CN220300741U - Device for rapidly extracting nucleic acid - Google Patents
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- CN220300741U CN220300741U CN202222967190.7U CN202222967190U CN220300741U CN 220300741 U CN220300741 U CN 220300741U CN 202222967190 U CN202222967190 U CN 202222967190U CN 220300741 U CN220300741 U CN 220300741U
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Abstract
The utility model belongs to the field of nucleic acid extraction, and discloses a device for rapidly extracting nucleic acid, which comprises the following components: the syringe, syringe needle filter, application of sample needle and magnet, both ends of syringe needle filter all can link to each other with syringe and application of sample needle, and magnet can overlap on the syringe. The device of the utility model can be used for extracting nucleic acid without special instruments, and the extraction process is simple, convenient and rapid.
Description
Technical Field
The utility model relates to the technical field of nucleic acid extraction, in particular to a device for rapidly extracting nucleic acid.
Background
Nucleic acid is a generic term for deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), a type of biopolymer polymerized from many nucleotide monomers, one of the most basic substances for life, the essential constituent substance of all known life forms, and the most important substance in all biomolecules. Nucleic acids are widely found in all animal and plant cells and microorganisms. The nucleic acid consists of nucleotides, while the nucleotide monomers consist of five carbon sugars, phosphate groups and nitrogen-containing bases. If the five carbon sugar is ribose, then the polymer formed is RNA; if the pentose is deoxyribose, the polymer formed is DNA. Nucleic acids carry genetic information and play an extremely important role in the inheritance, variation and biosynthesis of proteins in organisms.
Nucleic acid extraction, which refers to the process of separating nucleic acid from a sample by physical, chemical or other methods, has become an essential basic means in life science research. The principle or requirement of nucleic acid extraction is to ensure the integrity of the primary structure of nucleic acid, eliminate the pollution of other molecules (such as RNA interference during DNA extraction), and avoid the existence of organic solvent and high concentration metal ion with inhibiting effect on enzyme in nucleic acid sample to reduce protein, polysaccharide, lipid and other macromolecular matters as far as possible. The nucleic acid extraction methods in the market are a spin column method and a magnetic bead method. The simple spin column method requires the use of a high-speed centrifuge, the magnetic bead method requires the use of a nucleic acid extractor, and the resource-deficient places lack these instruments, and the operation of the instruments is complex, requiring specialized training to put on duty.
Therefore, there is a need for a device that can perform nucleic acid extraction with a simple magnetic rod or magnet without using special instruments during the entire process.
Disclosure of Invention
In view of the above, the present utility model aims to provide a device for rapidly extracting nucleic acid, which mainly adopts a combination device of magnetic beads, a syringe, a needle filter and a sample adding needle, and can obtain pure nucleic acid solution for on-site detection after steps such as nasopharyngeal swab sample cleavage treatment, syringe suction, magnetic bead adsorption of nucleic acid, eluting nucleic acid with eluent, and the like, so as to solve the problems that a high-speed centrifuge, a nucleic acid extractor, and the like are needed and a professional is needed in the prior art.
The purpose of the utility model and the solution of the technical problems thereof can be realized by adopting the following technical proposal.
In one aspect, the present utility model provides a device for rapid extraction of nucleic acids, comprising: the syringe, syringe needle filter, application of sample needle and magnet, both ends of syringe needle filter all can link to each other with syringe and application of sample needle, and magnet can overlap on the syringe.
In embodiments of the utility model, the syringe, needle filter, and loading needle may be disposable.
In embodiments of the utility model, the device for rapid extraction of nucleic acids may further comprise a nucleic acid extraction kit.
In an embodiment of the utility model, the nucleic acid extraction kit comprises a lysate, a binding solution, magnetic beads, a rinse solution, and an eluent.
In an embodiment of the utility model, the lysate comprises: 10-30mmol/L Tris-HCl, 5-20mmol/L EDTA, 20-50mmol/L glucose, deionized water as solvent, pH=4.0-5.5.
In an embodiment of the utility model, the binding fluid comprises: 1-5mol/L guanidine hydrochloride, 5-25mmol/L sodium citrate, 1-2% Triton X-100, 0.5-5mmol/L EDTA, deionized water as solvent, and pH=4.0-5.5.
In embodiments of the utility model, the magnetic beads may have a diameter of 300-400nm, such as 310nm, 320nm, 330nm, 340nm, 350nm, 360nm, 370nm, 380nm, 390nm, 400nm. In a specific embodiment of the present utility model, a suspension of magnetic beads having a concentration of 10-30mg/ml is preferably used. In a specific embodiment of the present utility model, the magnetic bead modifying group employed in the present utility model may be a carboxyl group or a silane group.
In an embodiment of the present utility model, the rinse solution includes rinse solution 1 and rinse solution 2. The rinse liquid 1 includes: 1-5mol/L NaAc, 20-40% absolute ethanol (V/V), deionized water as solvent, and pH=4.0-5.5, wherein the rinsing liquid 2 comprises: 10-30mmol/L Tris-HCl, 0.5-5mmol/L EDTA, 5-25mmol/L sodium citrate and 0.3-0.7L absolute ethyl alcohol, wherein the solvent is deionized water, and the PH=8.0-8.5.
In embodiments of the utility model, the eluent may be deionized water, RNase-free ultrapure water or TE buffer (TE buffer).
In an embodiment of the utility model, if the nucleic acid to be extracted is RNA, the device of the utility model further comprises a capture RNA solution and an rnase inhibitor.
In embodiments of the utility model, the capture RNA solution is well known to those skilled in the art and may be, for example, 2-5. Mu.g/. Mu.L carrier RNA solution.
In embodiments of the utility model, the RNase inhibitor is well known to those skilled in the art and may be, for example, diethyl pyrophosphate, guanidine isothiocyanate, vanadyl ribonucleoside complex, and the like.
In an embodiment of the utility model, a magnet is sleeved on the syringe at the end near the needle filter to enable the magnetic beads to be adsorbed at the end of the syringe cavity near the needle filter.
Compared with the prior art, the utility model has obvious beneficial effects. By means of the technical scheme, the utility model has at least the following beneficial technical effects: the utility model combines the magnetic beads, the injector, the needle filter and the magnet, and obtains pure nucleic acid solution for on-site detection after the steps of sample splitting treatment, injector suction, nucleic acid adsorption by the magnetic beads, nucleic acid elution by eluent and the like. The whole extraction process is quick and simple, no special instrument is needed, and the nucleic acid extraction can be completed by using a simple magnetic rod or magnet.
Drawings
FIG. 1 shows a device for rapid nucleic acid extraction according to the present utility model.
FIG. 2 is a schematic diagram showing the construction and an exploded view of a device for rapid nucleic acid isolation according to the present utility model.
FIG. 3 is a combination diagram of the device for rapid nucleic acid isolation of the present utility model when the device is used for pushing.
FIG. 4 shows the result of the fluorescent quantitative PCR amplification assay according to embodiment 4 of the present utility model.
Wherein, 1-syringe, 2-syringe needle filter, 3-magnet, 4-application of sample needle.
Detailed Description
Preferred embodiments of the present utility model will be described in detail below with reference to examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present utility model. Various modifications and substitutions may be made by those skilled in the art without departing from the spirit and scope of the utility model, all such modifications and substitutions being within the scope of the utility model as set forth in the appended claims.
The experimental methods used in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The utility model provides a rapid nucleic acid extraction device which is shown in figures 1-3. FIG. 1 shows a rapid nucleic acid extraction device according to the present utility model, which comprises a syringe 1, a needle filter 2, a magnet 3, and a sample injection needle 4, wherein both ends of the needle filter 2 are connected to the syringe 1 and the sample injection needle 4, and the magnet 3 is sleeved on the syringe 1. The syringe 1, needle filter 2, and loading needle 4 may be disposable. FIG. 2 is a view showing the construction and the exploded view of the rapid nucleic acid isolation apparatus of the present utility model, and FIG. 3 is a view showing the combination of the rapid nucleic acid isolation apparatus of the present utility model when the rapid nucleic acid isolation apparatus performs the pushing function by which the liquid can be discharged from the syringe chamber.
Example 1
In this embodiment, the apparatus of the present utility model is used to extract DNA from Shigella pallidum (Shigella bogdii), as shown in fig. 1, and comprises: including syringe 1, syringe needle filter 2, magnet 3 and application of sample needle 4, the both ends of syringe needle filter 2 all can link to each other with syringe 1 and application of sample needle 4, and magnet 3 can overlap on syringe 1, and syringe 1, syringe needle filter 2, application of sample needle 4 are disposable, and the extraction process is as follows:
1) Cracking: taking 1.0ml of fresh shigella pallidum culture solution, putting into a 2.0ml centrifuge tube, adding 200 mu L of lysate, and incubating in a water bath at 45-65 ℃ for 5min, wherein the lysate comprises 10mmol/L Tris-HCl, 20mmol/L EDTA and 50mmol/L glucose, the solvent is deionized water, and the PH=5.5;
2) Combination 1: standing and cooling to room temperature, transferring the solution in the step 1) to a device connected with a syringe 1 and a needle filter 2 (adopting a 2.0mL syringe), adding 20 mu L of proteinase K into a cavity of the syringe, uniformly mixing, adding 200 mu L of a binding solution, uniformly mixing, and standing for 5min, wherein the binding solution comprises 1mol/L guanidine hydrochloride, 25mmol/L sodium citrate, 2% Triton X-100, 1mmol/L EDTA, the solvent is deionized water, and the pH=4.0;
3) Combination 2: adding 200 mu L of absolute ethyl alcohol into the solution in the step 2), uniformly mixing, standing for 5min, adding 20 mu L of magnetic bead suspension (the concentration of the magnetic bead suspension is 10-30mg/mL, and the diameter of the magnetic bead is 320 nm), uniformly mixing, sleeving a magnet 3 at one end of a syringe 1 close to a needle filter 2, enabling the magnetic bead to be adsorbed at one end of a syringe cavity close to the needle filter 2, standing for 30s, removing the liquid by virtue of the pushing function of the syringe 1, and only retaining the magnetic bead;
4) Rinsing: taking down the device in the step 3) from the magnet 3, adding 300 mu L of rinsing liquid 1, sleeving the magnet 3 at one end of the injector 1 close to the needle filter 2 after uniformly mixing, removing the liquid by means of the pushing function of the injector 1 after standing for 30s, retaining magnetic beads as shown in fig. 3, adding 500 mu L of rinsing liquid 2, sleeving the magnet 3 on the injector 1 after uniformly mixing, removing the liquid by means of the pushing function of the injector 1 after standing for 30s, and standing for 5min at room temperature, wherein:
the rinse liquid 1 includes: 5mol/L NaAc, 20% absolute ethanol (V/V), deionized water as solvent, pH=4.0,
the rinse liquid 2 includes: 30mmol/L Tris-HCl, 0.5mmol/L EDTA, 5mmol/L sodium citrate and 0.7L/L absolute ethyl alcohol, the solvent is deionized water, pH=8.5;
5) Eluting: taking the device of the step 4) off the magnet 3, replacing the needle filter 2, connecting the sample adding needle 4, adding a proper amount of deionized water as eluent, sleeving the magnet 3 on the injector 1 after uniformly mixing, standing for 30s, extruding the eluent dissolved with nucleic acid by means of the pushing function of the injector 1 to obtain DNA, completing the whole extraction process within 1 hour, and then measuring the concentration and purity of the extracted DNA by adopting the NanoDrop 1000, wherein the result is shown in Table 1.
TABLE 1
| DNA concentration (ng/. Mu.L) | OD 260 /OD 280 | |
| Test sample 1 | 625.7 | 1.82 |
| Detection sample 2 | 691.6 | 1.83 |
The results of the measurement show that the concentration of the extracted DNA is greater than600 ng/. Mu.L, the purity is about 1.8. Pure DNA: OD (optical density) 260 /OD 280 And (3) about 1.8, so that the purity and concentration of the extracted DNA meet the detection requirements.
Example 2
In this example, the device of the present utility model was used to extract RNA from 293T cells (purchased from Beijing co-Hospital), comprising: including syringe 1, syringe needle filter 2, magnet 3 and application of sample needle 4, the both ends of syringe needle filter 2 all can link to each other with syringe 1 and application of sample needle 4, and magnet 3 can overlap on syringe 1, and syringe 1, syringe needle filter 2, application of sample needle 4 are disposable, and the extraction process is as follows:
1) Cracking: 200 mu L of 293T cell culture solution is put into a 2.0mL centrifuge tube, 200 mu L of lysate is added, 3 mu L of capture RNA solution (Shanghai Biyun biotechnology Co., ltd., product No. R0036-1 mg) and 4 mu L of RNase inhibitor (Beijing Soy Bao technology Co., ltd., product No. D8210) are incubated for 5min at 45-65 ℃ in a water bath, wherein the lysate comprises 30mmol/L Tris-HCl, 10mmol/L EDTA, 20mmol/L glucose, and the solvent is deionized water, and the PH=4.5;
2) Combination 1: standing and cooling to room temperature, transferring the solution in the step 1) to a device shown in fig. 1 (using a 2.0mL syringe), adding 20 mu L of proteinase K into a cavity of the syringe, uniformly mixing, adding 200 mu L of a binding solution, uniformly mixing, standing for 5min, wherein the binding solution comprises 5mol/L guanidine hydrochloride, 5mmol/L sodium citrate, 1% Triton X-100 and 5mmol/L EDTA, and the solvent is deionized water with pH=4.0;
3) Combination 2: adding 200 mu L of absolute ethyl alcohol into the solution in the step 2), uniformly mixing, standing for 5min, adding 20 mu L of magnetic bead suspension (the concentration of the magnetic bead suspension is 10-30mg/mL, and the diameter of the magnetic bead is 320 nm), uniformly mixing, sleeving a magnet 3 on a syringe 1, enabling the magnetic bead to be adsorbed at one end of a syringe cavity close to a needle filter 2, standing for 30s, removing liquid by virtue of the pushing function of the syringe 1, and only retaining the magnetic bead;
4) Rinsing: taking down the device in the step 3) from the magnet 3, adding 300 mu L of rinsing liquid 1, uniformly mixing, placing on the magnet 3, standing for 30s, removing liquid by means of the pushing function of the injector 1, retaining magnetic beads, adding 500 mu L of rinsing liquid 2, uniformly mixing, sleeving the magnet 3 on the injector 1, standing for 30s, removing liquid by means of the pushing function of the injector 1, and standing for 5min at room temperature, wherein:
the rinse liquid 1 includes: 1mol/L NaAc, 40% absolute ethanol (V/V), deionized water as solvent, pH=5.5,
the rinse liquid 2 includes: 10mmol/L Tris-HCl, 5mmol/L EDTA, 25mmol/L sodium citrate and 0.3L absolute ethyl alcohol, the solvent is deionized water, and the PH=8.0;
5) Eluting: taking the device of the step 4) off the magnet 3, replacing the needle filter 2, connecting the sample adding needle 4, adding a proper amount of ultrapure water without RNase as eluent, sleeving the magnet 3 on the injector 1 after uniformly mixing, standing for 30s, extruding the eluent dissolved with nucleic acid by virtue of the pushing function of the injector to obtain RNA, completing the whole extraction process within 1 hour, and then measuring the concentration and purity of the extracted RNA by adopting the NanoDrop 1000, wherein the result is shown in Table 2.
TABLE 2
| RNA concentration (ng/. Mu.L) | OD 260 /OD 280 | |
| Test sample 1 | 1568.5 | 1.93 |
| Detection sample 2 | 1572.1 | 1.90 |
The result of measurement shows that the concentration of the extracted RNA is more than 1500 ng/. Mu.L, and the purity is about 1.9. Pure RNA: OD 1.7 260 /OD 280 And less than 2.0, so that the purity and concentration of the extracted RNA meet the detection requirements.
Example 3
Bidding analysis
The device (extraction method is the same as that of example 2) of the utility model is compared with the extraction method of a commercial centrifugal column kit (Tiangen biochemical technology (Beijing) limited company, product number: DP 302), the operation is carried out according to the specification of the kit, an extracted sample is 293T cells, the concentration and purity of the extracted RNA are measured by using NanoDrop 1000 after the extraction is completed, and the result is shown in Table 3:
TABLE 3 Table 3
| RNA concentration (ng/. Mu.L) | OD 260 /OD 280 | |
| The device of the utility model | 1582.6 | 1.91 |
| Centrifugal column method | 1046.2 | 2.03 |
The results of the measurement show that the concentration of RNA extracted by the device of the utility model is more than 1500 ng/. Mu.L, and the purity is about 1.9. RNA concentration extracted by centrifugal column methodThe degree is about 1000 ng/. Mu.L, and the purity is about 1.9. Pure RNA: OD 1.7 260 /OD 280 The RNA extracted by the device of the utility model has higher purity than the commercial centrifugal column type method, and the concentration of the extracted RNA is higher than the commercial centrifugal column type method, so that the nucleic acid extracted by the device of the utility model meets the requirement and is better than the nucleic acid extracted by the commercial centrifugal column type method.
Example 4
The RNA extracted by the device of the utility model is used for downstream fluorescent quantitative PCR experiment.
Reverse transcription reaction: the reverse transcription experiment was performed in two steps, first taking 5ng of total RNA of the extracted 293T cells, adding 1. Mu.L of dNTP (10 mM), 1. Mu.L of Oligo dT Primer and nucleic-Free H 2 O to a total volume of 18. Mu.L of the reaction mixture, after which the mixture was incubated at 70℃for 5min, and then placed on ice and kept for 2min. Next, 5. Mu.L of 5 xRT Buffer, 1. Mu. L M-MLV Reverse Transcriptase (RNase H) was added to the above mixture - 200U/. Mu.L) and 1. Mu.L of an RNase inhibitor (40U/. Mu.L), followed by incubation at 42℃for 60min, followed by incubation at 85℃for 5min to terminate the reaction, and cooling on ice. Reverse transcription experiments were performed using the M-MLV first strand cDNA kit (Bio-Tekg, inc. of America, cat# TQ 2501) and following the instructions.
| Forward primer | 1μL |
| cDNA(200ng/μL) | 0.5μL |
| 20×master mix | 1μL |
| Nuclease-Free H 2 O | 16.5μL |
| Reverse primer | 1μL |
| Totals to | 20μL |
Real-time PCR experiments were performed using a FastStart essential DNA green master kit (Roche, inc., USA, cat# 6402712001) and according to the instructions. The real-time detection procedure was run on a StepOne plus Real-time PCR instrument (ABI Co., USA) as follows: at 94℃for 10 minutes, then 45 cycles (95 ℃,20 seconds, 60 ℃,30 seconds and 72 ℃,20 seconds) were performed; the reaction system was further incubated at 72℃for 10 minutes to extend any incomplete products, and the amplification results are shown in FIG. 4. As can be seen from FIG. 4, both positive control and extracted sample RNA were amplified, while negative control was not amplified, demonstrating that RNA extracted using the device of the present utility model can be used for downstream sample RNA detection.
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present utility model, and are not limiting. Although the utility model has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: modifications may be made to the technical solutions described in the foregoing embodiments, or equivalents may be substituted for some or all of the technical features thereof, without departing from the concept, spirit and scope of the utility model; and such modifications or substitutions fall within the scope of the appended claims.
Claims (2)
1. A device for rapid extraction of nucleic acids, comprising: the kit comprises a syringe, a needle head filter, a sample adding needle, a magnet and a nucleic acid extraction kit, wherein both ends of the needle head filter are connected with the syringe and the sample adding needle, and the magnet is sleeved on the syringe, and the nucleic acid extraction kit comprises a lysate, a binding solution, magnetic beads, a rinsing solution and an eluent.
2. The device for rapid nucleic acid extraction according to claim 1, wherein the syringe, needle filter, and sample addition needle are disposable.
Priority Applications (1)
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| CN202222967190.7U CN220300741U (en) | 2022-11-08 | 2022-11-08 | Device for rapidly extracting nucleic acid |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202222967190.7U CN220300741U (en) | 2022-11-08 | 2022-11-08 | Device for rapidly extracting nucleic acid |
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