Polymerase chain reaction device capable of detecting more than one fluorescent signal in real timeTechnical Field
The present invention relates to a device for polymerase chain reaction. More particularly, the present invention relates to a polymerase chain reaction apparatus capable of quantifying more than one fluorescent signal in real time.
Background
The polymerase chain reaction (Polymerase Chain Reaction, hereinafter referred to as PCR) is a technology for amplifying DNA signals rapidly, and its principle and main operation steps are (a) denaturation (denaturation) in which double-stranded DNA is dissociated into single-stranded DNA by high temperature (90-95 ℃) and then single-stranded DNA is used as a template for replication, (b) primer adhesion (PRIMER ANNEALING) in which the primer adheres to the correct target gene position when the temperature is reduced to a proper temperature, (C) primer extension (primer extension) in which the reaction temperature is corrected to 72 ℃, and DNA polymerase sequentially adheres deoxyribonucleoside triphosphates (deoxy-ribonucleotide triphosphate, hereinafter referred to as dNTPs) to as primers to synthesize another new DNA fragment.
The nucleic acid signal amplification is continuously repeated through three steps of denaturation, primer adhesion and primer extension, the number of target genes can be doubled every time the three steps are repeated, if the three steps are set to be operated for 40 times, the number of the target genes can be amplified by approximately 109 times, and the PCR can obtain a large number of target gene fragments in vitro, so that the PCR can be used as one of molecular diagnosis technologies used for clinical diagnosis at present, and can be applied to projects including genetic disease diagnosis, pathogenic bacteria diagnosis, diagnosis prognosis evaluation of tumor and cancer, basic research and the like, and is also used as a technology used for clinical diagnosis at present.
In response to the recent development demands, the Real-time polymerase chain reaction (Real-time polymerase chain reaction), also known as quantitative Real-time polymerase chain reaction (Quantitative Real-time polymerase chain reaction, abbreviated as Q-PCR), has been developed. The Real-time PCR and the traditional PCR both utilize the thermal cycle step to amplify the trace DNA of the target gene to achieve the purpose of amplification, but the difference between the Real-time PCR and the traditional PCR is that the Real-time PCR is characterized in that a non-specific fluorescent substance or a specific fluorescent probe is added in the PCR reaction, after each PCR amplification cycle, the target gene DNA is amplified and simultaneously generates a fluorescent signal, 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, a reaction curve graph can be obtained, the product generation condition of each cycle in the PCR reaction is completely presented, and then the Real-time quantitative result is achieved through the built-in program analysis.
The currently used non-specific fluorescent dye is SYBR Green 1, which releases fluorescence after binding to the minor groove (DNA), so that the fluorescence intensity can be measured at the end of the primer extension step of each cycle during PCR, and thus how much PCR product is generated in each cycle can be known. However, SYBR Green I binds to all double-stranded DNA, so that specific products and nonspecific products cannot be distinguished, and therefore, the specificity of SYBR Green I to the products is poor, and sometimes false positive test results appear.
The currently commonly used specific fluorescent probe is TaqMan probe, which is an oligonucleotide (oligonucleotide) synthesized artificially, has specificity to the target gene sequence, and is labeled with different fluorescent substances at two ends of the oligonucleotide, wherein the 5 '-end fluorescent is called a reporter and the 3' -end fluorescent is called a quencher (quencher). If the specific probe is in a free state, interaction of the information group and the quencher can shield the fluorescence of the other party, so that no fluorescence is generated, but when the PCR product is generated, the effect of shielding the quencher by the quencher is lost after the specific probe is hydrolyzed, so that the fluorescence of the reporter can be detected. Since the specific probe is an oligonucleotide specific to the target gene only, it will not bind to other non-specific products. The fluorescent dye currently used in practice in combination with TaqMan probe comprises FAM、VIC、HEX、ROX、CY3、CY5、CY5.5、JOE、TET、Texas Red、TAMRO、NED、Quasar705、Alexa488、Alexa546、Alexa594、Alexa633、Alexa643、Alexa680.
The fluorescent dyes have the optimal absorption wavelength range and the scattering wavelength range, but the optimal absorption wavelength range and the scattering wavelength range among the fluorescent dyes are overlapped, so that if more than two fluorescent dyes are needed to be used for simultaneously quantifying more than two different target genes of the same sample in the same test tube, the fluorescent dyes which do not generate high signal overlap (cross talk) are actually selected to be used in combination, and the obtained data are analyzed in a traditional data mode to finally obtain the fluorescent signal with resolution to assist in interpretation, and the other method is to test one target gene of the sample in one test tube at a time, and simultaneously detect different target genes of the same sample in a plurality of test tubes, and finally combine detection results of a plurality of detection targets of the sample to avoid the situation that the data cannot be analyzed due to the overlapping of the fluorescent signals. The former has the advantages that the test can be carried out in the same test tube no matter how different target genes are tested, the waste of consumable materials is avoided, the defect is that the signal overlapping phenomenon is generated due to various fluorescent signal sources, the data analysis is wrong, or the fact that whether the signal overlapping part belongs to which target gene is very easy to cause the erroneous judgment of experimental results cannot be effectively identified, and the latter can effectively solve the problem of signal overlapping because only one target gene is tested in one test tube at a time and then a plurality of test results are combined and judged, the signal judgment accuracy is much higher than that of the former, but the waste of consumable materials is caused, and the required quantity of the sample is also much. In practice, the latter method is mostly adopted when a plurality of target genes are to be quantified for the same sample.
At present, most of devices used in laboratories for performing real-time PCR use temperature-controlled metal as a heater, and utilize the characteristic of rapid heating and cooling to perform repeated heating and cooling operations so as to achieve the reaction temperatures of three steps of denaturation, primer adhesion and primer extension, and the effect of amplifying target gene signals and detecting fluorescent signals is achieved by transferring heat to reagents and reactants (fragments containing target genes) in test tubes through a reagent container made of heated plastic. However, such a machine using the temperature-controlled metal to repeatedly raise and lower the temperature is generally larger in volume, which is required for effective temperature control, the whole temperature control system must have larger volume and heat capacity ratio, and according to the design of the current machine, most of the time is used for waiting for the temperature of the temperature-controlled metal to raise or lower to the reaction temperature, for example, about 30-35 times of the cycle time required by the general test, the reaction time required by the conventional machine is about two to three hours, which results in difficult reduction of the reaction time, and thus the machine cannot be applied to the test requiring the result to be known in a very short time.
In order to improve the problems of the conventional devices, researchers have developed the application of real-time PCR to microfluidic wafer technology. The advantage of this technique is that the microfluidic chip can reduce the volume of reagents or reactants and the overall heat capacity ratio, thus reducing the reaction time and the consumption of reagents, but the technique still has the problem of excessively long temperature rise and fall time because the technique still needs to repeatedly rise and fall in three different temperature ranges.
Another developed real-time PCR microfluidic chip eliminates the setting of repeated heating and cooling of the heater, but uses a specially designed driving force to pressurize the reactant and reagent in the flow channel, so that the reactant and reagent repeatedly flow through three different temperature ranges in the flow channel designed by special processing, thereby completing the effect of amplifying the target gene and detecting the signal. The real-time PCR can eliminate the time consumption caused by temperature rise and drop, but the development of the technology is indirectly limited because the system of the technology needs to comprise a complex pressurizing system and a liquid driving system, and the liquid driving system is closely related to the viscosity of the volume of liquid, so that the system and the instrument are difficult to manufacture and regulate.
Researchers have developed another technique for real-time PCR using thermal convection cycling to solve the problems of high capacitance ratio and high time consumption of conventional machines. The technology utilizes two heat sources with different temperatures to heat the upper and lower ends of a closed test tube containing reagents and reactants, and drives the reagents and reactants in the test tube to flow through different temperature ranges by the temperature difference of the upper and lower ends so as to perform real-time PCR reaction. The technology overcomes the time consumption of the heater caused by repeated temperature rise and fall, and does not need to drive the liquid in the test tube to circulate in an external pressurizing mode, but because the heater is mostly made of massive metal, the volume of the machine cannot be reduced because the heat capacity ratio cannot be reduced, and the complicated temperature control mechanism and the metal heating system also make the manufacturing cost high.
The problems described above with respect to the fluorescent probe assembly and the machine are further emphasized when a plurality of target genes or targets are to be quantified for the same sample. In order to obtain a fluorescent signal value with higher reliability, in practice, only one target gene is tested in one test tube at a time, and then a plurality of test results are combined and judged, so that the test of each sample needs a plurality of test tubes, if the device is a machine commonly used in the laboratory or a microfluidic chip repeatedly heated and cooled in different temperature ranges, the problem of longer test time is still existed, and besides the problem of longer test time, the problem of waste of consumables and more required sample amount is also existed, even if the test is performed on the microfluidic chip designed in a special flow channel or a device for performing real-time PCR test by using thermal circulation, the problem of overlong test time can be eliminated, and the problems of waste of consumables and more required sample amount can not be eliminated.
In view of the above, the present invention discloses a pcr device capable of solving the problems of time consumption caused by excessive volume of the machine and repeated temperature rise and drop, waste of consumables, and large amount of required samples.
Disclosure of Invention
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, wherein the reagent container accommodating space is formed by a heat-resistant substance, the surface layer of the heat-resistant substance can comprise a heat-resistant insulating substance or a conductive film coated by a conductive substance (conductive material) or both, and the reagent container accommodating space can be designed according to the external profile of the reagent container and the structure design of the reagent container accommodating space and the temperature control condition, and other conductive accessories such as a circuit board or a conductive metal accessory can be added for facilitating operation. The heat-resistant material can be metal or nonmetal, and can be aluminum sheet, copper sheet or other heat-resistant metal if the heat-resistant material is metal, glass, plastic or ceramic if the heat-resistant material is nonmetal, and a layer of heat-resistant insulating material such as aluminum oxide, teflon, polyimide or the like is coated on the metal if the heat-resistant material is metal.
Whether the heat-resistant material is metal or not, whether a conductive film is coated on the surface layer of the heat-resistant material to provide a specific resistance value or not depends on the design of a heating mechanism of the accommodating space of the reagent container. If the heating mechanism design only comprises a conductive substance made of metal, and no other accessories can provide heating, at this time, the device disclosed by the invention can coat a conductive film on the surface layer of the heat-resistant substance, and the conductive film has a certain resistance value, and heating can be started after receiving current from the conductive substance, otherwise, if the heating mechanism design directly comprises an electric heating device, such as a circuit board welded with an electric heating element, the heating can be started once the electric heating device is electrified, and a layer of conductive film is not required to be coated on the heat-resistant substance. The conductive material of the present invention may be tin oxide, indium oxide, zinc oxide, indium tin oxide, chromium, titanium, tantalum, or copper, and the electrical heating element of the present invention may be a resistor or a circuit wiring (PCB layout). The heating mechanism design disclosed by the invention can greatly reduce the volume of the heating mechanism, and has more advantages and derivability in volume compared with the traditional PCR machine.
The position where the reagent container accommodating space contacts with the reagent container is provided with at least one temperature sensor, the placement position and the number of the temperature sensors can be determined according to the position where the temperature change is relatively easy to detect and the reaction is not influenced when the actual reagent container is placed in the reagent container accommodating space, and the temperature sensors are used for monitoring and reporting the temperature of the position.
The invention also comprises a power supply, a heat dissipation device and a processor. The power supply is used for supplying the temperature rise and fall of the reagent container accommodating space and the power required by the operation of the whole device, the heat dissipation device is used for cooling the system, and the processor is preloaded with a program to quantitatively analyze more than one fluorescent signal by using an algorithm, wherein the algorithm can be an algorithm for setting parameters according to a least squares method.
The processor can be used for analyzing fluorescent signals and controlling the time point when the power supply is started to provide power for the accommodating space of the reagent container, the heat dissipation device is started and the system light source is switched on and off. The power supply is connected with the accommodating space of the reagent container, more precisely, the connector of the power supply can be connected with the conductive film on the surface layer of the accommodating space of the reagent container or connected with other electric heating devices in the accommodating space of the reagent container. When the power supply starts to supply power, the reagent container accommodating space starts to heat up to a preset temperature, at the moment, the temperature sensor arranged in the reagent container accommodating space starts to detect the temperature and report the temperature to the processor, and when the temperature sensor detects that the temperature of the reagent container accommodating space exceeds the preset highest temperature of the system, the heat radiating device is started to start to cool down to a preset low temperature range of the system in a preset time. The installation of the heat sink or the position relative to the accommodating space of the reagent container is not specific, as long as the temperature of the device can be reduced rapidly and effectively. In the temperature setting of the present invention, the temperature range in which the temperature of the reagent container accommodation space is allowed to rise is 85 ℃ to 130 ℃, and the temperature range in which the temperature of the reagent container accommodation space is allowed to fall is 50 ℃ to 75 ℃.
In a preferred embodiment of the present invention, the heat dissipating device may be a fan, and a thermoelectric cooler (TE cooler) may be added or one or more fans may be combined with the heat dissipating device to achieve the effect of rapid and effective temperature dissipation, and the heat-resistant material forming the accommodating space of the reagent container may also include a plurality of through holes to accelerate the heat dissipating effect.
The disclosed device is equipped with at least one light source, a spectrometer, a photoexcitation lens (light illumination lens), and a photodetection lens (light detection lens). The light source is used for exciting fluorescent dye or fluorescent probe to generate detectable fluorescence, and the device disclosed by the invention can also be combined with a plurality of light sources with different excitation light wavelengths. The light emitted by the light source can be emitted into the reagent container through the light excitation lens at a specific angle, and the fluorescent material is excited to generate a fluorescent signal, and the generated fluorescent signal passes through the light detection lens and the spectrometer through the fluorescent signal emission port to send the signal to the processor for analysis. The light source applicable in the invention comprises an LED lamp, a laser lamp or other light sources with the wavelength consistent with that of fluorescent dye or fluorescent probe, and can be used in combination, and the light excitation lens and the light detection lens applicable in the invention can be a biconvex lens, a plano-convex lens, a biconvex lens, an aspherical lens, an achromatic lens, an aberration-eliminating lens, a Fresnel lens, a plano-concave lens, a biconcave lens, a positive/negative meniscus lens, an axicon, a gradient refractive index lens, a microlens array, a cylindrical lens, a diffractive optical element and a holographic optical element, or can be a combination of the lenses, the elements and the arrays. The processor can control the on and off time of the light source, and can also control whether the spectrometer and the light source start to detect the fluorescent signal in the same time period.
The invention discloses a device, wherein the arrangement mode of a light source, a spectrometer, a light excitation lens, a light detection lens and a reagent container accommodating space is not particularly limited, basically, only the light source, a light ray emission path and the light excitation lens are confirmed to be positioned at one side of the reagent container accommodating space, the light rays emitted by the light source can be guided into the reagent container through the light excitation lens to effectively excite fluorescent dyes or fluorescent probes in the reagent, and the other part can be used for transmitting fluorescent signals to the spectrometer through the light detection lens after the fluorescent signals are generated and then transmitting the fluorescent signals to a processor for analyzing related signals. In a preferred embodiment of the present invention, the light source and the light excitation lens are located vertically below the reagent container, and the light detection lens and the spectrometer are arranged at an angle of 90 degrees with respect to the light source and the light excitation lens. In a preferred embodiment of the present invention, the reagent container receiving space is provided with a fluorescent signal emitting port at the fluorescent light emitting path position, so as to facilitate the implementation of the apparatus of the present invention.
In the device disclosed by the invention, the fluorescence signal detectable by the spectrometer can be in the range of 340nm to 850 nm. The fluorescence dye or fluorescence probe used in the present invention has an emission spectrum (Emission Wavelength) of 340nm to 850nm, including but not limited to :FAM、VIC、HEX、ROX、CY3、CY5、CY5.5、JOE、TET、SYBR、Texas Red、TAMRO、NED、Quasar705、Alexa488、Alexa545、Alexa594、Alexa633、Alexa643、Alexa680., which can be used in the present invention if the emission spectrum of other fluorescence dye falls between 340nm to 850 nm.
The present invention discloses a device capable of detecting multiple target genes or targets in the same reagent container, i.e. adding more than one fluorescent dye or fluorescent probe to the same reagent container to perform real-time PCR, collecting fluorescent signals of each reaction, and analyzing the fluorescent signals by a preloaded algorithm of a processor to achieve the qualitative and quantitative effects of the multiple target genes or targets. If more than one fluorescent dye or fluorescent probe is added, the fluorescent signals will generate the signal overlapping condition, in order to effectively detect and identify more than two fluorescent signals in the same reagent container in real time, firstly, for the fluorescent dye or fluorescent probe which is expected to be used, the standard fluorescent spectrum is input into the algorithm preloaded by the processor, and at the same time, the standard spectrum of the excitation light source used by the device is input into the processor, the two are used as the reference standard value of the algorithm, and then the measured original fluorescent signals and the standard spectrum are compared through the algorithm operation, so as to obtain the fluorescent signal value. For example, if six different target genes or targets are to be detected in the same reagent container, six different fluorescent dyes or fluorescent probes are to be used, so that the standard spectra of the six fluorescent dyes or fluorescent probes are first input into the pre-loaded algorithm of the processor, and the standard spectra of the excitation light source are also first input into the pre-loaded algorithm of the processor, and then real-time PCR is started. After the procedure is completed, the processor will obtain the measured spectrum raw data, which is the total accumulated fluorescent signals of the six fluorescent dyes or fluorescent probes, then deduct the background value, calculate the respective ratio of the six fluorescent signals in the fluorescent spectrum of each PCR cycle by using the algorithm, separate and obtain the real-time fluorescent signal values of each fluorescent dye or fluorescent probe through the operation, and obtain the qualitative or quantitative results of six different target genes or targets through the conversion.
When the device disclosed by the invention is operated, the power supply is started first, the conductive substance is electrified immediately, and the accommodating space of the reagent container is heated. The reagent container added with the reactant and the reagent is placed in the reagent container accommodating space, and the reactant and the reagent begin to be denatured (density), primer adhesion (PRIMER ANNEALING), primer extension (primer extension) and other steps through rapid and repeated heating and cooling of the reagent container accommodating space. At this time, the processor will start the light source switch, the emitted excitation light source will emit into the reagent container through the light excitation lens and excite the fluorescent substance in the reagent container to emit fluorescence, the generated fluorescence passing detection lens receives the signal by the spectrometer, the signal is then transmitted to the processor for signal analysis, finally the qualitative or quantitative analysis can be carried out on the target gene or target to be detected in the reagent container.
The device disclosed by the invention utilizes the conductive film or the electric heating element to provide a rapid temperature rise and reduction function by combining a small-volume heating mechanism, so that the PCR reaction time is effectively shortened. Meanwhile, the fluorescence value of more than one fluorescence signal can be analyzed in real time through a built-in algorithm, so as to achieve the function of qualitative and quantitative targeting genes in a short time. In order to achieve the foregoing objects, a preferred embodiment is provided according to the present invention.
Drawings
Fig. 1 is a schematic view of the configuration of the components of a preferred embodiment device of the present invention.
FIGS. 2-1 and 2-2 show a reagent vessel containing space, heat dissipation holes, a reagent vessel according to a preferred embodiment of the present invention,
Schematic diagram of temperature sensor.
FIGS. 2-3 are top views of reagent vessel receiving spaces according to a preferred embodiment of the present invention.
FIGS. 2-4 illustrate an exemplary embodiment of the present invention on the side of the reagent vessel receiving space.
FIG. 3 is a diagram showing the original fluorescent signal according to a preferred embodiment of the present invention.
FIG. 4 is a graph showing the result of fluorescent signal analysis according to a preferred embodiment of the present invention.
FIG. 5-1 is a schematic view showing a reagent vessel in accordance with another preferred embodiment of the present invention.
Fig. 5-2 is a top view of a reagent vessel housing space according to another preferred embodiment of the present invention.
FIG. 6-1 is a schematic view showing a reagent vessel in accordance with another preferred embodiment of the present invention.
FIG. 6-2 is a schematic view showing a reagent vessel in accordance with another preferred embodiment of the present invention.
Detailed Description
The structure and function of a preferred embodiment of the present invention will be described in detail with reference to the accompanying drawings. In addition, the description of the mechanism or its portion in its position is covered with "front", "rear", "left", "right", "upper", "lower", etc. corresponding to the spatial relationship of the user when operating the present preferred embodiment.
Referring to fig. 1, the arrangement of the component parts of a device (1) according to a preferred embodiment of the present invention is shown. The device (1) of a preferred embodiment of the 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 for accommodating the reagent container (20) and providing a heating field for heating the reagent and reactants, the power supply (100) is directly connected with the reagent container accommodating space (10), and the fan (70) and the thermoelectric cooler (80) are used for cooling the device (1).
Referring to fig. 2-1 to 2-4, a reagent vessel accommodating space (10) according to a preferred embodiment of the present invention is shown. Comprises a first substrate (101) with a first concave surface (1011), a second substrate (102) with 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 emitting port (106), and a first temperature sensor (108), wherein the first temperature sensor (108) is used for detecting the temperature of the accommodating space (10) of the test agent container and reporting the temperature to the processor (90). The second substrate (102) has only the first heat dissipation hole (105) and the second heat dissipation hole (107).
The first substrate (101) and the second substrate (102) are arranged in parallel, so that the first concave surface (1011) and the second concave surface (1021) are coupled with 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 with the first concave surface (1011) and the second concave surface (1021). The shape of the first concave surface (1011) and the second concave surface (1021) is not particularly limited as long as they substantially conform to the reagent container (20), and in this embodiment, the first concave surface (1011) and the second concave surface (1021) are coupled to provide a space shape for placing the reagent container (20) and a shape of an outer wall of the test tube because the reagent container (20) is a 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 positioned at two sides of the first substrate (101) and the second substrate (102). The first metal sheet (103) and the second metal sheet (104) are connected to the power supply (100) respectively.
In this embodiment, the base materials of the first substrate (101) and the second substrate (102) are Aluminum, the base materials are first anodized, a layer of Aluminum Oxide is plated thereon, and then a layer of conductive film is plated thereon, wherein the conductive film of this embodiment may be tin Oxide, indium Oxide, zinc Oxide, indium tin Oxide, chromium, titanium, tantalum or copper, and the first metal sheet (103) and the second metal sheet (104) are copper in this embodiment.
Referring to FIG. 1, in the present embodiment, the light source (40) is a laser diode that can be used to excite a fluorescent dye or a fluorescent probe to generate a detectable fluorescent signal. The light source (40) may also be changed to a laser light set or an LED if other needs exist. The first lens (30) is a telecentric lens for directing light from the light source (40) toward the reagent container (20). In the embodiment, the reagent container (20) is placed in the reagent container accommodating space (10), the light source (40) and the first lens (30) are located below the reagent container (20) and vertically arranged 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 excitation light emitted from the light source (40) is directed by the first lens (30) towards the bottom of the reagent vessel (20) to excite the fluorescent dye or fluorescent probe within the reagent vessel (20).
Referring to fig. 1, in the present embodiment, the second lens (50) is a conjugate focal lens for transmitting the generated fluorescent signal to the spectrometer (60), and the spectrometer (60) is then transmitted 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 emitting port (106) are arranged horizontally as much as possible, so that the fluorescent signal excited in the reagent container (20) can be emitted in a straight line through the first fluorescent signal emitting port (106) and the second lens (50) successively and detected by the spectrometer (60). In the present embodiment, the second lens (50) and the spectrometer (60) are located at the right side of the reagent container accommodating space (10), and the arrangement of the second lens (50) and the spectrometer (60) is at an angle approximately perpendicular to the arrangement of the first lens (30) and the light source (40).
Referring to fig. 1, in the present embodiment, the fan (70) is a heat dissipating device and is combined with the thermoelectric cooler (80) for use, and the location thereof is not particularly limited, in the present embodiment, the fan (70) is located on the side of the reagent container accommodating space (10) closer to the second substrate (102), and the thermoelectric cooler (80) is located above the side, and when the device (1) begins to cool down, the first heat dissipating hole (105) and the second heat dissipating hole (107) will cooperate to dissipate heat. One embodiment includes a processor (90) in which a minimum flatness algorithm is preloaded for fluorescent signal analysis. In this embodiment, the standard spectrum of the fluorescent dye or fluorescent probe used in advance and the wavelength of the light source (40) are inputted in advance for the subsequent data analysis.
The processor (90) also receives the signal from the first temperature sensor (108), and when the temperature of the reagent container accommodating space (10) is higher than the system set temperature range, the fan (70) and the thermoelectric cooler (80) are started to cool the reagent container accommodating space (10), and when the temperature of the reagent container accommodating space (10) is higher than the system set temperature range, the power supply (100) starts to warm the device (1). In this embodiment, to coordinate with the temperature-raising speed control, the processor (90) may further control to simultaneously perform the electrical heating on the first metal sheet (103) and the second metal sheet (104), or to perform the single heating on the first metal sheet (103) or the second metal sheet (104).
The embodiment also comprises a power supply (100) for supplying the power required by the whole device (1). When the power supply (100) is started, current is transmitted to the first metal sheet (103) and the second metal sheet (104), and the first metal sheet (103) and the second metal sheet (104) are made of copper, so that current can be conducted to the contact part of the first substrate (101) and the second substrate (102), and the surface layers of the first substrate (101) and the second substrate (102) are coated with conductive films which have a certain resistance value, so that the temperature of the reagent container accommodating space (10) starts to rise to a temperature range of 95 ℃ to 100 ℃, wherein the temperature range is the temperature rising temperature range set by the embodiment, and in the embodiment, the processor (90) sets the temperature rising temperature range to be about 6 seconds to 15 seconds for the reagent container accommodating space (10) to provide real-time PCR reaction in the reagent container (20). The processor (90) then starts the fan (70) and the thermoelectric cooler (80) to cool the reagent container accommodating space (10) to a temperature range of 60 ℃ to 62 ℃, which is the low temperature range set in this embodiment, and in this embodiment, the processor (90) sets the reagent container accommodating space (10) to maintain the low temperature range for about 1 second to 5 seconds to provide real-time PCR reaction in the reagent container (20). In order to provide effective and sufficient reaction time and temperature, the processor (90) also regulates and controls the temperature of the reagent container accommodating space (10) to be repeatedly circulated in the temperature rising temperature interval and the low temperature interval until the reaction is finished when the reaction is carried out, and reactants and reagents in the reagent container (20) can reach three temperature intervals required by real-time PCR when the temperature of the reagent container accommodating space (10) is stably lifted in this way.
When the Real-time PCR reaction starts, the reagent and reactant at the contact part of the reagent container (20) and the reagent accommodating space (10) start to be heated first, and when the reactant and reagent at the contact part are heated to 95 ℃, the reactant and reagent at the contact part start to be denatured, and the temperature of the primer adhesion (PRIMER ANNEALING) and primer extension (primer extension) is achieved by controlling the temperature cycle of the reagent container accommodating space (10).
At this time, the processor (90) will start the switch of the light source (40), the emitted excitation light enters the reagent container (20) through the first lens (30) and excites the fluorescent substance in the reagent container to emit fluorescence, the generated fluorescence is received by the spectrometer (60) through the second lens (50) and is transmitted to the processor (90), the background light signal value is subtracted by the preloaded algorithm in the processor (90), the data is verified and analyzed with the standard spectrum of the fluorescent dye or fluorescent probe input in advance, finally the proportion value occupied by each fluorescent dye or fluorescent probe is obtained, and qualitative or quantitative analysis can be carried out on the target gene or target to be detected in the reagent container (20) through conversion.
Through the above setting of this embodiment, if four fluorescent probes with different concentrations are added into four reagent containers (20), and each reagent container (20) contains four fluorescent probes with the same concentration FAM, VIC, alexa594 94 and Alexa647, the mixed raw fluorescent data in the four reagent containers (20) can be obtained through the implementation of the device (1) as shown in fig. 3, and then the concentration analysis of each fluorescent probe in the four reagent containers (20) can be obtained through the built-in algorithm and analysis by using the least squares method as shown in fig. 4.
Referring to fig. 5-1 and 5-2, in another preferred embodiment, the device (1) may also use another reagent container receiving space (210) instead of the reagent container receiving space (10), and the rest of the device is the same as the previous embodiment. The reagent container accommodating space (210) comprises a third substrate (201), a fourth substrate (202) and an insulating sheet (204). The third substrate (2101) has a temperature sensor (205) thereon, and the temperature sensor (2105) is used for detecting the temperature of the accommodating space (210) of the test agent container and reporting the temperature to the processor (90). The third substrate (201) and the fourth substrate (202) are made of the same heat-resistant material, and are symmetrically arranged after being bent at a specific angle, and the bent parts form a reagent container accommodating part (203). The shape of the reagent container accommodation site (203) is not particularly limited, and basically, it is only required to conform to the reagent container (220). In this embodiment, the reagent container (220) is a test tube, and thus the reagent container accommodation site (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 with the power supply (100).
In this embodiment, the base materials of the third substrate (201) and the fourth substrate (202) are Aluminum, and the base materials are first anodized, then a layer of Aluminum Oxide (Aluminum Oxide) is plated thereon, and then a layer of conductive film is plated thereon, wherein the conductive film can be tin Oxide, indium Oxide, zinc Oxide, indium tin Oxide, chromium, titanium, tantalum or copper. The power supply (100) is respectively connected with the third substrate (201) and the fourth substrate (202) for power supply, the third substrate (201) and the fourth substrate (202) are respectively positive and negative, and the positive and negative electrodes are prevented from touching through the insulating sheet (204).
Referring to fig. 6-1 and 6-2, in another preferred embodiment, the device (1) may also use another reagent container receiving space (310) instead of the reagent container receiving space (10), and the parts and configuration of the rest of the device (1) are the same as those of the above disclosed embodiments. The reagent container accommodating space (310) comprises a fifth substrate (301) with a third concave surface (3011), a sixth substrate (102) with 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) further has a second fluorescent signal emitting port (306) and a second temperature sensor (308), wherein the second temperature sensor (308) is used for detecting the temperature of the accommodating space (310) of the test agent container and reporting the temperature to the processor (90). The sixth substrate (302) has only the third heat dissipation hole (305) and the fourth heat dissipation hole (307).
The fifth substrate (301) and the sixth substrate (302) are arranged in parallel, so that the third concave surface (3011) and the fourth concave surface (3021) are coupled with 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 with the third concave surface (3011) and the fourth concave surface (3021). The shape of the third concave surface 3011 and the fourth concave surface 3021 is not particularly limited as long as they substantially conform to the shape of the reagent container 20, and in this embodiment, the third concave surface 3011 and the fourth concave surface 3021 are coupled to provide a space shape in which the reagent container 20 is placed and a shape of an outer wall of the test tube because the reagent container 20 is a 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 positioned at two 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 the front and back sides, and are connected with the fifth substrate (301) and the sixth substrate (302). The first circuit board (303) and the second circuit board (304) are also connected to the power supply (100), respectively. In this embodiment, the base materials of the fifth substrate (301) and the sixth substrate (302) are Aluminum, and the base materials are anodized to be plated with a layer of Aluminum Oxide (Aluminum Oxide).
When the power supply (100) is started, current is transmitted to the first circuit board (303) and the second circuit board (304), at this time, the resistor (309) is electrified to start heating, and drives the fifth substrate (301) and the sixth substrate (302) to start heating, so that the reagent container accommodating space (310) starts heating to a temperature region of 95 ℃ to 100 ℃, the real-time PCR reaction starts to be performed, the second temperature sensor (308) detects the temperature of the reagent container accommodating space (310) and returns the temperature to the processor, and when the temperature exceeds a temperature rising temperature region set by the system, the third heat dissipation hole (305) and the fourth heat dissipation hole (307) assist the fan (70) and the thermoelectric refrigerator (80) to cool down. After the real-time PCR reaction is performed to generate a fluorescent signal, the fluorescent signal is emitted through the fluorescent signal emission port (306) and detected by the second lens (50) and the spectrometer (60).
Symbol description
1. Device 10 reagent vessel accommodation space
101. First substrate 102 second substrate
1011. First concave surface 1021 second concave surface
103. First metal sheet 104 second metal sheet
105. First fluorescent signal emission port of first heat dissipation hole 106
107. Second heat dissipation hole 108 first temperature sensor
20. First lens of reagent vessel 30
40. Light source 50 second lens
60. Spectrometer 70 fan
80. Thermoelectric cooler 90 processor
100. Power supply 210 reagent container accommodation space
201. Third substrate 202 fourth substrate
203. Insulating sheet at reagent container holding position 204
205. Temperature sensor 220 reagent container
301. Fifth substrate 302 sixth substrate
3011. Third concave surface 3021 fourth concave surface
303. First circuit board 304 second circuit board
305. Third heat dissipation hole 306 fluorescent signal emission port
307. Fourth heat dissipation hole 308 second temperature sensor
309. Resistor 310 reagent container accommodation space