substrate, the first conductive accessory is located on one side of the reagent container accommodating space, and the second conductive accessory is located on the other side of the reagent container accommodating space, and the heat-resistant insulating material on the surface of the first substrate and the second substrate comprises a conductive film; wherein the first substrate and the second substrate are arranged parallel to each other so that the first concave surface and the second concave surface are coupled to each other along the corresponding coupling surface in a direction perpendicular to the horizontal plane to form the reagent container accommodating space; and wherein the outer wall of the reagent container is respectively connected to the first concave surface and the second concave surface; A temperature sensor is placed at a location where the reagent container accommodating space contacts the reagent container, and is used to measure the temperature of the reagent container accommodating space; A light source is located at one side of the reagent container accommodating space, wherein the light emitted by the light source enters the reagent container at a specific angle and excites the fluorescent probe or fluorescent dye to generate the one or more fluorescent signals; A first lens is located between the reagent container accommodating space and the light source, directing the light source to the reagent container and exciting the fluorescent probe or fluorescent dye to generate the one or more fluorescent signals; A spectrometer for detecting and receiving one or more of the aforementioned fluorescent signals; A second lens is located between the reagent container accommodating space and the spectrometer, and is used to aggregate the one or more fluorescent signals and guide them to the spectrometer; A heat sink for dissipating heat; A power supply device, used to provide the power required for the temperature rise and fall of the reagent container storage space, and also to supply the power required by the device; wherein the power supply device is respectively connected to the first conductive component and the second conductive component and is energized separately or simultaneously; A processor is preloaded with an algorithm to quantify the above-mentioned one or more fluorescent signals. The above-mentioned processor also controls the above-mentioned power supply to provide power to the above-mentioned reagent container storage space to increase and decrease the temperature cycle and the timing of the above-mentioned light source switch; wherein, The power supply is connected to the reagent container storage space and starts to be powered on for heating, and the reagent container also starts to heat up accordingly. The processor controls the temperature of the reagent container storage space to cycle between the first temperature range and the second temperature range. At this time, the reactant and the reagent immediately start to perform a polymerase chain reaction. The light emitted by the light source passes through the first lens to excite the fluorescent probe or fluorescent dye to generate the one or more fluorescent signals. After the one or more fluorescent signals pass through the second lens and are detected by the spectrometer, the one or more fluorescent signals are sent to the processor for analysis. When the temperature sensor measures that the temperature of the reagent container storage space has exceeded the first temperature range, it will provide feedback to the processor, and the processor will start the heat dissipation device to cool down; when the temperature sensor measures that the temperature of the reagent container storage space is lower than the second temperature range, it will provide feedback to the processor, and the processor will start powering on and start heating.

2. The device as claimed in claim 1, wherein the heat-resistant material is metal or non-metal.

3. The device as claimed in claim 2, wherein the heat-resistant material is a metal, and is an aluminum sheet, a copper sheet, or other metals with fast heat conduction.

4. The device as claimed in claim 2, wherein the heat-resistant material is non-metallic and is glass, plastic or ceramic.

5. The device as claimed in claim 1, wherein the heat-resistant insulating material is aluminum oxide, Teflon or polyimide.

6. The device as claimed in claim 1, wherein the algorithm is an algorithm using a least squares method.

7. The device as claimed in claim 1, wherein the conductive film is made of tin oxide, indium oxide, zinc oxide, indium tin oxide, chromium, titanium, tantalum or copper.

8. The device as described in claim 1, wherein the light source is an LED, a laser light, or one or more LED light groups or laser light groups with different spectra, and the light source, the first lens, and the heating device are arranged vertically below the reagent container storage space, wherein the LED light group or laser light group further includes a light source that can filter out light sources that are not related to the one or more fluorescent signals.

9. The device of claim 1 , wherein the first lens and the second lens are biconvex lenses, plano-convex lenses, double lenses, aspheric lenses, achromatic lenses, aberration-free lenses, Fresnel lenses, plano-concave lenses, biconcave lenses, positive / negative meniscus lenses, axicons, gradient index lenses, microlens arrays, cylindrical lenses, diffractive optical elements, or holographic optical elements.

10. The device as claimed in claim 1, wherein the second lens is arranged in parallel with the spectrometer and the heating device and are located on the same horizontal plane.

11. The device as claimed in claim 1, wherein the first substrate has a fluorescent signal emission port, and the first substrate is located between the second substrate and the second lens.

12. The device of claim 1, wherein the processor controls the light source to turn on or off at a fixed time interval, and wherein the spectrometer detects the one or more fluorescent signals synchronously with the light source turning on period.

13. The device as claimed in claim 1, wherein the range detected by the spectrometer is between 340nm and 850nm.

14. The device of claim 13, wherein the spectrometer further comprises another light source for filtering out the one or more fluorescent signal wavelengths that are not detected.

15. The device as claimed in claim 1, wherein the aforementioned fluorescent probe or fluorescent dye is FAM, VIC, HEX, ROX, CY3, CY5, CY5.5, JOE, TET, SYBR, Texas Red, TAMRA, NED, Quasar705, Alexa488, Alexa546, Alexa594, Alexa633, Alexa643, Alexa680 or other aforementioned fluorescent probes or fluorescent dyes with a wavelength between 340nm and 850nm.

16. The device of claim 1, wherein the first temperature range is 85°C to 130°C, and the second temperature range is 50°C to 75°C.

17. The device as described in claim 16 further comprises a third temperature range different from the first temperature range and the second temperature range, and the processor controls the temperature of the reagent container storage space to repeatedly cycle between the first temperature range, the second temperature range and the third temperature range.

18. The device as claimed in claim 1, wherein the heat dissipation device is a fan.

19. The device as claimed in claim 18, wherein the device further comprises a thermoelectric cooler or one or more fans, which are combined with the fan to accelerate the cooling speed of the device.

20. The device as claimed in claim 1, wherein the heat-resistant material has more than one heat dissipation hole.

21. The device as claimed in claim 1, wherein the temperature sensor is used to measure the temperature of the reagent container accommodating space in real time.

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