CN116004371A - Amplification and hybridization integrated gene chip and nucleic acid amplification and hybridization method - Google Patents
Amplification and hybridization integrated gene chip and nucleic acid amplification and hybridization method Download PDFInfo
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Abstract
The invention discloses an amplification hybridization integrated gene chip and a nucleic acid amplification and hybridization method, and relates to the technical field of molecular biology. The gene chip integrates the reaction chambers of the gene amplification instrument and the hybridization instrument into a totally-enclosed three-dimensional chip mechanism, thereby effectively avoiding aerosol pollution. The amplification hybridization integrated gene chip has the advantages of simple structure, convenient operation, no need of complex structures such as microfluidic channels, valve structures and the like, and more stable and reliable results after nucleic acid amplification and hybridization reaction through the simple structure. The amplification hybridization integrated gene chip provided by the invention has a three-dimensional structure, and the three-dimensional structure is more convenient for accurately controlling nucleic acid amplification and hybridization reactions. This avoids the lack of microfluidic control.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to an amplification and hybridization integrated gene chip and a nucleic acid amplification and hybridization method.
Background
Polymerase Chain Reaction (PCR) is a molecular biological technique that increases the amount of specific DNA fragments significantly. The PCR utilizes the fact that DNA becomes single-stranded at a high temperature of 95 ℃ in vitro, the primer and the single-stranded are combined according to the base complementary pairing principle at about 60 ℃, the temperature is regulated to about 72 ℃ which is the optimal reaction temperature of DNA polymerase, and the DNA polymerase synthesizes complementary strand along the direction from phosphoric acid to five-carbon sugar (5 '-3'). The PCR instrument based on the polymerase chain reaction is actually a temperature control device, and can well switch and control between denaturation temperature, renaturation temperature and extension temperature.
The nucleic acid hybridization method is to detect a specific target sequence based on the known sequence of the probe used, namely: the extracted and purified nucleic acid sample is subjected to PCR polymerase chain reaction by matching with corresponding reagents on an amplification instrument, then hybridized on a hybridization instrument, and chromogenic reagent is added for chromogenic reading.
In the third region of the molecular laboratory where PCR and hybridization are performed, two semi-or fully automated instruments are typically used for the experiment. At present, there are also technical platforms for integrating PCR and hybridization methods by microfluidic technology, but the following drawbacks exist:
(1) The cost of the microfluidic chip integrated with amplification and hybridization is very high, and a piece of microfluidic chip usually needs tens of pieces of RMB;
(2) The microfluidic chip integrated with amplification and hybridization has poor experimental effect, and because a plurality of reaction chambers, valve structures, microfluidic channels and the like are integrated on a planar glass or other substrate, the technical precision is very high, and poor experimental effect is easy to occur;
(3) The common gene amplification instrument and hybridization instrument are mainly manual or semi-automatic, have complicated process and difficult operation, and have the problems of easy corrosion of a reaction chamber, rust of each structural part and the like after long-term use, thus influencing experimental effect;
(4) Whether the traditional amplification hybridization method or the microfluidic hybridization method is adopted, aerosol pollution is easy to generate due to the open experimental process, and the method is the most critical technical defect.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide an amplification and hybridization integrated gene chip and a nucleic acid amplification and hybridization method, which are used for reducing the cost of the amplification and hybridization integrated chip, improving the hybridization quality effect of the amplification and hybridization integration, simplifying the operation flow and avoiding the pollution of aerosol.
In general, in a hybridization apparatus of 1 to 30 persons, even 60 persons, 90 persons, factors such as the movement of samples from a PCR apparatus to a hybridization apparatus, the addition of various reaction reagents, etc. may occur in the experimental process due to the problems of apparatus stability, pollution, etc., and the individual hybridization effects may be unsatisfactory. The integrated chip provided by the invention is only one person and disposable, so that the pollution problem is basically avoided. And the whole process from PCR to hybridization is completed in one chip, the liquid flow is shorter, the temperature rise and fall are faster, the stability is higher, and the overall effect is better. Shallow spots, less quality problems common to non-specificity and the like.
The invention is realized in the following way:
in a first aspect, the present invention provides an amplification hybridization integrated gene chip comprising: the gene chip body is provided with a PCR reaction chamber and a hybridization reaction chamber which are communicated through a groove;
the bottom of the hybridization reaction chamber is sequentially provided with a drainage interface and a hybridization liquid outlet along the liquid outlet direction, the hybridization reaction chamber is internally provided with a hybridization film supporting plate, and a hybridization film is placed on the hybridization film supporting plate;
a chip cover is buckled above the gene chip body, a sealing space is formed by the chip cover, the PCR reaction chamber and the hybridization reaction chamber, and a liquid injection hole is formed above the PCR reaction chamber on the chip cover;
the bottom of the PCR reaction chamber is an inclined plane, and the depth of one end of the PCR reaction chamber close to the hybridization reaction chamber is greater than the depth of one end far away from the hybridization reaction chamber.
The gene chip integrates the reaction chambers of the gene amplification instrument and the hybridization instrument into a totally-enclosed three-dimensional chip mechanism, thereby effectively avoiding aerosol pollution. Specifically, the chip cover, the PCR reaction chamber and the hybridization reaction chamber enclose a fully sealed space, so that aerosol pollution is avoided.
The amplification hybridization integrated gene chip provided by the invention has the advantages of simple structure, convenience in operation, no need of complex structures such as microfluidic channels, valve structures and the like, and more stable and reliable results after nucleic acid amplification and hybridization reaction can be realized through the simple structure.
The existing microfluidic chip is in a planar structure, and the accuracy requirements for reagents and temperature are high because of few reaction liquids in the nucleic acid amplification and hybridization processes. The operation precision of liquid pipetting is micro-upgraded, the temperature is accurate to 0.1 degree, and the liquid volume precision and the temperature control precision are difficult to accurately control by the existing manual or semi-automatic instrument or micro-fluidic chip and other equipment. The integrated three-dimensional structure of the amplification and hybridization integrated gene chip provided by the invention is more convenient for accurately controlling nucleic acid amplification and hybridization reaction. This avoids the lack of microfluidic control. In addition, the space of the chip is three-dimensional, and the dip-dyeing type hybridization method is more complete than the surface hybridization reaction.
The bottom of the PCR reaction chamber is provided with an inclined plane, and the depth of one end of the PCR reaction chamber close to the hybridization reaction chamber is larger than the depth of one end far away from the hybridization reaction chamber, so that amplification products in the PCR reaction chamber can flow into the hybridization reaction chamber from the PCR reaction chamber. The inclined surface is inclined at an angle of 5-40 degrees compared with the horizontal plane.
The PCR reagent, the hybridization solution, the blocking solution and the enzyme-labeled solution are injected through the injection hole. The liquid injection hole is tightly attached to the gun head of the liquid transfer device.
The inventor only sets up the liquid injection hole above the PCR reaction chamber, but does not set up the liquid injection hole above the hybridization reaction chamber at the same time, so set up can simplify the design, promote PCR liquid to transfer to the hybridization chamber through drainage (for example through the pump suction) in the drainage export. In an alternative embodiment, the volume of the PCR reaction chamber is substantially less than the volume of the hybridization reaction chamber.
In a preferred embodiment of the present invention, the hybridization membrane support plate is located at the bottom of the hybridization reaction chamber, or the hybridization membrane support plate abuts against the inner wall of the hybridization reaction chamber.
In a preferred embodiment of the present invention, a drainage hole for facilitating drainage is provided in the middle of the hybridization membrane support plate. The drainage holes are arranged to facilitate the hybridization reaction liquid to flow downwards from the hybridization membrane support plate.
In an alternative embodiment, the shape of the hybridization membrane support plate includes, but is not limited to, a rice shape, a back shape, a field shape, a circular shape, an oval shape, or a square shape. The rice shape is beneficial to maintaining the temperature balance.
In a preferred implementation mode of the invention, a sealing ring mounting groove is formed above the gene chip main body along the outer edges of the PCR reaction tank and the hybridization reaction tank, a sealing ring is arranged in the sealing ring mounting groove, a buckle is arranged on one surface of the chip cover, which is close to the sealing ring, and the chip cover is matched with the sealing ring through the buckle so that the chip cover is in sealing connection with the gene chip main body.
The number of the buckles can be 1 or more, so long as the sealing ring can be clamped into the sealing ring mounting groove, and the sealing connection between the chip cover and the gene chip main body is ensured to be feasible.
In an alternative embodiment, the chip cover is a transparent chip cover. The transparent chip cover is arranged to be convenient for monitoring sample application, sample adding, drainage and reading of the chip hybridization result.
In a preferred embodiment of the invention, the drainage interface of the hybridization reaction chamber is connected with the liquid inlet end of the peripheral drainage pump. The drainage pump is used for pumping out the PCR reaction liquid and the hybridization liquid and sucking amplified products into the hybridization reaction chamber. The liquid flow in the experimental process is jointly formed by a drainage pump liquid and a liquid injection pressure of a liquid injection hole.
In a preferred embodiment of the present invention, the main body of the gene chip is injection molded from polypropylene, polyethylene, polystyrene or polyurethane material. The adoption of the material for one-time injection molding is beneficial to saving the cost to a certain extent (only a few pieces of hair are needed for each piece).
In a preferred embodiment of the invention, the PCR reaction chamber is a cuboid with the height of 3-4mm, the volume of 25-35 microliters and the wall thickness of 0.1-0.2mm, and the periphery and the outer wall of the PCR reaction chamber are provided with heat conducting materials; in an alternative embodiment, the thermally conductive material is a metal oxide or metal nitride. A thermally conductive material is provided to facilitate rapid heat transfer.
In an alternative embodiment, the metal oxide is selected from at least one of aluminum oxide, magnesium oxide, and zinc oxide, and the metal nitride is selected from at least one of aluminum nitride, magnesium nitride, and zinc nitride.
In a preferred embodiment of the invention, the hybridization reaction chamber is 14.1mm to 15mm long; the width is 11.3mm-12mm, and the height is 4mm-5mm; a volume of 600-700 microliters; the bottom and the periphery of the hybridization reaction chamber are provided with heat conducting materials;
in an alternative embodiment, the thermally conductive material is a metal oxide or metal nitride;
in an alternative embodiment, the metal oxide is selected from at least one of aluminum oxide, magnesium oxide, and zinc oxide, and the metal nitride is selected from at least one of aluminum nitride, magnesium nitride, and zinc nitride.
In a second aspect, the present invention also provides a method for amplifying and hybridizing nucleic acids using the amplification and hybridization integrated gene chip, comprising the steps of:
injecting preheated hybridization solution into the hybridization reaction chamber from the solution injection hole, and injecting a nucleic acid sample and an amplification reagent into the PCR reaction chamber from the solution injection hole;
adding volatile-preventing oil (such as paraffin) into the hybridization reaction chamber and the PCR reaction chamber, or sealing the liquid injection hole;
controlling the temperature of the PCR reaction chamber to perform PCR reaction, leading out part of hybridization solution of the hybridization reaction chamber from a hybridization solution outlet after the PCR reaction is finished, sucking amplified products into the hybridization reaction chamber, and simultaneously injecting the hybridization solution from a solution injection hole to enable the nucleic acid amplification products to enter the hybridization reaction chamber for hybridization reaction;
after hybridization reaction, adding hybridization solution from the solution injection hole, and then pumping the hybridization solution;
adding a blocking liquid from the liquid injection hole, and removing the blocking liquid after incubation;
removing the enzyme-labeled liquid from the injected Kong Jiaru enzyme-labeled liquid after incubation;
after washing, the hybridization results were read.
The liquid paraffin is added to prevent the sample from volatilizing from the liquid injection hole after heating, so that aerosol pollution is formed. In addition, other volatile-preventing oils may be optionally added. In other embodiments, a cap or plug may be provided to seal the fill port.
In an alternative embodiment, 600 microliters of hybridization solution preheated to 45 ° is injected from the injection port, the drainage pump is started, the hybridization solution is sucked into the hybridization reaction chamber, and the drainage pump is shut down.
The PCR reaction procedure was as follows: the chip PCR reaction chamber was heated to 95 °, maintained for 30 seconds, then cooled to 65 °, and maintained for 30 seconds. The heating and cooling cycle is repeated for more than 35 times.
In a preferred embodiment of the invention, when the preheated hybridization solution is introduced into the hybridization reaction chamber, the method further comprises the steps of opening a drainage pump, and closing the drainage pump after drainage is finished;
when the nucleic acid amplification product is introduced into the hybridization reaction chamber, the drainage pump is turned on, 500 microliters of hybridization solution in the hybridization reaction chamber is led out from the hybridization solution outlet, the amplified product is sucked into the hybridization reaction chamber, and 500 microliters of hybridization solution is injected from the solution injection hole, so that the nucleic acid amplification product enters the hybridization reaction chamber. Closing the drainage pump, carrying out hybridization reaction for 10 minutes, starting the pump, simultaneously injecting 1000 microliters of 25 DEG hybridization solution into the solution injection hole twice, pumping the hybridization solution, and closing the pump.
The invention has the following beneficial effects:
the invention provides an amplification hybridization integrated gene chip, which integrates a reaction chamber of a gene amplification instrument and a reaction chamber of a hybridization instrument into a totally-enclosed three-dimensional chip mechanism, thereby effectively avoiding aerosol pollution.
The amplification hybridization integrated gene chip has the advantages of simple structure, convenient operation, no need of complex structures such as microfluidic channels, valve structures and the like, and more stable and reliable results after nucleic acid amplification and hybridization reaction through the simple structure.
The existing microfluidic chip is in a planar structure, and the amplification and hybridization integrated gene chip provided by the invention is in a three-dimensional structure, so that the three-dimensional structure is more convenient for accurately controlling nucleic acid amplification and hybridization reactions. This avoids the lack of microfluidic control. The space of the three-dimensional chip is more fully and thoroughly compared with the surface hybridization reaction in the dip dyeing type hybridization mode.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a top view of an amplification hybridization integrated gene chip;
FIG. 2 is a side view of an amplification hybridization integrated gene chip;
FIG. 3 is a schematic structural diagram of a hybridization membrane;
FIG. 4 is a perspective view of an amplification hybridization integrated gene chip;
FIG. 5 is a perspective side view of an amplification hybridization integrated gene chip;
FIG. 6 is a perspective bottom view of an amplification hybridization integrated gene chip;
FIG. 7 is a size diagram of an amplification hybridization integrated gene chip;
FIG. 8 is a graph showing the result of hybridization reaction on a hybridization membrane.
Reference numerals: 1-a PCR reaction chamber; 2-hybridization reaction chamber; 3-hybridization membrane; 4-a sealing ring; 5-a liquid injection hole; 6-a transparent upper cover; 7-drainage interfaces; 8-m-shaped supporting plates; 9-grooves; 10-gene chip body.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
Example 1
The present embodiment provides an amplification hybridization integrated gene chip, the structure of which is shown with reference to fig. 1, 2 and 4, comprising: the gene chip body 10, the gene chip body 10 is provided with a PCR reaction chamber 1 and a hybridization reaction chamber 2, and the PCR reaction chamber 1 is communicated with the hybridization reaction chamber 2 through a groove 9. Nucleic acid amplification is performed in the PCR reaction chamber 1, and then hybridization reaction is performed in the hybridization reaction chamber 2.
The hybridization reaction chamber 2 is provided with a drainage interface 7 and a hybridization liquid outlet at the bottom along the liquid outlet direction in sequence, the drainage interface 7 is connected with an external drainage pump, and liquid in the hybridization reaction chamber 2 is discharged from the hybridization liquid outlet through the drainage interface 7 under the suction effect of the drainage pump.
Referring to fig. 4, a rice-shaped support plate 8 (i.e., a hybridization membrane support plate) is provided in the hybridization reaction chamber 2, and a hybridization membrane 3 is placed on the rice-shaped support plate 8. The structure of the hybridization membrane 3 is shown in FIG. 3. The rice-shaped supporting plate 8 in the embodiment is in a rice shape, and a drainage hole convenient for drainage is formed in the middle. The drainage holes are arranged to facilitate the hybridization reaction liquid to flow downwards from the hybridization membrane support plate.
A chip cover is fastened above the gene chip body, and this embodiment is a transparent upper cover 6. And the transparent upper cover 6, the PCR reaction chamber 1 and the hybridization reaction chamber 2 enclose a sealed space to avoid aerosol pollution.
The sealing ring mounting groove is formed in the upper side of the gene chip main body along the outer edges of the PCR reaction tank 1 and the hybridization reaction tank 2, the liquid injection hole is formed in the sealing ring mounting groove, the side, close to the sealing ring, of the transparent upper cover 6 is provided with a buckle, and the transparent upper cover 6 is matched with the sealing ring 4 through the buckle so that the transparent upper cover 6 is in sealing connection with the gene chip main body 10.
The number of the buckles can be 1 or more, so long as the sealing ring 4 can be clamped into the sealing ring mounting groove, and sealing connection between the transparent upper cover 6 and the gene chip main body 10 is ensured to be feasible.
Referring to fig. 2, the transparent upper cover 6 is further provided with a liquid injection hole 5 above the PCR reaction chamber, and the liquid injection hole 5 can be in close contact with the tip of the pipette, i.e. the liquid injection hole 5 can be anastomotic with the tip of the pipette, which is beneficial to injecting a reagent through the pipette and also beneficial to preventing pollution. The PCR reagent, the hybridization solution, the blocking solution and the enzyme-labeled solution are all injected through the liquid injection hole 5.
The bottom of the PCR reaction chamber 1 is an inclined plane, and the depth of one end of the PCR reaction chamber 1 close to the hybridization reaction chamber 2 is larger than the depth of one end far away from the hybridization reaction chamber 2. This facilitates the flow of the amplification products in the PCR reaction chamber 1 from the PCR reaction chamber 1 into the hybridization reaction chamber 2. The inclined surface is inclined at an angle of 5-40 degrees compared with the horizontal plane.
The amplification hybridization integrated gene chip is of a three-dimensional structure (shown in fig. 5-6), is formed by one-step injection molding of polypropylene materials, and comprises a PCR reaction chamber 1 with the volume of 25-35 microlitres and a hybridization reaction chamber 2 with the volume of 600-700 microlitres.
The dimension chart is shown in FIG. 7, the PCR reaction chamber 1 is a cuboid with a height of 3-4mm, a volume of 25-35 microlitres and a wall thickness of 0.1 mm, and the periphery and the outer wall are closely attached to a heat conducting material, which is aluminum in the embodiment.
The length of the hybridization reaction chamber 2 is 14.1mm to 15mm; 11.3mm to 12mm wide and 4mm to 5mm high; a volume of 600-700 microliters; the bottom and the surrounding bacteria of the hybridization reaction chamber 2 are provided with heat conducting materials, which are aluminum materials in the embodiment.
Example 2
This example exemplifies an HPV37 typing hybridization assay.
The purpose of the experiment is as follows: testing the experimental process of the miniature three-dimensional nucleic acid amplification hybridization integrated chip in an HPV37 typing hybridization method.
Experiment field: PCR laboratory.
Experiment platform: miniature three-dimensional nucleic acid amplification hybridization integrated chip instrument.
Experimental principle: the method comprises the steps of selecting a Human Papillomavirus (HPV) genome conserved sequence as an amplification target sequence, designing universal primers and specific probes (37 types in total), performing PCR amplification to obtain a product, hybridizing the product and the specific probes fixed on a hybridization membrane according to a base complementary pairing principle, and then presenting a hybridization result through a chromogenic reagent. If the hybridization reaction is paired with the probe, the hybridization reaction is carried out at the position of the corresponding probe on the hybridization membrane after hybridization and washing, and if the hybridization reaction is not paired with the probe, the hybridization reaction is not carried out.
Experiment preparation:
(1) Sample preparation: two positive quality control products, three negative quality control products and one HPV51 typing false virus positive specimen are selected as experimental samples.
(2) Hybridization membrane preparation: kaplan organism HPV37 typing hybrid membrane is selected.
(3) Reagent: 37 human papilloma virus typing detection kit (PCR+flow guide hybridization method), kappy Biochemical Co., ltd., national mechanical injection 20143401891.
(4) Experiment preparation: conventional HPV experimental equipment and reagent preparation.
The experimental operation process comprises the following steps:
(1) 600 microliters of hybridization solution preheated to 45 degrees is injected from the injection hole, a drainage pump is started, the hybridization solution is sucked into the hybridization reaction chamber, and the drainage pump is closed;
(2) Spotting: adding the prepared nucleic acid sample into a PCR reagent tube;
(3) Injecting a total of 20 microliters of the prepared nucleic acid sample and amplification reagent from the injection well by a pipette;
(5) Injecting 5 microlitres of paraffin;
(6) The chip PCR reaction chamber was heated to 95 °, maintained for 30 seconds, then cooled to 65 °, and maintained for 30 seconds. Repeating the heating and cooling cycle for 40 times;
(7) Starting a drainage pump, leading 500 microliters of hybridization solution in the hybridization reaction chamber out of a hybridization solution outlet, sucking amplified products into the hybridization reaction chamber, simultaneously injecting 500 microliters of hybridization solution into a solution injection hole, and pushing nucleic acid amplification products into the hybridization reaction chamber;
(8) Closing the drainage pump, carrying out hybridization reaction for 10 minutes, starting the pump, simultaneously injecting 1000 microliters of 25 DEG hybridization solution into the solution injection hole twice, pumping the hybridization solution, and closing the pump.
(9) Injecting 500 microliters of blocking liquid into the liquid injection hole, incubating for 5 minutes, and then draining by starting a pump;
(10) Injecting 500 microliters of enzyme-labeled liquid into the liquid injection hole, incubating for 5 minutes, and then draining by starting a pump;
(11) Injecting 1500 microlitres of distilled water into the liquid injection hole for three times, and draining by starting a pump;
(12) The chip hybridization results were read through the lid.
Interpretation of results
(1) Positive determination criteria: the corresponding sites on the hybridization membrane are blue dots with clear edges.
(2) Negative criterion: the hybridization membrane has no blue dots with clear edges at the corresponding sites.
Conclusion: the experimental results completely meet the expected effect of the design.
Experimental example 1
And (5) performing accuracy verification.
The reagent is used: 37 human papilloma virus typing detection kit (PCR+flow guide hybridization method), kappy Biochemical Co., ltd., national mechanical injection 20143401891
Sample source: the department of health clinical examination is a ventricular interstitial evaluation sample, a human papillomavirus whole genome typing reference provided by manufacturers and a clinical sample with a determined type.
The using device comprises: and the miniature three-dimensional nucleic acid amplification hybridization integrated chip is matched with an instrument.
Control device: gene amplification apparatus, hangzhou Bo technology Co., ltd., model: TC-96/G/H (B) B;
medical nucleic acid molecule hybridization instrument, model number of Guangdong Kaipu biotechnology Co., ltd.): HB-2012A;
the verification method comprises the following steps: and respectively carrying out performance accuracy verification on the integrated chip and the comparison equipment according to the invention by using the same sample.
The verification scheme is as follows:
and 5 negative samples and 10 positive samples are selected, and 15 samples are taken for detection. Wherein sample number 1 is positive for 16 types; sample No. 2 is negative; sample No. 3 was positive for type 18; sample No. 4 was negative; sample No. 5 was positive for 16 types; sample No. 6 was positive for 16 types; sample No. 7 was negative; sample No. 8 was positive for 16 types; sample No. 9 was positive for 16 types; sample number 10 was negative; sample No. 11 was positive for type 16; sample No. 12 was 52 typing positive; sample No. 13 was negative; sample No. 14 was positive for type 6; sample No. 15 was positive for type 51.
The experimental results are shown in table 1 below:
table 1 accuracy test results table
| Sample numbering | Integrated chip results | Comparing the experimental results of the |
| 1 | 16+ | 16+ |
| 2 | Negative of | Negative of |
| 3 | 18+ | 18+ |
| 4 | Negative of | Negative of |
| 5 | 16+ | 16+ |
| 6 | 18+ | 18+ |
| 7 | Negative of | Negative of |
| 8 | 16+ | 16+ |
| 9 | 16+ | 16+ |
| 10 | Negative of | Negative of |
| 11 | 16+ | 16+ |
| 12 | 52+ | 52+ |
| 13 | Negative of | Negative of |
| 14 | 6+ | 6+ |
| 15 | 51+ | 51+ |
Conclusion: the coincidence rate of the experimental result of the miniature three-dimensional nucleic acid amplification hybridization integrated chip and the experimental result of a control instrument is 100%.
Experimental example 2
Lower limit of measurement and repeatability experiment
The verification scheme is as follows:
the total of 5 samples of human papillomavirus whole genome typing standard substance provided by manufacturers are J1: HPV type 16, J2: HPV type 18, J3: HPV type 51, J4: HPV52 type and J5: HPV6 type, diluting to 5000 copies/reaction (the concentration of interfering substances in the diluent provided by the manufacturer is required to be within the range stated by the manufacturer, so as to ensure the accuracy of the diluted concentration), repeatedly detecting 5 times respectively, and verifying whether a positive result can be detected when the lower limit concentration is measured. The results are shown in the table 2, and the results show that the amplification hybridization integrated gene chip provided by the invention has good repeatability and high precision.
Table 2 table of test lower limit results
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. An amplification hybridization integrated gene chip, characterized in that it comprises: the gene chip body is provided with a PCR reaction chamber and a hybridization reaction chamber, and the PCR reaction chamber is communicated with the hybridization reaction chamber through a groove;
the hybridization reaction chamber is sequentially provided with a drainage interface and a hybridization liquid outlet at the bottom along the liquid outlet direction, a hybridization membrane supporting plate is arranged in the hybridization reaction chamber, and a hybridization membrane is arranged on the hybridization membrane supporting plate;
a chip cover is buckled above the gene chip body, a sealing space is formed by the chip cover, the PCR reaction chamber and the hybridization reaction chamber, and a liquid injection hole is formed above the PCR reaction chamber on the chip cover;
the bottom of the PCR reaction chamber is an inclined plane, and the depth of one end of the PCR reaction chamber close to the hybridization reaction chamber is greater than the depth of one end far away from the hybridization reaction chamber.
2. The amplification hybridization integrated gene chip according to claim 1, wherein the hybridization membrane support plate is located at the bottom of the hybridization reaction chamber or the hybridization membrane support plate abuts against the inner wall of the hybridization reaction chamber.
3. The amplification hybridization integrated gene chip according to claim 2, wherein a drainage hole for facilitating drainage is formed in the middle of the hybridization membrane support plate;
preferably, the hybridization membrane support plate is in a shape of a Chinese character 'mi', a Chinese character 'hui', a Chinese character 'tian', a circle, an ellipse or a square.
4. The amplification hybridization integrated gene chip according to claim 1, wherein a sealing ring mounting groove is formed above the gene chip main body along the outer edges of the PCR reaction tank and the hybridization reaction tank, a sealing ring is arranged in the sealing ring mounting groove, a buckle is arranged on one surface of the chip cover, which is close to the sealing ring, and the chip cover is matched with the sealing ring through the buckle so that the chip cover is in sealing connection with the gene chip main body;
preferably, the chip cover is a transparent chip cover.
5. The amplification hybridization integrated gene chip according to claim 4, wherein the drainage interface of the hybridization reaction chamber is connected with the liquid inlet end of a peripheral drainage pump.
6. The integrated gene chip of claim 1, wherein the main body of the gene chip is formed by one-time injection molding of polypropylene, polyethylene, polystyrene or polyurethane.
7. The integrated gene chip of claim 6, wherein the PCR reaction chamber is a cuboid with a height of 3-4mm, a volume of 25-35 microliters and a wall thickness of 0.1-0.2mm, and the periphery and the outer wall of the PCR reaction chamber are provided with heat conducting materials; preferably, the thermally conductive material is a metal oxide or a metal nitride;
preferably, the metal oxide is selected from at least one of aluminum oxide, magnesium oxide, and zinc oxide, and the metal nitride is selected from at least one of aluminum nitride, magnesium nitride, and zinc nitride.
8. The amplification hybridization integrated gene chip according to claim 6, wherein the hybridization reaction chamber is 14.1mm to 15mm long; the width is 11.3mm-12mm, and the height is 4mm-5mm; a volume of 600-700 microliters; the bottom and the periphery of the hybridization reaction chamber are provided with heat conducting materials;
preferably, the thermally conductive material is a metal oxide or a metal nitride;
preferably, the metal oxide is selected from at least one of aluminum oxide, magnesium oxide, and zinc oxide, and the metal nitride is selected from at least one of aluminum nitride, magnesium nitride, and zinc nitride.
9. A method for nucleic acid amplification and hybridization using the amplification and hybridization integrated gene chip according to any one of claims 1 to 8, comprising the steps of:
injecting preheated hybridization solution into the hybridization reaction chamber from the solution injection hole, and injecting a nucleic acid sample and an amplification reagent into the PCR reaction chamber from the solution injection hole;
adding volatile oil into the hybridization reaction chamber and the PCR reaction chamber, or sealing the liquid injection hole;
controlling the temperature of the PCR reaction chamber to perform PCR reaction, leading out part of hybridization solution of the hybridization reaction chamber from a hybridization solution outlet after the PCR reaction is finished, sucking amplified products into the hybridization reaction chamber, and simultaneously injecting the hybridization solution from a solution injection hole to enable the nucleic acid amplification products to enter the hybridization reaction chamber for hybridization reaction;
after hybridization reaction, adding hybridization solution from the solution injection hole, and then pumping the hybridization solution;
adding a blocking liquid from the liquid injection hole, and removing the blocking liquid after incubation;
removing the enzyme-labeled liquid from the injected Kong Jiaru enzyme-labeled liquid after incubation;
after washing, the hybridization results were read.
10. The method of claim 9, further comprising turning on a drain pump when introducing the preheated hybridization solution into the hybridization reaction chamber, and turning off the drain pump after draining is completed;
when the nucleic acid amplification product is introduced into the hybridization reaction chamber, the drainage pump is turned on, 500 microliters of hybridization solution in the hybridization reaction chamber is led out from the hybridization solution outlet, the amplified product is sucked into the hybridization reaction chamber, and 500 microliters of hybridization solution is injected from the solution injection hole, so that the nucleic acid amplification product enters the hybridization reaction chamber.
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| CN116694452A (en) * | 2023-07-24 | 2023-09-05 | 广东凯普科技智造有限公司 | Nucleic acid PCR amplification hybridization reaction instrument and amplification hybridization all-in-one machine |
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| CN104498620A (en) * | 2014-11-17 | 2015-04-08 | 中华人民共和国上海出入境检验检疫局 | Method for detecting microfluid gene chip for epidemic type B encephalitis virus |
| CN113512611A (en) * | 2021-06-10 | 2021-10-19 | 四川华汉三创生物科技有限公司 | Gene membrane chip-based method for detecting various respiratory viruses |
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| CN104498620A (en) * | 2014-11-17 | 2015-04-08 | 中华人民共和国上海出入境检验检疫局 | Method for detecting microfluid gene chip for epidemic type B encephalitis virus |
| CN113512611A (en) * | 2021-06-10 | 2021-10-19 | 四川华汉三创生物科技有限公司 | Gene membrane chip-based method for detecting various respiratory viruses |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116694452A (en) * | 2023-07-24 | 2023-09-05 | 广东凯普科技智造有限公司 | Nucleic acid PCR amplification hybridization reaction instrument and amplification hybridization all-in-one machine |
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