CN112251318A - Full-automatic nucleic acid extraction hybridization capture detection device based on magnetic separation - Google Patents
Full-automatic nucleic acid extraction hybridization capture detection device based on magnetic separation Download PDFInfo
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- CN112251318A CN112251318A CN201910663553.8A CN201910663553A CN112251318A CN 112251318 A CN112251318 A CN 112251318A CN 201910663553 A CN201910663553 A CN 201910663553A CN 112251318 A CN112251318 A CN 112251318A
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- 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
- C12Q1/6813—Hybridisation assays
- C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
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- 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
Abstract
The invention relates to an integrated full-automatic post-nucleic acid extraction liquid-phase hybridization capture detection device, which integrates the following core modules: the device comprises a nucleic acid pretreatment module, a reagent storage module, a nucleic acid hybridization module, a magnetic separation module, a pipetting module and a detection module. All modules surround: the task flows of nucleic acid extraction, nucleic acid denaturation, nucleic acid hybridization, signal generation and detection are operated, and the target of full-automatic nucleic acid liquid phase hybridization capture detection is finally realized.
Description
Technical Field
The invention relates to an integrated full-automatic post-nucleic acid extraction liquid-phase hybridization capture detection device, which integrates the following core modules: the device comprises a nucleic acid pretreatment module, a reagent storage module, a nucleic acid hybridization module, a magnetic separation module, a pipetting module and a detection module. All modules surround: the task flows of nucleic acid extraction, nucleic acid denaturation, nucleic acid hybridization, signal generation and detection are operated, and the target of full-automatic nucleic acid liquid phase hybridization capture detection is finally realized.
Background
Nucleic acid hybridization detection is one of the earliest means of nucleic acid detection. The principle is the theory of nucleic acid denaturation and renaturation, and in the renaturation process of the denatured nucleic acid, complementary nucleotide sequences (DNA and DNA, DNA and RNA, RNA and the like) are paired with each other through Watson-Crick bases to form non-covalent bonds, so that a stable homoduplex or heteroduplex molecule is formed. The classical nucleic acid hybridization method is that DNA or RNA is transferred and fixed on a nitrocellulose or nylon membrane carrier, a complementary single-stranded DNA or RNA probe is labeled with radioactivity or non-radioactivity, the probe is combined with a complementary target sequence through hydrogen bonds during hybridization on the membrane, unbound free probe is washed away, and the specifically bound probe is detected through autoradiography or color reaction.
The nucleic acid hybridization method mainly comprises the following steps: southern blot hybridization (Southern blot), Northern blot hybridization (Northern blot), Dot blot, in situ hybridization (FISH), and PCR reverse hybridization. The above-mentioned nucleic acid hybridization methods all require the use of a solid phase carrier to immobilize the target nucleic acid to be detected or to immobilize a nucleic acid probe, and then to hybridize with the probe having a signal label, thereby detecting the target nucleic acid sequence. The solid phase carrier can be prepared through a complicated experiment process, the operation process of the experiment is complicated, the result judgment mode is difficult to unify, the standard interpretation cannot be carried out, and the full-automatic detection is difficult to realize. Furthermore, since the nucleic acid sequence on the solid phase carrier is immobilized, the probability of contact and collision with the target nucleic acid molecule in the liquid phase during hybridization is limited, and the sensitivity is affected. Many disadvantages limit the application of hybridization methods in many situations, such as in vitro diagnostics, food safety, etc., where accurate and rapid results under uniform criteria are required, and hybridization methods only account for a small proportion.
Nucleic acid detection is carried out by a nucleic acid hybridization capture-based method, and the method is commercially applied at present. The German Qiagen HC2 method HPV nucleic acid detection kit is a typical representative. The kit is a method for detecting HPV nucleic acid based on a nucleic acid hybridization capture method, and can judge the existence of target nucleic acid to be detected by coating an anti-nucleic acid antibody on an enzyme label plate, adding reaction liquid containing a nucleic acid sequence to be detected and a probe nucleic acid sequence complementary with the nucleic acid sequence to be detected, hybridizing the nucleic acid sequence and the probe nucleic acid sequence to form a double-chain compound, capturing the double-chain compound by the antibody on the enzyme label plate, adding a second anti-double-chain nucleic acid antibody linked with alkaline phosphatase, and performing luminous color development by a substrate. The DH2 and DH3 kits of Hangzhou Desheng biology GmbH also adopt similar technical principles to detect HPV nucleic acid.
Another hybridization diagnostic technique widely used in the medical diagnostic field is the FISH fluorescence in situ hybridization technique, which is used for the diagnosis of diseases associated with chromosomal abnormalities. After the hybridization probe with the fluorescent label is hybridized with the lesion specific nucleic acid sequence fixed in the cell, the composition of a fluorescent signal is observed through a fluorescent microscope, and whether the gene abnormality exists on the chromosome is judged. The operation steps of the technology are very complicated, the whole detection process takes 1-3 days, the diagnosis efficiency is low, and even if the full-automatic FISH detection device is applied, the detection completion of the full-flow operation of the technology takes a long time.
Although hybridization techniques have been successfully commercialized for the field of disease diagnosis, current hybridization diagnostic techniques, whether HC2, DH2, DH3 or FISH, have short panels in common: the detection of full-automatic continuous samples cannot be realized, and the application field of the detection is limited. HC2, DH2 and DH3 are only applied to detection of HPV, and FISH technology is only applied to detection of solid phase cells as carriers. In the wider fields of infectious diseases, clinical microorganism identification and the like, the development and application of mature nucleic acid hybridization detection products are not available at present. Particularly in the field of identification of clinical microorganisms, morphological identification can be carried out only by culturing for 2-3 days in a microorganism culture medium in the conventional operation, and if the device is used for carrying out hybridization capture detection on a microorganism specific gene sequence, the time consumption is short, the efficiency is high, and the accuracy is high.
Disclosure of Invention
The invention provides a device capable of fully automatically carrying out nucleic acid extraction, hybridization, capture and detection. The device comprises the following core modules: the device comprises a nucleic acid pretreatment module, a reagent storage module, a nucleic acid hybridization module, a magnetic separation module, a pipetting module and a detection module.
A nucleic acid pretreatment module: the module is changed into a magnetic bead method nucleic acid extraction device, and the nucleic acid molecules in the sample to be detected are enriched and purified in the module through steps of cracking, adsorption, elution and the like.
A reagent storage module: the module has the function of storing various reaction systems required by full-automatic nucleic acid extraction and hybridization reaction, including lysis solution, magnetic beads for nucleic acid extraction, magnetic particle probes, signal probe reagents, buffer solution, washing solution, nucleic acid eluent, substrate solution and the like.
A nucleic acid hybridization module: the module comprises two sub-modules, wherein the first sub-module is a temperature control module, and the second sub-module is a hybridization reaction container module; the temperature control module realizes the accurate control of the temperature of the reaction system during hybridization, and the hybridization reaction container module is mutually embedded with the temperature control module to realize the control of the temperature of the hybridization reaction system in the reaction container module by the temperature control module. Under the control of temperature, a hybrid complex of a magnetic particle probe, a target nucleic acid sequence and a signal probe is generated through a hybridization reaction.
A magnetic separation module: the module functions to generate a magnetic field force for separating the magnetic particle hybridization complex from the liquid phase environment. The specific implementation way can be selected from two schemes. The first approach is to embed a permanent magnetic material in the module, wherein the permanent magnetic material is provided with magnetic field force; the second way is electromagnetic field force, which is generated when the electromagnetic device is powered on and disappears when the power is powered off. When the magnetic field force selects the built-in permanent magnetic material, the interaction between the magnetic field force and the nucleic acid hybridization module realizes the influence of the magnetic field force of the module on the nucleic acid hybridization module through the mechanical displacement of the mechanical transfer device; when the magnetic field force selects the electromagnetic field force, the module can be directly attached to the nucleic acid hybridization module, and the power supply is switched on or off as required, so that the influence of the module on the magnetic field force of the nucleic acid hybridization module is realized.
A pipetting module: the module functions to realize the transfer of various reaction liquids and/or reaction vessel components inside the apparatus, and the path of the transfer is set according to the program, including the transfer of the nucleic acid hybridization reaction system between the following modules: the nucleic acid pretreatment module, the reagent storage module, the nucleic acid hybridization module, the detection module, and the reagents in the reagent storage module are transferred to the respective modules. The implementation mode of liquid transfer comprises two modes, namely: temporarily sucking liquid from one carrier of one module into a channel by using the channel with the blowing and sucking functions, and blowing the temporarily sucked liquid out of the channel after transferring the liquid to another carrier of another module; the second method comprises the following steps: the container carrying the liquid-phase reaction system and the liquid-phase reaction system in the container are transferred together to another module using a mechanical moving arm, a conveyor belt, or a combination of a mechanical arm and a conveyor belt.
A detection module: the functional target is the capture and discrimination of a target signal, including the capture and discrimination of one or more of the following signals in combination: color signals and intensity thereof, photon signals and intensity thereof.
The working process among the modules of the detection device is as follows:
a) the nucleic acid pretreatment module extracts target nucleic acid in a sample to be detected by using magnetic beads and releases molecules of the nucleic acid to be detected into a liquid phase reaction system;
b) transferring a reaction system where target nucleic acid to be detected is located to a nucleic acid hybridization module by a liquid transfer module, starting a temperature control program by the hybridization module, and changing the temperature of the hybridization reaction system to denature target nucleic acid molecules;
c) the pipetting module sucks the magnetic particle hybridization probe and the signal probe from the reagent storage module into a hybridization reaction system, and adjusts the temperature of the nucleic acid hybridization module to ensure that the magnetic particle probe and the signal probe are hybridized with target nucleic acid to form a hybridization complex of the magnetic particle probe, the target nucleic acid molecule and the signal probe;
d) starting the magnetic separation module, stripping the hybrid complex from the liquid-phase reaction system by using a magnetic particle probe under the action of magnetic field force, transferring the residual liquid to a waste liquid pool by using a liquid transfer module, stopping the magnetic separation module, sucking washing liquid from a reagent storage module by the liquid transfer module, injecting the washing liquid into a container containing the magnetic particle hybrid complex for cleaning twice, starting the magnetic separation module again, separating the magnetic particle complex, and removing and discarding the waste liquid;
e) the pipetting module sucks the substrate from the reagent storage module, adds the substrate into a container where the magnetic particle complex is positioned, and completes the generation of a signal to be detected at a specified time and temperature;
f) and transferring the hybridization reaction system and the reaction carrier to a detection module, wherein the detection module converts the signal to be detected into a photoelectric signal and then presents the photoelectric signal in a numerical mode.
Drawings
FIG. 1: the functional regions of each module of the invention are schematic diagrams, and each functional region is as follows:
1-sample storage area; 2-sample storage unit of sample storage area; 3-nucleic acid pretreatment module; 4-reagent storage module; 5-magnetic separation module; 6-nucleic acid hybridization Module; 7-temperature control means in the nucleic acid hybridization module; 8-detection module; 9-mechanical pipetting module; 10-waste liquid storehouse; 11-hybridization reaction vessel (reaction cup, reaction tube)
The schematic diagram is only a functional overview of the modules of the present invention, and the structure and spatial arrangement of the modules are only used for illustrating the combination and association existing between the modules of the present invention, and are not limited by the structure and spatial position of the modules, and those skilled in the art can derive numerous combinations of spatial positions according to the schematic diagram.
Detailed Description
For further explanation of the association of the functional modules described in the present invention, the following embodiment describes the operation flow of each module in detail, and the flow described in the embodiment is only an illustration of the operation mechanism of the present invention, and the disclosure is not a limitation of the present invention, and those skilled in the art can derive various possible combinations according to the present invention.
Examples
As shown in fig. 1, the main unit of the full automatic detection device of the present invention at least comprises the following parts: the device comprises a sample storage area, a sample storage unit of the sample storage area, a nucleic acid front module, a reagent storage module, a magnetic separation module, a nucleic acid hybridization module, a temperature control device in the nucleic acid hybridization module, a detection module, a mechanical pipetting module and a waste liquid bin.
The sample storage area is used for storing samples, the code scanning recognition device is arranged in the sample storage area and can be used for recognizing identity information of the samples, and after sample information is recognized, when the nucleic acid pretreatment module is not in operation, the samples are transferred to the nucleic acid pretreatment module one by one through the mechanical transfer device to be subjected to nucleic acid extraction operation. In the nucleic acid pretreatment module, a plurality of fixed sample positions and a plurality of random sample positions are arranged, the fixed sample positions operate and process the conventional samples according to a set detection flow, and the random sample positions are used for performing emergency sample detection. When a sample to be detected is placed in the nucleic acid extraction working position in the module, the pipetting module is started, lysate is sucked from the reagent storage module and transferred to the sample working position, the oscillator attached to the whole module is started, after the oscillation operation is completed according to the program, the pipetting module is started, nucleic acid adsorption magnetic beads are sucked from the reagent storage module and added into the sample, and after oscillation is performed again, the magnetic separation module is started. The magnetic separation module is transferred to a working position of the nucleic acid pretreatment module from a standby position, and the two working modules are matched through a space structure and then started to separate magnetic beads. And the liquid transferring module is restarted to suck the waste liquid where the magnetic beads are positioned to be completely transferred to the waste liquid bin, and the liquid transferring module is restarted to suck eluent from the reagent storage module to clean the magnetic particles so as to lead the nucleic acid molecules adsorbed on the magnetic particles to fall off. And starting a sample transfer program, and transferring the sample after the nucleic acid extraction is finished to a corresponding working position of the nucleic acid hybridization module. Starting a temperature control device to control the temperature of a nucleic acid hybridization reaction system to a required temperature, starting a liquid transfer module to absorb a magnetic separation probe reagent and a signal probe reagent from a reagent storage module and add the reagents and the signal probe reagent into a nucleic acid hybridization position sample, after hybridization is completed according to time and temperature required by a program, starting the magnetic separation module to strip a hybridization complex from a liquid phase, simultaneously starting the liquid transfer module to transfer waste liquid to a waste liquid bin, starting a cleaning program, absorbing cleaning liquid from the reagent storage module and injecting the cleaning liquid into a magnetic particle reactor, starting the magnetic separation device again to separate the hybridization complex, and absorbing and transferring the waste cleaning liquid to the waste liquid bin. And starting a pipetting program, and sucking the substrate solution from the reagent storage module to the reaction vessel where the hybridization complex is positioned. Transferring the reactor with the injected substrate to a detection module, starting the detection device according to a set program, and collecting a reaction signal of the sample.
Claims (10)
1. A full-automatic nucleic acid extraction, hybridization, capture and detection device based on magnetic separation is characterized by comprising the following core modules: the device comprises a nucleic acid pretreatment module, a reagent storage module, a nucleic acid hybridization module, a magnetic separation module, a pipetting module and a detection module.
2. The nucleic acid pretreatment module of claim 1, which is used for purifying and extracting nucleic acid from a sample to be tested, so as to enrich and separate target nucleic acid molecules from a complex background environment of the sample, and is specifically a magnetic bead nucleic acid extraction device.
3. The reagent storage module of claim 1, which is capable of storing a lysis solution for hybridization, a magnetic bead for nucleic acid extraction, a magnetic particle probe, a signal probe reagent, a buffer solution, a washing solution, a nucleic acid eluent, and a substrate solution.
4. The nucleic acid hybridization module according to claim 1, which comprises two submodules, one being a temperature control module and the second being a hybridization reaction vessel module; the temperature control module realizes the accurate control of the temperature of the reaction system during hybridization, and the hybridization reaction container module is mutually embedded with the temperature control module to realize the control of the temperature of the hybridization reaction system in the reaction container module by the temperature control module.
5. A magnetic separation module according to claim 1, which is capable of generating magnetic force by embedding a permanent magnetic material within the module, the permanent magnetic material carrying the magnetic force itself; and the second way is electromagnetic field force, the module generates magnetic field force through an electromagnetic device, the magnetic field force is generated when the power supply is switched on, and the magnetic field force disappears when the power supply is switched off.
6. The magnetic separation module according to claim 5, which is linked to the nucleic acid hybridization module in a different manner according to the magnetic force generation pathway; when the magnetic field force selects the built-in permanent magnetic material, the interaction between the magnetic field force and the nucleic acid hybridization module realizes the influence of the magnetic field force of the module on the nucleic acid hybridization module through the mechanical displacement of the mechanical transfer device; when the magnetic field force selects the electromagnetic field force, the module can be directly attached to the nucleic acid hybridization module, and the power supply is switched on or off according to the requirement, so that the influence of the module on the magnetic field force of the nucleic acid hybridization module is realized.
7. The pipette module according to claim 1, which functions to effect transfer of a nucleic acid hybridization liquid phase reaction system by a path including transferring the nucleic acid hybridization reaction system to each other among: the device comprises a nucleic acid pretreatment module, a reagent storage module, a nucleic acid hybridization module and a detection module.
8. The pipetting module of claim 7, wherein the mode of transferring liquid between the modules comprises two modes, one: temporarily sucking liquid from one carrier of one module into a channel by using the channel with the blowing and sucking functions, and blowing the temporarily sucked liquid out of the channel after transferring the liquid to another carrier of another module; the second method comprises the following steps: and (3) transferring the container carrying the liquid phase reaction system and the liquid phase reaction system in the container to a corresponding physical position on the other module together by using a mechanical moving arm, a conveyor belt or a combination of the mechanical arm and the conveyor belt.
9. The detection module of claim 1, which functions as capture and discrimination of a target signal, including capture and discrimination of a combination of one or more of: color signal and its intensity, light signal and its intensity.
10. The detection apparatus according to claim 1, wherein the workflow between the modules is as follows:
(1) the nucleic acid pretreatment module extracts target nucleic acid in a sample to be detected by using magnetic beads and releases molecules of the nucleic acid to be detected into a liquid phase reaction system;
(2) transferring a reaction system where target nucleic acid to be detected is located to a nucleic acid hybridization module by a liquid transfer module, starting a temperature control program by the hybridization module, and changing the temperature of the hybridization reaction system to denature target nucleic acid molecules;
(3) the pipetting module sucks the magnetic particle hybridization probe and the signal probe from the reagent storage module into a hybridization reaction system, and adjusts the temperature of the nucleic acid hybridization module to ensure that the magnetic particle probe and the signal probe are hybridized with target nucleic acid to form a hybridization complex of the magnetic particle probe, the target nucleic acid molecule and the signal probe;
(4) starting the magnetic separation module, stripping the hybrid complex from the liquid-phase reaction system by using a magnetic particle probe under the action of magnetic field force, transferring the residual liquid to a waste liquid pool by using a liquid transfer module, stopping the magnetic separation module, sucking washing liquid from a reagent storage module by the liquid transfer module, injecting the washing liquid into a container containing the magnetic particle hybrid complex for cleaning twice, starting the magnetic separation module again, separating the magnetic particle complex, and removing and discarding the waste liquid;
(5) the pipetting module sucks the substrate from the reagent storage module, adds the substrate into a container where the magnetic particle complex is positioned, and completes the generation of a signal to be detected at a specified time and temperature;
(6) and transferring the hybridization reaction system and the reaction carrier to a detection module, wherein the detection module converts the signal to be detected into a photoelectric signal and then presents the photoelectric signal in a numerical mode.
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| US20050123933A1 (en) * | 2003-12-09 | 2005-06-09 | Asiagen Corporation | Assay systems, kits and methods for detecting microorganisms |
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