RU2691763C2 - Device for simultaneous monitoring in real time scale of nucleic acid amplifications - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to devices for qualitative and quantitative analysis of nucleic acids (DNA and RNA), which can be used in medical practice in diagnosing infectious, oncological and genetic human and animal diseases for research purposes in molecular biological, genetic studies, monitoring gene expression. Device uses the polymerase chain reaction (PCR) method in real time. Device for simultaneous monitoring in real time of multiple amplifications of nucleic acid comprises thermocycler including heat-conducting element with located in it recesses for test tubes with reaction mixtures, thermal cover, heat-insulating partition and device for automatic control of temperature mode, optical system, which includes radiation source, coaxial fiber-optic light guide for transmitting excitation light from the source to the test tubes and from the reaction mixture-containing tubes to the detector for detecting fluorescence, and a detector for detecting fluorescence, as well as a microprocessor control device and a personal computer with software. Device is provided with a correcting system comprising a database, an approximating device, a device for determining thermal parameters of the test tubes and a correction device, wherein the reaction mixtures can contain fluorescent probes or analyzed samples, the database input is connected to the output of the optical system, data base is connected by two-way communication with approximating device, device for determining thermal parameters of the test tubes and correction device, and output of database is connected to input of automatic control of temperature mode and input of personal computer, wherein correcting system provides formation, storage and transmission of temperature correction signals to automatic temperature control device, and automatic control device of temperature mode performs heat correction element temperature correction operation.

EFFECT: high homogeneity of temperature of the heat-conducting element with recesses for test tubes with reaction mixtures, reduced error of quantitative measurements in cyclic mode and reduced error of determining melting point of analyzed samples in each test tube.

9 cl, 5 dwg

Description

The present invention relates to devices for the qualitative and quantitative analysis of nucleic acids (DNA and RNA), which can be used in medical practice in the diagnosis of infectious, oncological and genetic diseases of humans and animals, for research purposes in molecular biological, genetic studies, in monitoring gene expression. The device used the method of polymerase chain reaction (PNR) in real time.

To implement the PCR method, the samples under study, after appropriate preparation, are introduced into test tubes containing the reaction mixture with fluorescent dyes. Then the tubes are placed in a device containing a thermal cycler that provides PCR, and a fluorescent detector, the output of which receives signals from fluorescent dyes, which are used to estimate the number of PCR products at any stage of the reaction.

The main stages of the analysis of nucleic acids are amplification in a cyclic mode (for example, from 60 to 95 ° C) and melting in the mode of changing the temperature of the samples (in increments of 0.5 or 1 ° C).

The DNA melting curve of the test samples (graphical representation of the fluorescence as a function of the sample temperature) can be obtained by registering discrete fluorescence signals of the dye that is connected to the DNA double strand of the test sample. When the temperature reaches the melting point of the T _m double-stranded DNA, a significant change in fluorescence is observed. The melting point is a function of the length and ratio of the nucleotides of the GC / AT DNA sequence. DNA melting curve can be obtained on the basis of the effect of quenching the fluorescence of the probe with a primer carrying a non-fluorescent fluorescence quencher. The DNA melting curve uniquely characterizes the product of a biochemical reaction, it is used to compare specific nucleotide sequences and detect nucleotide substitutions in the nucleic acid sequences studied.

In devices for the qualitative and quantitative analysis of nucleic acids by the PCR method, the traditional executive elements of the device for automatic temperature control are Peltier elements. The most common Peltier elements are 40 × 40 mm. Peltier elements have thermal contact with the heat-conducting element of the thermal cycler, in which the tubes are placed. For a device with 32 or 96 tubes, it is necessary to use 2 or 6 Peltier elements, while it is difficult to select Peltier elements with equal thermal performance. To measure the heterogeneity and temperature equalization of the heat-conducting element, contact sensors can be used. However, contact sensors have significant drawbacks: such sensors have a significant mass and, placed in test tubes, introduce distortions of the thermal field, connecting wires create additional inconveniences, the number of contact sensors is limited.

In the proposed device for measuring heterogeneity and temperature equalization of the heat-conducting element, non-contact sensors are used in the form of test tubes for PCR based on quenched fluorescent probes. The radiation intensity of such probes increases with increasing temperature. The melting point T _{m of the} probe corresponds to the maximum value at the peak of the differential melting curve. For probes near the melting point, the coefficient of the temperature dependence of fluorescence is significant (about 10% / ° C). In order to increase the temperature uniformity of the heat-conducting element and test tubes with reaction mixtures installed in the proposed device, the melting temperature of the probes is used to preliminarily determine the deviation of the temperature of each tube from the average value, generating control signals of the automatic temperature control device for each Peltier element

A known patent for the system and method for processing the DNA melting curve (DIFFERENTIAL DISSOCIATION AND MELTING CURVE PEAK DETECTION, US Patent No. 0037117 A1, class G06F 19/00, G06F 15/00, Feb. 05/02/2009).

In this system, microprocessors (for example, general-purpose digital signal processors, programmable controllers) are used to improve the appearance of the melting curve and determine the melting temperature. They perform differentiation, filtering, and detection of one or several peaks.

The melting point T _m , corresponding to the maximum value at the peak of the differential melting curve, characterizes the product of the biochemical reaction. A sample with several amplification products corresponds to a melting curve with several peaks.

The disadvantage of this system and method for processing the melting curve is that the results of determining the melting temperature are used only to characterize the products of the biochemical reaction in the studied samples, while there are no technical solutions for instrumental support for obtaining the melting curve.

There is a method of homogeneous detection of at least one single-chain amplification product (patent for invention of the Russian Federation No. 2460804 IPC C12Q 1/68, C12P 19/34, C12N 15/11, publ. 10.09.2012. Bull. No. 25).

In this method, the melting point of primers, probes and amplicons T _m means the temperature at which half of the test substance exists in double-stranded and half in single-stranded form.

The absorbable fluorescent probes corresponding to this invention preferably comprise a fluorophore at one end thereof and a non-fluorescent quenching group at the other end.

The disadvantage of this method is that the analysis of melting curves makes it possible to distinguish between products and to obtain a quantitative characteristic of their concentrations, while there are no technical solutions for instrumentation to obtain a melting curve.

A known method for the detection of specific nucleotide sequences and nucleotide substitutions using PCR in real time with the effect of quenching the fluorescence of the probe with a primer (patent for the invention of the Russian Federation No. 2451086, IPC C12Q 1/68, publ. 20.05.2012. Bull. No. 14).

This method uses the detection of specific nucleotide sequences by real-time PCR with the effect of quenching the fluorescence of the probe with a primer. To do this, the selection of nucleic acids from the samples under study, the selection of appropriate oligonucleotide probes and primers, PCR amplification of one or more necessary target regions of the nucleotide sequence, analysis of the melting curves of the probes after PCR. After amplification, melting curves are obtained by an initial incubation of 2 min at 45 ° C and a subsequent temperature increase of 1 ° C every 10 seconds to 80 ° C with constant fluorescence detection on the FAM, JOE, ROX and Cy5 channels. The spatial separation of the fluorophore and quencher as a result of the melting of the probe-DNA target complex leads to an increase in fluorescence. After the amplification is completed, the melting curves F from T are analyzed. The amplification products are identified by the T _m values corresponding to the melting curve peaks on the dF / dT graph from T.

The described method solves the problem of increasing the efficiency and expanding the possibilities of analyzing specific nucleotide sequences and nucleotide polymorphisms using the PCR-RV method by applying the effect of quenching the probe fluorescence with a primer carrying a non-fluorescent fluorescence quencher.

This known method has the following main disadvantage: the results of its implementation are obtained using a known PCR system in real time and are not used to improve the parameters of this system. Another disadvantage is the significant error in determining the melting temperature T _m , which is caused by the discreteness of the temperature increase by 1 ° C every 10 seconds, noise and drift of the zero line when the probe fluorescence is detected.

Known patent for the device and method for automated heat treatment of samples of liquids (US patent No. 8797526, B2, CL G01N 1/10, 08/05/2014). The device contains a thermoblock, equipped with a temperature control system and a container for a variety of test tubes with samples, while the design of the container ensures the thermal contact of the container with the test tubes installed in it. The container has thermal contact through the base plate with the upper surface of the thermoelectric element (Peltier element). The bottom surface of the thermoelectric element is connected to a heat exchanger (radiator). Above the sample cells is a heating plate with a heating element (thermal cover). In the holes of the thermal cover are located the optical fibers of the excitation and reception of radiation.

In some versions, a control unit designed to control the automatic heat treatment of samples is built in the form of a microprocessor controller that executes a machine-readable program consisting of commands to perform operations in accordance with a predetermined sequence of steps.

In some versions, several thermoelectric devices based on the Peltier effect are used. In some versions, the device can be used for heat treatment of samples in accordance with the melting curves.

The disadvantage of this device and method is the lack of technical solutions to the problem of measuring and correcting temperature irregularities in test tubes.

A device for simultaneous real-time monitoring of multiple nucleic acid amplification is known (patent for invention of the Russian Federation No. 2304277, IPC G01N 21/63, publ. 10.08.2007, Bull. No. 22).

The device comprises a thermal cycler with a heat-conducting element, a thermal lining, an automatic temperature control device and an optical system. In the heat-conducting element there are depressions for test tubes with reaction mixtures. The optical system includes a radiation source, fiber-optic fibers for transmitting excitation light from the source and fluorescence emission from test tubes and a detector. The light guides are made in the form of optical fibers with a coaxial central part for transmitting excitation light and a peripheral part for collecting fluorescence. The central part of the fiber is aperturely matched with the amount of the reaction mixture in test tubes.

The disadvantage of this device is the lack of technical solutions to the problem of measuring and adjusting temperature irregularities in test tubes.

The closest of the known technical essence and purpose is a device for simultaneous control in real time of multiple nucleic acid amplification (patent for invention of the Russian Federation No. 2418289, IPC G01N 21/64, publ. 10.05.2011. Bull. No. 13).

The known device solves the problem of reducing the time of analysis, increasing the sensitivity of the device and reducing the amount of reaction mixture required for PCR.

This device consists of an amplificator comprising a device for thermal cycling (thermal cycler) with a sample section, a fluorimetric detector with a radiation source and a radiation receiver, a microprocessor control device and a personal computer with software.

The device contains a thermal cycler, including a heat-conducting element with recesses located in it for test tubes with reaction mixtures, a thermal lining and an automatic temperature control device, as well as an optical system including a radiation source, coaxial fiber-optical fibers for transmitting excitation light from the source and emitting fluorescence from tubes, and a detector for detecting fluorescence, the central transmitting light excitation part of the fiber is aperturely matched to the number of The reaction mixture in test tubes installed in the recesses of the heat-conducting element, the volume of the tubes corresponds to the maximum volume of the reaction mixture, a replaceable heat-insulating partition with holes is installed between the tubes and the thermal lid.

A disadvantage of the known device can be recognized the lack of technical solutions to improve the temperature uniformity of the heat-conducting element with recesses located in it for test tubes with reaction mixtures and to reduce the error in determining the melting temperature T _{m of} samples, which is caused by the discreteness of temperature increase, noise and zero-line drift during probe detection .

The present invention solves the problem of increasing the temperature uniformity of the heat-conducting element with recesses located therein for test tubes with reaction mixtures, reducing the error of quantitative measurements as a result of amplification, cyclic mode and reducing the error of determining the melting temperature of the samples under study in each test tube.

This problem is solved due to the fact that the known device containing a thermal cycler, including a heat-conducting element with recesses located in it for test tubes with reaction mixtures, a thermal lining, a heat-insulating partition and an automatic temperature control device, an optical system that includes a radiation source, a coaxial fiber-optic light guide for transmitting excitation light from a source to test tubes and from test tubes with reaction mixtures to a detector for detecting fluorescence , and a detector for fluorescence detection, as well as a microprocessor control device and a personal computer with software, are equipped with a correction system containing a database, an approximation device, a device for determining thermal parameters of test tubes, and an adjustment device. Atomic reaction mixtures may contain fluorescent probes or test samples , the database input is connected with the output of the optical system, the database is connected by two-way communication with the approximation device, with the triple of determining the thermal parameters of the tubes and the correction device, and the output of the database is connected to the input of the automatic temperature control device and the input of the personal computer.

The database ensures the preservation of discrete signals of the melting curve F from T of each tube, the derivative of the melting curve dF / dT from T and real-time PCR signals from the detector output and from the outputs of other corrective system devices, the transmission of stored control signals to the automatic temperature control device to correct the temperature heterogeneity of the heat-conducting element, as well as transfer the stored signals to the input of the personal computer to observe the signals on the monitor screen torus.

The approximation device performs the operation of approximation of the derived melting curve dF / dT from T of each tube stored in the database with a continuous function, for example, a polynomial or a derivative of the sigmoidal function, as well as storing the continuous function in the database.

The device for determining the temperature parameters of the tubes ensures the operation of determining the values of the temperature parameters of each tube, and also ensures that these values are stored in the database. "

The correction device performs signal generation operations corresponding to the average temperature parameters of all test tubes stored in the database, determining deviations of temperature parameters of each test tube from the average value, generating temperature correction signals for the automatic temperature control device corresponding to the average value of temperature deviations in all clusters test tubes, the number of which is equal to the number of Peltier elements, as well as with control signals wounded in the database.

The correction device performs temperature correction signal generation operations for an automatic temperature control device corresponding to several probes differing in melting point and approximating these signals with a polynomial of the first or higher orders.

The automatic temperature control device performs the operation of adjusting the temperature of the heat-conducting element.

The correction device provides the operation of determining the values of the standard deviation of the temperature parameters of each tube from the average value.

The proposed device, after adjusting the temperature of the heat-conducting element, provides the operation of determining the values of threshold cycles of PCR signals in real time from the samples in each test tube, which are used in quantitative measurements.

The proposed device, after adjusting the temperature of the heat-conducting element, ensures the determination of the melting point of the samples under investigation in each tube.

The invention is illustrated by drawings, on which:

in fig. 1 is a schematic diagram of the inventive device for the simultaneous control of multiple nucleic acid amplifications;

in fig. 2 graphs of discrete signals of the melting curve of the probe (Row 1, left axis — relative fluorescence intensity) and the derivative of the melting curve of the probe (Row 2, right axis — relative intensity of the fluorescence derivative) at the input of the approximation device. The horizontal axis is the temperature ° С;

in fig. 3 - graphs of the approximating function (Row 1, left axis - relative signal intensity) and its derivative (Row 2, right axis - relative signal intensity) at the output of the approximation device. The horizontal axis is the temperature ° С;

in fig. 4 - an example of the histogram of temperature parameters of test tubes;

in fig. 5 is an example of a histogram of the temperature of the tubes.

The claimed device (FIG. 1) comprises a thermal cycler 1 with a sample section 2, an optical system 3 comprising a radiation source 4 and a fluorescence detector 5, a microprocessor control device 6, a personal computer 7 with software and a correction system 8.

The optical system 3 consists of a radiation source 9, two two-lens condensers 10 and 11, between the lenses of which interferential excitation optical filters 12 and emission 13 are installed, a photodetector 14 and a light-guide bundle 15.

Thermal cycler 1 contains a thermal lining 16, a heat-conducting element 17 with recesses located in it for test tubes with reaction mixtures, a replaceable thermal insulating partition 18 is installed between the thermal lining 16 and the thermally conductive element 17. Thermal cycler 1 also contains a device for automatic control of the temperature regime 19.

The correction system 8 contains a database 20, an approximation device 21, a device for determining thermal parameters 22 and a correction device 23.

The optical system 3 is connected to the microprocessor control device 6, the corrective system 8 and the database 20.

The microprocessor control unit 6 is connected to a personal computer 7, a thermal cycler 1 and an automatic temperature control device 19.

The outputs of the correction system 8 and the database 20 are connected to the inputs of the thermal cycler 1, the automatic temperature control device 18 and the personal computer 7.

The database 20 is connected by two-way communication with the approximation device 21, the device for determining thermal parameters 22 and the adjustment device 23.

All or some of the functional devices of the correction system of the proposed device can be made in the form of integrated circuits. The proposed device works as follows.

Test tubes with reaction mixtures that contain fluorescent probes or test samples are installed in the recesses of the heat-conducting element.

The discrete signals of the melting curve of each tube F from T, the derivative of the melting curve dF / dT from T, and the real-time PCR signals of the samples under study come from the detector output to the database and are stored in it. The graphs of the melting curve and the derivative of the melting curve of one of the tubes with a fluorescent probe are shown in FIG. 2

The signals of the derivative of the melting curve of the probe from the database using an approximation device are used to perform the operations of approximation of the derived signals by a continuous function, for example, a polynomial or a derivative of a sigmoidal function.

As an example, the result of approximation of the derivative of the melting curve of the probe dF / dT from T to a second-order polynomial F ⁽¹⁾ can be represented as formula 1:

where the values A and B are the coefficients of the polynomial, C is the constant component.

Graphs of the approximation function at the output of the approximation device are shown in FIG. 3

As an example, the result of the approximation of the derivative of the melting curve of the probe dF / dT from the T derivative of the sigmoidal function F ⁽¹⁾ can be represented as formula 2:

where F _m is the intensity (range) of the melting site;

T _m - fractional value of the melting point;

k - coefficient, which is determined by the slope of the curve.

The terms ƒ ₁ + ƒ ₂ * T take into account the displacement, as well as the linear dependence of the zero line of the derivative on temperature. The negative value of the derivative makes it possible to obtain its graphs in the region of positive values.

The approximating function is stored in the database.

Graphs of the approximation function at the output of the approximation device are shown in FIG. 3 (Row 1).

In the device for determining thermal parameters of test tubes, the data of a continuous function in the form of a polynomial stored in a database are used to perform the operations of calculating the derivative of a continuous function F ⁽² .

The temperature range of the continuous function is limited, based on the criterion of nonlinearity of the derivative of the continuous function F ⁽²⁾ , which can be represented as formula 3:

The thermal parameter of each tube T _m can be represented as the result of calculating the zero value of the derivative of the continuous function F ⁽²⁾ = 0 as formula 4:

Graphs of the derivative of the approximating function at the output of the approximating device are shown in FIG. 3 (Row 2).

In the device for determining the thermal parameters of the tubes, the data of the continuous function as a derivative of the sigmoidal function stored in the database are used without additional calculations: the temperature parameters are assumed to be equal to the values of T _m determined during the approximation using formula 2.

The values of temperature parameters of all tubes are stored in the database. The temperature parameters of the tubes with elevated temperatures have lower values compared with the temperature parameters of the tubes with lower temperatures. An example of a histogram of temperature parameters of 32 tubes is shown in FIG. 4. It can be seen that due to the edge effect, tubes A1, A8, D1 and D8 have maximum values of temperature parameters.

The values of temperature parameters of all test tubes stored in the database using a correction device are used to perform the operations of determining the average temperature parameters of all test tubes stored in the database, determining the deviation of the temperature of each test tube from the average value and the standard deviation of the test tubes, generating correction signals temperature for an automatic temperature control device corresponding to the mean deviation temperature, tubes parameters in all clusters, the number of which equals the number of Peltier elements, as well as maintaining the control signal in the database.

The value of the standard deviation can serve as a criterion for the initial non-uniformity of the thermal parameters of the test tubes.

All performed operations can be applied to signals from several probes with different melting curve temperatures to generate temperature correction signals in the operating temperature range. In this case, correction signals are generated by performing an operation of approximating the melting point of several probes with a polynomial of the first or higher orders.

Correction signals are used in the automatic temperature control device to significantly reduce the difference in average temperatures of test tubes of clusters belonging to different Peltier elements. To do this, in the automatic temperature control device the control signal of the microprocessor control device of each Peltier element is changed according to the corresponding correction signal.

After performing temperature correction using an automatic temperature control device, the proposed device can be used to re-perform operations to determine the deviation of the temperature of each tube from the average value and the RMS value of the temperature parameters of the test tubes. The value of the standard deviation can serve as a criterion for the residual non-uniformity of the thermal parameters of the test tubes.

The signals from the outputs of the detector, microprocessor control unit and the database of the correction system are fed to the inputs of a personal computer to observe the signals on the monitor screen.

After performing temperature correction using an automatic temperature control device, the proposed device can be used to perform operations for determining the values of threshold cycles of real-time PCR signals from the samples in each test tube, which are used in quantitative measurements.

Reducing the error of quantitative measurements is achieved by increasing the temperature uniformity of the heat-conducting element in a cyclic mode, while reducing the scatter of the values of the threshold cycles of the samples.

After performing temperature adjustment using an automatic temperature control device, the proposed device can be used to determine the melting temperature of the test samples in each tube.

An example of the temperature histogram of the test tubes is shown in FIG. 5. It can be seen that due to the edge effect, tubes A1, A8, D1 and D8 have lower temperature values compared to tubes in the central part of the heat-conducting element. In this case, the redefined values of the deviation of the temperature of each tube from the average value of the probes can be used as corrections to reduce the error in determining the melting temperature of the samples in each test tube.

The approximation of the derivative of the melting curve by a polynomial or a derivative of the sigmoidal function can significantly reduce the error in determining the melting temperature: the discretization error is eliminated, the influence of noise is averaged, and the zero line drift is compensated.

The proposed device allows to increase the temperature uniformity of the heat-conducting element with recesses located in it for test tubes with reaction mixtures, to reduce the error of quantitative measurements in a cyclic mode and to reduce the error in determining the melting temperature of the samples in each test tube.

Information sources

1. US patent No. 0037117 A1. Differential dissociation and melting curve peak detection. IPC G06F 19/00, G06F 15/00, 05.02.2009 r.

2. Patent for the invention of the Russian Federation No. 2460804. The method of homogeneous detection of at least one single-stranded amplification product. IPC C12Q 1/68, C12P 19/34, C12N 15/11, publ. September 10, 2012 Byul. №25.

3. Patent for the invention of the Russian Federation No. 2451086. The method of detection of specific nucleotide sequences and nucleotide substitutions using real-time PCR with the effect of quenching the fluorescence of the probe with a primer. IPC C12Q 1/68, publ. May 20, 2012 Byul. №14.

4. US patent number 8797526 B2. Instrument and method of thermal treatment of liquid samples, cl. G01N 1/10, 05.08,2014

5. Patent for the invention of the Russian Federation No. 2304277. A device for simultaneous real-time monitoring of multiple nucleic acid amplifications. IPC G01N 21/63, publ. August 10, 2007 Byul. №22.

6. Patent for the invention of the Russian Federation No. 2418289. A device for simultaneous real-time control of multiple amplification of nucleic acid IPC G01N 21/64, publ. 05.10.2011 Byul. №13.

Claims (9)

1. A device for simultaneous real-time monitoring of multiple nucleic acid amplifications containing a thermal cycler, including a heat-conducting element with recesses for test-tubes with reaction mixtures located in it, a thermal cover, a heat-insulating partition and an automatic temperature control device, an optical system including a radiation source, coaxial fiber optic light guide for transmitting excitation light from the source to the test tubes and from test tubes with reaction mixtures detector for fluorescence detection, and a detector for fluorescence detection, as well as a microprocessor control device and a personal computer with software, characterized in that it is equipped with a correction system containing a database, an approximation device, a device for determining thermal parameters of test tubes and a correction device, at the same time, the reaction mixtures may contain fluorescent probes or test samples, the database input is associated with the output of the optical system, the bases data is connected by two-way communication with an approximate device, a device for determining thermal parameters of test tubes and a correction device, and the database output is connected to the input of an automatic temperature control device and an input of a personal computer, while the correction system provides the formation, storage and transmission of temperature correction signals to an automatic control device temperature control, and the automatic temperature control device produces nyaet corrections Activity heat conductive element temperature. 2. The device according to claim 1, characterized in that all or some of the functional devices of the correction system of the device proposed are made in the form of integrated circuits. 3. The device according to claim 2, characterized in that the database provides for the preservation of discrete signals of the melting curve of each tube F from T, the derivative of the melting curve dF / dT from T and PCR signals in real time, coming from the detector output and from the outputs of other devices the correction system, the transmission of the stored control signals to the automatic temperature control device to correct the temperature heterogeneity of the heat-conducting element, as well as the transmission of the stored signals to the input of the personal computer Era to observe the signals on the monitor screen. 4. The device according to p. 3, characterized in that the approximation device provides an operation of approximation stored in the database derived melting curve of each tube with a continuous function, such as a polynomial or derivative of the sigmoidal function, as well as the preservation of a continuous function in the database. 5. The device according to claim 4, characterized in that the device for determining the temperature parameters of the tubes provides the operation of determining the values of temperature parameters of each tube, and also ensures that these values are stored in the database. 6. The device according to claim 5, characterized in that the correction device performs signal generation operations corresponding to the average temperature parameters of all test tubes stored in the database, determining deviations of temperature parameters of each tube from the average value, generating temperature correction signals for the automatic control device temperature regime corresponding to the average value of the deviation of temperature parameters in all clusters of test tubes, the number of which is the number of Peltier elements, as well as the storage of control signals in the database. 7. The device according to claim 6, characterized in that the automatic temperature control device performs the operation of adjusting the temperature of the clusters of the heat-conducting element. 8. The device according to claim 7, characterized in that the proposed device provides the operation of determining the values of threshold cycles of real-time PCR signals from the samples in each test tube, which are used in quantitative measurements. 9. The device according to claim 8, characterized in that the proposed device provides for the determination of the melting point of the investigated samples in each tube.

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