JPS6363067B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for identifying virus serotypes. More specifically, the present invention is based on the restriction enzyme cleavage pattern of total DNA of DNA virus-infected cells.
The present invention relates to a method for identifying virus serotypes. In order to appropriately diagnose, treat and prevent diseases caused by viruses, it is necessary to accurately identify the serotype of the virus. For example, adenovirus group viruses are the causative agents of diseases such as acute febrile pharyngitis, acute respiratory tract disease, pharyngoconjunctival fever, epidemic keratoconjunctivitis, and pertussis-like syndrome; There is a type (viral DNA
For the appropriate diagnosis, treatment, and prevention of diseases, it is necessary to accurately identify the virus serotype. Traditionally, for the identification of serotypes of viruses, e.g. adenoviruses, complement fixation and immunofluorescence have been used to detect common antigens of adenoviruses, while hemagglutination inhibition and neutralization reactions have been used to detect common antigens of adenoviruses. has been used to determine types. However, in the identification based on the hemagglutination inhibition reaction and the neutralization reaction, cross-reactivity may be observed between the types, and therefore, there are cases in which the accuracy is questionable.
Furthermore, it was necessary to prepare antiserum for each type, which caused problems in terms of simplicity. Recently, a method of cutting herpesvirus DNA with restriction enzymes and identifying the serotype from the cut pattern has been introduced (The Journal of Infectious Diseases).
Infectious Diseases) Volume 138, No. 4, No. 488
−498 pages (1978)). However, in this method, the herpesvirus is isolated and purified, and purification of the virus requires a large amount of infected cells and a complicated method, which requires a great deal of time and effort. This method had practical problems, especially in terms of diagnosis and treatment. The present inventors conducted intensive research on a method to easily and accurately identify the serotype of a DNA virus using a small amount of infected cells, and as a result, extracted total DNA from DNA virus-infected cells and Well, if you cut it with a restriction enzyme to form a cut pattern (without separating the viral DNA and cellular DNA), the virus will be removed without being affected by the cellular DNA.
The present invention was achieved based on the discovery that DNA cleavage patterns can be clearly distinguished and the serotype can be easily and accurately identified. That is, the present invention involves infecting DNA virus-susceptible cells with a DNA virus, culturing the infected cells, extracting and purifying the total DNA of the infected cells, and then extracting and purifying the extracted and purified total DNA without separating the viral DNA and the cellular DNA. DNA is cut with restriction enzymes and the cut pattern is compared to DNA viruses of known serotypes.
This is a method for identifying virus serotypes, which is characterized by comparison with DNA restriction enzyme cleavage patterns. There are no particular restrictions on the DNA viruses whose serotypes can be identified using the method of the present invention, and the principle can be applied to any DNA virus, including adenovirus group viruses and herpes group viruses. It can be particularly suitably applied to. Any DNA virus-susceptible cell may be used as long as it can be infected with a DNA virus and multiply, but in the case of adenovirus group viruses, KB cells, Hela cells, and human fetal kidney cells are preferable; Hep in case of virus
Cells, Hela cells or Velo cells are preferred, and in the case of varicella virus among the herpes group viruses, human fetal lung cells and fibroblasts are more preferred. Infected cells infected with a DNA virus can be cultured by known methods. Suitable media include, for example, Eagle Minimum Essential Medium (MEM) containing 5% calf serum. As for the culture conditions, for example, 1 to 7 days at 37° C. under 5% CO ₂ aeration is sufficient. If the infected cells are destroyed and virus particles are released, it will be inconvenient for subsequent treatment, so it is most preferable to stop the culture at the stage when a clear cytopathic effect appears. Subsequently, from such infected cells, the entire DNA of the infected cells, i.e. the viral DNA and the cell
Extract and purify DNA. The DNA extraction and purification method is not particularly limited, but the following phenol method and diethylpyrocarbonate (DEPC) method are preferred. The latter is particularly suitable when treating small amounts of cells. (1) An example of the phenol method preferably used in the present invention is as follows. Virus-infected cells (3 x 10 ^cells ) were washed once with phosphate buffered saline (PBS) and incubated at 1500 rpm.
Centrifuge the cells for 5 minutes at
7.5) and leave at room temperature for 10 minutes.
To this, pronase was added at a rate of 500 μg/ml and treated at 37°C for 3 hours, and then an equal volume of phenol was added and mixed thoroughly.
Centrifuge at 2000 rpm for 10 minutes to extract nucleic acids into the aqueous layer (repeat this operation 3 times). Add sodium acetate to the resulting aqueous layer to a concentration of 0.3M. Thereafter, this solution was cooled on ice with 2
Add ~2.5 times the volume of ethanol dropwise and incubate at −70°C for 10
After leaving for a minute, centrifuge at 15,000 rpm for 5 minutes,
The resulting precipitate is air-dried. And this precipitate,
Resuspend in an aqueous solution of 0.015M sodium chloride and 0.0015M sodium citrate, add 100 units/ml of RNase (previously heat-treated at 90°C for 10 minutes), and treat at 37°C for 2 hours. Decompose the RNA to obtain the desired purified total DNA. (2) An example of the diethylpyrocarbonate method is as follows. Cells obtained in the same manner as in the case of the phenol method in (1) above are suspended in 0.05M Tris-HCl buffer (PH7.5) containing 1.0% sodium dodecyl sulfate and 0.1 to 0.5% DEPC, Stir gently for 20 minutes at 0°C. After that, an equal amount of ethyl ether was added to remove DEPC, and the resulting lower layer (aqueous layer) was centrifuged at 1500 rpm for 3 to 5 minutes.
Add sodium acetate to 0.3M. Thereafter, cooled ethanol is added dropwise to the obtained solution under ice-cooling, and the subsequent treatment is carried out in the same manner as in the phenol method described in (1) above. The restriction enzyme used in the present invention is DNA
There is no particular limitation as long as it is an enzyme that recognizes a specific nucleotide sequence and cuts DNA at a specific site. These are commonly called type restriction enzymes, such as BamH, Hind, EcoR,
There are Sal and Hpa. However, in order to specifically apply the DNA cleavage pattern by such restriction enzymes to the identification of individual virus serotypes, methods such as those described in Examples may be used depending on the specific virus or specific serotype. It is necessary to select the most suitable restriction enzyme from among various types of restriction enzymes. For example, a restriction enzyme suitable for serotyping adenovirus DNA is a restriction enzyme that first cuts adenovirus DNA into an analyzable number of specific fragments, e.g., a restriction enzyme that recognizes a six-base sequence. Hind, EcoR, BamH, Sal and
Select from Hpa. Furthermore, it is necessary to select restriction enzymes from among these that have as few common cleavage sites as possible among different serotypes. For example, the cleavage patterns of adenovirus types 3 and 7 cannot be clearly distinguished using Hind, EcoR, or Sal, but can be clearly distinguished when the cleavage patterns of BamH are compared. Therefore, in order to distinguish between adenovirus types 3 and 7, it is found that BamH is optimally used as a restriction enzyme. By the method described above, the restriction enzyme most suitable for type identification of a specific virus serotype can be appropriately selected. Cutting DNA with restriction enzymes and creating a cut pattern can be carried out, for example, by the following method (Virology, Vol. 82, Vol.
(1977), pp. 509-512). Sufficient amount for DNA (e.g. per 1μg of DNA)
Add 3-5 units of restriction enzyme (0.2-1.0 units) at 37°C.
Allow time to react. The total volume of the reaction solution is 20-70μ, and an appropriate buffer is used. A DNA amount of 0.4 to 10 μg is sufficient to obtain a clear cut pattern of DNA. After the reaction is completed, the reaction solution is electrophoresed in a migration buffer using, for example, a 1.4% agarose plate gel, stained with ethidium bromide, and the cutting pattern is observed and photographed under ultraviolet light. Comparison of cleavage patterns by restriction enzymes was performed using Southern's blotting method (J.MOl.Biol.,
98, 503-517 (1975)). That is, infected cell DNA was cut with restriction enzymes as described above, subjected to agarose gel electrophoresis, each DNA fragment in the agarose gel was made into a single strand by alkaline treatment, and then transferred onto a membrane filter according to Southern's method. , ^32P or ^3H labeled probe DNA and hybridization is formed and detected. By using Southern's blotting method, the amount of infected cell DNA required for comparative examination of cleavage patterns can be reduced, and even from infected cells before clear cytopathic effects have appeared,
Clear cutting patterns can be obtained. Comparison of cutting patterns can also be performed by the DBM paper blot method (Proc. Natl.
Acad.Sci.USA74, 5350-5354). That is, DNA is transferred onto 3 paper treated with diazobenzyloxymethyl (DBM) and detected by forming a hybrid with probe DNA labeled with ³² P or ³ H. In the method of the present invention, spot hybridization (Gann, 70,
239-243 (1979)).
Identification based on DNA restriction enzyme cleavage patterns may be easier. For example, the total DNA of adenovirus-infected cells is treated with alkali, spotted on a membrane filter, and hybridized with ³² P-labeled adenovirus subgroup DNA to determine the presence or absence of homology by autoradiography. and determine the subgroup of the infecting virus in advance. After that, the infected cell's entire DNA
Virus serotypes can be easily identified by comparing the cleavage pattern of the virus with the cleavage patterns of DNA viruses within the same subgroup. According to the method of the present invention, a small number of virus-infected cells are used (the required number of infected cells is
(approximately 1/40 less than when using DNA), viruses
Since it is possible to easily and accurately fix virus serotypes without separating DNA from cellular DNA, the present invention has great utility in the diagnosis, treatment, and prevention of viral diseases, and in medical research related to viruses. is extremely large. Hereinafter, the present invention will be explained in detail with reference to Examples. Example 1 In this example, whole cells were isolated from DNA virus-infected cells.
Extract the DNA and use it as is (viral DNA
When cutting with a restriction enzyme (without separating into cellular DNA) and creating a cutting pattern, the resulting cutting pattern of viral DNA is similar to that of cellular DNA.
purified virus without being affected by the presence of
This exemplifies that the same cleavage pattern as in the case of using DNA is shown. (1) Culture of cells infected with DNA virus (adenovirus type 7) KB cells were cultured at 5%
Virus was cultured in a monolayer (approximately 1.5 x ¹⁰ cells) in Eagle Minimum Essential Medium (MEM) containing calf serum.
The cells were inoculated at a moi of 10-50 PFU/cell and cultured at 37°C for about 3 days until a clear cytopathic effect was observed. (2) Extraction and purification of virus (adenovirus type 7) DNA Based on the method of Green and Pina (Proc. Natl. Acad. Sci. 51, 1251-
1259 (1964) and Virology 20, 199-207 (1963)
), the infected cells obtained in (1) above were disrupted, cell-derived proteins were denatured and removed with an organic solvent, and virus particles were purified by cesium chloride density gradient equilibrium centrifugation. Thereafter, the phenol method was applied to extract and purify the viral DNA. (3) Extraction and purification of total DNA from infected cells The DNA virus-infected cells obtained in (1) above,
The cells were treated according to the phenol method (the phenol treatment was repeated twice), and the total DNA of the infected cells was extracted and purified. (4) Cutting the total DNA of infected cells with restriction enzymes and cutting pattern The total DNA of infected cells obtained in (3) above was diluted with 10μ
A sufficient amount of Hind, BamH, Sal or EcoR was added as a restriction enzyme per 1 μg of DNA, and the reaction was carried out at 37° C. for 5 hours. In addition, in the case of Hind, the reaction buffer is 20m
MTris-HCl (PH7.4), 7mM _MgCl2 , 60m
MNaCl, 2mM2-mercaptoethanol system, in the case of BamH, 6mM TrisHCl (PH7.5),
A system of 6mM MgCl ₂ , 20mM KCl, 6mM2-mercaptoethanol was added to
MTris-HCl (PH7.5), _10mMgCl2 system,
For EcoR, 100 mMTris-HCl (PH7.5),
A system of 10mM MgCl ₂ and 50mM NaCl was used, and the total amount of the reaction mixture was 50μ. After the reaction, the reaction mixture was transferred to a 1.4% agarose plate gel (2 x 160 x 220 mm) using running buffer - 36mM Tris-HCl (PH8.1) for 32m
MKH ₂ PO ₄ in 0.1 m MEDTA for 13 hours;
Electrophoresis was performed at 50 V, and the cells were stained with ethidium bromide (0.5 μg/ml) for 15 minutes, and the cutting patterns were observed and photographed under external light. The cleavage pattern for each restriction enzyme used is shown as a in FIG. 1, respectively. Exactly the same way as above (but with the virus
(use 1 μg of DNA), determine the restriction enzyme cleavage pattern of only the viral DNA obtained in (2) above,
It is shown as b in FIG. As is clear from Figure 1, the total DNA of infected cells
In the cutting pattern (a), there are cells in the background.
Although a continuous pattern of DNA appears faintly (indicated by diagonal lines in a of Figure 1, omitted in Figures 2 and 3), this is not enough to hinder the identification of the virus. I understand that. And also, the cleavage pattern of the viral DNA thus formed is the cleavage pattern of the purified viral DNA.
It can be seen that this is completely consistent with (b). The above facts indicate that in order to investigate the restriction enzyme cleavage pattern of viral DNA, it is not necessary to prepare a sample by performing the complicated operations of isolating and purifying virus particles from infected cells and extracting and purifying the viral DNA. This shows that it is possible to directly extract and purify total DNA from infected cells and use it as a sample. Furthermore, as is clear from Figure 1, even if the serotype of the virus is the same, the cleavage pattern differs depending on the restriction enzyme used. Therefore, in order to identify a specific serotype of a specific virus, it is necessary to use the most suitable restriction enzyme. It is desirable to select . Example 2 This example shows that adenovirus types 3 and 7 (both belong to group B and have high DNA homology) can be distinguished from the restriction enzyme cleavage pattern of total DNA of infected cells. Adenovirus types 3 and 7 were used as viruses, and various restriction enzymes (Hind, BamH, Sal
, EcoR) was obtained. The results are shown in Figure 2. As is clear from Figure 2, the cleavage patterns of adenovirus types 3 and 7 are Hind, Sal and
When EcoR is used, they are very similar and difficult to distinguish, but on the other hand, when BamH is used,
The number and position of bands are different between the two, and they can be clearly distinguished. Example 3 This example shows that when BamH is used, the restriction enzyme cleavage pattern clearly differs depending on the type of adenovirus. As adenoviruses, 12 and 31 (more than A
Group), 3, 7 (Group B), 2, 5 (Group C), 9,
Cleavage patterns were obtained in the same manner as in Examples (1), (3) and (4) using viruses of each type 13 (group D) and 4 (group E) and BamH as the restriction enzyme. Ta. The results are shown in Figure 3. From Figure 3, 12 type has 8 pieces, 31 type has 11 pieces, 3 type has 9 pieces, 7 type has 9 pieces, 2 type has 4 pieces, 5 type has 2 pieces, 9 type has 4 pieces,
It can be seen that 4 bands for type 13 and 8 bands for type 4 can be clearly observed, and they can be distinguished from each other. Next, the adenovirus type 7 DNA fragment cut and formed by EcoR, the type 12 DNA fragment cut and formed by Hind, and
Relationship curve between relative mobility of DNA fragments in electrophoresis and molecular weight (daltons) obtained using type 2 DNA fragments cleaved by BamH as size markers (4th
The molecular weight of each band (DNA fragment) of each adenovirus was calculated based on the above-mentioned BamH, and the results are shown in Table 1.

【table】

[Table] From Table 1, it can be seen that a unique restriction enzyme cleavage pattern for each type can be obtained as a numerical value, and this can be used for identification of each type.

[Brief explanation of drawings]

Figure 1 shows various restriction enzymes (Hind, BamH,
The cleavage pattern (a) of total DNA of adenovirus type 7-infected cells by Sal and EcoR) and the cleavage pattern of adenovirus type 7 DNA (b) are shown. Second
The figure shows various restriction enzymes (Hind, BamH, Sal
A comparison of the total DNA cleavage pattern of cells infected with adenovirus type 3 and the total DNA cleavage pattern of cells infected with adenovirus type 7 by adenovirus type 3 and EcoR) is shown. Figure 3 shows the total amount of cells infected with each type of adenovirus using BamH as a restriction enzyme.
Showing DNA cleavage patterns. FIG. 4 shows a relationship curve between relative mobility of DNA fragments in electrophoresis and molecular weight (Dalton).

Claims (1)

[Claims] 1. Infect DNA virus-susceptible cells with a DNA virus, culture the infected cells, and then
DNA is extracted and purified, then viral DNA and cells
All the extracted and purified products without separating the DNA.
DNA is cut with a restriction enzyme, and the cut pattern is
A method for identifying virus serotypes, which is characterized by comparing the DNA restriction enzyme cleavage patterns of DNA viruses with known serotypes. 2 DNA virus-susceptible cells are KB cells,
The method for identifying virus serotypes according to claim 1, which is Hela cells, human fetal kidney cells, or Velo cells. 3. The method for identifying virus serotypes according to claim 1, wherein the infected cells are cultured until a clear cytopathic effect of the infected cells appears. 4 Restriction enzymes are BamH, Hind, EcoR,
The method for identifying a virus serotype according to claim 1, which is Sal or Hpa. 5. The method for identifying virus serotypes according to claim 1, wherein the restriction enzyme cleavage pattern is a cleavage pattern determined by electrophoresis. 6 Restriction enzyme cleavage patterns can be compared using ethidium bromide staining or Southern
The method for identifying virus serotypes according to claim 1, which is carried out by the DBM paper blotting method or the DBM paper blotting method.