JPH0779776A - Oligonucleotide for detection of epstein-barr virus(ebv) and its use - Google Patents

Abstract

PURPOSE:To provide a new oligonucleotide having a specific nucleic acid sequence, capable of easily, quickly and surely detecting Epstein-Barr virus(EBV) and useful e.g. for the diagnosis of diseases caused by EBV such as Burkitt's lymphoma. CONSTITUTION:This new oligonucleotide for the detection of EBV contains a nucleic acid sequence consisting of at least 15 consecutive residues in 20 kinds of nucleic acid sequences expressed e.g. by the formula or nucleic acid sequences complimentary to these sequence. The oligonucleotide can detect exclusively EBV from the supernatant of a cultured liquid of various herpes viruses to enable easy and quick detection of EBV in high sensitivity and is useful e.g. for the diagnosis of diseases producing detectable antigen and DNA originated from EBV such as Burkitt's lymphoma and epipharynx carcinoma. These oligonucleotides can be synthesized by a phosphoamidite process using a DNA synthesizer.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an EBV detecting oligonucleotide for detecting Epstein-Barr virus (EBV) simply and rapidly, a method for detecting EBV using the oligonucleotide and a reagent therefor.

[0002]

2. Description of the Related Art Epstein-Barr virus (hereinafter
(Abbreviated as EBV) is a DNA belonging to the herpesvirus family
It is a type of virus, and the majority of adults are infected with it, and almost all of them are antibody-positive. Many people are infected by the age of 3 years of age, but since there are many subclinical infections, no particular symptoms occur. However, if the host immune system is reduced, E
BV may be reactivated. For example, a polyclonal B lymphoproliferative disease caused by EBV that occurs in a state of immunocompetence such as after splenectomy or renal transplant has become a problem (Canser Res., 41, 4262-4279 (1981). ). In addition, EBV reactivation is observed in patients with malignant tumors and AIDS, and infectious mononucleosis may develop and the prognosis may worsen, which may be a fatal symptom. In addition, EBV from cells of Burkitt lymphoma and nasopharyngeal carcinoma that frequently occur in specific areas
Since the antigen and DNA derived from these are detected, it is considered to be the cause of these tumors (Prog. Med. Viol., 30, 87--106 (198
Four)). Therefore, for the early treatment of the above diseases, rapid E
The establishment of a BV detection method is clinically very important.

The current methods for detecting EBV include a method of separating and identifying EBV from biological fluids (blood, saliva, etc.), tissues or cells, and viral capsid antigen (VCA) or nuclear antigen (EBNA) in serum. ) Etc. are used immunological test methods. However, in the diagnosis for separating viruses, it takes time to obtain a result, and there are problems that an antibody non-specific reaction occurs and high sensitivity cannot be obtained.

[0004]

SUMMARY OF THE INVENTION Therefore, an object of the present invention is to provide an oligonucleotide capable of detecting EBV simply, rapidly, specifically and with high sensitivity.

[0005]

As a result of various investigations concerning the gene of EBV, the present inventors have focused on the sequence of the DNA polymerase gene, and are able to detect EBV in a simple, rapid, specific and highly sensitive manner. The present invention has been completed by obtaining an oligonucleotide having the following formula and establishing a detection method using the oligonucleotide.

That is, the present invention contains a nucleic acid sequence portion consisting of at least 15 contiguous residues of the nucleic acid sequences shown in SEQ ID NO: 1 to SEQ ID NO: 20 or the nucleic acid sequences complementary to the sequences. It is a characteristic EBV detection oligonucleotide.

The present invention also provides an oligonucleotide containing a nucleic acid sequence portion consisting of at least 15 contiguous residues of the nucleic acid sequences shown in SEQ ID NO: 1 to SEQ ID NO: 20 or the nucleic acid sequences complementary to the sequences. Is an oligonucleotide modified with a modified product, which is a modified oligonucleotide for EBV detection.

Further, the present invention is a nucleic acid sequence containing a nucleic acid sequence portion consisting of at least 15 contiguous residues of the nucleic acid sequences shown in SEQ ID NO: 1 to SEQ ID NO: 20 or the nucleic acid sequences complementary to the sequences. Is a method for detecting EBV in a sample, which comprises hybridizing an EBV detection oligonucleotide modified with a modification with a nucleic acid in the sample, and measuring the modified product of the hybridized conjugate.

Furthermore, the present invention provides EBV detection containing a nucleic acid sequence portion consisting of at least 15 contiguous residues of the nucleic acid sequences shown in SEQ ID NO: 1 to SEQ ID NO: 20 or the nucleic acid sequences complementary to the sequences. The oligonucleotide for use is bound to a solid phase carrier, hybridized with the nucleic acid in the sample, and the nucleic acid in the immobilized sample is complementary to the nucleic acid sequence shown in SEQ ID NO: 1 to SEQ ID NO: 20 of the Sequence Listing. Among the nucleic acid sequences, an oligonucleotide containing a nucleic acid sequence portion consisting of at least 15 contiguous residues is hybridized with an EBV detection oligonucleotide which is an oligonucleotide modified with the modified product, and the hybridized modified product is measured. It is a method for detecting EBV in a characteristic sample.

The present invention is a sequence listing: SEQ ID NO: 1 to SEQ ID NO: 2
For detecting EBV, an oligonucleotide for detecting EBV containing a nucleic acid sequence portion consisting of at least 15 contiguous residues of the nucleic acid sequence shown in 0 or a nucleic acid sequence complementary to the sequence is modified by the first modification The oligonucleotide is bound to a solid phase carrier by utilizing the property of the first modified product, hybridized with the nucleic acid in the sample, and the immobilized nucleic acid in the sample is represented by Sequence Listing An EBV detection modification in which an EBV detection oligonucleotide containing a nucleic acid sequence portion consisting of at least 15 contiguous residues of the nucleic acid sequence shown in 1 or a nucleic acid sequence complementary to the sequence is modified with a second modified product. A method for detecting EBV in a sample, which comprises hybridizing with an oligonucleotide and measuring a second modified product of the hybridized conjugate.

According to the present invention, the nucleic acid in the sample is a nucleic acid sequence portion consisting of at least 15 contiguous residues of the nucleic acid sequences shown in SEQ ID NO: 1 to SEQ ID NO: 20 or the nucleic acid sequence complementary to the sequence. The method for detecting EBV is characterized in that amplification is carried out using the contained EBV detection oligonucleotide, and the obtained amplification product is used as a sample.

The present invention also provides a sequence listing having a promoter sequence of RNA polymerase on the 5'-terminal side.
An EBV detection oligonucleotide containing a nucleic acid sequence portion consisting of at least 15 contiguous residues of the nucleic acid sequence represented by SEQ ID NO: 20 or a nucleic acid sequence complementary to the sequence.

In the present invention, among the nucleic acid sequences shown in SEQ ID NO: 1 to SEQ ID NO: 20 of the sequence listing having the nucleic acid in the sample with the promoter sequence of RNA polymerase on the 5'end side, or the nucleic acid sequence complementary to the sequence, At least one continuous
The above-mentioned EBV detection method is characterized in that amplification is performed using an EBV detection oligonucleotide containing a nucleic acid sequence portion consisting of 5 residues, and the obtained amplification product is used as a sample.

The present invention is a sequence listing: SEQ ID NO: 1 to SEQ ID NO: 2
In the nucleic acid sequence shown in 0 or a nucleic acid sequence complementary to the sequence, an EBV detection oligonucleotide containing a nucleic acid sequence part consisting of at least 15 contiguous residues and / or sequence listing / SEQ ID NO: 1 to SEQ ID NO: 20 A modified EBV detection oligonucleotide, which is a modified EBV detection oligonucleotide containing a nucleic acid sequence portion consisting of at least 15 contiguous residues of the nucleic acid sequence shown in FIG. It is a reagent kit for EBV detection characterized by containing.

Further, the modified product is a radioisotope,
It is a modified oligonucleotide for EBV detection, which is a fluorescent substance, an enzyme, a nucleotide having a linker arm, a promoter sequence of biotin or RNA polymerase.

The terms used in the present invention will be explained below. "Modification" means adding or substituting other nucleotides to the sequence portion of an oligonucleotide. Addition is the attachment of a nucleotide having biotin, a linker arm, a fluorescent substance, etc. to the 5'end or 3'end of an oligonucleotide sequence. It also includes binding a promoter sequence of RNA polymerase or the like to the 5'end or 3'end of the oligonucleotide sequence. Substitution means introducing a nucleotide having biotin, a linker arm, a fluorescent substance, etc., in place of the nucleotide in the oligonucleotide sequence. It also includes the introduction of known oligonucleotide labels such as radioactive substances, enzymes and fluorescent substances. For example, when an oligonucleotide is enzymatically labeled, a nucleotide having a linker arm is chemically synthesized from deoxyuridine by the synthetic method disclosed in JP-A-60-500717, and after substitution or addition as a member of the oligonucleotide sequence, the literature (Nucleic
Acids Research; Vol. 14, p. 6115, 1986).

The "modified oligonucleotide for detecting EBV modified with the first modification" is an oligonucleotide for detecting EBV that has been modified to facilitate the immobilization of the oligonucleotide for detecting EBV on a solid phase carrier. Is. First
As the modified product of, preferably, a linker arm, biotin and the like can be mentioned.

"Modified oligonucleotide for detection of EBV modified with a second modification" means for detection of EBV modified / labeled with a radioisotope, a fluorescent substance, an enzyme or the like which is a modification for facilitating detection. A modified oligonucleotide may be used. The second modified product preferably includes a radioisotope, a fluorescent substance, an enzyme and the like.

By "sample" is meant any nucleic acid inclusion. Samples include clinical samples and virus-infected cell cultures. Depending on the case, a treatment for facilitating hybridization, detection, fixation and the like, for example, a treatment with a proteolytic enzyme, a treatment with phenol / chloroform, a precipitation with ethanol and the like to decompose and remove contaminating proteins may be performed. In addition, the sample used here is amplified by a DNA polymerase in order to increase the detection sensitivity (Japanese Patent Application Laid-Open No. S60-18753).
No. 61-274697; hereinafter abbreviated as “PCR”) or a method for amplification with RNA polymerase (Japanese Patent Laid-Open Nos. 2-5864 and 4-501057, etc .; hereinafter abbreviated as “NASBA”) It may be amplified.

The details of the present invention will be described below. The EBV detection oligonucleotide of the present invention refers to a nucleic acid sequence portion consisting of at least 15 contiguous residues of the nucleic acid sequences shown in SEQ ID NO: 1 to SEQ ID NO: 20 or the nucleic acid sequence complementary to the sequence. contains. The oligonucleotide can usually be prepared by chemical synthesis. The oligonucleotide of the present invention may be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). In the case of ribonucleic acid, it goes without saying that the thymidine residue (T) is read as the uridine residue (U). Alternatively, a DNA containing a uridine residue may be used by synthesizing by changing T at any position to U at the time of synthesis. Similarly, it may be RNA containing a thymidine residue in which U at any position is changed to T. In addition, point mutations such as deletion, insertion or substitution in the oligonucleotide,
There may be modified nucleotides. Since the EBV detection oligonucleotide of the present invention is a synthetic product, it is possible to obtain an oligonucleotide of constant quality easily, in large quantities, and at low cost, as compared with a cloned oligonucleotide or polynucleotide.

The modified oligonucleotide for EBV detection of the present invention refers to at least 15 consecutive residues of the nucleic acid sequences shown in SEQ ID NO: 1 to SEQ ID NO: 20 or the nucleic acid sequence complementary to the sequence. Is an oligonucleotide in which an oligonucleotide for detecting EBV containing a nucleic acid sequence portion is modified with a modified product. What is a modified product?
Radioisotopes, fluorescent substances, enzymes, nucleotides with linker arms, biotin or RNA polymerase promoter sequences.

Using the EBV detection oligonucleotide of the present invention, the EBV gene can be amplified to improve the detection sensitivity. EBV gene nucleic acid by PCR or NASBA method using an oligonucleotide for detecting EBV, which comprises a nucleic acid sequence part consisting of at least 15 contiguous residues of the sequence listing SEQ ID NOS: 1 to 20 You just need to amplify the sequence. Preferably, the sequence listing
The sequences of SEQ ID NOS: 3 to 16 may be used as a forward side primer, and the sequences of sequence listing / SEQ ID NOS: 17 to 20 may be used as a reverse side primer. When performing the NASBA method,
An RNA polymerase promoter sequence is attached to the 5'end.

Further, EBV in a sample can be detected using the modified oligonucleotide for detecting EBV of the present invention. Sequence of the modified oligonucleotide for detection
Of the nucleic acid sequences represented by SEQ ID NOs: 1 to 20 or the nucleic acid sequences complementary to the sequences, the nucleic acid sequence portion comprising at least 15 contiguous residues may be contained. Preferably, the nucleic acid sequences shown in SEQ ID NOS: 1 and 2 of the Sequence Listing or nucleic acid sequences complementary to the sequences are good.

The EBV detection method of the present invention is a method in which a modified oligonucleotide for EBV detection is hybridized with a nucleic acid in a sample, and the modified product of the hybridized conjugate is measured to measure the E in the sample.
This is a method of detecting BV. More specifically, after nucleic acid in a sample is subjected to agarose gel electrophoresis, the nucleic acid is immobilized on a membrane by Southern blotting and hybridized with a modified oligonucleotide for EBV detection modified with radiation, a fluorescent substance, an enzyme or the like. The detection may be performed by a known detection method according to the property of the modified product. Alternatively, the detection operation may be performed after fixing the nucleic acid as it is and denaturing with alkali.

Further, the EBV detecting oligonucleotide or the modified EBV detecting modified oligonucleotide modified with the first modified product is bound to a solid-phase carrier, hybridized with the nucleic acid in the sample, and the nucleic acid in the immobilized sample is mixed. Is hybridized with a modified oligonucleotide for detecting EBV modified with the second modified product, and the modified product of the hybridized conjugate is measured to detect EBV in the sample. More specifically, EB
Modified oligonucleotide for detection of EBV modified with a second modification or an oligonucleotide for detection of V or modified oligonucleotide for detection of EBV modified with a first modification is used as a capture probe by binding to a solid-phase carrier. As a detection probe, and sandwich assay (Japanese Patent Laid-Open No. 58-400
No. 99 publication). In this case, it is desirable that the sequence used for immobilization and the sequence used for detection be different.

The presence of the EBV gene in a sample can be easily detected using the detection method of the present invention.

[0027]

EXAMPLES The present invention will be described below with reference to examples. Example 1 (1) Synthesis of Modified Oligonucleotide for EBV Detection Using a DNA synthesizer type 392 manufactured by ABI Co., a phosphoamidite method was used. 1-2
No. 5 of the sequence having SEQ ID NO. Oligonucleotides each having a sequence having 1 to 25 were synthesized. At this time, a uridine having a linker arm at the 5-position prepared by chemical synthesis from deoxyuridine was introduced to the 5'end by the synthetic method described in JP-A-60-500717. Hereinafter, they will be referred to as modified oligonucleotides 1 and 2 for EBV detection.

The synthesized product was subjected to deprotection treatment with ammonia water at 50 ° C. overnight and then purified with FPLC manufactured by Pharmacia using an anion exchange column.

(2) Labeling with alkaline phosphatase The binding of the above modified oligonucleotides 1 and 2 for EBV detection and alkaline phosphatase via the linker arm thereof is described in a literature (Nucleic Acids Research; Vol. 14, 6115).
P. 1986). That is, modified oligonucleotides 1 and 2 1.5 A260 for EBV detection were mixed with 0.2 M NaHCO ₃
It was dissolved in 12.5 μl, and 25 μl of 10 mg / ml disuccinimidyl suberate (DSS) was added thereto and reacted at room temperature for 2 minutes. Se obtained by equilibrating the reaction solution with 1 mM CH ₃ COONa (pH 5.0)
The excess DSS was removed by gel filtration with a phadex G-25 (Pharmacia) column (1 cmφx30 cm). Modified oligonucleotides 1 and 2 for detection of EBV in which the terminal amino group is activated
2 times the molar amount of alkaline phosphatase (100 mM N
a) (dissolved in aHCO ₃ , 3M NaCl) at room temperature for 16 hours to obtain modified EBV detection oligonucleotides 1 and 2 labeled with alkaline phosphatase. Hereinafter, they will be referred to as detection probes 1 and 2.

The detection probes 1 and 2 thus obtained were purified by Pharmacia FPLC using an anion exchange column. Fractions containing detection probes 1 and 2 were collected and concentrated by an ultrafiltration method using Centricon 30K (Amicon).

(3) Preparation of Specimens Five kinds of 5x Protain were added to various herpesvirus culture supernatants.
ase K buffer (50 mM Tris-HCl (pH7.5), 50 mM EDTA, 75
0 mM NaCl, 2% SDS, 0.5 mg / ml proteinase K) was added and mixed, and the mixture was left overnight at 65 ° C. Contaminant proteins were removed with a phenol / chloroform solution, precipitated with ethanol, and redissolved in TE buffer.

(4) Detection Gene extraction products from various herpesviruses were dropped on a nylon membrane and fixed under alkaline conditions. As a control, 1 μg of the gene from human placenta was similarly immobilized on the membrane. After neutralizing this membrane, the membrane was transferred to a hybridization bag (BRL) and the hybridization buffer containing detection probe 1 or 2 labeled with alkaline phosphatase (5xSSC, 0.5% bovine serum albumin, 0.5% polyvinylpyrrolidone). , 1% sodium dodecyl sulphate) and seal with a policyr at 50 ℃
Hybridization was performed for 15 minutes.

The membrane was taken out from the hybridization bag, and washing solution 1 (1xSSC, 1% sodium dodecyl sulfate) was used.
It was washed by shaking at 50 ° C. for 10 minutes. Furthermore, cleaning liquid 2 (1xS
SC, 0.5% TritonX-100) was washed with shaking at room temperature for 10 minutes. Finally, washing with washing solution 3 (1xSSC) at room temperature for 5 minutes with shaking was performed. Transfer the membrane to a new hybridization bag,
Substrate solution (0.1M Tris-HCl, 0.1M NaCl, 0.1M MgCl ₂ , 0.3mg /
ml nitrotetrazolium blue 0.3 mg / ml and bromchloroindoferryl phosphate pH 7.5) were added, and the mixture was sealed with a policyr and incubated at 37 ° C. for 1 hour.

(5) Results Spots of purple dye generated by alkaline phosphatase were visually determined. Those with confirmed spots were regarded as positive. The results are shown in Table 1. The detection probe 1 and the detection probe 2 selectively detect only EBV,
It can be seen that these detection probes are functioning normally. Positive is indicated by +, and negative is indicated by-.

Example 2 (1) Synthesis of Oligonucleotides for EBV Amplification 18 kinds of oligonucleotides having the sequences shown in Sequence Listing / SEQ ID NOS: 3 to 20 by the phosphoamidite method using ABI DNA synthesizer type 392 Was synthesized. The method was according to the ABI manual, and deprotection of various oligonucleotides was carried out with ammonia water at 55 ° C. overnight. Purification was carried out by FPLC manufactured by Pharmacia on an anion exchange column.

(2) PCR reaction solution composition is based on the forward primer (SEQ ID NOS: 3 to 1).
6 out of 6), reverse primer (SEQ ID NO: 17)
~ 1 out of 20) 1 μM each, 10 mM Tris-HCl (pH 8.9), 1.
5 mM MgCl ₂ , 80 mM KCl, 500 μg / ml BSA, 0.1% sodium cholate, 0.1% TritonX-100, 0.2 mM dAT each
40 units / ml of P, dCTP, dGTP, dTTP and Tth DNA polymerase (manufactured by Toyobo) and 1 μl of a diluted solution of various herpes gene extract products. The reaction conditions were as follows and repeated 30 times. Thermal denaturation: 94 ° C., 1 minute Annealing: 55 ° C., 2 minutes Polymerization reaction: 75 ° C., 1.5 minutes These operations are perkin-elmer / cetus (Perkin-
This was performed using a DNA thermal cycler from Elmer / Cetus).

(3) Detection Amplification products from various herpes genes were dropped on a nylon membrane, and then the same procedure as in Example 1 was performed. (4) Results Spots of purple dye generated by alkaline phosphatase were visually determined. The same results were obtained with all possible combinations of oligonucleotides for EBV amplification, and are shown together in Table 1. The detection probe 1 and the detection probe 2 selectively detect only EBV,
It can be seen that these probes and the EBV amplification oligonucleotide are functioning normally. Positive is +, negative is-
Was displayed.

[0038]

[Table 1]

Example 3 (1) Preparation of capture probe Using a DNA synthesizer type 392 manufactured by ABI Co., a phosphoamidite method was used. 1-2
A sequence having 5 was synthesized and used as a capture probe 2-1. In addition, at this time, 5'of uridine having a linker arm at the 5-position prepared by chemical synthesis from deoxyuridine by the synthetic method described in JP-A-60-500717 is used.
It was introduced into the terminal and a capture probe 2-2 was synthesized.

(2) Detection The capture probe 2-1 or 2-2 synthesized in (1) was immobilized on the membrane and blocked with salmon testes DNA. This membrane was transferred to hybridization, and N of the PCR product obtained in Example 2 (2) was used.
Add neutralization solution after aOH denaturation, detection probe 1 and hybridization buffer (5 x SSC, 5% bovine serum albumin, 0.5% polyvinylpyrrolidone, 1% sodium dodecyl sulfate) and seal with a policyr. Fifty
Hybridization was performed at 15 ° C for 15 minutes.

The membrane was taken out from the hybridization and washed with Washing Solution 1 (1 × SSC, 1% sodium dodecyl sulfate) at 50 ° C. for 10 minutes with shaking. Furthermore, cleaning liquid 2 (1 x S
It was washed by shaking with SC, 0.5% Triton X-100) at room temperature for 10 minutes. Finally, washing with washing liquid 3 (1 × SSC) at room temperature for 5 minutes with shaking was performed. Transfer the membrane to a new hybridization bag,
Substrate solution (0.1M Tris-HCl, 0.1M NaCl, 0.1M MgCl ₂ , 0.3mg
/ ml nitrotetrazolium blue, 0.3 mg / ml bromine chloroindoferryl phosphate pH 7.5) was added, and the mixture was sealed with a policyr and incubated at 37 ° C. for 1 hour.

(4) Results When the spot of the purple dye generated by alkaline phosphatase was visually determined, the capture probe 2-
Only EBV was detected when either 1 or 2 was used. The same results were obtained with all possible combinations of oligonucleotides for EBV amplification, and are shown together in Table 2. Positive is indicated by +, and negative is indicated by-.

[0043]

[Table 2]

Example 4 (1) Synthesis of Amplification Oligonucleotide for NASBA 14 kinds of oligonucleotides having the sequences shown in the sequence listing and SEQ ID NOS: 3 to 16 by the phosphoamidite method using ABI DNA synthesizer type 392 Was synthesized. In addition, 5 kinds of oligonucleotides having the sequences shown in SEQ ID NOS: 17 to 20 have T7 RNA polymerase promoter sequences (5'-AATTCT) at their 5'ends.
AATACGACTCACTAGGGAG-3 ') was included. The synthetic method was according to the manual of ABI, and various oligonucleotides were deprotected with ammonia water at 55 ° C. overnight. Purification was carried out by FPLC (Pharmacia) using an anion exchange column.

(2) NASBA The NASBA method was carried out based on Japanese Patent Application Laid-Open No. 2-5864.
1 μl of a diluted solution of each herpes gene extract product was used as a forward primer (one of SEQ ID NOS: 3 to 16) 0.35
μM, reverse primer (one of SEQ ID NOs: 16 to 20) 0.35 μM, 50 mM Tris-HCl (pH8.3), 3.6 mM MgC
l ₂ , 40 mM KCl, 1 mM deoxyribonucleotide (Pharmac
ia) and heated to 95 ° C. for 3 minutes, then placed on ice. T7 DNA Polymerase (United States Biochemic
als Sequenase ^™ ) was added to a final concentration of 0.5 units / μl and incubated at 37 ° C. for 15 minutes. The mixture was heated to 95 ° C for 3 minutes, then cooled,
0.5 mM ribonucleotide (Pharmacia), 10 mM DT
T, 40 units reverse transcriptase (Toyobo), 0.4 units RN
aseH (manufactured by Toyobo) and 20 units of T7 DNA polymerase (manufactured by Stratagene) were added and reacted at 42 ° C. for 3 hours.

(3) Detection Amplification products from various herpes genes were dropped on a nylon membrane, and then the same method as in Example 1 was performed. (4) Results Spots of purple dye generated by alkaline phosphatase were visually determined. NBV for NASBA
The same results were obtained with all possible combinations of amplification oligonucleotides and are summarized in Table 3 below.
Only the detection probe and the detection probe 2 selectively detect, and it can be seen that these probes are functioning normally. Positive is indicated by +, and negative is indicated by-.

[0047]

[Table 3]

Example 5 (1) Preparation of biotin-modified capture probe No. 2 of Sequence Listing / SEQ ID NO: 2 was prepared by a phosphoamidite method using a DNA synthesizer type 392 manufactured by ABI. 1-2
A sequence having 5 was synthesized. Also, at this time, special table Sho-60-5
After introducing a uridine having a linker arm at the 5-position prepared by chemical synthesis from deoxyuridine into the 5'end by the synthesis method described in 00717, and leaving it in ammonia water at room temperature for 24 hours for deprotection. , N-hydroxysuccinimidylbiotin was bound (CF Barbara et al .; DNA. Vol. 4, p. 327 (1985)) to synthesize a biotin-modified capture probe.

(2) Synthesis of ³² P-labeled probe Using a DNA synthesizer type 392 manufactured by ABI, the No. 1 of Sequence Listing / SEQ ID NO: 1 was prepared by the phosphoamidite method. 1-2
A sequence having 5 was synthesized. The compound is ammonia water 5
After deprotection at 0 ° C overnight, Pharmacia F
Purified on PLC using anion exchange column. Thereafter, the 5'end was labeled with ³² P-ATP and T4 polynucleotide kinase to give a ³² P-labeled probe.

(3) PCR Using the sequence listing / SEQ ID NO: 10 synthesized in Example 2 (1) as the forward side primer and the sequence listing / SEQ ID NO: 18 as the reverse side primer, the PCR method was performed according to the example 2 (2). Carried out. The PCR product was denatured with 0.3 N NaOH and then neutralized.

(4) Detection Streptavidin magnetic beads (manufactured by Dynal), biotin-modified capture probe, ³² P-labeled probe, denaturing neutralization solution of PCR product, hybridization buffer (5
X SSC, 0.5% bovine serum albumin, 0.5% polyvinylpyrrolidone, 1% sodium dodecyl sulfate) and mix
Hybridization was performed at 2 ° C. for 2 hours. afterwards,
The magnetic beads were collected with a magnet and washed 3 times with 1 × SSC at 50 ° C. The radioactivity of the magnetic beads was measured by liquid scintillation counting using Beckman Ready Solve solution.

As shown in Table 4, the results show that only EBV was detected. As for the detection results of all PCR products obtained in Example 2 (2), only EBV was detected as in the case of Example 5.

[0053]

[Table 4]

[0054]

According to the present invention, it is possible to determine EBV directly, easily, quickly and surely. The oligonucleotide of the present invention can be used as a probe by labeling and as a capture probe by modification, and can significantly shorten the time as compared with the conventional immunization method, and can obtain a simple and reproducible result. Therefore, its clinical significance is great.

[0055]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 25 Sequence type: Nucleic acid Number of strands: Single-stranded sequence TGCTAAACCA TTTCGTGATC CACGT 25

SEQ ID NO: 2 Sequence length: 25 Sequence type: Nucleic acid Number of strands: Single-stranded sequence TAGAGATTGC CAAGATCGCT CACAT 25

SEQ ID NO: 3 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single-stranded sequence GCACATCTAC AGCATCAACC 20

SEQ ID NO: 4 Sequence length: 19 Sequence type: Nucleic acid Number of strands: Single-stranded sequence GGACATGCTC TACGCCTTC 19

SEQ ID NO: 5 Sequence length: 18 Sequence type: Nucleic acid Number of strands: Single-stranded sequence TGACCGGCTA CAACGTGG 18

SEQ ID NO: 6 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single-stranded sequence CAGGCACATC TACAGCATCA 20

SEQ ID NO: 7 Sequence length: 19 Sequence type: Nucleic acid Number of strands: Single-stranded sequence GGACACAGTA GTCAGGCAC 19

SEQ ID NO: 8 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single-stranded sequence GGACAAGCTC AGCCTCTCAG 20

SEQ ID NO: 9 Sequence length: 18 Sequence type: Nucleic acid Number of strands: Single-stranded sequence GAGATTCCTC GCCTCTTT 18

SEQ ID NO: 10 Sequence length: 18 Sequence type: Nucleic acid Number of strands: Single-stranded sequence TTTGGCCACC ACCAAGGT 18

SEQ ID NO: 11 Sequence length: 16 Sequence type: Nucleic acid Number of strands: Single-stranded sequence AGGGCCACCA CCAAGG 16

SEQ ID NO: 12 Sequence length: 23 Sequence type: Nucleic acid Number of strands: Single-stranded sequence GGTTTGATCC TTATCGACAT GTA 23

SEQ ID NO: 13 Sequence length: 22 Sequence type: Nucleic acid Number of strands: Single-stranded sequence CTGGTCTGAT CCTTATCGAC AT 22

SEQ ID NO: 14 Sequence length: 23 Sequence type: Nucleic acid Number of strands: Single-stranded sequence AAGGATGATC TGTATTACAA GGA 23

SEQ ID NO: 15 Sequence length: 23 Sequence type: Nucleic acid Number of strands: Single-stranded sequence AAGGATGATT TGTATTACAA GGA 23

SEQ ID NO: 16 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single-stranded sequence GCCTTTACTG TGTGCAGGAC 20

SEQ ID NO: 17 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single-stranded sequence GAGTTGTAGA ATCCGGACAG 20

SEQ ID NO: 18 Sequence length: 19 Sequence type: Nucleic acid Number of strands: Single-stranded sequence AGTGCTTGGG CAGGATAAA 19

SEQ ID NO: 19 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single-stranded sequence CGGCGTTATC ATGGTAGAAT 20

SEQ ID NO: 20 Sequence length: 19 Sequence type: Nucleic acid Number of strands: Single-stranded sequence AGAAGACACG GATCTGCTG 19

Claims (11)

[Claims] 1. A nucleic acid sequence represented by SEQ ID NO: 1 to SEQ ID NO: 20 or a nucleic acid sequence complementary to the sequence, which contains a nucleic acid sequence portion consisting of at least 15 contiguous residues. An EBV detection oligonucleotide. 2. A modified oligonucleotide for EBV detection, wherein the oligonucleotide according to claim 1 is an oligonucleotide modified with a modified product. 3. The modified oligonucleotide for EBV detection according to claim 2, wherein the modified product is a radioisotope, a fluorescent substance, an enzyme, a nucleotide having a linker arm, biotin or an RNA polymerase promoter sequence. 4. A method for detecting EBV in a sample, which comprises hybridizing the modified EBV detection oligonucleotide according to claim 2 with a nucleic acid in the sample, and measuring the modified product of the hybridized conjugate. 5. The EBV-detecting oligonucleotide according to claim 1 is bound to a solid phase carrier to hybridize with the nucleic acid in the sample, and the nucleic acid in the immobilized sample is mixed with the nucleic acid in the sample. Crossed with a modified oligonucleotide for detection,
A method for detecting EBV in a sample, which comprises measuring a modified product of a hybridized conjugate. 6. A sample in which the modified EBV detection oligonucleotide according to claim 2 is bound to a solid phase carrier by utilizing the property of the first modified product, hybridized with the nucleic acid in the sample, and immobilized. In the sample, the nucleic acid therein is hybridized with the modified oligonucleotide for detecting EBV modified with the second modified product according to claim 2, and the second modified product of the hybridized complex is measured. EBV detection method. 7. The sample according to claim 6, wherein the first modified product is a nucleotide having a linker arm and the second modified product is a radioisotope, a fluorescent substance, an enzyme, or biotin. EBV detection method. 8. The nucleic acid in the sample according to claim 1
6. Amplification using the oligonucleotide for BV detection, and the obtained amplification product is used as a sample.
Or the method for detecting EBV described in 6 above. 9. A nucleic acid sequence represented by SEQ ID NO: 3 to SEQ ID NO: 20 in the sequence listing according to claim 1 having an RNA polymerase promoter sequence at the 5'end side, or at least a continuous nucleic acid sequence complementary to the sequence. EBV containing a nucleic acid sequence portion consisting of 15 residues
Modified oligonucleotide for detection. 10. The nucleic acid in the sample is amplified using the EBV detection oligonucleotide according to claim 1, and the obtained amplification product is used as a sample.
The method for detecting EBV according to 5 or 6. 11. The EBV detection oligonucleotide according to claim 1 and / or the EB according to claim 2.
An EBV detection reagent kit comprising a modified oligonucleotide for V detection.