KR20060035684A - Method for extracting nucleic acids from human body fluids and compositions there for - Google Patents

Abstract

        The present invention relates to an extract composition for rapidly extracting genomic DNA from human body fluids. In particular, the present invention can be easily extracted genomic DNA and does not affect the polymerase chain reaction (Polymerase Chain Reaction, PCR) 0.01 / 0.01M, pH 7.6 Tris-HCl, 0.01 that can determine the presence of the virus quickly or accurately Adding a dissolution solution consisting of M EDTA and 0.5% SDS to elute the nuclei in various body fluids; A second step of separating genomic DNA and nuclear protein by adding a protein precipitation solution consisting of 6M and sodium chloride solution to the eluted nucleus; And a third step of recovering the genomic DNA by adding a buffer solution kit for genomic DNA recovery consisting of solution 3 to the genomic DNA separated from the above.

Nucleic acid, extraction, virus, PCR

Description

Method for extracting nucleic acids from human body fluids and compositions there for}

1A Chlamydia using a nucleic acid extraction composition of the present invention trachomatis nucleic acid is an electrophoresis picture showing the amplification by PCR analysis,

1B is Trichomonas Vaginalis nucleic acid is extracted using the nucleic acid extraction composition of the present invention and then electrophoresis picture showing the amplification by PCR analysis

1C is Ureaplasma The urealyticum nucleic acid is extracted using the nucleic acid extracting composition of the present invention and then electrophoresed to show amplification by PCR analysis.

1D is Neisseria The gonorrhoeae nucleic acid is extracted using the nucleic acid extracting composition of the present invention, and then electrophoresis showing amplification by PCR analysis.

1E is Mycoplasma The genitalium nucleic acid is extracted using the nucleic acid extracting composition of the present invention, and then electrophoresis showing amplification by PCR analysis.

1F is Treponema The pallidum nucleic acid is extracted using the nucleic acid extracting composition of the present invention and then electrophoresed to show amplification by PCR analysis.

1g Candida Albicans nucleic acid is extracted using the nucleic acid extraction composition of the present invention and then electrophoresis showing the amplification by PCR analysis.

Figure 1h is an electrophoresis picture showing the amplification through a multiplex polymerase chain reaction carried out to determine the possibility of amplifying a total of seven bacterial nucleic acids in a single PCR.

[TECHNICAL FIELD OF THE INVENTION]

        The present invention relates to an extract composition for extracting nucleic acids from a variety of human-derived specimens, and more particularly, for genomic DNA extraction, including sodium chloride that can quickly and accurately extract and purify genomic DNA from a variety of human-derived specimens. It relates to a composition.

[Private Technology]

        All animals, including humans, are not only exposed to various viruses, but are also easily infected, and these viruses carry various diseases. In order to quickly confirm the presence of these viruses, the polymerase chain reaction method should be performed. A problem that must be performed is a prerequisite to extract the nucleic acid of the virus.

        Various methods to confirm the existence of various viruses have been developed so far, and among these, molecular biological methods, which are recently attracting attention, are beginning to stand out in confirming the presence and presence of virus infection. Representative molecular biological methods include polymerase chain reaction (PCR), developed in 1983 by Carrie Mullis.

        The rapid diagnosis of virus presence through polymerase chain reaction is confirmed by PCR based on the presence of viral nucleic acid in a sample. This method is a method of viral DNA from various samples before PCR is performed among various molecular biological methods. Or extraction of RNA must be preceded. However, in the process of purifying the viral nucleic acid from the sample, a number of problems may occur, which may affect the PCR reaction, and a lot of time, expense, and reagents are required to separate the nucleic acid from a large number of samples, respectively.

        First, it is a problem that can occur in the process of purifying viral nucleic acid, which can cause denaturation of nucleic acid by excessive physical treatment. The physical treatment here refers to mechanical bead beating by destroying viral cell wall and extracting nucleic acid. These methods can cause denaturation of nucleic acid, and even if PCR analysis is performed using a virus-infected sample, the nucleic acid is not amplified and thus can be misdiagnosed.

        Secondly, the use of excessive organic solvents must be overlooked. Organic solvents such as phenol and chloroform are traditionally used to extract nucleic acids from prokaryotic and eukaryotic cells. Since nucleic acid extraction is denatured by proteolytic enzymes, cells are crushed with an ionic surfactant, and then the phenol or phenol / chloroform mixture is mixed to separate the organic solvent phase and the water phase, and only the water phase containing the nucleic acid is recovered and obtained through alcohol precipitation. The phenol and phenol / chloroform mixtures used here are substances that destroy human skin tissue and are considered hazardous wastes and therefore require special handling.

        Third, substances capable of inhibiting the PCR reaction may be included in the purified viral nucleic acid. PCR inhibitors can interfere with the action of polymerases and lead to unsatisfactory results in clinical trials for subsequent diagnosis and can result in huge losses. In particular, the serum contains a large amount of PCR inhibitors, through which the polymerase is heavily influenced in performing the final PCR reaction, so that the proper analysis cannot be performed. In addition, since PCR inhibitors are extracted and coexist from the extraction stage of nucleic acids, they must be removed to perform molecular biological processes.

        Various types of nucleic acid purification methods have been developed so far, but methods for completely removing interferences are still incomplete. Commonly used methods were to sequentially perform processes such as chemical treatment with phenol, electrophoresis on agarose gel, cleavage and extraction purification of gel. However, this method also has a problem, depending on the type of sample, the removal efficiency may vary, or the amount of extracted nucleic acids may decrease or increase the experiment time.

        The present invention has been made to solve the problems of the prior art, the present invention is not only to safely extract a variety of genomic DNA from human body fluid, but also does not affect the PCR reaction and to provide a nucleic acid extraction composition that is not harmful to the human body It is done.

        In another aspect, the present invention is to provide a genomic DNA extraction composition that can quickly and accurately extract DNA from the sample to shorten the diagnostic process, saving time and money.

        In order to achieve the above invention, the kit comprises a first step of eluting nuclei in various body fluids by adding a solution for dissolution consisting of 0.01M, pH 7.6, Tris-HCl, 0.01M EDTA, and 0.5% SDS; A second step of separating genomic DNA and nuclear protein by adding a protein precipitation solution consisting of 6M and sodium chloride solution to the eluted nucleus; And a third step of recovering the genomic DNA by adding a buffer solution kit for genomic DNA recovery consisting of solution 3 to the genomic DNA separated from the above.

Hereinafter, the present invention will be described in detail.

        Nucleic acid extraction composition of the present invention is a buffer solution consisting of 0.01M, pH 7.6 Tris-HCl, 0.01M EDTA and 0.5% SDS; 6M in the eluted nucleus, a buffer solution kit for protein precipitation consisting of sodium chloride solution 2 and solution 3 (sterile distilled water) in the separated genomic DNA.

        In the above process, the use of the buffer solution 2 must clearly reflect its composition, and if the concentration of the sodium collide is less than 6M, nucleic acid extraction may not be performed efficiently. The preferred concentration of sodium chloride is 6M because it may adversely affect or affect subsequent PCR reactions.

There nucleic acid extraction composition of the present invention from all the genomic DNA and the normal of the virus that can be isolated from human body fluids can also extract the DNA, in the present invention as a typical property spread diseases related bacteria, Chlamydia trachomatis, Trichomonas vaginalis , Ureaplasma urealyticum , Neisseria gonorrhoeae , Mycoplasma genitalium , Treponema pallidum , and Candida Nucleic acid of albicans was used.

        The nucleic acid extracting composition of the present invention can extract bacterial nucleic acids contained in these samples using animal tissues, cells or saliva, including humans. More specifically, the nucleic acid extraction buffer solution 3 is added to the samples used in the clinical trials, especially serum, urine, saliva, and tears, followed by heat at 95 ° C. for 5 minutes, followed by electrophoresis for amplification through purification. DNA and viral DNA can be extracted quickly.

        The nucleic acid extracting composition of the present invention can extract genomic DNA quickly and accurately, and is used in a diagnostic method for quickly confirming infection by electrophoresis through amplification using a viral nucleic acid from a sample, thereby determining the diagnosis time, cost and accuracy. Can be improved.

In another aspect, the present invention comprises the steps of collecting a sample from an animal including a human, mixing the nucleic acid extracting composition to the sample and applying heat, performing a PCR to the sample immediately after the ethanol purification of the heated mixture and Checking the PCR results by electrophoresis provides a virus detection method comprising the step of determining whether the virus infection.

The PCR process can be carried out by a conventional method, and there is no need to change the composition of the PCR conditions and enzymes used in the PCR according to the type of virus to be tested and can be optimized under the same conditions in the current clinical trials I think it will be useful to you.

Hereinafter, examples of the present invention will be described. The following examples are only for illustrating the present invention, but the protection scope of the present invention is not limited to the following examples.

Example 1 Preparation of Nucleic Acid Extraction Solution

        Nucleic acid extraction solution 1 was prepared by mixing a dissolution solution consisting of 0.01M, pH 7.6, Tris-HCl, 0.01M EDTA, and 0.5% SDS, and then used to elute nuclei in various body fluids; Solution 2 prepared 6M, sodium chloride buffer solution 2 in the nucleus extracted above as a solution for precipitating proteins and other substances, and prepared a buffer solution for recovering genomic DNA consisting of solution 3 (sterile distilled water). The final volume is 100 ml each. The nucleic acid extraction solution was stored at room temperature.

Example 2: Template DNA Extraction from Clinical Specimens

2-1. Cervical Exfoliated Cells and Urine

        After 200 μl each of cervical exfoliated cells and urine, 600 μl of Tris-HCl buffer solution with pH 7.6 of 0.01M was added and vortex mixed, followed by boiling for 5 minutes in a 95 ° C. heat block to elute the nuclei of cells. 300 μl of 6M sodium chloride was added to the tube in which the cell nucleus was eluted, followed by vortex mixing, 800 μl of the same amount of ethanol was added thereto, followed by well mixing to precipitate genomic DNA. Centrifuge the tube at 16,000 x g for 2 minutes in a microcentrifuge, carefully discard the supernatant and remove residual residue in the genomic DNA pellets at the bottom of the tube with absorbent paper and then at room temperature for 10 minutes. Dried over. After drying, 50 µl of sterile distilled water was added to dissolve the genomic DNA pellet and stored at -20 to -80 ° C for long-term storage of the dissolved genomic DNA. The process took 20-30 minutes in total.

Experimental Example

        In order to assay the nucleic acid extraction efficiency in the case of extraction by the method of Examples 1 and 2, PCR was performed using this.

Experimental Example 1. STD PCR Analysis

Chlamydia isolated from each patient trachomatis , Trichomonas vaginalis , Ureaplasma urealyticum , Neisseria gonorrhoeae , Mycoplasma genitalium , Treponema pallidum And Candida albicans After 3 times nucleic acid extraction solution 1 was mixed with the sample and thermally denatured, the nucleic acid extraction solution 2 was added thereto, followed by addition of 2 times ethanol. Primer for diagnosing holy wave disease was made species-specific and the composition for PCR analysis was performed according to the standard protocol shown in Table 1 below.

             Table 1 Composition for PCR Reaction

PCR components Volumr (ul) 10X buffer solution 2.5 10 mM dNTPs 0.5 10 mM MgCl2 3.75 Taq DNA polymerase 0.5 (1 unit) species specific primers 1.0 (10pmol) each DW 3.75 Template DNA 8.0 Total 20

        PCR reaction conditions were first denatured at 95 ℃ for 15 minutes (94 ℃, 30 seconds-> 60 ℃, 1 minute 30 seconds-> 72 ℃, 1 minute 30 seconds), 40 times-> 72 ℃, 5 minutes reaction It was finished. PCR products were electrophoresed for 15 to 20 minutes at 100 V on 2% agarose gel to confirm whether the DNA extracted with the nucleic acid extraction solution of the present invention affects PCR amplification.

Figure 1 is an electrophoresis picture showing the results of PCR after extracting the nucleic acid of various temple-related bacteria related nucleic acid extraction solution of the present invention. In Figure 1, lane 1 is Chlamydia trachomatis Would use the DNA extracted from cervical cells eliminated and urine were collected from infected patient, Lane 2 times Trichomonas vaginalis Cervical decidual cells and urine extracted DNA from infected patients were used. Lane 3 is Ureaplasma urealyticum. Cervical decidual cells and urine extracted DNA from infected patients were used. Neisseria gonorrhoeae Cervical decidual cells and urine extracted DNA from infected patients were used. Mycoplasma Cervical decidual cells from genitalium infected patients and DNA extracted from urine were used. Lane 6 is Treponema. pallidum DNA extracted from urine collected from infected patients was used. Lane 7 was Candida. albicans Using DNA extracted from urine collected from infected patients, we confirmed that it was specifically amplified in all infected patients, and mixed 7 bacterial DNAs as shown in lane 8 to efficiently analyze a small amount of samples. As a result of PCR amplification using multiple polymerase chain reaction using one sample, very useful results were obtained. Therefore, the nucleic acid extracting solvent of the present invention efficiently extracts nucleic acids from various samples, and it can be confirmed that there is no interference effect on the PCR reaction.

        As mentioned above, the nucleic acid extracting solution of the present invention can extract nucleic acids easily, quickly and accurately compared to organic solvents and various commercial kits, and can perform PCR analysis immediately after extraction. In addition, it can be applied to various clinical tests because it does not interfere with the PCR reaction and can lead to quick and accurate results.

Claims (3)

        The method of claim 1,         (a) a solution kit for cell lysis consisting of Tris-HCl buffer solution at pH 7.6 of 0.01 M, EDTA at 0.01 M and 0.5% SDS; (b) a solution kit for protein precipitation consisting of 6 M sodium chloride; And (c) buffer solution kit for genomic DNA recovery consisting of sterile distilled water; Reagent kit for genomic DNA extraction from human body fluid consisting of.         The method of claim 2,        Eluting a nucleus from human body fluids; Separating the genomic DNA and nuclear protein from the eluted nucleus; And a third step of recovering the separated genomic DNA. A method of extracting genomic DNA from human body fluid.         The method of claim 3, wherein        (a) collecting a sample from an animal, including a human; (b) mixing and heating a nucleic acid extracting composition according to any one of claims 1 to 2 to a sample; (c) performing PCR on the heated mixture with a sample; And (d) checking the PCR result by electrophoresis to determine whether a virus is infected.

Images