JPH05309000A - Method for detecting epstein-barr virus - Google Patents

Abstract

PURPOSE:To obtain a method for rapidly and accurately detecting Epstein-Barr virus. CONSTITUTION:A mixture solution containing at least one of 4 combinations of the first DNA primer containing an oligonucleotide part of specific >=15 bases with the second DNA primer, a DNA polymerase and an aqueous liquid test sample is subjected to a DNA amplifying step and the resultant reactional solution is then subjected to a DNA inspecting step. Furthermore, at least one of four probes, containing an oligonucleotide part of specific >=10 bases and carrying a label is brought into contact with a test sample to detect a signal from the label.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for detecting Epstein-Barr virus (hereinafter sometimes referred to as EBV).

[0002]

2. Description of the Related Art Epstein-Barr virus (EB
V) is a kind of virus belonging to the herpes family. The majority of adults are infected with this virus, and almost all of them are antibody-positive. Many people are infected by the age of 3 years old, but since there are many subclinical infections, no particular medical condition appears. However, EBV may be reactivated when the immune function of the host is reduced. In particular, reactivation of EBV is seen in patients with malignant tumors, AIDS patients, and those who have undergone bone marrow transplantation, and the pathological conditions include the occurrence of infectious mononucleosis and the worsening of prognosis. is there. Therefore, it is clinically very important to definitively diagnose EBV infections such as nasopharyngeal cancer, Burkitt's lymphoma or opportunistic lymphoma.

Conventionally, as a method for detecting EBV, a method of separating and identifying EBV from a biological fluid (blood, saliva, etc.), a tissue or a cell, a virus capsid antigen (VCA) or a nuclear antigen in serum ( Immunological test methods such as EBNA) are used. However, it takes time to obtain a result in the diagnosis to isolate the virus, and there are problems that antibody nonspecific reaction occurs with immunological methods, high sensitivity cannot be obtained, and antibody titer changes. The drawback is that it is difficult to track the spread of the virus. Therefore, not only in the case of organ transplantation or bone marrow transplantation, but also in the diagnosis of diseases, it has been desired to develop a method and an in-vitro diagnostic agent that enable accurate diagnosis of EBV in a short time.

[0004]

DISCLOSURE OF THE INVENTION In order to detect EBV rapidly and accurately, the present inventor has found that EBV DNA or R
When various nucleotide sequences specific to NA were searched, DNA fragments complementary to the sequences were synthesized and evaluated as primers and probes for EBV, they specifically hybridized with DNA or RNA of EBV. We have found what can be used as a primer or a probe. The present invention is based on such findings.

[0005]

SUMMARY OF THE INVENTION Therefore, the present invention provides
(A) A first DNA primer containing an oligonucleotide portion consisting of at least 15 bases of the base sequence represented by the sequence represented by SEQ ID NO: 1 in the sequence listing [hereinafter sometimes referred to as the first primer (1a)] At least one of the nucleotide sequences represented by the sequence of SEQ ID NO: 2 in the sequence listing
Second containing an oligonucleotide portion consisting of 5 bases
DNA primer [hereinafter sometimes referred to as the second primer (2a)], (b) at least 15 of the nucleotide sequences represented by the sequence of SEQ ID NO: 3 in the sequence listing.
A first DNA primer containing an oligonucleotide portion consisting of bases (hereinafter sometimes referred to as the first primer (1b)) and at least 15 bases of the base sequence represented by the sequence of SEQ ID NO: 4 in the sequence listing. Second DNA primer containing an oligonucleotide portion [hereinafter, sometimes referred to as second primer (2b)]
And (c) a first DNA primer containing an oligonucleotide part consisting of at least 15 bases of the base sequence represented by the sequence of SEQ ID NO: 5 in the sequence listing [hereinafter referred to as the first primer (1c) There is]
And a second DNA primer containing an oligonucleotide portion consisting of at least 15 bases of the base sequence represented by the sequence of SEQ ID NO: 6 in the sequence listing [hereinafter, sometimes referred to as second primer (2c)] , And (d) a first DNA primer containing an oligonucleotide portion consisting of at least 15 bases of the base sequence represented by the sequence of SEQ ID NO: 7 in the sequence listing [hereinafter, sometimes referred to as first primer (1d) ] And at least one of the nucleotide sequences represented by the sequence of SEQ ID NO: 8 in the sequence listing
Second containing an oligonucleotide portion consisting of 5 bases
Selected from the group consisting of a DNA primer [hereinafter sometimes referred to as a second primer (2d)],
A mixture containing at least one combination of the first primer and the second primer, a DNA polymerase and an aqueous liquid test sample is subjected to a DNA amplification step, and then the obtained reaction solution is subjected to a DNA inspection step. The present invention relates to a method for detecting Epstein-Barr virus. Furthermore,
The present invention provides SEQ ID NO: 9, SEQ ID NO: 10,
A probe containing a oligonucleotide containing at least 10 bases of the base sequence represented by SEQ ID NO: 11 or SEQ ID NO: 12 and carrying a label is contacted with a test sample, and a signal from the label is detected. The present invention also relates to a method for detecting Epstein-Barr virus. In the nucleotide sequences of the present specification, A means adenine, C means cytosine, G means guanine, and T means thymine.

The sample used in the method of the present invention is not particularly limited as long as it is a sample suspected of containing EBV. For example, extracts from saliva, blood or tissues can be used.

EB of the invention using a combination of primers
The V detection method mainly comprises (1) a DNA amplification step and (2)
DNA detection step. In the EBV detection method of the present invention, it is preferable to first perform the DNA amplification step.

In the DNA amplification step (1) of the present invention, PC
R (Polymerase chain react)
on) method can be used. By using the PCR method, it is possible to automatically amplify only a target DNA region from a small amount of DNA up to about 1,000,000 times (Science).
e, 239: 487-491, 1988). In the PCR method, two types of DNA primers are used, a primer for the + strand (hereinafter referred to as the first primer) and a primer for the − strand (hereinafter referred to as the second primer) with the DNA region to be amplified interposed therebetween.

The combination of the first primer and the second primer used in the DNA amplification step (1) of the present invention includes (a) the first primer (1a) and the second primer (2).
a) (b) First primer (1b) and second primer (2
b) (c) First primer (1c) and second primer (2
c) (d) First primer (1d) and second primer (2
There are four combinations of d), and any one of these combinations can be used alone, or two or more can be used simultaneously.

When the combination (a) or (b) of each of the above-mentioned primers according to the present invention is used, DN of only the BamHI W region (repeat region) in the entire DNA sequence of EBV is used.
The A region can be specifically amplified. Here, the region amplified by the primer combination (a) is referred to as BamHIW-1,
Then, the region amplified by the primer combination (b) is
amHIW-2. Specifically, BamHIW-1
The 195 bp portion corresponding to BamHIW-2 or the 134 bp portion corresponding to BamHIW-2 is specifically amplified in large amounts. When the primer combination (c) is used, the DNA region of only the EBER1 region can be specifically amplified in the entire EBV DNA sequence. Here, the region amplified by the primer combination (c) is referred to as EBER.
Specifically, the 157 bp portion corresponding to EBER is specifically amplified in a large amount. Finally, using primer combination (d), LMV in the total DNA sequence of EBV
P region (latent membrane prote
in) only the DNA region can be specifically amplified. Here, the region amplified by the primer combination (d) is LMP
Called. Specifically, the 318 bp portion corresponding to LMP is specifically amplified in a large amount.

The first primer and the second primer may be 15 mer to 30 mer, but generally 20 mer to 25 mer is preferable. When it is less than 15 mer, the specificity of the base sequence is lowered, and when it exceeds 30 mer, the specificity is increased but the synthesis cost is increased. Each base constituting each of the first primer and the second primer used in the method of the present invention is
It may be modified (for example, biotinylated or labeled with a luminescent substance) in any known manner. The respective first and second primers according to the invention are
It can be prepared by a known DNA synthesis method (for example, the fossoamidide method) using an ordinary automatic DNA synthesizer (for example, Applied Biosystems).

In the DNA amplification step (1) of the present invention, the first
The amplification cycle is repeated using a DNA polymerase, especially a thermostable polymerase, with the primer and the second primer. As a thermostable DNA polymerase, DNA capable of maintaining its activity especially at a temperature of up to 95 ° C
Polymerases such as the commercially available Taq polymerase can be used.

In the DNA amplification step of the present invention, the first
A mixture of a specific primer combination of a primer and a second primer, a DNA polymerase and a liquid test sample is used. The amounts of the first primer, the second primer and the DNA polymerase used vary depending on the type of the liquid test sample, but can be easily determined within a range in which the DNA amplification step by the PCR method can be carried out. The mixture may optionally contain a buffer (eg Tris-HCl buffer), a stabilizer (eg gelatin), or salts (eg sodium chloride, magnesium chloride).

In the method of the present invention, P
A CR method amplification cycle is performed. The amplification cycle is
(I) DNA denaturation step (about 90 ° C to 95 ° C, about 10 seconds to 2 minutes), (ii) Annealing step of single-stranded DNA with the first primer and the second primer (about 37 ° C to 70 ° C)
C., about 30 seconds to 3 minutes), and (iii) a DNA synthesis step by a DNA polymerase (about 65 ° C. to 80 ° C., about 30 seconds to 5 minutes).

The amount of DNA is doubled every cycle, and after n cycles, it is amplified 2 ⁿ times. In the present invention, the number of amplification cycles is 10 to 60 times, preferably 2 times.
Repeat 0-40 times. In the final cycle, the heating time was extended by about 5 to 10 minutes under the condition of step (iii) to perform DN.
It is preferred that the A synthesis be complete. When EBV-DNA is present in the test sample, a large amount of about 50 to 500 bp of DNA is synthesized after the amplification cycle is completed in the amplification step. This DNA is detected by the following DNA inspection step.

As the DNA inspection step, a method utilizing ethidium bromide staining after gel electrophoresis, a method of transferring to a filter after electrophoresis and carrying out a Southern blot hybrid method, a blotted amplified DNA as it is to a filter and a dot blot hybrid The method of performing the method, the nucleotide sequence determination method by the dideoxy method, or the like can be used. When performing the gel electrophoresis method, for example, submarine-type electrophoresis using an agarose gel as a carrier, or slab-type electrophoresis using acrylamide can be used.

When the Southern blot hybrid method, the dot blot hybrid method, or the in situ hybrid method is performed, a non-radioactive probe (for example, an enzyme-labeled probe, a biotinylated probe, a digoxigenylated probe, or a chemiluminescent substance, a fluorescent substance) is used. Probe labeled with.

Further, when utilizing the nucleotide sequence determination method by the dideoxy method, a DNA autosequencer (for example, Applied Biosystems) using a fluorescent label can be used.

In the method of the present invention using various combinations of the first primer and the second primer, only in the case where EBV-DNA is present in the test sample, a part of the BamHI W region depending on the combination, EBE
Part of the R1 region and the base sequence part corresponding to part of the LMP region are specifically amplified and synthesized in a large amount within a short time. Therefore, EBV-DNA in the test sample
The presence of can be detected very specifically. Furthermore, even if the amount of EBV-DNA in the test sample is very small, D
High sensitivity because NA is amplified and synthesized.

The present invention also provides four kinds of DNA probes which can be used for specific detection of EBV-DNA. These DNA probes
(A) a probe containing an oligonucleotide portion consisting of at least 10 bases of the base sequence represented by the sequence represented by SEQ ID NO: 9 in the sequence listing (hereinafter sometimes referred to as probe A), (B) the sequence listing in the sequence listing A probe containing an oligonucleotide portion consisting of at least 10 bases of the base sequence represented by the sequence of 10 (hereinafter sometimes referred to as probe B), (C) the base represented by the sequence of SEQ ID NO: 11 in the sequence listing. A probe containing an oligonucleotide portion consisting of at least 10 bases of the sequence (hereinafter, also referred to as probe C), (D) an oligo consisting of at least 10 bases of the base sequence represented by the sequence of SEQ ID NO: 12 in the sequence listing. A probe containing a nucleotide portion (hereinafter sometimes referred to as probe D). These probes can be used alone or simultaneously.

The probe A is specific to the DNA region of the BamHI W region (repeat region), and the length of the probe A differs depending on the type of pretreatment on the test sample. If the test sample has undergone a DNA amplification step by a PCR method using the first primer (1a) and the second primer (2a) of the combination (a), 10
It can range from bp to the size of the DNA fragment amplified by the PCR method. In addition, the test sample is DN of PCR method.
A probe A was inserted instead of going through the A amplification process.
When used in the u-hybrid method, the length of the probe A can be from 10 bp to the size of the BamHI W region (about 3 kbp). 40-base DNA probe (40mer) represented by the sequence of SEQ ID NO: 9 in the sequence listing
Is preferable because it can be used in any case whether the test sample has undergone the DNA amplification step by the PCR method or in the case of the in situ hybrid method.

The probe B is specific to the DNA region of the BamHI W region (repeat region), and the length of the probe B differs depending on the type of pretreatment on the test sample. 10 when the test sample has undergone a DNA amplification step by the PCR method using the first primer (1b) and the second primer (2b) of the combination (b)
It can range from bp to the size of the DNA fragment amplified by the PCR method. In addition, the test sample is DN of PCR method.
A probe B was inserted in place of the A amplification step.
When used in the u-hybrid method, the length of the probe B can be from 10 bp to the size of the BamHI W region (about 3 kbp). 40-base DNA probe (40mer) represented by the sequence of SEQ ID NO: 10 in the sequence listing
Is preferable because it can be used in any case whether the test sample has undergone the DNA amplification step by the PCR method or in the case of the in situ hybrid method.

The probe C is DN in the EBER1 region.
It is specific to the A region, and the length of the probe C differs depending on the type of pretreatment for the test sample. When the test sample has undergone a DNA amplification step by a PCR method using the first primer (1c) and the second primer (2c) of the combination (c), DNA amplified by the PCR method from 10 bp It can be up to the size of the fragment. Further, when the test sample is not one that has undergone the DNA amplification step of the PCR method and the probe C is used for the in situ hybrid method, the length of the probe C is 10 bp to EBE.
The size of the R1 region (173 bp) is possible. D of 40 bases represented by the sequence of SEQ ID NO: 11 in the sequence listing
The NA probe (40 mer) is used even if the test sample has undergone the DNA amplification step by the PCR method, or
Even in the case of the in situ hybrid method, it can be used in any method, which is preferable.

The probe D is an LMP (laten).
It is specific to the DNA region of the gene region encoding t membrane protein, and the length of the probe D differs depending on the type of pretreatment for the test sample.
The test sample is the first primer (1d) of the combination (d).
By the PCR method using the DNA and the second primer (2d)
When it has been subjected to the NA amplification step, it can be from 10 bp to the size of the DNA fragment amplified by the PCR method. When the test sample is not one that has undergone the DNA amplification step of the PCR method and the probe D is used for the in situ hybrid method, the length of the probe D is 10
The size of bp to LMP region (about 2 kbp) is possible. 4 represented by the sequence of SEQ ID NO: 12 in the sequence listing
The 0-base DNA probe (40 mer) can be used even if the test sample has undergone the DNA amplification step by the PCR method.
Alternatively, even in the case of the in situ hybrid method, it can be used in any method, which is preferable.

The probes A, B, C, and D are prepared by a method using succinimide (eg, disuccimidyl suberate), a maleimide method, an active halogen method, and a photoreaction with an azide compound. Alternatively, the carbodiimide method can be used. As the labeling substance for the probes A, B, C, and D, any conventionally known substance can be used.
Preferably, a non-radioactive substance (for example, an enzyme, biotin,
Digoxigenin, chemiluminescent substance, or fluorescent substance) is used.

The method of synthesizing the labeled DNA is roughly classified into (1) a method of directly preparing the labeled DNA in the process of synthesizing DNA, and (2) synthesizing a DNA to which a linker is bound, There is a method in which this is isolated and a labeling reagent is allowed to act thereon. Particularly, the method (2) is preferable because the labeling substance can be easily changed according to the purpose and it is excellent in application. As the linker used in the method (2), 5'-dimethoxytriethyl-5- [N- (trifluoroacetylaminohexyl) -3-acrylimide] -2'-deoxyuridine-3 '-[(2-cyanoethyl) is used. Examples include-(N, N-diisopropyl)] phosphoramidite and the like. As a method of generating a signal from the labeling substance bound to the probe and further measuring the signal, any conventionally known method can be used.

In the method of the present invention using the above-mentioned probe, EBV-DN (optionally amplified by PCR)
Since a labeled DNA probe specific to A is used, EBV-DNA can be detected extremely accurately.

[0028]

The present invention will be described in detail below with reference to examples, but these do not limit the scope of the present invention. Example 1: Synthesis of primer Adenine CPG column was attached to 381A type automatic DNA synthesizer (Applied Biosystems), and SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 6 in the sequence listing.
A primer (25 ') having 25 bases described in each sequence of
(1b ′), (1c ′) and (2c ′), and a primer of 23 bases (1
d ′) and the base 22 described in the sequence of SEQ ID NO: 8 in the sequence listing
Primer (2d ′) was synthesized, a guanine CPG column was mounted to synthesize a 25-base primer (2a ′) described in the sequence of SEQ ID NO: 2 in the sequence listing, and a cytosine CPG column was mounted. The 25-base primer (2b ′) described in the sequence of SEQ ID NO: 4 in the sequence listing was synthesized. A disposable syringe (2.5 ml) containing 2.5 ml of ammonia water (about 30%) was added to the CPG for which synthesis was completed.
It was connected to a column and aqueous ammonia was pushed out into the column to elute the synthesized DNA fragment. Close the vial bottle containing the recovered DNA ammonia solution, and
After heating at 5 ° C for 6 hours, the mixture was cooled to room temperature and concentrated. The concentrate was completely dried and dissolved in 10 mM triethylene ammonium acetate (pH 7.4) (hereinafter referred to as TEA-A). On the other hand, in advance, Nensorb ^™ Pre
p (NEN Research Products B
iotechnology Systems) Column 1
The column was equilibrated by washing with 0 ml of methanol and then adding 5 ml of TEA-A. Pass the above primer solution through this column and TEA containing 10% acetonitrile.
After washing the column with -A (10 ml), 25 ml of 0.5% trifluoroacetic acid was passed through the column. Furthermore, TE
After washing the column with AA (10 ml), the primer adsorbed on the column was eluted with 5 ml of 35% methanol. The eluted primer solution was dried under reduced pressure, stored, and used in Example 3 and the like described later.

Example 2 Preparation of Enzyme-Labeled Probe Four types of DNA probe were synthesized using the same apparatus as in Example 1 under the same conditions except that the sequence of SEQ ID NO: 9 in the sequence listing was used. In the synthesis of the probe A ′ having 40 bases described, the 30th T from the 5 ′ end was analyzed using an adenine CPG column, and the base 4 described in the sequence of SEQ ID NO: 10
The 0 probe B ′ was the 15th T from the 5 ′ end using an adenine CPG column, and the 40-base probe C ′ described in the sequence of SEQ ID NO: 11 in the sequence listing was cytosine CP.
Using the G column, the 10th T from the 5 ′ end, and the probe D ′ having 40 bases described in the sequence of SEQ ID NO: 12 in the sequence listing, uses the guanine CPG column to obtain the 20th T from the 5 ′ end. 5'-dimethoxytriethyl-
5- [N- (trifluoroacetylaminohexyl)-
3-acrylimide] -2'-deoxyuridine-3 '
It was replaced with-[(2-cyanoethyl)-(N, N-diisopropyl)] phosphoramidite (Grain Research Co., Ltd.) (hereinafter referred to as linker). The position of the linker is not limited to this site, and can be replaced with another thymine site. Purification of the probe with linker was also performed under the conditions described in Example 1.

3 nmole of the purified probe with linker was dissolved in 8 μl of sterilized water. 0.2M-Na in this solution
After addition of HCO ₃ / 4mM-EDTA solution 8 [mu] l, disuccinimidyl suberate rate (Pierce Co.) (hereinafter,
A solution of DSS) in dimethyl sulfoxide (10 mg /
50 ml) was added, and the mixture was reacted at room temperature in the dark for 15 minutes. After the reaction was completed, 30 μl of sterilized water was added to the reaction solution, the mixture was centrifuged, and the supernatant was subjected to HPLC (Toso-G3000PW column; mobile phase = water) to separate the DNA bound to DSS (hereinafter referred to as modified linker DNA). It was taken and freeze-dried. Alkaline phosphatase (reagent prepared by doubling the EIA reagent of Boehringer Mannheim Co., Ltd.) on the dried modified linker DNA: 20 mg / ml: hereinafter referred to as AP)
40 μl was added, and the mixture was reacted at room temperature in the dark for 16 hours.
After completion of the reaction, unreacted AP was removed with 0.1 M Tris-HCl buffer (pH 8.4), and 0.33 M-NaCl was added.
Target AP labeled D with 1M Tris-HCl buffer (pH 8.4)
NA was eluted. The eluate (about 6 ml) of the collected purified AP-labeled DNA was dialyzed against 0.1 M Tris-HCl buffer (pH 8.4) containing 3M-NaCl and then concentrated to 1 ml. This concentrate was used as an AP-labeled probe in Example 5 and the like described below.

Example 3: EBV-DNA by PCR method
Amplification and detection sensitivity of purified EBV-DNA [EBV infected cell line (preserved by Tokyo Medical and Dental University Intractable Disease Research Institute; No. MDH0601)
The DNA sequence collected from the above was purified] (5 pg ~
0.00005 pg) was prepared and used as a sample. Sample 5μ
l GeneAmp DNA Amplificat
Ion kit (Takara Shuzo) 10x PCR buffer 5μ
1, 8 μl of dNTP mixed solution, 5 μl of Taq polymerase (AmpliTaq DNA polymerase, Cetus) solution diluted 20 times, 0.4 μM of each primer prepared in Example 1 was added, and the total amount was adjusted to 50 μl with sterilized water. DNA thermal cycler (Perkin
Using Elmer Cetus, one cycle of (1) 94 ° C. for 1 minute, (2) 60 ° C. for 2 minutes, and (3) 72 ° C. for 3 minutes is performed for 30 cycles.
Finally, the program is to perform a 7 minute treatment at 72 ° C for DNA.
Was amplified.

Example 4: Confirmation of amplified DNA by electrophoresis
Gel for sure electrophoresis Tris base 5.4 g, boric acid 2.7
g, and 2 ml of 0.5 M EDTA (pH 8.
0) Agarose 1.8% or 0.7% was melt | dissolved in 1 liter (henceforth 0.5 * TBE) 1 liter, and it flowed and prepared on the gel plate of Mupid (Advance). Next, in Example 3, the solution in which the PCR reaction was completed was 10 μm.
2 μl of a solution of 0.25% promophenol blue and 15% Ficoll (Pharmacia) was mixed with 1 and the total amount of the mixed solution was put into a sample well. 0.5 x T
BE was placed in an electrode tank and electrophoresed at room temperature and 100 V for 45 minutes. After the electrophoresis, the agarose was stained with ethidium bromide, and the results of the electrophoresis were observed under UV illuminator irradiation.
Bands could be confirmed at p, 157 bp and 318 bp. The results are shown in Figure 1. BamHI-1 in FIG.
[Upper] uses the first primer (1a ′) and the second primer (2a ′), and BamHI-2 [upper] uses the first primer (1b ′) and the second primer (2b ′). Is used when EBER [upper] is the first primer (1c ′) and the second primer (2c ′), and LMP [upper] is the first primer (1d ′) and the second primer. This corresponds to the case of using (2d ′) and. With ethidium bromide staining, the combination of primer (1a ′) and primer (2a ′) and primer (1b ′) and primer (2b ′) can detect up to 0.005 pg, and primer (1c ′) and primer (2c ′) ), And the combination of the primer (1d ′) and the primer (2d ′), detection was possible up to 0.05 pg.

Example 5: Southern blot hybrid method
Confirmation of amplified DNA by 1M-NaCl after confirmation of electrophoresis in Example 4
The agarose gel is soaked in 500 ml of 0.5N-NaOH solution containing 30 minutes, and then transferred to 500 ml of 0.5M Tris-HCl buffer (pH 7.4) containing 1.5M-NaCl for 30 minutes. I made it The neutralized agarose gel is placed on a filter paper (Whatman) wetted with 20 × SSC (both ends of the filter paper are dipped in 20 × SSC), and a nylon filter (Hybond) wetted with sterile water is placed on the agarose gel. -N ⁺ , Amersham Co., Ltd., and Kim Towel (Jujo Kimberley Co., Ltd.) at a height of about 5 cm, and leave a load of about 500 g for 16 hours at room temperature to transfer the DNA from the agarose gel to the filter. Blotted. Filter under reduced pressure 1
After being dried for an hour, it was baked at 80 ° C. for 1 hour. Next, 5 × S containing 0.1% sarcosine, 0.5% SDS, 5% blocking agent for nucleic acid hybridization (Boehringer Mannheim), and 30% formamide.
SC (hereinafter referred to as hybridization buffer)
After allowing the above filter to stand at 37 ° C. for 1 hour, the above filter was transferred to 5 ml of a hybridization buffer containing the AP-labeled probe (3 pmol) prepared in Example 2 and allowed to react overnight at 37 ° C. It was The filter that has been hybridized with the AP-labeled probe is washed 3 times with 100 ml of 2 × SSC at room temperature for 5 minutes each time, and then left still in 100 ml of 2 × SSC containing 0.1% SDS at 42 ° C. for 45 minutes. did. Furthermore, 2 x SSC100
Washing was carried out 3 times for 5 minutes at room temperature in ml. Finally, 0.
The filter was immersed for 30 seconds to 1 minute in a 0.1 M Tris-hydrochloric acid buffer solution (pH 9.5) containing 1 M-NaCl and 10 mM-MgCl ₂ (hereinafter, referred to as a color-developing solution) to allow it to adapt.

A solution of 75 mg of nitroblue tetrazolium (Boehringer Mannheim) dissolved in 1 ml of a mixture of 30% sterile water and 70% dimethylformamide (hereinafter referred to as NB
T solution). Also, 5-bromo-4-
A solution (hereinafter referred to as a BCIP solution) was prepared by dissolving 50 mg of chloro-3-indolyl phosphoric acid (Boehringer Mannheim) in 1 ml of a mixed solution of dimethylformamide.
40 μl of NBT solution and 40 μl of BCIP are placed in a plastic bag containing a filter that is adapted to the coloring solution.
10 ml of the color-developing solution to which 1 was added was added and allowed to stand at 37 ° C. for 5 minutes to 5 hours for color development. AP reaction is 10 mM-EDT
The filter was stopped by immersing the filter in 50 ml of 10 mM Tris-HCl buffer (pH 7.5) containing A. As a result, 1
The 95 bp band was hybridized with probe A'and the 134 bp band was hybridized with probe B '.
Probe C ′ was added to the 57 bp band, and 318 bp was added.
It was confirmed that the probe D ′ was hybridized to each band. The results are shown in Figure 1. BamHI-1 [bottom row] in FIG. 1 is probe A ′, BamH
I-2 [lower] corresponds to probe B ', EBER [lower] corresponds to probe C', and LMP [lower] corresponds to probe D '. In the Southern blot hybrid method, the primer (1a ′) and the primer (2
a '), and the primer (1b') and the primer (2
When the DNA amplified in combination with b ') is reacted with probe A'and probe B', up to 0.0005 pg can be detected, and the primer (1c ')
And a primer (2c ′), and a DN amplified by a combination of the primer (1d ′) and the primer (2d ′)
When A was reacted with probe C'and probe D ', up to 0.005 pg could be detected.

Example 6: For EBV and other viruses
Specificity of the primer for the specificity of the primer for EBV amplification according to the present invention was investigated. As the virus, human cytomegalovirus (H
CMV: Towne strain), herpes simplex virus-type I (HSV-I: HF strain), herpes simplex virus-type II (HSV-II: UW268 strain), Varicello-Zoster virus (VZV: zonular foam shin isolate). : H-N3
Epstein-Barr virus (EBV: B95)
-8 strain) and HHV-6 (exudative rash isolated strain) were used to perform DNA amplification by the PCR method under the conditions described in Example 3 to examine the presence or absence of cross reaction. The results are shown in Figure 2. BamHI-1 of FIG. 2 is BamHI-2 when the first primer (1a) and the second primer (2a) are used.
Is the first primer (1b) and the second primer (2
b), EBER uses the first primer (1c) and the second primer (2c), and LMP uses the first primer (1d) and the second primer (2d). It corresponds to each case. EB
Specific D amplified with each primer for V-DNA only
The NA band was detected, indicating that it did not cross-react with viral DNA other than EBV.

Example 7: EBV-non-infected cell line and EBV
Specificity of the primer using the isolate The specificity of the primer was examined using the EBV non-infected cell line and the EBV isolate. As EBV non-infected cells, BJ
AB strain (Tokyo Medical and Dental University, Research Institute for Intractable Diseases, Conservation No. M
DH0101) and Molt4 strain (Tokyo Medical and Dental University, Research Institute for Intractable Diseases, No. MDH0200).
As BV-infected cells, Raji strain (Tokyo Medical and Dental University, Research Institute for Intractable Diseases, No. MDH0301), P3HR
-1 strain (Tokyo Medical and Dental University Research Institute for Intractable Diseases, Save No. M
DH0401), B95-8 strain (Tokyo Medical and Dental University, Research Institute for Intractable Diseases, No. MDH0601) and AKATA
Strain (Tokyo Medical and Dental University, Research Institute for Intractable Diseases, Conservation No. MDH
Using 0501), DNA amplification was carried out by the PCR method under the conditions described in Example 3 to examine the presence or absence of cross-reactivity with DNA of human lymphocytes and whether or not EBV-DNA in virus-infected cells can be detected. Results are shown in FIG. Figure 3
BamHI-1, BamHI-2, EBER, and L
MP has the same meaning as in FIG. No DNA band due to a specific reaction was detected in the DNAs of BJAB and Molt4 cell lines which were not infected with EBV. Also, E
In BV-infected cells, specific DNA bands were detected with all primer combinations, indicating that these primers according to the present invention were set in a region where EBV-DNA was well conserved.

Example 8: E in samples from patients with EBV infection
Detection of BV-DNA The following test samples were prepared. That is, a mononuclear cell fraction (1 ×) in blood collected from an infectious mononucleosis patient containing EBV.
10 ⁶ pieces) was added with 1 ml of sterilized water to prepare a sample. Sample 5μ
As a result of carrying out the same operation as in Examples 3 to 5 using l, according to ethidium bromide staining, 195 bp, 134
Bands could be observed at bp, 157 bp and 318 bp. Furthermore, according to the Southern blot hybrid method, the probe A'at 195 bp, the probe B'at 134 bp, the probe C'at 157 bp, 31
It could be confirmed that the probe D ′ hybridized to 8 bp.

Example 9: EBV with AP labeled probe
-Detection of DNA 10 B95-8 cell lines (Tokyo Medical and Dental University Research Institute for Intractable Diseases No. MDH0601), which are EBV producing cells, were used.
RPMI1640 medium containing 15% fetal bovine serum (Nissui Pharmaceutical)
The cells were subcultured at 37 ° C. for 1 week in the presence of 5% CO ₂ . The cultured B95-8 cell line was centrifuged to collect the cells, and 1 × 1
0 ⁶ were suspended in PBS to cells / ml, after placing 10μl glass slides and air dried. The dried slide glass was washed with PBS at room temperature for 5 minutes and further dried. The slide glass was immersed in 4% formalin / 0.1 M sodium phosphate buffer (pH 7.2) [hereinafter, 0.1 M sodium phosphate buffer (pH 7.2) is referred to as NaPB] for 5 minutes at room temperature. PBS with 0.1M-NaPB, washed 3 times for 3 minutes each at room temperature, immersed in 2 × SSC (pH 7.0) at room temperature for 5 minutes, and dissolving 2 mg of RNase (Takara Shuzo).
Spread 1 ml on the cells and leave at 37 ° C for 1 hour in a moist chamber.
The plate was washed with 0.1 M NaPB for 3 minutes at room temperature, and immersed in 4% formalin / NaPB for 5 minutes at room temperature. Next, 0.1M
-Wash with NaPB for 3 minutes each at room temperature 3 times, 0.2N-
After soaking in HCl for 5 minutes at room temperature, 0.1M-NaPB
Washed for 3 minutes. Then 70%, 90%, and 100
% Ethanol for 3 minutes at room temperature for dehydration, and chloroform for 10 minutes at room temperature. Finally, it was immersed in 100% ethanol twice at room temperature for 3 minutes each and air dried. The dried cells were immersed in 0.07N-NaOH for 2 minutes and finally washed with 0.1M-NaPB for 3 minutes at room temperature.

10% dextran sulfate sodium salt, 3
0% formamide, 0.025% salmon sperm DNA, and 1
2 pmol of the AP-labeled probe (probe A ′ or probe B ′) prepared in Example 2 was dissolved in 100 μl of 2 × SSC containing × Denhardt's solution, spread on a slide glass, and kept in a wet box at 37 ° C. for about 16 hours. I let it stand. Then 2x
Wash with SSC at room temperature for 10 minutes, then 0.1 mM-ZnCl ₂
And left at 45 ° C. for 1.5 to 2 hours.
It was replaced with a fresh washing solution every 30 minutes. 0.1 mM-ZnC
After immersed for 10 minutes at room temperature with 1 × SSC containing l _2,
It was immersed for 3 minutes in 0.1 M Tris-hydrochloric acid buffer solution (pH 9.5) (hereinafter referred to as AP buffer solution) containing 0.1 mM-ZnCl ₂ and 10 mM-MgCl ₂ .

4 μl of NTB solution and B in 1 ml of AP buffer
CIP solution (4 μl) was added and mixed, and the mixed solution was about 20
Spread 0 μl on the slide glass and place in a wet box for 25
The reaction was carried out at 30 ° C to 30 ° C for about 24 hours. Color development is 10 mM-E
5 mM with 10 mM Tris-HCl buffer (pH 7.5) containing DTA
The cells were treated for a minute, stopped, and sealed with a 90% glycerol solution containing 10 mM-EDTA and 10 mM Tris-hydrochloric acid buffer (pH 7.5) and observed under a microscope. The results are shown in FIG. The B95-8 cell line contains EBV-DNA,
It was shown that EBV-DNA was detected using either probe A'or probe B '.

[0041]

According to the method of the present invention, since EBV-DNA-specific primers and probes are used, EBV can be measured rapidly and accurately.

[0042]

[Sequence list]

SEQ ID NO: 1 Sequence length: 25 Sequence type: Nucleic acid Number of strands: Single strand Sequence: GTGCAGTAAC AGGTAATCTC TGGTA

SEQ ID NO: 2 Sequence length: 25 Sequence type: Nucleic acid Number of strands: Single strand Sequence: ATAGCAGCAG CGCAGCCAAC CATAG

SEQ ID NO: 3 Sequence length: 25 Sequence type: Nucleic acid Number of strands: Single strand Sequence: CAAGAACCCA GACGAGTCCG TAGAA

SEQ ID NO: 4 Sequence length: 25 Sequence type: Nucleic acid Number of strands: Single strand Sequence: AAGAAGCATG TATACTAAGC CTCCC

SEQ ID NO: 5 Sequence length: 25 Sequence type: Nucleic acid Number of strands: Single strand Sequence: CTACGCTGCC CTAGAGGTTT TGCTA

SEQ ID NO: 6 Sequence length: 25 Sequence type: Nucleic acid Number of strands: Single strand Sequence: ATGCGGACCA CCAGCTGGTA CTTGA

SEQ ID NO: 7 Sequence length: 23 Sequence type: Nucleic acid Number of strands: Single strand Sequence: TTATGAGTGA CTGGACTGGA GGA

SEQ ID NO: 8 Sequence length: 22 Sequence type: Nucleic acid Number of strands: Single strand Sequence: GTTAGATCTT ACCAAGTAAG CA

SEQ ID NO: 9 Sequence length: 40 Sequence type: Nucleic acid Number of strands: Single strand Sequence: AAAGTCCTCC AGAGCTCTAA AGTGTCAGAT TTCGGGTCCA

SEQ ID NO: 10 Sequence length: 40 Sequence type: Nucleic acid Number of strands: Single strand Sequence: GAGGTCAGGT TACTTACCCC TGAAGGTGAA CCGCTTACCA

SEQ ID NO: 11 Sequence length: 40 Sequence type: Nucleic acid Number of strands: Single strand Sequence: AGCAGAGTCT GGGAAGACAA CCACAGACAC CGTCCTCACC

SEQ ID NO: 12 Sequence length: 40 Sequence type: Nucleic acid Number of strands: Single strand Sequence: AATTCCAAGG AACAATGCCT GTCCGTGCAA ATTCCAGAGA

[Brief description of drawings]

FIG. 1 is a photograph instead of a drawing showing the results of electrophoresis and its Southern blot hybrid in which the detection sensitivity of EBV-DNA by the PCR method was examined. Lane 1 is EBV-
DNA 5 pg, lane 2 is EBV-DNA 0.5 pg,
Lane 3 has EBV-DNA of 0.05 pg and lane 4 has E.
0.005 pg of BV-DNA, EBV-DN in lane 5
A 0.0005 pg, lane 6 is EBV-DNA 0.0
0005 pg and M are PHY DNA molecular weight markers.

FIG. 2 is a photograph as a substitute for a drawing, which shows the result of electrophoresis in which the specificity of a primer was examined using DNAs of EBV and other viruses. Lane 1 is EBV-DNA, lane 2 is HSV-I-DNA, lane 3 is HSV-II-D.
NA, lane 4 is HCMV-DNA, lane 5 is VZV
-DNA, lane 6 is HHV-6-DNA, M is PHY
It is a DNA molecular weight marker.

FIG. 3: D of EBV uninfected cell line and EBV infected cell line
It is a photograph instead of a drawing, which shows the result of electrophoresis in which the specificity of a primer was examined using NA. Lane 1 is BJAB
Cell line, lane 2 is Molt4 cell line, lane 3 is Ra
ji cell line, lane 4 is B95-8 cell line, lane 5 is P3HR-1 cell line, lane 6 is AKATA cell line, M
Is a PHY DNA molecular weight marker.

FIG. 4 is a photograph instead of a drawing, which shows the result of detecting EBV-DNA with probes A ′ and B ′. (A) is a probe A ′, (B) is a probe B ′ in situ
Detection of EBV-DNA in B95-8 cell line by u hybridization.

Claims (2)

[Claims] 1. A first DNA primer containing an oligonucleotide portion consisting of at least 15 bases of the base sequence represented by the sequence represented by SEQ ID NO: 1 in the sequence listing and the sequence represented by the sequence represented by SEQ ID NO: 2 in the sequence listing. With a second DNA primer containing an oligonucleotide portion consisting of at least 15 bases of the nucleotide sequence shown in (b).
A first DNA primer containing an oligonucleotide portion consisting of at least 15 bases of the base sequence represented by the sequence number 3 in the sequence listing and at least 15 bases of the base sequence represented by the sequence number 4 in the sequence listing A second DN containing an oligonucleotide moiety consisting of
Combination with A primer, (c) First DNA containing an oligonucleotide portion consisting of at least 15 bases of the base sequence represented by the sequence of SEQ ID NO: 5 in the sequence listing
A combination of a primer and a second DNA primer containing an oligonucleotide portion consisting of at least 15 bases of the base sequence represented by the sequence of SEQ ID NO: 6 in the sequence listing, and (d) the sequence of SEQ ID NO: 7 in the sequence listing A first DNA primer containing an oligonucleotide part consisting of at least 15 bases of the represented base sequence and a first DNA primer containing an oligonucleotide part consisting of at least 15 bases of the base sequence represented by the sequence of SEQ ID NO: 8 in the sequence listing; A mixture of at least one combination of a first primer and a second primer selected from the group consisting of the combination of 2 DNA primers, a DNA polymerase and an aqueous liquid test sample is subjected to a DNA amplification step, Epstein, characterized in that the obtained reaction solution is subjected to a DNA inspection step. Detection method of Barr virus. 2. SEQ ID NO: 9 and SEQ ID NO: 1 in the sequence listing
0, SEQ ID NO: 11 or SEQ ID NO: 12 containing an oligonucleotide portion consisting of at least 10 bases of the base sequence, the probe carrying the label is contacted with the test sample, the signal from the label A method for detecting Epstein-Barr virus, which comprises detecting.