CN118028098A

CN118028098A - Temperature control device and biological sample detection system

Info

Publication number: CN118028098A
Application number: CN202311815278.XA
Authority: CN
Inventors: 冉宇成; 王洪兵; 肖体明; 罗严; 王晓涛
Original assignee: Maccura Medical Electronics Co Ltd
Current assignee: Maccura Medical Electronics Co Ltd
Priority date: 2023-12-26
Filing date: 2023-12-26
Publication date: 2024-05-14

Abstract

The invention provides a temperature control device and a biological sample detection system, wherein the device comprises a device main body, a first temperature control component and a second temperature control component; the first temperature control assembly is provided with a placing groove for inserting a reaction cavity of a tubular structure, and the reaction cavity is internally provided with a reaction system and an upper oil layer; the first temperature control component is used for increasing and decreasing the temperature of the reaction system in the reaction cavity at a first variable temperature so that the reaction system sequentially reaches a denaturation temperature, an annealing temperature and an extension temperature; the second temperature control component is used for raising and lowering the temperature of the upper oil layer at a second variable temperature so as to enable the upper oil layer to compensate the raising and lowering temperature rate of the liquid at the part, close to the liquid level, of the upper part of the reaction system. Based on the technical scheme of the invention, temperature compensation can be performed near the liquid level of the reaction system, the distribution of temperature gradients is improved, the uniformity of the internal temperature of the reaction system is improved, the method is fully suitable for high temperature rise and fall rate, and the nucleic acid detection time can be shortened while the amplification efficiency and the detection signal intensity are ensured.

Description

Temperature control device and biological sample detection system

Technical Field

The invention relates to the technical field of medical equipment, in particular to a temperature control device and a biological sample detection system.

Background

Nucleic acid detection has found widespread use in many fields of disease diagnosis and prognosis, public health, food safety, molecular breeding, forensic identification, and the like. The nucleic acid detection process comprises two main steps: nucleic acid extraction and nucleic acid amplification. Nucleic acid extraction is divided into two methods, purification-free extraction and full extraction with purified nucleic acid. Nucleic acid amplification is divided into two modes of isothermal amplification and PCR variable-temperature amplification. In order to improve the sensitivity of nucleic acid detection, the nucleic acid detection is generally performed by adopting a mode of full extraction of nucleic acid and PCR variable-temperature amplification. Polymerase Chain Reaction (PCR) is a molecular biological technique for amplifying specific DNA fragments, which can be regarded as specific DNA replication in vitro, and the greatest feature of PCR is the ability to greatly increase minute amounts of DNA. The basic PCR reaction steps are divided into three steps: 1. denaturation: the reaction temperature is maintained at the first temperature for a certain period of time, and the high temperature breaks the hydrogen bonds between the double strands of the template DNA to form two single strands. 2. Annealing (renaturation): the reaction temperature is reduced from the first temperature to the second temperature, and the temperature is maintained for a certain time, so that the primer and the template DNA single strand are combined according to the base complementary pairing principle. 3. Extension: the second temperature is adjusted to the third temperature for a certain period of time, and a new DNA strand complementary to the template strand is formed in the presence of DNA polymerase, 4 dNTPs, magnesium ions, and the like. For positive samples, the amount of target DNA fragments doubles for every three phases of PCR temperature cycles. PCR amplification is typically performed at a temperature of 30-45 cycles. By adding fluorescent groups into the PCR amplification reaction, the change of the amount of amplified products in each cycle in the PCR amplification process can be detected in real time by utilizing the change of fluorescent signals, and quantitative analysis can be performed.

The current mainstream practice of domestic nucleic acid detection relies on a standard PCR laboratory, and is performed by a professional operator who holds a PCR on duty. The foremost logic here is also to effectively circumvent cross-contamination by means of a PCR compartmentalized laboratory. The molecular POCT has the remarkable characteristics that the instrument is small, the functions of nucleic acid extraction and nucleic acid amplification are integrated, and the nucleic acid extraction and amplification are fully automatic and fully sealed, so that the theory is independent of a PCR laboratory. And because the simple operation can give the detection result (sample to answer-sample in and out), no professional operator is needed in theory.

For molecular POCT products, in order to shorten the nucleic acid detection time and realize the purpose of rapidly outputting the detection result, the main technical route at present is to design a PCR amplification reaction chamber to be flat and thin, reduce the PCR temperature cycle time by improving the heat transfer area and the heat conduction efficiency, reduce the PCR amplification time and improve the detection speed. However, the PCR amplification reaction chamber is designed to be flat and thin, the clear aperture for fluorescent signal detection is reduced, the fluorescent signal intensity is greatly reduced, and the signal detection sensitivity is reduced, so that the overall performance of the instrument is deteriorated. To ensure the fluorescence signal intensity, the PCR amplification reaction chamber may be configured in a tubular configuration. However, at present, manufacturers adopting tubular structure amplification cavities for PCR variable-temperature amplification reaction generally have amplification reaction time of more than 1 hour and long nucleic acid detection time.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a temperature control device and a biological sample detection system, which can ensure the detection intensity of fluorescent signals based on a PCR amplification reaction cavity with a tubular structure, reduce the detection time of nucleic acid and improve the detection speed of nucleic acid on the premise of ensuring the amplification efficiency of PCR.

In a first aspect, the present invention provides a temperature control device for heating and cooling a biological sample processing member, the biological sample processing member including a reaction chamber having a tubular structure, the temperature control device comprising:

A device body; and

The device comprises a device main body, a first temperature control assembly and a second temperature control assembly, wherein the first temperature control assembly and the second temperature control assembly are arranged on the device main body, the second temperature control assembly is positioned above the first temperature control assembly, the first temperature control assembly is provided with a placing groove for inserting a reaction cavity of a tubular structure, and a reaction system and an upper oil layer floating above the liquid level of the reaction system are accommodated in the reaction cavity;

the first temperature control component is used for heating and cooling the reaction system in the reaction cavity at a first variable temperature so that the reaction system sequentially reaches a denaturation temperature, an annealing temperature and an extension temperature;

The second temperature control component is used for increasing and decreasing the temperature of the upper oil layer at a second variable temperature so that the upper oil layer compensates the increasing and decreasing rate of the liquid at the part, close to the liquid level, of the upper part of the reaction system.

In one embodiment, the first temperature control assembly and the second temperature control assembly are configured to independently control operation; the second variable temperature and the first variable temperature synchronously change in a lifting manner, and the second variable temperature at any moment is larger than the first variable temperature.

In one embodiment, the first temperature control component and the second temperature control component respectively comprise a first heating block and a second heating block, the placing groove is configured on the first heating block, and a through hole corresponding to a notch of the placing groove is configured at the center of the second heating block so that the reaction cavity can pass through and be inserted into the placing groove.

In one embodiment, the height of the notch position of the placing groove is higher than the height of the liquid level of the reaction system in the reaction cavity.

In one embodiment, the first temperature control component and the second temperature control component further comprise a first peltier and a second peltier respectively, and the first peltier and the second peltier are respectively attached to the first heating block and the second heating block.

In one embodiment, the device main body comprises two heat dissipation blocks, the first temperature control component is clamped between the two heat dissipation blocks, and the second temperature control component is arranged on a first end face of the device main body, which is formed by top end faces of the two heat dissipation blocks;

The first heating block and the two first peltier devices arranged on the two sides of the first heating block are integrally clamped between the two heat dissipation blocks, and the first peltier devices are attached to the inner side surfaces of the corresponding heat dissipation blocks; the two second peltier devices are respectively arranged on top end surfaces of the two heat dissipation blocks, and the second heating blocks are constructed into a plate-shaped structure and cover the two second peltier devices.

In one embodiment, the first heating block is internally configured as a hollow structure, and the hollow structure does not extend to the groove wall of the placing groove and the side surface of the first heating block, which is attached to the first peltier.

In one embodiment, the first heating block and the second heating block are both made of heat-conducting metal blocks, and heat-conducting media are arranged between the first heating block and the first peltier and between the second heating block and the second peltier.

In one embodiment, the device body further comprises two fixing pieces, the two fixing pieces are respectively covered on two sides of the second heating block, and the through hole is positioned between the two fixing pieces;

The fixing piece is fixedly connected with the top end face of the corresponding heat dissipation block body through a fastening piece, so that the second heating block and the second Peltier are pressed on the first end face.

In one embodiment, the surfaces on both sides of the second heating block are respectively provided with a positioning groove, and the bottom surface of the fixing piece is provided with a positioning protrusion capable of being matched into the positioning groove and a pressing protrusion capable of contacting the surface of the second heating block.

In one embodiment, the portion of the through hole is configured as a boss protruding relative to the surface of the second heating block, and the boss is located between the two second peltier devices to limit the two second peltier devices.

In one embodiment, the bottom end surfaces of the two heat dissipation blocks form a second end surface of the device body, and the second end surface is provided with a heat dissipation fan.

In one embodiment, a first temperature sensing unit and a second temperature sensing unit are further disposed in the first temperature control component and the second temperature control component, respectively, and the first temperature sensing unit and the second temperature sensing unit are used for detecting the first variable temperature and the second variable temperature, respectively.

In one embodiment, a first assembly hole corresponding to the bottom of the reaction cavity is arranged at the bottom of the placing groove and used for installing an excitation optical fiber for exciting the reaction system to generate a fluorescence signal;

And a second assembly hole corresponding to the side surface of the reaction cavity is formed in the side wall of the placing groove and is used for installing and detecting a light receiving optical fiber of a fluorescent signal generated by the reaction system.

In one embodiment, the first variable temperature comprises a first high point temperature and a first low point temperature, and the second variable temperature comprises a second high point temperature and a second low point temperature;

The first temperature control component is configured to be capable of increasing the temperature of the reaction system from the first low point temperature to the first high point temperature according to a first rate of increase in temperature to a denaturation temperature; the second temperature control assembly is configured to be capable of warming from the second low point temperature to the second high point temperature according to a second warming rate to warm the overlying oil layer;

The first temperature control component is configured to be capable of cooling from the first high point temperature to the first low point temperature according to a first cooling rate, so that the reaction system passes through an annealing temperature and reaches an extension temperature reaction system; the second temperature control assembly is configured to be capable of cooling from the second high point temperature to the second low point temperature according to a second cooling rate to cool the overlying oil layer.

In one embodiment, the first temperature control assembly and the second temperature control assembly are configured to have the same warm-up duration and cool-down duration;

The second temperature rising rate is calculated and determined according to the temperature difference between the second high-point temperature and the second low-point temperature and the temperature rising duration of the first temperature control component at the first temperature rising rate; the second cooling rate is computationally determined in the same manner from the first cooling rate.

In one embodiment, the temperature difference between the second low point temperature and the first low point temperature is greater than the temperature difference between the second high point temperature and the first high point temperature.

In one embodiment, the first temperature control assembly is configured to maintain a first preset duration after warming to the first high point temperature, and to maintain a second preset duration after cooling to the first low point temperature;

The second temperature control assembly is configured to maintain the first preset duration after warming to the second high point temperature and maintain the second preset duration after cooling to the second low point temperature.

In one embodiment, the value range of the first heating rate and the first cooling rate is 5 ℃/s-13 ℃/s.

In a second aspect, the present invention provides a biological sample detection system, which includes:

The biological sample treatment component comprises a cracking cavity, a cleaning cavity and a reaction cavity with a tubular structure, which are sequentially communicated; the cracking cavity is used for receiving the sample to be treated and cracking the sample to be treated so as to release target substances in the sample to be treated; the cleaning cavity is used for cleaning target substances of the sample to be processed; the reaction cavity is used for amplification reaction of target substances of a sample to be treated;

the magnetic conduction piece is accommodated in the biological sample treatment component and used for adsorbing target substances in the sample to be treated and carrying the target substances under the action of an external magnetic field;

a magnet abutting against an outer side wall of the biological sample processing member for providing the external magnetic field; and

The temperature control device.

The above-described features may be combined in various suitable ways or replaced by equivalent features as long as the object of the present invention can be achieved.

Compared with the prior art, the temperature control device and the biological sample detection system provided by the invention have the following beneficial effects:

According to the temperature control device and the biological sample detection system, two independent temperature control components are utilized to heat the reaction cavity, so that the heating temperature rate of liquid at the upper part of the reaction system can be compensated by utilizing the heated upper oil layer while the reaction system is heated, the distribution of temperature gradients is improved, the uniformity of the internal temperature of the reaction system is improved, the reaction system can adapt to higher heating temperature rate, and the amplification efficiency of the reaction system can be ensured under the higher heating temperature rate; thereby shortening the nucleic acid detection time while ensuring the amplification efficiency and the detection signal intensity.

Drawings

The invention will be described in more detail hereinafter on the basis of embodiments and with reference to the accompanying drawings. Wherein:

FIG. 1 is a schematic view showing the overall structure of a temperature control apparatus of the present invention;

FIG. 2 shows an exploded view of the structure of the temperature control apparatus of the present invention;

FIG. 3 is a schematic view showing the structure of a first heating block of the temperature control apparatus of the present invention;

FIG. 4 is a schematic diagram showing the structure of a second heating block of the temperature control apparatus of the present invention;

FIG. 5 shows a schematic structural view of a fixing member of the temperature control device of the present invention;

FIG. 6 shows a front cross-sectional view of a partial structure at the heating assembly of FIG. 1;

FIG. 7 shows a side cross-sectional view of a partial structure at the heating assembly of FIG. 1;

FIG. 8 is a schematic view showing the temperature gradient distribution of the reaction system in the reaction chamber for temperature control based on the temperature control device of the present invention;

FIG. 9 is a schematic diagram showing the temperature gradient distribution of a reaction system in a reaction chamber for temperature control using the prior art.

In the drawings, like parts are designated with like reference numerals. The figures are not to scale.

Reference numerals:

1-device main body, 11-heat dissipation block, 12-mounting, 121-positioning protrusion, 122-compaction protrusion, 2-first temperature control component, 21-first heating block, 211-placing groove, 212-first assembly hole, 213-second assembly hole, 214-first temperature measuring groove, 215-hollow structure, 22-first peltier, 3-second temperature control component, 31-second heating block, 311-through hole, 312-positioning groove, 313-second temperature measuring groove, 314-boss, 32-second peltier, 4-heat dissipation fan, 5-reaction chamber, 51-reaction system, 52-overlying oil layer.

Detailed Description

Features and exemplary embodiments of various aspects of the application are described in detail below. In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the application. It will be apparent, however, to one skilled in the art that embodiments of the application may be practiced without some of these specific details. The following description of the embodiments is merely intended to provide a better understanding of the application by showing examples of the application.

In the description of the present application, it should be understood that the direction or positional relationship indicated with respect to the description of the orientation, such as up, down, back, etc., is based on the direction or positional relationship shown in the drawings, and is merely for convenience in describing the embodiments of the present application and simplifying the description, and does not indicate or imply that the apparatus or element referred to must have a specific orientation, be configured and operated in a specific orientation, and thus should not be construed as limiting the embodiments of the present application.

In the description of the embodiments of the present application, plural means two or more, and greater than, less than, etc. are understood to exclude this number. The description of the first and second is for the purpose of distinguishing between technical features only and should not be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated or implicitly indicating the precedence of the technical features indicated.

In the description of the embodiments of the present application, unless explicitly defined otherwise, terms such as arrangement, installation, connection, etc. should be construed broadly, and those skilled in the art may reasonably ascertain the specific meaning of the terms in the present application by combining the specific contents of the technical solutions.

It should be noted that, without conflict, the embodiments of the present application and features of the embodiments may be combined with each other. The embodiments will be described in detail below with reference to the accompanying drawings.

Molecular POCT is a detection mode which is carried out on a sampling site and can rapidly obtain a detection result by using a portable analysis instrument and a matched reagent, and the main standard is a detection site which does not need to be fixed, and the reagent and the instrument are portable and can be operated in time. Molecular POCT is carried out by integrating cleavage, washing, amplification and fluorescence detection into different chambers of the same consumable cartridge,

Currently, for molecular POCT products, the technical scheme adopted in the industry for performing PCR temperature-variable amplification reaction based on a reaction chamber (i.e., a consumable tube with a tubular structure) generally has an amplification reaction time of more than 1 hour, and the whole nucleic acid detection time is long, resulting in low detection efficiency. Therefore, the temperature rate of the temperature control module is increased to reduce the PCR temperature cycle time, thereby achieving the purpose of shortening the nucleic acid amplification reaction time. However, at present, manufacturers adopting tubular reaction chambers for PCR amplification reaction in the industry cannot achieve the rapid temperature rise and fall rate of the temperature control module, and the temperature rise and fall rate is generally not more than 4 ℃/s. The inventors found that the reason for this is that, due to the limitation of the thermal conductivity of the reaction system itself and the reaction chamber having a tubular structure, a certain process is required for transferring heat from the bottom to the central region and the upper region of the reaction system when the reaction chamber is heated from the bottom, and therefore the temperature rise and fall rate of the upper liquid of the reaction system is lower than that of the lower liquid, and a temperature difference gradient is easily present in the vertical height of the reaction system, which affects the effect and amplification efficiency of the PCR reaction. Therefore, in order to ensure the PCR amplification efficiency, the prior art can only be carried out at a lower temperature rise and fall rate, and the lower temperature rise and fall rate is utilized to relatively prolong the heat conduction time, thereby ensuring the PCR amplification efficiency.

If the temperature rise and fall rate is directly increased on the basis of the prior art scheme, the temperature rise and fall rate of the upper liquid of the reaction system is lower than that of the lower liquid, and the temperature rise and fall rate of the liquid of the reaction system in the vertical direction is inconsistent, so that the temperature in the reaction system is uneven and the temperature gradient is larger; as shown in figure 9 of the drawings, when the temperature control module carries out temperature rise and fall control on the reaction system at a high temperature rise and fall rate of 10 ℃/s, the temperature gradient in the reaction system is large, the amplification efficiency of the PCR reaction is low, and finally, an effective nucleic acid detection result cannot be obtained.

The invention will be further described with reference to the accompanying drawings.

Embodiments of the present application provide a biological sample detection system that includes a biological sample processing member, a magnetically permeable member, a magnet, and a temperature control device. The biological sample treatment component comprises a cracking cavity, a cleaning cavity and a reaction cavity 5 with a tubular structure, which are sequentially communicated; the cracking cavity is used for receiving the sample to be treated and cracking the sample to be treated so as to release target substances in the sample to be treated; the cleaning cavity is used for cleaning a target substance of a sample to be processed; the reaction chamber 5 is used for amplification reaction of a target substance of a sample to be treated. The magnetic conduction piece is accommodated in the biological sample treatment component and is used for adsorbing target substances in a sample to be treated and carrying the target substances under the action of an external magnetic field; the number of the magnetic conduction pieces can be multiple, and the target substances are adsorbed simultaneously by utilizing the magnetic conduction pieces, so that the adsorption and transfer efficiency of the target substances is improved; illustratively, in some embodiments of the application, the magnetically permeable member may be superparamagnetic magnetic particles. The magnet is abutted to the outer side wall of the biological sample processing component and used for providing an external magnetic field; the temperature control device is attached to the outer side wall of the reaction cavity 5 and is used for performing temperature cycle control on the PCR amplification reaction of a reaction system 51 in the reaction cavity 5, wherein the reaction system 51 is mixed liquid of target substances of a sample to be processed and PCR reaction reagents; in this embodiment, the target substance is a nucleic acid.

Furthermore, in order to prevent the reaction system 51 from evaporating under the heating of the temperature control device, so as to avoid the influence of the liquid of the reaction system 51 on the detection result due to the decrease of the liquid, and to prevent the reaction system 51 from evaporating to condense on the inner wall of the consumable tube, so as to avoid the influence of the formation of condensed water on the collection of fluorescent signals, an upper oil coating layer 52 is arranged on the liquid surface of the reaction system 51 in the reaction chamber 5 of the invention; the upper oil layer 52 is a lipophilic substance, which may be a lipophilic material such as paraffin oil or oil. The lipophilic material is not compatible with the reaction system 51, and it is possible that the lipophilic material does not react with the reaction system 51 in any chemical way, and the lipophilic material is layered with the reaction system 51, i.e., the lipophilic material floats above the liquid surface of the reaction system 51, and does not form an emulsion under natural conditions.

The embodiment of the invention also provides a temperature control device which is used for heating and cooling a biological sample treatment component, wherein the biological sample treatment component comprises a reaction cavity 5 with a tubular structure, and the temperature control device comprises a device main body 1, and a first temperature control component 2 and a second temperature control component 3 which are both arranged on the device main body 1; the second temperature control assembly 3 is positioned above the first temperature control assembly 2, the first temperature control assembly 2 is provided with a placing groove 211 for inserting a reaction cavity 5 with a tubular structure, and the reaction cavity 5 is internally provided with a reaction system 51 and an upper oil coating layer 52 floating above the liquid level of the reaction system 51;

The first temperature control component 2 is configured to raise and lower the temperature of the reaction system 51 in the reaction chamber 5 at a first variable temperature, so that the reaction system 51 sequentially reaches a denaturation temperature, an annealing temperature and an extension temperature; the second temperature control assembly 3 is used for raising and lowering the temperature of the upper oil layer 52 at a second variable temperature, so that the upper oil layer 52 compensates the raising and lowering rate of the liquid at the part, close to the liquid surface, of the upper part of the reaction system 51. By adopting the structure, the temperature rise and fall rate of the liquid at the upper part of the reaction system 51 and the temperature rise and fall rate of the liquid at the lower part are more consistent, the temperature difference gradient on the vertical height of the reaction system 51 is improved, the temperature uniformity on the vertical height of the inside of the reaction system 51 is improved, the first temperature control component 2 is prevented from improving the temperature difference gradient on the vertical height of the reaction system at a lower temperature rise and fall rate to ensure the PCR amplification efficiency, the reaction system 51 can adapt to the high temperature rise and fall rate of the first temperature control component 2, the PCR temperature cycle time is reduced, the nucleic acid amplification time is reduced, the nucleic acid detection time is reduced, and the nucleic acid detection speed is further improved.

Specifically, the invention is based on the PCR reaction of a reaction chamber 5 having a tubular structure, wherein the reaction chamber 5 is integrally formed as a part of a biological sample processing member, and the reaction chamber 5 may be a cylindrical tube or a tapered tube, preferably a tapered tube, as shown in FIG. 2 of the drawings. Compared with a cylindrical tube, the reaction cavity 5 adopts a conical tube, so that the liquid level of the reaction system 51 with the same volume is higher, the position of a receiving optical fiber for detecting fluorescent signals is more easily reached, the normal detection of the fluorescent signals is ensured, and meanwhile, the structural strength of the reaction cavity 5 can be enhanced. The invention mainly designs the structure of a temperature control module of a temperature control device, based on a device main body 1, designs the temperature control module into a first temperature control assembly 2 and a second temperature control assembly 3 which independently control operation, and further based on the arrangement of an upper oil coating layer 52 (the components are lipophilic substances, such as paraffin oil) above the liquid level of a reaction system 51 in a reaction cavity 5, so that the first temperature control assembly 2 mainly corresponds to the reaction system 51, and the second temperature control assembly 3 mainly corresponds to the upper oil coating layer 52, thereby controlling the temperature of the reaction system 51 from multiple aspects. Wherein, the first temperature control component 2 meets the temperature rise and fall control required by the reaction system 51; and the second temperature control component 3 can compensate the temperature rising and falling rate of the liquid on the upper part of the reaction system 51 based on the temperature rising and falling control of the upper oil layer 52.

Further, as shown in fig. 6 of the drawings, the first temperature control component 2 is provided with a placement groove 211, the lower portion of the reaction chamber 5 can be inserted into the placement groove 211, the height of the notch position of the placement groove 211 is preferably higher than the height of the liquid level of the reaction system 51 in the reaction chamber 5, so that the reaction system 51 at the bottommost part of the inner space of the reaction chamber 5 is completely located in the placement groove 211, and effective heating and temperature control can be obtained by the first temperature control component 2, the first temperature control component 2 provides a main heating source for the reaction system 51, and of course, the first temperature control component 2 also corresponds to the part of the overlying oil layer 52 close to the liquid level of the reaction system 51, so that the overlying oil layer 52 can be heated to a certain extent. The second temperature control assembly 3 is disposed above the first temperature control assembly 2, and corresponds to a main portion of the overlying oil layer 52, and mainly provides a heat source for the overlying oil layer 52. The second temperature control assembly 3 can heat the upper oil layer 52 above the reaction system 51, and the upper oil layer 52 is utilized to compensate the temperature rising and falling rate of the liquid above the reaction system 51, so that the temperature rising and falling rates of the liquid above the reaction system 51 and the liquid below the reaction system 51 are more consistent, the temperature rising and falling rates of the vertical height of the reaction system 51 are more consistent, the temperature change of the vertical height of the reaction system 51 is more consistent, the temperature is more uniform, the liquid of the reaction system 51 can adapt to the higher temperature rising and falling rate, the amplification reaction time can be reduced, the nucleic acid detection time can be reduced, and the nucleic acid detection speed can be improved.

Therefore, based on the temperature control device of the present invention, the uniformity of the temperature distribution inside the reaction system 51 can be ensured also at a high temperature rise and drop rate; in the actual use process of the temperature control device, based on the reaction cavity 5 with the tubular structure, the temperature gradient in the reaction system 51 is shown in the figure 8 at the temperature rising and falling rate of 10 ℃/s, and compared with the temperature gradient in the figure 9, the temperature gradient is greatly improved, and the temperature change of different areas in the reaction system 51 is more consistent. Therefore, based on the structural design of the invention, the uniformity of the temperature of the reaction system 51 at the vertical height can be improved by adopting two temperature control components, so that the reaction system 51 can adapt to higher temperature rise and drop rate, thereby shortening the nucleic acid amplification reaction time and shortening the nucleic acid detection time. The temperature control device is practically applied to molecular POCT, the duration of the PCR reaction process can be effectively shortened to 30-40 minutes, and the duration of the whole detection process can be controlled within 60 minutes.

The first variable temperature and the second variable temperature refer to the temperatures output by the first temperature control unit 2 and the second temperature control unit 3, respectively, that is, the temperatures of the members of the first temperature control unit 2 and the second temperature control unit 3 for directly conducting heat with the reaction chamber (corresponding heat transfer members are attached to the outer wall of the reaction chamber), but not the temperatures of the reaction system 51 and the overlying oil layer 52 in the reaction chamber 5.

In one embodiment, the first temperature control assembly 2 and the second temperature control assembly are configured 3 to independently control operation; the second variable temperature and the first variable temperature synchronously change in a lifting manner, and the second variable temperature at any time is larger than the first variable temperature.

Specifically, the independent control operation means that the two temperature control components are independently controlled in structure and do not affect each other, but in the operation, a synchronous operation mode is adopted, and the temperature ranges of the two temperature control components are different, so that the temperature rise and fall rates are different, the temperature rise and fall rate of 10 ℃/s is mainly the first temperature control component 2, and the specific temperature rise and fall rate of the second temperature control component 3 can be calculated based on the first temperature control component 2. Meanwhile, because the temperature control device is limited in volume, the space for assembling the second temperature control component 3 is limited, so that the heat transfer area of the second temperature control component 3 is limited, and the amount of the actual upper oil coating layer 52 is large, so that the heating temperature of the second temperature control component 3 is relatively increased, namely, the second variable temperature at any moment is larger than the first variable temperature, so that the second temperature control component 3 can raise and lower the upper oil coating layer 52 by using a smaller heat transfer area, and the upper oil coating layer 52 can effectively compensate the raising and lowering temperature rate of the liquid at the upper part of the reaction system 51. The second variable temperature of the second temperature control assembly 3 may coincide with the first variable temperature of the first temperature control assembly 2 if the second temperature control assembly 3 is capable of being allowed to increase the heat transfer area or the amount of overlying oil layer 52 is small.

In one embodiment, the first temperature control assembly 2 and the second temperature control assembly 3 respectively include a first heating block 21 and a second heating block 31, the placement groove 211 is configured on the first heating block 21, and a through hole 311 corresponding to a notch of the placement groove 211 is configured at a center of the second heating block 31 for the reaction chamber 5 to pass through and be inserted into the placement groove 211.

Specifically, as shown in fig. 3 and 4 of the accompanying drawings, the placement groove 211 is disposed on the first heating block 21, and its shape matches the shape of the reaction chamber 5, for example, in this embodiment, the reaction chamber 5 adopts a structure with a conical bottom, and the placement groove 211 is correspondingly configured as a conical groove as shown in fig. 6 of the accompanying drawings; the center of the second heating block 31 is provided with a through hole 311 for the reaction cavity 5 to penetrate, and can be sleeved on the reaction cavity 5 relatively; the inner walls of the placing grooves 211 and the through holes 311 are contacted and attached with the outer wall of the reaction chamber 5. The first heating block 21 and the second heating block 31 correspond to the region of the reaction chamber 5 to be heated in the circumferential direction and the axial direction, and the heating effect on the reaction chamber 5 can be improved.

In one embodiment, the first temperature control component 2 and the second temperature control component 3 further include a first peltier 22 and a second peltier 32, respectively, and the first peltier 22 and the second peltier 32 are attached to the first heating block 21 and the second heating block 31, respectively. The peltier is used as a heat source, and the heat of the peltier is transferred to the reaction cavity 5 through the heating block; the peltier has the advantages of rapid temperature rise and fall and simple temperature rise and fall control.

In one embodiment, the device main body 1 includes two heat dissipation blocks 11, the first temperature control assembly 2 is sandwiched between the two heat dissipation blocks 11, and the second temperature control assembly 3 is disposed on a first end face of the device main body 1 formed by top end faces of the two heat dissipation blocks 11;

The first heating block 21 and two first peltier devices 22 arranged on two sides of the first heating block are integrally clamped between the two heat dissipation blocks 11, and the first peltier devices 22 are attached to the inner side surfaces of the corresponding heat dissipation blocks 11; the two second peltier devices 32 are respectively disposed on the top end surfaces of the two heat dissipation blocks 11, and the second heating block 31 is configured in a plate-like structure and covers the two second peltier devices 32.

Specifically, the device main body 1 is used for providing a mounting foundation for the temperature control module, and also is used for radiating heat of the peltier serving as a heat source in the temperature raising and lowering process, and has a large surface area, so that a radiating effect is ensured. The apparatus main body 1 is constituted by two heat dissipation blocks 11 split left and right in structure, the two heat dissipation blocks 11 are substantially identical in structure and symmetrical with respect to the split surface, the top end surfaces of the two heat dissipation blocks 11 constitute a first end surface of the apparatus main body 1, and the bottom end surfaces of the two heat dissipation blocks 11 constitute a second end surface of the apparatus main body 1.

The second temperature control component 3 is directly installed on the first end face of the device main body 1, and the first temperature control component 2 is clamped between the two heat dissipation blocks 11 and is close to the first end face, and the two temperature control components adopt different installation orientations, so that the compactness of the structure can be improved. While the opposite sides of the second heating block 31 of the second temperature control assembly 3 located in the radial direction of the through hole 311 are configured as plate-shaped structures, which are fully adapted to the limited space in the height direction, and the two sides of the second heating block 31 respectively correspond to the two heat dissipation blocks 11.

Preferably, in order to further improve the heat radiation effect of the device body 1 on the peltier, a heat radiation fan 4 is provided on the second end surface of the device body 1.

In one embodiment, the first heating block 21 is internally configured as a hollow structure 215, and the hollow structure 215 does not extend to the wall of the placement groove 211 and the side surface of the first heating block 21 attached to the first peltier 22.

Specifically, as shown in fig. 3 and 7 of the drawings, the first heating block 21 is internally configured as a hollow structure 215 composed of a plurality of cavities, and the main purpose of the hollow structure is to reduce the mass of the first heating block 21 on the basis of ensuring the heat transfer area, and the mass of the first heating block 21 affects the temperature rise and fall rate thereof, so that the temperature rise and fall rate of the first heating block 21 is improved by reducing the mass of the first heating block 21. It should be noted that, the hollow structure 215 does not extend to the groove wall of the placement groove 211 and the side surface of the first heating block 21 attached to the first peltier 22, i.e. the hollow structure 215 does not form holes on the groove wall of the placement groove 211 and the corresponding side surface of the first heating block 21, so as not to reduce the original heat transfer area.

In one embodiment, the first heating block 21 and the second heating block 31 are made of heat conductive metal blocks, and a heat conductive medium is disposed between the first heating block 21 and the first peltier 22, and between the second heating block 31 and the second peltier 32.

Specifically, in this embodiment, the heating block is made of aluminum, which has good heat conduction performance and light weight, and may be selected from metals such as copper, tungsten, molybdenum, etc.; besides metals, other nonmetallic materials with high temperature resistance and good thermal conductivity can be adopted. The heat conducting medium is used for improving the heat transfer effect at the contact surface, and can be high heat conducting materials such as heat conducting silicone grease, heat conducting silica gel, carbon film and the like.

In one embodiment, the device body 1 further includes two fixing members 12, the two fixing members 12 are respectively covered on two sides of the second heating block 31, and the through hole 311 is located between the two fixing members 12; the fixing piece 12 is fixedly connected with the top end surface of the corresponding heat dissipation block 11 through a fastener, so that the second heating block 31 and the second peltier 32 are pressed on the first end surface.

Specifically, the fixing member 12 of the device body 1 is mainly used for fixing the second temperature control assembly 3, as shown in fig. 2 and 6 of the accompanying drawings, the fixing member 12 compresses the second heating block 31 and the second peltier 32 together on the top end surface of the corresponding heat dissipation block 11, that is, the first end surface of the device body 1, and the contact surface is made to be closely contacted to ensure the heat transfer effect, and the fixing member 12 is fixedly connected with the heat dissipation block 11 through a fastener such as a screw.

In one embodiment, the surfaces of both sides of the second heating block 31 are respectively configured with positioning grooves 312, and the bottom surface of the fixing member 12 is configured with positioning protrusions 121 that can be fitted into the positioning grooves 312 and pressing protrusions 122 that can contact the surface of the second heating block 31.

Specifically, as shown in fig. 4 and 5 of the drawings, the second heating block 31 and the fixing member 12 are further limited by the positioning groove 312 and the positioning protrusion 121, so as to avoid the second heating block 31 and the fixing member 12 from moving relatively in a direction perpendicular to the pressing direction; of course, the positioning protrusion 121 may also contact the bottom of the positioning groove 312 to have the same pressing effect as the pressing protrusion 122. In addition, when the fixing piece 12 compresses the second heating block 31, the plurality of compressing protrusions 122 are in contact with the surface of the second heating block 31, the compressing effect is further ensured through multipoint contact, the condition that gaps exist between the second heating block 31 and the second peltier 32 due to the fact that the second heating block 31 is compressed only through the positioning protrusions 121 is avoided, the heat conduction between the second peltier 32 and the second heating block 31 is prevented from being influenced, the second heating block 31 is fully contacted with the second peltier 32, and the heat conduction effect is ensured; meanwhile, the multipoint contact can reduce the contact area between the fixing piece 12 and the second heating block 31, so that excessive heat of the second heating block 31 is prevented from being transmitted to the fixing piece 12, the temperature dissipation of the second heating block 31 is reduced, and the power load of the second peltier 32 is reduced.

In one embodiment, the portion of the through hole 311 is configured as a boss 314 protruding relative to the surface of the second heating block 31, and the boss 314 is located between the two second peltier devices 32 to limit the two second peltier devices 32.

Specifically, as shown in fig. 4 and 6 of the drawings, a boss 314 is provided at the center of the bottom surface of the second heating block 31, which is attached to the second peltier 32, and a through hole 311 is formed in the boss 314. On the one hand, the boss 314 increases the contact area with the wall of the reaction cavity 5 in the axial direction of the through hole 311, so that the heat transfer effect can be improved; on the other hand, the boss 314 can limit the positions of the second peltier devices 32 on both sides, so as to facilitate the installation of the second temperature control assembly 3.

In one embodiment, the first temperature control component 2 and the second temperature control component 3 are further provided with a first temperature sensing unit and a second temperature sensing unit, respectively, which are used for detecting the first variable temperature and the second variable temperature, respectively.

Specifically, the first temperature sensing unit and the second temperature sensing unit (not shown in the drawings) are configured to detect the temperatures of the first heating block 21 and the second heating block 31, i.e. detect the first variable temperature and the second variable temperature, respectively, so as to provide a basis for temperature control. As shown in fig. 7 of the drawings, a first temperature measuring groove 214 for installing a first temperature sensing unit is provided on the first heating block 21, and the groove bottom of the first temperature measuring groove 214 is close to the groove wall of the placing groove 211 inside the first heating block 21. As shown in fig. 4 of the drawings, the upper surface of the second heating block 31 is provided with a second temperature measuring groove 313 for mounting the second temperature sensing unit, the second temperature measuring groove 313 is in a long strip shape extending along the radial direction of the through hole 311, and the tail end thereof is close to the region where the through hole 311 is located.

In one embodiment, as shown in fig. 6 and 7 of the drawings, a first assembly hole 212 corresponding to the bottom of the reaction chamber 5 is arranged at the bottom of the placement groove 211, and is used for installing an excitation optical fiber for exciting the reaction system 51 to generate a fluorescence signal; the side wall of the placing groove 211 is provided with a second assembly hole 213 corresponding to the side surface of the reaction chamber 5 for installing a light receiving optical fiber for detecting the fluorescent signal generated by the reaction system 51.

Specifically, the present invention excites and detects and collects fluorescent signals to the reaction system 51 based on the optical fiber, thereby obtaining a detection result. The invention can ensure the intensity of the fluorescence signal while rapidly increasing and decreasing the temperature, namely the initial nucleic acid amount in the reaction system 51 can be obtained based on the detection result of the fluorescence signal.

In one embodiment, the design and numerical calculation determination mode of the specific temperature control range, the temperature rise and fall rate and other aspects of the temperature control device of the present invention is described by the following PCR amplification process, which is:

S000: determining a first variable temperature of the first temperature control assembly 2, and a first heating rate and a second cooling rate, wherein the first variable temperature comprises a first high-point temperature and a first low-point temperature, and the value ranges of the first heating rate and the second cooling rate are 5 ℃/s-13 ℃/s;

Specifically, the first variable temperature, that is, the temperature control range corresponding to the first temperature control component 2, in this embodiment, the first high-point temperature of the temperature control range is selected to be 95 ℃, the first low-point temperature is selected to be 55 ℃, and in this embodiment, it is determined that the first temperature rising rate and the first temperature reducing rate of the first temperature control component 2 are both 10 ℃/s, and then the temperature rising duration and the temperature reducing duration can be calculated according to the temperature difference between the first high-point temperature and the first low-point temperature, the first temperature rising rate and the first temperature reducing rate respectively. In this embodiment, the second high point temperature of the temperature control range is 105 ℃ and the second low point temperature is 75 ℃, and the second temperature rising rate and the second temperature reducing rate of the second temperature control component 3 can be calculated by combining the heating duration and the cooling duration of the first temperature control component 2. In the embodiment, the first heating rate and the first cooling rate are 10 ℃/s, and the detection of the nucleic acid has higher accuracy under the heating rate; in addition, the first heating rate and the second cooling rate can also be selected from values of 5 ℃/s, 6 ℃/s, 7 ℃/s, 8 ℃/s, 9 ℃/s, 11 ℃/s, 12 ℃/s, 13 ℃/s and the like in the value range according to the requirements of specific conditions.

In this embodiment, the temperature difference between the second low-point temperature and the first low-point temperature is greater than the temperature difference between the second high-point temperature and the first high-point temperature, so that the accuracy of the detection result can be improved. The temperature difference between the second low-point temperature and the first low-point temperature is larger than the temperature difference between the second high-point temperature and the first high-point temperature, so that the corresponding temperature control ranges of the first variable temperature and the second variable temperature are different, and the temperature rising and falling rates of the first temperature control component 2 and the second temperature control component 3 are different. The above-described specific high-point temperature and low-point temperature may be adjusted according to the specific requirements of the reaction system 51.

S100: heating the first temperature control assembly 2 from a first low point temperature to a first high point temperature according to a first heating rate, and enabling the reaction system 51 to reach a denaturation temperature; the second temperature control assembly 3 is heated from the second low point temperature to the second high point temperature according to the second heating rate so as to heat the overlying oil layer 52 for a first preset period of time.

Specifically, in this embodiment, the first temperature control assembly 2 is heated to 95 ℃ at a first heating rate of 10 ℃/s, and the second temperature control assembly 3 is synchronously heated to 105 ℃ at a calculated and determined second heating rate, and the first preset time period is maintained at this temperature, which is 1s in this embodiment, and this maintenance time period is the time for waiting for the reaction system 51 to denature.

S200: the first temperature control component 2 is cooled from a first high-point temperature to a first low-point temperature according to a first cooling rate, and the reaction system 51 passes through an annealing temperature and reaches an extension temperature; cooling the second temperature control assembly 3 from a second high point temperature to a second low point temperature according to a second cooling rate to cool the overlying oil layer 52; and (5) completing one round of temperature control.

Specifically, in this embodiment, the first temperature control component is cooled from 95 ℃ to 55 ℃ at a first cooling rate of 10 ℃/s, and the second temperature control component is cooled from 105 ℃ to 75 ℃ at a calculated second cooling rate, and the second preset time period is maintained at this temperature for 5 seconds, and this maintenance time period is the time period for waiting for the reaction system 51 to extend. One round of temperature control is completed through S100 and S200, namely, one round of amplification is completed on the nucleic acid in the reaction system 51, and the number is doubled. In this embodiment, there is no waiting time in the annealing stage of the reaction system 51, and the PCR temperature cycle time can be further shortened, and the amplification reaction time can be shortened.

S300: repeating the steps according to the amplification requirement to complete the temperature control of the preset number of rounds.

In the description of the present invention, it should be understood that the terms "upper," "lower," "bottom," "top," "front," "rear," "inner," "outer," "left," "right," and the like indicate or are based on the orientation or positional relationship shown in the drawings, merely to facilitate description of the present invention and to simplify the description, and do not indicate or imply that the devices or elements referred to must have a particular orientation, be configured and operated in a particular orientation, and thus should not be construed as limiting the present invention.

Although the invention herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present invention. It is therefore to be understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the present invention as defined by the appended claims. It should be understood that the different dependent claims and the features described herein may be combined in ways other than as described in the original claims. It is also to be understood that features described in connection with separate embodiments may be used in other described embodiments.

Claims

1. A temperature control device for heating and cooling a biological sample processing member, the biological sample processing member comprising a reaction chamber of tubular configuration, the temperature control device comprising:

A device body; and

2. The temperature control device of claim 1, wherein the first temperature control assembly and the second temperature control assembly are configured to independently control operation; the second variable temperature and the first variable temperature synchronously change in a lifting manner, and the second variable temperature at any moment is larger than the first variable temperature.

3. The temperature control device according to claim 1, wherein the first temperature control assembly and the second temperature control assembly respectively comprise a first heating block and a second heating block, the placement groove is formed in the first heating block, and a through hole corresponding to a notch of the placement groove is formed in the center of the second heating block so that the reaction chamber can pass through and be inserted into the placement groove.

4. A temperature control apparatus according to claim 1 or 3, wherein the height of the notch position of the placement groove is higher than the height of the liquid surface of the reaction system in the reaction chamber.

5. The temperature control device of claim 3, wherein the first and second temperature control assemblies further comprise first and second peltier devices, respectively, the first and second peltier devices being in registry with the first and second heating blocks, respectively.

6. The temperature control device according to claim 5, wherein the device main body includes two heat dissipation blocks, the first temperature control assembly is sandwiched between the two heat dissipation blocks, and the second temperature control assembly is provided on a first end face of the device main body constituted by top end faces of the two heat dissipation blocks;

7. The temperature control device of claim 5, wherein the first heating block is internally configured as a hollow structure, and the hollow structure does not extend to a wall of the placement groove and a side surface of the first heating block, which is attached to the first peltier.

8. The temperature control device of claim 5, wherein the first heating block and the second heating block are both made of heat-conducting metal blocks, and a heat-conducting medium is arranged between the first heating block and the first peltier and between the second heating block and the second peltier.

9. The temperature control device according to claim 6, wherein the device body further comprises two fixing members, the two fixing members are respectively covered on both sides of the second heating block, and the through hole is located between the two fixing members;

10. The temperature control device according to claim 9, wherein the surfaces of both sides of the second heating block are respectively configured with positioning grooves, and the bottom surface of the fixing member is configured with positioning protrusions capable of being fitted into the positioning grooves and pressing protrusions capable of contacting the surface of the second heating block.

11. The temperature control device of claim 6, wherein the portion of the through hole is configured as a boss protruding relative to the surface of the second heating block, the boss being located between the two second peltier devices to limit the two second peltier devices.

12. The temperature control device according to claim 6, wherein bottom end surfaces of the two heat dissipation blocks constitute a second end surface of the device body, and a heat dissipation fan is provided on the second end surface.

13. The temperature control device of claim 1, wherein a first temperature sensing unit and a second temperature sensing unit are further disposed in the first temperature control assembly and the second temperature control assembly, respectively, and the first temperature sensing unit and the second temperature sensing unit are configured to detect the first variable temperature and the second variable temperature, respectively.

14. The temperature control device according to claim 1, wherein a first assembly hole corresponding to the bottom of the reaction cavity is arranged at the bottom of the placing groove, and is used for installing an excitation optical fiber for exciting the reaction system to generate a fluorescent signal;

15. The temperature control device of claim 1, wherein the first variable temperature comprises a first high point temperature and a first low point temperature, and the second variable temperature comprises a second high point temperature and a second low point temperature;

16. The temperature control device of claim 15, wherein the first temperature control assembly and the second temperature control assembly are configured to have the same warm-up duration and cool-down duration;

17. The temperature control device according to claim 15 or 16, characterized in that a temperature difference between the second low-point temperature and the first low-point temperature is larger than a temperature difference between the second high-point temperature and the first high-point temperature.

18. The temperature control device of claim 15, wherein the first temperature control assembly is configured to maintain a first preset length of time after warming to the first high point temperature and a second preset length of time after cooling to the first low point temperature;

19. The temperature control device of claim 15, wherein the first heating rate and the first cooling rate range from 5 ℃/s to 13 ℃/s.

20. A biological sample detection system, comprising:

A temperature control device as claimed in any one of claims 1 to 19.