KR100426544B1 - Method for extracting viral nucleic acids from serum or plasma and compositions therefor - Google Patents

Abstract

The present invention relates to a method for extracting a nucleic acid of a virus from human or animal serum or plasma, and to a composition used in the method. The extract composition is mixed with serum or plasma to extract the viral nucleic acid, the mixture is heated and cooled, and centrifuged to extract the nucleic acid. The present invention also provides a nucleic acid extracting composition used for the extraction of the viral nucleic acid.

Description

Method for extracting viral nucleic acids from serum or plasma and compositions therefor}

The present invention relates to a method for extracting a nucleic acid of a virus from the serum or plasma of humans or animals, and a composition for extracting nucleic acids used in the extraction method, in detail, Dithiothreithol (DTT) and ethyl phenol polyethylene Human or animal serum, comprising mixing a composition for extracting nucleic acids containing glycoether (Ethylphenolpolyethyleneglycoether, Nonidet P40) with serum or plasma to extract viral nucleic acids, heating and cooling the mixture and centrifuging the mixture Or it relates to a method for extracting a nucleic acid of a virus from plasma and a composition for extracting nucleic acids used in the extraction method.

Many viruses can be present in the blood of humans or animals. Viruses that can be diagnosed using serum and plasma rather than leukocytes or erythrocytes in the blood using the polymerase chain reaction (PCR) method are hepatitis A virus (HAV) and type B. Hepatitis Virus (HBV), Hepatitis C Virus (HCV), Hepatitis D Virus (HDV), Hepatitis E Virus (HEV), Hepatitis G Virus (HGV), AIDS Virus (HIV), Rubella Virus (Rubella) , Influenza virus. At this time, in order to perform fish, the virus must be extracted only if the nucleic acid, ie, DNA or RNA, of the virus is extracted.

Conventionally, organic solvents such as phenol and chloroform have traditionally been used to extract nucleic acids from prokaryotic and eukaryotic or complex biological samples. Nucleic acid extraction using the enzyme is digested enzymatically with proteolytic enzymes, cells are broken with ionic surfactant, and nucleic acid is extracted with phenol or phenol / chloroform mixture. According to the extraction, the organic solvent phase and the water phase are separated, and the extracted nucleic acid can be separated by precipitating the nucleic acid distributed toward the water with alcohol. However, phenol or phenol / chloroform mixtures destroy human skin and are considered hazardous wastes and should be handled with care and disposed of well. In addition, the extraction process is time consuming and cumbersome. Marmur is J. Mol. Biol. 3: 208-218 (1961) describes the process of extracting and purifying DNA from prokaryotes using enzyme treatment, surfactant addition, and organic solvents such as phenol or phenol / chloroform.

On the other hand, due to the fact that chaotrophic salts inhibit nuclease and protease, guanidium thiocyanate (GnSCN) when breaking cells and secreting nucleic acids in solution Kiotropic materials such as) are widely used. In contrast, Chirgwin et al. Homogenized cells under guanidium thiocyanate and 2-mercaptoethanol in Biochemistry 18: 5294-5 299 (1979), and either ethanol precipitation or cesium chloride (CsCl). The isolation of RNA by precipitation is described. However, it has been found that ionic surfactants and kiotrops are incompatible and that nucleic acids are difficult to separate into the aqueous phase when using high molar concentrations of salts, making it difficult to separate nucleic acids from these kiotropic salts. lost.

The ability to effectively inhibit nucleases during the nucleic acid isolation process is particularly important when the starting material is complex, such as blood. Brownhill et al. Reported that bentonite was an inhibitor of nuclease in Biochem. J. 73: 434 (1959), and Fraenkel-Conrat et al. Purified the tobacco mosaic virus in Virology 14: 54-58 (1961). We have developed a method to use bentonite to inhibit ribonuclease. The researchers then reported the use of bentonite in combination with phenol and chloroform to reduce ribonuclease activity in the purification of RNA.

However, the use of toxic substances such as phenol or phenol / chloroform in the above methods have a dangerous effect on life or the environment, and when using a kiotropic substance, it is not easy to separate nucleic acids from these substances. In addition, in the case of using the above methods, there was a problem that the extraction process consumes a lot of time and effort.

It is an object of the present invention to provide a composition for extracting nucleic acids for simple and rapid extraction of viral nucleic acids from non-cellular biological fluids, preferably plasma or serum.

Another object of the present invention is to provide a method for simply and quickly extracting viral nucleic acid from a non-cellular biological fluid, preferably plasma or serum, using the nucleic acid extracting composition.

Another object of the present invention is to provide a method for easily diagnosing a nucleic acid of a virus by applying a nucleic acid extracted simply and quickly by the above method to a fish.

Figure 1 is a picture of the results of fish (PCR) of hepatitis B virus.

Figure 2 is a picture of the results of PCAL (PCR) of hepatitis C virus.

In order to achieve the above object, one aspect of the present invention is a polythioethylene sorbitan monolaurate or polyoxyethylene sorbitan monooleate which is dithiothitol, ethyl phenol polyethylene glycol ether and / or nonionic surfactant It provides a nucleic acid extracting composition for extracting a nucleic acid from the blood containing.

The composition can preferably be used to extract viral nucleic acids from serum or plasma of humans or animals.

Viruses that can be extracted with the composition are hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), hepatitis E virus (HEV), hepatitis G Viruses (HGV), AIDS virus (HIV), Rubella virus (Rubella), and Influenza virus (Influenza), and the like, but are not necessarily limited thereto.

Specifically, the composition contains 0.5 mol% to 5.0 mol% of 1 mol dithiothreitol, and 0.5 mass% to 5.0 volume% of 10% ethylphenol polyethylene glycol ether.

When the composition further comprises polyoxyethylene sorbitan monolaurate or polyoxyethylene sorbitan monoorate, their final concentration is preferably 0.5 to 5.0% by volume, respectively.

When the viral nucleic acid is DNA, it is preferable to dilute the composition in distilled water, and when the viral nucleic acid is RNA, the composition is diluted in diethyl water treated with Diethyl Pyrocarbonate (DEPC).

If the viral nucleic acid is RNA, the reason for using distilled water treated with Dipci is that RNA degrading enzyme is stable for various treatments, but its activity is removed when Dipci is treated, so it is used for the stability of the extracted RNA.

According to another aspect of the present invention, the serum or plasma to be extracted from the nucleic acid is mixed with the nucleic acid extracting composition according to the present invention described above, the mixed solution is heated, the heated solution is cooled, and the cooled solution is Provided are methods for extracting viral nucleic acids from serum or plasma, comprising centrifugation.

As described above, the method for extracting the viral nucleic acid from the serum or plasma according to the present invention, instead of the process of digesting with a conventional proteolytic enzyme, simply mixing the heating nucleic acid extract composition according to the invention with serum or plasma and heating And extracting nucleic acids directly therefrom by cooling and centrifuging the heated solution without the addition of a separate extract, which greatly simplifies the overall process for nucleic acid extraction, and thus time and This is a way to save effort.

In the method according to the present invention, the step of heating the mixture of serum or plasma and nucleic acid extraction composition is heated for 2 to 10 minutes at a temperature of 80 ~ 120 ℃, the centrifugation step is 2 ~ at 10,000 ~ 15,000rpm Centrifugation for 10 minutes is preferred.

Hereinafter, the present invention will be described in detail with reference to Examples.

The following examples are provided only to aid the understanding of the present invention, and the present invention is not limited to the following examples.

Unless otherwise specified in the following examples, it is carried out at room temperature.

Example 1

(Preparation of composition for extracting viral DNA nucleic acid)

The following dithiothitol was dissolved in 0.01 M sodium acetate (pH 5.2) and used by filtration. Sodium acetate was used by autoclaving after adjusting the pH of the sodium acetate to acetic acid in tertiary distilled water to 5.2.

The following ethyl phenol polyethylene glycol ether is to stabilize protein dissolution and Taq polymerase (Polymerase), 10% stock solution was used.

The following polyoxyethylene sorbitan monolaurate and polyoxyethylene sorbitan monolaurate are nonionic surfactants, and a stock solution was used.

Example 1-1

10 μl of 1M dithiothreitol and 10 μl of 10% ethylphenol polyethyleneglycoether stock solution were diluted in distilled water to make a total volume of 1000 μl and frozen.

Example 1-2

Dilute 10M of 1M dithiothritol, 10µl of 10% ethylphenolpolyethyleneglycoether stock solution, and 10µl of polyoxyethylene sorbi tan monolaurate (Tween 20) stock in distilled water. Was made to 1000 μl and then frozen.

Example 1-3

Dilute 10M of 1M dithiothreitol, 10µl of 10% ethylphenolpolyethyleneglycoether stock solution, and 10µl of polyoxyethylene sorbitan monooleate (Tween 80) stock in distilled water. After making 1000 μl and frozen.

Example 2

(Preparation of a composition for extracting a viral RNA nucleic acid)

Example 2-1

10 μl of 1M dithiothreitol and 10 μl of 10% ethylphenol polyethyleneglycoether stock were diluted in distilled water to make a total volume of 1000 μl and frozen.

Example 2-2

After diluting 10 µl of 1M dithiothitol, 10 µl of 10% ethylphenol polyethylene glycol ether, and 10 µl of polyoxyethylene sorbitan monolaurate in distilled water, the total volume was changed to 1000 µl. Frozen.

Example 2-3

10 μl of 1 M dithiothitol, 10 μl of 10% ethylphenol polyethylene glycol ether, and 10 μl of polyoxyethylene sorbitan monooleate were diluted in distilled water to make a total volume of 1000 μl. And then frozen.

Example 3

(Extraction method of viral nucleic acid)

1) The blood of a patient infected with hepatitis B virus and hepatitis C virus is collected and centrifuged to remove serum or plasma solids such as red blood cells, leukocytes, and the like. Put well and mix well.

2) The container containing the liquid mixture of the said serum or plasma and the nucleic acid extracting composition was put into the water tank which adjusted the temperature to 95 degreeC, and it heated for 2 minutes.

3) The heated mixed solution was taken out and cooled in the prepared ice.

4) The separated supernatant was obtained by centrifuging for 4 minutes at 13,000 rpm.

Example 4

(Fish)

A portion of the supernatant obtained through the above process was used for fish or the fish was added by directly adding the fish solution to the container.

The composition and reaction conditions of the fish were as shown in the table below.

Example 4-1 : Fishes of Hepatitis B Virus

Table 1 relates to the composition and reaction conditions of the fishes of the hepatitis B virus.

[Table 1] Fish Composition and Reaction Conditions of Hepatitis B Virus

Example 4-2 Alti-pisal of Hepatitis C Virus

Table 2 relates to fish's composition and reaction conditions of hepatitis C virus. Fishes of hepatitis C virus were subjected to fishes using reverse transcriptase from RNA with Alti-Pisal (RT-PCR).

TABLE 2 Alti-Phial Composition and Reaction Conditions

Fish was carried out by the above method and the result was examined through agarose gel electrophoresis. Electrophoresis was performed at 100V, 3 minutes in 2% agarose gel.

After electrophoresis, the presence or absence of the band was examined whether the nucleic acid of hepatitis B virus or hepatitis C virus was extracted.

The results of separating the nucleic acid of the virus by the above process are shown in Figs.

Figure 1 shows a picture of the results of the fish hepatitis B virus. M represents the molecular weight standard marker of ФX 174 / Hae III, lanes 1-10 represent HBV positive samples of 256 bp and hepatitis B virus negative samples 11-15. As shown in FIG. 1, DNA was extracted using the present invention, and when a fish virus of hepatitis B virus, which is a DNA virus, was tested, all of them were positive in 10 positive (No. 1-10) and negative. Five controls (No. 11-15) showed negative results.

In addition, Fig. 2 shows a picture of the results of the hepatitis C virus fish. As shown in FIG. 2, RNA was extracted using the present invention, and HCV (HCV), which was RNA virus, was used to examine all 10 positive cells. 5 negative controls (No. 11-15) showed negative results.

The time required to extract the nucleic acid of the virus using the nucleic acid extracting composition according to the embodiment was about 5-10 minutes. This takes about 60 minutes or more for RNA extraction using commercially available extraction kits such as the TRI REAGENT BD kit from Molecular Research Center, Inc. and 30 minutes or more for DNA extraction. In comparison, the nucleic acid can be extracted in about 1/8 time. Therefore, the nucleic acid extraction method according to the present invention has the advantage that the extraction process is quick and simple to minimize the abnormality of the results due to technology and contamination, and the overall good nucleic acid can be obtained due to the relatively small influence on the nucleic acid enzyme. There is also.

On the other hand, when all three of the above materials of dithio thritol, ethyl phenol polyethylene glycoether and polyoxyethylene sorbitan monolaurate or polyoxyethylene sorbitan monoorate were used to extract the nucleic acid, the most effective Nucleic acid extraction is easy even when a mixture of ostoleitol and ethyl phenol polyethylene glycol ether is used. Could get

According to the constitution of the present invention, the nucleic acid extracting composition according to the present invention is simply mixed with serum or plasma, heated, cooled and centrifuged, and the supernatant is directly applied to the PSI reaction. In particular, the presence or absence of viral nucleic acids associated with disease can be determined simply and quickly.

In addition, the nucleic acid extraction method according to the present invention not only simplifies the procedure but also reduces the exposure time to nuclease, thereby contributing to stabilization of the nucleic acid, thereby increasing the yield of nucleic acid having a more complete nucleic acid sequence.

Claims (8)

0.5M to 5.0% by volume of 1M dithiothrate, 0.5% to 5.0% by volume of 10% ethylphenolpolyethyleneglycoether, and A composition for extracting nucleic acids containing the remaining amount of distilled water or dipfish treated distilled water. The nucleic acid extracting composition according to claim 1, further comprising 0.5 to 5.0% by volume of polyoxyethylene sorbitan monolaurate or polyoxyethylene sorbitan monolaurate. The composition of claim 1, wherein the composition is used to extract viral nucleic acid from serum or plasma of a human or animal. 4. The virus of claim 3, wherein the virus is hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus (HDV), hepatitis E virus (HEV), hepatitis G virus (HGV), AIDS virus. Nucleic acid extraction composition is selected from the group consisting of, Rubella virus, and influenza virus. 4. The nucleic acid extracting composition according to claim 3, wherein when the viral nucleic acid is DNA, the composition contains dithiothritol, ethylphenol polyethylene glycol ether, and distilled water. 4. The nucleic acid extracting composition according to claim 3, wherein when the viral nucleic acid is RNA, the composition contains dithiothitol, ethylphenol polyethyleneglycoether and distilled water. a) mixing the serum or plasma with the nucleic acid extracting composition according to any one of claims 1 to 6, b) heating the mixed solution at a temperature of 80-120 ° C. for 2-10 minutes, c) cooling the heating solution, and d) centrifuging the cooled solution at 10,000-15,000 rpm for 2-10 minutes. delete