CN111518669A - Nucleic acid detection micro-fluidic chip and application thereof - Google Patents
Nucleic acid detection micro-fluidic chip and application thereof Download PDFInfo
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- 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
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Abstract
The invention provides a nucleic acid detection micro-fluidic chip and application thereof, belonging to the technical field of nucleic acid detection, wherein the nucleic acid detection micro-fluidic chip comprises an RT-RPA reaction area and a test paper detection area which are communicated; the RT-RPA reaction area comprises a nucleic acid sample adding area, a first buffer liquid pool, a second buffer liquid pool, an RT-RPA reaction liquid pool, an isothermal amplification pool and an amplification product pool which are sequentially communicated; the test paper detection area comprises a test paper buffer solution pool and a test paper storage pool. The nucleic acid detection microfluidic chip can integrate multi-step reactions including nucleic acid isothermal amplification and test paper detection into a closed system, and can complete the detection in one step, so that the automatic and visual detection of nucleic acid is realized.
Description
Technical Field
The invention belongs to the technical field of nucleic acid detection, and particularly relates to a nucleic acid detection micro-fluidic chip and application thereof.
Background
The microfluidic chip can integrate and integrate multi-step biological reactions into a production line, and automatically complete complex multi-step reactions in a closed system. Since nucleic acid detection generally requires amplification of signals, test strip detection of nucleic acids has a technical problem, and isothermal amplification technology is a simple nucleic acid amplification technology which is newly developed in recent years, wherein the reaction process is always maintained at a constant temperature, and the purpose of rapidly amplifying nucleic acids is achieved by adding enzymes with different temperature activities and specific primers.
Compared with PCR, the isothermal amplification greatly simplifies the requirements of instruments even does not need the instruments, greatly shortens the reaction time, and can better meet the requirements of quick, simple and convenient detection. At present, isothermal amplification is a more common technique, loop-mediated isothermal amplification (LAMP) and Recombinase Polymerase Amplification (RPA). The loop-mediated isothermal amplification is usually carried out at 60-65 ℃, the reaction time is about 40 minutes, and the result is judged by turbidity index or SYBR Green fluorescence; in the other method, Recombinase Polymerase Amplification (RPA) is generally carried out at 37-42 ℃, the measurement time is short (3-15 minutes), the sensitivity is high, the system components are stable and easy to store, the reading result is diversified, and the method can be operated on portable equipment. Recently, reverse transcription recombinase polymerase isothermal amplification technology (RT-RPA) has been used to identify nucleic acid targets, which requires neither high temperatures nor cycle control, and thus RPA isothermal amplification is a good alternative to PCR.
In the reaction process of the RT-RPA experiment, a plurality of components need to be mixed in advance and then amplified in an isothermal environment, so the steps are complicated, the operation is difficult, and the experiment can be carried out by professional personnel. In addition, in the test paper detection of nucleic acid, RT-RPA amplification products need to be mixed with test paper detection buffer solution, then the mixture is dripped on a sample pad of a test paper strip, and the final result observation is carried out.
Disclosure of Invention
In view of the above, the present invention provides a nucleic acid detection microfluidic chip and applications thereof; the nucleic acid detection microfluidic chip can integrate multi-step reactions including nucleic acid isothermal amplification and test paper detection into a closed system, and can complete the detection in one step, so that the automatic and visual detection of nucleic acid is realized.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a nucleic acid detection microfluidic chip, which comprises an RT-RPA reaction area and a test paper detection area which are communicated;
the RT-RPA reaction area comprises a nucleic acid sample adding area, a first buffer liquid pool, a second buffer liquid pool, an RT-RPA reaction liquid pool, an isothermal amplification pool and an amplification product pool which are sequentially communicated;
the test paper detection area comprises a test paper buffer solution pool and a test paper storage pool.
Preferably, the RT-RPA reaction area is communicated with the test paper detection area through a micro-pipeline.
Preferably, the width of the micro-pipeline is 180-220 μm, and the depth of the micro-pipeline is 80-120 μm.
Preferably, a first buffer solution is added into the first buffer solution pool; the first buffer solution takes water as a solvent and comprises the following components in concentration: 80-120 ng/mu L of muscular creatine and creatinase, 45-55 mmol/L, ATP 2-4 mmol/L of phosphocreatine, 4-6 wt% of polyethylene glycol, 1-3 mmol/L of dithiothreitol, 45-55 mmol/L of trihydroxymethyl aminomethane, 1 mu mol/L of specific primer and 80-120 mmol/L of potassium acetate.
Preferably, a second buffer solution is added into the second buffer solution pool; the second buffer solution is 10-20 mmol/L magnesium acetate aqueous solution.
Preferably, RT-RPA is added into the RT-RPA reaction liquid poolReacting; the RT-RPA reaction system comprises the following components in concentration: moloney mouse leukemia virus recombinant reverse transcriptase M-MoLV RT 25-35 ng/. mu. L, T4UvsX recombinant protein 110-130 ng/. mu. L, T4 UvsY recombinant protein 55-65 ng/. mu. L, T4 gp32 recombinant protein 550-650 ng/. mu.L, Bacillus subtilis DNA recombinant polymerase Bsu DNA polymerase 25-35 ng/. mu. L, dNTP 700-900. mu. mol/. mu. L, poly (A) 0.08-0.12 mmol/L and oligo (dT)12-180.08～0.12mmol/L。
Preferably, the addition amount of the RT-RPA reaction system is 40-60 mu L.
Preferably, the RT-RPA reaction system is added as a dry powder.
Preferably, the test paper storage pool is used for placing nucleic acid detection test paper.
The invention provides application of the nucleic acid detection microfluidic chip in nucleic acid detection.
The invention has the beneficial effects that: the nucleic acid detection microfluidic chip provided by the invention comprises an RT-RPA reaction area and a test paper detection area which are communicated; the RT-RPA reaction and the test strip detection are combined in the same microfluidic chip, only a sample to be detected needs to be added, and the subsequent reaction is completed in one step in the chip, so that the automatic and visual detection of nucleic acid is realized; the operation is simple and convenient.
Drawings
FIG. 1 is a schematic structural diagram of a nucleic acid detection microfluidic chip provided by the invention.
Detailed Description
The invention provides a nucleic acid detection microfluidic chip, which comprises an RT-RPA reaction area and a test paper detection area which are communicated; the RT-RPA reaction area comprises a nucleic acid sample adding area, a first buffer liquid pool, a second buffer liquid pool, an RT-RPA reaction liquid pool, an isothermal amplification pool and an amplification product pool which are sequentially communicated; the test paper detection area comprises a test paper buffer solution pool and a test paper storage pool.
In the invention, the RT-RPA reaction area and the test paper detection area are preferably communicated through a micro-pipeline; the reaction tanks in the RT-RPA reaction area are preferably communicated through a micro-pipeline; the test paper buffer solution pool and the test paper storage pool are preferably communicated through a micro-pipeline. In the invention, the width of the micro-pipeline is preferably 180-220 μm, and more preferably 200 μm; the depth of the micro-pipeline is preferably 80-120 mu m, and more preferably 100 mu m.
In the invention, a first buffer solution is added into the first buffer solution pool; the first buffer solution uses water as a solvent, and preferably comprises the following components in concentration: 80-120 ng/mu L of myocreatine, 45-55 mmol/L, ATP 2-4 mmol/L of phosphocreatine, 4-6 wt% of polyethylene glycol, 1-3 mmol/L of dithiothreitol, 45-55 mmol/L of trihydroxymethyl aminomethane, 1 mu mol/L of specific primer and 80-120 mmol/L of potassium acetate; more preferred are compositions comprising the following concentrations: 100 ng/mu L of creatine myo-creatine, 50mmol/L, ATP 3mmol/L of phosphocreatine, 20M 5 wt% of polyethylene glycol, 2mmol/L of dithiothreitol, 50mmol/L of tris (hydroxymethyl) aminomethane, 1 mu mol/L of specific primer and 100mmol/L of potassium acetate.
In the invention, a second buffer solution is added into the second buffer solution pool; the second buffer solution is preferably 10-20 mmol/L magnesium acetate aqueous solution, and more preferably 15mmol/L magnesium acetate aqueous solution.
In the invention, an RT-RPA reaction system is added into the RT-RPA reaction liquid pool; the RT-RPA reaction system comprises the following components in concentration: moloney mouse leukemia virus recombinant reverse transcriptase M-MoLV RT 25-35 ng/. mu. L, T4UvsX recombinant protein 110-130 ng/. mu. L, T4 UvsY recombinant protein 55-65 ng/. mu. L, T4 gp32 recombinant protein 550-650 ng/. mu.L, bacillus subtilis DNA recombinant polymerase Bsu DNApolymerase 25-35 ng/. mu. L, dNTP 700-900. mu. mol/. mu. L, poly (A) 0.08-0.12 mmol/L and oligo (dT)12-180.08-0.12 mmol/L, more preferably comprises the following components in concentration: moloney mouse leukemia virus recombinant reverse transcriptase M-MoLV RT 30 ng/. mu. L, T4UvsX recombinant protein 120 ng/. mu. L, T4 UvsY recombinant protein 60 ng/. mu. L, T4 gp32 recombinant protein 600 ng/. mu.L, Bacillus subtilis DNA recombinant polymerase Bsu DNApolymerase 30 ng/. mu. L, dNTP 800. mu.mol/L, poly (A)0.1mmol/L and oligo (dT)12- 180.1 mmol/L. In the present invention, the dNTPs include A, T, C and G; the concentration ratio of A, T, C to G is preferably 1:1:1: 1.
In the invention, the addition amount of the RT-RPA reaction system in the RT-RPA reaction liquid pool is preferably 40-60 muL, and more preferably 50 muL; in the invention, the RT-RPA reaction system is added in the form of dry powder, namely, the RT-RPA reaction system with the diameter of 40-60 mu LRT-RPA is added into an RT-RPA reaction liquid pool after being prepared into the dry powder.
In the invention, the test paper detection area comprises a test paper buffer solution pool and a test paper storage pool. In the present invention, a test buffer is added to the test buffer pool, and the test buffer is preferably 1/4SSC + 2% BSA + 2% PVP. In the invention, the test paper storage pool is used for placing nucleic acid detection test paper.
In the invention, the nucleic acid detection test paper comprises a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad which are sequentially overlapped.
In the invention, the binding region of the nucleic acid detection test paper adsorbs the gold-coated silicon sphere nano-particle Si02@ Au modified detection probes; the detection area for nucleic acid detection comprises a detection line and a quality control line; a capture probe is sprayed on the detection line, and a quality control probe is sprayed on the quality control line; and the quality control probe and the detection probe are complementarily paired.
In the present invention, the capture probe is preferably a biotin-or streptavidin-labeled capture probe. In the invention, the spraying concentration of the capture probe and the quality control probe is preferably 1.2-1.8 muL/cm, and more preferably 1.5 muL/cm. The specific method of the spraying is not particularly limited in the present invention, and a conventional spraying method in the art can be adopted.
In the invention, a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad are sequentially lapped on a bottom plate to obtain the nucleic acid detection test paper. In the present invention, the material of the sample pad is preferably a glass fiber membrane. The sample pad has the function of slowing down the migration speed of a sample to be detected, is beneficial to the uniform distribution of liquid to be detected on the sample pad, and creates a precondition for better flowing to the combination pad. In the invention, the material of the bonding pad is preferably a glass fiber film; the bonding pad is used for adsorbing gold-coated silicon ball nanoparticles (Si 0)2@ Au) in the hairUniformly conveying the sample solution to be detected to an NC membrane at a certain speed under the action of fine adsorption force; the stability and the integrity of subsequent marker particles are maintained, the background signal is reduced, and the stability and the repeatability of detection are improved. In the present invention, the absorbent pad preferably uses absorbent paper having high absorption efficiency, large capacity and good stability. The absorption pad aims to promote the migration of liquid on the cross flow test strip to smoothly and completely reach the absorption pad, ensure that an object (liquid) to be detected can cross an NC membrane through the last step of siphoning action, improve the output value of a detection signal and reduce the signal-to-noise ratio.
In the practice of the present invention, it is preferred that the nitrocellulose membrane (NC membrane) is first adhered to the PVC base plate, then the conjugate pad is fixed in place and secured in overlap with the NC membrane, then the sample pad is adhered in place and secured in overlap with the conjugate pad, and finally the absorbent pad is adhered and secured in overlap with the NC membrane to ensure smooth liquid flow. In the present invention, the length of the overlap is preferably 2 mm.
The invention also provides application of the nucleic acid detection microfluidic chip in nucleic acid detection. In the present invention, the detection sample includes, but is not limited to, nucleic acids, i.e., DNA or RNA, of viruses, bacteria, mycoplasma, parasites, and the like. In the invention, after a nucleic acid sample to be detected is added to a nucleic acid sample adding area, the nucleic acid detection microfluidic chip is inserted into a detector, the nucleic acid detection microfluidic chip automatically flows and is heated for reaction, and the nucleic acid detection microfluidic chip is taken out after 30min, and the result is observed. In the invention, a nucleic acid sample to be detected flows through a first buffer solution pool, a second buffer solution pool and an RT-RPA reaction solution pool, amplification is finally carried out in an isothermal amplification pool to obtain an amplification product, the amplification product is mixed with a test paper buffer solution, and then the amplification product flows into sample pads in two test paper storage pools to realize test paper detection; and observing a test paper reaction band in the test paper detection area, wherein 2 reaction bands are positive, and 1 reaction band is negative.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
A nucleic acid detection microfluidic chip is shown in figure 1; the whole chip comprises an RT-RPA reaction area and a test paper detection area, wherein the RT-RPA reaction area comprises a nucleic acid sample adding area, a first buffer solution pool, a second buffer solution pool, an RT-RPA reaction solution pool, an isothermal amplification pool and an amplification product pool which are sequentially communicated; the test paper detection area comprises a test paper buffer solution pool and a test paper storage pool.
In the invention, the RT-RPA reaction area and the test paper detection area are preferably communicated through a micro-pipeline; the reaction tanks in the RT-RPA reaction area are preferably communicated through a micro-pipeline; the test paper buffer solution pool and the test paper storage pool are preferably communicated through a micro-pipeline.
A preparation method of a nucleic acid detection micro-fluidic chip comprises the following steps:
the chip is made of PDMS materials, and is manufactured after being reasonably designed, and the manufacturing method comprises the following steps:
1) preparing glue: glue A: and B glue is 10: 1. The larger the ratio of glue A to glue B, the softer the glue formulated.
2) Glue homogenizing: and opening the vacuum defoaming stirrer, adding the weighed glue, vacuumizing, and stirring and mixing the glue.
3) Modification: and (3) putting the treated silicon wafer into a volatilization cylinder, and dropping 1-2 drops of a modifier (methyl chlorosilane) for modification for about 3 min.
4) Pouring glue: spreading the tinfoil in a dish, putting the dish into a silicon wafer mould, slightly compacting the silicon wafer, and pouring glue, wherein the glue on the silicon wafer is free of bubbles.
5) And (3) drying: drying in a constant-temperature drying oven at 85 ℃ for about 30 min.
6) Stripping glue: after cooling slightly, the tinfoil is torn off, the solidified PDMS is separated from the silicon chip, and the PDMS is carefully torn off, so that the silicon chip is not damaged.
7) Cutting: carefully cut along the outer frame of the chip by a cutter, and the cutting is neat.
8) Punching: punching with a puncher, and paying attention to the punching position and the hole diameter.
9) Cleaning: and cleaning the chip.
10) Microscopic examination: and observing whether the chip channel is qualified or not by using a microscope.
A first buffer solution:
1. creatine myogenic enzyme 100 ng/. mu.l
2. Creatine phosphate 50mM
3.ATP 3mM
4. Polyethylene glycol 20M 5% concentration
5. Dithiothreitol 2mM
6. Trimethylolaminomethane 50mM
7. Potassium acetate 100mM
8. Primer 1 mu mol/L
Detection primers: ORF1ab sequence detection primer of the novel coronavirus RNA:
Orf1ab F：5-cccccccccc-C3-ccctgtgggttttacacttaaaaac-3 (SEQ ID No.1)
Orf1ab R：5-tttttttttt-C3-acgattgtgcatcagctgactgaag-3 (SEQ ID No.2)
where C3 is not a base and C3 is preceded by an extended sequence.
A second buffer solution: magnesium acetate 15mM
RT-RPA reaction solution:
1. moloney mouse leukemia virus recombinant reverse transcriptase (M-MoLVRT) 30ng/ul
T4UvsX recombinant protein 120 ng/. mu.l
T4 UvsY recombinant protein 60 ng/. mu.l
T4 gp32 recombinant protein 600 ng/. mu.l
5. Bacillus subtilis DNA recombinant polymerase (Bsu DNApolymerase) 30 ng/. mu.l
dNTPs (A, T, C, G) 200. mu.M each
7.poly(A) 0.1mM
8.Oligo(dT)12-180.1mM
Test paper buffer pool: 1/4SSC + 2% BSA + 2% PVP
Nucleic acid detection test paper strip-novel coronavirus nucleic acid detection test paper strip
Detecting a sample: novel coronavirus nucleic acids
Preparation of nucleic acid detection test paper:
SiO2preparation of @ Au nano material:
firstly, preparing SiO with the diameter of 150nm2Silicon sphere liquid: 128mL of absolute ethanol, 18mL of deionized water and 3mL of ammonia water were mixed, and the mixture was stirred at 30 ℃ for 10 min. Then 3mL of tetraethyl orthosilicate TEOS is added, and the mixture is stirred and reacts for 2 hours. Centrifuging at 15000rpm for 20min, collecting precipitate, washing with ethanol twice, washing with water twice, centrifuging, and suspending the precipitate in 50mL deionized water or ethanol to obtain SiO2And (4) silicon ball liquid.
Preparing a gold cluster solution: 1mL of 1% HAuCl4+50mL of double distilled water +0.03M of sodium citrate, mixing for 20s, and +1mL of 0.1M of sodium borohydride.
Ratio of gold cluster liquid to silicon sphere liquid: 2mL (5mg) of silica sphere solution was slowly added dropwise to 8mL of gold clusters.
The specific adsorption conditions are as follows: stirring for 1h at room temperature on a magnetic stirrer.
Preparing the test strip:
(1) sample pad: the material is glass fiber film. The device has the function of slowing down the migration speed of a sample to be detected, is favorable for uniform distribution of liquid to be detected in a sample pad, and creates a precondition for better flowing to a combination pad.
(2) Combining the pads: a glass fiber membrane. The bonding pad is used for adsorbing gold-coated silicon sphere nano particles (Si 0)2@ Au), detection probe 1: orf1abdP 5-aaaaaaaaaaaaaaa-3-SH (SEQ ID No. 3); and (3) detection probe 2: 5-gagagcgggttcacg ttt-3-SH (SEQ ID No.4) and uniformly conveying the sample solution to be detected to an NC membrane at a certain speed under the action of capillary adsorption force; the stability and the integrity of subsequent marker particles are maintained, the background signal is reduced, and the stability and the repeatability of detection are improved.
DNA Probe labeling Si02@ Au step: 1mL of 3-fold concentrated Si0 was taken2@ Au solution, 10. mu.L of 1mM dATP was added thereto, shaking was carried out at room temperature for 20min with a shaker, 15. mu.L of 1% SDS was then added thereto, after shaking culture was carried out for 10min, 50. mu.L of 0.2M NaCl was added thereto (rate was controlled to be 2. mu.L per 2 to 3 min), 1OD detection probe was then added thereto, reaction was carried out at 60 ℃ for 3 hours, centrifugation was carried out with a centrifuge (rotation speed: 12,000rpm, 10min), the supernatant was removed, washing was carried out 3 times with PBS buffer (pH 7.2 to 7.4), and finally the precipitate was dissolved in 1mL of nanoparticle stock solution (20 mmol/LNa)3PO4·12H2O, 5% BSA, 0.25% Tween 20, 10% sucrose), and storing the conjugate solution in a refrigerator at 4 ℃ for later use.
(3) NC membrane (Nitrocellulose membrane): comprises a detection line 1, a detection line 2 and a quality control line. The detection center region of the lateral flow strip was mainly concentrated on the nitrocellulose membrane. Generally, the detection result is measured by a detection line and a quality control line, and the color on the detection line can be subjected to semi-quantitative or quantitative and qualitative analysis.
The quality control line is used for verifying the detection effectiveness of the test strip. If the quality control line has no color, the test result of the test strip is not credible.
Detection line 1 spray capture probe 1: orf1abcP:5-ggggggggggggggg-3-Biotin (SEQ ID No.5)
Quality control line spraying: 5-tttttttttt-3-Biotin (SEQ ID No.7) and 5-cgtgaacccg-3-Biotin (SEQ ID No. 8).
The spraying concentration is as follows: 1.5. mu.L/cm.
And (3) processing a capture probe: after incubation of 50nM biotin-labeled capture probe mixed with 200. mu.L of 2.5mg/ml streptavidin (Shanghai Biotech) in 0.01M PBS for 1h, the mixture was transferred to a dialysis tube (cut-off 30000) and centrifuged (6000rpm, 20min, 4 ℃) in a refrigerated centrifuge to remove unbound aptamer probe. The above procedure was repeated twice with PBS, and the centrifuged solution (Yes, trapped solution) was collected to 600. mu.L. And finally, spraying a mixture solution of biotin-labeled DNA and streptavidin on the test strip.
(4) An absorption pad: the absorbent paper with high absorption efficiency, large capacity and good stability is used. The absorption pad is used for promoting the migration of liquid on the cross flow test strip to smoothly and completely reach the absorption pad, ensuring that an object (liquid) to be detected can cross the NC membrane through the last step of siphoning action, improving the output value of a detection signal and reducing the signal-to-noise ratio.
The position relation is as follows: a nitrocellulose membrane (NC membrane) was first attached to the PVC base plate, then the conjugate pad was fixed in place and ensured to have a 2mm overlap with the NC membrane, then the sample pad was attached in place and ensured to have a 2mm overlap with the conjugate pad, and finally the absorbent pad was attached and ensured to have a 2mm overlap with the NC membrane to ensure smooth liquid flow.
And (3) detection process: after 2. mu.L of the novel coronavirus nucleic acid sample was added to the nucleic acid sample application zone, the mixture was flowed and mixed at a time: the amounts of the first buffer solution 41.5 mu L and the second buffer solution 2.5 mu L, RT-RPA reaction solution 50 mu L are made into dry powder, and the final mixture flows into an isothermal amplification pool to be heated and amplified for 20min at 37 ℃. The amplification product then flows into the RT-RPA amplification product pool with the volume of 10 mu L, then 10 mu L of amplification product is mixed with the test paper buffer solution and flows into the sample pad in the two test paper storage pools. And observing the test result of the test paper after 10 min.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Anhui science and technology institute
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Claims (10)
1. A nucleic acid detection microfluidic chip is characterized by comprising an RT-RPA reaction area and a test paper detection area which are communicated;
the RT-RPA reaction area comprises a nucleic acid sample adding area, a first buffer liquid pool, a second buffer liquid pool, an RT-RPA reaction liquid pool, an isothermal amplification pool and an amplification product pool which are sequentially communicated;
the test paper detection area comprises a test paper buffer solution pool and a test paper storage pool.
2. The microfluidic chip for nucleic acid detection according to claim 1, wherein the RT-RPA reaction region and the test paper detection region are communicated through a micro-channel.
3. The microfluidic chip for nucleic acid detection according to claim 1, wherein the width of the microchannel is 180 to 220 μm, and the depth of the microchannel is 80 to 120 μm.
4. The nucleic acid detection microfluidic chip according to claim 1, wherein a first buffer solution is added to the first buffer solution pool; the first buffer solution takes water as a solvent and comprises the following components in concentration: 80-120 ng/mu L of muscular creatine and creatinase, 45-55 mmol/L, ATP 2-4 mmol/L of phosphocreatine, 4-6 wt% of polyethylene glycol, 1-3 mmol/L of dithiothreitol, 45-55 mmol/L of trihydroxymethyl aminomethane, 1 mu mol/L of specific primer and 80-120 mmol/L of potassium acetate.
5. The nucleic acid detection microfluidic chip according to claim 1, wherein a second buffer solution is added to the second buffer solution pool; the second buffer solution is 10-20 mmol/L magnesium acetate aqueous solution.
6. The nucleic acid detection microfluidic chip according to claim 1, wherein an RT-RPA reaction system is added to the RT-RPA reaction liquid pool; the RT-RPA reaction system comprises the following components in concentration: moloney mouse leukemia virus recombinant reverse transcriptase M-MoLV RT 25-35 ng/. mu. L, T4UvsX recombinant protein 110-130 ng/. mu. L, T4 UvsY recombinant protein 55-65 ng/. mu. L, T4 gp32 recombinant protein 550-650 ng/. mu.L, bacillus subtilis DNA recombinant polymerase Bsu DNApolymerase 25-35 ng/. mu. L, dNTP 700-900. mu. mol/. mu. L, poly (A) 0.08-0.12 mmol/L and oligo (dT)12-180.08～0.12mmol/L。
7. The nucleic acid detection microfluidic chip according to claim 6, wherein the RT-RPA reaction system is added in an amount of 40-60 μ L.
8. The nucleic acid detection microfluidic chip according to claim 6 or 7, wherein the RT-RPA reaction system is added in the form of dry powder.
9. The microfluidic chip for nucleic acid detection according to claim 1, wherein the test paper storage pool is used for placing nucleic acid detection test paper.
10. The use of the nucleic acid detecting microfluidic chip according to any one of claims 1 to 9 in nucleic acid detection.
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| CN115228517A (en) * | 2022-03-17 | 2022-10-25 | 烟台大学 | Rotary micro-fluidic paper chip for detecting virus based on frame nucleic acid and preparation method thereof |
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