KR101803371B1

KR101803371B1 - Gene purification kit for prompt diagnosis

Info

Publication number: KR101803371B1
Application number: KR1020160026350A
Authority: KR
Inventors: 피경태; 최주연
Original assignee: 주식회사 바이오솔루션
Priority date: 2016-03-04
Filing date: 2016-03-04
Publication date: 2017-11-30
Also published as: KR20170103433A

Abstract

Problems to be solved: Most of the conventional diagnostic kits require a short time of 5 hours or 12 hours to diagnose a disease. Experiments must be carried out by the hands of experts, , There is a possibility of secondary infection during sample transfer. As the diagnosis becomes delayed, the damage of infection increases, so it is necessary to perform quick and easy field diagnosis or field gene isolation.
Problem Solution: After a specific solvent and salt are added to a gene, it can be easily separated by filtering with a syringe filter or a vacuum filter, so that the gene can be purified and separated in a short time on the spot.

Description

[0002] Gene purification kit for prompt diagnosis [

TECHNICAL FIELD The present invention relates to a gene purification kit for rapid diagnosis, and more particularly, to a kit for gene purification for rapidly diagnosing diseases such as avian flu, SARS, foot and mouth disease, H1N1 and tuberculosis, And a method of shortening a sample lysis time by passing through a fine tunnel and a method of attaching an adsorption filter to a syringe filter to purify a gene in a sample.

Recently, various diseases such as tuberculosis, SARS, bird flu, H1N1, and foot-and-mouth disease have repeatedly occurred in humans and livestock. Although the authorities are trying to reduce the damage by a lot of efforts, the same damage is repeatedly occurring and it is expected to continue to occur in the future. Thus, the incidence of disease is increasing, lethal, and the propagation speed is getting faster, but side effects are frequently caused by lack of initial response, such as secondary infection due to failure to diagnose and take action in the field.

The market for gene-based molecular diagnostics is expected to reach about US $ 15 billion by 2014, and the cost of genetic purification in this area is estimated to be about US $ 5 billion (Biona Inc., 2012 business report).

Hereinafter, some methods for extracting DNA from sputum will be discussed.

In the process of isolating DNA from sputum disclosed in Korean Journal of Industrial and Applied Biology, Vol. 10, No. 1 (February 1998), 0.5-3 ml of sputum and 2% NaOH in the same amount were added and mixed for 1 minute with a shaker. Let stand for 15 minutes while shaking in a water bath every 5 minutes. Then, supernatant was removed by centrifugation, and TEN buffer (10 mM Tris-HCl, 1 mM EDTA, 150 mM NaCl) was added to float, 25 ul of phenol and CIA (chloroform isoamylalcohol) Shake for 2 minutes with a bead-beater. The supernatant was again centrifuged, and the supernatant was transferred to another test tube. Then, the same amount of CIA was injected and centrifuged again. The supernatant was added with 0.1 M 3M sodium acetate and 3 times anhydrous cold ethanol. It stays. The residue of the formed DNA is allowed to dry at room temperature, and then the DNA is dissolved in 30 ul of sterilized distilled water and used as a template for PCR.

LG Life Science AdvanSure TB / NTM real-time PCR kit (Korean Journal of Diagnostic and Investigative Medicine, Vol. 28, No. 1, 2008) is as follows. Add 3 ml of pretreatment solution I to 3 ml of sputum or bronchial washing solution, mix vigorously twice for 30 seconds at 1 minute intervals, centrifuge at 3,000 rpm for 20 minutes, and remove supernatant. Add the pretreatment solution I to the sample, mix the precipitate with a pipette, transfer to a 1.5-ml microtube, mix vigorously for 5 minutes for 30 seconds at 1 minute intervals, and centrifuge to remove the supernatant. This procedure is repeated 3-5 times until the viscosity of the sample disappears. The remaining precipitate is used for DNA extraction.

Transfer the precipitate to a 1.5 ml microtube, add 1 ml of sample pretreatment solution II, vigorously stir and mix, centrifuge at 13,000 rpm for 3 minutes, and remove supernatant. After repeating this process twice, 50 μl of extraction buffer is added and the mixture is heated at 100 ° C for 20 minutes. After centrifugation at 13,000 rpm for 1 minute, vigorous mixing for 10 seconds, centrifugation at 13,000 rpm for 3 minutes and use 5 ul of the separated supernatant for PCR.

In addition, the extraction method, physical method, boiling method for 10-15 minutes, Veterinarni Medicina, 51, 2006 (5) 180-192, method of extracting with protein K or phenol-chloroform after the silicon bead ultrasonication, And other chemical methods.

It is an object of the present invention to provide a gene purification kit capable of solving the problems of the prior art and capable of purifying genes easily and quickly.

Although the inventors have developed and commercialized a large number of diagnostic kits for a specific disease, they have not been able to diagnose locally-occurring livestock or human diseases and have been interested in methods for improving them. The present inventor has focused on the fact that the area where the disease occurs is not completely shut down until the diagnosis of the disease, so that when the diagnosis is made quickly in the area where the disease occurs, or when only the gene that is not a pathogen in the area is purified and brought to the laboratory, The present invention aims to provide a kit that can quickly isolate and purify genes in the field, taking into account the fact that diagnosis can be made and the problem of secondary contamination can be solved.

In order to successfully develop a kit for rapidly isolating and purifying genes, the present inventor has searched for existing diagnostic kits and it is necessary to shortly 5 hours, 12 hours long to diagnose diseases using most conventional diagnostic kits I realized that the experiment had to be carried out by a professional hand. Accordingly, the object of the present invention is to provide a kit that can easily refine a gene and request a diagnosis when a disease suspect case arises, and a gene that is necessary for molecular diagnosis can be purified within 30 minutes on the spot will be.

The present invention relates to a cytolytic solution composition for dissolving a specimen sample, a method of connecting two syringes and connecting them with an adapter having a small diameter, passing the specimen sample through an adapter to shorten the sample lysis time, To thereby purify the gene in the sample.

In the molecular diagnosis using PCR, gene purification is an essential experiment, and the reagents and kits necessary for this are now largely dependent on imports. Current methods of gene purification use a silica membrane or resin. The adsorption filter method is widely used for small gene purification, and it is a method that uses the property that the (-) charge of the gene binds to the (+) charge of silica when the salt concentration is high in a specific environment. The adsorption filter method has advantages in that it can rapidly purify the gene and is generally used because it can obtain a pure gene. Most gene purification products developed and marketed in Korea utilize this method. A method of gene purification using a resin is a method of purifying a gene by binding a (+) charge molecule capable of adsorbing a gene to the surface of a solid bead, and it can purify the gene to the purest one , And Qiagen's products account for 90% of the total market. It is used for purification of genes used for experiments requiring high purity such as mass gene purification or transfection.

The inventors of the present invention focused on the fact that crystals were made in a specific environment, that is, ethanol and a high salt concentration, and the size thereof became large and heavy (FIG. 1) When passed, the liquid phase passed through the filter, but the experiment was carried out with the expectation that the combination of the gene and the salt would not pass through the filter and would be filtered.

Experimental results show that 99% of the genes present in the solution can be purified regardless of the type of filter when passing through a syringe filter having a pore size of 0.1 ~ 1.0 ㎛. In the case of the adsorption filter, about 25 ug, and in the case of the resin type, 100 and 500 ug of the gene can be obtained depending on the resin capacity. It is possible to secure only a certain amount of genes regardless of the amount of genes on the cell lysate. However, in the present invention, it was confirmed that most of the genes present in the cell lysate can be secured.

In addition, pretreatment of the sample is essential for gene purification. The pretreatment of the sample exposes the gene in the cell by treating the sample for a long time at a high temperature using a reagent such as Protease K. These exposed genes are purified by adsorption filters or resin columns in specific environments, such as high salt concentrations or specific pH ranges.

The inventor of the present invention has developed a device as shown in FIG. 1A by focusing on the fact that samples used for diagnosis of diseases are mostly liquid, such as runny nose, saliva, blood, etc., and confirmed that the gene can be exposed to the outside by rapidly breaking up the sample cells. Using this experimental method, we examined whether the cells were broken by using the most difficult to break down tuberculosis samples. In addition, we attempted molecular diagnosis by securing the genes in the pseudotuberculosis samples. It was confirmed that the purified genes from the pseudotuberculosis samples were well diagnosed even at dilution of 1/100.

It was confirmed that genes necessary for diagnosis of diseases can be purified within about 15-30 minutes when one kit is used by combining the cell disruption method of FIG. 1A and the gene purification method of FIG. 1B. This means that the sample preparation process can be performed in a remarkably short period of time, compared to about 4 hours for the sample processing time in case of the L gene diagnosis product of the domestic company.

The present invention

A) The cell lysate composition is added to a specimen sample and stirred. Two syringes are placed against each other and connected to each other by an adapter made of a tube having a diameter of 0.1 to 2 mm. The pistons are reciprocated to disrupt the cells in the specimen sample. Extracting;

(B) adding alcohol having 1 to 3 carbon atoms and a chaotropic salt to the gene extracted in step (a), stirring the solution, and passing the solution through a filter;

C) washing the filter after step b); And

D) eluting the gene attached to the filter after step c) washing.

In laminating the cells, two syringes are connected to each other with an adapter, and the piston is easily reciprocated to disrupt the cells and extract the internal gene. At this time, the adapter has a tubular shape with a diameter of 0.1 to 2 mm, which is suitable for cell disruption. If the diameter is too small, the cells will not break up quickly and smoothly. If they are too large, the cells will not be broken.

Further, the present invention relates to a method for purifying gene for rapid diagnosis, wherein the filter is a syringe filter equipped with a vacuum filter or a filtration membrane.

The present invention also relates to a method for purifying a gene for rapid diagnosis, wherein the alcohol in step b) is at least one selected from the group consisting of methanol, ethanol, propanol and isopropanol. The alcohols lower the solubility of the gene in the gene solution.

The present invention is also characterized in that the chaotropic salt in step b) is at least one selected from the group consisting of sodium salt, LiClO ₄ , CH ₃ COOLi, MgCl ₂ , thiourea, guanidine hydrochloride and GuSCN (guanidine thiocyanate) And a gene purification method for rapid diagnosis. The above chaotropic salts are not limited to the salts listed above, but usually the salts are well-complexed with nucleic acids.

Further, the present invention relates to a method for purifying gene for rapid diagnosis, wherein the filtration membrane of the filter in step b) is a selected one of a silica membrane and a glass membrane. Silica membranes and glass membranes are remarkably capable of adsorbing genes.

In addition, the present invention relates to a method for purifying gene for rapid diagnosis, characterized in that the filtration membrane pore of the filter is 0.1 to 5.0 mu m. If the pore size is less than 0.1 탆, the amount of the gene obtained by filtration becomes too small. If it exceeds 5.0 탆, the gene is not filtered and passes through the membrane.

The present invention also relates to a method for producing a cell lysate composition, wherein the cell lysate composition in step (a) is selected from the group consisting of chaotropic salts selected from the group consisting of guanidine cyanocyanate and potassium acetate of 1.5M to 7M, 5-10% The present invention relates to a gene purification method for rapid diagnosis, which comprises a surfactant.

The present invention also relates to a method for preparing a cell lysate composition, which comprises two syringes connected to each other by an adapter made of a tube having a diameter of 0.1 to 2 mm, a cell lysate composition, an alcohol having 1 to 3 carbon atoms, a chaotropic salt for gene purification, And a buffer for elution.

The present invention also relates to a gene purification kit for rapid diagnosis, wherein the alcohol having 1 to 3 carbon atoms is at least one selected from the group consisting of methanol, ethanol, propanol and isopropanol.

In addition, the present invention provides a method for purifying a gene, wherein the chaotropic salt for gene purification is at least one selected from the group consisting of sodium salt, LiClO ₄ , CH ₃ COOLi, MgCl ₂ , Thiourea, guanidine hydrochloride and GuSCN (guanidine thiocyanate) To a diagnostic gene purification kit.

The present invention also relates to a gene purification kit for rapid diagnosis, characterized in that the filtration membrane of the filter is a selected one of a silica membrane and a glass membrane.

Further, the present invention relates to a gene purification kit for rapid diagnosis, characterized in that the pore of the filter filtration membrane is 0.1 to 5.0 m.

Further, the present invention relates to a gene purification kit for rapid diagnosis, characterized in that the gene purification kit for rapid diagnosis is used for on-site diagnosis.

Further, the present invention relates to a gene purification kit for rapid diagnosis, characterized in that the filter is a syringe filter equipped with a vacuum filter or a filter membrane.

The gene purification kit of the present invention eliminates the risk of secondary infection due to the direct movement of the pathogen by a kit that can diagnose the disease on the spot without moving the pathogen when the disease occurs, .

In addition, the gene purification kit of the present invention can be utilized for mass purification of genes used in a kit for gene purification for laboratory research, gene therapy and the like. It is possible to purify as many genes as desired by overcoming the limitation of purification amount, which is the limit of the adsorption filter or resin type gene kit.

In addition, the kit of the present invention permits early diagnosis of disease occurrence by allowing the gene to be purified within 10 minutes to one hour in the field so that an experiment for diagnosis can be performed.

In addition, using the kit of the present invention allows non-specialists to readily refine the gene and request diagnosis outside when the disease is suspected in the farm, thereby reducing the diagnosis time and reducing the spread of the disease.

In addition, when the kit of the present invention is used for the purification of a gene for research, a large amount of genes can be obtained in a short time as compared with gene purification using an existing product.

Further, when the kit of the present invention is used, more than 90% of the genes can be purified.

In addition, by using the kit of the present invention, trace genes in the sample can be purified.

Further, when the kit of the present invention is used, no separate experimental equipment is required for sample dissolution and gene purification, and the gene purification time can be shortened to about 10 minutes to 1 hour.

1A and 1B are conceptual diagrams showing the principle of the present invention.
FIG. 2 is an experiment for confirming whether it is applicable to the field of diagnosis of diseases. FIG. 2 shows the result of purification of a gene and PCR diagnosis using a gene purification kit and a kit of the present invention which have been previously marketed from a sample of M. tuberculosis.

Hereinafter, the configuration of the present invention will be described in more detail with reference to the drawings and specific embodiments. However, it is apparent to those skilled in the art that the scope of the present invention is not limited to the description of the embodiments.

Comparative Example : According to the conventional method Artificial sputum Mycobacteria ( Mycobacterium tuberculosis ) Gene purification

Prepare a mixture of 4% NaOH and 2.9% sodium citrate in a 1: 1 ratio. The mixed solution is added three times the weight of the sample, mixed vigorously for 1 minute, and left at room temperature for 20 minutes. Mix 1-2 times in the middle. Transfer the specimen to a 50 ml tube, add 45 ml of PBS and mix well. Centrifuge at 4000 rpm, 4 ° C for 20 minutes. Remove all supernatants.

1 ml of NaOH is added to the sample solution in which the supernatant is removed, and the mixture is boiled in boiling water for 15 minutes. Transfer the supernatant to a centrifuge tube and add Proteinase K solution. Add 200 μl of the above solution and 200 μl of tissue lysis buffer and mix for 5 seconds. The reaction was carried out in a constant temperature water bath at 60 ° C for 1 hour. Add the same amount of PCI (phenol: chloroform: isoamyl alcohol = 25: 24: 1) and vigorously mix and centrifuge. Transfer the supernatant to a 1.5 ml centrifuge tube, add 30 μl of 2 M sodium acetate and 900 μl of cold 100% ethanol and mix gently. Leave at -20 ° C for 30 minutes and centrifuge at 13,000 rpm for 5 minutes. Discard the supernatant, mix with 500 μl of 70% ethanol, centrifuge at 13,000 rpm for 5 minutes and remove supernatant. After drying for 1 hour in the oven, add 50 μl of buffer solution. The isolated DNA is immediately used for experiments or stored at 4 ° C and stored at -20 ° C for long-term storage.

Example : By the method of the present invention Artificial sputum Mycobacteria ( Mycobacterium tuberculosis ) Gene purification

3 ml of artificial sputum and various amounts of Mycobacterium tuberculosis are mixed and syringe is sucked. The same amount of the cell lysate composition is sucked into another syringe. Wherein the cell lysate composition comprises a chaotropic salt selected from the group consisting of guanidine cyanosanate and potassium acetate from 1.5 M to 7 M, a 5 to 10% alcohol having 1 to 3 carbon atoms (e.g., methanol, ethanol, propanol, isopropanol ), 10% sodium lauryl salicylate.

Combine the two syringes across the adapter. Push the two syringes alternately on both sides to allow the two solutions to mix well. Repeat 20 times and leave for 5 minutes. Push the solution through one syringe, disconnect the adapter, and transfer it to the centrifuge tube. Centrifuge at 3,500 rpm for 5 minutes and pass through the spin column. After washing with 500 μl of Wash Buffer I and II, it is dried, added with 30 μl of elution buffer, left for 1 minute, and centrifuged for 30 seconds to obtain purified DNA. This is used as a template for PCR experiments. The wash buffer I is a mixture of 5 M guanidine hydrochloric acid and 30% isopropyl alcohol, and the wash buffer II is 80% ethanol.

PCR exam

The H37Ra bacteria cultured in 3 ml and 1 ml of artificial sputum were added at the ratios of 1/10, 1/100, and 1/1000, respectively, and the genes were purified by the methods of the above Examples and Comparative Examples. And tested with a PCR diagnostic kit. 1 to 6 samples were genes purified by the examples, 7-10 samples were purified using Qiagen kit, PCR primers were 1-8 lane IS6110 F / IS6110 R, 9-10 lanes were RV1908v F516 / R668 Were used.

1A and 1B are conceptual diagrams showing the principle of the present invention. When a chaotropic salt and an alcohol having 1 to 3 carbon atoms, preferably ethanol, are added to a sample containing a gene, a crystal is formed and the gene binds to the salt to increase the size of the conjugate and become heavy. When it is passed through a filter having micro- Pass the filter, but the combination of the gene and the chaotropic salt can not be passed through the filter and can be separated and purified by a simple process. 1A is a conceptual diagram showing a method of disrupting cells in a liquid specimen sample. A sample of liquid specimen is put into a syringe, and an adapter made of a tube having a small diameter such as a capillary tube is interposed between two syringes, and the piston is reciprocated at a high speed to reciprocate right and left. And nucleic acid such as DNA is extracted. 1B is a schematic view showing that a gene in a cell lysate is attached to a filter membrane of a filter using a syringe filter.

FIG. 2 is a graph showing the result of purification of a gene and PCR diagnosis using a gene purification kit and a kit of the present invention, which were previously marketed from a sample of M. tuberculosis, to confirm whether the method of the present invention is applicable to the field of disease diagnosis. DNA samples were extracted by using a syringe filter (lane 1-6), or extracted using a kit of Q company (lane 7-10) by using the method of the present invention, or samples of sputum mycobacteria (artificial sputum containing a certain amount of Mycobacterium tuberculosis) ), And the results were confirmed by PCR.

Claims

A) A sample of the sample is treated with proteolytic enzyme, a chaotropic salt selected from the group consisting of guanidine cyanosanate and potassium acetate of 1.5M to 7M, an alcohol of 1 to 3 carbon atoms of 1 to 3% and a surfactant Adding and stirring the cell lysate composition;
B) The sample solution to which the cell lysate composition obtained in the above step (a) is added is injected into the space of two syringes which are connected to each other by an adapter made of a tube having a diameter of 0.1 to 2 mm and the piston of the two syringes is reciprocated Separating the cells in the specimen sample by allowing the specimen to pass through the adapter several times and extracting the genes;
(C) one or more alcohol and sodium salts of 1 to 3 carbon atoms in methanol, ethanol, propanol and isopropanol, LiClO ₄ , CH ₃ COOLi, MgCl ₂ , thiourea, , Guanidine hydrochloride, and guanidine thiocyanate (GuSCN) to a sample containing the extracted gene, followed by stirring.
D) passing the sample stirred in step c) through a silica membrane or glass membrane filter having a pore size of 0.1 to 5.0 m;
E) washing the filter after step d); And
(F) eluting the gene attached to the filter after the step (e) washing.

The method according to claim 1,
Wherein the filter is a syringe filter equipped with a vacuum filter or a filtration membrane.

Two syringes connected to each other with an adapter made of a tube having a diameter of 0.1 to 2 mm;
A chaotropic salt selected from the group consisting of guanidine cyanosanate of 1.5M to 7M and potassium acetate, an alcohol of 5 to 10% of 1 to 3 carbon atoms, and a surfactant, and dissolving the cell solution Composition;
At least one alcohol of 1 to 3 carbon atoms in methanol, ethanol, propanol and isopropanol;
A chaotropic salt for purifying at least one selected from sodium salt, LiClO ₄ , CH ₃ COOLi, MgCl ₂ , thiourea, guanidine hydrochloride and guanidine thiocyanate (GuSCN); And
And a filter having a pore size of 0.1 to 5.0 탆 and having a filtration membrane for purification of a gene selected from a silica membrane and a glass membrane, and which does not contain a proteolytic enzyme.

The method of claim 3,
Wherein the filter is a syringe filter equipped with a vacuum filter or a filtration membrane.

The method of claim 3,
Wherein the rapid diagnosis gene purification kit is used for on-site diagnosis.

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