CN115851422A - Integrated small-sized nucleic acid detection device and nucleic acid detection method - Google Patents
Integrated small-sized nucleic acid detection device and nucleic acid detection method Download PDFInfo
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- CN115851422A CN115851422A CN202211258538.3A CN202211258538A CN115851422A CN 115851422 A CN115851422 A CN 115851422A CN 202211258538 A CN202211258538 A CN 202211258538A CN 115851422 A CN115851422 A CN 115851422A
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Abstract
The invention discloses an integrated small-sized nucleic acid detection device, which comprises a closed card box consisting of a sleeve, a shell and a base, wherein the upper half part of the inner wall of the shell is provided with two centrosymmetric spiral tracks; two symmetrical vertical guide rails are arranged on two sides of the sleeve wall; the puncture suspension rod is positioned in the sleeve, two bulges are arranged at two ends of the top end of the puncture suspension rod, the two bulges penetrate through a guide rail on the wall of the sleeve and are embedded into a spiral track on the inner wall of the shell, so that the puncture suspension rod vertically moves up and down along with the rotation of the sleeve, and the tail end of the puncture suspension rod is an arrow; the device also comprises a reagent bin which is arranged at the lower half part of the shell; the top of the shell is connected with the sleeve, and the bottom of the shell is connected with the base in a sealing mode. The invention provides a small nucleic acid detection device integrating nucleic acid extraction, amplification and detection, which can be used for completing the whole processes of nucleic acid extraction, target sequence amplification, detection and the like of a sample, thereby greatly reducing the requirements on detection places and technicians.
Description
Technical Field
The invention belongs to the field of nucleic acid detection, and relates to an integrated small nucleic acid detection device and a nucleic acid detection method.
Background
Nucleic acid detection is the most sensitive and accurate method for pathogen detection, and many pathogen detection technologies based on nucleic acid amplification, such as fluorescent quantitative PCR technology, loop-mediated isothermal amplification technology, nucleic acid sequencing technology, etc., have been developed in recent years. However, these techniques require steps such as sample nucleic acid extraction, target amplification, and detection of amplification products during pathogen detection. Each step requires specialized instrumentation and requires the manipulation of specialized personnel familiar with the skill of molecular biology experiments. The biggest problem in nucleic acid detection is false positive result caused by cross contamination of amplification products, and usually, the steps of nucleic acid extraction, amplification, detection and the like must be carried out in a completely physically isolated partitioned laboratory, so that rapid detection in the field is difficult to realize. In order to realize the field detection of pathogens, an optimal mode is to develop an integrated device and an instrument integrating nucleic acid extraction, amplification and detection, and overcome the defect that a partition laboratory is required for the existing pathogen nucleic acid detection.
Disclosure of Invention
The invention discloses an integrated small-sized nucleic acid detection device, which integrates the steps of nucleic acid extraction, amplification and detection into a small-sized device for completion.
The purpose of the invention is realized by the following technical scheme:
an integrated small nucleic acid detecting device, characterized in that: the sealing device comprises a sleeve, a shell and a base, wherein the upper half part of the inner wall of the shell comprises two centrosymmetric spiral tracks;
the sleeve comprises an integrated sleeve knob and a sleeve wall, the sleeve wall extends into the shell, the sleeve knob is embedded at the upper end of the shell, and two symmetrical vertical guide rails are arranged on two sides of the sleeve wall;
the puncture suspension rod is positioned in the sleeve, two bulges are arranged at two ends of the top end of the puncture suspension rod and penetrate through a guide rail on the wall of the sleeve to be embedded into a spiral track on the inner wall of the shell, so that the puncture suspension rod vertically moves up and down along with the rotation of the sleeve, and the tail end of the puncture suspension rod is an arrow;
the device also comprises a reagent bin which is arranged at the lower half part of the shell;
the top of the shell is connected with the sleeve, and the bottom of the shell is connected with the base in a sealing mode.
Preferably, the reagent silo comprises a plurality of reagent pools and a plurality of suction pools, and generally, the number of the suction pools is less than that of the reagent pools. The reagent pool is filled with reagents, and the top surface and the bottom surface of the reagent pool are both easy-to-pierce films. The water absorption pool is hollow at the top, a hollow round hole is formed in the center of the water absorption pool, a plurality of fences are arranged around the round hole, and water absorption cotton is filled in a cavity outside the fences. Each layer of water absorption pool is tightly combined below the corresponding reagent pool and is used for collecting waste liquid.
Preferably, the puncture hanging rod further comprises a magnet, and the magnet is positioned in the cylinder above the arrow at the tail end of the puncture hanging rod.
Preferably, the shell is provided with a sample adding hole and a matched sample adding hole cover at a position between the reagent bin and the sleeve wall.
Preferably, the wall thicknesses of the upper part, the middle part and the lower part of the shell are different, the wall thickness of the part of the lower part corresponding to the reagent bin is the thinnest, the wall thickness of the part of the middle part corresponding to the sampling hole is thicker, and the wall thickness difference forms a limiting effect on the reagent bin; the wall thickness of the upper part corresponding to the sleeve is the thickest, and the wall thickness difference forms a limit for the lower edge of the sleeve wall.
Preferably, the reagent pool is provided with a plurality of reagent pools, wherein the reagent pools sequentially contain suspension of magnetic particles and cell lysis solution, cleaning solution, eluent and nucleic acid amplification reaction reagent from top to bottom.
The invention also discloses a nucleic acid detection method based on the integrated small-sized nucleic acid detection device, which is characterized by comprising the following steps:
(1) Puncturing the top surface film of the first layer of reagent pool through a sample adding hole on the card box shell, and adding a sample solution of nucleic acid to be extracted into the top surface film;
(2) By rotating the sleeve, the magnet on the suspension rod moves downwards to the first layer of reagent pool, so that the magnetic particles combined with the sample nucleic acid are all adsorbed to the surface of the cylinder containing the magnet above the arrow;
(3) Continuing rotating the sleeve to enable the arrow to puncture the bottom film of the first layer of reagent pool, so that the liquid in the reagent pool is completely absorbed by the absorbent cotton after being discharged;
(4) Continuing rotating the sleeve to enable the arrows to sequentially enter the next layer or layers of reagent pools, cleaning the magnetic particles on the surface of the magnet by using the cleaning liquid in each reagent pool, and absorbing the cleaned waste liquid by absorbent cotton;
(5) Continuing rotating the sleeve to enable the arrow to enter the next layer of reagent pool, and immersing the magnetic particles in the eluent in the reagent pool to enable the nucleic acid on the magnetic particles to be eluted;
(6) Continuously rotating the sleeve, injecting the extracted nucleic acid into the reagent pool at the bottom layer, and mixing the nucleic acid with the nucleic acid amplification reagent in the reagent pool;
(7) And continuing rotating the sleeve, and injecting the mixed liquid into the base to perform amplification detection.
The invention has the following advantages:
the invention provides an integrated small-sized nucleic acid detection device, which integrates the steps of nucleic acid extraction, amplification and detection into one device to be completed, thereby greatly reducing the requirements on detection places and technicians. The device is disposable, and each sample to be detected is detected by using an independent device, so that cross contamination among different samples can be effectively avoided. In addition, the device adopts a fully-closed design, so that the dissipation of nucleic acid amplification products can be effectively avoided, and a false positive result is prevented. In summary, the present invention can provide an integrated small nucleic acid detecting apparatus and a method of using the same.
Drawings
FIG. 1 is a view showing the structure of an apparatus of the present invention, (A) is an overall sectional view, (B) is a schematic view before operation with an arrow at the upper part, and (C) is a schematic view after operation with the arrow moved into a base reaction tube.
FIG. 2 is a view showing the construction of a housing of the apparatus of the present invention, wherein (A) is an overall perspective view and (B) is an overall sectional view.
FIG. 3 is a diagram of the sleeve configuration of the apparatus of the present invention.
FIG. 4 is a diagram showing the structure of a reagent cartridge of the apparatus of the present invention, wherein (A) is a perspective view of a main body of the reagent cartridge, (B) is a perspective view of a cross section of the reagent cartridge, (C) is a diagram showing the structure of a reagent reservoir, and (D) is a diagram showing the structure of a water-absorbing reservoir.
FIG. 5 is a view showing the structure of the base of the apparatus of the present invention.
Detailed Description
Example 1
The integrated small-sized nucleic acid detection device shown in the invention is shown in figure 1 and comprises a tubular base 1, a cylindrical shell 2 and a sleeve 3, wherein the top of the sleeve 3 is provided with a sleeve knob 4, the wall of the sleeve extends into the shell, the sleeve knob is embedded at the upper end of the shell, and meanwhile, the sleeve knob can rotate under the action of external force so as to drive the wall of the sleeve to rotate. The junction of the tubular base 1, the cylindrical housing 2 and the sleeve 3 remains closed, forming a closed cavity. The sleeve 3 contains a suspension rod 5 with an arrow-shaped end, and a magnet 6 is contained in a column body above the arrow. The lower part of the housing 2 contains a reagent chamber 7 for containing reagents required for nucleic acid extraction and purification. The tubular base 1 is used for carrying out a nucleic acid amplification reaction.
Wherein the specific configuration of the cylindrical housing 2 is shown in figure 2. The upper half of the inner wall of the housing 2 contains two centrosymmetric helical tracks 8-1 and 8-2 for limiting the movement path of the housing 2 and the sleeve 3 when they move relative to each other. The middle part of the shell 2 comprises a sample adding hole 9 and a sample adding hole cover 10 of the accessory thereof, which are used for adding a sample to be detected, and the sample adding hole cover 10 is used for sealing the sample adding hole 9 after the sample is added, so that a closed cavity is formed inside the device. The wall thicknesses of the upper part, the middle part and the lower part of the shell are different, the wall thickness of the part of the lower part corresponding to the reagent bin is the thinnest, the wall thickness of the part of the middle part corresponding to the sampling hole is thicker, and the wall thickness difference forms the limiting effect on the reagent bin; the wall thickness of the upper part corresponding to the sleeve is the thickest, and the wall thickness difference forms a limit for the lower edge of the sleeve wall.
The specific construction of the sleeve 3 is shown in fig. 3, the lower part of the sleeve 3 is provided on both sides with two symmetrical vertical guides 11 and 12, and the boom 5 is provided on both sides at its top end with two projections which can be inserted through the guides 11 and 12 in the wall of the sleeve into the helical track 8 in the inner wall of the housing 2. When the device is used, the lower sleeve 3 is driven by external force to rotate relative to the shell 2, the suspension rod 5 vertically moves up and down along with the external force, and the tail end of the suspension rod 5 is an arrow 13. At least one magnet 6 is arranged in the boom cylinder above arrow 13. When in use, the magnetic particles for nucleic acid extraction are adsorbed on the surface of the suspension rod corresponding to the magnet 6.
The specific structure of the reagent cabin 7 is shown in fig. 4, and comprises a reagent pool 14 and a water suction pool 15. The reagent pools 14-1 to 14-5 and the water absorption pools 15-1 to 15-4 are distributed at intervals to form a nested structure. When in use, a nucleic acid extraction reagent is pre-stored in each of the reagent pools 14-1 to 14-5, a nucleic acid amplification reagent is pre-stored in the reagent pool 14-6, and the top surface and the bottom surface of the reagent pool are sealed by films. The top of the water absorption pool is empty, the center of the water absorption pool is a hollow round hole, a plurality of fences 16 are arranged around the round hole, and water absorption materials such as water absorption cotton are pre-installed in a cavity outside the fences and used for collecting waste liquid. The film can be made of common aluminum foil, waterproof paper film or other materials easy to puncture.
The specific structure of the base is shown in FIG. 5, the upper part 1-1 is tightly connected with the bottom of the shell through screw threads, and the lower part 1-2 is tubular and is used for carrying out nucleic acid amplification detection reaction.
When in use, an operator punctures the top film of the reagent pool 14-1 through the sample adding hole 9 on the shell of the card box, adds samples comprising nasopharyngeal swab leachate, blood, sputum, urine, tissues, excrement and the like, and covers the sample adding hole cover 10; the sleeve 3 is rotated to make the arrow 13 at the end of the suspender 5 sequentially pierce the top and bottom films of each reagent pool, the magnetic particles for nucleic acid extraction are adsorbed on the surface of the suspender corresponding to the magnet 6, and the waste liquid is completely collected by the water absorbing material in the water absorbing pool after being discharged. Finally, the extracted nucleic acid and the amplification reaction reagent flow into the tube of the base 1 together for amplification detection. The extraction, purification and mixing of the amplification reaction reagents of the sample nucleic acid are accomplished by rotating the cartridge in forward and reverse directions.
Example 2
A method for detecting nucleic acid using the above-mentioned apparatus, comprising the steps of:
(1) Puncturing the aluminum foil on the top surface of the reagent pool 14-1 from the sample adding hole 9 on the shell, adding a sample to be detected, performing cell lysis, releasing nucleic acid, and adsorbing nucleic acid by magnetic beads;
(2) Rotating the sleeve 3 to enable the suspender 5 to move downwards until the suspender section corresponding to the magnet 6 is immersed in the liquid in the reagent pool 1, and adsorbing the magnetic beads on the surface of the suspender corresponding to the magnet 6;
(3) Continuing rotating the sleeve 3, puncturing the aluminum foil on the bottom surface of the reagent pool 14-1 by using an arrow 13 at the tail end of the suspender, discharging the liquid to the liquid absorption pool 15-1, and completely absorbing the liquid by absorbent cotton;
(4) Continuing rotating the sleeve 3, using an arrow 13 at the tail end of the suspender to puncture the aluminum foil on the top surface of the reagent pool 14-2, and immersing the suspender section corresponding to the magnet 6 in the cleaning solution 1 in the reagent pool 14-2;
(5) Rotating the sleeve 3 repeatedly in the forward direction/reverse direction for more than three times, and cleaning magnetic beads on the surface of the suspender corresponding to the magnet 6;
(6) Continuing rotating the sleeve 3, puncturing the aluminum foil on the bottom surface of the reagent pool 14-2 by using an arrow 13 at the tail end of the suspender, discharging the liquid to the liquid absorption pool 15-2, and completely absorbing the liquid by absorbent cotton;
(7) Continuing rotating the sleeve 3, using an arrow 13 at the tail end of the suspender to pierce the aluminum foil on the top surface of the reagent pool 14-3, and immersing the suspender section corresponding to the magnet 6 in the cleaning solution 1 in the reagent pool 14-3; (ii) a
(8) Rotating the sleeve 3 repeatedly in the forward direction/reverse direction for more than three times, and cleaning magnetic beads on the surface of the suspender corresponding to the magnet 6;
(9) Continuing rotating the sleeve 3, puncturing the aluminum foil on the bottom surface of the reagent pool 14-3 by using an arrow 13 at the tail end of the suspender, discharging the liquid to the liquid absorption pool 15-3, and completely absorbing the liquid by absorbent cotton;
(10) Continuing to rotate the sleeve 3, using an arrow 13 at the tail end of the suspender to puncture the aluminum foil on the top surface of the reagent pool 14-4, and immersing the suspender section corresponding to the magnet 6 in the cleaning solution 2 in the reagent pool 14-4;
(11) Rotating the sleeve 3 repeatedly in the forward direction/reverse direction for more than three times, and cleaning magnetic beads on the surface of the suspender corresponding to the magnet 6;
(12) Continuing rotating the sleeve 3, using an arrow 13 at the tail end of the suspender to puncture the aluminum foil on the bottom surface of the reagent pool 14-4, discharging the liquid to the liquid absorption pool 15-4, and completely absorbing the liquid by absorbent cotton;
(13) Standing for at least 1min to evaporate residual liquid on the surfaces of the magnetic beads;
(14) Continuing to rotate the sleeve 3, using an arrow 13 at the tail end of the suspender to puncture the aluminum foil on the top surface of the reagent pool 14-5, and immersing the suspender section corresponding to the magnet 6 in the eluent in the reagent pool 14-5;
(15) Standing for at least 2 minutes to dissolve the nucleic acid on the surface of the magnetic beads into the eluent;
(16) Continuing to rotate the sleeve 3, puncturing the aluminum foil on the top surface of the reagent pool 14-6 by using an arrow 13 at the tail end of the suspender, discharging the liquid in the reagent pool 14-5, and dissolving the nucleic acid amplification reaction freeze-drying reagent in the reagent pool 14-6;
(17) Rotating the sleeve forward/backward for more than 3 times repeatedly, and uniformly mixing the liquid in the reagent pool 14-6;
(18) The cartridge 3 is further rotated, and the aluminum foil on the bottom surface of the reagent tank 14-6 is pierced by the arrow 13 at the end of the hanger rod, so that the liquid in the reagent tank 14-6 is discharged into the reaction tube in the base 1, and the nucleic acid amplification reaction is performed.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (7)
1. An integrated small nucleic acid detecting device, characterized in that: the device comprises a sealing device consisting of a sleeve, a shell and a base, wherein the upper half part of the inner wall of the shell is provided with two centrosymmetric spiral tracks;
the sleeve comprises an integrated sleeve knob and a sleeve wall, the sleeve wall extends into the shell, the sleeve knob is clamped at the upper end of the shell, and two symmetrical vertical guide rails are arranged on two sides of the sleeve wall;
the puncture suspension rod is positioned in the sleeve, two bulges are arranged at two ends of the top end of the puncture suspension rod and penetrate through a guide rail on the wall of the sleeve to be embedded into a spiral track on the inner wall of the shell, so that the puncture suspension rod vertically moves up and down along with the rotation of the sleeve, and the tail end of the puncture suspension rod is an arrow;
the device also comprises a reagent bin which is arranged at the lower half part of the shell;
the top of the shell is connected with the sleeve, and the bottom of the shell is connected with the base in a sealing mode.
2. The integrated small nucleic acid detecting device according to claim 1, characterized in that: the reagent storehouse includes a plurality of reagent ponds and water absorption tank, the reagent pond is equipped with reagent, and its top surface and bottom surface are the film that easily punctures, and water absorption tank sets up in the below of corresponding reagent pond, water absorption tank top is empty, and its center is the fretwork round hole, and there are many fences around the round hole, and the cotton that absorbs water is filled in the cavity in the fence outside.
3. The integrated small nucleic acid detecting device according to claim 1 or 2, characterized in that: the puncture suspension rod further comprises a magnet, and the magnet is positioned in the column body above an arrow at the tail end of the puncture suspension rod.
4. The integrated small nucleic acid detecting device according to claim 3, characterized in that: and a sample adding hole and a matched sample adding hole cover are arranged at the position of the shell between the reagent bin and the sleeve wall.
5. The integrated small nucleic acid detecting device according to claim 3, characterized in that: the wall thicknesses of the upper part, the middle part and the lower part of the shell are different, the wall thickness of the part of the lower part corresponding to the reagent bin is the thinnest, the wall thickness of the part of the middle part corresponding to the sampling hole is larger than that of the part corresponding to the reagent bin, and the wall thickness of the part of the upper part corresponding to the sleeve is the thickest.
6. The integrated small nucleic acid detecting device according to claim 3, characterized in that: the reagent pool is provided with a plurality of suspension, cleaning solution, eluent and nucleic acid amplification reaction reagent which are sequentially filled with magnetic particles and cell lysis solution from top to bottom, wherein a water absorption pool is arranged below the reagent pool filled with the suspension and the cleaning solution.
7. A method for detecting nucleic acid based on the integrated small nucleic acid detecting apparatus according to any one of claims 1 to 6, characterized by comprising the steps of:
(1) Puncturing the top surface film of the first layer of reagent pool through a sample adding hole on the card box shell, and adding a sample solution of nucleic acid to be extracted into the top surface film;
(2) By rotating the sleeve, the magnet on the suspension rod moves downwards to the first layer of reagent pool, so that the magnetic particles combined with the sample nucleic acid are all adsorbed to the surface of the cylinder containing the magnet above the arrow;
(3) Continuing rotating the sleeve to enable the arrow to puncture the bottom film of the first layer of reagent pool, so that the liquid in the reagent pool is completely absorbed by the absorbent cotton after being discharged;
(4) Continuing rotating the sleeve to enable the arrows to sequentially enter the next layer or layers of reagent pools, cleaning the magnetic particles on the surface of the magnet by using the cleaning liquid in each reagent pool, and absorbing the cleaned waste liquid by absorbent cotton;
(5) Continuing rotating the sleeve to enable the arrow to enter the next layer of reagent pool, and immersing the magnetic particles in the eluent in the reagent pool to enable the nucleic acid on the magnetic particles to be eluted;
(6) Continuing rotating the sleeve, injecting the extracted nucleic acid into the reagent pool at the bottom layer, and mixing the nucleic acid with the nucleic acid amplification reagent in the reagent pool;
(7) And continuing rotating the sleeve, and injecting the mixed liquid into the base to perform amplification detection.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211258538.3A CN115851422A (en) | 2022-10-14 | 2022-10-14 | Integrated small-sized nucleic acid detection device and nucleic acid detection method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211258538.3A CN115851422A (en) | 2022-10-14 | 2022-10-14 | Integrated small-sized nucleic acid detection device and nucleic acid detection method |
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| Publication Number | Publication Date |
|---|---|
| CN115851422A true CN115851422A (en) | 2023-03-28 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211258538.3A Pending CN115851422A (en) | 2022-10-14 | 2022-10-14 | Integrated small-sized nucleic acid detection device and nucleic acid detection method |
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| Country | Link |
|---|---|
| CN (1) | CN115851422A (en) |
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- 2022-10-14 CN CN202211258538.3A patent/CN115851422A/en active Pending
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