CN117327577A - Gene amplification extraction tube, nucleic acid amplification extraction device, and gene identification method - Google Patents
Gene amplification extraction tube, nucleic acid amplification extraction device, and gene identification method Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Abstract
The invention discloses a gene amplification extraction tube which is used for being placed in a PCR instrument to realize the amplification and extraction integrated operation of a gene sample, wherein the gene amplification extraction tube comprises a cracking cavity and a reaction cavity, the cracking cavity can be embedded into the reaction cavity, and the cracking cavity is detachably connected with the reaction cavity; the lysis cavity is provided with a first accommodating cavity for accommodating a gene sample and performing tissue lysis reaction, and the reaction cavity is provided with a second accommodating cavity for accommodating PCR reaction liquid and can be used for performing PCR amplification reaction; the bottom or the side wall of the first accommodating cavity is provided with a plurality of small holes, a plurality of iron particles are arranged in the first accommodating cavity, the iron particles are provided with rough surfaces, and the iron particles can pass through the small holes from the first accommodating cavity to bring a gene sample in the first accommodating cavity into the second accommodating cavity under the action of an external magnetic field; the method can solve the technical problem of how to improve the sample processing efficiency and reduce the pollution risk in the genotype identification process.
Description
Technical Field
The invention relates to the field of gene extraction and nucleic acid amplification, in particular to a gene amplification extraction tube, a nucleic acid amplification extraction device and a gene identification method.
Background
Experimental animals play a vital role in life medicine research. In particular in the medical and life sciences, genetically edited laboratory animals have become an integral part of research. However, with intensive studies on genome, higher demands are put on experimental animal models. Genotyping is an essential step in genetically modified animal studies, a common method of determining whether an individual experimental animal is our desired model, which usually requires the handling of large numbers of samples. Therefore, simplifying the genotyping process is critical to the scientific researchers and can save a lot of time and effort for the scientific researchers.
Current genotyping methods cover four main steps: tissue sample collection, DNA extraction, nucleic acid amplification, and gel imaging, wherein DNA extraction and PCR amplification are critical steps in the overall process.
First, in the DNA extraction stage, the genome is extracted from cells or tissues, typically by using enzymatic methods, alkaline lysis methods, and lipid-dissolving reagents, such as proteinase K, SDS.
Subsequently, the specific gene or DNA fragment is amplified by PCR or other nucleic acid amplification techniques. Among them, PCR amplification (i.e., polymerase chain reaction, polymerase Chain Reaction) is widely used for replicating DNA sequences in vitro. The principle of PCR amplification is based on the natural replication process of DNA, so that even a small amount of DNA will produce a very distinct DNA band after PCR amplification. This requires very stringent sample loading during the course of the experiment.
The current genotyping method mainly relies on the manual extraction of DNA, and then the DNA-containing solutions are added one by one into the PCR reaction system one by one. However, since the DNA storage container is usually separated from the PCR reaction container, this process is liable to cause contamination between samples.
Although related methods have allowed for the direct addition of tissue samples to PCR reaction systems for amplification to reduce the probability of sample contamination, there are still some limitations to these methods. Each sample can only construct one PCR reaction system, in the same PCR reaction system, the DNA extracting solution can be used as the PCR of a new system, namely, the PCR reaction does not affect the original template, and some other nucleic acids and the PCR reaction system directly add the sample into the reaction solution, but the mode can only identify one type, the wild type and the mutant type are difficult to identify at the same time, and the tissue sample is difficult to extract and use the DNA again.
Therefore, there is a need to solve the technical problem of how to increase the sample processing efficiency while reducing the risk of contamination during genotyping.
Disclosure of Invention
The invention aims to provide a gene amplification extraction tube, a nucleic acid amplification extraction device and a gene identification method, so as to solve the technical problem of how to improve sample processing efficiency and reduce pollution risk in the genotype identification process.
In order to solve the technical problems, the invention adopts the following technical scheme:
in a first aspect, the invention discloses a gene amplification extraction tube which is used for being placed in a PCR instrument to realize amplification and extraction integrated operation of a gene sample, wherein the gene amplification extraction tube comprises a cracking cavity and a reaction cavity, the cracking cavity can be embedded into the reaction cavity, and the cracking cavity is detachably connected with the reaction cavity; the lysis cavity is provided with a first accommodating cavity for accommodating a gene sample and performing tissue lysis reaction, and the reaction cavity is provided with a second accommodating cavity for accommodating PCR reaction liquid and can be used for performing PCR amplification reaction; the bottom or the side wall of the first accommodating cavity is provided with a plurality of small holes, a plurality of iron particles are arranged in the first accommodating cavity, the iron particles are provided with rough surfaces, and the iron particles can penetrate through the small holes from the first accommodating cavity to bring a gene sample in the first accommodating cavity into the second accommodating cavity under the action of an external magnetic field.
The working principle of the scheme is as follows: when the gene amplification extraction tube disclosed by the invention is used for carrying out gene amplification, an electromagnetic field or a magnet is required to be matched for use, a PCR reaction liquid is firstly placed in the second accommodating cavity, iron particles and a gene sample are placed in the first accommodating cavity, a tissue lysis liquid is added, then the lysis cavity and the reaction cavity are nested and connected, the mixture is placed in a PCR instrument, and meanwhile, a magnetic field is applied to the gene amplification extraction tube, so that the iron particles carry the gene sample into the second accommodating cavity, the gene sample is contacted with the PCR reaction liquid, and then the PCR instrument is opened for carrying out PCR amplification, thereby realizing the integration of extraction and amplification of the gene sample.
Aiming at simultaneous amplification and extraction of two identification systems of a genotype wild type and a genotype mutant type, improving a cracking cavity of a gene amplification extraction tube, wherein the cracking cavity further comprises a third accommodating cavity, small holes are formed in the bottoms or the side walls of the third accommodating cavity and the first accommodating cavity, and a fourth accommodating cavity is further arranged in a corresponding reaction cavity and is separated from the second accommodating cavity by a partition board;
the third accommodating cavity corresponds to the fourth accommodating cavity, the third accommodating cavity can be embedded into the fourth accommodating cavity, the first accommodating cavity corresponds to the second accommodating cavity, and the first accommodating cavity can be embedded into the second accommodating cavity.
Therefore, the interior of a single gene amplification extraction tube can be divided into two reaction tanks, and two identification systems of the wild type and the mutant type of the genotype can be respectively amplified and extracted at the same time, so that the amplification and extraction efficiency of a gene sample can be improved.
Preferably, the iron particles have a particle size in the range of less than 1.0. 1.0 mm.
Preferably, the surface of the iron particles is provided with a plurality of concave structures, and the concave structures of the iron particles can carry the gene sample in the first accommodating cavity.
The surface area of iron particle can be increased to the concave structure simultaneously, because the existence of surface tension to and the viscosity of gene sample self, the concave structure can the certain degree restriction get into the gene sample in the concave structure and drop, make the gene sample can more stable existence in the concave structure, just so make the iron particle can make the gene sample carry the gene sample through adsorption and the carrying effect of concave structure more simultaneously, when applying the magnetic field to the gene amplification extraction tube, the magnetic field can make the iron particle that carries more have the gene sample pass the aperture gets into the second holding intracavity from first holding chamber, increases the total amount of the gene sample that gets into the second holding chamber, and then improves gene sample extraction efficiency, and then can improve measuring result's accuracy.
Preferably, the surface of the iron particle is provided with a plurality of protrusions, and the protrusions are surrounded to form a plurality of narrow slit structures, and the narrow slit structures can carry the gene sample in the first accommodating cavity.
The principle of increasing the carrying amount of the gene sample into the second accommodation cavity by using the narrow slit structure is that the fluid itself has viscosity, i.e. can be adsorbed on the surface of the object, the fluid usually has surface tension, and the presence of the protrusions and slits increases the total area of the surface of the iron particles. Since the surface tension tends to reduce the surface area of the fluid surface, fluid molecules are more closely arranged between the protrusions and the slits, and since a plurality of slit structures can accommodate more gene samples, the plurality of slit structures generated on the surface of the iron particles due to the surface tension can carry more gene samples per unit surface area, thereby improving the sample carrying capacity.
Preferably, the iron particle has a cavity therein, the surface of the iron particle has a plurality of holes extending from the surface of the iron particle toward the cavity, and the cavity and the holes are capable of carrying a gene sample located in the first accommodation cavity.
Preferably, the cracking cavity and the reaction cavity are detachably connected through a connecting structure;
the connecting structure comprises a sample adding cavity, the radial dimension of the sample adding cavity is matched with the radial dimension of the opening of the second accommodating cavity, a clamping groove is formed in the bottom edge of the sample adding cavity, a buckle is arranged at the edge of the opening of the second accommodating cavity, and the buckle is clamped with the clamping groove to realize detachable connection of the cracking cavity and the reaction cavity;
the first holding chamber fixed connection in loading chamber bottom center department, just first holding chamber with loading intracavity portion is linked together, the top of loading chamber is provided with sealed lid, sealed lid with loading chamber detachable connection for open the loading chamber, to the interior loading of first holding chamber.
In a second aspect, the invention also discloses a nucleic acid amplification and extraction device, which comprises the gene amplification and extraction tube, wherein an electromagnetic coil is sleeved outside the gene amplification and extraction tube and used for generating a magnetic field in a fixed direction under the action of electric power of an external power supply, and the magnetic field can enable the iron particles carrying a gene sample to pass through the small hole and enter the second accommodating cavity from the first accommodating cavity.
The electromagnetic coil is sleeved outside the gene amplification extraction tube, so that the whole nucleic acid amplification extraction device is integrated, the use is more convenient, the electromagnetic coil does not block the bottom and the side wall areas of the reaction cavity of the gene amplification extraction tube, the gene amplification extraction tube is convenient to put into a PCR instrument, and the follow-up sampling and identification of amplified gene samples are also convenient.
Preferably, the nucleic acid amplification and extraction device comprises an electromagnetic frame, wherein a plurality of electromagnetic coils are arranged on the electromagnetic frame, each electromagnetic coil can generate a magnetic field in a fixed direction under the action of electric power of an external power supply, and the directions of the magnetic fields generated by the electromagnetic coils are the same; a gene amplification extraction tube can be placed in each electromagnetic coil.
Therefore, the efficiency of nucleic acid amplification and extraction can be improved, and the gene samples in the first accommodating cavity in the plurality of gene amplification and extraction tubes can be simultaneously transferred to the second accommodating cavity along with the iron particles.
In a third aspect, the present invention also discloses a gene identification method using the nucleic acid amplification and extraction device as described above, comprising the steps of:
s1, preparing work before identification; taking a gene amplification extraction tube, adding a uniformly mixed PCR reaction solution into a second accommodating cavity of a reaction cavity for subsequent PCR reaction, then nesting a cracking cavity back into the reaction cavity, and placing the reaction cavity in a refrigerator for standby;
s2, preprocessing a gene sample; separating a cracking cavity and a reaction cavity of the gene amplification extraction tube, adding a gene sample into a first accommodating cavity of the cracking cavity, and temporarily placing the separated reaction cavity comprising PCR reaction liquid in a 4-DEG environment for later use;
s3, tissue lysis of the gene sample; the first accommodating cavity is internally provided with iron particles, tissue lysate is added into the first accommodating cavity, and after shaking, the tissue lysate is embedded into the reaction cavity;
s4, amplifying and extracting a gene sample; placing the gene amplification extraction tube obtained in the step S3 into a PCR instrument, executing a PCR digestion program, installing an electromagnetic coil on the periphery of the gene amplification extraction tube, then switching on an external power supply of the electromagnetic coil, transferring microbeads in tissue fluid into a reaction cavity, and entering the PCR amplification program;
s5, identifying a gene sample; and carrying out gene identification on PCR amplification products in a gene amplification extraction tube after amplification and extraction of the gene sample.
The invention has the following beneficial effects:
1. the gene amplification extraction tube provided by the invention is used for carrying out the steps of digestion and amplification in intervals by separating the reaction cavity on the gene amplification extraction tube, so that the extraction and the amplification are respectively carried out in independent spaces arranged in the same space, then the independent spaces arranged in the same space are subjected to substance transmission and communication by the action of iron particles and a magnetic field, the integrated operation of nucleic acid amplification and extraction can be realized, the amplification and the extraction can be completed without carrying out secondary operation in the middle, the possibility of polluting samples in the process can be reduced, the extraction efficiency of gene samples is improved, and the friction force between the iron particles and the gene samples can be increased due to the rough surface of the iron particles, so that more gene samples can be carried into the second accommodating cavity, and the accuracy of measurement results can be improved.
2. The nucleic acid amplification device provided by the invention can integrate the whole nucleic acid amplification extraction device, is more convenient in use, and simultaneously, the electromagnetic coil does not block the areas of the bottom and the side wall of the reaction cavity of the gene amplification extraction tube, so that the nucleic acid amplification device is convenient to put into a PCR instrument, and is also convenient for subsequent sampling and identification of amplified gene samples.
3. The gene identification method provided by the invention can reduce the pollution of PCR sample addition, reduce the manual operation, combine the two parts of gene sample amplification and extraction, improve the efficiency of gene sample extraction and amplification, and further reduce the identification time; in view of cost, the method can simply improve the existing used instrument or material, is compatible with the existing PCR instrument, has lower cost, does not need higher cost, and has the advantages of low cost, high feasibility, wide application range and high efficiency.
Drawings
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings, in which:
FIG. 1 is a schematic diagram of the structure of a gene amplification extraction tube according to the present invention.
FIG. 2 is a schematic diagram of the optimized structure of the gene amplification extraction tube of the present invention.
Fig. 3 is a schematic view of the electromagnetic coil installation of the present invention.
Fig. 4 is a schematic structural view of an electromagnetic frame according to the present invention.
FIG. 5 is a flow chart of the gene identification method of the present invention.
FIG. 6 is a schematic diagram showing the positional relationship of the nucleic acid amplification and extraction apparatus of the present invention in a PCR instrument.
FIG. 7 is a schematic diagram showing the placement positions of a gene amplification extraction tube and a gel electrophoresis apparatus according to the present invention including a sampling rack.
Reference numerals illustrate:
100. a gene amplification extraction tube; 101. a lysing chamber; 102. a reaction chamber; 103. a first accommodation chamber; 104. a second accommodation chamber; 105. iron particles; 107. a third accommodation chamber; 108. a fourth accommodation chamber; 109. a partition plate; 110. a sample adding cavity; 111. sealing cover; 200. a nucleic acid amplification and extraction device; 201. an electromagnetic coil; 202. an electromagnetic frame; 300. a PCR instrument; 400. a sampling frame; 401. needle structure.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.
It should be noted that: like reference numerals and letters denote like items in the following figures, and thus once an item is defined in one figure, no further definition or explanation thereof is necessary in the following figures. In the description of the present invention, it should be noted that, directions or positional relationships indicated by terms such as "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc., are directions or positional relationships based on those shown in the drawings, or are directions or positional relationships conventionally put in use of the inventive product, are merely for convenience of describing the present invention and simplifying the description, and are not indicative or implying that the apparatus or element to be referred to must have a specific direction, be constructed and operated in a specific direction, and thus should not be construed as limiting the present invention. Furthermore, the terms "first," "second," "third," and the like are used merely to distinguish between descriptions and should not be construed as indicating or implying relative importance. Furthermore, the terms "horizontal," "vertical," and the like do not denote a requirement that the component be absolutely horizontal or overhang, but rather may be slightly inclined. As "horizontal" merely means that its direction is more horizontal than "vertical", and does not mean that the structure must be perfectly horizontal, but may be slightly inclined. In the description of the present invention, it should also be noted that, unless explicitly specified and limited otherwise, the terms "disposed," "mounted," "connected," and "connected" are to be construed broadly, and may be, for example, fixedly connected, detachably connected, or integrally connected; can be mechanically or electrically connected; can be directly connected or indirectly connected through an intermediate medium, and can be communication between two elements. The specific meaning of the above terms in the present invention will be understood in specific cases by those of ordinary skill in the art.
The method is mainly applied to genotype identification of experimental animals in life medicine research, and solves the technical problem how to improve sample processing efficiency and reduce pollution risk in the genotype identification process.
Referring to fig. 1, based on the above-mentioned technical problems, the present invention discloses a gene amplification extraction tube 100 for being placed in a PCR instrument to realize integrated operations of amplification and extraction of a gene sample, wherein the gene amplification extraction tube 100 includes a cleavage cavity 101 and a reaction cavity 102, the cleavage cavity 101 can be embedded in the reaction cavity 102, and the cleavage cavity 101 and the reaction cavity 102 are detachably connected; the lysis cavity 101 is provided with a first accommodating cavity 103 for accommodating a gene sample and performing tissue lysis reaction, and the reaction cavity 102 is provided with a second accommodating cavity 104 for accommodating PCR reaction liquid and can be used for performing PCR amplification reaction; a plurality of small holes are formed in the bottom or the side wall of the first accommodating cavity 103, a plurality of iron particles 105 are arranged in the first accommodating cavity 103, the iron particles 105 have a rough surface, and the iron particles 105 can penetrate through the small holes from the first accommodating cavity 103 to bring a gene sample in the first accommodating cavity 103 into the second accommodating cavity 104 under the action of an external magnetic field.
The working principle of the scheme is as follows: when the gene amplification extraction tube 100 disclosed by the invention is used for carrying out gene amplification, an electromagnetic field or a magnet is required to be matched for use, a PCR reaction liquid is firstly placed in the second accommodating cavity 104, iron particles 105 and a gene sample are placed in the first accommodating cavity 103, a tissue lysis liquid is added, then the lysis cavity 101 and the reaction cavity 102 are nested and connected, the mixture is placed in the PCR instrument 300, and meanwhile, a magnetic field is applied to the gene amplification extraction tube 100, so that the gene sample carried by the iron particles 105 enters the second accommodating cavity 104, the gene sample contacts with the PCR reaction liquid, and then the PCR instrument 300 is opened for PCR amplification, so that the extraction and amplification integration of the gene sample are realized.
The integrated operation of nucleic acid amplification and extraction can be realized, secondary operation is not needed in the middle, amplification and extraction can be completed by using one set of equipment, the possibility of sample pollution in the process can be reduced, and the friction force between the iron particles and the gene sample can be increased because the iron particles have rough surfaces, so that more gene samples can be carried into the second accommodating cavity, the extraction efficiency of the gene sample can be improved, and the accuracy of a measurement result can be improved.
For simultaneous amplification and extraction of two identification systems of a genotype, the lysis chamber 101 of the gene amplification extraction tube 100 is improved, referring to fig. 2, the lysis chamber 101 further includes a third accommodating chamber 107, small holes are formed at the bottoms or sidewalls of the third accommodating chamber 107 and the first accommodating chamber 103, the corresponding reaction chamber 102 further includes a fourth accommodating chamber 108, and the second accommodating chamber 104 and the fourth accommodating chamber 108 are spatially separated by a partition plate 109;
the third accommodating cavity 107 corresponds to the fourth accommodating cavity 108, the third accommodating cavity 107 can be embedded into the fourth accommodating cavity 108, the first accommodating cavity 103 corresponds to the second accommodating cavity 104, and the first accommodating cavity 103 can be embedded into the second accommodating cavity 104.
Thus, the interior of the single gene amplification extraction tube 100 can be divided into two reaction tanks, and two identification systems of the wild type and the mutant type of the genotype can be respectively amplified and extracted at the same time, so that the amplification and extraction efficiency of the gene sample can be improved, and the identification efficiency of the gene sample can be improved.
Preferably, the surface of the iron particles is provided with a plurality of concave structures, and the concave structures of the iron particles can carry the gene sample in the first accommodating cavity.
The surface area of iron particle can be increased to the concave structure simultaneously, because the existence of surface tension to and the viscosity of gene sample self, the concave structure can the certain degree restriction get into the gene sample in the concave structure and drop, make the gene sample can more stable existence in the concave structure, just so make the iron particle can make the gene sample carry the gene sample through adsorption and the carrying effect of concave structure more simultaneously, when applying the magnetic field to the gene amplification extraction tube, the magnetic field can make the iron particle that carries more have the gene sample pass the aperture gets into the second holding intracavity from first holding chamber, increases the total amount of the gene sample that gets into the second holding chamber, and then improves gene sample extraction efficiency, and then can improve measuring result's accuracy.
Preferably, the iron particles have a particle size in the range of less than 1.0. 1.0 mm.
Preferably, the recess structure is an irregular recess structure.
Specifically, the irregular concave structure can be processed by adopting a micro-machining mode, a laser or electron beam processing mode, or can be processed by adopting chemical etching (strong acid or weak acid), electrochemical etching, photoetching technology (commonly used laser or ultraviolet light) or ion bombardment technology (commonly used argon ion Ar+ or krypton ion Kr+); the method of physical polishing or forging can also be adopted for processing, and the molding conditions need to be controlled when the physical polishing or forging is carried out.
Preferably, the surface of the iron particle is provided with a plurality of protrusions, and the protrusions are surrounded to form a plurality of narrow slit structures, and the narrow slit structures can carry the gene sample in the first accommodating cavity. The narrow slit structure can carry more gene samples by using the viscosity and surface tension of the fluid itself.
Specifically, the narrow slit structure is an irregular narrow slit structure.
The principle of increasing the carrying amount of the gene sample into the second accommodation cavity by using the narrow slit structure is that the fluid itself has viscosity, i.e. can be adsorbed on the surface of the object, the fluid usually has surface tension, and the presence of the protrusions and slits increases the total area of the surface of the iron particles. Since the surface tension tends to reduce the surface area of the fluid surface, fluid molecules are more closely arranged between the protrusions and the slits, and since a plurality of slit structures can accommodate more gene samples, the plurality of slit structures generated on the surface of the iron particles due to the surface tension can carry more gene samples per unit surface area, thereby improving the sample carrying capacity.
The narrow slit structure can be manufactured by firstly sputtering inert metal particles, such as gold or silver, on the surface of the iron particles by adopting a magnetron sputtering method, then placing the iron particles with the inert metal particles on the surface into a chemical etching solution for etching, wherein the chemical etching solution can adopt sulfuric acid or nitric acid or hydrochloric acid, and removing the inert metal particles on the surface after etching is finished, so that the iron particles with the narrow slit structure on the surface can be obtained.
Preferably, the iron particle has a cavity therein, the surface of the iron particle has a plurality of holes extending from the surface of the iron particle toward the cavity, and the cavity and the holes are capable of carrying a gene sample located in the first accommodation cavity. The iron particles with the cavities inside can be purchased in the market, and are not required to be manufactured, and are not described herein.
Specifically, the number of small holes arranged on the first accommodating cavity can be adjusted according to actual needs, so that the small holes are matched with the orders of magnitude of the gene samples and the orders of magnitude of the iron particles, and therefore, the gene samples can enter the second accommodating cavity as much as possible.
Specifically, the gene amplification extraction tube is made of high-temperature-resistant plastic or other materials which do not influence the PCR and other reaction systems.
In particular, the shape of the iron particles is not limited to a sphere, and other shapes with roughened surfaces for loading are within the scope.
Preferably, referring to FIGS. 1 and 2, the cleavage chamber 101 and the reaction chamber 102 are detachably connected by a connection structure; the specific manner of sealing connection is that the connection structure includes a sample adding cavity 110, the radial dimension of the sample adding cavity 110 is adapted to the radial dimension of the opening of the second accommodating cavity 104, a clamping groove is provided at the bottom edge of the sample adding cavity 110, a buckle is provided at the edge of the opening of the second accommodating cavity 104, and the buckle is clamped with the clamping groove to realize detachable connection of the cracking cavity 101 and the reaction cavity 102;
the first accommodating cavity 103 is fixedly connected to the center of the bottom of the sample adding cavity 110, the first accommodating cavity 103 is communicated with the inside of the sample adding cavity 110, a sealing cover 111 is arranged at the top end of the sample adding cavity 110, and the sealing cover 111 is detachably connected with the sample adding cavity 110 and used for opening the sample adding cavity 110 and adding samples into the first accommodating cavity 103.
In order to make the connection between the cleavage cavity 101 and the reaction cavity 102 more compact, prevent the leakage of the PCR reaction solution in the reaction cavity 102, a sealing ring is disposed in the clamping groove, so that the sealing connection between the cleavage cavity 101 and the reaction cavity 102 can be realized.
The gene amplification extraction tube provided by the invention can integrate the extraction and amplification of a gene sample, and reduces the artificial sample adding step so as to reduce the pollution probability in the sample adding process; the digestion and amplification steps are compartmentalized by separating the reaction cavity on the gene amplification extraction tube, so that the extraction and amplification can be respectively carried out in independent spaces arranged in the same space, and then the substance transmission and communication are generated between the independent spaces arranged in the same space under the action of iron particles and a magnetic field. The integrated operation of nucleic acid amplification and extraction can be realized, secondary operation is not needed in the middle, amplification and extraction can be completed by using one set of equipment, the possibility of sample pollution in the process can be reduced, and the friction force between the iron particles and the gene sample can be increased because the iron particles have rough surfaces, so that more gene samples can be carried into the second accommodating cavity, the extraction efficiency of the gene samples can be improved, and the accuracy of measurement results can be improved.
In a second aspect, referring to fig. 3, the present invention further discloses a nucleic acid amplification and extraction apparatus 200, which includes the gene amplification and extraction tube 100 as described above, wherein an electromagnetic coil 201 is sleeved outside the gene amplification and extraction tube 100, and is used for generating a magnetic field in a fixed direction under the action of electric power of an external power source, and the magnetic field can enable the iron particles 105 carrying the gene sample to pass through the small hole and enter the second accommodating cavity 104 from the first accommodating cavity 103.
The electromagnetic coil 201 is sleeved outside the gene amplification extraction tube 100, so that the whole nucleic acid amplification extraction device 200 is integrated, the use is more convenient, meanwhile, the electromagnetic coil 201 does not block the bottom and side wall areas of the reaction cavity 102 of the gene amplification extraction tube 100, and the gene amplification extraction tube is convenient to put into a PCR instrument, and is also convenient for subsequent sampling and identification of amplified gene samples.
Preferably, referring to fig. 4, the nucleic acid amplification and extraction apparatus 200 includes an electromagnetic rack 202, wherein a plurality of electromagnetic coils 201 are disposed on the electromagnetic rack 202, each electromagnetic coil 201 can generate a magnetic field in a fixed direction under the action of electric power of an external power source, and the directions of the magnetic fields generated by the electromagnetic coils 201 are the same; one gene amplification extraction tube 100 can be placed within each electromagnetic coil 201.
This can improve the efficiency of nucleic acid amplification and extraction, and can simultaneously transfer the gene samples in the first accommodation chamber 103 in the plurality of gene amplification and extraction tubes 100 to the second accommodation chamber 104 along with the iron microparticles 105.
Specifically, the magnetic field is generated by an electromagnet, and the direction of the magnetic field can be adjusted according to the opening position of the small hole, so as to achieve that the iron particles 105 can pass through the small hole to enter the second accommodating cavity 104. The main function of the magnetic field is to overcome the surface tension caused by the liquid and bring the iron particles 105 into the second accommodation chamber 104 without affecting the reaction system.
In a third aspect, referring to FIG. 5, the present invention also discloses a gene identification method using the nucleic acid amplification and extraction device as described above, comprising the steps of:
s1, preparing work before identification; taking a gene amplification extraction tube, adding a uniformly mixed PCR reaction solution into a second accommodating cavity of a reaction cavity for subsequent PCR reaction, then nesting a cracking cavity back into the reaction cavity, and placing the reaction cavity in a refrigerator for standby;
s2, preprocessing a gene sample; separating a cracking cavity and a reaction cavity of the gene amplification extraction tube, adding a gene sample into a first accommodating cavity of the cracking cavity, and temporarily placing the separated reaction cavity comprising PCR reaction liquid in a 4-DEG environment for later use;
s3, tissue lysis of the gene sample; the first accommodating cavity is internally provided with iron particles, tissue lysate is added into the first accommodating cavity, and after shaking, the tissue lysate is embedded into the reaction cavity;
s4, amplifying and extracting a gene sample; placing the gene amplification extraction tube obtained in the step S3 into a PCR instrument, executing a PCR digestion program, installing an electromagnetic coil on the periphery of the gene amplification extraction tube, then switching on an external power supply of the electromagnetic coil, transferring microbeads in tissue fluid into a reaction cavity, and entering the PCR amplification program;
s5, identifying a gene sample; and carrying out gene identification on PCR amplification products in a gene amplification extraction tube after amplification and extraction of the gene sample.
Specifically, in step S1, the PCR solution includes a primer, a polymerase, dntps, and magnesium ions, which are components conventionally used in the prior art, so as to achieve a PCR amplification effect.
Specifically, in step S3, the iron particles may be optionally placed in the first accommodating cavity in advance, or may be added into the first accommodating cavity when in use, and then be embedded back into the reaction cavity.
Specifically, in step S3, the tissue lysis is performed by using proteinase K as an example, the amount of the digestion solution containing proteinase is 10 μl to 50 μl, the specific amount of the digestion solution is determined according to the volume of the reaction chamber, the digestion is performed for 15min at 55 to 65 ℃ and then for 3 to 5min at 95 ℃.
Specifically, in step S4, referring to fig. 6, the PCR apparatus is a laboratory apparatus for polymerase chain reaction (Polymerase Chain Reaction, PCR). PCR is a molecular biological technique used to amplify, replicate and analyze DNA fragments. The function of the PCR instrument is to provide a constant temperature environment for performing the different steps of the PCR reaction, including denaturation, annealing and extension.
Specifically, in step S4, referring to fig. 6, the height of the gene amplification extraction tube is not higher than the maximum allowable height of the PCR instrument, and is not more than 2.0. 2.0 cm.
Specifically, in step S5, referring to fig. 7, the method of performing gene identification on the PCR amplification product in the gene amplification extraction tube after amplification and extraction of the gene sample employs a gel electrophoresis method, the gel electrophoresis method employs a gel electrophoresis apparatus, the gel electrophoresis apparatus includes a sampling frame 400, the sampling frame 400 is provided with a plurality of glue holes containing needle structures 401, the needle structures 401 are hollow needle structures 401, the outer wall of the second accommodating cavity 104 of the gene amplification extraction tube 100, which is close to the bottom, has a thin-wall structure, and the hollow needle structures 401 can penetrate through the side wall of the second accommodating cavity 104 to enter the second accommodating cavity 104 for sampling.
Gel electrophoresis by running the PCR products in an agarose gel you can separate DNA fragments of different sizes, can be used to determine the size of the PCR products, detect the presence of the products, and estimate their relative concentrations. This is very useful for primary screening and identification of PCR products, but gel electrophoresis has a relatively limited ability to quantify at low concentrations.
Therefore, in step S5, preferably, q-PCR (real-time fluorescent quantitative PCR), which is a method for precisely measuring the number of PCR products, is directly used for gene identification, and usually, a specific fluorescent marker is used to label DNA fragments, and PCR products are quantified by real-time monitoring of fluorescent signals, so that amplification, extraction and identification of gene samples can be simultaneously achieved, and PCR amplification products can be automatically analyzed. The fluorescent quantitation method has high quantitative sensitivity and can detect DNA at very low concentration.
Specifically, in step S5, the identification of the gene sample can also be read by using an enzyme-labeled instrument, so that the identification result is easier to interpret.
The gene identification method provided by the invention can reduce the pollution of PCR sample addition, reduce manual operation, improve the efficiency of gene sample extraction and amplification, and further reduce the identification time; experiments prove that even though iron particles with higher density (glass beads with smaller density) are difficult to spontaneously pass through small holes to enter a second accommodating cavity under the limited condition, iron particles with overlarge volume (diameter of 1.0 mm) can cause that most of tissue lysate in a first accommodating cavity is mixed into the second accommodating cavity in the reaction process to pollute PCR reaction liquid and influence the progress of PCR reaction, the genotype identification method provided by the invention combines the two parts of gene sample amplification and extraction on the premise of not polluting the extracted gene sample result, so that manpower and time are reduced to a certain extent; in view of cost, the method can simply improve the existing used instrument or material, is compatible with the existing PCR instrument, has lower cost, does not need higher cost, has the advantages of low cost, high feasibility and high efficiency, and has the advantage of wide application range because the genotyping is the routine operation of experimental animals.
It will be understood that the invention has been described in terms of several embodiments, and that various changes and equivalents may be made to these features and embodiments by those skilled in the art without departing from the spirit and scope of the invention. Modifications may be made to the features and embodiments of the invention in light of the teachings of the invention to adapt a particular situation or material to the teachings of the invention without departing from the essential scope thereof. The described embodiments of the invention are some, but not all, embodiments of the invention. The components of the embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations. Thus, the following detailed description of the embodiments of the invention, as presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed, but that the invention will include all other embodiments falling within the scope of the invention as defined by the appended claims, as interpreted according to the breadth to which they are fairly set forth in the claims.
Claims (10)
1. The gene amplification extraction tube is characterized by being placed in a PCR instrument to realize amplification and extraction integrated operation of a gene sample, and comprises a cracking cavity and a reaction cavity, wherein the cracking cavity can be embedded into the reaction cavity, and the cracking cavity is detachably connected with the reaction cavity; the lysis cavity is provided with a first accommodating cavity for accommodating a gene sample and performing tissue lysis reaction, and the reaction cavity is provided with a second accommodating cavity for accommodating PCR reaction liquid and can be used for performing PCR amplification reaction; the bottom or the side wall of the first accommodating cavity is provided with a plurality of small holes, a plurality of iron particles are arranged in the first accommodating cavity, the iron particles are provided with rough surfaces, and the iron particles can penetrate through the small holes from the first accommodating cavity to bring a gene sample in the first accommodating cavity into the second accommodating cavity under the action of an external magnetic field.
2. The gene amplification extraction tube of claim 1, wherein the lysis chamber further comprises a third accommodating chamber, small holes are formed in the bottoms or the side walls of the third accommodating chamber and the first accommodating chamber, the corresponding reaction chamber further comprises a fourth accommodating chamber, and the second accommodating chamber and the fourth accommodating chamber are spatially separated by a partition plate;
the third accommodating cavity corresponds to the fourth accommodating cavity, the third accommodating cavity can be embedded into the fourth accommodating cavity, the first accommodating cavity corresponds to the second accommodating cavity, and the first accommodating cavity can be embedded into the second accommodating cavity.
3. The gene amplification extraction tube of claim 1 or 2, wherein the particle size range of the iron microparticles is controlled to be less than 1.0 mm.
4. The gene amplification extraction tube of claim 3, wherein the surface of the iron microparticles has a plurality of recessed structures, and the recessed structures of the iron microparticles are capable of carrying a gene sample located in the first receiving chamber.
5. The gene amplification extraction tube of claim 3, wherein the surface of the iron microparticles has a plurality of protrusions, and the plurality of protrusions enclose a plurality of slit structures with each other, and the slit structures are capable of carrying the gene sample located in the first receiving chamber.
6. The gene amplification extraction tube of claim 3, wherein the iron microparticles have a cavity therein, the surface of the iron microparticles having a plurality of holes extending from the surface of the iron microparticles toward the cavity, the cavity and holes being capable of carrying a gene sample within the first receiving chamber.
7. The gene amplification extraction tube of claim 1 or 2, wherein the cleavage cavity and the reaction cavity are detachably connected by a connection structure;
the connecting structure comprises a sample adding cavity, the radial dimension of the sample adding cavity is matched with the radial dimension of the opening of the second accommodating cavity, a clamping groove is formed in the bottom edge of the sample adding cavity, a buckle is arranged at the edge of the opening of the second accommodating cavity, and the buckle is clamped with the clamping groove to realize detachable connection of the cracking cavity and the reaction cavity;
the first holding chamber fixed connection in loading chamber bottom center department, just first holding chamber with loading intracavity portion is linked together, the top of loading chamber is provided with sealed lid, sealed lid with loading chamber detachable connection for open the loading chamber, to the interior loading of first holding chamber.
8. A nucleic acid amplification and extraction device comprising the gene amplification and extraction tube according to any one of claims 1 to 7, wherein an electromagnetic coil is sleeved outside the gene amplification and extraction tube and is used for generating a magnetic field in a fixed direction under the action of electric power of an external power supply, and the magnetic field can enable iron particles carrying a gene sample to pass through the small hole and enter the second accommodating cavity from the first accommodating cavity.
9. The nucleic acid amplification and extraction device according to claim 8, wherein the nucleic acid amplification and extraction device comprises an electromagnetic frame, a plurality of electromagnetic coils are arranged on the electromagnetic frame, each electromagnetic coil can generate a magnetic field in a fixed direction under the action of electric power of an external power supply, and the directions of the magnetic fields generated by the electromagnetic coils are the same; a gene amplification extraction tube can be placed in each electromagnetic coil.
10. A gene identification method using the nucleic acid amplification extraction apparatus according to claim 8 or 9, characterized by comprising the steps of:
s1, preparing work before identification; taking a gene amplification extraction tube, adding a uniformly mixed PCR reaction solution into a second accommodating cavity of a reaction cavity for subsequent PCR reaction, then nesting a cracking cavity back into the reaction cavity, and placing the reaction cavity in a refrigerator for standby;
s2, preprocessing a gene sample; separating a cracking cavity and a reaction cavity of the gene amplification extraction tube, adding a gene sample into a first accommodating cavity of the cracking cavity, and temporarily placing the separated reaction cavity comprising PCR reaction liquid in a 4-DEG environment for later use;
s3, tissue lysis of the gene sample; the first accommodating cavity is internally provided with iron particles, tissue lysate is added into the first accommodating cavity, and after shaking, the tissue lysate is embedded into the reaction cavity;
s4, amplifying and extracting a gene sample; placing the gene amplification extraction tube obtained in the step S3 into a PCR instrument, executing a PCR digestion program, installing an electromagnetic coil on the periphery of the gene amplification extraction tube, then switching on an external power supply of the electromagnetic coil, transferring microbeads in tissue fluid into a reaction cavity, and entering the PCR amplification program;
s5, identifying a gene sample; and carrying out gene identification on PCR amplification products in a gene amplification extraction tube after amplification and extraction of the gene sample.
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