CN112760208A - Constant-temperature PCR reaction tube and use method thereof - Google Patents
Constant-temperature PCR reaction tube and use method thereof Download PDFInfo
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- CN112760208A CN112760208A CN202011641800.3A CN202011641800A CN112760208A CN 112760208 A CN112760208 A CN 112760208A CN 202011641800 A CN202011641800 A CN 202011641800A CN 112760208 A CN112760208 A CN 112760208A
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- 238000000034 method Methods 0.000 title claims description 9
- 239000007788 liquid Substances 0.000 claims abstract description 164
- 238000001514 detection method Methods 0.000 claims abstract description 45
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 23
- 238000007789 sealing Methods 0.000 claims abstract description 20
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 7
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 7
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 7
- 239000002131 composite material Substances 0.000 claims abstract description 6
- 239000012528 membrane Substances 0.000 claims abstract description 5
- 238000000926 separation method Methods 0.000 claims description 5
- 230000009471 action Effects 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 238000010438 heat treatment Methods 0.000 claims description 2
- 230000008569 process Effects 0.000 claims description 2
- 230000000694 effects Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 1
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- C12M23/00—Constructional details, e.g. recesses, hinges
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
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Abstract
The invention relates to a constant-temperature PCR reaction tube, which comprises a liquid storage tube cover, a liquid storage tube and a reaction tube which are connected in sequence; the liquid storage pipe cover comprises a cover plate, a pipe cap and a sealing ring; the liquid storage pipe comprises a liquid feeding opening, an external thread, a liquid storage bin, a clamping hook and a liquid leakage hole; the reaction tube comprises a guide groove, a positioning groove, a liquid adding bin, a low-temperature detection bin, an alternating current channel, a high-temperature bin and a composite membrane. The constant-temperature PCR reaction tube can lead the sample to be in continuous high-temperature and low-temperature self-circulation, the reaction is quicker, and simultaneously, the operations of reagent storage, swab collection, uniform mixing, sample receiving, nucleic acid detection and the like are all completed in the reaction tube, so the operation is simpler and more convenient.
Description
Technical Field
The invention relates to the technical field of medical detection, in particular to a constant-temperature PCR reaction tube and a using method thereof.
Background
At present, when the reaction tube on the market is subjected to temperature change detection, a complex thermal cycler is often required, the detection cost is high, and reagent storage, swab collection, uniform mixing and sample receiving are mostly subjected to step-by-step operation, so that the operation is complex and not convenient enough, and a professional organization or a professional person is required to operate.
Therefore, improvement on the basis of the traditional PCR reaction tube is urgently needed, so that a complex thermal cycler is not needed, the reagent can complete thermal cycle under the thermodynamic action, and multi-flux rapid detection is realized; meanwhile, the swab collection and the sample receiving are synchronously completed, and the hand-held PCR is matched for use, so that the nucleic acid detection is more convenient.
Disclosure of Invention
The invention aims to provide a constant-temperature PCR reaction tube and a using method thereof, aiming at the defects in the prior art.
In order to achieve the purpose, the invention adopts the technical scheme that:
the invention provides a constant temperature PCR reaction tube, which comprises a liquid storage tube cover, a liquid storage tube and a reaction tube which are connected in sequence;
wherein, the top end of the liquid storage tube cover is embedded with a cover plate, the upper part is provided with a tube cap, and the lower part is provided with a sealing ring for sealing;
the liquid storage pipe is hollow to form a liquid storage bin, a liquid feeding opening is formed in the top of the liquid storage pipe, the aperture of the lower portion of the liquid storage pipe is narrowed, a pair of clamping hooks is arranged on the outer wall of the liquid storage pipe, the bottom of the liquid storage pipe is conical, and a liquid leakage hole with two through sides is formed in the center of the liquid storage;
a guide groove matched with the width of the clamping hook is arranged on the inner wall of the top of the reaction tube, a digital positioning mark is arranged on the outer wall of the reaction tube, a positioning groove matched with the height of the clamping hook is formed in the side wall below the guide groove and the digital positioning mark, a liquid feeding bin is arranged at the upper part of the reaction tube, a low-temperature detection bin is arranged at the middle part of the reaction tube, and a high-temperature bin communicated with the low-temperature detection bin through an alternating current channel is arranged at; composite membranes are sealed on the front side and the rear side of the reaction tube.
Preferably, the cover plate is colored with information representing the stored liquid, or/and
the surface is printed with a two-dimensional code or a text description indicating the information of the stored liquid.
Preferably, the outer wall of the pipe cap is provided with a plurality of cylindrical protrusions with anti-skidding effect, and the inner wall of the pipe cap is provided with internal threads.
Preferably, the liquid feeding port is a bell mouth with a small inside and a large outside (namely, the end close to the liquid storage bin is small, and the end communicated with the outside is large) and plays a guiding role, and an external thread matched with the internal thread is arranged on the outer wall of the bell mouth.
Preferably, a sealing edge for sealing is arranged at the joint of the lower part and the bottom of the liquid storage pipe.
Preferably, the digital positioning identification comprises "1", "0" and "2"; the position of the digital positioning mark '0' corresponds to the position of the guide groove, and the digital positioning mark '1' and the digital positioning mark '2' are respectively positioned at two sides of the digital positioning mark '0'.
Preferably, barbs for stopping positions are arranged at trisection points of the positioning grooves.
Preferably, the filling bin comprises: the liquid adding groove is matched with the bottom of the liquid storage pipe in shape, the liquid channel is communicated with the side wall of the liquid adding groove, and the liquid separating channel is formed by extending from the bottom of the liquid channel to two sides; the liquid channel and the digital positioning mark 2 are positioned on the same vertical surface; the liquid separation channel is communicated with the low-temperature detection bin.
Preferably, the low-temperature detection bin is semicircular or semi-elliptical.
Preferably, the low temperature detection bin is semi-elliptical, and the arc surface is a reflecting surface to play a role in reflecting light.
Preferably, the high temperature bin is triangular.
Preferably, the angle between the exchange channel and the vertical direction is between 15 ° and 85 °, the width is between 0.5mm and 3mm, and the length is between 2mm and 10 mm; the volume enclosed by the exchange channels formed by every two seals is more than 10% of the volume of the high-temperature chamber and less than 80% of the volume of the high-temperature chamber.
Preferably, the volumes of the low-temperature detection chamber and the high-temperature chamber are both between 15 microliters and 50 microliters, and the volume of the low-temperature detection chamber is 10% -100% of the volume of the high-temperature chamber.
The second aspect of the present invention provides a method for using the isothermal PCR reaction tube, comprising the steps of:
s1, the liquid storage tube and the reaction tube are connected in a rotating and sealing manner through the clamping hook and the positioning groove, and the clamping hook corresponds to the digital positioning mark '0';
s2, clockwise rotating the liquid storage pipe to enable the hook to correspond to the digital positioning mark '1', placing a liquid reagent or a freeze-dried reagent in the liquid storage bin, and hermetically connecting the liquid storage pipe cover and the liquid storage pipe through the internal thread and the external thread;
s3, collecting swabs; unscrewing the liquid storage tube cover from the liquid storage tube, and placing the swab into the liquid storage tube; if the reagent is lyophilized in step S2, adding an appropriate amount of water into the liquid storage tube; screwing the liquid storage tube cover on the liquid storage tube to be tightly sealed, and forcibly shaking the liquid storage tube to uniformly mix the sample and the reagent;
s4, rotating the liquid storage tube anticlockwise to enable the hook to correspond to the digital positioning mark '2', wherein the liquid leakage hole is communicated with the liquid channel, namely the liquid storage tube is communicated with the reaction tube; forcibly shaking the liquid storage tube, or releasing the uniformly mixed sample and reagent in the liquid storage tube to the low-temperature detection bin, the alternating-current channel and the high-temperature bin through the liquid leakage hole and the liquid separation channel in sequence by means of a centrifugal machine; rotating the liquid storage pipe (B) clockwise to enable the clamping hook (B-4) to correspond to the digital positioning mark '1' so as to close the liquid channel (A-3-2);
s5, inserting the constant-temperature PCR reaction tube into a nucleic acid detector for constant-temperature PCR detection:
simultaneously heating the low-temperature detection bin and the high-temperature bin to 50 ℃ and maintaining for 2 minutes;
after the low-temperature detection chamber is heated to 95 ℃ and maintained for 1 minute, the temperature is reduced to 55 ℃ within 1 minute, and meanwhile, the high-temperature chamber is heated to 95 ℃ and maintained for 2 minutes;
the low-temperature detection chamber is maintained at 55 ℃, and the high-temperature chamber is maintained at 95 ℃ for 1 minute and at 92 ℃ for 1 minute in a circulating manner; in the process, the sample and the reagent circulate between the low-temperature detection bin and the high-temperature bin through the alternating current channel under the thermodynamic action, and at the moment, signal collection can be carried out in the low-temperature detection bin.
By adopting the technical scheme, compared with the prior art, the invention has the following technical effects:
the constant-temperature PCR reaction tube can lead the sample to be in continuous high-temperature and low-temperature self-circulation, the reaction is quicker, and simultaneously, the operations of reagent storage, swab collection, uniform mixing, sample receiving, nucleic acid detection and the like are all completed in the reaction tube, so the operation is simpler and more convenient.
Drawings
FIG. 1 is a schematic structural view of a constant temperature PCR reaction tube according to the present invention;
FIG. 2 is a schematic view of the structure of a reaction tube according to the present invention;
FIG. 3 is a sectional view showing the structure of a reaction tube according to the present invention;
FIG. 4 is a schematic view of a liquid storage tube according to the present invention;
FIG. 5 is a schematic view of a liquid storage cap according to the present invention;
wherein the reference numerals include: a reaction tube A; a guide groove A-1; a positioning groove A-2; a liquid adding bin A-3; a liquid adding groove A-3-1; a liquid channel A-3-2; a liquid separation channel A-3-3; a low-temperature detection bin A-4; an alternating current channel A-5; a high-temperature bin A-6; composite film A-7; a liquid storage pipe B; a liquid adding port B-1; external threads B-2; a liquid storage bin B-3; a hook B-4; sealing edge B-5; a weep hole B-6; a liquid storage tube cover C; a cover plate C-1; a nut C-2; and a sealing ring C-3.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict.
The invention is further described with reference to the following drawings and specific examples, which are not intended to be limiting.
Examples
This embodiment provides a constant temperature PCR reaction tube, as shown in fig. 1, including a liquid storage tube cover C, a liquid storage tube B and a reaction tube a connected in sequence;
as shown in fig. 5, a cover plate C-1 is embedded at the top end of the liquid storage tube cap C, a tube cap C-2 is arranged at the upper part, and a sealing ring C-3 for sealing is arranged at the lower part;
as shown in fig. 4, the liquid storage tube B is hollow to form a liquid storage bin B-3, the top is provided with a liquid feeding port B-1, the aperture of the lower part is narrowed, the outer wall is provided with a pair of hooks B-4, the bottom is conical, and the center is provided with a liquid leakage hole B-6 with two through sides;
as shown in fig. 2, a guide groove a-1 which is matched with the width of the hook B-4 is arranged on the inner wall of the top of the reaction tube a, a digital positioning mark is arranged on the outer wall, a positioning groove a-2 which is matched with the height of the hook B-4 is arranged on the guide groove a-1 and the side wall below the digital positioning mark, a liquid feeding bin a-3 is arranged at the upper part, a low temperature detection bin a-4 is arranged at the middle part, and a high temperature bin a-6 which is communicated with the low temperature detection bin a-4 through an alternating current channel a-5 is arranged at the lower part; and the front side and the rear side of the reaction tube A are sealed with composite membranes A-7.
Preferably, the cover plate C-1 is colored to represent the information of the stored liquid, or/and
the surface is printed with a two-dimensional code or a text description indicating the information of the stored liquid.
Preferably, the outer wall of the pipe cap C-2 is provided with a plurality of cylindrical protrusions with anti-skidding function, and the inner wall of the pipe cap C-2 is provided with internal threads.
Preferably, the liquid filling opening B-1 is a bell mouth with a small inner part and a large outer part for guiding, and an external thread B-2 matched with the internal thread is arranged on the outer wall of the bell mouth.
Preferably, a sealing edge B-5 for sealing is arranged at the joint of the lower part and the bottom of the liquid storage tube B.
Preferably, the digital positioning identification comprises "1", "0" and "2"; the position of the digital positioning mark '0' corresponds to the position of the guide groove A-1, and the digital positioning mark '1' and the digital positioning mark '2' are respectively positioned at two sides of the digital positioning mark '0'.
Preferably, barbs for stopping positions are arranged at trisection points of the positioning grooves A-2.
As shown in fig. 3, the liquid adding bin a-3 includes: a liquid adding groove A-3-1 matched with the bottom of the liquid storage pipe B in shape, a liquid channel A-3-2 communicated with the side wall of the liquid adding groove A-3-1 and a liquid separating channel A-3-3 formed by extending from the bottom of the liquid channel A-3-2 to two sides; the liquid channel A-3-2 and the digital positioning mark 2 are positioned on the same vertical surface; the liquid separation channel A-3-3 is communicated with the low-temperature detection bin A-4.
Preferably, the low-temperature detection chamber A-4 is a semi-ellipse, and the arc surface is a reflecting surface to play a role in reflecting light.
Preferably, the high-temperature bin A-6 is triangular.
As can be seen in the figure, the liquid channel a-3-2 extends downwards from two sides of the liquid feeding groove a-3-1 until the liquid channel a-3-2 is combined into a whole, the bottom of the liquid channel a-3-2 extends towards two sides to form 2 liquid separating channels a-3-3 on the left and the right respectively, and the liquid separating channels a-3-3 are grouped in pairs and converge at the upper ends of the low-temperature detection bins a-4 on two sides after extending respectively; the two groups of the liquid separating channel A-3-3, the low-temperature detection bin A-4, the alternating current channel A-5 and the high-temperature bin A-6 are symmetrical left and right and are symmetrical front and back respectively, and a closed cavity is formed by sealing the composite membrane A-7.
Preferably, the angle between the exchange channel A-5 and the vertical direction is between 15 degrees and 85 degrees, the width is between 0.5mm and 3mm, and the length is between 2mm and 10 mm; the volume enclosed by the exchange channel A-5 formed by every two seals is more than 10% of the volume of the high-temperature chamber A-6 and less than 80% of the volume of the high-temperature chamber A-6.
Preferably, the volumes of the low-temperature detection chamber A-4 and the high-temperature chamber A-6 are both between 15 microliters and 50 microliters, and the volume of the low-temperature detection chamber A-4 is 10% -100% of the volume of the high-temperature chamber A-6.
This example further provides a method for using the isothermal PCR reaction tube, comprising the steps of:
s1, the liquid storage tube B and the reaction tube A are connected in a rotating and sealing mode through the hook B-4 and the positioning groove A-2, and the hook B-4 corresponds to the digital positioning mark '0';
s2, clockwise rotating the liquid storage tube B to enable the hook B-3 to correspond to the digital positioning mark '1', placing a liquid reagent or a freeze-dried reagent in the liquid storage bin B-2, and connecting the liquid storage tube cover C and the liquid storage tube B through the internal thread and the external thread B-2 in a threaded sealing manner;
s3, collecting swabs; unscrewing the liquid storage tube cover C from the liquid storage tube B, and putting the swab into the liquid storage tube B; if the reagent is lyophilized in step S2, adding a proper amount of water into the liquid storage tube B; screwing the liquid storage tube cover C on the liquid storage tube B tightly, and shaking the liquid storage tube B forcibly to uniformly mix the sample and the reagent;
s4, rotating the liquid storage tube B counterclockwise to enable the hook B-4 to correspond to the digital positioning mark '2', wherein the liquid leakage hole B-6 is communicated with the liquid channel A-3-2, namely the liquid storage tube B is communicated with the reaction tube A; forcibly shaking the liquid storage tube B, or releasing the uniformly mixed sample and reagent in the liquid storage tube B into the low-temperature detection bin A-4, the alternating-current channel A-5 and the high-temperature bin A-6 through the liquid channel A-3-2 and the liquid distribution channel A-3-3 in sequence by means of a centrifuge through the liquid leakage hole B-6; rotating the liquid storage pipe (B) clockwise to enable the clamping hook (B-4) to correspond to the digital positioning mark '1' so as to close the liquid channel (A-3-2);
s5, inserting the constant-temperature PCR reaction tube into a nucleic acid detector for constant-temperature PCR detection:
in step S5-3, the sample and reagent are thermodynamically circulated between the low temperature test chamber a-4 and the high temperature test chamber a-6 through the ac channel a-5, and signal collection is performed in the low temperature test chamber a-4.
The constant-temperature PCR reaction tube can lead the sample to be in continuous high-temperature and low-temperature self-circulation, the reaction is quicker, and simultaneously, the operations of reagent storage, swab collection, uniform mixing, sample receiving, nucleic acid detection and the like are all completed in the reaction tube, so the operation is simpler and more convenient.
While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention.
Claims (9)
1. A constant temperature PCR reaction tube is characterized by comprising a liquid storage tube cover (C), a liquid storage tube (B) and a reaction tube (A) which are connected in sequence;
wherein, the top end of the liquid storage tube cover (C) is embedded with a cover plate (C-1), the upper part is provided with a tube cap (C-2), and the lower part is provided with a sealing ring (C-3);
the liquid storage pipe (B) is hollow to form a liquid storage bin (B-3), the top of the liquid storage pipe (B) is provided with a liquid feeding port (B-1), the aperture of the lower part of the liquid storage pipe (B) is narrowed, the outer wall of the liquid storage pipe (B) is provided with a pair of clamping hooks (B-4), the bottom of the liquid storage pipe (B) is conical, and the center of the liquid storage pipe (B;
a guide groove (A-1) which is matched with the clamping hook (B-4) in width is arranged on the inner wall of the top of the reaction tube (A), a digital positioning mark is arranged on the outer wall, a positioning groove (A-2) which is matched with the clamping hook (B-4) in height is formed in the guide groove (A-1) and the side wall below the digital positioning mark, a liquid adding bin (A-3) is arranged at the upper part, a low-temperature detection bin (A-4) is arranged at the middle part, and a high-temperature bin (A-6) which is communicated with the low-temperature detection bin (A-4) through an alternating current channel (A-5) is arranged at the lower part; and the front side and the rear side of the reaction tube (A) are sealed with composite membranes (A-7).
2. The isothermal PCR reaction tube according to claim 1, wherein the cover plate (C-1) is colored with information on the volume of the solution to be stored, and/or
The surface is printed with a two-dimensional code or a text description indicating the information of the stored liquid.
3. The isothermal PCR reaction tube according to claim 1, wherein the cap (C-2) has a plurality of cylindrical protrusions on the outer wall and an internal thread on the inner wall.
4. The isothermal PCR reaction tube according to claim 2, wherein the filling port (B-1) is a bell-mouth with a small inside and a large outside, and the outer wall of the filling port is provided with an external thread (B-2) matching the internal thread.
5. The isothermal PCR reaction tube according to claim 1, wherein a sealing edge (B-5) is provided at the junction of the lower part and the bottom of the reservoir tube (B).
6. The isothermal PCR reaction tube of claim 1, wherein the digital location indicator comprises "1", "0", and "2"; the position of the digital positioning mark '0' corresponds to the position of the guide groove (A-1), and the digital positioning mark '1' and the digital positioning mark '2' are respectively positioned at two sides of the digital positioning mark '0'.
7. The isothermal PCR reaction tube according to claim 1, wherein barbs for stopping are provided at the trisections of the positioning grooves (A-2).
8. The isothermal PCR reaction tube according to claim 1, wherein the liquid addition compartment (A-3) comprises: a liquid adding groove (A-3-1) matched with the bottom shape of the liquid storage pipe (B), a liquid channel (A-3-2) communicated with the side wall of the liquid adding groove (A-3-1) and a liquid separating channel (A-3-3) formed by extending from the bottom of the liquid channel (A-3-2) to two sides; the liquid channel (A-3-2) and the digital positioning mark (2) are positioned on the same vertical surface; the liquid separation channel (A-3-3) is communicated with the low-temperature detection bin (A-4).
9. A method of using a isothermal PCR reaction tube according to any of claims 1 to 8, comprising the steps of:
s1, the liquid storage tube (B) and the reaction tube (A) are connected in a rotating and sealing mode through the clamping hook (B-4) and the positioning groove (A-2), and the clamping hook (B-4) corresponds to the digital positioning mark '0';
s2, clockwise rotating the liquid storage tube (B) to enable the clamping hook (B-3) to correspond to the digital positioning mark '1', placing a liquid reagent or a freeze-dried reagent into the liquid storage bin (B-2), and connecting the liquid storage tube cover (C) and the liquid storage tube (B) through the internal thread and the external thread (B-2) in a threaded sealing manner;
s3, collecting swabs; unscrewing the liquid storage tube cover (C) from the liquid storage tube (B), and placing the swab into the liquid storage tube (B); if the reagent is lyophilized in step S2, adding a proper amount of water into the liquid storage tube (B); screwing the liquid storage tube cover (C) on the liquid storage tube (B) tightly for sealing, and forcibly shaking the liquid storage tube (B) to uniformly mix the sample and the reagent;
s4, rotating the liquid storage pipe (B) anticlockwise to enable the clamping hook (B-4) to correspond to the digital positioning mark '2', wherein the liquid leakage hole (B-6) is communicated with the liquid channel (A-3-2), namely the liquid storage pipe (B) is communicated with the reaction pipe (A); forcibly shaking the liquid storage tube (B), or releasing the uniformly mixed sample and reagent in the liquid storage tube (B) to the low-temperature detection bin (A-4), the alternating-current channel (A-5) and the high-temperature bin (A-6) through the liquid channel (A-3-2) and the liquid distribution channel (A-3-3) in sequence by means of a centrifuge; rotating the liquid storage pipe (B) clockwise to enable the clamping hook (B-4) to correspond to the digital positioning mark '1' so as to close the liquid channel (A-3-2);
s5, inserting the constant-temperature PCR reaction tube into a nucleic acid detector for constant-temperature PCR detection:
simultaneously heating the low-temperature detection chamber (A-4) and the high-temperature chamber (A-6) to 50 ℃ and maintaining for 2 minutes;
after the low-temperature detection chamber (A-4) is heated to 95 ℃ and maintained for 1 minute, the temperature is reduced to 55 ℃ within 1 minute, and meanwhile, the high-temperature chamber (A-6) is heated to 95 ℃ and maintained for 2 minutes;
the low temperature detection chamber (A-4) is maintained at 55 ℃, and the high temperature chamber (A-6) is maintained at 95 ℃ for 1 minute and at 92 ℃ for 1 minute in a circulating manner; in the process, the sample and the reagent circulate between the low-temperature detection chamber (A-4) and the high-temperature detection chamber (A-6) through the alternating current channel (A-5) under the thermodynamic action, and the signal collection can be carried out in the low-temperature detection chamber (A-4).
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| CN202011641800.3A CN112760208A (en) | 2020-12-31 | 2020-12-31 | Constant-temperature PCR reaction tube and use method thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023178711A1 (en) * | 2022-03-23 | 2023-09-28 | 无锡百泰克生物技术有限公司 | Single-part rapid pcr chip |
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