JP3113321B2 - Epstein Barr virus detection method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION The present invention relates to an Epstein Barr Virus (hereinafter referred to as Epstein Barr Virus).
EBV) ( non-infected, subclinical infection or
The present invention relates to a method for detecting overt infection .

[0002]

2. Description of the Related Art Epstein-Barr virus (EBV) is a type of virus belonging to the herpes family, which is infected by the majority of adults, and almost all are antibody-positive. Although many people become infected by the age of three years, their symptoms do not appear because of many subclinical infections. However, EBV may be reactivated when the immune function of the host is reduced. In particular, EBV is reactivated in patients with malignant tumors, AIDS, and those who have undergone bone marrow transplantation, and the pathology includes infectious mononucleosis and deterioration of prognosis. is there. Therefore, it is of crucial clinical importance to definitively diagnose EBV infections such as nasopharyngeal carcinoma, Burkitt's lymphoma or opportunistic lymphoma.

[0003] Conventionally, EBV has been detected by a method of separating and identifying EBV from biological fluids (blood, saliva, etc.) or tissues or cells, a method of detecting viral capsid antigen (VCA) or nuclear antigen (VCA) in serum. An immunological test method for measuring EBNA) or the like is used. However, it takes a long time to obtain a result in virus isolation diagnosis, and immunological methods have a problem that antibody nonspecific reaction occurs, high sensitivity cannot be obtained, and antibody titer changes. There was a drawback that it was difficult to track the fate of the virus. Therefore, not only in the case of organ transplantation and bone marrow transplantation, but also for the diagnosis of disease, there has been a demand for a method and an in vitro diagnostic agent that enable accurate and accurate EBV diagnosis in a short time.

[0004]

SUMMARY OF THE INVENTION In order to detect EBV quickly and accurately, the present inventor has determined that EBV DNA or R
Various nucleotide sequences specific to NA were searched, a DNA fragment complementary to the sequence was synthesized, and evaluated as an EBV probe. A probe that specifically hybridized with EBV DNA or RNA Found something that can be used as The present invention is based on such findings.

[0005]

Accordingly, the present invention provides an EB
It is intended to provide three types of DNA probes capable of specifically detecting V-DNA or RNA. These three types of DNA probes include (1) a DNA probe containing an oligonucleotide portion consisting of a base sequence represented by the sequence of SEQ ID NO: 1 in the sequence listing (hereinafter sometimes referred to as probe A), and (2) A probe containing an oligonucleotide portion consisting of the nucleotide sequence represented by SEQ ID NO: 2 in the sequence listing (hereinafter sometimes referred to as probe B), and (3) SEQ ID NO: 3 in the sequence listing
(Hereinafter, may be referred to as probe C) containing an oligonucleotide portion consisting of a base sequence represented by the following sequence:

The base sequence constituting the core of the probe A consisting of 40 bases represented by the sequence of SEQ ID NO: 1 in the sequence listing corresponds to the antisense of the EBER1 region of EBVＥDNA, Specific to the EBER1 region. The EBER1 region is abundantly present in cells of an EBV-infected host, and is not translated into protein.
Is an area for coding. Probe B comprising 40 bases represented by the sequence of SEQ ID NO: 2 in the sequence listing
The base sequence that constitutes the nucleus is LMP of EBV─DNA.
LMP of EBV─DNA, corresponding to the antisense region
Region specific. The LMP region is a region that encodes a protein that is expressed in a host cell during EBV latent infection. Furthermore, the nucleotide sequence constituting the core of the probe C, consisting of 41 nucleotides represented by the sequence of SEQ ID NO: 3 in the sequence listing, is the gp220 region and g
EBV─DNA, corresponding to the antisense of the p350 region
Are specific to the gp220 and gp350 regions.
The gp220 and gp350 regions are regions that encode glycoproteins that are expressed late in infection.

[0007] Probes A to C correspond to bases 4 represented by the sequences of SEQ ID NOS: 1 to 3 in the sequence listing, respectively.
It may contain a base sequence consisting of 0 or 41, and there is no particular upper limit, but it is preferable that the base sequence consists of 40 to 50 bases. In addition, as long as it does not affect the hybridization with the test sample, the base 40 contained in the base sequence represented by the sequence of SEQ ID NO: 1 to SEQ ID NO: 3 in the sequence listing
Or 41 bases may have some deletions or substitutions in some of the bases.

[0008] Probe A, probe B and probe C can be synthesized by a known method. Examples of the preparation method include a method using succinoimide (for example, disuccinimidyl suberate), a maleimide method, an active halogen method, a photoreaction using an azide, and a carbodiimide method.

As a labeling substance for the probe A, the probe B and the probe C, any conventionally known substance can be used. Preferably, a non-radioactive substance (enzyme, fluorescent dye, luminescent substance, biotin, etc.) is used. The methods of synthesizing labeled DNA are roughly classified into (1) a method of directly preparing a labeled DNA in the process of DNA synthesis, and (2) a method of synthesizing a DNA to which a linker is bound and then isolating the DNA. There is a method in which a labeling reagent is allowed to act, and in particular, method (2) is preferable because the type of label can be easily changed according to the purpose and is excellent in application. As the linker used in the method (2), 5′-dimethoxytrityl-5- [N
-(Trifluoroacetylaminohexyl) -3-acrylimide] -2'-deoxyuridine-3 '-[(2
-Cyanoethyl)-(N, N-diisopropyl)] phosphoramidite.

[0010] The sample to be subjected to the method of the present invention is not particularly limited as long as it is a sample which may contain EBV.
In particular, host cells or tissues that may be infected with EBV, for example, human tonsillar tissues, nasopharyngeal carcinoma tissues, liver tissues, and lymph node tissues that may be infected with EBV can be mentioned. .

In the method of the present invention, EBV
(4) DNA or mRNA expressed in host cells is taken out by a known method to prepare a test sample that can be hybridized with each probe of the present invention. To remove DNA, for example, after treating the specimen with an SDS solution containing protease E, the specimen is further treated with chloroform / phenol to precipitate the DNA with ethanol. Collected DN
A is directly immobilized on a suitable carrier (for example, a nylon filter) and a test sample for dot blot hybridization is prepared, or is digested with a suitable restriction enzyme (for example, BamHI or EcoRI),
A test sample can be prepared by Southern blotting. In order to remove mRNA, for example, it is extracted by ultracentrifugation in a guanidine solution under a cesium chloride concentration gradient. The mRNA can be directly immobilized on a suitable carrier and used as a test sample for dot blot hybridization, or can be used as a test sample by Northern blot hybridization using electrophoresis. If necessary, DNA or RNA in the sample can be amplified by, for example, a PCR method.

In the method of the present invention, the contact between the label-carrying probe and the DNA or RNA in the test sample is performed by a conventionally known method, for example, by using a filter after electrophoresis or a slide glass on which cells are fixed. It can be done by doing. That is, when both chains meet with a complementary chain during molecular movement in a solution, a double chain is formed by hydrogen bonding between the bases.

As a method for generating a signal from the label bound to the probe and measuring the signal, any conventionally known method can be used. For example, when a radioisotope is used as a label, after hybridization, it is exposed on a radiation emulsion or an X-ray film, developed, and its signal is detected. When biotin is used as a label, after hybridization, peroxidase-labeled avidin is reacted to form a biotin-avidin complex, and the color development of peroxidase is detected. According to the method of the present invention, the time required from collection of a specimen to obtaining a test result is at most 24 hours,
The results can be obtained very quickly.

[0014]

[Function] Since probe A has a specific nucleotide sequence in the region encoding RNA transcribed during latent infection in host cells infected with EBV, the method of the present invention using probe A should be performed. The result is positive only when EBV is present in a specimen such as a tissue or a cell, and it can be determined whether the tissue or the cell is infected with EBV. That is, not only superficial infections but also subclinical infections can be detected. Further, since the probe B has a specific nucleotide sequence in a region encoding a protein expressed at the time of latent infection in a host cell infected with EBV, when the method of the present invention is performed using the probe B, tissue It becomes positive only when EBV is present in a specimen such as cells or cells, and it can be determined whether or not a tissue or cell is infected with EBV. That is, not only superficial infections but also subclinical infections can be detected. Further, probe C
Has a specific nucleotide sequence in a region encoding a glycoprotein that is expressed late in host cells infected with EBV, and thus is positive only when EBV is proliferating in a specimen such as a tissue or a cell. Thus, it can be determined whether or not EBV is proliferating in a tissue or a cell. That is,
When the method of the present invention is carried out using the probe C, overt infection can be detected.

[0015]

The present invention will be described in more detail with reference to the following examples, which do not limit the scope of the present invention.

Example 1 Preparation of Enzyme-Labeled Probe A cytosine CPG column was attached to a type 381A automatic DNA synthesizer (Applied Biosystems), and a probe consisting of 40 bases described in the sequence of SEQ ID NO: 1 in the sequence listing. a, and an adenine CPG column was attached to synthesize a probe b consisting of 40 bases described in the sequence of SEQ ID NO: 2 in the sequence listing.
The probe c consisting of 41 bases described in the sequence of SEQ ID NO: 3 in the sequence listing was attached with the PG column attached.
However, in the synthesis of probe a, the 10th T
In the synthesis of probe b, the 20th T
And in the synthesis of probe c, the T at the 23rd position from the 5 ′ end is 5′-dimethoxytrityl-5- [N
-(Trifluoroacetylaminohexyl) -3-acrylimide] -2'-deoxyuridine-3 '-[(2
-Cyanoethyl)-(N, N-diisopropyl)] phosphoramidite (Glen Research) (hereinafter referred to as a linker). The position of the linker is not limited to this site, and it is also possible to replace another thymine site.

A disposable syringe containing 2.5 ml of aqueous ammonia (about 30%) was connected to the CPG column in which the synthesis step was completed, and the aqueous ammonia was extruded into the column to elute the synthesized DNA fragment. Recovered DNA
Seal the vial containing the ammonia solution,
After heating for 6 hours, the mixture was cooled to room temperature and concentrated.
The concentrate is lyophilized, dissolved in 10 mM triethylammonium acetate (hereinafter, referred to as TEA-A) (pH 7.4), and the precipitate is removed to remove the precipitate. Then, an L-6200 type high performance liquid chromatography apparatus (Hitachi, Ltd.) , HPLC
) And a separation column (YMC-Pack ODS-A)
M313: YMC) and purified using a concentration gradient of 95 mM TEA-A containing 5% acetonitrile and acetonitrile. The main peak was collected, dried under reduced pressure, and the obtained residue was suspended by adding 80% acetic acid (adjusted with acetonitrile), kept at room temperature for 30 minutes, and dried under reduced pressure. Dissolve the dried product in 10 mM TEA-A,
After extraction with diethyl ether, the extract was dried under reduced pressure. The dried DNA sample was dissolved in TEA-A, and after removing the precipitate, the second purification by HPLC was performed, and the main peak was collected. The purified DNA probe thus obtained was dried under reduced pressure and stored.

3 nmol of the purified probe with a linker was dissolved in 8 μl of sterile water. 0.2M-N
After addition of aHCO ₃ / 4mM-EDTA solution 8 [mu] l, disuccinimidyl suberate rate (Pierce Co.) (hereinafter,
DSS) in dimethyl sulfoxide (10 mg /
ml), and reacted at room temperature for 15 minutes in the dark. After completion of the reaction, 30 μl of sterilized water was added to the reaction solution, mixed, and centrifuged. The obtained supernatant was passed through HPLC (column: Tosoh G3000PW, mobile phase: water), and DNA to which DSS was bound (hereinafter, referred to as modified linker DNA) was fractionated and freeze-dried. A reagent (20 mg / m 2) prepared by concentrating a reagent for alkaline phosphatase EIA (Boehringer Mannheim) twice to the dried modified linker DNA.
l) 40 μl (hereinafter referred to as AP) was added, and the mixture was allowed to react at room temperature in the dark for 16 hours. After completion of the reaction, 2.5 ml of 0.1 M Tris-HCl buffer (pH 8.4) was added, and HPLC was performed.
(Column: Tosoh DEAE-5PW). That is, first, unreacted AP was removed with a 0.1 M Tris buffer (pH 8.4) containing 0.1 M NaCl, and 0.33 M
-0.1 M Tris buffer containing NaCl (pH 8.4)
Eluted the target AP-labeled DNA. Fractionated purified AP
About 6 ml of the eluate of the labeled DNA was dialyzed against 0.1 M Tris buffer (pH 8.4) containing 3 M NaCl, and then concentrated to 1 ml. This concentrate was used as an AP-labeled probe in Example 2 described below.

Example 2: Northern hybridization
BJAB cells (Tokyo Medical and Dental University, intractable disease Institute: MDH0101) not infected with detection EBV of EBV by the emissions and have persistently infected with EBV are produced viral particles P3HR-
1 cell (Tokyo Medical and Dental University, Institute for Intractable Diseases: MDH0
401) was cultured in PRMI1640 medium and collected by centrifugation (1,200 rpm for 5 minutes). After adding 16 ml of a 4 M guanidine solution containing 0.7% mercaptoethanol to these BJAB cells and P3HR-1 cells and stirring to break the cells, a part (3.6 ml) of the cell lysate was removed. The solution was overlaid on 1.3 ml of a 5.7 M cesium chloride solution in a polyaroma centrifuge tube (Beckman) and centrifuged at 40,000 rpm for 15 hours (Beckman, L5-50 ultracentrifuge, SW50Ti rotor). The supernatant was discarded, and the precipitated RNA was washed with 10 mM Tris-HCl buffer (pH 7.5) containing 1 mM EDTA (hereinafter referred to as T
This was dissolved in 400 μl, transferred to a 1.5 ml Eppendorf tube, and mixed with 200 μl of chloroform and 200 μl of TE-saturated phenol. 15,
Centrifuged at 000 rpm. The supernatant was transferred to another Eppendorf tube, 40 μl of 3 M sodium acetate and 1 ml of ethanol were added and mixed, left at −80 ° C. for 30 minutes, and then centrifuged at 15,000 rpm for 10 minutes. 500 μl of 80% ethanol was added to the obtained RNA precipitate, centrifuged (15,000 rpm, 5 minutes), washed, and dried. 20 μl of sterilized water was added to the dried precipitate to dissolve the precipitate, and this was used as an extracted RNA sample.

50 mM sodium acetate and 10 mM EDTA
And 2 μl of 0.2 M-3- (N-morpholino) propanesulfonic acid buffer (pH 7.0) (hereinafter referred to as MOPS), 3.5 μl of formaldehyde, and 10 μl of formaldehyde in 1 μl of the above extracted RNA solution (R
(Equivalent to 5 μg NA), and the total volume was adjusted to 20 μl with sterile water. After heating this RNA solution at 60 ° C. for 5 minutes, it was quenched in ice water, and 2 μl of a 50% glycerol solution containing 0.4% bromophenol blue and 1 mM-EDTA was added to prepare an RNA sample for electrophoresis. .

RNA for electrophoresis was placed on a 1% agarose gel containing 10-fold diluted MOPS and 5% formaldehyde.
A sample (20 μl) was placed thereon, and electrophoresed at 100 mA / cm for 2 hours. A PHY DNA marker (Takara Shuzo) was simultaneously electrophoresed as a molecular weight marker. After the electrophoresis is completed, the gel is separated from the gel, and ethidium bromide (100 μg
(/ Ml) in an aqueous solution for 10 minutes, and the movement distance from the well was measured. On the agarose gel after electrophoresis,
A nylon filter (Amersham, Hydon-N) moistened with 20 × SSC was placed thereon, and blotted at room temperature for 16 hours. After the blotting was completed, the nylon filter was dried under reduced pressure for 1 hour, and baked at 80 ° C. for 1 hour. The baked filter is
5 × SSC containing 5% sarcosine, 0.2% SDS, 5% blocking agent for nucleic acid hybridization (Boehringer Mannheim) and 30% formamide (hereinafter, referred to as
42 ° C. in a hybridization buffer).
, And replaced with 5 ml of hybridization buffer containing 3 picomoles of each of the probes a to c.
Treated overnight at 42 ° C. The filter after hybridization with each probe was immersed in 50 ml of 2 × SSC (pH 7.0) containing 0.1 mM zinc chloride (hereinafter referred to as “washing solution I”) at room temperature for 5 minutes, and further 0.1 mM. 2 × SSC containing zinc chloride and 0.1% SDS (pH 7.
0) The washing treatment performed by immersing in 50 ml at 42 ° C. for 20 minutes was repeated three times. Further, the filter was washed with washing solution I (50
ml) at room temperature for 10 minutes and washing was repeated three times. Finally, 0.1 M Tris-HCl buffer containing 0.1 M sodium chloride and 10 mM magnesium chloride (pH
9.5) (hereinafter referred to as a coloring solution) for 1 minute.

75 mg of nitro blue tetrazolium (Boehringer Mannheim) was dissolved in a mixture of 30% sterilized water / 70% dimethylformamide (hereinafter referred to as NBT solution). 50 mg of 5-bromo-4-chloro-3-indolyl phosphoric acid (Boehringer Mannheim) was dissolved in dimethylformamide (hereinafter referred to as BCIP solution). NB
The filter immersed in the coloring solution was transferred to 10 ml of a coloring solution (hereinafter referred to as an AP buffer) to which 40 μl of the T solution and 40 μl of the BCIP solution were added, and left at 37 ° C. for 4 hours. The color reaction was stopped by immersing the filter in Tris-HCl buffer (pH 7.5) containing 10 mM EDTA. The molecular weight of the target RNA was calculated by comparing the position of the colored band with the marker.

FIG. 1 shows the results of Northern hybridization. In FIG. 1, A is a probe a and 1
67b is a specific band, B is probe b, 2.5
a specific band at kb and C is probe c;
Specific bands are shown at 3.2 kb and 2.5 kb, respectively. X indicates the migration position of a molecular weight marker (Takara Shuzo), and Y indicates the position of a well. As is clear from FIG. 1, the probe a, the probe b and the probe c each hybridized with the small RNA extracted from the P3HR-1 cells persistently infected with EBV,
R extracted from BJAB cells not infected with EBV
It did not react with NA. On the other hand, when a similar test was conducted by preparing probes on the sense side complementary to the probes a, b and c, none of the probes hybridized with RNA extracted from each cell.
Therefore, it can be seen that probe a, probe b, and probe c are probes specific to EBV mRNA.

Example 3 In-Situ Hybridization
Uninfected evaluation EBV probes by Shon BJAB cells (used in Example 2), Akata cells although persistently infected with EBV does not produce viral particles (Tokyo Medical and Dental University,
Intractable disease research institute: MDH0501) and P3HR-1 cells producing virus particles (used in Example 2) were cultured under the same conditions as in Example 2, collected by centrifugation, and suspended in 200 µl of PBS. Was. Take 5 μl of the suspension, spread it on a slide glass, air-dry the cells, and immerse in 0.1 M sodium phosphate buffer (pH 7.2) containing 4% formaldehyde (hereinafter abbreviated as NaPB) at room temperature for 10 minutes. After fixing, first, 0.1M-N
A washing process of immersing in aPB for 3 minutes was performed three times. Next, three times at room temperature in the order of 70% ethyl alcohol, 90% ethyl alcohol and 100% ethyl alcohol.
Treated for minutes and dehydrated. Finally, it was degreased by immersion in chloroform for 10 minutes, dehydrated again with 100% ethyl alcohol, and air-dried at room temperature.

AP labeled probe a, AP labeled probe b
And 2 pmoles of each of the AP-labeled probe c and 10
% Dextran sulfate sodium salt, 30% formamide, 0.025% salmon sperm DNA and 1 × Denhardt's solution in 4 × SSC (100 μl), and spread the resulting solution on a slide glass on which cells were fixed. Was. next,
The slide glass was transferred to a wet box, and left at 37 ° C. for 12 hours for hybridization. First, slide the glass slide that has completed hybridization with the probe.
After immersion in XSSC for 10 minutes, washing was performed by immersion at 45 ° C. for 2 hours while replacing 1 × SSC containing 0.1 mM zinc chloride every 30 minutes.
C for 3 minutes at room temperature. The washed slide glass was immersed in the coloring solution prepared in Example 2 for 3 minutes.

On a slide glass equilibrated with the color developing solution used in Example 2, the AP prepared in the same manner as in Example 2 was used.
200 μl of the buffer was spread and left at 25 ° C. for 15 hours to develop color. Color development was stopped by treating the slide glass with a reaction stop solution for 5 minutes.

As shown in Table 1, none of the probes a to c hybridized with RNA extracted from BJAB cells, but the probes a and b did not hybridize with RNA extracted from Akata cells and P3HR-1 cells. Each was hybridized. Probe c hybridized only with RNA extracted from P3HR-1 cells. The AP-labeled probe having a nucleotide sequence complementary to the probes a to c used was negative for all cells. These results are thought to reflect the status of EBV infection. That is, probe a and probe b can be used to determine whether or not EBV has been infected, and probe c can be used to determine whether or not EBV has proliferated.

Example 4: EBV production opening of Akata cells
Changes in Specificity for Probes at the Beginning The Akata cells used in Example 2 were cultured under the conditions described in Example 2, collected by centrifugation, and the cell number was 1 × 10 ⁶
/ Ml to make RPMI1640 [Nissui Pharmaceutical Co., Ltd.]
It was suspended in 0.9 ml. The cell suspension was divided into two, one of which was an anti-human IgG antibody (6.65 mg / ml) (Ipel)
100 µl was added, and each was further cultured for 24 hours without adding to the other. EBV-infected Akata cells produce EBV particles when treated with anti-human IgG antibodies. Next, these cells are collected by centrifugation,
After washing once with PBS, the cell number was again 1 × 10 ⁶ /
The cells were suspended in PBS so as to obtain ml. Each suspension 5
After taking μl, placing it on a slide glass and air-drying, the cells were fixed under the conditions described in Example 3, and in situ hybridization was performed.

The results are shown in Table 2. Probe a and probe b are always positive regardless of the presence or absence of EBV particle production in Akata cells. On the other hand, the probe c
Only positive for Akata cells producing V particles. Also in the case of this example, the AP-labeled probes having the nucleotide sequences complementary to the probe a, the probe b and the probe c were all negative regardless of the state of the Akata cells.

Example 5: Specificity of Probes Clone # 9 cells (Tokyo Medical and Dental University, Institute for Intractable Diseases: MDH0411) and Raji cells (Tokyo Medical Dentistry) which are persistently infected with EBV but do not produce virus particles University, Institute for Intractable Diseases: MDH0301); EBV
HLN-STL-C cells producing particles (Shimane Medical University, First Pathology: null isolated from ATL patient blood
Cells); and M of T cells not infected with human EBV
olt3 cells (Tokyo Medical and Dental University, Institute for Intractable Diseases: M
DH200) was cultured under the conditions described in Example 2, and the cells were fixed under the conditions described in Example 3;
In situ hybridization was performed.

The results are shown in Table 3. Probe a and probe b were positive for EBV-infected cells with or without EBV particle production. Probe c is positive for HLN-STL-C cells producing EBV,
Even for V persistently infected cells, Clone # 9 cells and Raji cells that did not produce virus particles were negative. Also in this example, none of the probes having a base sequence complementary to each of the probes a to c showed a positive result. Molt3, an EBV-uninfected T cell
Cells were negative for all probes used.

Example 6: Probe a, probe b and probe
Diagnosis by Lobe c A tonsil tissue section of a healthy person, a tumor tissue section of a nasopharyngeal carcinoma patient, and a liver tissue section of a lymphoma patient were respectively placed on a slide glass, and a sample fixed by the method described in Example 3 and a probe a, probe b and probe c were used for in situ hybridization.

As is clear from Table 4 showing the results, the tonsils of the healthy subject were probe a, probe b and probe c.
Were negative for any of the above. That is, it shows that the tissue of a healthy person is not infected with EBV. On the other hand, liver tissue foci of nasopharyngeal cancer patients and lymphoma patients were positive for probe a and probe b and negative for probe c. In other words, it indicates that EBV is present in the tumor tissue foci but is not growing.

In Tables 1, 3 and 4 below, + indicates positive and-indicates negative. The evaluations in Table 2 are as follows. ++: EBV positive rate of cells is 80% or more, +: EBV positive rate of cells is 40% or more,-: EBV positive rate of cells is less than 0.5%.

[0035]

[Table 1]

[0036]

[Table 2]

[0037]

[Table 3]

[0038]

[Table 4]

[0039]

According to the method of the present invention, EBV can be contained in a sample by using the aforementioned probe A or probe B.
Can be determined quickly and accurately. Further, by using the probe C, E
It is possible to quickly and accurately determine whether or not BV is growing. Further, by using the probe A and / or the probe B in combination with the probe C, non-infection, subclinical infection, and overt infection can be quickly and accurately determined with respect to the EBV infection state.

[0040]

[Sequence list]

SEQ ID NO: 1 Sequence length: 40 Sequence type: Number of nucleic acid strands: Single strand Sequence: AGCAGAGTCT GGGAAGACAA CCACAGACAC CGTCCTCACC

SEQ ID NO: 2 Sequence length: 40 Sequence type: Number of nucleic acid strands: Single strand Sequence: AATTCCAAGG AACAATGCCT GTCCGTGCAA ATTCCAGAGA

SEQ ID NO: 3 Sequence length: 41 Sequence type: nucleic acid Number of strands: single-stranded Sequence: TCGTTGTATT GGGATCAGCA AATCCAGTTG TATTCAAGGT A

[Brief description of the drawings]

FIG. 1 is a drawing showing the results of Northern hybridization.

Continued on the front page (72) Inventor Yuki Yamaguchi 1-11-4 Higashikanda, Chiyoda-ku, Tokyo Inside Yatron Co., Ltd. (72) Inventor Hiroshi Kita 1-14-1 Higashikanda, Chiyoda-ku, Tokyo Inside Yatron Co., Ltd. (56) References JP-A-61-257188 (JP, A) Nucleic Acids Res. , Vol. 10 (1982), p. 3407-3425 Virol. , Vol. 41, (1982), p. 376-389 (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 15/00-15/90 C12Q 1/00-1/70 G01N 33/53 G01N 33/566 BIOSIS (DIALOG) GenBank / EMBL / DDBJ (G ENETYX) MEDLINE (STN) WPI (DIALOG)

Claims (3)

(57) [Claims] 1. (1) A probe containing an oligonucleotide portion consisting of a base sequence represented by the sequence of SEQ ID NO: 1 or SEQ ID NO: 2 in the sequence listing and carrying a label is brought into contact with a test sample, Detecting a signal from the label, and (2) contacting a probe carrying a label with a probe containing an oligonucleotide portion consisting of a base sequence represented by SEQ ID NO: 3 in the sequence listing and a test sample And detecting a signal from the label. A method for detecting non-infection, occult infection, or evident infection of Epstein-Barr virus. 2. A probe containing an oligonucleotide portion comprising a base sequence represented by the sequence of SEQ ID NO: 1 or SEQ ID NO: 2 in a sequence listing and carrying a label, and a test sample are brought into contact with each other. A method for detecting subclinical infection or overt infection of Epstein-Barr virus, characterized by detecting the following signal: 3. A probe containing an oligonucleotide portion consisting of a base sequence represented by the sequence of SEQ ID NO: 3 in the sequence listing and carrying a label is brought into contact with a test sample, and a signal from the label is detected. Characterized in that
Method for detecting overt Epstein-Barr virus infection.