CN112342128B

CN112342128B - Nucleic acid extraction amplification test tube and working method thereof

Info

Publication number: CN112342128B
Application number: CN202011256843.XA
Authority: CN
Inventors: 王威; 梅宗羲; 聂晶
Original assignee: Suzhou Molarray Biotechnology Co ltd
Current assignee: Suzhou Yarui Biotechnology Co.,Ltd.
Priority date: 2020-11-11
Filing date: 2020-11-11
Publication date: 2021-07-20
Anticipated expiration: 2040-11-11
Also published as: CN112342128A

Abstract

The invention discloses a nucleic acid extraction amplification test tube and a working method thereof, belonging to the field of molecular diagnosis and detection instruments, and the design key points are that the nucleic acid extraction amplification test tube comprises the following components: the device comprises a PCR reaction tube, an extraction tube assembly lower tube, an extraction tube assembly middle tube, an extraction tube assembly upper tube, a top cover, a piston rod, a piston ring, a first valve core, a second spring, a second valve core and a third spring; the upper pipe component of the extraction pipe component, the middle pipe of the extraction pipe component, the lower pipe of the extraction pipe component and the PCR reaction pipe are sequentially connected from top to bottom; the extraction tube assembly upper tube assembly comprises: two vertical parallel tubes: an extraction tube, a piston tube; a top cover is arranged at the top end of the extraction pipe; the piston rod is arranged in the piston tube. The invention aims to provide a nucleic acid extraction amplification test tube and a working method thereof, which greatly improve the detection efficiency.

Description

Nucleic acid extraction amplification test tube and working method thereof

Technical Field

The invention relates to the field of medical examination and inspection instruments and molecular diagnosis and detection instruments, in particular to a nucleic acid extraction and amplification test tube and a working method thereof.

Background

Current methods for detecting and diagnosing microbial infections include the use of quantitative polymerase chain reaction (qPCR) to amplify nucleic acids (RNA/DNA) in patient samples to confirm the presence of pathogens. Furthermore, these tests must be done by a test laboratory, where two steps must be performed to make a diagnosis: nucleic acid extraction followed by qPCR. The two processes may also be performed by different devices, which increases the complexity and length of time required between sample collection and diagnosis. Finally, these laboratory tests are typically done manually, take up physical labor, and also have potential additional hazards in handling pathogen positive samples.

Currently, the prior art already exists for automatic nucleic acid extraction machines, such as:

reference 1: CN 102472695a discloses a nucleic acid extraction device in which an extraction cartridge is transferred to the upper part of a waste liquid well, and blood added to the sample well is sucked up by a dispensing pipette. Then, the blood is transferred to a lysis solution in the reagent well, and the blood is mixed with the lysis solution to elute the nucleic acid (DNA) from the blood cells. By adopting a method of dispensing by breaking the protective film on the reagent well by the tip of the dispensing pipette, the sealing property of the reagent can be maintained until the time of operation, and therefore, the extraction operation can be realized without causing contamination or deterioration. Blood is added to a reagent well filled with a chaotropic agent (a dissolving solution) for dissolving a biological material by a dispensing pipette. In the reagent well, the mixed solution of the lysis solution and blood is repeatedly discharged and sucked by using a pipette to be completely mixed, and the biological material is lysed.

Reference 2: CN 205687906U has proposed a PCR short-term test kit, also is a nucleic acid extraction element, is equipped with the layer board that the level set up in the inner chamber of box body, is equipped with the centrifuge tube hole that a plurality of interval set up on the layer board, and the downthehole centrifuging tube that is equipped with the DNA extract of placing of centrifuging tube is equipped with swing joint and the container that is equipped with prefabricated PCR detect reagent on the mouth of pipe of centrifuging tube, is equipped with the container hole on the lower terminal surface of container, is equipped with extruding means on the container.

Reference 3: CN 103789201A proposes an instrument for nucleic acid isolation, comprising a 1 st wick composed of a 1 st oil, a 2 nd wick composed of a 1 st washing liquid for washing a nucleic acid-binding solid phase carrier to which a nucleic acid is bound, which is separated from an oil phase, a 3 rd wick composed of a 2 nd oil, a 4 th wick composed of a reverse transcription reaction liquid for performing a reverse transcription reaction, which is separated from an oil phase, a 5 th wick composed of a 3 rd oil, a 6 th wick composed of an eluent separated from an oil phase for eluting the nucleic acid from a nucleic acid-binding solid phase carrier to which a nucleic acid is bound, and a 7 th wick composed of a 4 th oil.

Reference 4: CN 105331525 a provides a biological sample processing apparatus, which comprises a column having a first and a second tube body parts, wherein the column is hollow and has two open ends, so that a flow channel is formed in the column, and an open end of the first tube body part forms a first opening, and an open end of the second tube body part forms a second opening, wherein the inner diameter of the column gradually decreases from the first opening to the second opening along an axial direction, so that the average inner diameter of the first tube body part is larger than that of the second tube body part, and a holder having a plurality of through holes; providing a solid biological sample, and placing the solid biological sample on the bracket; providing a lysis reagent, making the lysis reagent pass through the second opening of the second tube part and pass through the through holes on the bracket to enter the first tube part, so as to obtain a nucleic acid product.

These are relatively compact machines that can handle the extraction of multiple samples with minimal human intervention, and yet are small enough to be used in an environment outside the laboratory when necessary. The most common sample collectors take the form of swabs (nasopharynx, throat, etc.) which are then stored in sample containers and then transported to the testing facility. Although some available techniques are more likely to be used for commercial research and testing, qPCR instruments are typically not automated in a testing facility.

Particularly in the current new coronavirus epidemic situation, SARS-CoV2 needs to be detected quickly and accurately, and the quantity of nucleic acid extraction kits is large and the supply is short. Extraction-free techniques developed during this time are becoming more and more common, eliminating the need for a separate nucleic acid extraction step. However, these techniques still require a separate transfer step from sample collection to the test equipment running qPCR. Again, this includes possible exposure and cross-contamination risks, as the sample must be opened to manually place it into the qPCR machine.

In response to the above problems, functional test kits/tubes for nucleic acid extraction AND amplification were developed (using "C12M 1/IC AND nucleic acid AND extraction AND amplification" in HIMMPAT).

For example:

reference 5: CN 111235008A a closed integrated pathogen detection card box, mainly including stock solution base, inhale and put liquid subassembly and closing cap, the stock solution base can fix and settle in external metal heat trap device, rotates along with the rotation of external metal heat trap device to inhale the imbibition head in putting the liquid subassembly and can get into every reservoir. Reference 5 is a closed system for extracting, amplifying and detecting nucleic acid to be detected. Automatic control, only adding samples manually in the whole operation process; but the types of reagents are as many as 17, and the operation needs pumping and rotation, which is more complicated; also, it requires the use of magnetic separation.

From this, most of the studies in the prior art still remain on how to extract nucleic acids; however, the small amount of research on the integrated functions of nucleic acid extraction and amplification has the problems of complicated operation and complicated equipment design, and cannot meet the requirement of SARS-CoV2 detection.

Disclosure of Invention

The present invention has been made in view of the above-mentioned problems of the prior art, and an object of the present invention is to provide a nucleic acid isolation and amplification test tube.

Another object of the present invention is to provide a method for operating a nucleic acid amplification reaction tube, which overcomes the above-mentioned disadvantages of the prior art.

The technical scheme of the invention is as follows:

a nucleic acid extraction amplification test tube comprising: the device comprises a PCR reaction tube, an extraction tube assembly lower tube, an extraction tube assembly middle tube, an extraction tube assembly upper tube, a top cover, a piston rod, a piston ring, a first valve core, a second spring, a second valve core and a third spring;

wherein, the upper tube component of the extraction tube component, the middle tube of the extraction tube component, the lower tube of the extraction tube component and the PCR reaction tube are sequentially connected from top to bottom;

wherein, the extraction tube assembly upper tube assembly includes: two vertical parallel tubes: an extraction tube, a piston tube; a top cover is arranged at the top end of the extraction pipe; the piston rod is arranged in the piston tube;

wherein, still include: a first air hole and a second air hole; the first air hole is used for communicating the extraction tube with the outside air, and the second air hole is used for communicating the PCR reaction tube with the outside air;

the joint of the lower pipe of the extraction pipe assembly and the middle pipe of the extraction pipe assembly is provided with: a second valve core and a third spring; the junction of the upper pipe component of the extraction pipe component and the middle pipe of the extraction pipe component is provided with: a first valve core and a second spring.

Furthermore, the top of the PCR reaction tube is provided with an opening, and the opening at the top of the PCR reaction tube is connected with the bottom end of the lower tube of the extraction tube component.

Further, wherein the extraction tube assembly lower tube comprises: the pipe comprises a first pipe and a second pipe, wherein a flange is arranged at the bottom end of the first pipe inwards, and the lower side of the flange is connected with the second pipe; the first pipe and the second pipe are connected into a whole, the central axes of the first pipe and the second pipe are overlapped, the first pipe is arranged at the upper end of the second pipe, and the radius of the inner surface of the first pipe is larger than that of the outer surface of the second pipe; i.e. the width of the flange-the radius of the inner surface of the first tube-the radius of the outer surface of the second tube.

Wherein, the extraction tube subassembly middle part pipe includes: an upper member tube, a lower member tube, a flange member; the upper member tube includes: an extraction tube corresponding portion and a piston tube corresponding portion; the upper surfaces of the corresponding parts of the extraction pipe and the piston pipe are flush, and the lower surface of the corresponding part of the extraction pipe is lower than the lower surface of the corresponding part of the piston pipe; the bottom of the corresponding part of the extraction pipe is provided with a flange component inwards, and the lower side of the flange component is connected with a lower component pipe;

the bottom of the corresponding part of the piston pipe is closed.

Further, the extraction tube assembly upper tube assembly comprises: two vertical parallel tubes: the extraction pipe and the piston pipe are connected into a whole; the extraction tube comprises: the extraction pipe comprises an extraction pipe body, an extraction pipe insertion pipe and a flange body, wherein the flange body is arranged inwards at the bottom of the extraction pipe body, and the extraction pipe insertion pipe is arranged at the lower part of the flange body; the piston tube is provided with a tube body extending to the piston tube corresponding part of the middle tube of the extraction tube assembly; the bottom of the extraction tube insertion tube is lower than the bottom of the piston tube.

Further, still include: the lower end part of the piston rod is fixedly provided with a piston ring, and the piston ring is used for sealing; a push rod is arranged at the upper end part of the piston rod; one end of the first spring is connected with the upper surface of the piston tube, and the other end of the first spring is connected with the lower surface of the push rod of the piston rod.

Further, the mode that sets up second case, third spring in the junction of extraction pipe subassembly lower part pipe, extraction pipe subassembly middle part pipe does:

the width of the flange of the lower pipe of the extraction pipe assembly is larger than the thickness of the lower component pipe of the middle pipe of the extraction pipe assembly; the width of the flange member of the middle tube of the extraction tube assembly is greater than the thickness of the lower member tube of the middle tube of the extraction tube assembly; a second accommodating space is formed at the upper part of the flange of the lower pipe of the extraction pipe assembly, the inner part of the lower member pipe of the middle pipe of the extraction pipe assembly and the lower part of the flange member of the middle pipe of the extraction pipe assembly, and the third spring and the second valve core are arranged in the second accommodating space; and a third spring is arranged on a flange of the lower pipe of the extraction pipe assembly, the third spring is arranged in the lower component pipe of the middle pipe of the extraction pipe assembly, and a second valve core is sleeved on the third spring.

Further, the second valve core comprises a core print and a core rod, wherein the upper surface of the core print is in a cone frustum shape and is gradually increased in radius from top to bottom; the lower surface of the core head is fixedly connected with a core rod, and the sectional area of the lower surface of the core head is larger than that of the core rod; the core bar is inserted into a third spring, namely the third spring surrounds the core bar, one end of the third spring is fixedly connected to the flange of the lower pipe of the extraction pipe assembly, and the other end of the third spring is fixedly connected to the lower surface of the core head; the surface of the core of the second valve core is form-fitted with the lower surface of the flange member of the extraction pipe assembly middle tube, i.e. holes are formed between the flange members of the extraction pipe assembly middle tube, which can be blocked by the second valve core.

Further, be provided with at the junction of extraction tube subassembly upper portion pipe assembly, extraction tube subassembly middle part pipe: the first valve core and the second spring are in the following modes:

the width of the upper surface of the flange member of the middle pipe of the extraction pipe assembly is larger than the thickness of the extraction pipe insertion pipe of the upper pipe assembly of the extraction pipe assembly;

a first accommodating space is formed at the upper part of the flange component of the middle pipe of the extraction pipe assembly, the inner part of the extraction pipe inserting pipe of the upper pipe assembly of the extraction pipe assembly and the lower part of the flange body of the upper pipe assembly of the extraction pipe assembly, and the first valve core and the second spring are arranged in the first accommodating space;

a second spring is arranged on the upper part of the flange component of the middle pipe of the extraction pipe assembly, the second spring is arranged in the extraction pipe insertion pipe of the upper pipe assembly of the extraction pipe assembly, and a first valve core is sleeved on the second spring;

the first valve core comprises a core print and a core rod, wherein the upper surface of the core print is in a cone frustum shape and is gradually increased in radius from top to bottom; the lower surface of the core head is fixedly connected with a core rod, and the sectional area of the lower surface of the core head is larger than that of the core rod; the core rod is inserted into a second spring, namely the second spring surrounds the core rod, one end of the second spring is fixedly connected to the upper surface of the flange member of the middle pipe of the extraction pipe assembly, and the other end of the second spring is fixedly connected to the lower surface of the core head; the surface of the core of the first valve core is matched with the shape of the lower surface of the flange body of the upper pipe assembly of the extraction pipe assembly, namely, holes are formed between the flange bodies of the upper pipe assembly of the extraction pipe assembly, and the first valve core can block the holes.

Further, the connection of the lower pipe of the extraction pipe assembly and the PCR reaction pipe is as follows: the second tube of the lower tube of the extraction tube assembly can be inserted into the PCR reaction tube, namely the outer surface of the second tube of the lower tube of the extraction tube assembly is matched with the inner surface of the PCR reaction tube in shape, and the diameter of the outer surface of the second tube of the lower tube of the extraction tube assembly is equal to that of the inner surface of the PCR reaction tube;

the extraction tube assembly middle pipe and the extraction tube assembly lower pipe are connected in a matched manner: the lower member pipe of the middle pipe of the extraction pipe assembly is inserted into the first pipe of the lower pipe of the extraction pipe assembly, namely the outer surface of the lower member pipe of the middle pipe of the extraction pipe assembly and the inner surface of the first pipe of the lower pipe of the extraction pipe assembly are matched in shape;

wherein, the extraction tube subassembly upper portion pipe assembly is connected with extraction tube subassembly middle part pipe matched with is: the extraction pipe insertion pipe is inserted into the extraction pipe corresponding part of the upper component pipe of the extraction pipe assembly middle pipe, namely the outer surface of the extraction pipe insertion pipe is matched with the inner surface shape of the extraction pipe corresponding part; the piston pipe is inserted into the piston pipe corresponding part of the upper component pipe of the middle pipe of the extraction pipe assembly, namely the outer surface of the piston pipe is matched with the inner surface of the piston pipe corresponding part in shape; the lower surface of the extraction tube insertion tube is in contact with the flange member; the piston tube is just contacted with the bottom of the corresponding part of the piston tube;

the space among the piston tube corresponding part, the piston tube and the piston rod forms a third accommodating space, namely, the space among the bottom of the piston tube corresponding part, the interior of the piston tube and the lower part of the piston rod forms a third accommodating space.

Further, the first accommodation space communicates with the third accommodation space, specifically, an opening is provided in a region of the extraction tube insertion tube opposite to the piston tube, and a height below the opening of the extraction tube insertion tube is not higher than a bottom of the piston tube corresponding portion; an opening is arranged at the bottom of the piston tube relative to the area of the extraction tube insertion tube; correspondingly, the corresponding parts of the piston pipe and the extraction pipe are also provided with openings, namely: the opening of the extraction tube insertion tube, the opening of the extraction tube corresponding portion, the opening of the piston tube corresponding portion, and the opening of the piston tube are communicated with each other, so that the first accommodation space is communicated with the third accommodation space;

the inside of the extraction pipe body forms a fourth accommodation space.

Further, the nucleic acid extraction amplification test tube is characterized in that in an initial state, the second spring and the third spring are in a compressed state, namely the first valve core and the second valve core respectively block a hole formed between the flange members of the middle tube of the extraction tube assembly and a hole formed between the flange bodies of the upper tube assembly of the extraction tube assembly;

in an initial state, reaction enzyme is preset in a PCR reaction tube; reaction liquid is preset in the first accommodating space and the third accommodating space; a lysis solution is pre-disposed in the fourth receiving space.

Further, the first air hole is: be provided with first gas port at the top of extraction tube, the top cap includes: the top cover comprises a top cover body and a top cover convex edge, wherein the top cover convex edge is also provided with a connecting hole corresponding to the first air hole; namely, the opening/closing of the first air hole is realized by rotating the top cover;

or the top of the extraction pipe is provided with a first air hole, and the top cover is not provided with a connecting hole corresponding to the first air hole; the position of the first air hole is lower than the height of the convex edge of the top cover;

or the top of the extraction tube is not provided with a hole, a first air hole is arranged on the convex edge of the top cover, the convex edge of the top cover is made of elastic materials, and the sectional area of the convex edge of the top cover is larger than the opening area of the extraction tube body in the initial state; when the extraction tube is used, the top cover is placed at the upper end of the extraction tube body, and at the moment, the first air hole is formed in the convex edge of the top cover and does not enter the extraction tube body; when first gas pocket needs to be sealed, press the top cap down, with the protruding edge of top cap impressed extraction tube body, first gas pocket is also impressed in the extraction tube body promptly to seal first gas pocket.

Further, the second air hole is: the top of the PCR reaction tube is not provided with a hole, a second tube of the lower tube of the extraction tube assembly is provided with an L-shaped second air hole, namely a vertical hole and a horizontal hole, the horizontal hole is communicated to the outer side of the wall of the second tube, and the vertical hole is communicated with the lower side of the second tube and the horizontal hole; the second tube of the lower tube of the extraction tube assembly is made of elastic material, and the sectional area of the second tube is larger than the opening area of the PVC reaction tube in the initial state;

or, the top of the PCR reaction tube is provided with a second air hole, the end part of the second tube of the lower tube of the extraction tube assembly is matched with the second air hole to be provided with a connecting hole, namely, the opening/closing of the second air hole is realized by rotating the PVC reaction tube.

A working method of a nucleic acid extraction amplification test tube comprises the following steps:

s1, sample adding:

s1-1, opening the top cover, and adding the test sample into the fourth accommodating space;

s1-2, covering the top cover and rotating the top cover to enable the connecting hole of the convex edge of the top cover to correspond to the first air hole so as to ensure that the air hole is open, namely, the fourth accommodating space is kept to be communicated with the outside air;

s1-3, uniformly shaking the upper pipe assembly of the extraction pipe assembly;

s2, imbibition: lifting the piston rod, due to the action of negative pressure: the first valve core moves downwards, the hole formed between the flange bodies of the pipe assembly on the upper part of the extraction pipe assembly is not blocked, and the solution in the fourth accommodating space is sucked into the first accommodating space; the second valve spool will keep a tendency to rise due to the negative pressure, i.e. the holes formed between the flange members of the central tube 3 of the extraction tube assembly are blocked;

s3, pressing the liquid into the PCR reaction tube: the piston rod is pressed down, the third containing space is under high pressure to form a high-pressure cavity, and the solution in the first containing space can enter the lower pipe of the extraction pipe assembly and further enter the PCR reaction pipe; air in the PCR reaction tube is discharged from the second air hole;

s4, finishing the extraction: the piston rod is pushed to the lowest position, and then the first air hole and the second air hole are sealed; all solutions in the first accommodating space and the third accommodating space enter the PCR reaction tube; while the solution in the fourth receiving space remains in the extraction tube.

The beneficial effect of this application lies in:

(1) the basic idea of the application is as follows: the implementation of the automated processing of samples in a closed system with minimal human intervention in multiple steps: by providing successive chambers in the sample collector, each initially separated by a one-way valve; allowing the three reagents to be combined in a specific order according to the treatment protocol without having to open the system during the treatment; the piston rod is pulled and pushed to generate negative pressure and positive pressure so as to open and close the designated one-way valve and realize the mixing and flowing of the liquid; the proper volume is ensured to be obtained by calculating the push-pull distance of the piston rod;

meanwhile, when the piston rod is completely pressed down by the cylinder, all the solution is mixed with the final reagent and flows into the PCR tube. One-way valves seal the respective receiving spaces (respective tubes) in the last step to prevent the reaction from evaporating during PCR; in addition, specific airways are designed in the present invention: when the piston rod is pushed and pulled, the hole is required to be opened to release air pressure, so that the movement is facilitated; after the piston movement is finished, the air channel needs to be blocked when the PCR reaction is started, so that the cross contamination between the reaction of the reactant inside and the external environment is avoided.

That is, the design of the double check valve and the design of the air channel of the application have a synergistic effect and are an integral technical characteristic.

(2) The second invention of the present application is to provide: a working method of a nucleic acid extraction amplification test tube. In fact, the device-method of the present application is extremely relevant; namely, the inventors have obtained the following requirements: a test tube is designed, and the extraction and amplification of nucleic acid can be completed without special training.

Specifically, when the test tube is used, the extraction and amplification of nucleic acid can be completed only by pushing and pulling the piston rod.

On the basis of the using method, the structural requirements are provided, namely, the structural design of 'double one-way valves + piston rod push-pull' is innovatively provided, particularly, the design target can be realized without 'randomly setting the double one-way valves + piston rod push-pull', and the extraction and amplification of nucleic acid are realized; instead, the design target can be finished only by adopting the design of the application on the position of the piston rod and the structural design of the double-one-way valve. That is, the structural design of the present application is original and unique.

(3) The third invention of the present application is to provide: the application also provides a one-step POCT instant nucleic acid detection device (the invention conception is lack of unicity with the nucleic acid extraction amplification test tube, so that the invention is applied for another case), which is suitable for processing the nucleic acid extraction amplification test tube in batch;

in particular, in order to satisfy the operation method of the instant nucleic acid detection device for POCT in one step, it is necessary to provide: the nucleic acid extraction amplification test tube is uniquely designed as follows:

first, the design of the first air vent 16-1: the scheme shown in fig. 5, 6, 7 and 8 is necessary, namely, no hole is arranged on the top of the extraction tube 4-1, a first air hole 16-1 is arranged on the top cover convex edge of the top cover 5, the top cover convex edge is made of elastic material, and the sectional area of the top cover convex edge is larger than the opening area of the extraction tube body 4-1-1 in the initial state; when in use, the top cover is lightly placed at the upper end of the extraction tube body 4-1-1, and at the moment, the convex edge of the top cover is provided with the first air hole 16-1 which does not enter the extraction tube body 4-1-1; when the first air hole 16-1 needs to be sealed, the top cover is pressed down, the convex edge of the top cover is pressed into the extraction tube body 4-1-1, namely, the first air hole 16-1 is also pressed into the extraction tube body 4-1-1, so that the first air hole 16-1 is sealed

Secondly, the design of the second air hole 16-2 needs to adopt the scheme of figures 5, 6, 7 and 8, the top of the PCR reaction tube is not provided with a hole, the second tube 2-2 of the lower tube of the extraction tube component is provided with an L-shaped second air hole 16-2, namely a vertical hole and a horizontal hole, the horizontal hole is communicated with the outer side of the tube wall of the second tube 2-2, and the vertical hole is communicated with the lower side of the second tube 2-2 and the horizontal hole; the second tube 2-2 of the lower tube of the extraction tube assembly is made of elastic material and has a cross-sectional area larger than the opening area of the PVC reaction tube in the initial state. When the device is used, in an initial state, the second pipe 2-2 of the lower pipe of the extraction pipe assembly is inserted into the PVC reaction pipe, at the moment, the height of the horizontal hole of the second air hole 16-2 is larger than the height of the pipe orifice of the PVC reaction pipe, and the second air hole 16-2 is kept open; when the second air hole 16-2 needs to be closed, the second tube 2-2 of the lower tube of the extraction tube assembly is pressed down, so that the second air hole 16-2 is closed by the tube wall of the PVC reaction tube. The design of the first air hole 16-1 and the second air hole 16-2 is coordinated with the working process of the one-step POCT instant nucleic acid detection device.

Thirdly, the upper end of the piston rod 6 is provided with a protruding lower bearing part 6-11, the lower bearing part 6-11 is arranged above the top cover 5, and the projection of the lower bearing part 6-11 and the top cover 5 on the horizontal plane is overlapped, namely, when the piston rod 6 is pushed to descend to a certain distance, the top cover 5 can be pushed to descend.

Fourthly, the fourth invention of the present application is to provide a working method of the instant nucleic acid detecting device of the one-step POCT, which has the following difficulties: how to keep the coordination between the mechanical structure of a nucleic acid extraction amplification test tube and a one-step POCT (point of care testing) instant nucleic acid detection device and a working method.

Drawings

The invention will be further described in detail with reference to examples of embodiments shown in the drawings to which, however, the invention is not restricted.

FIG. 1 is a schematic three-dimensional design of a nucleic acid extraction amplification test tube according to the first embodiment.

Fig. 2 is a schematic view of an upper tube assembly and a middle tube assembly of the extraction tube assembly according to the first embodiment.

Fig. 3 is a combined design of the upper tube assembly of the extraction tube assembly of the first embodiment.

Fig. 4 is an exploded view of the upper tube assembly of the extraction tube assembly of the first embodiment.

FIG. 5 is a schematic view showing the arrangement of the solution in the nucleic acid amplification test tube according to the first embodiment.

FIG. 6 is a schematic diagram showing the nucleic acid extraction amplification test tube in the first embodiment during sample addition and liquid extraction (the third containing space is in a negative pressure state and becomes a negative pressure chamber; the first check valve 15-1 is opened, and the second check valve 15-2 is closed).

FIG. 7 is a schematic view showing the nucleic acid amplification test tube of the first embodiment when a pressure liquid is pushed into the PCR reaction tube (the third containing space is in a high-pressure state and becomes a high-pressure chamber; the first check valve 15-1 is closed and the second check valve 15-2 is opened).

FIG. 8 is a schematic view of the nucleic acid amplification test tube of example I at the end of sample extraction (first check valve 15-1 closed, second check valve 15-2 closed; first inlet hole 16-1 closed, second inlet hole 16-2 closed).

Fig. 9 is a state diagram in which the first air hole corresponds to the connection hole (for ventilation) of the protruding edge of the top cover.

FIG. 10 is a schematic three-dimensional design diagram of the instant nucleic acid detection device for one-step POCT according to the second embodiment.

FIG. 11 is a schematic three-dimensional design diagram of the instant nucleic acid detection device for one-step POCT according to the second embodiment at another angle.

FIG. 12 is a schematic diagram showing the design of the instant nucleic acid detecting device for POCT filled with nucleic acid extraction and amplification tubes in the second embodiment.

Fig. 13 is a schematic design view of the second air hole 16-2 of the second embodiment.

FIG. 14 is a diagram showing the instant nucleic acid detecting device for one-step POCT according to the second embodiment in a state where the device is turned on.

FIG. 15 is a diagram showing the instant nucleic acid detecting apparatus for one-step POCT according to the second embodiment at the end of the examination.

The reference numerals are explained below:

the device comprises a PCR reaction tube 1, an extraction tube assembly lower tube 2, an extraction tube assembly middle tube 3, an extraction tube assembly upper tube assembly 4, a top cover 5, a piston rod 6, a first spring 7, a piston ring 8, a first valve core 9, a second spring 10, a second valve core 11 and a third spring 12;

a first tube 2-1, a second tube 2-2, a flange 2-3;

an upper component pipe 3-1, a lower component pipe 3-2, a corresponding part 3-1-1 of the extraction pipe, and a corresponding part 3-1-2 of the piston pipe;

an extraction tube 4-1, a piston tube 4-2, an extraction tube body 4-1-1, an extraction tube insertion tube 4-1-2, and a flange body 4-1-3;

the outer end of the extraction pipe 4-1 is provided with a protruding upper bearing part 4-10, and the outer end of the extraction pipe 4-1 is provided with a protruding lower bearing part 4-11;

the upper end part of the piston rod 6 is provided with a protruded upper bearing part 6-10, and the upper end part of the piston rod 6 is provided with a protruded lower bearing part 6-11;

a first accommodating space 13-1, a second accommodating space 13-2, a third accommodating space 13-3 and a fourth accommodating space 13-4;

lysis solution 14-1, reaction solution 14-2 and reaction enzyme 14-3;

a first check valve 15-1 and a second check valve 15-2;

a first air hole 16-1, a second air hole 16-2;

a sample 20;

the device comprises a PCR instrument 101, an extraction tube fixing plate 102, a piston

rod fixing plate

103, 2 motors 104, a coupling 105, a

bearing seat

106, 2 circular guide rails 107, a ball screw 108, a screw nut 109 and a linear bearing 110;

an extraction tube sliding placement groove 102-1 and an extraction tube placement section 102-2;

a piston sliding placing groove 103-1 and a piston placing part 103-2.

Detailed Description

In a first embodiment, referring to FIG. 1, a nucleic acid extraction amplification test tube comprises: the device comprises a PCR reaction tube 1, an extraction tube assembly lower tube 2, an extraction tube assembly middle tube 3, an extraction tube assembly upper tube assembly 4, a top cover 5, a piston rod 6, a first spring 7, a piston ring 8, a first valve core 9, a second spring 10, a second valve core 11 and a third spring 12;

wherein, the top of the PCR reaction tube 1 is provided with an opening, and the opening at the top of the PCR reaction tube 1 is connected with the bottom end of the lower tube 2 of the extraction tube component;

the upper pipe component 4 of the extraction pipe component, the middle pipe 3 of the extraction pipe component and the lower pipe 2 of the extraction pipe component are sequentially connected into a whole from top to bottom; the top end of the extraction pipe of the upper pipe component 4 of the extraction pipe component is connected with a top cover 5;

the joint of the lower pipe 2 of the extraction pipe assembly and the middle pipe 3 of the extraction pipe assembly is provided with: a second spool 11, a third spring 12; the joint of the upper pipe component 4 and the middle pipe 3 of the extraction pipe component is provided with: a first spool 9 and a second spring 10.

Wherein the extraction duct assembly lower tube 2 comprises: the pipe comprises a first pipe 2-1 (round pipe) and a second pipe 2-2 (round pipe), wherein a flange 2-3 is arranged at the bottom end of the first pipe 2-1 inwards, and the lower side of the flange is connected with the second pipe 2-2; the first pipe and the second pipe are connected into a whole, the central axes of the first pipe and the second pipe are overlapped, the first pipe 2-1 is arranged at the upper end of the second pipe 2-2, and the radius of the inner surface of the first pipe is larger than that of the outer surface of the second pipe; i.e. the width of the flange-the radius of the inner surface of the first tube-the radius of the outer surface of the second tube.

Wherein the extraction tube assembly middle tube 3 comprises: an upper member pipe 3-1, a lower member pipe 3-2 (circular pipe), and a flange member 3-3;

the upper member tube 3-1 includes: a corresponding part 3-1-1 of the extraction tube and a corresponding part 3-1-2 of the piston tube; the upper surfaces of the corresponding part 3-1-1 of the extraction pipe and the corresponding part 3-1-2 of the piston pipe are flush, and the lower surface of the corresponding part 3-1-1 of the extraction pipe is lower than the lower surface of the corresponding part 3-1-2 of the piston pipe; the bottom of the corresponding part 3-1-1 of the extraction pipe is provided with a flange component 3-3 inwards, and the lower side of the flange component 3-3 is connected with a lower component pipe 3-2;

the bottom of the corresponding part 3-1-2 of the piston pipe is closed.

Wherein the extraction tube assembly upper tube assembly 4 comprises: two vertical parallel tubes: an extraction tube 4-1 (round tube) and a piston tube 4-2 (round tube), wherein the extraction tube 4-1 and the piston tube 4-2 are connected into a whole; the extraction tube 4-1 includes: an extraction pipe body 4-1-1, an extraction pipe insertion pipe 4-1-2 and a flange body 4-1-3, wherein the flange body 4-1-3 is arranged inwards at the bottom of the extraction pipe body 4-1-1, and the extraction pipe insertion pipe 4-1-2 is arranged at the lower part of the flange body 4-1-3;

the piston tube 4-2 is provided with a tube body extending to the piston tube corresponding part 3-1-2 of the middle tube 3 of the extraction tube component;

specifically, the bottom of the extraction tube insertion tube 4-1-2 is lower than the bottom of the piston tube 4-2;

the lower end part of the piston rod 6 is fixedly provided with a piston ring 8, the piston is arranged in the piston tube 4-2, and the piston ring 8 is used for sealing; a push rod is arranged at the upper end part of the piston rod 6; one end of the first spring 7 is connected to the upper surface of the piston tube 4-2, and the other end is connected to the lower surface of the push rod of the piston rod 6 (specifically, to the lower surface of the lower receiving portion of the piston rod 6).

Wherein, the mode that sets up second case 11, third spring 12 in the junction of extraction pipe subassembly lower part pipe 2, extraction pipe subassembly middle part pipe 3 does:

the width of the flange 2-3 of the lower tube 2 of the extraction tube assembly is larger than the thickness of the lower member tube 3-2 of the middle tube 3 of the extraction tube assembly; the width of the flange member 3-3 of the middle tube 3 of the extraction tube assembly is greater than the thickness of the lower member tube 3-2 of the middle tube 3 of the extraction tube assembly; a second accommodating space 13-2 is formed at the upper part of the flange 2-3 of the lower pipe 2 of the extraction pipe assembly, the inner part of the lower member pipe 3-2 of the middle pipe 3 of the extraction pipe assembly and the lower part of the flange member 3-3 of the middle pipe 3 of the extraction pipe assembly, and the third spring 12 and the second valve core 11 are arranged in the second accommodating space 13-2; a third spring 12 is arranged on a flange 2-3 of the lower pipe 2 of the extraction pipe component, the third spring 12 is arranged inside a lower component pipe 3-2 of the middle pipe 3 of the extraction pipe component, and a second valve core 11 is sleeved on the third spring 12;

particularly, the second valve core 11 comprises a core print and a core rod, wherein the upper surface of the core print is in a cone frustum shape and gradually increases in radius from top to bottom; the lower surface of the core head is fixedly connected with a core rod, and the sectional area of the lower surface of the core head is larger than that of the core rod; the core rod is inserted into a third spring 12 (namely the third spring 12 surrounds the core rod), one end of the third spring is fixedly connected to the flange 2-3 of the lower tube 2 of the extraction tube component, and the other end of the third spring is fixedly connected to the lower surface of the core head;

in particular, the surface of the core of the second valve spool 11 is form-fitted to the lower surface of the flange member 3-3 of the extraction pipe assembly central tube 3, i.e. holes are formed between the flange members 3-3 of the extraction pipe assembly central tube 3, which holes can be blocked by the second valve spool 11 (the upper surface of the second valve spool 11 is flush with the upper surface of the flange member 3-3 of the extraction pipe assembly central tube 3).

Wherein, be provided with at the junction of extraction tube subassembly upper portion pipe assembly 4, extraction tube subassembly middle part pipe 3: the first spool 9 and the second spring 10 are formed by:

the width of the upper surface of the flange member 3-3 of the middle tube 3 of the extraction tube assembly is larger than the thickness of the extraction tube insertion tube 4-1-2 of the upper tube assembly 4 of the extraction tube assembly;

a first accommodating space is formed at the upper part of a flange component 3-3 of the middle pipe 3 of the extraction pipe assembly, the inner part of an extraction pipe inserting pipe 4-1-2 of the upper pipe assembly 4 of the extraction pipe assembly and the lower part of a flange body 4-1-3 of the upper pipe assembly 4 of the extraction pipe assembly, and the first valve core 9 and the second spring 10 are arranged in the first accommodating space 13-1;

a second spring 10 is arranged on the upper part of a flange component 3-3 of the middle pipe 3 of the extraction pipe component, the second spring 10 is arranged inside an extraction pipe insertion pipe 4-1-2 of the upper pipe component 4 of the extraction pipe component, and the second spring 10 is sleeved with a first valve core 9;

particularly, the first valve core 9 comprises a core print and a core rod, wherein the upper surface of the core print is in a cone frustum shape and gradually increases in radius from top to bottom; the lower surface of the core head is fixedly connected with a core rod, and the sectional area of the lower surface of the core head is larger than that of the core rod; the core rod is inserted into the second spring 10 (i.e. the second spring 10 surrounds the core rod), one end of the second spring 10 is fixedly connected to the upper surface of the flange member 3-3 of the middle tube 3 of the extraction tube assembly, and the other end is fixedly connected to the lower surface of the core head;

in particular, the surface of the core of the first valve core 9 is matched with the shape of the lower surface of the flange body 4-1-3 of the upper pipe assembly 4 of the extraction pipe assembly, namely, holes are formed between the flange bodies 4-1-3 of the upper pipe assembly 4 of the extraction pipe assembly, and the first valve core 9 can block the holes (the upper surface of the first valve core 9 is flush with the upper surface of the flange body 4-1-3 of the upper pipe assembly 4 of the extraction pipe assembly).

Wherein, the connection of the lower tube 2 of the extraction tube component and the PCR reaction tube 1 is as follows: the second tube 2-2 of the lower tube 2 of the extraction tube assembly can be inserted into the PCR reaction tube 1 (i.e. the outer surface of the second tube 2-2 of the lower tube 2 of the extraction tube assembly is adapted to the inner surface of the PCR reaction tube 1, and the diameter of the outer surface of the second tube 2-2 of the lower tube 2 of the extraction tube assembly is equal to the diameter of the inner surface of the PCR reaction tube 1).

The connection of the middle pipe 3 of the extraction pipe component and the lower pipe 2 of the extraction pipe component is as follows: the lower member tube 3-2 of the middle tube 3 of the extraction tube assembly is inserted into the first tube 2-1 of the lower tube 2 of the extraction tube assembly (i.e. the outer surface of the lower member tube 3-2 of the middle tube 3 of the extraction tube assembly, the inner surface of the first tube 2-1 of the lower tube 2 of the extraction tube assembly are form-fitted).

Wherein, the extraction tube assembly upper portion pipe assembly 4 is connected with extraction tube assembly middle part pipe 3 matched with is:

the extraction tube insertion tube 4-1-2 is inserted into the extraction tube corresponding portion 3-1-1 of the upper member tube 3-1 of the extraction tube assembly middle tube 3; (i.e., the outer surface of the extraction tube insertion tube 4-1-2 is fitted to the inner surface of the extraction tube corresponding portion 3-1-1 in shape)

The piston tube 4-2 is inserted into the piston tube corresponding part 3-1-2 of the upper component tube 3-1 of the middle tube 3 of the extraction tube assembly; (i.e., the outer surface of the piston tube 4-2 is form-fitted to the inner surface of the piston tube counterpart 3-1-2);

specifically, the lower surface of the extraction tube insertion tube 4-1-2 is in contact with the flange member 3-3; the piston tube 4-2 is just contacted with the bottom of the corresponding part 3-1-2 of the piston tube;

the piston tube corresponding part 3-1-2, the piston tube 4-2 and the piston rod 6, and the space among the three parts forms a third accommodating space, namely, the space among the bottom of the piston tube corresponding part 3-1-2, the interior of the piston tube 4-2 and the lower part of the piston rod 6 forms a third accommodating space 13-3;

more specifically, the first accommodation space 13-1 communicates with the third accommodation space 13-3, and specifically, an opening is provided in a region of the extraction tube insertion tube 4-1-2 opposite to the piston tube 4-2, and a height of the bottom of the corresponding piston tube portion 3-1-2 is not higher below the opening of the extraction tube insertion tube 4-1-2; an opening is arranged at the bottom of the piston tube 4-2 relative to the area of the extraction tube insertion tube 4-1-2; correspondingly, the corresponding parts 3-1-2 of the piston pipe and the corresponding parts 3-1-1 of the extraction pipe are also provided with openings, namely: the opening of the extraction tube insertion tube 4-1-2, the opening of the extraction tube corresponding portion 3-1-1, the opening of the piston tube corresponding portion 3-1-2, and the opening of the piston tube 4-2 communicate with each other, so that the first accommodation space 13-1 communicates with the third accommodation space 13-3.

More specifically, the interior of the extraction tube body 4-1-1 forms a fourth accommodation space 13-4;

a nucleic acid extraction amplification test tube, wherein in an initial state, the second spring 10 and the third spring 12 are both in a compressed state, namely, the first valve core 9 and the second valve core 11 respectively block holes formed between the flange members 3-3 of the middle tube 3 of the extraction tube assembly and holes formed between the flange bodies 4-1-3 of the upper tube 4 of the extraction tube assembly;

in an initial state, reaction enzyme 14-3 is preset in the PCR reaction tube 1; reaction liquid 14-2 is preset in the first accommodating space 13-1 and the third accommodating space 13-3; a lysis solution 14-1 is placed in the fourth accommodation space 13-4.

Further, the technical solution (also belonging to the necessary technical features) further comprises: a first air hole 16-1 and a second air hole 16-2; the first air hole 16-1 is used for communicating the fourth accommodating space 13-4 with the outside, and the second air hole 16-2 is used for communicating the PCR reaction tube with the outside.

The first vent 16-1 is variously designed:

as shown in fig. 9, a first air hole 16-1 is formed at the top of the extraction pipe 4-1, and the top cover 5 includes: the top cover comprises a top cover body and a top cover convex edge, wherein the top cover convex edge is also provided with a connecting hole corresponding to the first air hole 16-1; that is, the opening/closing of the first air hole 16-1 is accomplished by rotating the top cover 5.

It can also be: it is also feasible that the top of the extraction pipe 4-1 is provided with a first air hole 16-1, and the top cover 5 is not provided with a connecting hole corresponding to the first air hole 16-1: for example, the first air hole 16-1 is located at a position lower than the height of the protruding edge of the top cover 5, and other blocking objects are used to open and close the first air hole 16-1.

It can also be (schemes of fig. 5, 6, 7, 8): the top of the extraction tube 4-1 is not provided with a hole, a top cover convex edge of the top cover 5 is provided with a first air hole 16-1, the top cover convex edge is made of elastic materials, and the sectional area of the top cover convex edge is larger than the opening area of the extraction tube body 4-1-1 in an initial state;

when in use, the top cover is lightly placed at the upper end of the extraction tube body 4-1-1, and at the moment, the convex edge of the top cover is provided with the first air hole 16-1 which does not enter the extraction tube body 4-1-1; when the first air hole 16-1 needs to be closed, the top cover is pressed down, and the convex edge of the top cover is pressed into the extraction tube body 4-1-1, namely the first air hole 16-1 is also pressed into the extraction tube body 4-1-1, so that the first air hole 16-1 is closed.

The design of the second vent 16-2 is varied as follows:

the arrangements shown in figures 5-8 may be used,

the top of the PCR reaction tube is not provided with a hole, a second tube 2-2 of the lower tube of the extraction tube assembly is provided with an L-shaped second air hole 16-2, namely a vertical hole and a horizontal hole, the horizontal hole is communicated with the outer side of the tube wall of the second tube 2-2, and the vertical hole is communicated with the lower side of the second tube 2-2 and the horizontal hole; the second tube 2-2 of the lower tube of the extraction tube assembly is made of elastic material and has a cross-sectional area larger than the opening area of the PVC reaction tube in the initial state.

Of course, it may be: the top of the PCR reaction tube is provided with a second air hole, the end part of the second tube 2-2 of the lower tube of the extraction tube component is matched with the second air hole to be provided with a connecting hole, namely, the opening/closing of the second air hole is realized by rotating the PVC reaction tube.

A working method of a nucleic acid extraction amplification test tube comprises the following steps,

s1, sample adding:

s1-1, opening the top cover 5, and adding the test sample into the fourth containing space;

s1-3, uniformly shaking the upper pipe assembly 4 of the extraction pipe assembly;

s2, imbibition: lifting the piston rod 6, due to the effect of the negative pressure: the first valve spool 9 is moved downward, the hole formed between the flange bodies 4-1-3 of the upper tube assembly 4 of the extraction tube assembly is not blocked, and the solution in the fourth receiving space is sucked into the first receiving space 13-1; the second valve spool 11 will keep a tendency to rise due to the negative pressure, i.e. the hole formed between the flange members 3-3 of the central tube 3 of the extraction tube assembly is blocked;

s3, pressing the liquid into the PCR reaction tube 1: the piston rod 6 is pressed down, the third containing space 13-3 is under high pressure to form a high-pressure cavity, and the solution in the first containing space 13-1 can enter the lower pipe 2 of the extraction pipe assembly and further enter the PCR reaction pipe 1; air in the PCR reaction tube 1 is discharged from the second air hole 16-2;

s4, finishing the extraction: the piston rod 6 is pushed to the lowest position, and then the first air hole and the second air hole are sealed; the solutions in the first containing space and the third containing space all enter the PCR reaction tube 1; while the solution in the fourth receiving space remains in the extraction duct 4-1.

Specifically, 150ul of lysis solution, 30ul of reaction solution and PCR reaction enzyme dry powder are preset in a nucleic acid extraction amplification test tube; opening the top cover, adding a saliva sample to be detected into the lysate, covering the top cover, and keeping the first air hole 16-1 and the second air hole 16-2 in an open state, namely keeping the air passages of the fourth accommodating space (the upper tube component 4 of the extraction tube component) and the lower PCR reaction tube 1 in an open state; then, shaking the homogeneous lysate;

then, the piston rod is pulled up, 10ul of extracting solution is extracted and mixed with the reaction solution evenly;

and thirdly, pressing down the piston rod, adding 40ul of mixed liquor into the reaction enzyme for mixing to perform PCR reaction, closing the upper air passage and the lower air passage (as shown in figure 8), sealing the first air hole and the second air hole, namely closing the top air passage and the lower PCR tube air passage, and performing PCR reaction.

The second embodiment aims to realize full automation of the process from sample collection to qPCR result output;

instant nucleic acid detection device of one-step POCT includes: the device comprises a PCR instrument 101, an extraction tube fixing plate 102, a piston

rod fixing plate

103, 2 motors 104, a coupling 105, a

bearing seat

106, 2 circular guide rails 107, a ball screw 108, a screw nut 109, a linear bearing 110 and a PCR reaction tube fixing seat;

the upper surface of the PCR instrument 101 is provided with a plurality of PCR reaction tube fixing seats, and the PCR reaction tube fixing seats are matched with the shapes of the PCR reaction tubes;

the output shaft of 2 motors is connected with a coupling 105, and the coupling 105 is connected with a ball screw 108;

the 2 circular guide rails 107 are arranged in parallel, and the axis direction is vertical;

the extraction tube fixing plate 102 and the piston rod fixing plate 103 are arranged in parallel up and down, and the 2 circular guide rails 107 penetrate through the extraction tube fixing plate 102 and the piston rod fixing plate 103, so that the extraction tube fixing plate 102 and the piston rod fixing plate 103 move up and down along the 2 circular guide rails 107; linear bearings 110 matched with the circular guide rails are arranged on the extraction tube fixing plate 102 and the piston rod fixing plate 103;

the piston rod fixing plate 103 and the extraction tube fixing plate 102 are both fixedly provided with a screw rod nut 109, the 2 screw rod nuts 109 respectively correspond to the 2 screw rods 108, namely 1 motor 104 rotates to drive the screw rod 108 connected with the motor to rotate, and further drive the extraction tube fixing plate 102 to move up and down; the other 1 motor 104 rotates to drive the screw rod 108 connected with the motor to rotate, and further drives the piston rod fixing plate 103 to move up and down;

a fixing plate is fixedly disposed on the

PCR instrument

101, and 2 bearing seats 106 are disposed on the fixing plate, and the 2 ball screws 108 are disposed in the bearing seats 106 to bear the weight of the ball screws 108.

The PCR instrument 101 is used for detecting RNA (ribonucleic acid) of a virus by real-time fluorescent RT-PCR. The detection principle is to design primers and probes by utilizing the specificity of nucleic acid sequences in virus bases to distinguish from other viruses. When the nucleic acid sequence of the specific region of the virus is in targeted matching with the designed primer and probe, the PCR amplification result of the specimen to be detected can be judged to be positive, and the virus to be detected exists in the specimen.

Wherein, the front end of the extraction tube fixing plate 102 is provided with a plurality of horizontal extraction tube sliding placing grooves 102-1, and the two adjacent horizontal extraction tube sliding placing grooves 102-1 are extraction tube placing parts 102-2;

the outer end of the extraction pipe 4-1 of the upper pipe component 4 of the extraction pipe component is provided with a protruding upper bearing part 4-10 and a protruding lower bearing part 4-11, and the upper bearing part and the lower bearing part are in arc shapes (or in other shapes);

the extraction tube 4-1 of the upper tube component 4 of the extraction tube component can be inserted into the extraction tube sliding placement groove 102-1, the upper bearing part 4-10 and the lower bearing part 4-11 which are arranged at the outer end of the extraction tube 4-1 of the upper tube component 4 of the extraction tube component are clamped on the extraction tube placement part 102-2, namely when the extraction tube fixing plate 102 moves up and down, the nucleic acid extraction amplification test tube can be synchronously driven to move up and down;

wherein, the front end of the piston rod fixing plate 103 is provided with a plurality of horizontal piston sliding placing grooves 103-1, and the two adjacent horizontal piston sliding placing grooves 103-1 are piston placing parts 103-2;

the upper end part of the piston rod 6 is provided with a protruding upper bearing part 6-10 and a protruding lower bearing part 6-11, and the upper bearing part and the lower bearing part are circular (of course, the upper bearing part and the lower bearing part can be in other shapes);

the piston rod 6 can be inserted into the piston sliding placement groove 103-1, and the bearing part 6-10 of the piston rod 6 is clamped on the piston placement part 103-2, namely the piston rod 6 is synchronously driven to move up and down when the piston rod fixing plate 103 moves up and down;

in order to cooperate with the instant nucleic acid detection device of the one-step POCT of the second embodiment, the nucleic acid extraction and amplification test tube of the first embodiment needs to be modified as follows:

first, the design of the first air vent 16-1: the scheme shown in fig. 5, 6, 7 and 8 is necessary, namely, no hole is arranged on the top of the extraction tube 4-1, a first air hole 16-1 is arranged on the top cover convex edge of the top cover 5, the top cover convex edge is made of elastic material, and the sectional area of the top cover convex edge is larger than the opening area of the extraction tube body 4-1-1 in the initial state;

when in use, the top cover is lightly placed at the upper end of the extraction tube body 4-1-1, and at the moment, the convex edge of the top cover is provided with the first air hole 16-1 which does not enter the extraction tube body 4-1-1; when the first air hole 16-1 needs to be sealed, the top cover is pressed down, the convex edge of the top cover is pressed into the extraction tube body 4-1-1, namely, the first air hole 16-1 is also pressed into the extraction tube body 4-1-1, so that the first air hole 16-1 is sealed

Secondly, the design of the second air hole 16-2 needs to adopt the scheme of figures 5, 6, 7 and 8, the top of the PCR reaction tube is not provided with a hole, the second tube 2-2 of the lower tube of the extraction tube component is provided with an L-shaped second air hole 16-2, namely a vertical hole and a horizontal hole, the horizontal hole is communicated with the outer side of the tube wall of the second tube 2-2, and the vertical hole is communicated with the lower side of the second tube 2-2 and the horizontal hole; the second tube 2-2 of the lower tube of the extraction tube assembly is made of elastic material and has a cross-sectional area larger than the opening area of the PVC reaction tube in the initial state.

When the device is used, in an initial state, the second pipe 2-2 of the lower pipe of the extraction pipe assembly is inserted into the PVC reaction pipe, at the moment, the height of the horizontal hole of the second air hole 16-2 is larger than the height of the pipe orifice of the PVC reaction pipe, and the second air hole 16-2 is kept open;

when the second air hole 16-2 needs to be closed, the second tube 2-2 of the lower tube of the extraction tube assembly is pressed down, so that the second air hole 16-2 is closed by the tube wall of the PVC reaction tube.

The design of the first air hole 16-1 and the second air hole 16-2 is coordinated with the working process of the one-step POCT instant nucleic acid detection device.

The working method of the one-step POCT instant nucleic acid detection device comprises the following steps:

firstly, presetting lysis solution, reaction solution and PCR reaction enzyme dry powder in an instant nucleic acid extraction amplification test tube;

secondly, opening the top cover, adding the saliva sample to be detected into the lysate, and covering the top cover, wherein the first air hole and the second air hole are kept open;

thirdly, loading: after the lysate-sample liquid of the nucleic acid extraction amplification test tube is uniformly shaken, the heights of the extraction tube fixing plate 102 and the piston rod fixing plate 103 are adjusted so as to place the nucleic acid extraction amplification test tube on the one-step POCT instant nucleic acid detection device;

fourthly, positioning: the piston rod fixing plate 103 and the extraction tube fixing plate 102 are descended at the same speed until the PCR reaction tube is fixed in the PCR reaction tube fixing seat;

and fifthly, pulling up the piston rod, extracting 10ul of extracting solution and uniformly mixing with the reaction solution: the extraction tube fixing plate 102 is stationary and the piston rod fixing plate 103 is raised.

Sixthly, pressing down the piston rod, adding 40ul of mixed solution into the reaction enzyme for mixing, and closing the upper air channel (the first air hole): the extraction tube fixing plate 102 is not moved, the piston rod fixing plate 103 descends until the piston rod fixing plate 103 drives the piston rod 6 to completely press the top cover 5 into the upper tube assembly 4, the first air hole is closed, and the piston rod also reaches the deepest part 3-1-2 corresponding to the piston tube;

seventh, close the lower airway (second vent): the extraction tube fixing plate 102 descends and the piston rod fixing plate 103 descends at the same speed until the extraction tube fixing plate 102 completely presses the second tube 2-2 of the lower tube of the extraction tube assembly into the PCR reaction tube 1, the second air hole is blocked and closed, and the extraction tube fixing plate 102 and the piston rod fixing plate 103 do not descend any more;

and eighth, carrying out PCR reaction.

The above-mentioned embodiments are only for convenience of description, and are not intended to limit the present invention in any way, and those skilled in the art will understand that the technical features of the present invention can be modified or changed by other equivalent embodiments without departing from the scope of the present invention.

Claims

1. A nucleic acid extraction amplification test tube comprising: the device comprises a PCR reaction tube, an extraction tube assembly lower tube, an extraction tube assembly middle tube, an extraction tube assembly upper tube, a top cover, a piston rod, a piston ring, a first valve core, a second spring, a second valve core and a third spring;

the joint of the lower pipe of the extraction pipe assembly and the middle pipe of the extraction pipe assembly is provided with: a second valve core and a third spring; the junction of the upper pipe component of the extraction pipe component and the middle pipe of the extraction pipe component is provided with: a first valve core, a second spring;

the second valve core comprises a core print and a core rod, wherein the upper surface of the core print is in a cone frustum shape and is gradually increased in radius from top to bottom; the lower surface of the core head is fixedly connected with a core rod, and the sectional area of the lower surface of the core head is larger than that of the core rod; the core bar is inserted into a third spring, namely the third spring surrounds the core bar, one end of the third spring is fixedly connected to the flange of the lower pipe of the extraction pipe assembly, and the other end of the third spring is fixedly connected to the lower surface of the core head; the surface of the core print of the second valve core is matched with the shape of the lower surface of the flange component of the middle pipe of the extraction pipe component, namely, holes are formed between the flange components of the middle pipe of the extraction pipe component, and the second valve core can block the holes;

the first valve core comprises a core print and a core rod, wherein the upper surface of the core print is in a cone frustum shape and is gradually increased in radius from top to bottom; the lower surface of the core head is fixedly connected with a core rod, and the sectional area of the lower surface of the core head is larger than that of the core rod; the core rod is inserted into a second spring, namely the second spring surrounds the core rod, one end of the second spring is fixedly connected to the upper surface of the flange member of the middle pipe of the extraction pipe assembly, and the other end of the second spring is fixedly connected to the lower surface of the core head; the surface of the core print of the first valve core is matched with the shape of the lower surface of the flange body of the upper pipe assembly of the extraction pipe assembly, namely, holes are formed between the flange bodies of the upper pipe assembly of the extraction pipe assembly, and the first valve core can block the holes;

wherein, the extraction tube subassembly middle part pipe includes: an upper member tube, a lower member tube, a flange member; the upper member tube includes: an extraction tube corresponding portion and a piston tube corresponding portion;

the space among the corresponding part of the piston pipe, the piston pipe and the piston rod forms a third accommodating space, namely the space among the bottom of the corresponding part of the piston pipe, the interior of the piston pipe and the lower part of the piston rod forms a third accommodating space;

the first accommodating space is communicated with the third accommodating space, specifically, an opening is arranged in the area of the extraction pipe insertion pipe relative to the piston pipe, and the lower part of the opening of the extraction pipe insertion pipe is not higher than the height of the bottom of the corresponding part of the piston pipe; an opening is arranged at the bottom of the piston tube relative to the area of the extraction tube insertion tube; correspondingly, the corresponding parts of the piston pipe and the extraction pipe are also provided with openings, namely: the opening of the extraction tube insertion tube, the opening of the extraction tube corresponding portion, the opening of the piston tube corresponding portion, and the opening of the piston tube communicate with each other, so that the first accommodation space communicates with the third accommodation space.

2. The tube for nucleic acid extraction and amplification test of claim 1, wherein the top of the PCR tube is provided with an opening, and the opening at the top of the PCR tube is connected to the bottom end of the lower tube of the extraction tube assembly.

3. The nucleic acid extraction and amplification test tube of claim 1, wherein the lower tube of the extraction tube assembly comprises: the pipe comprises a first pipe and a second pipe, wherein a flange is arranged at the bottom end of the first pipe inwards, and the lower side of the flange is connected with the second pipe; the first pipe and the second pipe are connected into a whole, the central axes of the first pipe and the second pipe are overlapped, the first pipe is arranged at the upper end of the second pipe, and the radius of the inner surface of the first pipe is larger than that of the inner surface of the second pipe; i.e. the width of the flange = radius of the inner surface of the first tube-radius of the inner surface of the second tube;

the upper surfaces of the corresponding parts of the extraction pipe and the piston pipe are flush, and the lower surface of the corresponding part of the extraction pipe is lower than the lower surface of the corresponding part of the piston pipe; the bottom of the corresponding part of the extraction pipe is provided with a flange component inwards, and the lower side of the flange component is connected with a lower component pipe;

the bottom of the corresponding part of the piston pipe is closed.

4. The nucleic acid extraction and amplification test tube of claim 1, wherein the upper tube assembly of the extraction tube assembly comprises: two vertical parallel tubes: the extraction pipe and the piston pipe are connected into a whole; the extraction tube comprises: the extraction pipe comprises an extraction pipe body, an extraction pipe insertion pipe and a flange body, wherein the flange body is arranged inwards at the bottom of the extraction pipe body, and the extraction pipe insertion pipe is arranged at the lower part of the flange body; the piston tube is provided with a tube body extending to the piston tube corresponding part of the middle tube of the extraction tube assembly; the bottom of the extraction tube insertion tube is lower than the bottom of the piston tube.

5. The nucleic acid extraction and amplification test tube of claim 1, further comprising: the lower end part of the piston rod is fixedly provided with a piston ring, and the piston ring is used for sealing; a push rod is arranged at the upper end part of the piston rod; one end of the first spring is connected with the upper surface of the piston tube, and the other end of the first spring is connected with the lower surface of the push rod of the piston rod.

6. The nucleic acid amplification test tube of claim 1, wherein the second valve element and the third spring are disposed at the connection between the lower tube of the extraction tube assembly and the middle tube of the extraction tube assembly in a manner that:

7. The nucleic acid amplification test tube according to claim 1, wherein at the junction of the upper tube assembly and the middle tube assembly of the extraction tube assembly: the first valve core and the second spring are in the following modes:

the connection of the lower pipe of the extraction pipe component and the PCR reaction pipe is as follows: the second tube of the lower tube of the extraction tube assembly can be inserted into the PCR reaction tube, namely the outer surface of the second tube of the lower tube of the extraction tube assembly is matched with the inner surface of the PCR reaction tube in shape, and the diameter of the outer surface of the second tube of the lower tube of the extraction tube assembly is equal to that of the inner surface of the PCR reaction tube;

a fourth accommodating space is formed inside the extraction pipe body;

in the initial state of the nucleic acid extraction amplification test tube, the second spring and the third spring are in a compressed state, namely the first valve core and the second valve core respectively block a hole formed between flange members of the middle tube of the extraction tube assembly and a hole formed between flange bodies of the upper tube assembly of the extraction tube assembly;

8. The nucleic acid extraction and amplification test tube of claim 1, wherein the first pore is: be provided with first gas port at the top of extraction tube, the top cap includes: the top cover comprises a top cover body and a top cover convex edge, wherein the top cover convex edge is also provided with a connecting hole corresponding to the first air hole; namely, the opening/closing of the first air hole is realized by rotating the top cover;

or the top of the extraction tube is not provided with a hole, a first air hole is arranged on the convex edge of the top cover, the convex edge of the top cover is made of elastic materials, and the sectional area of the convex edge of the top cover is larger than the opening area of the extraction tube body in the initial state; when the extraction tube is used, the top cover is placed at the upper end of the extraction tube body, and at the moment, the first air hole is formed in the convex edge of the top cover and does not enter the extraction tube body; when the first air hole needs to be sealed, the top cover is pressed down, and the convex edge of the top cover is pressed into the extraction tube body, namely the first air hole is also pressed into the extraction tube body, so that the first air hole is sealed;

the second air hole is as follows: the top of the PCR reaction tube is not provided with a hole, a second tube of the lower tube of the extraction tube assembly is provided with an L-shaped second air hole, namely a vertical hole and a horizontal hole, the horizontal hole is communicated to the outer side of the wall of the second tube, and the vertical hole is communicated with the lower side of the second tube and the horizontal hole; the second tube of the lower tube of the extraction tube assembly is made of elastic material, and the sectional area of the second tube is larger than the opening area of the PCR reaction tube in the initial state;

or the top of the PCR reaction tube is provided with a second air hole, the end part of the second tube of the lower tube of the extraction tube assembly is matched with the second air hole to be provided with a connecting hole, namely, the opening/closing of the second air hole is realized by rotating the PCR reaction tube.

9. A working method of a nucleic acid extraction amplification test tube, which is the nucleic acid extraction amplification test tube according to any one of claims 1 to 8, comprising the steps of:

s1, sample adding: