CN115261183A - Instant gene detection kit - Google Patents
Instant gene detection kit Download PDFInfo
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- CN115261183A CN115261183A CN202210612756.6A CN202210612756A CN115261183A CN 115261183 A CN115261183 A CN 115261183A CN 202210612756 A CN202210612756 A CN 202210612756A CN 115261183 A CN115261183 A CN 115261183A
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- 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
Abstract
The invention relates to a ready-to-use gene detection package, which comprises: 1. sampling tools, flocked or rough-surface swabs; 2. the device comprises a sample processing tube, a sample collecting tube and a sample collecting tube, wherein the sample processing tube contains lysis solution and magnetic beads; 3. the sample adding tool at least comprises a sample adsorption part and a sealing layer puncture part. 4. The amplification reaction tube is internally provided with a gene amplification detection liquid reaction system, the surface of the gene amplification detection liquid reaction system is provided with a sealing layer, and the sealing layer seals the gene amplification detection liquid reaction system in a fixed space in the amplification reaction tube; the instant gene detection kit of the invention can extract and detect genes without any professional instrument and equipment.
Description
Technical Field
The invention relates to the field of gene detection, in particular to a ready-to-use gene detection kit.
Background
In 1985, mullis et al, the institute of human genetics, PE-Centus, USA, invented the Polymerase Chain Reaction (PCR) technology, and the gene diagnosis technology became the most potential field, and the PCR technology has also taken the monopoly of the nucleic acid amplification reaction technology, and the technology has gained Nobel medical prize in 2003. However, the PCR technology is dependent on a fine instrument, and nucleic acid amplification cannot be performed without the instrument, which limits the wider application of the PCR technology. Under these circumstances, in recent years, researchers have developed a series of isothermal nucleic acid amplification techniques. Compared with PCR, the isothermal amplification of nucleic acid has the advantages of high speed and efficiency and no need of special instrument. Verification isothermal amplification techniques have been rapidly developed since their birth. Some techniques are mature and are gradually widely applied in the fields of inspection and quarantine, medicine, law and the like.
The mainstream constant temperature amplification technology of nucleic acid at present comprises: helicase dependent isothermal amplification (HAD), strand Displacement Amplification (SDA), cross-primer amplification (CPA), loop-mediated isothermal amplification of nucleic acids (LAMP), rolling Circle Amplification (RCA), single Primer Isothermal Amplification (SPIA). Researchers further develop a visual detection method of a constant-temperature amplification technology, for example, LAMP technology can directly judge results according to turbidity in a tube, and fluorescent dyes such as SYBR Green, pico Green, gene Finder, calcein and the like can be added, so HNB is also a common visual detection dye. With these means, nucleic acid detection can be achieved under very common experimental conditions, for example, under laboratory conditions with water baths, common nucleic acid sample processing equipment such as centrifuges, water baths, and pipettes.
However, based on the limitation of professional knowledge of home test equipment and operators, and since a home generally does not have an accurate pipetting tool, a centrifuge, etc., it is difficult to complete detection and achieve the purpose of judgment in a general home. Only by developing a nucleic acid detection tool which is easier for ordinary non-professional people to operate, nucleic acid detection can go into thousands of households.
Disclosure of Invention
The invention aims to solve the defects of the prior art and provide a simpler and easier-to-use nucleic acid detection tool platform which can be used for detecting nucleic acid in ordinary families or in the field without any special instrument and equipment, and of course, the invention can also reduce the gene detection cost of hospitals.
The invention first provides a ready-to-use gene detection kit, comprising:
sampling tools, flocked or rough-surface swabs; the device comprises a sample processing tube, a sample collecting tube and a sample collecting tube, wherein the sample processing tube contains lysis solution and magnetic beads; the sample adding tool at least comprises a sample adsorption part and a sealing layer puncture part. The amplification reaction tube is internally provided with a gene amplification detection liquid reaction system, the surface of the gene amplification detection liquid reaction system is provided with a sealing layer, and the sealing layer seals the gene amplification detection liquid reaction system in a fixed space in the amplification reaction tube; the instant gene detection kit of the invention can extract and detect genes without any professional instrument and equipment.
The sampling tool is used for wiping the sampling tool from the surface of the oral cavity, the nostril or an object to dip a sample to be detected.
The sample processing tube is used for cracking and releasing sample nucleic acid.
The magnetic beads in the sample processing tube adsorb nucleic acid in lysis solution and release nucleic acid in reaction solution.
The isothermal gene amplification detection liquid reaction system is sealed at the lower part of the isothermal amplification reaction tube by a sealing layer, and can be a layer or a plurality of layers.
The sample adding tool comprises a sample adsorption part and a sealing layer puncture part; preferably, the sample adsorption part and the sealing layer puncture part are located on the same member to constitute a sample-bearing puncture device.
And the sample adsorption part of the sample adding tool is used for adsorbing magnetic beads in the lysate.
The solid-liquid conversion starting point temperature of the sealing layer is above room temperature and below the amplification reaction temperature, and the density of the sealing layer at the amplification temperature is lower than that of the gene amplification detection liquid reaction system at the same temperature.
The material of the blocking layer may be selected from, but is not limited to: rubber, plastic, paraffin, biological glue or resin, etc.
The amplification reaction tube may be a single tube or a tube group formed by connecting a plurality of single tubes. In preferred embodiments, the individual tubes in the stack may be arranged in a linear or array.
The sealing component of the amplification reaction tube is not limited in form, and can be detachable or non-detachable, and the detachable sealing component includes but is not limited to: plug-in closures, envelopes, screw-on closures, and the like. The sample can be added after the sealing component which can not be disassembled is damaged, for example, the sample can be added after puncture.
The sample application tool can pierce the sealing layer to bring the gene of the sample into the reaction solution.
The gene amplification reaction may be isothermal or variable temperature.
The sample adsorption member is selected from: one or more of a sampling swab, a straw, a plastic stick or a cotton swab; the sample-adsorbing portion of the sample-carrying puncturing member is selected from: a straw structure composed of an air bag and a tube cavity communicated with the air bag, a sampling swab structure with a core material externally wrapped by a sample attaching material, or a sampling rod-shaped structure directly made of the sample attaching material; the closure layer piercing member or the closure layer piercing portion of the sample carrying piercing member includes one or more piercing tips.
When the sample adsorption part and the sealing layer puncture part are positioned on the same part, a sample bearing puncture piece is formed, and the sample adsorption part and the sealing layer puncture part can be integrally formed; or the sample adsorption part and the sealing layer puncture part are fixedly connected or detachably connected components. Specifically, the sample adsorption part may be any structure capable of bearing a sample, including but not limited to: a straw structure composed of an air bag and a same tube cavity, a sampling swab structure with a core material externally wrapped by a sample attaching material, a sampling rod-shaped structure directly made of the sample attaching material, and the like. The sample attachment material is suitable for attaching a sample, including but not limited to: cotton, wool, plastic, bamboo, wood, metal, and the like. When the isothermal amplification reaction tube is a tube group, in order to further simplify the operation steps, the sample adding tool can be set to be an assembly formed by connecting a plurality of parallel sample bearing piercing elements corresponding to the arrangement mode of the single tubes in the tube group.
Compared with the prior art, the invention has the following beneficial effects: 1. the nucleic acid detection kit does not need other special equipment, is convenient to detect, has low cost, is suitable for families and fields, can be used as samples at present, and does not relate to complex sample storage and transportation. 2. The nucleic acid detection package of the invention does not affect the detection accuracy even though jolting during transportation, and the detection steps are particularly simple, and is especially suitable for non-professionals to operate 3. The nucleic acid detection package of the invention has rapid and high-efficiency amplification, and can complete the steps of nucleic acid sampling and nucleic acid extraction within a few minutes. 4. The nucleic acid sample obtained by the nucleic acid detection kit has high purity, and is suitable for further carrying out gene amplification at constant temperature or variable temperature. 5. The domestic nucleic acid detection kit simplifies the preparation process of the sample, and makes the detection kit possible to be moved to the home. 6. The household nucleic acid detection package provided by the invention completely adopts the sealed disposable small tube, so that the possibility of aerosol pollution is reduced.
Drawings
FIG. 1 is a schematic view showing the detachment of an isothermal amplification reaction tube in a ready-to-use gene detection kit according to an embodiment of the present invention.
FIG. 2 is a schematic diagram showing the disassembly of a isothermal amplification reaction tube in a ready-to-use gene detection kit according to a second embodiment of the present invention.
FIG. 3 is a schematic view showing the detachment of a third embodiment of the isothermal amplification reaction tube in the ready-to-use gene detection kit according to the present invention.
FIG. 4 is a schematic view showing the detachment of a fourth embodiment of the isothermal amplification reaction tube in the ready-to-use gene detection kit according to the present invention.
FIG. 5 is a schematic view showing a tube set form of the isothermal amplification reaction tube in the ready-to-use type gene detection kit according to the present invention
FIG. 6 is a schematic view showing another embodiment of the tube set of the isothermal amplification reaction tube in the ready-to-use type gene detection kit according to the present invention
FIG. 7 is a schematic view showing a tube array format of the isothermal amplification reaction tube in the ready-to-use type gene detection kit according to the present invention
FIG. 8 is a schematic diagram showing another array format of the tube set of the isothermal amplification reaction tube in the ready-to-use type gene detection kit according to the present invention
FIG. 9 is a schematic view of a sealing layer piercing member of the sampling tool of the present invention
FIG. 10 is a schematic view of a sample adsorption member of the sampling tool of the present invention
FIG. 11 is a schematic view of a sample-bearing piercing member according to an embodiment of the present invention
FIG. 12 is a schematic view of another embodiment of a sample-carrying piercing member of the present invention
Description of the element reference numerals
1. Constant temperature amplification reaction tube
11. Sealing member
12. Constant temperature gene amplification detection liquid reaction system
13. Sealing layer
14. Connection structure
21. Sample adsorption part
22. Sealing layer puncture part
23. Hand-held part
Detailed Description
The following description of the embodiments of the present invention is provided for illustrative purposes, and other advantages and effects of the present invention will become apparent to those skilled in the art from the present disclosure.
Please refer to fig. 1 to 12. It should be understood that the structures, ratios, sizes, and the like shown in the drawings and described in the specification are only used for matching with the disclosure of the specification, so as to be understood and read by those skilled in the art, and are not used to limit the conditions under which the present invention can be implemented, so that the present invention has no technical significance, and any structural modification, ratio relationship change, or size adjustment should still fall within the scope of the present invention without affecting the efficacy and the achievable purpose of the present invention. In addition, the terms "upper", "lower", "left", "right", "middle" and "one" used in the present specification are for clarity of description, and are not intended to limit the scope of the present invention, and the relative relationship between the terms and the terms is not to be construed as a scope of the present invention.
One embodiment of the present invention provides a ready-to-use gene testing kit, comprising:
sampling tools, flocked swabs.
The sample processing tube adopts a common 1-15ml centrifuge tube and contains 1-10ml of lysis solution and magnetic beads. The lysis solution comprises tris-hcl, guanidine thiocyanate, edta, triton-100 and peg-6000; (ssds); isopropyl alcohol; wherein tris-hcl is 0.1m-0.5m; guanidine thiocyanate 3-5m; edta is 10-40mm; peg-6000 accounts for 2-5% of the total mass; the sds accounts for 1% -3% of the total mass; triton-100 accounts for 4.5-9% of the total mass; the isopropanol accounts for 5-15% of the total mass; the concentration of the magnetic beads is 20-50mg/ml.
The isothermal amplification reaction tube 1, as shown in fig. 1-8, includes a sealing part 11, a isothermal gene amplification detection liquid reaction system 12 is installed in the isothermal amplification reaction tube, a sealing layer 13 is disposed on the surface of the isothermal gene amplification detection liquid reaction system, and the sealing layer seals the isothermal gene amplification detection liquid reaction system in a fixed space in the isothermal amplification reaction tube.
As shown in FIGS. 8 to 12, the sample addition tool includes at least a sample adsorption part 21 and a sealing layer piercing part 22.
Based on unpredictable jolts in transportation and handling processes, liquid reaction reagents in the reaction tubes are easily and randomly distributed at any positions of tube walls, and for families, the detection problem caused by inaccurate volume of the reaction reagents cannot be overcome for pre-assembled reaction reagents without centrifuges or accurate liquid transferring tools. The invention utilizes the sealing layer to seal the constant temperature gene amplification detection liquid reaction system in the fixed space in the constant temperature amplification reaction tube, on one hand, the liquid reagent is prevented from being randomly distributed in the transportation and carrying process, and on the other hand, the volatilization of the reaction reagent can be prevented in the constant temperature amplification reaction.
Further preferably, the temperature of the solid-liquid transition starting point of the blocking layer is not lower than room temperature and not higher than the isothermal amplification reaction temperature. Considering the room temperature in most regions, the temperature at which isothermal amplification reaction is carried out is usually 65 ℃, and therefore, the preferable solid-liquid transition starting temperature range is 35 to 65 ℃. In order to adapt to a more hot environment, the lower limit of the solid-liquid transition starting point of the low melting point sealing layer may be further selected from any temperature of 35 ℃ to 50 ℃, for example, 35 ℃, 36 ℃, 37 ℃, 38 ℃, 39 ℃, 40 ℃, 41 ℃, 42 ℃, 43 ℃, 44 ℃, 45 ℃, 46 ℃, 47 ℃, 48 ℃, 49 ℃, 50 ℃ and the like. In order to achieve more rapid solid-liquid transition of the sealing layer during isothermal amplification, the upper limit of the solid-liquid transition starting point of the low-melting-point sealing layer is more preferably less than 65 ℃, for example, 64 ℃, 63 ℃, 62 ℃, 61 ℃, 60 ℃, 59 ℃, 58 ℃, 57 ℃, 56 ℃, 55 ℃, 54 ℃, 53 ℃, 52 ℃, 51 ℃ and the like. The solid-liquid conversion starting point temperature of the sealing layer can be properly selected according to the temperature conditions of the production and sale places of the ready-to-use gene detection package.
The starting point temperature of solid-liquid conversion of the sealing layer is higher than room temperature, so that the solid state can be maintained under the conditions of room temperature transportation and storage, and the purpose of sealing the detection reagent is achieved. The temperature of the solid-liquid conversion starting point is lower than the temperature of the isothermal amplification reaction, so that the sealing layer is partially or completely liquefied during the isothermal amplification detection reaction, even if the sealing state of the sealing layer is damaged during sample adding, the liquefied sealing layer can still recover the sealing state based on the tension of the liquefied part during the isothermal amplification reaction, and the reaction system is isolated from the outside, so that the requirements on operators and the operating environment are reduced, and the detection error caused by manual or external conditions is reduced. And after the reaction is finished and the room temperature is recovered, the low-melting-point sealing layer recovers the solid state again, so that the solution after the reaction is well fixed and is not influenced by factors such as carrying conditions and placing conditions, and a user can conveniently observe the detection result at any time.
Still further preferably, the density of the sealing layer at the isothermal amplification temperature is lower than that of the isothermal gene amplification detection liquid reaction system at the same temperature. The density of the sealing layer is lower, so that even if part of the sealing layer material is brought into the detection reagent in the sample adding process, the sealing layer material can easily float to the surface of the detection reagent, on one hand, the clarity of the reaction reagent can be ensured, and on the other hand, the sealing layer material is also favorable for sealing the reaction liquid level again.
The material of the sealing layer may be selected from, but not limited to: rubber, plastic, paraffin, biological glue or resin, etc.
The tube housing of the isothermal amplification reaction tube 1 may be a conventional reaction tube. The sealing layer seals the constant temperature gene amplification detection liquid reaction system in a fixed space in the constant temperature amplification reaction tube, and the sealing layer is characterized in that: through the sealing of the sealing layer, the isothermal gene amplification detection liquid reaction system is fixed at the position of the isothermal amplification reaction tube, and even if the liquid state exists, the liquid state is limited in a closed space and cannot flow to other positions of the isothermal amplification reaction tube at will. Preferably, the isothermal gene amplification detection liquid reaction system is sealed at the lower part of the isothermal amplification reaction tube 1 by a sealing layer. The bottom of the isothermal amplification reaction tube can be of various bottom structures such as a round bottom, a pointed bottom, a flat bottom and the like. In a preferred embodiment, as shown in FIGS. 1 to 4, the lower portion of the reaction tube has a cross section gradually decreasing toward the end. The reaction system of the isothermal amplification reaction is generally a micro system, the total volume of the system is generally below 500 mu l, and the cross section of the cavity is gradually reduced from top to bottom, so that the operation and observation are facilitated.
The isothermal amplification reaction tube can be a single tube or a tube group formed by connecting a plurality of single tubes. Generally, a single tube corresponds to the detection of one base at a SNP site. The gene detection kit may comprise a plurality of single tubes, thereby enabling detection of various base types of one SNP site or further detection of various base types of a plurality of SNP sites. A plurality of single tubes are connected to form a tube group, which is convenient for operation.
In the preferred embodiment shown in fig. 5-8, the individual tubes in the tube set may be arranged in a linear or array arrangement, including but not limited to a rectangular array, a circular array, etc. In the tube group, the single tubes are parallel to each other. In the tube group, the tube walls of the adjacent single tubes may be connected by the connecting structure 14, as shown in fig. 5, or may share the same tube wall, as shown in fig. 6.
The number of single tubes in the instant gene test kit is mainly determined by the detectable sites of the test kit, and generally one site corresponds to 1-3 single tubes, and for the test kit capable of detecting a plurality of sites, there may be 12, 18, 24, 32, 36, 40, 48, 60, 72, 84, 96 single tubes or the tube group consisting of them.
The sealing component of the isothermal amplification reaction tube is not limited in form, and can be used for sealing, as shown in fig. 1-4, including but not limited to: plug-in closures, envelopes, screw-threaded closures, etc. Preferably, the sealing part is flexibly connected with the pipe body.
The sample adsorption part and the sealing layer puncture part in the sample adding tool can be positioned on different parts and also can be positioned on the same part.
The sample adsorption part is used for bearing a sample. The sample adsorption member includes a sample-receiving chamber or a sample adsorption material.
The closure layer piercing section is adapted to pierce the closure layer, and preferably the closure layer piercing section comprises at least one piercing tip.
As shown in fig. 9 and 10, when the sample adsorbing portion and the sealing layer piercing portion are provided on different members, the sample adding tool includes a sample adsorbing member including a sample adsorbing portion 21 and a sealing layer piercing member including a sealing layer piercing portion 22. The sample adsorbing member may be any conventional member that can carry a sample, including but not limited to: sampling swabs, straws, plastic sticks, cotton swabs, and the like. The closure layer piercing member may be any member that can pierce a closure layer, and may have one or more piercing tips for piercing the closure layer. When the isothermal amplification reaction tube is a tube group, the sample adsorption part may also be an assembly formed by connecting a plurality of parallel sample adsorption parts corresponding to the arrangement of the single tubes in the tube group, in order to further simplify the operation steps.
When the sample adsorption part and the sealing layer puncture part are positioned on the same part, a sample bearing puncture piece is formed, and the sample adsorption part 21 and the sealing layer puncture part 22 can be integrally formed; alternatively, the sample adsorbing portion 21 and the sealing layer piercing portion 22 may be fixedly connected or detachably connected. Specifically, the sample adsorption part may be any structure capable of bearing a sample, including but not limited to: a straw structure composed of an air bag and a tube cavity communicated with the air bag, a sampling swab structure with a core material externally wrapped with a sample attaching material, a sampling rod structure directly made of the sample attaching material, and the like. The sample attachment material is suitable for attaching a sample, including but not limited to: cotton, wool, plastic, bamboo, wood, metal, and the like. When the isothermal amplification reaction tube is a tube group, in order to further simplify the operation steps, the sample adding tool can be set to be an assembly formed by connecting a plurality of parallel sample bearing piercing elements corresponding to the arrangement mode of the single tubes in the tube group.
For ease of handling, the sample application tool can include a hand-held portion 23 that can be located at an end or a middle portion of the sample application tool.
In a preferred embodiment of the sample-receiving piercing member shown in FIG. 11, the sample-adsorbing portion 21 and the sealing layer-piercing portion 22 are located at both ends of the sample-receiving piercing member.
In another preferred embodiment of the sample-carrying piercing member, the sample-adsorbing portion 21 is formed integrally with the closure layer-piercing portion 22, at the same end as the sample-carrying piercing member.
In a preferred embodiment of the sample-carrying piercing member shown in fig. 12, a plastic rod and a piercing tip are integrally formed as a sample-adsorbing portion and a sealing layer piercing portion at one end of the sample application tool, and a hand-held portion 23 is formed at the other end. The application means is particularly suitable for body fluid samples, such as saliva. The user only needs to hold the plastic rod or place the plastic rod in the liquid sample for a moment and can adsorb a certain amount of sample on the plastic rod, is the non-infiltration material based on plastics, and plastics itself has certain adsorption to the sample again, and the adsorbed sample in surface can not be too much or too little, consequently adds the appearance volume accurately. The plastic rod and the puncture tip are integrally formed, and the puncture and the sample adding can be finished at one time. The design makes the sampling and sample adding become very simple and easy to operate.
The sample may be in various sample forms, for example, a raw sample, more commonly saliva, or a diluted sample. Saliva contains broken oral epithelial cells, and the quantity of nucleic acid templates required by isothermal gene amplification is very low, so that saliva stock solution can meet the sampling requirement. In addition, the oral epithelial cells can also be scraped to serve as an original sample, and the sample can be diluted to serve as a detection sample. For the case of dilution after sampling, the detection packet may further include: and (4) independently packaging the sample diluent so as to facilitate the sampling after the sample is diluted. The sample diluent can be selected from conventional diluents, such as sterile water, buffer solution, etc.
The isothermal gene amplification detection liquid reaction system comprises an isothermal gene amplification reaction reagent mixed solution. The constant temperature gene amplification reaction reagent mixed solution is added into a sample and then placed at a proper temperature to carry out constant temperature gene amplification reaction on the sample containing the gene locus to be detected. The isothermal gene amplification reaction reagent mixed solution can be designed based on an isothermal gene amplification technology, which belongs to the prior art and is known to commonly comprise: helicase-dependent isothermal amplification (HAD), strand Displacement Amplification (SDA), cross-primer amplification (CPA), loop-mediated isothermal amplification of nucleic acids (LAMP), rolling Circle Amplification (RCA), single-primer isothermal amplification (SPIA), and the like. The isothermal gene amplification detection liquid reaction system generally contains an enzyme for promoting isothermal gene amplification reaction, a buffer system, a specific primer of a target site of a gene to be detected and nucleotide, and can perform the gene amplification reaction under the condition of a reaction temperature suitable for isothermal gene amplification. Based on different isothermal amplification techniques, the skilled person can select suitable enzymes and buffer systems according to the prior art. The specific primer can be preliminarily designed by utilizing the prior art such as primer design software and the like, and can be verified whether the specific primer is suitable or not by adopting a conventional means. Any constant-temperature gene amplification detection liquid reaction system which can effectively detect a specific gene locus and is verified by a professional method is suitable for being used as a constant-temperature gene amplification detection liquid reaction system pre-installed in a constant-temperature amplification reaction tube to prepare the detection kit, so that a non-professional person can obtain an accurate detection result without professional tools and professional skills. In addition, the isothermal gene amplification reaction is carried out at a higher temperature, a cell lysate is not needed, and the temperature of the isothermal gene amplification reaction can promote the cell lysis. Preferably, a substance for promoting cell lysis may be further added to the reaction system to accelerate the reaction.
In order to facilitate the better stability, longer shelf life and convenient transportation and storage of the instant gene detection package, the enzyme required by gene amplification can be freeze-dried on the upper part of the sealing layer, or the enzyme is freeze-dried on the bottom, and the sealing layer is added in the middle. Some color indicators are also unstable and the same strategy can be used.
For the convenience of the operator, preferably, the instant gene testing kit further comprises: and the temperature indicating element is used for indicating the temperature. The temperature indicating element can be a thermometer or a temperature measuring and displaying unit in a water cup or a pot with temperature indication. Also preferably, the instant gene testing kit may further comprise: and (5) a constant-temperature amplification reaction tube rack. The isothermal amplification reaction tube holder can be used for bearing a reaction tube, so that the position of the isothermal amplification reaction tube in the water bath is relatively fixed. Preferably, a floating frame can be used, which enables the isothermal amplification reaction tube to vertically float in the water bath. The floating frame is generally made of light materials and can float on the water surface. The floating frame can be provided with through holes and the like, so that the constant-temperature amplification reaction tube can be conveniently inserted. Also preferably, the instant gene testing kit may further comprise: and the heat preservation device or the constant temperature device is used for providing a constant reaction temperature environment. The heat-insulating device may be, for example, a heat-insulating cup, a heat-insulating pot, or any other device that functions as a heat-insulating water bath container, and the thermostatic device may be, for example, a thermostatic box, a thermostat, or any other device that provides a constant temperature environment. Based on common family stock of thermometer, thermos cup, and general foam material, leaf can be used as floating frame, and the family uses the complete local material of can getting and need not special matching, therefore temperature indicating element, floating frame, heat preservation device or constant temperature equipment are not the indispensible accessory of detection package, can dispose as required.
The instant gene detection kit of the invention adopts the following steps when in use:
1. samples were collected with swabs in the genetic test pack.
2. Placing the swab in a sample processing tube releases the nucleic acid of the sample.
3. And adsorbing the magnetic beads in the sample processing tube by using a sample adsorption part of the sample adding tool.
4. Puncturing a sealing layer of the constant-temperature amplification reaction tube by using a puncturing part of the sample adding tool, and then immersing a sample adsorption part into a gene amplification detection liquid reaction system;
5. reacting the amplification reaction tube at the amplification reaction temperature for a proper time;
6. and judging the reaction result.
The appropriate reaction time varies depending on the reaction system, and can be determined using a positive standard and a negative standard, and a reaction time range in which the positive standard can significantly differ from the negative standard is generally selected as the appropriate reaction time range. Generally, the reaction time is between 5 and 120 minutes. More commonly, it is generally between 15 and 60 minutes.
The isothermal amplification reaction temperature environment may be prepared in advance by using an incubator or a thermostat. In the present invention, the term "isothermal temperature" is not an absolute isothermal temperature, but a relative concept, as long as it is within a temperature range in which isothermal gene amplification reaction can occur, for example, 55 to 65 ℃. For example, some vacuum cups used in the present invention may be 65 degrees at the beginning, and the temperature will gradually decrease with time, generally not lower than 55 degrees, and the reaction will proceed normally. The isothermal reactions involved in the present invention can therefore allow the temperature to fluctuate over a range.
Taking the detection of the N gene of the new coronavirus as an example, the N gene instant detection kit is provided and comprises the following components:
1. sampling tools, flocked swabs.
2. The sample processing tube adopts a common 2ml centrifuge tube and contains 1ml of lysis solution and magnetic beads. The lysis solution comprises tris-hcl, guanidine thiocyanate, edta, triton-100 and peg-6000; (ds); isopropyl alcohol; wherein tris-hcl is 0.1m-0.5m; guanidine thiocyanate 3-5m; edta is 10-40mm; peg-6000 accounts for 2-5% of the total mass; the sds accounts for 1-3% of the total mass; triton-100 accounts for 4.5-9% of the total mass; the isopropanol accounts for 5-15% of the total mass; the concentration of the magnetic beads is 20-50mg/ml.
3. A isothermal amplification reaction tube is a 200-microliter small tube with a cover, a isothermal gene amplification detection liquid reaction system is preloaded in the isothermal amplification reaction tube, the isothermal amplification reaction tube is purchased from Shanghai Baiyan Biotech company (product number COVN 0010), 25ul of the reaction system is added into the reaction tube, and then 30ul of melted paraffin is added.
4. As shown in FIG. 12, the sample adding tool has a plastic rod at one end and a pricking tip integrally formed, and a hand-held portion at the other end.
The using method of the N gene instant detection packet comprises the following steps:
a. the test pack is opened, hot water is added into the heat preservation cup, and the temperature is adjusted to 65 ℃ by a thermometer.
b. The inner wall of the oral cavity is scraped by using an oral swab, and the swab is put into a sample processing tube to release the nucleic acid of the sample.
c. And adsorbing the magnetic beads in the sample processing tube by using a sample adsorption part of the sample adding tool, puncturing a sealing layer of the constant-temperature amplification reaction tube by using a puncture part of the sample adding tool, and immersing the sample adsorption part into a gene amplification detection liquid reaction system.
d. The reaction tube is covered tightly and put into a thermos cup for reaction for 15-60 minutes.
e. The reaction tube was observed for color change, with a light blue positive and a light red negative.
The positive control of the experiment was an N gene plasmid purchased from Shanghai Baiyan Biotech company (Commodity number COVN 1010). 100copy plasmid was added to the swab of the positive control sample and an equal volume of sterile water was added to the negative control.
The pre-detected positive sample and the pre-detected negative sample are detected by adopting the ready-to-use detection kit, and after 30 minutes of reaction, the positive sample shows clear light blue, and the negative sample shows clear light red. This indicates that the design of the sampling tool fully meets the sampling volume requirements.
In order to verify whether the kit is suitable for non-professional use, a comparison test between professional operation and the test pack operation is performed under the condition that the used detection reagents are completely consistent. A professional adopts a pipette to temporarily configure a reaction system in a 200ul eppendorf tube according to the formula of the reaction system, the reaction system is covered after centrifugation, and a professional water bath kettle is adopted to react at 65 ℃. Non-professionals adopt the ready-to-use detection package to carry out detection according to the method of the description, and tools such as a centrifuge, a pipettor and the like are not used in the process. And after the operation is finished, comparing the consistency of the detection results of the two.
The result shows that whether the sample is a negative sample, a positive sample or other samples, the detection results of the professional and the non-professional are completely consistent for the same sample. By adopting the detection package, the detection result can be still observed within 72 hours after the reaction is finished based on the sealing effect of the paraffin.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which may be made by those skilled in the art without departing from the spirit and scope of the present invention as defined in the appended claims.
Claims (10)
1. A ready-to-use gene testing kit, comprising:
sampling tools, flocked or rough-surface swabs; the sample processing tube contains lysis solution and magnetic beads; the sample adding tool at least comprises a sample adsorption part and a sealing layer puncture part. The amplification reaction tube is internally provided with a gene amplification detection liquid reaction system, the surface of the gene amplification detection liquid reaction system is provided with a sealing layer, and the sealing layer seals the gene amplification detection liquid reaction system in a fixed space in the amplification reaction tube; the instant gene detection kit of the invention can extract and detect genes without any professional instrument and equipment.
2. The ready-to-use gene testing kit of claim 1, wherein said sampling tool can be wiped from the mouth, nostrils or surface of an object to dip the sample to be tested.
3. The ready-to-use gene detection kit of claim 1, wherein the magnetic beads in the sample processing tube adsorb nucleic acids in the lysis solution and release nucleic acids in the reaction solution.
4. The ready-to-use gene detection kit as claimed in claim 1, wherein the isothermal gene amplification detection liquid reaction system is enclosed by a sealing layer at the lower part of the isothermal amplification reaction tube, and may be a single layer or multiple layers.
5. The ready-to-use gene testing kit of claim 1, wherein the sample application means comprises a sample adsorption member and a blocking layer piercing member; or the sample adsorption part and the sealing layer puncture part are positioned on the same component to form a sample bearing puncture piece.
6. The ready-to-use genetic testing kit of claim 1, wherein the temperature of the solid-liquid transition starting point of the sealing layer is above room temperature but below the amplification reaction temperature, and the density of the sealing layer at the amplification temperature is lower than the density of the liquid reaction system for gene amplification testing at the same temperature.
7. The ready-to-use gene detection kit of claim 1, wherein the gene amplification reaction is either isothermal or isothermal.
8. The ready-to-use genetic test kit of claim 7, wherein the sample adsorption component is selected from the group consisting of: one or more of a sampling swab, a straw, a plastic stick or a cotton swab; the sample-adsorbing portion of the sample-carrying puncturing member is selected from: a straw structure composed of an air bag and a tube cavity communicated with the air bag, a sampling swab structure with a core material externally wrapped by a sample attaching material, or a sampling rod-shaped structure directly made of the sample attaching material; the closure layer piercing member or the closure layer piercing portion of the sample carrying piercing member includes one or more piercing tips.
9. The ready-to-use gene detection kit of claim 7, wherein the sample application means further comprises one or more of the following features:
A. the sample adding tool also comprises a handheld part;
B. the sample adsorption part and the sealing layer puncture part of the sample bearing puncture piece are respectively positioned at two ends of the sample bearing puncture piece or at the same end of the sample bearing puncture piece;
C. the sample adsorption part of the sample bearing puncture piece and the sealing layer puncture part are integrally formed.
10. An instant gene detection method, which is carried out by using the gene detection package of any one of claims 1 to 13, and comprises the following operation steps;
1) Samples were collected using a sampling tool in the genetic testing package.
2) Placing the sample into the sample processing tube releases the nucleic acid of the sample.
3) And adsorbing the magnetic beads in the sample processing tube by using a sample adsorption part of the sample adding tool.
4) Penetrating a sealing layer of the constant-temperature amplification reaction tube by using a puncture part of the sample adding tool, and immersing a sample adsorption part into a gene amplification detection liquid reaction system;
5) Reacting the amplification reaction tube at the amplification reaction temperature for a proper time;
6) And judging the reaction result.
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