CN113150961A - Integrated device for sampling, storing and rapidly extracting nucleic acid sample - Google Patents
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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
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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Abstract
The invention discloses a nucleic acid sample sampling, storing and rapid extracting integrated device, which comprises a sampling unit and a nucleic acid extracting unit, wherein the sampling unit consists of a sampling pipe, a sampling rod, a sampling pipe cap and a vertical isolation plate, and the vertical isolation plate separates the sampling unit from the nucleic acid extracting unit; the nucleic acid extraction unit consists of a nucleic acid extraction tube, a horizontal clapboard, a temporary storage cell penetrating through the horizontal clapboard, an adsorption filter membrane and an isolation film, wherein the adsorption filter membrane and the isolation film are embedded at the lower end of the vertical clapboard. After sampling, the sampling rod penetrates through the temporary storage small chamber, so that the reagent dry powder sealed in the temporary storage small chamber is released into a lysate, and a biological sample on the sampling rod is cracked to release nucleic acid; and (3) bursting the isolating film on the vertical isolating plate by using a sampling rod, so that a sample cracking product passes through the adsorption filter membrane and is filtered into the sampling tube to be directly used for nucleic acid detection. The invention reduces the complicated operation links in nucleic acid extraction, avoids cross contamination, has stable reagent storage and high purity of extracted nucleic acid, and is suitable for clinical detection and scientific research application.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an integrated device for sampling, storing and quickly extracting a nucleic acid sample.
Background
The PCR technology is used for directly detecting pathogen nucleic acid, has the advantages of high sensitivity and strong specificity in diagnosis of infectious diseases and screening of asymptomatic infectors, not only improves the accurate diagnosis level, but also is beneficial to early discovery of recessive infectors. When the novel coronavirus epidemic situation is rolled into the whole world, China screens and discovers early symptoms atypical and asymptomatic infectors through PCR (polymerase chain reaction) nucleic acid detection of people, effectively isolates patients and closely contacted people, and rapidly controls the development of the epidemic situation. However, the PCR nucleic acid detection needs a plurality of operation steps such as sampling, nucleic acid extraction, and nucleic acid amplification, wherein the collection, storage, and extraction of the nucleic acid sample are key steps for determining the detection efficiency, and professional personnel, specific equipment, a special environment, a long detection period, and risks of cross contamination and environmental pollution are required, which leads to high threshold and high cost of the nucleic acid detection technology, and severely restricts the comprehensive popularization of the nucleic acid detection technology.
Aiming at the technical problems, the number of times of opening the cover of the sample and the operation link in the collection and extraction of the nucleic acid sample are reduced, the formation and the scattering of aerosol can be greatly reduced, the risks of personnel infection and environmental pollution are reduced, and the detection time is shortened. Therefore, the nucleic acid sampling, storing and rapid extracting device with integrated functions is developed, so that the safety and the timeliness of nucleic acid detection can be improved, the investment of professionals and detection equipment can be reduced, and the detection cost is reduced.
Disclosure of Invention
The invention aims to provide an integrated device for sampling, storing and quickly extracting a nucleic acid sample, which simplifies the operation link of nucleic acid detection, shortens the detection period, removes impurities in the sample to the maximum extent, improves the nucleic acid detection efficiency, reduces the investment of professionals and equipment, and reduces the risks of cross contamination and environmental pollution.
A nucleic acid sample sampling, storing and rapid extracting device is characterized by comprising a sampling unit and a nucleic acid extracting unit; the sampling unit comprises a sampling pipe, a sampling rod, a vertical isolation plate, a sampling pipe cap, a clamping groove, a buckle, a sampling port positioned on the sampling pipe cap and a sampling port cover;
the nucleic acid extraction unit comprises a nucleic acid extraction tube, a horizontal isolation plate, a temporary storage chamber, an isolation film and an adsorption filter membrane, wherein the isolation film and the adsorption filter membrane are positioned on the vertical isolation plate;
the vertical isolation plate completely isolates the sampling unit from the nucleic acid extraction unit, an adsorption filter membrane is embedded under the vertical isolation plate 1/2, the side of the nucleic acid extraction unit of the adsorption filter membrane is hermetically covered by an isolation film, and the nucleic acid in a sample is prevented from contacting with a lysate before being completely released;
the sampling rod is connected with the sampling pipe cap through the clamping groove, is convenient to grasp as a whole during sampling, and is used for wiping the surfaces of swabs of the nose, the pharynx and the oral cavity, and various secretions, excretions and objects; after the sample is collected, the sampling pipe cap is rotated by 180 degrees, a sampling rod can penetrate through a temporary storage small chamber in the nucleic acid extraction unit and be inserted into the lysate, and meanwhile, the sampling port and the sampling port cover are rotated to the upper part of the sampling pipe;
the horizontal isolation plate in the nucleic acid extraction unit divides the nucleic acid extraction unit into an upper part and a lower part, a temporary storage chamber penetrating through the whole layer of the horizontal isolation plate is arranged on the horizontal isolation plate, reagent dry powder is sealed in the temporary storage chamber, and a lysis solution is filled in the nucleic acid extraction tube below the horizontal isolation plate;
the temporary storage small chamber is made of tin foil, aluminum foil and other membrane materials which are stable to acid and alkali and have large brittleness, wherein tin foil is preferred;
the reagent dry powder comprises protease K, streptokinase and other protein digestive enzymes, protein in a digested sample, DNA enzyme and RNA enzyme inhibition, and reducing reagents such as beta-mercaptoethanol, Dithiothreitol (DTT), phenylmethylsulfonyl fluoride (PMSF) and the like, so that the RNA enzyme activity is inhibited; the dry powder reagent can be a composition of one or more than two reagents, is prepared into a dry powder form by a conventional method, and is sealed in the temporary storage chamber; among them, proteinase K and DTT are preferred;
the reagents are easy to denature or oxidize and lose activity when exposed to air and in a solution state, are very stable in a dry powder state and are generally prepared for use; after sampling, the sampling rod penetrates through the temporary storage small chamber, the reagent dry powder is released and enters the lysate, the release of the nucleic acid in the sample is accelerated, and the nucleic acid is protected from being degraded;
the lysis solution does not contain phenol, does not precipitate DNA and RNA, precipitates or does not precipitate protein, and maintains the pH value within the range of 8.0-12.0, and the lysis solution can rapidly and completely lyse cells, bacteria and viruses to release nucleic acid and does not influence the activity of protease released from the temporary storage chamber;
the lysis solution comprises a composition of guanidine hydrochloride, guanidine isothiocyanate, urea, NaOH, Sodium Dodecyl Sulfate (SDS), hexadecyl trimethyl bromoethylamine (CTAB) and Tris-HCl buffer solution; the lysis solution may be a combination of one or more than two reagents, among which SDS and Tris-HCl buffer are preferred;
after the sample enters the lysis solution, cells, viruses, bacteria and the like in the sample are quickly lysed, proteins combined with nucleic acid are digested by protease to release free nucleic acid, the nucleic acid is kept in a dissolved state, and nuclease is inhibited from being degraded;
the isolating film is a film material which is stable to acid and alkali and has large brittleness, wherein tin foil is preferred; the isolating film covers one side of the adsorption filter membrane nucleic acid extraction unit, prevents the adsorption filter membrane from contacting with a lysate before a sample enters, and keeps the activity of the adsorption filter membrane;
the adsorption filter membrane is a cellulose membrane or hydroxyapatite filler which is subjected to surface modification and is connected with a chelating agent through a covalent bond, the chelating agent is preferably iminodiacetic acid (IDA), and the filter membrane is preferably a cellulose membrane;
the adsorption filter membrane can adsorb undigested residual protein in the lysate, chelate divalent metal ions such as Ca2+/Mg2+, and the like, block cell debris, mucus and the like, and does not adsorb nucleic acid molecules in the pH range of 8.0-12.0;
the purified nucleic acid solution is filtered into a sampling tube, and a nucleic acid sample is drawn from a sampling port for subsequent nucleic acid detection.
Drawings
FIG. 1 is a schematic structural view of an integrated device for sampling, storing and rapidly extracting nucleic acid samples
FIG. 2 is a schematic view of the working state of an integrated device for sampling, storing and rapidly extracting nucleic acid samples
FIG. 3 is a diagram showing the result of electrophoresis of PCR products of nucleic acids extracted from 6 samples according to an embodiment of the present invention
FIG. 4 is a diagram showing the results of electrophoresis of PCR products after the nucleic acid sample of example two of the present invention is left at room temperature for different periods of time
FIG. 5 is a diagram showing the results of electrophoresis of PCR products after the three nucleic acid samples of the example of the present invention are left for one week under different temperature conditions
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
In this example, the components of the formulation of the lysate were: Tris-HCl, SDS and NaCl, the concentration of Tris-HCl is 2.5mM, pH9.0, the concentration of SDS is 1% (M/v), the concentration of NaCl is 1M; the dry powder reagent comprises proteinase K and DTT, the dissolving concentration of the proteinase K in the lysis solution is 1mg/ml, and the concentration of the DTT is 1M; iminodiacetic acid (IDA) is covalently linked to the cellulose membrane.
PCR detection is carried out on the extracted nucleic acid sample by adopting PCR detection reagent (Premix Taq, RR901) of TaKaRa company, the reaction system is 50 microliter, wherein, 25 microliter of amplification reagent, 1 microliter of each of upstream primer and downstream primer, 13 microliter of sterilized water and 10 microliter of sample template are subjected to amplification reaction on a Bio-Rad PTC-200 PCR instrument, and the reaction conditions of PCR are as follows: the amplification products were electrophoresed on a 1% agarose gel for 34 cycles of 94 ℃ for 30 seconds, 56 ℃ for 30 seconds, and 72 ℃ for 45 seconds, and bands of interest were observed.
The first embodiment is as follows: after 6 healthy volunteers collect the nasal swab, the sampling rod penetrates through the temporary storage chamber and is inserted into the lysate, the dry powder reagent in the temporary storage chamber is simultaneously released into the lysate, the sampling rod is used for stirring and uniformly mixing the lysate, then the sampling rod is tightly attached to the isolation film, the isolation film is punctured from top to bottom, and the sampling pipe cap is tightly covered. And opening a cover of the sampling port, sucking 10 microliters of filtered nucleic acid samples from the sampling tube, adding the nucleic acid samples into the prepared PCR reaction system, and performing agarose gel electrophoresis after the PCR amplification is finished, wherein the result shows that the target gene fragments are amplified from 6 nucleic acid extraction samples, which indicates that the nucleic acid extraction in the embodiment is successful.
Example two: after the nucleic acid sample in the first embodiment is successfully extracted through PCR, one sample is selected, the sample is placed at room temperature for 1 week, 2 weeks and 3 weeks, 10 microliters of filtered nucleic acid sample is sucked from a sampling tube and added into a prepared PCR reaction system, and agarose gel electrophoresis is performed after PCR amplification is finished, so that the result shows that after the nucleic acid sample is placed at room temperature for 1 week, the amplified target gene band has no obvious difference from a fresh sample, and the amplified target gene band is slightly weakened after the sample is placed for 2 weeks and 3 weeks, which indicates that the extracted nucleic acid sample can be stably placed at room temperature for at least 3 weeks.
Example three: after the nucleic acid sample in the first embodiment is successfully extracted through PCR, one sample is selected, and after the samples are respectively placed at-80 ℃, 4 ℃, room temperature and 60 ℃ for one week, 10 microliters of filtered nucleic acid sample is sucked from a sampling tube and added into a prepared PCR reaction system, and agarose gel electrophoresis is performed after PCR amplification is finished, so that the result shows that the nucleic acid extracted sample can be stably maintained at-80 ℃, 4 ℃, room temperature and 60 ℃ for at least 1 week.
The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Therefore, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.
Claims (9)
1. A nucleic acid sample sampling, storing and rapid extracting integrated device is characterized by comprising a sampling unit 1 and a nucleic acid extracting unit 2;
the sampling unit 1 comprises a sampling pipe 11, a sampling rod 12, a vertical isolation plate 13, a sampling pipe cap 14, a clamping groove 15, a buckle 16, a sampling port 17 positioned on the sampling pipe cap 14 and a sampling port cover 18;
the nucleic acid extraction unit 2 comprises a nucleic acid extraction tube 21, a horizontal separation plate 22, a temporary storage chamber 23, a separation film 24 and an adsorption filter membrane 25 which are positioned on a vertical separation plate 13.
2. The integrated device for sampling, storing and quickly extracting nucleic acid samples as claimed in claim 1, wherein the vertical isolation plate 13 completely isolates the sampling unit 1 from the nucleic acid extraction unit 2, the sampling rod 12 is connected with the sampling tube cap 14 through the clamping groove 15, the sampling tube cap is rotated by 180 degrees after the samples are collected, the sampling rod 12 can be inserted into the nucleic acid extraction unit 2, and the sampling port 17 and the sampling port cover 18 are rotated to the upper part of the sampling tube 11.
3. The integrated device for sampling, storing and rapidly extracting nucleic acid samples according to claim 1, wherein the nucleic acid extracting unit 2 is divided into an upper part and a lower part by a horizontal isolation plate 22, a temporary storage chamber 23 penetrating through the whole layer of the horizontal isolation plate 22 is arranged on the horizontal isolation plate 22, and a lysis solution 27 is filled in the nucleic acid extracting tube 21 below.
4. The integrated device for sampling, storing and rapidly extracting nucleic acid sample according to claim 3, wherein the temporary storage chamber 23 is made of tin foil, aluminum foil or other membrane materials that are stable to acid and alkali and have high brittleness, preferably tin foil, and the sampling rod 12 can easily pass through the temporary storage chamber 23 and enter the lysis solution 27 after sampling.
5. The integrated device for sampling, storing and rapidly extracting nucleic acid sample according to claim 3, wherein the temporary storage chamber 23 contains dry reagent powder 26, which is protease such as proteinase K and streptokinase, can digest protein in the sample and inhibit DNase and RNAse, and reducing reagent such as beta-mercaptoethanol, Dithiothreitol (DTT) and phenylmethylsulfonyl fluoride (PMSF) to inhibit RNAse activity.
6. The integrated device for sampling, storing and rapidly extracting nucleic acid sample according to claim 5, wherein the dry reagent powder 26 is sealed in the temporary storage chamber, isolated from air and the lysis solution 27, released into the lysis solution 27 when the sampling rod 12 passes through the temporary storage chamber 23, and rapidly dissolved to exert activity, thereby promoting the release of nucleic acid and protecting nucleic acid from degradation.
7. The integrated device for sampling, storing and rapidly extracting nucleic acid sample according to claim 1, wherein the lysate 27 is phenol-free, does not precipitate DNA and RNA, precipitates or does not precipitate proteins, maintains pH in the range of 8.0-12.0, and can rapidly and completely cleave and release nucleic acid from exfoliated cells, bacteria and viruses 28, and maintain the activity of protease.
8. The integrated device for sampling, storing and rapidly extracting nucleic acid sample according to claim 1, wherein the adsorption filter membrane 25 is embedded on the vertical isolation plate 13, one side of the adsorption filter membrane is exposed to the sampling tube, the other side of the adsorption filter membrane is isolated from the lysate 27 by the isolation membrane 24, the activity of the adsorption filter membrane 25 is maintained, and the isolation membrane 24 is a membrane material with stability to acid and alkali and high fragility, preferably tin foil.
9. The integrated device for sampling, storing and rapidly extracting nucleic acid samples according to claim 1, wherein the adsorption filter membrane 25 is a cellulose membrane or hydroxyapatite filler, preferably a cellulose membrane, which is surface-modified and is linked with a chelating agent through covalent bonds, can adsorb divalent metal ions such as protein, chelated Ca2+, Mg2+ and does not adsorb nucleic acid molecules within the pH range of 8.0-12.0; the sampling rod 12 breaks the isolation film 24, the adsorption filter membrane 25 contacts with the lysate 27, residual proteins which are not digested in the lysate, excessive metal ions which are chelated, cell debris and mucus which are blocked and the like are adsorbed, the purified nucleic acid solution 29 is filtered into the sampling tube 11, and the nucleic acid solution 29 is sucked from the sampling port 17 and directly used for nucleic acid detection.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114045216A (en) * | 2022-01-11 | 2022-02-15 | 至美时代生物智能科技(北京)有限公司 | Air sampling bottle, air sampling system and air sampling method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114045216A (en) * | 2022-01-11 | 2022-02-15 | 至美时代生物智能科技(北京)有限公司 | Air sampling bottle, air sampling system and air sampling method |
| CN114045216B (en) * | 2022-01-11 | 2022-04-15 | 至美时代生物智能科技(北京)有限公司 | Air sampling bottle, air sampling system and air sampling method |
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