TW201809284A - Compact thermal cycling device and system comprising said thermal cycling device - Google Patents

Abstract

The present invention relates to a compact thermal cycling device (1) for heating and cooling at least one housing mount (2) for housing a lower portion of a reagent tube (3), adapted for conducting heat into or out of same; and a thermoelectric heater (6) coupled to said mount (2). Advantageously, the device (1) of the invention further comprises a heating block (8) arranged in a region located above the region of the mount (2), comprising at least one housing port (9) adapted for receiving an upper portion of the tube (3) located above the portion housed by the mount (2), being in thermal contact with said upper portion.

Description

Compact thermal cycler and system including the same

The invention belongs to the experimental fields of molecular biology, biochemistry and genomics. More specifically, the present invention relates to a thermal loop device for DNA sequence amplification, and a system related to the device.

Thermal loop devices, also known as PCR (polymerase chain reaction) devices, are preferably used in the field of molecular biology to loop polymerases at different temperatures, such as DNA amplification or sequencing reactions using the Sanger method device of. The most common model of the device consists of multiple holes, which are configured to hold test tubes, connected to a resistance block through a heating plate, which distributes uniform temperature into the holes for programming multiple times, Typical temperature ranges include between 4 ° C and 96 ° C.

Considering that the culture reaction in the test tube is usually performed in an aqueous solution, the thermal loop device usually also includes a heating cover, which is arranged to cover the test tube and is constantly heated at 104 ° C, so that the condensation of water is limited to the closed tube in which the reaction occurs. In this case, the concentration of the solute is reduced to prevent the optimal conditions of the polymerase from changing, and the required thermodynamics are maintained for the primer pairing used as a starting point for DNA replication.

In the thermal loop instrument, rapid and continuous temperature change loops are programmed to begin the process of separating and denaturing DNA strands at high temperatures, then annealing at low temperatures, and finally extending at high temperatures. This loop can be repeated multiple times to achieve the desired degree of amplification of the DNA fragment.

Various commercial thermal loop meters including heating devices based on Peltier technology using semiconductor properties have been available for several years. These materials provide better temperature uniformity and steeper temperature ramps up and down for better results during the PCR process. Also, in order to improve temperature accuracy, accuracy, and uniformity, some solutions on the market choose to use metals such as gold, silver, and other alloys in the hole heating block to achieve higher test stability and reproducibility.

However, even in thermal loop-based thermocyclers, the problem of condensation still exists, thus limiting the validity of its results. In this sense, the lid, which is a heating block in this type of thermal loop instrument, must be lowered or slid onto the upper part of the tube and placed on the test tube. This requires a complex structure that restricts access and manipulation of the test tube, and also places the focal point of heating (ie, the base of the test tube and the heater of the heated lid) too far away, which results in a higher degree of reagent condensation.

As mentioned in the previous paragraph, there is therefore a need to provide a solution that can be integrated into a thermocycler, which effectively reduces condensation in the samples in which the amplification reaction occurs, and also allows more direct access to the used containment tube. The present invention meets the needs through a novel compact thermal loop device, It has dual static and dynamic heating systems for heating the containment tube.

According to the information mentioned in the previous section, it is an object of the present invention to provide a device that can significantly reduce sample condensation compared to known thermal loop meters based on heated lid systems, in which the lid used is lowered or slid to the tube. The top, on their ends.

For the above purpose, the object of the present invention is achieved by a compact thermal loop device for heating and cooling at least one receiving base for receiving a lower part of a test tube, wherein the base is connected to a thermoelectric heater to The lower part of the test tube is heated / cooled.

Advantageously, the device of the invention further comprises a heating block thermally isolated from the base, the heating block being arranged in the upper area above the heating block, comprising at least one receiving port adapted to receive the upper part of the test tube and heating to be received by the base The upper area of the lower area.

The heating block preferably includes one or more resistance heating devices, which are adapted to be in thermal contact with the test tube when the test tube is contained in a thermal loop meter, so that the upper part of the test tube can be maintained at a constant temperature, thereby preventing Samples condensed on the wall of the test tube looped at different temperatures. The combination of the effect of the heating block and the thermoelectric heater allows a reduction in the number of times to obtain results, as well as a faster temperature increase and decrease (steeper slopes), which equates to a greater reaction efficiency, thus being comparable to other heat of the prior art Improved results compared to lap meters.

In a preferred embodiment of the invention, the thermoelectric heater comprises two Peltier elements facing each other and surrounding the base. This achieves thermally symmetrical distribution Home.

In another preferred embodiment of the present invention, the base includes a port for optically detecting or inspecting a reaction occurring in a test tube due to its heating and cooling.

In another preferred embodiment of the present invention, the resistance heating device is configured to keep the portion of the test tube contained in the receiving port at a constant temperature, and its value is preferably equal to or greater than the minimum condensation suppression temperature of the sample contained in the test tube. More preferably, the temperature is kept substantially at 104 ° C. There is therefore no need to lower or slide the device to its upper part like most prior art thermal loop devices. Therefore, it allows for a simpler structure that does not require automatic devices or user intervention, and maintains lateral heating of the test tube to provide a larger non-condensing surface, as well as higher temperature accuracy and uniformity in the test tube.

In another preferred embodiment of the present invention, the base and the heating block are separated from each other by a distance of 0.5 mm to 5.0 mm.

In another preferred embodiment of the present invention, the base includes an upper extension or a hole generally having a height of 0.1 mm to 5.0 mm. The range is suitable for preventing thermal contact between the Peltier heater and the heating block, especially if the test tube is made of a low thermal conductivity material such as a plastic material.

The hole may be an integrated part of the base, an extension thereof or an element in thermal contact therewith, whose main function is to enlarge the heat transfer surface of the thermoelectric heater in the lower part of the test tube. Therefore, the holes help to heat the tube above the reagent level, thereby preventing heat loss from the wall of the test tube near the level and uniformizing the temperature of the reagent.

In another preferred embodiment of the invention, the device includes a temperature sensor in thermal contact with the base, the temperature sensor being configured to control the The power provided by the heater and the temperature of the test tube contained in the base. More preferably, the temperature sensor is arranged in a plane of symmetry next to the base and / or in an area closest to the containing tube containing the reagent.

In another preferred embodiment of the present invention, the thermal loop device of the present invention includes a plurality of receiving bases for receiving test tubes, which have corresponding Peltier heaters, heating blocks, and holes (if used). As a result, a fully expandable thermal loop meter is obtained, and thus its related technology is not limited by the number of test tubes used.

Another object of the invention relates to a system comprising at least one thermal loop device according to any of the embodiments described herein in combination with one or more test tubes housed in one or more receiving bases of the thermal loop meter .

In a preferred embodiment of the system of the invention, the one or more test tubes include a stopper or an upper closure that enters until the height is substantially equal to or less than a height of 20 mm above the level of the reagent contained in the test tube. More preferably, the height is substantially equal to or less than 1 mm.

For the purpose of illustration, the following provides some of the main terms used in this description and their definitions in accordance with the scope of the present invention: The thermal loop device or PCR device must be interpreted as suitable for subjecting one or more test tubes containing reagents to different Temperature loop devices, such as DNA amplification or sequencing reactions using the Sanger method.

The base must be interpreted as a device for the lower or bottom of a thermocycler containing a test tube containing a reagent, said base being temperature controlled by one or more heating devices.

Test tubes containing reagents must be interpreted as any The device is shaped to fit in the base of the thermal lap device.

The hole must be interpreted as a thermal connection element or element that is part of the base and is arranged at a height higher than the base to accommodate the test tube containing the reagent. Therefore, the hole receives a part of the tube located above the part received by the base.

The optical inspection port must be interpreted as any entrance to the base, through which information related to light transmitted to or from the inside of the base can be obtained.

A thermoelectric heater must be interpreted as a device that controls the temperature of the base by a mechanism based on any known manifestation of the thermoelectric effect.

Thermoelectric Peltier elements must be interpreted as elements based on thermoelectric heaters whose temperature is controlled by the Peltier effect.

The temperature sensor must be interpreted as any mechanism having a device for measuring a temperature value inside the base or for measuring a change in said value.

The heating block must be interpreted as any resistance heating device for applying heat to the upper part of the test tube accommodated by the receiving port.

The lower part of the test tube must be interpreted as a continuous part of the test tube. When the test tube is contained in the base of the thermal loop instrument, the lower part of the test tube accordingly includes the volume of the test tube containing the reagents that have undergone temperature looping.

The upper part of the test tube must be interpreted as a continuous part of the test tube. When the tube is contained in the base, the upper part of the test tube is set higher than the lower part referred to in the previous paragraph.

The stopper or closure of the tube must be interpreted as any upper closing element of the test tube, the application of which limits the volume of reagents that can be contained therein.

The expression "thermal contact" between two or more elements must be interpreted within the scope of the present invention as close contact between the elements (this is understood as the direct contact in the absence of an interface medium such as air), As a result, heat transfer between the elements is allowed.

An expression "substantially" applied to a relationship between two or more orders of magnitude must be interpreted within the scope of the present invention to be equal to or contained within a range having a ± 10% change from the value in question.

The device of the invention acts on a longitudinal tube in a vertical position, with gravity as a reference for verticality. Therefore, the relevant terms above, below, above or below must be interpreted relative to the vertical direction. The vertical direction of the test tube is determined by the configuration of the test tube's accommodating area, so that the test tube adopts the correct position in its operating position in the thermal loop device.

When the expression "comprises" is applied to a relationship between a primary element and other secondary elements, it must be interpreted to include or include the secondary element, but does not exclude other additional elements.

When the expression "consists" is applied to the relationship between a primary element and other secondary elements, it must be interpreted as including or including the secondary element, excluding other additional elements.

1‧‧‧ thermal loop device

2‧‧‧ Lower base for receiving test tubes containing reagents

3‧‧‧ test tubes containing reagents

4‧‧‧hole or upper extension of base

5‧‧‧ Optical inspection port

6‧‧‧thermoelectric heater

6 ’, 6” ‧‧‧ Thermoelectric Peltier element

7‧‧‧ temperature sensor

8‧‧‧ heating block

9‧‧‧Receiving port for receiving the upper part of the test tube

10‧‧‧ Stopper or closure for test tube

FIG. 1 herein shows a cross-sectional view of a thermal loop meter of the present invention in which a plurality of test tubes are housed, one of which is truncated and visually entered, and according to its preferred embodiment, it describes a dynamic heating block and a static The relative position of the heating block, and the separation between the two.

Figures 2a-2b show two views of the base and the hole of the thermal cycler of the present invention, in which some main elements are shown in detail.

Figure 3 shows a part of a test tube that can be accommodated by the device of the invention, wherein the test tube is closed by a stopper.

The following provides a detailed description of the present invention with reference to the preferred embodiment of the present invention based on FIGS. 1-3, which is provided for non-limiting and illustrative purposes.

As mentioned in the previous sections, the purpose of the thermal loop device (1) (FIG. 1) of the present invention is to imagine a combined static heating (hot plate mode) and / or dynamic heating (thermal Looper mode) for accommodating test tubes (3), wherein each base (2) is optionally thermally connected or fitted to the hole (4) in an integral manner, which allows extension in the lower part of the test tube (3) Heat to a higher level than the volume occupied by the reagent. Therefore, the heating provided by the hole (4) makes the temperature of the sample contained in the test tube (3) uniform, thereby minimizing the thermal gradient in the interface between the reagent and air contained in the test tube (3), and Prevent condensation.

In more detail, the compact and preferred integrated thermal loop device (1) of the present invention includes at least the following elements:

-Base (2): it comprises a preferred metal structure through which heat is introduced or exported, which respectively causes heating or cooling of the lower region of the test tube (3) contained therein due to heat conduction. The base (2) also includes a port (5) for optical detection or inspection of reactions occurring in the test tube (3) due to heating and cooling (Figures 1 and 2a). To make the inspection easier, the base (2) preferably has a flat lower base. In turn, the upper part of the base (2) is preferably internally There is a frusto-conical portion for receiving a corresponding tube (3).

-Hole (4): it is an element for accommodating the test tube (3), the test tube is thermally connected to the base (2) or part of the base (2), and is arranged at a height higher than the base (2) . Therefore, the hole (4) receives a part of the test tube (3) located above the test tube part received by the base (2), and serves as an extension of the base.

Thermoelectric heater (6): The heater (6) preferably comprises two thermoelectric Peltier elements (6 ', 6 "), which are positioned opposite each other and on each side of the base (2) such that when A better thermal symmetry is obtained when the test tube (3) applies or removes heat from the test tube (3).

-Temperature sensor (7) (Figures 2a and 2b): preferably included in and in thermal contact with the base (2), which allows monitoring of the power provided by the thermoelectric heater (6), thereby enabling the test tube (3) Precise temperature control.

-Heating block (8): The block (8) is arranged in the upper area of the thermal loop device (1) above the base (2) and the hole (4) (if used). In a preferred embodiment, the blocks (8) are connected to the base (2), but they must be thermally separated and isolated from each other. In the same embodiment, the base (2) and the hole (4) are the same part, although they may be formed as different parts in thermal communication with each other. The heating block (8) also includes at least one receiving element (9) configured to receive a portion of the test tube (3) located above the portion of the test tube (3) received by the hole (4).

The heating block (8) includes one or more resistance heating devices (such as a "hot plate" type device) and a receiving port (9) for receiving the test tube (3) so as to be in thermal contact therewith, so that the port (9) The portion of the contained test tube (3) can be kept at a constant temperature, thereby preventing the sample from being trapped in the test tube (3) during the PCR reaction. Condensation on the wall. In addition, the combination of the effects of the heating block (8) and the thermoelectric heater (6) results in obtaining results in a shorter time, and the temperature rises and decreases more quickly (a steeper slope), which is equivalent to improving the reaction efficiency, This results in improved results compared to other thermal loop meters of the prior art.

Preferably, the static heating block (8) located on the upper part of the test tube (3) heats the test tube (3), and the temperature in the lateral area thereof is substantially 104 ° C, so that the block (8) does not have to be as in the prior art Most of the thermal loop devices drop or slide to the upper part of the test tube (3). This therefore allows for a simpler structure that does not require robotic or user intervention, and maintains the lateral heating of the test tube (3) to provide a larger non-condensing surface (i.e. non-condensing), and in Better temperature accuracy and uniformity.

In a preferred embodiment of the present invention, in order to provide greater independence of the dynamic and static heating schemes of the thermal loop device (1), the base (2) and the heating block (8) are separated such that they are thermally isolated from each other. Their separation range is preferably between 0.5 mm and 5.0 mm. The separation is considered to be suitable because the material of the test tube is usually plastic and is a poor thermal conductor, so there will be no thermal contact between the thermoelectric heater (6) and the heating block (8).

In turn, the configuration of the base (2) according to the invention is optimized in order to maximize thermal symmetry and minimize thermal inertia. This reduces the energy input required to reach the target temperature throughout the base, which helps improve the efficiency of the thermal loop meter (1).

In this sense, in a preferred embodiment of the present invention, the shape of the base (2) is designed such that the quality as a determining factor of thermal inertia increases as the distance to the main focus heating point of the thermal loop device (1) increases Less Small, the focus heating point is a thermoelectric heater (6). In view of this, the cross-sectional size of the base (2) is the largest, and the possible temperature gradient is reduced due to the increasing distance between the upper regions of the test tubes (3), which is beneficial to the thermoelectric heater (6). ) Transfer heat or transfer heat from the thermoelectric heater (6). Likewise, the smaller mass of the central area in contact with the test tube (3) minimizes the thermal inertia of the base (2). Given that this arrangement is symmetrical, the temperature uniformity in the portion of the test tube inserted into the base (2) is maximized.

In an embodiment of the invention, holes (4) are incorporated in the base (2), said holes (4) preferably having a height between 0.1 mm and 5.0 mm, which allows heating above the reagent level, This prevents loss through the wall of the test tube near the level and makes the temperature of the reagent uniform.

In another embodiment of the invention, the temperature sensor (7) is arranged in a plane of symmetry immediately adjacent to the base (2), i.e. in its coldest region. This allows to ensure that the minimum temperature in the test tube (3) is at a level as close as possible to the target temperature of each reaction. Alternatively or additionally, in another embodiment of the invention, the temperature sensor (7) is located in an area in contact with the test tube (3). This allows to ensure that the lowest temperature in the tube is as close as possible to the target temperature of each reaction.

The combination of the shape of the base (2) that provides the maximum thermal symmetry and the minimum thermal inertia and the position of the temperature sensor (7) in the plane of symmetry and / or in the area closest to the test tube (3) allows a thermal loop device (1) Optimized quality, ie heating / cooling uniformity and rate, while keeping energy consumption to a minimum.

The proposed invention is scalable and can be applied to a random number of test tubes (3), so that the thermal loop device (1) has a modular and reproducible structure, allowing May be used in single test tubes (3) and large-scale reactions. Based on this heating / cooling method or other heating / cooling methods (e.g. oil, air, etc.), it also relates to other differences for prior art devices designed for multiple test tubes (3). Reducing the conventional design specifically to a single base produces a base with uniform sections along its entire length, which is less efficient than the efficiency proposed by the present invention. Reducing the distribution of the heating / cooling elements specifically to a single base results in an asymmetric distribution around the test tube, which translates into worse uniformity than described herein.

Furthermore, in other embodiments of the invention, the thermal loop device (1) may include the use of a stopper (10) or a closure (Figure 3) in the test tube (3), which is preferably entered in the form of a plunger until the height is substantially It is equal to or less than 20 mm above the level of the reagent to be contained in the test tube (3). More preferably, the height is substantially equal to or less than 1 mm.

The use of the stopper (10) in the test tube (3) even improves the temperature uniformity and reduces the evaporation and condensation of the reagent. All these also increase the efficiency of the PCR reaction, because when the stopper (10) enters the tube (3), the stopper (10) is heated vertically by the static heating block (8) to the area close to the reagent, which helps Reduce heat loss and condensation. The plug (10) further reduces the volume of air in the test tube (3), thereby achieving a saturation pressure corresponding to the reaction temperature, and converting less liquid reagents into vapor. This minimizes the variation in reagent concentration, which is an additional advantage over other thermal loop meters of the prior art.

1‧‧‧ thermal loop device

2‧‧‧ Lower base for receiving test tubes containing reagents

3‧‧‧ test tubes containing reagents

4‧‧‧hole or upper extension of base

5‧‧‧ Optical inspection port

6‧‧‧thermoelectric heater

6 ’, 6” ‧‧‧ Thermoelectric Peltier element

8‧‧‧ heating block

9‧‧‧Receiving port for receiving the upper part of the test tube

Claims (12)

A compact thermal loop device (1) for heating and cooling at least one receiving base (2) for receiving a lower portion of a test tube (3), wherein the base (2) is connected to for heating / cooling the The lower part of the test tube (3) is a thermoelectric heater (6); and the device (1) is characterized in that the device (1) further comprises a thermal insulation from the base (2) and arranged above the base (2) A heating block (8) in an upper region, said heating block (8) including at least one receiving port (9) adapted to receive an upper portion of a test tube (3) and to heat a chamber above the lower region received by said base (2) Said upper part;-wherein said base (2) and said heating block (8) are separated from each other by a thermal isolation distance, said thermal isolation distance being 0.5mm to 5.0mm; and-wherein said heating block (8) comprises a Or a plurality of resistance heating devices configured to maintain a constant temperature of a portion of the test tube (3) received by the accommodation port (9), the temperature value being equal to or higher than that of the sample contained in the test tube (3) Minimum condensation suppression temperature. The compact thermal loop device (1) according to item 1 of the patent application scope, wherein the base (2) includes a hole (4) for receiving at least a portion of a lower portion of a test tube (3), wherein the hole (4) is an integrated part of the base (2), an extension or an element in thermal contact therewith, which is arranged in the upper area of the base (2) and is suitable for a thermoelectric heater to be placed in said lower part of the test tube (3) (6) The heat transfer surface is enlarged to a level higher than the reagent contained in the test tube (3). The compact thermal loop device (1) according to item 2 of the scope of patent application, wherein the hole (4) has a height of 0.1 mm to 5.0 mm. The compact thermal loop device (1) according to item 1 of the patent application scope, wherein the thermoelectric heater (6) includes two thermoelectric Peltier elements positioned opposite each other and on each side of the base (2) (6 ', 6 "). The compact thermal loop device (1) according to item 1 of the patent application scope, wherein the base (2) includes a port (5) for optical inspection or inspection in a test tube (3) due to heating and cooling The reaction occurred. According to the compact thermal loop device (1) according to item 1 of the patent application scope, wherein the temperature of the heating block (8) is substantially 104 ° C. The compact thermal loop device (1) according to item 1 of the scope of patent application, wherein the base (2) includes a temperature sensor (7), the temperature sensor (7) and the base ( 2) Thermal contact for controlling the power provided by the thermoelectric heater (6) and the temperature of the test tube (3). The compact thermal loop device (1) according to item 7 of the scope of patent application, wherein the temperature sensor (7) is arranged on a symmetrical plane of the base (2) and / or in contact with the test tube (3) In the area. Compact thermal return according to any of claims 1 to 8 of the patent application scope The ring device (1) includes a plurality of bases (2) for receiving test tubes (3), and is provided with corresponding thermoelectric heaters (6) and heating blocks (8). A system comprising at least one compact thermal loop device (1) according to any of claims 1 to 9 of the scope of the patent application, said system comprising one or more of the devices contained in said device (1) It is produced by combining one or more test tubes (3) in the base (2). The system according to item 10 of the scope of patent application, wherein one or more test tubes (3) include an upper closure or stopper (10) which enters until it is substantially equal to or smaller than said A height of 20 mm above the level of the reagent contained in the test tube (3). The system according to item 11 of the patent application scope, wherein the height is substantially equal to or less than 1 mm.