CN220265694U

CN220265694U - Thermal cycling device and detection device comprising thermal cycling device and sample container

Info

Publication number: CN220265694U
Application number: CN202320832779.8U
Authority: CN
Inventors: 盛广济
Original assignee: Suzhou Sinafu Medical Technology Co ltd
Current assignee: Suzhou Sinafu Medical Technology Co ltd
Priority date: 2023-04-14
Filing date: 2023-04-14
Publication date: 2023-12-29
Anticipated expiration: 2033-04-14

Abstract

The utility model provides a thermal cycling device for sample analysis and processing and a detection device comprising the thermal cycling device and a sample container. The thermal cycling device includes a plurality of temperature zones having surfaces matching the sample container and channels in fluid communication with a gas source. The thermal cycle device greatly improves the transfer speed of the sample solution and the speed of the sample solution reaching the target temperature, thereby greatly improving the nucleic acid amplification rate. The sample container includes: a substrate; and (3) a film. The thickness of the membrane used in the sample container of the utility model can be remarkably reduced, the thermal cycle rate is greatly improved, and the nucleic acid amplification rate is further greatly improved. In summary, by adopting the detection device comprising the sample container and the thermal cycling device provided by the utility model, the time for amplifying and detecting the whole nucleic acid can be effectively shortened, and the requirement of instant detection is met.

Description

Thermal cycling device and detection device comprising thermal cycling device and sample container

Technical Field

The present utility model relates to nucleic acid amplification and detection techniques, and more particularly to a thermal cycler for sample analysis and processing, and a detection device including a thermal cycler and a sample container.

Background

To accommodate the clinical rapid diagnostic need, many techniques have been developed to perform PCR (polymerase chain reaction) -based nucleic acid amplification at a higher rate, and on the one hand, a method of transferring PCR sample solutions between different spaces of a thermal cycler provided with different temperatures has been widely used. The method ensures that the PCR sample solution moves rapidly through the flow channels communicated with different spaces, thereby realizing denaturation and annealing/extension circulation in different spaces, avoiding heating/cooling circulation in the same space, and greatly improving the PCR reaction rate due to slow heating/cooling rate and long time required for completing the PCR reaction.

However, on one hand, the rate of PCR reaction is affected by the slow rate of transfer of the sample solution, and on the other hand, the rate of temperature change is quite slow when the sample solution changes temperature to a value close to the target temperature, which restricts the rate of final temperature rise to the target temperature, since the rate of heat exchange is proportional to the temperature difference, which also affects the rate of PCR reaction.

On the other hand, in order to improve the heat transfer efficiency, a technique of forming a nucleic acid amplification space by using a flexible film has also been developed. For example, CN115322888A discloses a nucleic acid amplification device having a double-layered film that is closely attached together before a PCR sample solution is injected, and after the sample solution is injected, the double-layered films are separated from each other at a reaction chamber (including a denaturation chamber, an annealing chamber, and an extension chamber) and elastically deformed to form a micro-pouch, and the sample solution can be transferred to each other between the reaction chambers via a connection channel by applying or withdrawing a pressing force. When the sample solution is discharged from the current chamber, the double-layer films are clung together again under the elastic action to form a closed valve. The microcapsule bag type reaction chambers are respectively provided with heaters correspondingly to provide respective preset temperatures, and the requirements of temperature rise and temperature reduction of the PCR reaction can be met by only controlling the time that the PCR sample solution needs to stay in each chamber, so that the PCR reaction time can be greatly shortened. As another example, CN112041073a discloses a high-speed PCR analysis plate having an elastic membrane covered on a substrate, the elastic membrane sealing a receiving area (i.e., a reaction chamber), and when a sample solution is injected, the pressure inside the elastic membrane sealing the receiving area increases, resulting in the surface protruding outwards, and when a heating block is in close contact with the protruding portion of the elastic membrane surface, close contact is formed between the elastic membrane and the heating block, thereby enabling a rapid PCR reaction.

However, either the above-mentioned double-layered film or single-layered elastic film is elastically deformed under tension to form a chamber, and then is contacted with a heating block, and considering that nucleic acid amplification usually involves 20 to 50 annealing/extension cycles, the film needs to withstand tens of elastic deformation processes, and in order to avoid the film yielding and cracking caused by multiple deformation, such film usually has a thickness of at least 0.1 to 0.5 mm. The film thickness directly affects the heat transfer efficiency of the heating block and thus ultimately affects the nucleic acid amplification rate. In the case of using a double-layered film, after filling the sample solution, the lower film becomes a plane from a hemispherical shape due to contact with the heating block, reducing the contact area with the heating block, and further reducing the heat transfer efficiency.

In view of the foregoing, there is a need in the art for an improved thermal cycling apparatus, a testing apparatus including a thermal cycling apparatus and a sample container, that overcomes the above-described deficiencies in the prior art.

Disclosure of Invention

One aspect of the present utility model provides a thermal cycle apparatus characterized in that: the thermal cycling device comprises a plurality of temperature areas, wherein the plurality of temperature areas at least comprise a first temperature area and a second temperature area; the plurality of temperature zones are arranged separately from each other; each temperature zone is provided with a surface matched with the sample container and a channel in fluid communication with a gas source; the sample solution in the sample container can be circulated between the surfaces of the plurality of temperature zones by means of the gas source and the channels provided by the plurality of temperature zones.

In some embodiments, the first temperature zone is for achieving a high target temperature for the sample solution within the sample container and the second temperature zone is for achieving a low target temperature for the sample solution within the sample container.

In some embodiments, the first temperature zone is within a first temperature interval and the second temperature zone is within a second temperature interval, the first temperature interval being a higher temperature interval than the second temperature interval.

In some embodiments, the sample solution comprises a sample to be tested having a target denaturation temperature and a target annealing temperature; the first temperature interval is higher than the target denaturation temperature; the second temperature interval is lower than the target annealing temperature.

In some embodiments, the sample solution comprises a sample to be tested having a target denaturation temperature and a target annealing temperature; the first temperature interval contains the target denaturation temperature; the second temperature interval includes the target annealing temperature.

In some embodiments, the sample solution comprises a sample to be tested having a target denaturation temperature and a target annealing temperature; the high target temperature is substantially equal to the target denaturation temperature and the low target temperature is substantially equal to the target annealing temperature.

In some embodiments, the plurality of temperature zones further comprises a third temperature zone, wherein: the third temperature zone is for obtaining an intermediate target temperature for the sample solution within the sample container, the intermediate target temperature being intermediate the high target temperature and the low target temperature.

In some embodiments, the plurality of temperature zones further comprises a third temperature zone, wherein: the third temperature zone is within a third temperature interval, the third temperature interval being a higher temperature interval than the first temperature interval.

In some embodiments, the plurality of temperature zones further comprises a third temperature zone, wherein: the third temperature zone is within a fourth temperature zone, the fourth temperature zone being a lower temperature zone than the second temperature zone.

In some embodiments, the plurality of temperature zones further comprises a third temperature zone, wherein: the third temperature zone is capable of providing a temperature within a third temperature interval and a temperature within a fourth temperature interval, and the third temperature interval is a higher temperature interval than the first temperature interval and the fourth temperature interval is a lower temperature interval than the second temperature interval.

In some embodiments, the plurality of temperature zones further comprises a third temperature zone and a fourth temperature zone, wherein: the third temperature zone is used for enabling the sample solution in the sample container to obtain a first intermediate target temperature, the fourth temperature zone is used for enabling the sample solution in the sample container to obtain a second intermediate target temperature, the first intermediate target temperature and the second intermediate target temperature are both between the high target temperature and the low target temperature, and the first intermediate target temperature is higher than the second intermediate target temperature.

In some embodiments, the third temperature zone is within a third temperature zone and the fourth temperature zone is within a fourth temperature zone, the third temperature zone being a higher temperature zone than the first temperature zone and the fourth temperature zone being a lower temperature zone than the second temperature zone.

One aspect of the present utility model provides a detection device including: the thermocycling device and sample container as described hereinbefore.

In some embodiments, the sample container comprises: a substrate having a first surface and an opposing second surface, the first surface having at least two surface areas; a membrane on the first surface of the substrate and comprising at least two membrane regions, each membrane region defining a chamber in cooperation with a corresponding said surface region on the substrate; a fluid channel, the membrane region being in fluid communication via the fluid channel.

In some embodiments, the at least two membrane regions are in fluid communication via the fluid channel in the absence of an external force.

In some embodiments, each of the film regions includes a peripheral region surrounding one of the at least two surface regions and a central region corresponding to the surface region, the peripheral region being fixedly bonded to the first surface of the substrate, the central region being separate from the surface region.

In some embodiments, the surface area of the central region is greater than the orthographic projected area of the surface region on the first surface in the absence of external forces and without undergoing plastic deformation.

In some embodiments, the central region has pleats that are capable of expanding under an external force without undergoing plastic deformation.

In some embodiments, each amplification chamber has a variable volume without external force and without plastic deformation of the central region.

In some embodiments, each of the membrane regions is capable of expanding or compressing under an external force, wherein the membrane regions are capable of expanding or compressing under the same external force without substantial deformation of the substrate.

In some embodiments, the film has a thickness of about 0.001mm to about 0.2mm, preferably about 0.001mm to about 0.1mm, more preferably about 0.01mm to about 0.1mm, for example about 0.01mm, about 0.03mm, about 0.05mm, or about 0.07mm.

In some embodiments, the surface area of the central region is about 10% to about 400%, preferably about 100% to about 300%, more preferably about 150% to about 250%, still more preferably about 200% greater than the orthographic projection area of the corresponding surface region on the first surface for each film region.

In some embodiments, the first surface of the substrate has three surface areas and the film includes three film areas.

In some embodiments, the first surface of the substrate has four surface areas and the film includes four film areas.

The thermal cycle device provided by the utility model is provided with the temperature zone capable of increasing the heat exchange driving force, so that the temperature rise and fall speed of the sample solution is improved, and the time required for completing the amplification cycle is shortened.

The present utility model provides a sample container comprising a central region of a membrane region having a surface area in an initial state of the sample container that is greater than a corresponding surface region such that the central region is not stretched or only minimally stretched when expanded by application of a negative pressure to form an amplification chamber, and thus the thickness of the membrane used in the present utility model may be significantly reduced, e.g. as low as 0.1mm, e.g. 0.001 to 0.10mm, e.g. 0.01mm, about 0.03mm, about 0.05mm or about 0.07mm, compared to a membrane that is subjected to stretching to form a chamber containing a nucleic acid sample, without rupture after undergoing up to 50 amplification cycles. The reduced thickness of the membrane allows for higher heat transfer efficiency between the membrane and the heat source, allowing the nucleic acid sample within the membrane to complete a denaturation or annealing extension reaction even with as little as 1 second of contact.

In addition, the detection device comprising the sample container and the thermal cycling device provided by the utility model has the advantages that in the first aspect, the sample solution is circularly moved between the membrane areas of the sample container through the air source, so that the transfer speed of the sample solution is improved, and the time required by the PCR reaction is shortened; in the second aspect, a plurality of temperature areas corresponding to each cavity of the sample container are arranged in the thermal circulation device to heat the sample container, the sample container and the thermal circulation device are relatively static, the sample container and the thermal circulation device are closely attached to each other without gaps basically, and meanwhile, the sample container uses a film with obviously reduced thickness, so that the heat exchange efficiency and the heat exchange rate are facilitated, the temperature rise and fall rate of the sample solution is further effectively improved, the reaction time is shortened, and the reaction efficiency is improved; in the third aspect, the temperature zone of the thermal circulation driving force can be increased in the thermal circulation device, so that the time required for changing the temperature of the sample solution to a target temperature value is further shortened, and the reaction efficiency is improved. In summary, by adopting the detection device comprising the sample container and the thermal cycling device provided by the utility model, the time for amplifying and detecting the whole nucleic acid can be effectively shortened, and the requirement of instant detection is met.

Drawings

The utility model will be described in more detail with reference to the accompanying drawings. It is noted that the illustrated embodiments are merely representative examples of the embodiments of the present utility model, and that elements in the drawings are not drawn to scale such as actual dimensions, the number of actual elements may vary, the relative positional relationship of the actual elements is substantially consistent with the illustration, and some elements are not shown in order to more clearly illustrate the details of the exemplary embodiments. Where multiple embodiments exist, while one or more features described in the previous embodiments may also be applied to another embodiment, for brevity, the latter embodiment or embodiments will not be described in further detail as having described such features, unless otherwise indicated. Those skilled in the art will appreciate upon reading the present disclosure that one or more features illustrated in one drawing may be combined with one or more features in another drawing to construct one or more alternative embodiments not specifically illustrated in the drawings, which also form a part of the present disclosure.

Fig. 1 is a schematic structural view of the thermal cycler 13.

Fig. 2 is a side perspective view of the thermal cycler 13.

Fig. 3 schematically shows a front view of a sample container according to a preferred embodiment of the utility model.

Fig. 4 schematically shows the structure of three different embodiments of the substrate of some embodiments of the utility model.

Fig. 5 shows a schematic structural view of a sample container according to two different embodiments of the present utility model.

Fig. 6 shows a schematic structural view of a sample container according to other embodiments of the present utility model.

Fig. 7 shows a schematic diagram of the cyclic transfer of sample solution in a sample vessel according to the utility model between the various temperature zones.

Detailed Description

In order that the above objects, features and advantages of the utility model will be readily understood, a more particular description of the utility model will be rendered by reference to the appended drawings. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present utility model. The utility model may be embodied in many other forms than described herein and similarly modified or substituted for those skilled in the art without departing from the spirit of the utility model, and therefore the utility model is not to be limited to the specific embodiments disclosed below.

In the description of the present utility model, it should be understood that the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", "clockwise", "counterclockwise", "axial", "radial", "circumferential", etc. indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings are merely for convenience in describing the present utility model and simplifying the description, and do not indicate or imply that the device or element being referred to must have a specific orientation, be configured and operated in a specific orientation, and therefore should not be construed as limiting the present utility model.

Furthermore, the terms "first," "second," and the like, are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include at least one such feature. In the description of the present utility model, the meaning of "plurality" means at least two, for example, two, three, etc., unless specifically defined otherwise.

In the present utility model, the terms "mounted," "connected," "secured," and the like are to be construed broadly, and may be, for example, fixedly connected, detachably connected, or integrally formed, mechanically connected, electrically connected, directly connected, or indirectly connected through an intermediary, or may be in communication with each other between two elements or the relationship between two elements unless explicitly defined or stated otherwise. The specific meaning of the above terms in the present utility model can be understood by those skilled in the art according to the specific circumstances.

In the present utility model, unless explicitly defined and stated otherwise, a first feature "up" or "down" a second feature may be that the first and second features are in direct contact, or that the first and second features are in indirect contact via an intermediary. Moreover, a first feature "above," "over" and "on" a second feature may be a first feature directly above or obliquely above the second feature, or simply indicate that the first feature is higher in level than the second feature. The words "under", "below" and "beneath" are used interchangeably.

It will be understood that when an element is referred to as being "fixed" or "disposed" on another element, it can be directly on the other element or intervening elements may also be present. When an element is referred to as being "connected" to another element, it can be directly connected to the other element or intervening elements may also be present. The terms "vertical," "horizontal," "upper," "lower," "left," "right," and the like are used herein for illustrative purposes only and are not meant to be the only embodiment.

Fig. 1 is a schematic structural view of the thermal cycler 13, and fig. 2 is a side perspective view of the thermal cycler 13. As shown in the figure, the thermal cycler 13 is provided with a plurality of temperature zones, a first temperature zone 131A, a second temperature zone 131B, and a third temperature zone 131C.

In some embodiments, the plurality of temperature zones of the thermal cycler 13 are also provided with surfaces that mate with the sample containers and channels 132 in fluid communication with the air source. In some embodiments, pneumatic pressure, such as positive or negative pressure, of a gas source that moves the sample solution in the sample container is provided through the channel 132. The plurality of temperature zones of the thermal cycle device 13 are provided with auxiliary passages 133 communicating with the passages 132, serving as auxiliary passages for pneumatic pressure supply. The auxiliary channel 133 is not limited in size and configuration and may be arcuate in shape extending along the inner surface of the warm zone as shown in fig. 1 and 2, annular in shape extending along the inner surface of the warm zone, or other shapes. The size and configuration of the channel 132 is also not limited and the pneumatic pressure of the air source required to effect the cyclic transfer of the sample solution within the sample container 100 between the surfaces of the plurality of temperature zones may be transferred to the sample container 100.

In some embodiments, the plurality of temperature zones of the thermal cycler 13 may include or consist of a plurality of heating blocks, may provide the required necessary temperatures, and may include various modules for maintaining the required necessary temperatures, such that each heating block may independently provide the respective required necessary temperatures.

The thermal cycler 13 shown in fig. 1 and 2 can be used with the sample vessel 100 shown in fig. 3, including 3 temperature zones, just matching the three amplification chambers of the sample vessel 100.

In some embodiments, the thermal cycler 13 is provided with a first temperature zone 131A, a second temperature zone 131B separate from the first temperature zone 131A, and a third temperature zone 131C separate from the first temperature zone 131A and the second temperature zone 131B. The thermal cycler 13 is a device for amplifying nucleic acid having a specific base sequence by PCR (polymerase chain reaction). For example, a thermal cycler for amplification of DNA (deoxyribonucleic acid) having a specific base sequence performs three steps: denaturation, annealing and extension, and can exponentially amplify DNA having a specific base sequence by repeating these three steps for, for example, 20 to 50 cycles: the denaturation step involves heating the sample solution including double-stranded DNA to a predetermined temperature, for example, about 95 ℃, to separate the double-stranded DNA into single-stranded DNA; the annealing step involves providing an oligonucleotide primer having a sequence complementary to the specific base sequence to be amplified to the sample solution, cooling the isolated single-stranded DNA together with the primer to a predetermined temperature, e.g., 55 ℃, thereby binding the primer to the specific base sequence of the single-stranded DNA, thereby forming a partial DNA-primer complex; and the extension (or amplification) step involves maintaining the temperature of the sample solution at an appropriate temperature, e.g., 72 ℃, after the annealing step, thereby forming double-stranded DNA by DNA polymerase action of primers based on the partial DNA-primer complex.

In some embodiments, the thermal cycling apparatus may also perform the annealing step and the extension (or amplification) step simultaneously, and in this regard, the PCR may complete one cycle by performing a two-step method consisting of the annealing step and the annealing and extension (or amplification) step. The thermal cycler 13 is provided with a first temperature zone 131A, and a second temperature zone 131B separated from the first temperature zone 131A.

In some embodiments, the first temperature zone 131A of the thermal cycler 13 includes a first heating block and the second temperature zone 131B includes a second heating block separate from the first heating block. The thermal cycling apparatus 13 further includes a thermal isolation member having physical and/or chemical properties that do not change due to heating or maintaining the temperature of the first and second heating blocks and that is constructed of any material through which heat exchange between the first and second heating blocks does not occur, e.g., the thermal isolation member may include or may be formed of a material such as plastic.

In some embodiments, the thermal cycler 13 sets the first and second temperature zones 131A, 131B to maintain temperatures for performing denaturation, annealing, and extension (or amplification) steps to amplify nucleic acids. The first temperature zone 131A and the second temperature zone 131B may provide the necessary temperatures required in each step. Thus, when the sample solution in the sample container 100 is transferred to the surface of one of the first and second temperature zones 131A and 131B, the entire surfaces of the first and second temperature zones 131A and 131B are heated or maintained at a desired temperature, so that the sample solution in the sample container 100 is uniformly heated and maintained at the desired temperature. Unlike the conventional thermal cycler using a single heating block, the thermal cycler 13 including the first and second temperature zones 131A and 131B according to one embodiment of the present utility model can significantly reduce PCR time.

In some embodiments, the first temperature zone 131A of the thermal cycler 13 may be maintained at a temperature suitable for performing the denaturation step, in some embodiments, 80 ℃ to 100 ℃ when the extension step is performed at the first temperature zone 131A, in some embodiments, 90 ℃ to 100 ℃ when the denaturation step is performed at the first temperature zone 131A, in some embodiments, 95 ℃ when the denaturation step is performed at the first temperature zone 131A; the second temperature zone 131B may be maintained at a temperature suitable for performing the annealing and extension (or amplification) steps. In some embodiments, the annealing and extension (or amplification) step may be performed at the second temperature zone 131B at 55 ℃ to 75 ℃, and in some embodiments, the annealing and extension (or amplification) step may be performed at the second temperature zone 131B at 72 ℃. However, the temperature is not limited thereto as long as the denaturation step or the annealing and extension (or amplification) step can be performed.

In some embodiments, the thermal cycler 13 sets the first and second temperature zones 131A, 131B to maintain temperatures for performing denaturation, annealing, and extension (or amplification) steps to amplify nucleic acids. The first temperature zone 131A and the second temperature zone 131B may provide the necessary temperatures required in each step. The first and second temperature zones 131A, 131B are also provided with surfaces matching the sample container, a channel 132 in fluid communication with the gas source, and an auxiliary channel 133 in communication with the channel 132. The sample solution in the sample container 100 can be rapidly circulated between the surfaces of the plurality of temperature zones by means of the gas source, the channel 132 and the auxiliary channel 133. Thus, when the sample solution in the sample container 100 is transferred to the surface of one of the first and second temperature zones 131A and 131B, the entire surfaces of the first and second temperature zones 131A and 131B are heated or maintained at a desired temperature, so that the sample solution in the sample container 100 is uniformly heated and maintained at the desired temperature. Unlike the conventional thermal cycler that uses mechanical means to transfer the sample solution, the thermal cycler 13 of one embodiment of the present utility model further significantly reduces PCR time by transferring the sample solution through pneumatic pressure.

In some embodiments, the thermal cycler 13 is configured with a first temperature zone 131A, a second temperature zone 131B, and a third temperature zone 131C to maintain temperatures for performing denaturation, annealing, and extension (or amplification) steps to amplify nucleic acids. The first temperature zone 131A of the thermal cycler 13 may be maintained at a temperature suitable for performing the denaturation step, in some embodiments 80 ℃ to 100 ℃ when the extension step is performed at the first temperature zone 131A, in some embodiments 90 ℃ to 100 ℃ when the denaturation step is performed at the first temperature zone 131A, in some embodiments 95 ℃ when the denaturation step is performed at the first temperature zone 131A; in some embodiments, the second temperature zone 131B may be maintained at a temperature suitable for performing the annealing step. In some embodiments, the annealing and extension (or amplification) step may be performed at the second temperature zone 131B at 55 ℃ to 75 ℃, and in some embodiments, the annealing and extension (or amplification) step may be performed at the second temperature zone 131B at 72 ℃. In some embodiments, the third temperature zone 131C may be maintained at a temperature suitable for performing the extension step, in some embodiments, 55 ℃ to 75 ℃ when the extension (or amplification) step is performed at the third temperature zone 131C, in some embodiments, 72 ℃ when the extension (or amplification) step is performed at the third temperature zone 131C. However, the temperature is not limited thereto, as long as a denaturation step or an annealing and extension (or amplification) step can be performed. It will be appreciated that the denaturation temperature, annealing extension temperature and the number of cycles vary depending on factors such as the kind of nucleic acid sample to be amplified, the nucleotide constitution of the primer, the kind of the synthase, the concentration of the reaction solution components, and the like.

In some embodiments, the thermal cycling device 13 is capable of driving the sample solution within the sample container 100 to circulate between the first temperature zone 131A and the second temperature zone 131B or between the first temperature zone 131A, the second temperature zone 131B and the third temperature zone 131C via the air source, the channel 132 and the auxiliary channel 133.

The gas source includes all devices that enable the sample solution in each amplification chamber of the sample container 100 to circulate between the surfaces of the first temperature zone 131A and the second temperature zone 131B or between the first temperature zone 131A, the second temperature zone 131B, and the third temperature zone 131C.

In some embodiments, the sample container 100 may contain a sample solution that includes nucleic acids, such as double-stranded DNA, oligonucleotide primers having a sequence complementary to a particular base sequence to be amplified, DNA polymerase, deoxyribonucleotide triphosphates (dntps), or PCR reaction buffers. As the sample solution in the sample container 100 is moved to the surface of one of the first, second and third temperature zones 131A, 131B and 131C, heat of the first, second and third temperature zones 131A, 131B and 131C is transferred to the sample solution in the sample container 100, whereby the sample solution contained in the chamber of the sample container 100 can be heated and maintained at a temperature.

In some embodiments, the first temperature zone 131A of the thermal cycler 13 may be maintained at a fixed temperature or temperature interval of 100-155 ℃ to increase the temperature difference between the first temperature zone 131A and the sample solution, increasing the driving force for heat exchange, so that the sample solution may reach the temperature required for denaturation, e.g., 95 ℃, more quickly when the denaturation step is performed at the first temperature zone 131A; the second temperature zone 131B may be maintained at a fixed temperature or temperature range of 10-50 c, and as such, the temperature difference between the second temperature zone 131B and the sample solution is increased, increasing the driving force for heat exchange, so that the sample solution reaches a temperature suitable for performing the annealing and extension (or amplification) steps, for example 55 c, more rapidly. Therefore, the time required by thermal cycle can be shortened, the heating efficiency and the cooling efficiency can be improved, and the PCR time can be further obviously reduced. However, the temperature is not limited thereto as long as the denaturation step or the annealing and extension (or amplification) step can be performed. It will be appreciated that the denaturation temperature, annealing extension temperature and the number of cycles vary depending on factors such as the kind of nucleic acid sample to be amplified, the nucleotide constitution of the primer, the kind of the synthase, the concentration of the reaction solution components, and the like.

In some embodiments, the third temperature zone 131C may maintain a fixed temperature or temperature interval of 100-155 ℃, and/or a fixed temperature or temperature interval of 10-50 ℃. The sample solution in the sample container 100 is rapidly heated to a temperature required for denaturation by the third temperature zone 131C, or the sample solution in the sample container 100 is rapidly cooled to a temperature required for annealing and extension (or amplification) by the third temperature zone 131C, whereby the time required for heat exchange can be shortened, heating efficiency and cooling efficiency can be improved, and PCR time can be further remarkably reduced. However, the temperature is not limited thereto as long as the denaturation step or the annealing and extension (or amplification) step can be performed. It will be appreciated that the denaturation temperature, annealing extension temperature and the number of cycles vary depending on factors such as the kind of nucleic acid sample to be amplified, the nucleotide constitution of the primer, the kind of the synthase, the concentration of the reaction solution components, and the like.

In some embodiments, the thermal cycler 13 further includes a fourth temperature zone, the third temperature zone 131C may maintain a fixed temperature or temperature interval of 100-155 ℃, and the fourth temperature zone may maintain a fixed temperature or temperature interval of 10-50 ℃. The sample solution in the sample container 100 is rapidly heated to a temperature required for denaturation through the third temperature zone 131C, and the sample solution in the sample container 100 is rapidly cooled to a temperature required for annealing and extension (or amplification) through the fourth temperature zone, whereby the time required for heat exchange can be shortened, heating efficiency and cooling efficiency can be improved, and PCR time can be further remarkably reduced. However, the temperature is not limited thereto as long as the denaturation step or the annealing and extension (or amplification) step can be performed. It will be appreciated that the denaturation temperature, annealing extension temperature and the number of cycles vary depending on factors such as the kind of nucleic acid sample to be amplified, the nucleotide constitution of the primer, the kind of the synthase, the concentration of the reaction solution components, and the like.

According to some embodiments of the present utility model, there is provided a sample container, and fig. 3 schematically illustrates a front view of a sample container 100 according to a preferred embodiment of the present utility model, which is supported by a base plate 102 and is divided into three regions, namely, a mixing region 130, an amplification region 140 and a positioning region 150, which are connected in order from top to bottom as shown.

The sample mixing region 130 is used for receiving a nucleic acid sample to be tested, and comprises a sample receiving cavity 112 and an end cap 120 sealing the sample receiving cavity 112. The nucleic acid sample to be measured may be a sample obtained by pretreating (e.g., releasing or diluting nucleic acid) the sample and mixing the reagents for the nucleic acid amplification reaction, or may be a sample obtained without the pretreatment and the mixing step. In the latter case, the lyophilized microspheres 114 of the reagents for the nucleic acid amplification reaction may be pre-disposed in the sample receiving chamber 112, and when the pre-treated sample is added to the sample receiving chamber 112, the sample flows through the lyophilized microspheres 114, dissolving and mixing the microspheres 114, forming a mixed solution for the subsequent amplification reaction. The lyophilized microspheres 114 for nucleic acid amplification typically comprise components necessary for nucleic acid amplification, such as buffers, enzymatic reaction solutions, and primer pairs. In some embodiments, the lyoprotectant is further included in the lyoprotectant 114. In some embodiments, the lyophilized microspheres 114 may further comprise a nucleic acid releasing agent. Such lyophilized microspheres 114 are known in the art, for example, as disclosed in CN111394346A, CN115029423a or CN110452972B. In some embodiments of the utility model, the sample container 100 does not contain the lyophilized microspheres 114. In other embodiments of the present utility model, sample container 100 comprises lyophilized microspheres 114. In some embodiments of the present utility model, the sample container 100 does not include a mixing zone 130. In such embodiments, the nucleic acid sample to be tested may be directly injected into the sample container 100 after being processed to perform the amplification reaction.

In some embodiments, the amplification zone 140 includes at least two amplification chambers (e.g., three amplification chambers, 106a, 106b, and 106c, are shown) with the amplification chambers 106 in series and in fluid communication via channels 108 (108 a and 108 b). As will be described in detail below, the amplification chamber 106 is defined by the substrate 102 and the membranes 104 (104 a, 104b, and 104 c) together. The mixing zone 130 is in fluid communication with the amplification zone 140 via a channel (only port 110 of which is shown). In some embodiments, the amplification zone 140 of the sample container 100 may include only two amplification chambers 106a and 106b, which are in fluid communication via the channel 108a, and the amplification chamber 106a may be in fluid communication with the mixing zone 130. In some embodiments, the amplification zone 140 of the sample container 100 may include four amplification chambers. The amplification region 140 may also include a stiffener 116 that covers and bonds to the region of the amplification region 140 other than the amplification chamber 106 to prevent unwanted peeling of the membrane 104 from the substrate 102.

In some embodiments, a positioning region 150 is provided at the bottom of the sample container 100, which is provided to allow accurate positioning of the sample container 100 relative to the thermal cycling apparatus. As shown, the positioning region 150 of the sample container 100 may include a positioning slot 118 to vertically position the sample container 100. The sample container 100 may also include other positioning elements as desired, such as positioning elements for positioning the sample container 100 in a lateral direction. In some embodiments, the sample container 100 may not include the positioning region 150, but rather a positioning element is provided in a thermal cycling device used in conjunction therewith to achieve accurate positioning of the sample container 100 relative to other amplification devices.

Referring to fig. 4, structures of three different embodiments of the substrate 102 according to some embodiments of the utility model are schematically shown. The substrate 102 defines the outline of the sample container 100, which is substantially sheet-shaped and formed of a hard material. By way of example, alternative hard materials are plastics, metals or metal alloys such as polypropylene, polystyrene, polyester, polycarbonate, polyethylene terephthalate (PET), polyvinyl chloride, polyvinyl dichloride, plexiglas, aluminum alloys, and the like. The hardness of substrate 102 may be, for example, greater than 40 shore HA, greater than 50 HA, greater than 60 HA, greater than 70 HA, greater than 80 HA, greater than 90 HA, or greater than 40HD of shore hardness, greater than 50 HD, greater than 60 HD, greater than 70 HD, greater than 80 HD, or greater than 90 HD. The hard material is selected such that the substrate 102 is subjected to pressure without deformation during the amplification reaction, and one skilled in the art can reasonably determine the desired hardness of the substrate 102 and select an appropriate material according to actual needs. For example, the substrate 102 may withstand the pressure generated when moving the sample solution between the various amplification chambers without deformation.

In some embodiments, the substrate 102 has a first surface 1021 and an opposite back surface (or second surface), the first surface 1021 having a plurality of surface areas (e.g., 2 or 3) 1023 (e.g., 1023a, 1023b, 1023 c) to locate a plurality of amplification chambers 106 (e.g., 106a, 106b, 106 c). The surface area 1023 may be circular, oval or rounded rectangular as shown in fig. 4. The surface area 1023 may be other suitable closed shape, but preferably has rounded corners to minimize the risk of reaction liquid residue. Each of the surface regions 1023a, 1023b, and 1023c may have the same or different shapes, but preferably have the same shape. Preferably, the surface area 1023 has a circular shape as shown in fig. 4.

In some embodiments, the area of each surface area 1023 may be set according to actual needs. For example, the surface areas 1023a, 1023b, and 1023c may be 10 to 100 mm ² For example 20 to 100 mm ² 30 to 100 mm ² 40 to 100 mm ² 50 to 100 mm ² 60 to 100 mm ² 70 to 100 mm ² 80 to 100 mm ² 90 to 100 mm ² 10 to 90 mm ² 10 to 80 mm ² 10 to 70 mm ² 10 to 60 mm ² 10 to 50 mm ² 10 to 40 mm ² 10 to 30 mm ² 10 to 20. 20 mm ² 20 to 90 mm ² 30 to 80 mm ² 40 to 70 mm ² 50 to 60 mm ² 20 to 40 mm ² 20 to 50 mm ² 20 to 60 mm ² 30 to 50 mm ² Or 30 to 40 mm ² . In a preferred embodiment, the surface areas 1023a, 1023b and 1023c may be 20 to 40 mm ² 20 to 50 mm ² 20 to 60 mm ² 30 to50 mm ² Or 30 to 40 mm ² . In a preferred embodiment, the surface areas 1023a, 1023b and 1023c may be 20 mm ² ，25 mm ² ，30 mm ² ，35 mm ² ，40 mm ² ，45 mm ² ，50 mm ² ，55 mm ² Or 60 mm ² 。

In some embodiments, substrate 102 may be provided with a channel 1024 in fluid communication with sample receiving chamber 112, with one end communicating with sample receiving chamber 112 and the other end 110 exposed to surface area 1023, preferably surface area 1023a closest to sample receiving chamber 112. The sample to be amplified in the sample receiving chamber 112 enters the surface area 1023a via the channel 1024 and the end 110. As noted above, sample receiving chamber 112 may also be absent. In this case, the end of the channel 1024 opposite the end 110 may be in direct communication with the outside world to directly receive the sample and introduce it through the end 110 into the surface area 1023a. The channels 1024 may be through holes through the substrate 102, or closed (e.g., by plastic film) grooves formed in the first surface 1021 of the substrate 102, or formed in other suitable ways. In a preferred embodiment, the channels 1024 are through holes that extend through the substrate 102.

In some embodiments, the number of surface regions 1023 is at least two to locate at least two amplification chambers to effect transfer and corresponding reaction of a sample to be amplified between the two amplification chambers. As shown in fig. 4, first surface 1021 includes only two surface areas 1023 (1023 a and 1023 b), and one end of channel 1024 communicates with sample receiving cavity 112, while the other end 110 is preferably exposed to surface area 1023a. In some embodiments, as shown in fig. 3, the surface areas 1023 are three to locate three amplification chambers (fig. 1, 106a, 106b, and 106 c). In some embodiments, the surface area 1023 is four to locate four amplification chambers. The location of each surface region 1023 is not particularly limited, but is preferably vertically aligned to facilitate transfer of the nucleic acid sample between each surface region 1023 (and thus the amplification chamber 106) in a shortest path.

In some embodiments, substrate 102 may include channels 108 that connect each surface region 1023 in series and enable fluid communication of each surface region 1023, such as channels 108a that fluidly communicate surface regions 1023a and 1023b and channels 108b that fluidly communicate surface regions 1023b and 1023 c. In a preferred embodiment, the surface regions are not in direct fluid communication with each other, i.e. one surface region is in direct fluid communication with only a further single surface region. For example, in various embodiments as shown in fig. 4, surface regions 1023a are not in direct fluid communication with surface regions 1023c, but are instead in indirect fluid communication only through surface regions 1023b, i.e., the "series" between the surface regions. Channels 108a and 108b are preferably vertically aligned and coaxial, and are preferably vertically coaxial with channel 1024.

In some embodiments, as shown in fig. 4, the channels 108a and 108b are through holes through the interior of the substrate 102 that are not visible (shown in phantom) at the first surface 1021 of the substrate 102. The through holes are respectively communicated with two adjacent surface areas at two ends. In other embodiments, as shown in fig. 4, channels 108a and 108b are grooves provided on first surface 1201 of substrate 102 that communicate with adjacent two surface areas at each end and are closed (e.g., covered by a membrane) on the surface exposed to first surface 1201 to prevent sample from escaping while flowing through the channel. In other embodiments, as shown in fig. 4, the channels 108a and 108b may be channels attached to the first surface 1021 of the substrate 102, such as channels formed by covering the first surface 1021 with a plastic film, the channels communicating with adjacent two surface areas at each end. Other suitable channel formation patterns are contemplated by those skilled in the art.

In some embodiments, the channels 1024, 108a, and 108b may have a suitable width. For example, the width is 0.1 mm to 2.0 mm, such as 0.1 mm to 1.8 mm,0.1 to 1.6 mm,0.1 to 1.4 mm,0.1 to 1.2 mm,0.1 to 1.0 mm,0.1 to 0.8 mm,0.1 to 0.6 mm,0.2 to 2.0 mm,0.4 to 2.0 mm,0.6 to 2.0 mm,0.8 to 2.0 mm,1.0 to 2.0 mm,1.2 to 2.0 mm,1.4 to 2.0 mm,1.6 to 2.0 mm,1.8 to 2.0 mm,0.1 to 1.0 mm,0.2 to 0 mm,0.8 to mm or 0.4 to mm to 0.8 mm. In a preferred embodiment, the width is from 0.1 mm to 1.0 mm, from 0.2 mm to 0.8 mm or from 0.4 mm to 0.8 mm, more preferably 0.6 mm. When the channel is a groove, the width is the cross-sectional length of the groove, and when the channel is a hole, the width is the inner diameter of the hole. When the channel is of other shape, the width is the maximum distance between the liquids in a direction perpendicular to the flow direction when the sample flows through the channel.

The surface area 1023 forms the bottom surface of the amplification chamber 106 in the present utility model. In some embodiments, surface regions 1023a, 1023b, and 1023c of first surface 1021 are substantially planar or planar. In such embodiments, surface region 1023 is preferably flush with first surface 1021, i.e., surface region 1023 does not form a recess or protrusion relative to first surface 1021. By "substantially" planar or planar, it is meant in the present utility model that the surface area 1023 has a surface roughness Ra of less than 1000 μm, such as less than Ra 800 μm, less than Ra 600 μm, less than Ra 300 μm, less than Ra 200 μm, less than Ra 100 μm, less than Ra 50 μm, less than Ra 20 μm, less than Ra 10 μm, less than Ra 5 μm, less than Ra 3 μm or less than Ra 1 μm. In other embodiments, surface regions 1023a, 1023b, and 1023c may have a greater surface roughness, e.g., a surface roughness Ra of greater than 300 μm, e.g., greater than Ra 400 μm, greater than Ra 500 μm, greater than Ra 600 μm, greater than Ra 700 μm, greater than Ra 800 μm, greater than Ra 900 μm, greater than Ra 1000 μm, greater than Ra 1500 μm, greater than Ra 2000 μm, greater than Ra 2500 μm, greater than Ra 3000 μm.

Fig. 5 shows a schematic structural view of two different embodiments of the sample container 100. The stiffener 116 is shown in phantom in its position and size, removed in the figure to more clearly show the structure of the sample container 100. The sample container 100 is covered with a membrane 104 on the first surface 1021 of the substrate 102, the membrane 104 comprising at least membrane areas 104a, 104b and 104c. Each film region surrounds a surface region and has a peripheral region surrounding the surface region and a central region corresponding to the surface region. The following description is mainly directed to the film region 104a, and the film regions 104b and 104c have the same or similar structural arrangement as 104a, and will not be repeated.

Specifically, the film region 104a has a peripheral region 1041a and a central region 1042a, wherein the peripheral region 1041a is disposed around the surface region 1023a of the first surface 1021 of the substrate 102 and fixedly bonded to the first surface 1021, and the central region 1042a corresponds to the surface region 1023a and is separable from the surface region 1023 a. The peripheral region 1041a is fixedly bonded to the first surface 1021 by a process such as welding (e.g., ultrasonic welding) or bonding (e.g., ultraviolet gluing, nanogluing), among others, which are known in the art. The central region 1042a can be separate from the surface region 1023a and thus not fixedly coupled to the first surface 1021 (i.e., the surface region 1023 a).

Peripheral region 1041a may together with surface region 1023a form, for example, a circular ring (as shown in fig. 5), a rectangular shape (as shown in fig. 5), or other suitable shape, so long as peripheral region 1041a is disposed around surface region 1023a and surrounds surface region 1023 a. Preferably, the area of peripheral region 1041a in combination with surface region 1023a is greater than the area of surface region 1023a by 5% to 250%, such as greater than 5% to 240%, greater than 5% to 220%, greater than 5% to 200%, greater than 5% to 180%, greater than 5% to 160%, greater than 5% to 140%, greater than 5% to 120%, greater than 5% to 100%, greater than 5% to 80%, greater than 5% to 60%, greater than 5% to 40%, greater than 5% to 20%, greater than 10% to 250%, greater than 20% to 250%, greater than 40% to 250%, greater than 60% to 250%, greater than 80% to 250%, greater than 100% to 250%, greater than 120% to 250%, greater than 140% to 250%, greater than 160% to 250%, greater than 180% to 250%, greater than 200% to 250%, greater than 220% to 250%, greater than 240% to 250%, or greater than 150% to 200%. Preferably, the area of peripheral region 1041a in combination with surface region 1023a is 100% to 250% greater than the area of surface region 1023a, 120% to 250% greater than the area of surface region 1023a, 140% to 250% greater than the area of surface region 1023a, 160% to 250% greater than the area of surface region 1023a, 180% to 250% greater than the area of surface region 1023a, 200% to 250% greater than the area of surface region 1023a, 220% to 250% greater than the area of surface region 1023a, 240% to 250% greater than the area of surface region 1023a, or 150% to 200% greater than the area of surface region 1023 a.

In the present utility model, the surface area of the central region of each film region is larger than that of the corresponding surface region without external force and without undergoing plastic deformation. The central region 1042a has a larger surface area than the surface region 1023a, so that folds, indicated by arrows in fig. 4, are formed in the central region 1042a of the film region 104a of the film 104. It is envisioned that when the surface area of the central region 1042a is equal to or less than the surface area of the surface region 1023a, there are no wrinkles in the central region 1042 a. However, when the surface area of the central region 1042a is larger than that of the surface region 1023a, the difference in area is hidden in the wrinkles, which may disappear or decrease due to the central region 1042a being applied with a negative pressure (as will be described later), because the central region 1042a is pulled up toward the reader direction (arranged as shown in fig. 5) after the negative pressure is applied, and thus the central region 1042a may extend over a larger space. It is noted that in the present utility model, the larger surface area of the central region 1042a than the surface region 1023a occurs in the case where the central region 1042a is not subjected to external force and is not subjected to plastic deformation. For example, this may be the initial state of the sample container 100, i.e. the sample container 100 has never been used. In some embodiments, the central region 1042a of the sample container 100 of the present utility model is also not subjected to or has not undergone plastic deformation when the sample container 100 is in or after use, and thus this may also occur with sample containers 100 that have undergone at least one use. Determining whether the central region 1042a has been subjected to plastic deformation may be accomplished by a variety of methods, such as comparing the surface area of the central region 1042a before and after the application of a force or comparing the film thickness of the central region 1042a, wherein an increase in surface area or a decrease in thickness after the application of a force may mean that the central region has been subjected to plastic deformation.

In some embodiments, the membrane regions 104a, 104b, and 104c of the membrane 104 used in the present utility model do not plastically deform during amplification. In some embodiments, membrane regions 104a, 104b, and 104c of membrane 104 used in the present utility model do not elastically deform during amplification even if expanded, which can be accomplished by rationally designing the difference in the surface area of membrane regions 104a, 104b, and 104c relative to the area of surface regions 1023a, 1023b, and 1023 c.

In some embodiments, the surface area of the central region 1042a is 10% to 400%, such as 10% to 380%, 10% to 360%, 10% to 340%, 10% to 320%, 10% to 300%, 10% to 280%, 10% to 260%, 10% to 240%, 10% to 220%, 10% to 200%, 10% to 180%, 10% to 160%, 10% to 140%, 10% to 120%, 10% to 100%, 10% to 80%, 10% to 60%, 10% to 40%, 10% to 30%, 10% to 20%, 20% to 400%, 40% to 400%, 60% to 400%, 80% to 400%, 100% to 400%, 120% to 400%, 140% to 400%, 160% to 400%, 180% to 400%, and 400% to 80% greater than the surface area of the surface area 1023a200% to 400%, 220% to 400%, 240% to 400%, 260% to 400%, 280% to 400%, 300% to 400%, 320% to 400%, 340% to 400%, 360% to 400%, 380% to 400%, 100% to 300%, 120% to 280%, 140% to 260%, 150% to 250%, 160% to 240%, 180% to 220%, or 200%. In a preferred embodiment, the surface area of the central region 1042a is 100% to 300% greater than the surface area 1023a, more preferably 150% to 250% greater, still more preferably 200% greater. For example, in some embodiments, the area of surface region 1023a is about 30 to 40 mm ² The surface area of the central region 1042a may be 60 to 80 mm ² 。

In the present utility model, the central region 1042a and the surface region 1023a together define an amplification chamber 106a (see FIG. 3). In some embodiments, each amplification chamber is defined only by a central region and a surface region. The surface area 1023a forms the bottom surface of the amplification chamber 106a, and the central area 1042a is pulled up by an external force (e.g., negative pressure) to separate from the surface area 1023a, forming the upper space of the amplification chamber 106a, which together define the space for receiving the amplified sample. When under positive pressure, the central region 1042a is pressed into engagement with the surface region 1023a, thereby expelling the sample through the channel. Thus, the amplification chamber 106a may have a variable volume without external forces and without plastic deformation of the central region 1042 a. When subjected to an external force, such as a negative pressure, the amplification chamber 106a may reach its maximum volume, and the amplification chamber 106a may shrink in volume to zero or close to zero under a positive pressure. The amplification chamber 106a thus has a variable volume, and the amplification chamber 106a can reach its maximum volume under negative pressure, and the amplification chamber 106a can shrink in volume under positive pressure until it is zero or close to zero. The maximum volume of the amplification chamber 106a depends on factors such as the area of the surface region 1023a, the surface area of the central region 1042a, the material of the central region 1042a, and the like. For example, the maximum volume of the amplification chamber 106a may be 5 to 100 microliters, such as 10 to 100 microliters, 15 to 100 microliters, 20 to 100 microliters, 25 to 100 microliters, 30 to 100 microliters, 35 to 100 microliters, 40 to 100 microliters, 45 to 100 microliters, 50 to 100 microliters, 55 to 100 microliters, 60 to 100 microliters, 65 to 100 microliters, 70 to 100 microliters, 75 to 100 microliters, 80 to 100 microliters, 85 to 100 microliters, 90 to 100 microliters, 95 to 100 microliters, 5 to 90 microliters, 5 to 80 microliters, 5 to 70 microliters, 5 to 60 microliters, 5 to 50 microliters, 5 to 40 microliters, 5 to 30 microliters, 5 to 20 microliters, 5 to 10 microliters, 10 to 50 microliters, 10 to 40 microliters, 10 to 35 microliters, 10 to 30 microliters, 10 to 25 microliters, 10 to 20 microliters, 10 to 15 microliters, 15 to 35 microliters, 20 to 30 microliters, or 25 microliters. In preferred embodiments, the maximum volume of the amplification chamber 106a is 10 to 50 microliters, more preferably 15 to 35 microliters, or 25 microliters.

In the present utility model, the boundary point of the central region 1042a and the peripheral region 1041a is defined by the boundary of the surface region 1023 a. As shown in fig. 5, the boundary of the surface region 1023a is circular, and thus the central region 1042a is bordered by the peripheral region 1041a along the circular boundary. The film material constituting the central region 1042a may be the same as the film material constituting the peripheral region 1041 a. In embodiments of the utility model, the membrane may or may not be elastic. Suitable film materials may be transparent or translucent film materials, such as films of polyethylene, polypropylene, thermoplastic elastomers, thermoplastic polyurethane, polymethyl methacrylate, polystyrene, polyvinyl chloride and polyethylene terephthalate. For example, the material may have a transmittance of at least 80%, preferably at least 90%, for incident light, such as laser light. Other suitable films also include silicone films or other elastic films. The film has a thickness of 0.01 mm to 0.20 mm, for example, 0.01 mm to 0.18 mm,0.01 mm to 0.16 mm,0.01 mm to 0.14 mm,0.01 mm to 0.12 mm,0.01 mm to 0.10 mm,0.01 mm to 0.08 mm,0.01 mm to 0.06 mm,0.01 mm to 0.05 mm,0.01 mm to 0.04 mm,0.01 mm to 0.02 mm,0.02 mm to 0.20 mm,0.03 mm to 0.20 mm,0.04 mm to 0.20 mm,0.05 mm to 0.20 mm,0.06 mm to 0.20 mm,0.07 mm to 0.20 mm,0.08 mm to 0.20 mm,0.10 mm to 0.20 mm,0.15 mm to 0.20 mm,0.03 mm to 0.10 mm,0.05 mm to 0.09 mm,0.06 mm to 0.62 mm. In preferred embodiments, the film has a thickness of 0.01 mm to 0.1 mm,0.03 to 0.10 mm,0.05 to 0.09 mm, or 0.06 mm to 0.08 mm, more preferably 0.07 mm.

In the present utility model, amplification chamber refers to a chamber for a nucleic acid amplification process, which includes a chamber in which amplification reactions (denaturation, annealing, extension) directly occur, as well as a chamber in which amplification is assisted for the purpose of carrying out the present utility model or obtaining one or more of its advantageous effects, for example, a chamber for effecting transfer of a sample in a different chamber. For example, as described in detail below, in the sample container 100 shown in FIG. 3, the amplification chamber 106a is continuously pressurized with positive pressure while the amplification reaction is occurring to block the channel 108a, so that the sample is transferred and amplified (denatured, annealed, and extended) only between the amplification chambers 106b and 106 c. In addition, the amplification chamber 106a may also be used as a reverse transcription reaction in the amplification of RNA nucleic acid to reverse transcribe RNA into cDNA and then perform an amplification process of cDNA, wherein the amplification process of cDNA may be performed in the amplification chambers 106b and 106c, or may also be performed in at least two of the amplification chambers 106a, 106b and 106 c.

Fig. 6 shows a schematic structural view of a sample container 100 according to further embodiments of the present utility model, wherein the reinforcement 116 is shown in phantom in its position and dimensions, removed in the figure to more clearly show the structure of the sample container 100. The sample container 100 shown in fig. 6 has a substantially similar structure as in fig. 5, except for the structure of the membrane region 104. In fig. 5, the film region 104 includes a peripheral region 1041 surrounding the surface regions 1023 and a central region 1042 corresponding to the surface regions 1023, the peripheral regions 1041 surrounding the respective surface regions 1023 no longer being independent of each other but connected as a whole. Thus, each membrane region 104 may be formed from a single sheet of membrane, with the peripheral region 1041 being continuous and non-segmented in material with each central region 1042. Methods of making such film regions may include providing respective molds, overlaying the films on the molds, and fixedly bonding (e.g., ultrasonic welding or heat staking) the films to the substrate at the peripheral regions.

As shown in fig. 6, the size and shape of the peripheral region 1041 may vary. The peripheral region 1041 is at least partially covered by the stiffener 116, preferably completely covered by the stiffener 116. Preferably, the stiffener 116 is sized larger than the peripheral region 1041, for example extending 1 to 5 cm outwardly from the boundary of the peripheral region 1041, to provide greater adhesion. The stiffener 116 does not cover the central region 1042 and thus has holes of the same contour, location and size as the surface region 1023. The presence of the stiffener 116 may prevent unwanted peeling of the film 104 from the substrate 102. The reinforcement 116 may be an additional film or formed of other hard materials such as plastic.

A nucleic acid amplification method according to an embodiment of the present utility model. The method comprises the following steps:

step 202: the sample container 100 of any of the above-described embodiments of the present utility model is provided such that the sample container 100 includes at least a first amplification chamber (e.g., 106 a) and a second amplification chamber (e.g., 106 b) that are vertically high to low. In a preferred embodiment, step 202 provides a sample container 100 of the present utility model having three amplification chambers (e.g., 106a, 106b, and 106 c) that are vertically from low to high, a first amplification chamber (e.g., 106 c), a second amplification chamber (e.g., 106 b), and a third amplification chamber (e.g., 106 a), respectively.

Step 204: the sample container 100 is vertically positioned. The vertical direction is a direction perpendicular to the horizontal direction. It will be appreciated that vertical positioning does not require that the sample container be in a strictly vertical orientation, for example the plane in which the base plate 102 of the sample container lies may have an angle of 1 ° to 45 °, preferably 1 ° to 30 °,1 ° to 10 °, or 1 ° to 5 °, or less than 1 °, or less than 0.1 ° to the vertical plane. As previously described, the sample container 100 may include a detent 118 to facilitate vertical positioning. The vertical positioning may also rely on other suitable positioning elements, as is known in the art. The vertically oriented sample container 100 has the sample receiving chamber 112, the amplification chamber 106a, the channel 108a, the amplification chamber 106b, the channel 108b, and the amplification chamber 106c arranged vertically from top to bottom. In the present utility model, the sample container 100 is still in a vertical orientation during the nucleic acid amplification procedure.

Step 206: a nucleic acid sample to be amplified is introduced into the sample container 100. The nucleic acid sample to be amplified may be a sample that has been subjected to nucleic acid sample release, which may be mixed with pre-set freeze-dried microspheres in a nucleic acid sample receiving chamber to form a reaction solution for an amplification procedure. Alternatively, the nucleic acid sample to be amplified may be a sample in which the essential components for nucleic acid amplification are released, diluted and mixed by the sample, and the sample receiving chamber does not require presetting of lyophilized microspheres. Alternatively, as previously described, the sample container 100 may be free of sample receiving chambers and the prepared nucleic acid sample to be amplified is directly injected through the channel to one of the amplification chambers.

Step 208: the nucleic acid amplification operation is performed according to a predetermined procedure. Preferably, the predetermined program is computer programmed. The nucleic acid sample is transferred between the amplification chambers by applying positive or negative pressure to the amplification chambers, and the different amplification chambers are provided with different predetermined temperatures, whereby the nucleic acid sample undergoes different temperature changes between the different amplification chambers, which temperature changes cause the nucleic acid to undergo denaturation, annealing and extension. The positive or negative pressure may be achieved by a variety of means, such as pneumatic pressure.

Step 210: step 208 is repeated such that the nucleic acid sample is subjected to a plurality (e.g., 20 to 50) of denaturation-annealing-extension cycles, thereby effecting amplification of the nucleic acid sample.

Preferably, after step 206, before step 208, the method 200 may include a step 212 of removing bubbles from the sample container.

FIG. 7 shows a schematic diagram of the transfer of sample solution between temperature zones according to one embodiment of the utility model. In this figure, the sample container 100 contains three amplification chambers. Wherein positive pressure is indicated by right arrow, negative pressure is indicated by left arrow, and nucleic acid sample reaction solution is indicated by gray filling.

Thanks to the sample vessel provided by the utility model, the central region of the membrane region comprised therein has a surface area which is larger than the corresponding surface area in the initial state of the sample vessel, such that the central region is not stretched or only minimally stretched when expanded by application of negative pressure, and thus is not plastically deformed, nor is elastically deformed or only minimally elastically deformed, and thus the thickness of the membrane used in the utility model may be significantly reduced compared to a membrane which is subjected to stretching to form a chamber containing a nucleic acid sample, e.g. may be as low as about 0.1 mm, e.g. about 0.001 to about 0.10 mm, e.g. about 0.01 mm, about 0.03 mm, about 0.05 mm or about 0.07 mm, without rupture after undergoing up to 50 amplification cycles. The reduced thickness of the membrane results in a higher heat transfer efficiency between it and the heat source, which greatly increases the rate of nucleic acid amplification.

Furthermore, the sample container of the present utility model does not comprise a mechanical valve. The sample receiving cavity and each amplification cavity are provided with only channels which can enable the sample receiving cavity and each amplification cavity to circulate mutually, no mechanical valve or other additional blocking structures are arranged, during detection, only positive and negative pressures corresponding to each amplification cavity are required to be controlled, the control scheme is simple, and the film cannot bear additional external force due to the fact that reaction liquid breaks through the valves or other blocking structures.

Thus, the present utility model provides a thermal cycling apparatus, a sample container and a detection apparatus comprising the same. Due to the inventive design of the present utility model, the detection time is effectively shortened, and the thermal cycler, the sample vessel and the detection device comprising the same according to the present utility model are particularly suitable for immediate detection.

In addition to the sample container 100 and the thermal cycling apparatus 13, the detection apparatus of the present utility model may also include a temperature control assembly, a pneumatic assembly, a positioning assembly, an optical assembly, a code scanning module, and/or a control module.

The foregoing is a representative example of embodiments of the present utility model and is provided for illustrative purposes only. The present utility model contemplates that one or more features used in one embodiment can be added to another embodiment to form an improved or alternative embodiment without departing from the purpose of the embodiment. Likewise, one or more features used in one embodiment may be omitted or substituted without departing from the purpose of the embodiment to form a substituted or simplified embodiment. Furthermore, one or more features used in one embodiment may be combined with one or more features of another embodiment to form improved or alternative embodiments without departing from the purpose of the embodiments. The present utility model is intended to include all such improved, alternative, and simplified embodiments.

Claims

1. A thermal cycling apparatus, characterized in that:

the thermal cycling device comprises a plurality of temperature areas, wherein the plurality of temperature areas at least comprise a first temperature area and a second temperature area;

the plurality of temperature zones are arranged separately from each other;

each temperature zone is provided with a surface matched with the sample container and a channel in fluid communication with a gas source;

the sample solution in the sample container can be circulated between the surfaces of the plurality of temperature zones by means of the gas source and the channels provided by the plurality of temperature zones.

2. The thermal cycler of claim 1, wherein:

the first temperature zone is used for enabling the sample solution in the sample container to obtain a high target temperature, and the second temperature zone is used for enabling the sample solution in the sample container to obtain a low target temperature; or alternatively

The first temperature zone is within a first temperature interval and the second temperature zone is within a second temperature interval, the first temperature interval being a higher temperature interval than the second temperature interval.

3. The thermal cycler of claim 2 wherein:

the sample solution comprises a sample to be tested having a target denaturation temperature and a target annealing temperature;

wherein:

the first temperature interval is higher than the target denaturation temperature; the second temperature interval is lower than the target annealing temperature; or alternatively

The first temperature interval contains the target denaturation temperature; the second temperature interval contains the target annealing temperature; or alternatively

The high target temperature is substantially equal to the target denaturation temperature and the low target temperature is substantially equal to the target annealing temperature.

4. A thermal cycling device in accordance with claim 2 or 3, characterised in that:

the plurality of temperature zones further includes a third temperature zone, wherein:

the third temperature zone is used for enabling the sample solution in the sample container to obtain an intermediate target temperature, and the intermediate target temperature is between the high target temperature and the low target temperature; or alternatively

The third temperature zone is within a third temperature interval, the third temperature interval being a higher temperature interval than the first temperature interval; or alternatively

The third temperature zone is within a fourth temperature zone, the fourth temperature zone being a lower temperature zone than the second temperature zone; or:

the third temperature zone is capable of providing a temperature within a third temperature interval and a temperature within a fourth temperature interval, and the third temperature interval is a higher temperature interval than the first temperature interval and the fourth temperature interval is a lower temperature interval than the second temperature interval.

5. A thermal cycling device in accordance with claim 2 or 3, characterised in that:

the plurality of temperature zones further includes a third temperature zone and a fourth temperature zone, wherein:

the third temperature zone is used for enabling the sample solution in the sample container to obtain a first intermediate target temperature, the fourth temperature zone is used for enabling the sample solution in the sample container to obtain a second intermediate target temperature, the first intermediate target temperature and the second intermediate target temperature are both between the high target temperature and the low target temperature, and the first intermediate target temperature is higher than the second intermediate target temperature; or alternatively

The third temperature zone is within a third temperature zone, the fourth temperature zone is within a fourth temperature zone, the third temperature zone is a higher temperature zone than the first temperature zone, and the fourth temperature zone is a lower temperature zone than the second temperature zone.

6. A detection apparatus, characterized by comprising:

the thermal cycler and sample vessel of any of claims 1-5.

7. The test device of claim 6, wherein the sample container comprises:

a substrate having a first surface and an opposing second surface, the first surface having at least two surface areas;

A membrane on the first surface of the substrate and comprising at least two membrane regions, each membrane region defining a chamber in cooperation with a corresponding said surface region on the substrate;

a fluid channel, the membrane region being in fluid communication via the fluid channel.

8. The device of claim 7, wherein the at least two membrane regions are in fluid communication via the fluid channel in the absence of an external force.

9. The detection apparatus according to claim 7, wherein:

each of the film regions includes a peripheral region surrounding one of the at least two surface regions and a central region corresponding to the surface region, the peripheral region being fixedly bonded to the first surface of the substrate, the central region being separate from the surface region;

the surface area of the central region is larger than the orthographic projection area of the surface region on the first surface under the condition that no external force is applied and plastic deformation is not carried out; or alternatively

The central zone having folds which can be unfolded under the action of an external force without undergoing plastic deformation; or alternatively

Each amplification chamber having a variable volume without external forces and without plastic deformation of the central zone; or alternatively

Each of the membrane regions is capable of expanding or compressing under an external force, wherein the membrane regions are capable of expanding or compressing under the same external force without substantial deformation of the substrate.

10. The detection apparatus according to claim 7, wherein:

the film has a thickness of 0.001 mm to 0.2 mm.

11. The detection apparatus according to claim 9, wherein:

the film has a thickness of 0.001 mm to 0.1 mm.

12. The detection apparatus according to claim 9, wherein:

the thickness of the film is 0.01 to mm a to 0.1 to mm a.

13. The detection apparatus according to claim 9, wherein:

the thickness of the film was 0.01 mm.

14. The detection apparatus according to claim 9, wherein:

the thickness of the film was 0.03 mm.

15. The detection apparatus according to claim 9, wherein:

the thickness of the film was 0.05 mm.

16. The detection apparatus according to claim 9, wherein:

the thickness of the film was 0.07 mm.

17. The detection apparatus according to claim 7, wherein:

for each film region, the surface area of the central region is 10% to 400% greater than the orthographic projected area of the corresponding surface region on the first surface.

18. The detection apparatus according to claim 16, wherein:

for each film region, the surface area of the central region is 100% to 300% greater than the orthographic projected area of the corresponding surface region on the first surface.

19. The detection apparatus according to claim 16, wherein:

for each film region, the surface area of the central region is 150% to 250% greater than the orthographic projected area of the corresponding surface region on the first surface.

20. The detection apparatus according to claim 16, wherein:

for each film region, the surface area of the central region is still more preferably 200% greater than the orthographic projected area of the corresponding surface region on the first surface.

21. The detection apparatus according to any one of claims 7 to 20, wherein:

the first surface of the substrate has three surface areas, the film comprising three film areas; or,

the first surface of the substrate has four surface areas and the film includes four film areas.