RU2640186C2 - Device for real time simultaneous control of plurality of nucleic acid amplifications - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: group of inventions refers to instruments for qualitative and quantitative analysis of nucleic acids (DNA and RNA) and can be used in medical practice in the diagnosis of infectious, cancer and genetic diseases of humans and animals, as well as for research purposes. Device for simultaneous control of multiple real-time nucleic acid amplifications contains a thermocycler, which includes a thermal element with recesses for tubes with reaction mixtures, a thermocover and automatic temperature control device, an optical system comprising a radiation source, fiber optic, coaxial, fiber optic lightguides for excitation light transmission from the source and radiation of fluorescence from tubes, a fluorescence detector, a microprocessor control device and a personal computer. The device has a pneumo-hydraulic system, which includes two tanks, partially filled with liquid, piping, an air compressor, four electromagnetic valves, radiators, a controller and air filters, while the thermal element has internal through channels connected by pipelines via solenoid valves controlled by a controller, with tanks, partly filled with liquid. The group of inventions also relates to an embodiment of the said device, the pneumatic hydraulic system of which comprises one container partially filled with a liquid.EFFECT: increased of temperature change in the cooling mode, improved performance and productivity by reducing the analysis time.2 cl, 6 dwg

Description

The invention relates to devices for qualitative and quantitative analysis of nucleic acids (DNA and RNA), which used the method of polymerase chain reaction (NDP) in real time. Such devices are widely used in medical practice and for research purposes:

- in the diagnosis of infectious, oncological and genetic diseases of humans and animals;

- in the analysis of products containing genetically modified organisms;

- when monitoring gene expression for diagnostic and research purposes, etc.

A patent is known for a device and method for automated heat treatment of liquid samples (US patent No. 8797526 B2, class G01N 1/10, 08/05/2014). The description of this device contains traditional technical solutions. The device contains a fuser equipped with a temperature control system and a container for many test tubes with samples, while the design of the container provides thermal contact of the container with the tubes installed in it. The device includes a module for detecting light emitted by the samples and a device for interfacing with a plurality of optical fiber optical fibers for transmitting the emitted light to the registration device. The device contains a device for moving the light registration module and the container, which allows you to vary the distance between them in order to install test tubes with samples in the container or to take them out, and also to register the light emitted by the samples contained in the test tubes installed in the container.

The container has thermal contact through the base plate with the upper surface of the thermoelectric element (Peltier element). The lower surface of the thermoelectric element is connected to a heat exchanger (radiator). A heating plate with a heating element (thermal cover) is located above the cells with samples. In the openings of the thermal cover are located optical fibers for exciting and receiving radiation.

According to the claims, a method for automated heat treatment of liquid samples is proposed, which includes a method for varying the distance between a temperature-controlled container for loading a plurality of test tubes with samples and ends of fiber optic fibers. Other distinctive features relate to the light registration module: each optical fiber has a first and second end, each first and every second end of the optical fiber are fixed relative to each other and serve to transmit light, varying the intercell distance allows loading and unloading tubes and recording light from samples, contained in one or more test tubes installed in the container, the second ends of the optical fibers being stochastic.

The disadvantage of this device and method is the lack of technical solutions aimed at increasing the heating and cooling rates of the samples.

The device The LightCycler® 480 Real-Time PCR System (www.roche-applied-science.com) is known, in which a layer such as a heat pump is used that provides efficient heat transfer between the Peltier elements and the radiator. The tube holder is made of silver.

The disadvantage of this device is the low heating and cooling rate of the samples, since the measures taken slightly increase the heating and cooling rates of the samples.

A well-known holder of test tubes with reagents with reduced mass and increased rate of temperature change of the samples used in thermal cycling during the polymer chain reaction (US patent No. 7632464 B2, class USA 422/99, 422/102, 422/130, 15.12.2009, ) Weight reduction is achieved by adding holes in two perpendicular directions.

The disadvantages of this device are a slight decrease in the mass and heat capacity of the tube holder and a slight increase in the rate of temperature change.

A device is known that has the name "Lightweight die matrix used in a PCR amplifier" (utility model patent of the Russian Federation No. 133835, IPC С12М 1/38, published on October 27, 2013). The matrix for dies with reagents used in thermal cycling during the polymerase chain reaction is characterized in that it is made of aluminum alloy, and triangular-shaped grooves are made between the wells for dies with reagents in mutually perpendicular directions.

The disadvantage of this device is to reduce the surface of the matrix for dies with reagents adjacent to the Peltier elements, which leads to a decrease in the heating and cooling rates of the samples.

A device for simultaneous real-time monitoring of multiple amplification of nucleic acids (patent for the invention of the Russian Federation No. 2304277, IPC G01N 21/63, publ. 08/10/2007).

The device comprises a thermal cycler with a heat-conducting element, a thermal cover, an automatic temperature control device, and an optical system. In the heat-conducting element there are recesses for tubes with reaction mixtures. The optical system includes a radiation source, fiber optic fibers for transmitting excitation light from the source and fluorescence radiation from the tubes and a detector. The optical fibers are made in the form of optical fibers with a coaxially located central part and a peripheral part for collecting fluorescence. The central part of the fiber is apertured to match the amount of reaction mixture in test tubes.

The disadvantage of this device is the lack of technical solutions that increase the rate of cyclic temperature changes for tubes with reaction mixtures. Another disadvantage of the known device is caused by the use of traditional reaction tubes for PCR, which have relatively long cylindrical and conical parts and are made of polymer material. The polymeric material of these tubes slows down the heat transfer from the heat-conducting element to the reaction mixture, which does not allow the implementation of fast thermal cycles.

The closest known by technical essence and purpose is a device for simultaneous real-time monitoring of multiple amplification of nucleic acids (patent for the invention of the Russian Federation No. 2418289, IPC G01N 21/64, published on 05/10/2011).

The device comprises a thermal cycler, including a heat-conducting element with recesses located in it for test tubes with reaction mixtures, a thermocover and an automatic temperature control device. The device also contains an optical system including a radiation source, coaxial fiber optic fibers for transmitting excitation light from the source and fluorescence radiation from the tubes, a detector for detecting fluorescence. In this case, the central transmitting light of the excitation part of the fiber is apertured to match the amount of the reaction mixture in test tubes installed in the recesses of the heat-conducting element. Moreover, the volume of the tubes corresponds to the maximum volume of the reaction mixture. In this case, a replaceable heat-insulating partition with holes, the axes of which coincide with the axes of the tubes, and the diameters equal to the outer diameter of the optical fibers for transmitting fluorescence radiation from the tubes, is installed between the tubes and the thermal cap.

The present invention solves the problem of reducing analysis time, increasing the sensitivity of the device and reducing the amount of reaction mixture necessary for PCR.

The disadvantage of this device is the lack of technical solutions for equalizing the heating and cooling rates of samples. In almost all known devices for automatic temperature control of devices that use the real-time PCR method, thermoelectric elements (Peltier elements) are used, which have the effect of a significant difference in the heating and cooling rates of the samples. This effect is explained by the fact that thermoelectric elements have a low thermal performance for the cooling mode (transfer of thermal energy per unit time). In the heating mode, the thermal performance of these elements is greatly increased due to the summation of the transferred thermal energy and the thermal energy of the elements received from the power source.

The present invention solves the problem of increasing the rate of temperature change in the cooling mode and thereby improves the speed and performance of this device by reducing the analysis time. The following are two options for solving this problem.

1. This problem is solved due to the fact that the known device for simultaneous real-time monitoring of the amplification set of a nucleic acid, comprising a thermal cycler including a heat-conducting element with recesses for test tubes with reaction mixtures located therein, a thermal cap and an automatic temperature control device, optical a system including a radiation source and a radiation receiver, coaxial fiber optic fibers for transmitting excitation light from a source and fluorescence emission from the tubes, the central part of the fiber transmitting the excitation light is aperture aperture matched with the amount of reaction mixture in the 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 thermocover, microprocessor control unit and a personal computer equipped with a pneumohydraulic system, which contains two containers, partially liquid pipelines, an air compressor, four solenoid valves, radiators and a controller, air filters, while the heat-conducting element has through internal channels, the first output of the first tank is connected by piping through the first electromagnetic valve to the environment, the air compressor is connected by pipelines to the first inlet of the first tank and with the inlet of the second electromagnetic valve, the first outlet of the second tank through an air filter is connected by a pipe to the surrounding environment, the output of the second electromagnetic valve is connected by a pipeline to the inputs of the internal channels of the heat-conducting element, the second output of the first tank is connected by pipelines through the third electromagnetic valve to the same inputs of the internal channels of the heat-conducting element, the outputs of the internal channels of the heat-conducting element are connected by a pipe to the input of the second capacity, the second output of the second tanks connected by pipelines through the fourth electromagnetic valve to the second input of the first tank, the first and second tanks hav e thermal contact with radiators, the controller is connected to an air compressor, four solenoid valves and microprocessor control device.

2. This problem is solved due to the fact that a device for simultaneous real-time monitoring of multiple amplifications of a nucleic acid containing a thermal cycler including a heat-conducting element with recesses for test tubes with reaction mixtures located therein, a thermal cap and an automatic temperature control device, an optical system comprising a radiation source and a radiation receiver, coaxial fiber optic optical fibers for transmitting excitation light from a source and radiation I of fluorescence from tubes, the central part of the fiber transmitting the excitation light is aperture apertured with the amount of reaction mixture in the 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 thermocover, microprocessor control unit and personal a computer, characterized in that the device is equipped with a pneumohydraulic system that contains a capacitance partly filled with liquid, four solenoid or check valves, two piston or membrane pumps and an air filter, while the heat-conducting element has through internal channels, the first inlet of the tank is connected through the filter to the environment, the first pump is piped through the first valve connected to the first outlet of the tank and through the second valve connected to the inputs of the internal channels of the heat-conducting element, the second pump through pipelines through the third valve is connected to the second output container and through the fourth valve is connected to the same internal channels of the heat conducting member inputs, outputs, internal channels of the heat conducting element connected to the second input conduit container, the controller is connected to an air compressor, four solenoid valves and microprocessor control device.

The invention is illustrated by drawings, in which:

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

in FIG. 2 is a diagram of the interface of one of the many tubes with reaction mixtures with coaxial fiber optic optical fibers of the claimed device;

in FIG. 3 is a diagram of the pneumohydraulic system of the first embodiment of a possible embodiment of the inventive device;

in FIG. 4 is a diagram of a pneumohydraulic system of a second embodiment of a possible embodiment of the inventive device;

in FIG. 5 is a diagram of a temperature change of a thermal cycler with test tubes during a temperature cycle;

in FIG. 6 is a table showing the operation of the components of the inventive device during the temperature cycle.

The claimed device for the simultaneous control of multiple amplifications of nucleic acid (Fig. 1) consists of a device for thermal cycling 1 (hereinafter referred to as the thermal cycler) with tubes 2, an optical system 3 with a radiation source 4 and a radiation receiver 5, a microprocessor control device 6, a personal computer with software providing 7 and a pneumohydraulic system 8. A microprocessor control device 6 is connected to a thermal cycler 1, an optical system 3, a personal computer 7, and a pneumohydraulic system 8. radiation source 4 and radiation receiver 5 contain a halogen lamp or a white LED 9, two-lens capacitors 10 and 11, between the lenses of which interference interference filters of excitation 12 and emission 13 are installed, a multi-channel photodetector 14 and a light guide bundle 15. Angle α is the angle between the extreme rays of the conical light beam optical system 3.

The thermal cycler 1 contains a heat-conducting element 16 with tubes 2 located in its recesses, an automatic temperature control device 17 and a thermal cover 18. A replaceable heat-insulating partition 19 is installed between the heat-conducting element 16 and the thermal cover 18.

Pneumohydraulic system 8 contains a controller 20 connected to a microprocessor control device 6. Pneumohydraulic system 8 is connected to a heat-conducting element 16.

One of the many tubes 2 is shown in enlarged view (Fig. 2). The test tube 2 with the reaction mixture 21 is closed by a cap 22 and installed in the recess of the heat-conducting element 16. In the thermal cap 18 there are holes in which the ends of the coaxial optical fibers with the central 23 and peripheral 24 parts are inserted.

The central parts of the optical fibers 23 are used to transmit the excitation light from the radiation source 4, and the peripheral parts 24 are used to transfer the radiation from the test tubes to the radiation receiver 5.

The heat-insulating partition 19 has openings whose axes coincide with the axes of the tubes, and the diameters are equal to the outer diameter of the peripheral part of the optical fibers 24. The central transmitting light of the excitation part of the optical fiber 23 is aperture-matched with the amount of reaction mixture 21 in the test tube 2: the partition 19 has a vertical size, which ensures the maximum angle α between the extreme rays of the conical light beam at the exit from the Central part of the fiber incident on the surface of the reaction mixture with a diameter d, simultaneously the minimal effect of the heat cap on the temperature of the reaction mixture is baked.

In FIG. 3 shows thermoelectric elements 25 (Peltier elements) and a radiator 26, which are included in the automatic temperature control device 17 and with which the cyclic temperature mode of the heat-conducting element 16 is provided.

Pneumohydraulic system 8 (first option) contains two containers 27, 28 partially filled with liquid, pipelines 29, air compressor 30, four solenoid valves 31-34, radiators 35, 36, controller 20 and air filters 38, 39. The heat-conducting element 16 has through internal channels.

The first outlet of the first tank 27 is connected by a pipe through the first solenoid valve 31 to the environment.

The air compressor 30 through the air filter 37 is connected by pipelines to the first input of the first tank 27 and to the input of the second electromagnetic valve 32.

The first outlet of the second container 28 through the air filter 38 is connected by a pipe to the environment.

The output of the second electromagnetic valve 32 is connected by a pipeline to the inputs of the internal channels of the heat-conducting element 16.

The second output of the first tank 27 is connected by pipelines through the third electromagnetic valve 33 with the same inputs of the internal channels of the heat-conducting element 16.

The outputs of the internal channels of the heat-conducting element 16 are connected by a pipe to the input of the second tank 28.

The second output of the second tank 28 is connected by pipelines through the fourth electromagnetic valve 34 to the second input of the first tank 27.

The first and second containers 27, 29 have thermal contact with radiators 35, 36.

The controller 20 is connected to the air compressor 30 and the electromagnetic valves 31-34.

In FIG. 4 shows thermoelectric elements 25 (Peltier elements) and a radiator 26, which are included in the automatic temperature control device 17 and with which the cyclic temperature mode of the heat-conducting element 16 is provided.

Pneumohydraulic system 8 (second option) contains a container 27 partially filled with liquid, four solenoid or check valves 31-34, two piston or diaphragm pumps 39, 40, a radiator 35, a controller 20, and an air filter 37. The heat-conducting element 16 has through internal channels .

The first inlet of the container 27 is connected through the air filter 37 to the environment.

The first pump 39 through pipelines through the first valve 31 is connected to the first output of the tank 27 and through the second valve 32 is connected to the inputs of the internal channels of the heat-conducting element 16.

The second pump 40 through pipelines through the third valve 33 is connected to the second output of the tank 27 and through the fourth valve 34 is connected to the same inputs of the internal channels of the heat-conducting element 16.

The outputs of the internal channels of the heat-conducting element 16 are connected by a pipe to the second input of the tank 27.

The controller 20 is connected to valves 31-34, pumps 39, 40.

The inventive device operates as follows.

During operation, many tubes 2 (FIG. 1) are placed in the recesses of the heat-conducting element 16. An example of the placement of one of these tubes is shown in FIG. 2. Test tube 2, which contains the reaction mixture 21 with a set of chemicals and a nucleic acid fragment, is hermetically closed by a cover 22. The thermocover 18 with a replaceable heat-insulating partition 19, restricting the air volume above the test tube, provides additional heating of the cap 22 of test tube 2 and prevents condensation on it water droplets. Using the microprocessor control device 6, the temperature cycle of thermal cycler 1 is set, which has four temperature conditions: heating in the time interval t1-t2, stabilization of temperature T2 at the upper level in the time interval t2-t3, cooling in the time interval t3-t5 and stabilization of the temperature T1 at the lower level in the time interval t5-t7 (Fig. 5).

The operation of the device is additionally illustrated by the table of the components of the inventive device during the temperature cycle (Fig. 6). The “+” sign indicates the time intervals when the compressor and pumps are in operation and the valves are open.

The microprocessor control device 6 transmits cyclic signals to synchronize the operation of the controller 20.

During operation of the pneumohydraulic system 8, the controller 20 (Fig. 3) using the air compressor 30 through the air filter 37 during the heating, temperature stabilization at the upper level and cooling creates and maintains increased air pressure in the first tank 27.

In cooling mode, the controller 20 opens the valve 33. From the first tank 27, liquid flows under pressure 29 to the inputs of the internal channels of the heat-conducting element 16. The fluid flows through the channels of the heat-conducting element 16 and the outputs of the internal channels of the heat-conducting element 16 through the pipe and enters the input of the second tank 28, in which normal pressure is maintained using a filter 38. The fluid flowing through the channels of the heat-conducting element significantly increases the rate of temperature change in cooling mode. At the end of the cooling mode at time t4, the controller 20 turns off the valve 33 and turns on the valve 32. The liquid from the internal channels of the heat-conducting element 16 is removed into the second tank 28. Radiators 36 and 35 provide two-stage cooling of the liquid heated during heat exchange with the heat-conducting element 16. The liquid in capacity 28 is cooled using a radiator 36.

In the temperature stabilization mode at the lower level, the controller 20 at the time t5 stops the air compressor 26, the air discharge stops. The controller 20 opens the valve 31 to reduce the pressure in the first tank 27, then at the time t6 turns on the valve 34, while the excess liquid from the second tank 28 is poured into the first tank 27. The liquid in the tank 27 is cooled by a radiator 35 to room temperature.

In the heating and temperature stabilization mode at the upper level, the controller 20 closes all the valves and turns on the air compressor 30 to create increased pressure in the first tank 27. Then, the temperature cycles are repeated many times.

During operation of the pneumohydraulic system 8 (Fig. 4) using the controller 20 through the valves 31, 33 during the time of stabilization of the temperature at the lower level, heating and stabilization of temperature at the upper level, the pumps 39, 40 are filled from the outputs of the tank 27, respectively, with air or liquid. In the cooling mode, the pump 40 through the valve 34 ensures that the channels of the heat-conducting element 16 are filled with liquid and flow through the channels 16. At the end of the cooling mode, at the time t4, the pump 40 stops, and the pump 39 through the valve 32 removes liquid from the channels of the heat-conducting element 16 to the tank 27. Then temperature cycles are repeated many times.

At all temperature conditions of the thermal cycler, thermoelectric elements 25 of the automatic temperature control device 17 are actively operating. In the cooling mode, the temperature of the heat-conducting element and tubes containing the reaction mixture should vary from 95 to 60 ° C.

In cooling mode, a fluid flow is provided through the internal channels of the heat-conducting element. The liquid filling the tank with the help of radiators is maintained at the level of the environment, which can be in the range of 10-35 ° С. When a fluid flows through the internal channels of a heat-conducting element, heat exchange actively occurs between the heated heat-conducting element with test tubes and a liquid having a much lower (room) temperature. The duration of the cooling mode is significantly reduced, while reducing the total analysis time.

In the modes of temperature stabilization at the lower level, heating and temperature stabilization at the upper level, there is no liquid in the internal channels of the heat-conducting element. Due to the internal channels, the weight and heat capacity of the heat-conducting element are reduced, while the duration of the heating mode is also reduced.

The rate of temperature change is defined as the temperature increment ° C per second. With a reduction in cooling time, the rate of temperature change increases.

The proposed design of the device allows to increase the rate of temperature change in the cooling mode and thereby increase the speed and performance of this device by reducing the analysis time.

Information sources

1. US patent No. 8797526 B2, cl. G01N 1/10, 08/05/2014

2. The LightCycler® 480 Real-Time PCR System (http://www.roche-applied-science.com).

3. US patent No. 7632464 B2, cl. USA 422/99, 422/102, 422/130, 12/15/2009

4. Patent for utility model of the Russian Federation No. 133835, MPK7 S12M 1/38, publ. 10/27/2013

5. Patent for the invention of the Russian Federation No. 2304277, IPC G01N 21/63, publ. 08/10/2007

6. Patent for the invention of the Russian Federation No. 2418289, IPC G01N 21/64, publ. 05/10/2011

Claims (2)

1. A device for simultaneous real-time monitoring of multiple amplifications of a nucleic acid, comprising a thermal cycler including a heat-conducting element with recesses for test tubes with reaction mixtures located therein, a thermal cap and an automatic temperature control device, an optical system including a radiation source and a radiation receiver, coaxial optical fibers for transmitting excitation light from a source and emitting fluorescence from test tubes, central ne The part of the optical fiber that excites the excitation light is apertured with the amount of reaction mixture in the 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 microprocessor, the microprocessor control device and a personal computer, characterized in that the device is equipped with a pneumohydraulic system, which contains two containers, partially filled with liquid, tr water pipes, an air compressor, four solenoid valves, radiators and a controller, air filters, while the heat-conducting element has through internal channels, the first output of the first tank is connected by piping through the first electromagnetic valve to the environment, the air compressor is connected by piping to the first input of the first capacity and with the input of the second electromagnetic valve, the first output of the second capacity through the air filter is connected by a pipe to the environment, the output of the second of the solenoid valve is connected by a pipeline to the inputs of the internal channels of the heat-conducting element, the second output of the first capacity is connected by pipelines through the third electromagnetic valve with the same inputs of the internal channels of the heat-conducting element, the outputs of the internal channels of the heat-conducting element are connected by a pipe to the input of the second capacity, the second output of the second capacity is connected by pipelines through the fourth solenoid valve with a second input of the first tank, the first and second tanks have a thermal co tact with radiators, the controller is connected to an air compressor, four solenoid valves and microprocessor control device. 2. A device for simultaneous real-time monitoring of multiple nucleic acid amplifications, comprising a thermal cycler, including a heat-conducting element with recesses for test tubes with reaction mixtures located therein, a thermal cap and an automatic temperature control device, an optical system including a radiation source and radiation receiver, coaxial optical fibers for transmitting excitation light from a source and emitting fluorescence from test tubes, central ne The part of the optical fiber that excites the excitation light is apertured with the amount of reaction mixture in the 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 microprocessor, the microprocessor control device and a personal computer, characterized in that the device is equipped with a pneumohydraulic system, which contains a container partially filled with liquid, four electromagnetic or non-return valves, two piston or diaphragm pumps and an air filter, while the heat-conducting element has through internal channels, the first inlet of the tank is connected through the filter to the environment, the first pump through pipelines through the first valve is connected to the first outlet of the tank and through the second valve connected to the inputs of the internal channels of the heat-conducting element, the second pump through pipelines through the third valve connected to the second output of the tank and through the fourth valve connected to the same the inputs of the internal channels of the heat-conducting element, the outputs of the internal channels of the heat-conducting element are connected by a pipe to the second input of the tank, the controller is connected to valves, pumps and a microprocessor control device.

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