CN116445258A - PCR tube and detection method thereof applied to target nucleic acid molecules - Google Patents
PCR tube and detection method thereof applied to target nucleic acid molecules 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention is used in the technical field of nucleic acid detection, in particular to a PCR tube and a detection method of the PCR tube applied to target nucleic acid molecules, wherein the PCR tube comprises a tube body; the first isolation block is arranged in the pipe body and is provided with a first notch; the second isolation block is arranged in the pipe body and positioned below the first isolation block, and a second notch is formed in the second isolation block; a cell lysate comprising magnetic cellulose microspheres; the cleaning buffer solution is arranged between the first isolation block and the second isolation block; PCR reaction reagent. Such PCR tubes can reduce the washing steps and time and the detection of fully enclosed nucleic acid amplification. A PCR tube is applied to a detection method of target nucleic acid molecules, and a sample liquid is added to a tube body; the magnet is utilized to drive the magnetic cellulose microsphere to enter the cleaning buffer solution; using a magnet to drive the magnetic cellulose microsphere to enter a PCR reaction reagent; the PCR tube is put into a PCR amplification instrument for amplification reaction, and the tube body is irradiated by a corresponding fluorescent probe to observe whether fluorescence is emitted in the PCR reaction reagent.
Description
Technical Field
The invention is used in the technical field of nucleic acid detection, and in particular relates to a PCR tube and a detection method of the PCR tube applied to target nucleic acid molecules.
Background
The existing PCR (Polymerase Chain Reaction) -based nucleic acid detection technology has important application value in the fields of basic biological research, disease diagnosis, judicial identification, food safety and the like. Typical PCR nucleic acid detection processes generally include multiple steps of sample pretreatment, nucleic acid extraction, and PCR amplification detection. The extraction of nucleic acid is a very critical step in the PCR detection process, and the operations of capturing and adsorbing nucleic acid molecules, cleaning and purifying the nucleic acid molecules and the like need to be completed, so that the quality and the efficiency of the nucleic acid directly influence the detection result. The conventional nucleic acid extraction method mainly comprises a silica gel membrane centrifugal column method and a silica magnetic bead method, and the magnetic bead method is easier to realize automation, so that the current commercialized nucleic acid extraction instrument adopts magnetic beads coated by silica to realize nucleic acid extraction, and the nucleic acid extraction method based on the silica magnetic beads usually involves a plurality of washing and eluting steps, and the whole flow operation is complex, consumes long time and has high requirements on environment and operators. Although automated nucleic acid extractors greatly reduce the intensity of experimenters, they tend to be more expensive and are not affordable to common laboratories, community hospitals and small clinics; meanwhile, the nucleic acid extraction method based on large instrument equipment cannot be applied to on-site instant detection.
In addition, at present, two steps of nucleic acid extraction and PCR amplification detection of laboratories and clinical laboratory are often separated, and in the operation process, a plurality of sample tubes need to be uncapped for sample transfer, reagent addition and other operations, and the uncapping operation is extremely easy to cause aerosol pollution, and when pathogenic bacteria or viruses are detected, certain safety risks exist for operators.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems existing in the prior art. Therefore, the invention provides a PCR tube and a detection method of the PCR tube applied to target nucleic acid molecules, and the adopted technical scheme is as follows.
A PCR tube comprises
The pipe body is provided with a pipe cover;
the first isolation block is arranged in the pipe body and is provided with a first notch;
the second isolation block is arranged in the pipe body, is positioned below the first isolation block and is spaced from the first isolation block, and a second notch is formed in the second isolation block;
a cell lysate, the cell lysate being disposed above the first spacer, the cell lysate comprising magnetic cellulose microspheres;
the cleaning buffer solution is arranged between the first isolation block and the second isolation block;
and the PCR reaction reagent is arranged below the second isolation block.
The PCR tube provided by the embodiment of the invention has at least the following beneficial effects: the cell lysate is used for realizing cell lysis of a sample to be tested so as to release nucleic acid molecules therein, the magnetic cellulose microspheres can quickly adsorb nucleic acid in the cell lysate, and can not be eluted when being washed in a washing buffer solution for a short time, dNTPs in the magnetic cellulose microspheres can promote the adsorbed nucleic acid to be released from the magnetic cellulose microspheres into a solution after the magnetic cellulose microspheres adsorbing the nucleic acid are transferred into a PCR reaction reagent, the nucleic acid is not required to be released in a mode of adjusting the pH value or the salt concentration of the buffer solution, the complex elution process is avoided, the experimental operation of the nucleic acid extraction process is greatly simplified, the integrated whole-process closed operation of nucleic acid separation and extraction and PCR amplification detection in the PCR detection process can be simply and quickly realized, the whole process does not depend on an expensive nucleic acid extractor, the washing step and the time are greatly reduced, the nucleic acid elution step is not required, and the sample liquid or the reagent transfer operation is not required.
According to other embodiments of the invention, the cell lysate is separated from the wash buffer by a spacer oil, and the wash buffer is separated from the PCR reagents by a spacer oil.
According to other embodiments of the present invention, the first notch and the second notch are staggered.
According to other embodiments of the invention, the magnetic cellulose microsphere is a cellulose particle comprising micro-nano particles of ferroferric oxide or iron powder.
According to the PCR tube of other embodiments of the present invention, the surface of the magnetic cellulose microsphere is porous.
According to still further embodiments of the present invention, when the sample to be detected is a mammalian cell, plant cell or bacterial sample, the cell lysate is a solution comprising one or more of guanidinium thiocyanate, lysozyme, proteinase K, ethylenediamine tetraacetic acid, sodium dodecyl sulfate and RNAse enzyme.
According to other embodiments of the present invention, the washing buffer is a solution of tris (hydroxymethyl) aminomethane containing a surfactant or a solution containing one or more of guanidine hydrochloride, nitroglucose, isopropanol, or an ethanol solution with a mass fraction of 50% or 70%, or pure water.
According to other embodiments of the invention, the PCR reaction reagent is a mixture comprising amplification primers, DNA polymerase, deoxyribonucleotides, fluorescent probes or fluorescent dyes, and PCR reaction buffer.
According to other embodiments of the present invention, the spacer oil is one or more of fluorinated oil, mineral oil, silicone oil, or vegetable oil.
A method for detecting a target nucleic acid molecule by using a PCR tube according to any one of the above, comprising the steps of:
opening the tube cover, adding the sample liquid into the cell lysate of the tube body, covering the tube cover, slightly shaking the tube body and incubating;
placing a magnet at one side of the tube body near the first notch, attracting the magnetic cellulose microspheres in the cell lysis solution through the magnet, enabling the magnetic cellulose microspheres to be concentrated at the tube wall of the first notch, moving the magnet, driving the magnetic cellulose microspheres to enter the cleaning buffer solution from the first notch by using the magnet, then removing the magnet, slightly shaking the tube body, and enabling the magnetic cellulose microspheres to be dispersed in the cleaning buffer solution;
placing a magnet at one side of the tube body near the second notch, attracting the magnetic cellulose microspheres in the cleaning buffer solution to concentrate to the tube wall of the second notch, moving the magnet, driving the magnetic cellulose microspheres to enter the PCR reaction reagent from the second notch by using the magnet, then removing the magnet, and shaking the tube body to disperse the magnetic cellulose microspheres in the PCR reaction reagent;
and (3) placing the PCR tube into a PCR amplification instrument for thermal cycle amplification reaction, irradiating the tube body by using monochromatic light with the excitation wavelength corresponding to the fluorescent probe after the amplification reaction is finished, and judging whether target nucleic acid molecules exist in the sample liquid by observing whether fluorescent light is emitted in the PCR reaction reagent of the tube body.
Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
FIG. 1 is a schematic diagram showing the structure of a PCR tube according to an embodiment of the present invention;
FIG. 2 is a schematic view of the first spacer and the second spacer in the embodiment of FIG. 1;
FIG. 3 is a flow chart of assembling a PCR tube in one embodiment of the present invention;
FIG. 4 is a flow chart of a method for detecting a target nucleic acid molecule according to an embodiment of the present invention.
Detailed Description
The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention.
In the description of the embodiments of the present invention, if an orientation description such as "upper", "lower", "front", "rear", "left", "right", etc. is referred to, it is merely for convenience of description and simplification of the description, and it is not indicated or implied that the apparatus or element referred to must have a specific orientation, be configured and operated in a specific orientation, and thus should not be construed as limiting the present invention.
In the description of the embodiments of the present invention, if a feature is referred to as being "disposed", "fixed", "connected" or "mounted" on another feature, it can be directly disposed, fixed or connected to the other feature or be indirectly disposed, fixed or connected or mounted on the other feature. In the description of the embodiments of the present invention, if "several" is referred to, it means more than one, if "multiple" is referred to, it is understood that the number is not included if "greater than", "less than", "exceeding", and it is understood that the number is included if "above", "below", "within" is referred to. If reference is made to "first", "second" it is to be understood as being used for distinguishing technical features and not as indicating or implying relative importance or implicitly indicating the number of technical features indicated or implicitly indicating the precedence of the technical features indicated.
The existing PCR (Polymerase Chain Reaction) -based nucleic acid detection technology has important application value in the fields of basic biological research, disease diagnosis, judicial identification, food safety and the like. Typical PCR nucleic acid detection processes generally include multiple steps of sample pretreatment, nucleic acid extraction, and PCR amplification detection. The extraction of nucleic acid is a very critical step in the PCR detection process, and the operations of capturing and adsorbing nucleic acid molecules, cleaning and purifying the nucleic acid molecules and the like need to be completed, so that the quality and the efficiency of the nucleic acid directly influence the detection result. The conventional nucleic acid extraction method mainly comprises a silica gel membrane centrifugal column method and a silica magnetic bead method, and the magnetic bead method is easier to realize automation, so that the current commercialized nucleic acid extraction instrument adopts magnetic beads coated by silica to realize nucleic acid extraction, and the nucleic acid extraction method based on the silica magnetic beads usually involves a plurality of washing and eluting steps, and the whole flow operation is complex, consumes long time and has high requirements on environment and operators. Although the automated nucleic acid extractor greatly reduces the working intensity of laboratory staff, the price is often expensive, common laboratories, community hospitals and small clinics are difficult to bear, and meanwhile, the nucleic acid extraction method based on large instrument equipment cannot be applied to on-site instant detection. Although automated nucleic acid extractors greatly reduce the intensity of experimenters, they tend to be more expensive and are not affordable to common laboratories, community hospitals and small clinics; meanwhile, the nucleic acid extraction method based on large instrument equipment cannot be applied to on-site instant detection.
In addition, at present, two steps of nucleic acid extraction and PCR amplification detection of laboratories and clinical laboratory are often separated, and in the operation process, a plurality of sample tubes need to be uncapped for sample transfer, reagent addition and other operations, and the uncapping operation is extremely easy to cause aerosol pollution, and when pathogenic bacteria or viruses are detected, certain safety risks exist for operators.
Referring to fig. 1 to 4, the invention provides a PCR tube, comprising a tube body 1, a first isolation block 2, a second isolation block 3, a cell lysate 4, a cleaning buffer 5 and a PCR reaction reagent 6, wherein a tube cover 7 is arranged on the tube body 1, the first isolation block 2 is arranged inside the tube body 1, a first notch 8 is arranged on the first isolation block 2, the second isolation block 3 is arranged inside the tube body 1, the second isolation block 3 is arranged below the first isolation block 2 and is mutually spaced from the first isolation block 2, a second notch 9 is arranged on the second isolation block 3, the cell lysate 4 is arranged above the first isolation block 2, the cell lysate 4 contains magnetic cellulose microspheres 10, the cleaning buffer 5 is arranged between the first isolation block 2 and the second isolation block 3, and the PCR reaction reagent 6 is arranged below the second isolation block 3.
The cell lysate 4 is used for realizing cell lysis of a sample to be tested so as to release nucleic acid molecules therein, the magnetic cellulose microsphere 10 can quickly adsorb nucleic acid in the cell lysate 4, but can not be eluted when the magnetic cellulose microsphere 10 adsorbing the nucleic acid is washed in the washing buffer solution 5 for a short time, dNTPs in the magnetic cellulose microsphere can promote the release of the adsorbed nucleic acid from the magnetic cellulose microsphere 10 into a solution after the magnetic cellulose microsphere 10 is transferred into the PCR reaction reagent 6, the nucleic acid is not required to be released in a mode of adjusting the pH value or the salt concentration of the buffer solution, the tedious elution process is avoided, the experimental operation of the nucleic acid extraction process is greatly simplified, the integrated operation of nucleic acid separation and extraction and PCR amplification detection in the PCR detection process and the whole-process closed operation can be simply and quickly realized, the whole process does not depend on an expensive nucleic acid extractor, the washing step and the time are greatly reduced, and the sample solution or the reagent transfer operation is not required.
The first isolation block 2 and the second isolation block 3 are cylindrical or round table-shaped elastic isolation blocks manufactured through a reverse molding or injection molding process, the first notch 8 and the second notch 9 are semi-open cone shapes formed on the side edges of the isolation blocks, the diameter of the cross section of each notch is 1-2 mm, and when the isolation blocks are placed in the pipe body 1, the side with the larger cross section of each conical notch faces upwards.
In some embodiments, the first notch 8 and the second notch 9 are offset.
Specifically, the first notch 8 faces the left side of the tube body 1, the second notch 9 faces the right side of the tube body 1, and the liquid flow path between the first notch 8 and the second notch 9 is extended.
In some embodiments, the magnetic cellulose microsphere 10 is a cellulose particle comprising micro-nano particles of ferroferric oxide or iron powder.
Specifically, the magnetic cellulose microsphere 10 is prepared by the technological processes of oil phase emulsification, regeneration precipitation, washing and freeze drying of cellulose solution mixed with ferroferric oxide micro-nano particles or iron powder, and the diameter of the magnetic cellulose microsphere 10 is between 5 and 100 mu m.
In some embodiments, the surface of the magnetic cellulose microsphere 10 is porous.
Because the cellulose material has stronger specific adsorption capacity to nucleic acid molecules, and the prepared magnetic cellulose microsphere 10 is of a porous structure and has extremely large effective adsorption surface area, the nucleic acid adsorption capacity of the magnetic cellulose microsphere 10 is stronger, and the dynamic process of capturing the nucleic acid molecules by the magnetic cellulose microsphere 10 has the characteristics of quick adsorption and slow release.
To prevent mixing of the cell lysate 4, the wash buffer 5 and the PCR reaction reagent 6, in some embodiments, the cell lysate 4 and the wash buffer 5 are separated by a spacer oil 11, and the wash buffer 5 and the PCR reaction reagent 6 are separated by a spacer oil 11. Thus, by utilizing the characteristic that the oil phase and the water are mutually insoluble and the action of the interfacial tension of the oil/water, the stable physical separation and layered storage of the cell lysate 4, the cleaning buffer 5 and the PCR reaction reagent 6 can be realized.
In some embodiments, barrier oil 11 is one or more of a fluorinated oil, a mineral oil, a silicone oil, or a vegetable oil.
In some embodiments, the wash buffer 5 is a solution of tris containing a surfactant or a solution containing one or more of guanidine hydrochloride, nitroglucose, isopropanol, or an ethanol solution with a mass fraction of 50% or 70%, or pure water. This allows rapid elution of non-specifically adsorbed proteins, salts or other impurities from the surface of the magnetic cellulose microsphere 10.
In some embodiments, where the sample to be tested is a mammalian cell, plant cell or bacterial sample, the cell lysate 4 is a solution comprising one or more of guanidinium thiocyanate, lysozyme, proteinase K, ethylenediamine tetraacetic acid, sodium dodecyl sulfate, and RNAse enzyme.
In some embodiments, PCR reaction reagent 6 is a mixture comprising amplification primers, DNA polymerase, deoxyribonucleotides, fluorescent probes or fluorescent dyes, PCR reaction buffer.
The assembly flow of the PCR tube is as follows:
as shown in FIG. 3 (a), a 200. Mu.L PCR tube sterilized at high temperature was taken, and 20 to 30. Mu.L of PCR reagent 6 was added dropwise to the tube;
as shown in fig. 3 (b), a second spacer 3 prepared and sterilized at high temperature is inserted into a PCR tube into which the PCR reaction reagent 6 has been introduced by tweezers, the bottom of the second spacer 3 is brought into contact with the upper surface of the PCR reaction reagent 6, the second spacer 3 is gently pressed, and air between the PCR reaction reagent 6 and the second spacer 3 is discharged through a second notch 9 of the second spacer 3;
as shown in fig. 3 (c), 30 μl of spacer oil 11 is dropped into the tube so as to submerge the second spacer 3;
as shown in FIG. 3 (d), 30. Mu.L of the washing buffer 5 was slowly dropped into the tube;
as shown in fig. 3 (e), the prefabricated and high-temperature sterilized first spacer 2 is inserted into the tube 1 to which the washing buffer 5 has been added by tweezers, the bottom of the first spacer 2 is brought into contact with the upper surface of the washing buffer 5, the first spacer 2 is gently pressed, and the air between the washing buffer 5 and the first spacer 2 is discharged through the first notch 8 of the first spacer 2;
as shown in fig. 3 (f), 20 μl of spacer oil 11 is dropped into the tube body 1 so as to submerge the first spacer block 2;
as shown in fig. 3 (g), 20 μl of the cell lysate 4 containing the magnetic cellulose microspheres 10 was slowly dropped into the tube body 1;
as shown in fig. 3 (h), the tube cover 7 is covered, and the assembled PCR tube is put into a refrigerator for low-temperature preservation, so that the PCR tube can be taken and used.
The invention also provides a detection method of the PCR tube applied to target nucleic acid molecules, which adopts any one of the PCR tubes and comprises the following steps:
opening the tube cover 7, adding the sample solution to the cell lysate 4 of the tube body 1, covering the tube cover 7, slightly shaking the tube body 1 and incubating;
placing a magnet at one side of the tube body 1 near the first notch 8, attracting the magnetic cellulose microspheres 10 in the cell lysate 4 through the magnet, enabling the magnetic cellulose microspheres 10 to be concentrated at the tube wall of the first notch 8, moving the magnet, driving the magnetic cellulose microspheres 10 to enter the cleaning buffer solution 5 from the first notch 8 by using the magnet, removing the magnet, and slightly shaking the tube body 1 to enable the magnetic cellulose microspheres 10 to be dispersed in the cleaning buffer solution 5;
placing a magnet at one side of the tube body 1 near the second notch 9, attracting the magnetic cellulose microspheres 10 in the cleaning buffer solution 5 to concentrate at the tube wall of the second notch 9, moving the magnet, driving the magnetic cellulose microspheres 10 to enter the PCR reaction reagent 6 from the second notch 9 by using the magnet, then removing the magnet, and shaking the tube body 1 to disperse the magnetic cellulose microspheres 10 in the PCR reaction reagent 6;
and (3) placing the PCR tube into a PCR amplification instrument for thermal cycle amplification reaction, irradiating the tube body 1 with monochromatic light corresponding to the excitation wavelength of the fluorescent probe after the amplification reaction is finished, and judging whether target nucleic acid molecules exist in the sample liquid by observing whether the PCR reaction reagent 6 in the tube body 1 emits fluorescence or not.
The method comprises the following specific steps:
(1) As shown in fig. 4 (a), taking an assembled PCR tube, opening the tube cover 7, adding 10-20 μl of blood sample liquid to be detected into the tube cover, covering the tube cover 7, slightly shaking the PCR tube, and standing for 1-5 minutes to complete the lysis of blood cells and the adsorption of nucleic acid by the magnetic cellulose microsphere 10;
(2) As shown in fig. 4 (b), a magnet is placed on one side, close to the first notch 8, of the outer side of the pipe body 1, so that the magnet gathers the magnetic cellulose microspheres 10 adsorbed with nucleic acid beside the pipe wall of the first notch 8, and then the magnet slowly moves downwards, so that the magnet pulls the magnetic cellulose microspheres 10 to break through interfacial tension between oil and water phases, and the magnetic cellulose microspheres enter the cleaning buffer solution 5 from the first notch 8 through the isolation oil 11;
(3) After the magnetic cellulose microspheres 10 enter the washing buffer 5, the magnet is removed and the tube 1 is gently shaken to disperse the magnetic cellulose microspheres 10 in the washing buffer 5, and then the PCR tube is left for about 10 to 30 seconds, as shown in FIG. 4 (c);
(4) As shown in fig. 4 (d), a magnet is placed on one side, close to the second notch 9, of the outer side of the tube body 1, so that the magnet gathers the cleaned magnetic cellulose microspheres 10 beside the tube wall of the second notch 9, and then the magnet slowly moves downwards, so that the magnet pulls the magnetic cellulose microspheres 10 to break through interfacial tension between oil and water phases, separates the oil 11 phase and enters the PCR reaction reagent 6 from the second notch 9;
(5) As shown in fig. 4 (e), the magnet is removed, the PCR tube is gently shaken to disperse the magnetic cellulose microspheres 10 in the PCR reaction reagent 6, and then the PCR tube is placed on a PCR amplification apparatus for thermal cycle amplification (fig. 4 (f));
(6) As shown in fig. 4 (g), after the PCR thermal cycle reaction is completed, the PCR tube is taken out, a laser pen with a light emission wavelength corresponding to the absorption wavelength of the fluorescent group in the PCR reaction reagent 6 is taken out, the PCR reaction reagent 6 of the PCR tube is aligned and irradiated, meanwhile, a control PCR tube, i.e. the sample liquid is pure water, is also taken out, and the PCR reaction reagent 6 is irradiated with the laser light as described above (fig. 4 (h)), and if the bottom layer of the PCR tube of the blood sample to be detected emits visible fluorescence, and the bottom layer of the control PCR tube has no visible fluorescence, the blood sample to be detected can be judged to contain the target DNA.
In the description of the present specification, reference to the terms "one embodiment," "some embodiments," "illustrative embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The detection method of the target nucleic acid molecule based on the PCR tube can rapidly realize the integrated and whole-process closed operation of nucleic acid separation and extraction and PCR amplification detection in the PCR detection process, and the whole process does not depend on an expensive nucleic acid extractor, so that the cleaning step and time are greatly reduced, the nucleic acid elution step is not needed, and the sample liquid or reagent transfer operation is also not needed.
While embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that: many changes, modifications, substitutions and variations may be made to the embodiments without departing from the spirit and principles of the invention, the scope of which is defined by the claims and their equivalents.
Claims (10)
1. A PCR tube, characterized in that: comprising
The pipe body is provided with a pipe cover;
the first isolation block is arranged in the pipe body and is provided with a first notch;
the second isolation block is arranged in the pipe body, is positioned below the first isolation block and is spaced from the first isolation block, and a second notch is formed in the second isolation block;
a cell lysate, the cell lysate being disposed above the first spacer, the cell lysate comprising magnetic cellulose microspheres;
the cleaning buffer solution is arranged between the first isolation block and the second isolation block;
and the PCR reaction reagent is arranged below the second isolation block.
2. The PCR tube of claim 1 wherein: the cell lysate is separated from the cleaning buffer solution by isolating oil, and the cleaning buffer solution is separated from the PCR reaction reagent by isolating oil.
3. The PCR tube of claim 1 wherein: the first notch and the second notch are staggered.
4. The PCR tube of claim 1 wherein: the magnetic cellulose microsphere is a cellulose particle containing ferroferric oxide micro-nano particles or iron powder.
5. The PCR tube according to claim 4, wherein: the surface of the magnetic cellulose microsphere is of a porous structure.
6. The PCR tube of claim 1 wherein: when the sample to be detected is a mammalian cell, plant cell or bacterial sample, the cell lysate is a solution containing one or more of guanidinium thiocyanate, lysozyme, proteinase K, ethylenediamine tetraacetic acid, sodium dodecyl sulfate and RNAse enzyme.
7. The PCR tube of claim 1 wherein: the cleaning buffer solution is a solution of trimethylol aminomethane containing a surfactant or a solution containing one or more of guanidine hydrochloride, nitroglucose and isopropanol or an ethanol solution with the mass fraction of 50% or 70% or pure water.
8. The PCR tube of claim 1 wherein: the PCR reaction reagent is a mixed solution containing an amplification primer, DNA polymerase, deoxyribonucleotide, a fluorescent probe or fluorescent dye and a PCR reaction buffer solution.
9. The PCR tube of claim 1 wherein: the isolating oil is one or more of fluorinated oil, mineral oil, silicone oil or vegetable oil.
10. A method for detecting a target nucleic acid molecule using a PCR tube according to any one of claims 1 to 9, comprising the steps of:
opening the tube cover, adding the sample liquid into the cell lysate of the tube body, covering the tube cover, slightly shaking the tube body and incubating; placing a magnet at one side of the tube body near the first notch, attracting the magnetic cellulose microspheres in the cell lysis solution through the magnet, enabling the magnetic cellulose microspheres to be concentrated at the tube wall of the first notch, moving the magnet, driving the magnetic cellulose microspheres to enter the cleaning buffer solution from the first notch by using the magnet, then removing the magnet, slightly shaking the tube body, and enabling the magnetic cellulose microspheres to be dispersed in the cleaning buffer solution;
placing a magnet at one side of the tube body near the second notch, attracting the magnetic cellulose microspheres in the cleaning buffer solution to concentrate at the tube wall of the second notch through the magnet, moving the magnet, driving the magnetic cellulose microspheres to enter the PCR reaction reagent from the second notch by using the magnet, then removing the magnet, and shaking the tube body to disperse the magnetic cellulose microspheres in the PCR reaction reagent;
and (3) placing the PCR tube into a PCR amplification instrument for thermal cycle amplification reaction, irradiating the tube body by using monochromatic light with the excitation wavelength corresponding to the fluorescent probe after the amplification reaction is finished, and judging whether target nucleic acid molecules exist in the sample liquid by observing whether fluorescent light is emitted in the PCR reaction reagent of the tube body.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117210309A (en) * | 2023-11-09 | 2023-12-12 | 迈克生物股份有限公司 | Biological sample processing device and biological detection system |
| CN117282483A (en) * | 2023-11-23 | 2023-12-26 | 中国科学院空天信息创新研究院 | Analysis test tube and analysis device |
| CN117282482A (en) * | 2023-11-23 | 2023-12-26 | 中国科学院空天信息创新研究院 | Method for preparing test tube and method for using test tube |
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2023
- 2023-02-27 CN CN202310173522.0A patent/CN116445258A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117210309A (en) * | 2023-11-09 | 2023-12-12 | 迈克生物股份有限公司 | Biological sample processing device and biological detection system |
| CN117210309B (en) * | 2023-11-09 | 2024-01-26 | 迈克生物股份有限公司 | Biological sample processing device and biological detection system |
| CN117282483A (en) * | 2023-11-23 | 2023-12-26 | 中国科学院空天信息创新研究院 | Analysis test tube and analysis device |
| CN117282482A (en) * | 2023-11-23 | 2023-12-26 | 中国科学院空天信息创新研究院 | Method for preparing test tube and method for using test tube |
| CN117282483B (en) * | 2023-11-23 | 2024-02-02 | 中国科学院空天信息创新研究院 | Analysis test tube and analysis device |
| CN117282482B (en) * | 2023-11-23 | 2024-02-13 | 中国科学院空天信息创新研究院 | Method for preparing test tube and method for using test tube |
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