JPH07250699A - Method for discriminatory detection of human herpes virus and reagent therefor - Google Patents

Abstract

PURPOSE:To accomplish the subject detection of plural human herpes virus easily, quickly, specifically and in high sensitivity, by amplifying plural kinds of human herpes virus nucleic acids in a specimen using plural amplifying primers followed by discriminatory determination using a specificity detective probe. CONSTITUTION:At least two kinds of human herpes virus nucleic acids in a specimen are amplified using at least four kinds of amplifying primers each containing an oligonucleotide having a homology of 70% or higher and nucleic acid sequence(s) selected from at least two sequences selected from a group composed of a group A of sequences of formulas I-VIII, a group B of sequences of formulas IX-XIV and a group C of sequences of formulas XV0XX, followed by detecting the virus by Southern blotting hybridization method, etc., using a specific detective probe, thus accomplishing the aimed discriminatory detection of plural or all human herpes virus easily, quickly, specifically and in high sensitivity.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for conveniently and rapidly fractionating human herpesvirus and a reagent therefor.

[0002]

2. Description of the Related Art Human herpes virus (hereinafter sometimes abbreviated as HHV) has hitherto been herpes simplex virus type 1,
Type 2 (HSV-1, HSV-2), Varicella-zoster virus (VZV), Epstein-Barr virus (EBV), Cytomegalovirus (CMV), h
uman herpes virus-6 type A
(HHV-6A), the same B (HHV-6B), and hu
man herpes virus-7 (HHV-7)
Has been found. All of these HHVs are characterized by latent infection after the initial infection. In general, the initial infection pattern of HHV is usually subclinical or mild, whereas in patients with immunodeficiency associated with organ transplantation, AIDS patients, cancer patients, etc., pneumonia caused by HHV,
Opportunistic infections such as hepatitis and encephalitis have become serious problems in recent years. HH such as acyclovir and ganciclovir
As the development of therapeutic agents effective against V progresses, HHV
There is an urgent need to establish a simple and quick early diagnosis method.

Diagnosis of HHV infection has so far focused on virus isolation by culture and immunological diagnosis. However, in the culturing method, from separation culture to identification, 5-2
It takes 0 days and cannot make a quick diagnosis. on the other hand,
In the case of immunological diagnostic methods, a method of testing an antigen cannot detect a latent virus, and in HHV with a severe mutation, a change in antigenicity is expected, and there are many practical problems. In addition, the method of testing an antibody generally only indicates that the virus has been affected in the past, and does not reflect the state at the time of testing. Furthermore, all of the HHVs, whether by culture or immunization,
It is difficult to separately detect the two simultaneously. Although the detection of HHV by a gene diagnosis method has been attempted with the recent progress of gene diagnosis technology, a system capable of differentially detecting all of the above HHV has not been reported yet.

[0004]

SUMMARY OF THE INVENTION Therefore, an object of the present invention is to detect plural or all HHVs separately, conveniently, rapidly, specifically and highly sensitively.

[0005]

Means for Solving the Problems As a result of various studies on the gene of HHV, the present inventors devised a method capable of differentially detecting HHV in a simple, rapid, specific and highly sensitive manner and used it. The detection method was established and the present invention was completed.

The present invention will be described in brief. A reaction for preamplifying a plurality or all of HHV nucleic acids suspected to be present in a sample is carried out, and then the resulting amplification product is tested with an oligonucleotide probe specific to the virus to be tested. The present invention relates to a method for detecting HHV in a sample, which is characterized by performing differential detection.

That is, the present invention is a method for differentially detecting HHV, which comprises amplifying at least two types of HHV nucleic acids in a sample in advance using at least four types of amplification primers and then performing differential detection using a specific detection probe. Is.

The present invention also provides a method for differential detection of HHV, which comprises amplifying all types of HHV nucleic acids in a sample in advance using at least four types of amplification primers and then performing differential detection using a specific detection probe. is there.

In the present invention, at least four kinds of amplification primers are sequence group A (SEQ ID NOS: 1 to 8) and sequence group B
(SEQ ID NOS: 9-14) and sequence group C (SEQ ID NOS: 15-
It is preferable to include an oligonucleotide having a homology of 70% or more with a nucleic acid sequence selected from at least two sequence groups selected from the group consisting of 20).

In the present invention, the specific detection probes are sequence group A '(SEQ ID NOS: 21-26), sequence group B' (SEQ ID NOS: 27-30) and sequence group C '(SEQ ID NOS: 31-3).
An oligonucleotide having a homology of 70% or more with a nucleic acid sequence selected from at least two sequence groups selected from the group consisting of 6), or an oligonucleotide having a complementary nucleic acid sequence thereof is preferable.

In the present invention, at least two kinds of H in the sample are used.
The HV nucleic acid is selected from at least two sequence groups selected from the group consisting of sequence group A (SEQ ID NOS: 1 to 8), sequence group B (SEQ ID NOS: 9 to 14) and sequence group C (SEQ ID NOS: 15 to 20). Sequence group A '(SEQ ID NOS: 21-26), sequence group B' (SEQ ID NO: 27) after amplification using at least four kinds of amplification primers containing an oligonucleotide having 70% or more homology with the nucleic acid sequence To 30) and a sequence group C ′ (SEQ ID NOS: 31 to 36), an oligonucleotide having a homology of 70% or more with a nucleic acid sequence selected from at least two sequence groups selected from the group consisting of SEQ ID NOS: 31 to 36, or a complementary nucleic acid sequence thereof. HHV is differentially detected using a specific detection probe which is an oligonucleotide having

Further, in the present invention, the HHV nucleic acids of all species in the sample are preliminarily sequence group A (SEQ ID NOS: 1-8) and sequence group B.
(SEQ ID NOS: 9-14) and sequence group C (SEQ ID NOS: 15-
20) Sequence group A ′ after amplification using at least 4 kinds of amplification primers containing an oligonucleotide having 70% or more homology with a nucleic acid sequence selected from at least two sequence groups selected from the group consisting of (SEQ ID NOs: 21 to
26), a nucleic acid sequence selected from at least two sequence groups selected from the group consisting of sequence group B ′ (SEQ ID NOS: 27 to 30) and sequence group C ′ (SEQ ID NOS: 31 to 36), and
HHV is differentially detected using a specific detection probe which is an oligonucleotide having a homology of not less than% or an oligonucleotide having a nucleic acid sequence complementary thereto.

Further, in the present invention, at least two kinds of HHV nucleic acids in a sample are used as amplification primers in advance and nucleic acid sequences selected from the sequence group A (SEQ ID NOS: 1 to 8) and 70
An oligonucleotide having a homology of at least% and a nucleic acid sequence selected from the sequence group B (SEQ ID NOS: 9 to 14);
After carrying out an amplification reaction using a mixture of oligonucleotides having a homology of 0% or more, as a specific detection probe, a sequence group A ′ (SEQ ID NOS: 21 to 26) and a sequence group B ′ (SEQ ID NOS: 27 to 30) are used. In the sample, HSV-1, HSV-2, VZV, E using an oligonucleotide having a homology of 70% or more with a nucleic acid sequence selected from
Separately detect BV or CMV.

Further, in the present invention, at least two kinds of HHV nucleic acids in a sample are used as amplification primers in advance and nucleic acid sequences selected from the sequence group A (SEQ ID NOS: 1 to 8) and 70
After performing an amplification reaction using a mixture of an oligonucleotide having a homology of not less than% and a nucleic acid sequence selected from the sequence group C (SEQ ID NOS: 15 to 20) and an oligonucleotide having a homology of not less than 70%, As a specific detection probe, an oligonucleotide having 70% or more homology with a nucleic acid sequence selected from the sequence group A ′ (SEQ ID NOS: 21 to 26) and the sequence group C ′ (SEQ ID NOS: 31 to 36), or its complement HSV-1, HSV-2, VZV, H in a sample using an oligonucleotide having a nucleic acid sequence
HV-6 (type A), HHV-6 (type B) or HHV-7 is differentially detected.

In the present invention, at least two kinds of HHV nucleic acids in a sample are used as amplification primers in advance and nucleic acid sequences selected from the sequence group B (SEQ ID NOs: 9 to 14) and 7
After carrying out an amplification reaction by using an oligonucleotide having 0% or more homology and an oligonucleotide having 70% or more homology with a nucleic acid sequence selected from sequence group C (SEQ ID NOS: 15 to 20) , As a specific detection probe, an oligonucleotide having 70% or more homology with a nucleic acid sequence selected from the sequence group B ′ (SEQ ID NOS: 27 to 30) and the sequence group C ′ (SEQ ID NOS: 31 to 36), or its complement EBV, CMV, HHV-6 (type A), HHV-6 (type B) or HHV-7 in the sample is differentially detected using an oligonucleotide having a specific nucleic acid sequence.

Further, in the present invention, at least two kinds of HHV nucleic acids in a sample are used as amplification primers in advance and a nucleic acid sequence selected from the sequence group A (SEQ ID NOS: 1 to 8) and 70
Oligonucleotides having a homology of at least%, sequence group B
70% with a nucleic acid sequence selected from (SEQ ID NOs: 9 to 14)
An oligonucleotide having the above homology and a nucleic acid sequence selected from the sequence group C (SEQ ID NOS: 15 to 20) and 7
After performing an amplification reaction using a mixture of oligonucleotides having a homology of 0% or more, sequence groups A ′ (SEQ ID NOS: 21 to 26) and sequence groups B ′ were used as specific detection probes.
(SEQ ID NOs: 27 to 30) and sequence group C ′ (SEQ ID NO: 3
1 to 36), an oligonucleotide having a homology of 70% or more with a nucleic acid sequence selected from 1 to 36), or an oligonucleotide having a complementary nucleic acid sequence thereof is used, and HSV-1, HSV-2, VZV, EBV in a sample is used. , CMV, H
HV-6 (type A), HHV-6 (type B) or HHV-7 is differentially detected.

Further, in a specific example of the present invention, at least two kinds of HHV nucleic acids in a sample are used as amplification primers in advance and an oligonucleotide having a nucleic acid sequence selected from sequence group A (SEQ ID NOS: 1 to 8) and sequence group B are used. After carrying out an amplification reaction using a mixture of oligonucleotides having a nucleic acid sequence selected from (SEQ ID NOS: 9 to 14), the sequence group A ′ (SEQ ID NOS: 21 to 2) is used as a specific detection probe.
6) and an oligonucleotide having a nucleic acid sequence selected from Sequence Group B ′ (SEQ ID NOS: 27 to 30) or a complementary nucleic acid sequence thereof, HSV-1, HV in the sample
SV-2, VZV, EBV or CMV is separately detected.

In another embodiment of the present invention, at least two HHV nucleic acids in a sample are used as amplification primers in advance, and an oligonucleotide having a nucleic acid sequence selected from the sequence group A (SEQ ID NOS: 1 to 8) and a sequence After carrying out an amplification reaction using a mixture of oligonucleotides having a nucleic acid sequence selected from group C (SEQ ID NOS: 15 to 20), a specific detection probe is sequence group A ′ (SEQ ID NO: 2).
1-26) and a sequence group C ′ (SEQ ID NOS: 31-36), or an oligonucleotide having a nucleic acid sequence complementary thereto, the HSV-
1, HSV-2, VZV, HHV-6 (Type A), H
Differentially detect HV-6 (type B) or HHV-7.

In another embodiment of the present invention, sequence group B (SEQ ID NOs: 9 to 14) is prepared by using at least two human herpesvirus nucleic acids in a sample as amplification primers in advance.
After carrying out an amplification reaction by mixing an oligonucleotide having a nucleic acid sequence selected more and an oligonucleotide having a nucleic acid sequence selected from Sequence Group C (SEQ ID NOS: 15 to 20), the sequence as a specific detection probe is used. An oligonucleotide having a nucleic acid sequence selected from the group B ′ (SEQ ID NOS: 27 to 30) and the sequence group C ′ (SEQ ID NOS: 31 to 36) or a complementary nucleic acid sequence thereof is used,
Human herpesvirus, EBV, CMV, HH in samples
V-6 (Type A), HHV-6 (Type B) or H
HV-7 is separately detected.

In another embodiment of the present invention, an oligonucleotide having a nucleic acid sequence selected from Sequence Group A (SEQ ID NOS: 1 to 8) is prepared by using at least two human herpesvirus nucleic acids in a sample as amplification primers in advance. , An oligonucleotide having a nucleic acid sequence selected from Sequence Group B (SEQ ID NOS: 9 to 14) and Sequence Group C (SEQ ID NO: 1)
5 to 20), an oligonucleotide having a nucleic acid sequence selected from 5 to 20) is mixed and used to carry out an amplification reaction.
6), a nucleic acid sequence selected from the sequence group B ′ (SEQ ID NOS: 27 to 30) and the sequence group C ′ (SEQ ID NOS: 31 to 36),
Alternatively, using an oligonucleotide having a complementary nucleic acid sequence thereof, HSV-1, HSV-2, VZ in a sample can be obtained.
V, EBV, CMV, HHV-6 (Type A), HHV
-6 (type B) or HHV-7 is detected separately.

The detection method of the present invention includes a detection method by sandwich hybridization, a detection method by Southern blot hybridization and a detection method by dot blot hybridization.

The HHV differential detection reagent of the present invention comprises (1) sequence group A (SEQ ID NOS: 1-8) and sequence group B (SEQ ID NOS: 9-1)
4) and sequence group C (SEQ ID NOs: 15 to 20), and at least four kinds of amplification primers containing an oligonucleotide having a homology of 70% or more with a nucleic acid sequence selected from at least two sequence groups selected from the group consisting of , (2) amplification enzyme, (3) amplification substrate, (4) amplification buffer, (5) sequence group A '(SEQ ID NOS: 21 to 26), sequence group B' (SEQ ID NO: 2)
7 to 30) and a sequence group C ′ (SEQ ID NOs: 31 to 36), and an oligonucleotide having a homology of 70% or more with a nucleic acid sequence selected from at least two sequence groups selected from the group consisting of sequence groups C ′ (SEQ ID NOS: 31 to 36), or a complementary nucleic acid thereof. It contains a specific detection probe which is an oligonucleotide having a sequence, (6) a detection buffer, (7) a detection washing solution, (8) a detection substrate, and the like.

The amplification enzyme of the present invention is a nucleic acid synthase selected from the group consisting of DNA polymerase, RNA polymerase and reverse transcriptase, and the amplification substrate is dNTP.
And / or rNTP.

The specific detection probe of the present invention has a label as
It is preferable to have a radioactive substance, an enzyme, a fluorescent substance, an antigen, a hapten, an enzyme substrate or an insoluble carrier.

Specific examples of the specific detection probe of the present invention include those having alkaline phosphatase as a label and the substrate for detection being a luminescent 1,2-dioxetane compound.

The present invention will be described in more detail below. Generally, in order to simultaneously amplify a plurality of target nucleic acids, it is achieved by mixing and using primers specific to each target nucleic acid. However, chaotic mixing of the primers increases the possibility that primer dimers, non-specific amplification products, etc. will be generated, and as a result the amplification reaction will not proceed normally, resulting in a large decrease in sensitivity and characteristics. Will be invited. Although it is theoretically possible to use a random primer to amplify all nucleic acids in a sample and then detect with a specific probe, it is also possible to amplify all nucleic acids having sequences unnecessary for detection such as human genome. Therefore, the background at the time of detection increases, and again, it is difficult to perform specific detection with high sensitivity.

Therefore, the present inventors designed and created primers for HHV amplification that do not affect each other, and have sequence groups A to
It was classified into C. Group A is HSV-1, HSV-2, VZ
Primers for amplifying V (SEQ ID NOS: 1 to 8). Similarly, Group B is a primer (SEQ ID NO: 9 to 14) for amplifying EBV and CMV, and Group C is H
Primers (SEQ ID NOs: 15 to 20) for amplifying HV-6A, HHV-6B and HHV-7. These primers do not interfere with each other even if the primers of different groups are mixed and used in the same group,
By mixing and using primers selected from two groups or all groups, multiple or all HH
It became possible to amplify V at the same time. The amplification primer of the present invention contains 70% of the nucleic acid sequences described in the above sequence group.
It includes an oligonucleotide having the above homology.

On the other hand, the HHV differential detection probe is designed and prepared for the purpose of separating the above HHV-derived amplification products,
They were classified into sequence group A ′ (SEQ ID NOS: 21 to 26), sequence group B ′ (SEQ ID NOS: 27 to 30) and sequence group C ′ (SEQ ID NOS: 31 to 36) according to the classification of the amplification primers. The HHV differential detection probe of the present invention has a homology of 70% or more with the nucleic acid sequence shown here or has a complementary sequence thereof, and is specific for detection of each HHV, It does not cross-react with HHV-derived amplification products and non-target nucleic acids such as the human genome.

The oligonucleotide used for the primer or probe of the present invention is a deoxyribonucleic acid (DN
It may be A) 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). In addition, in the synthesis, T at any position was changed to U to perform the synthesis, and D containing a uridine residue was used.
It may be NA. Similarly, it may be RNA containing a thymidine residue in which any U is changed to T. The oligonucleotide may have a mutation such as a deletion, insertion or substitution of several bases, or a modified nucleotide.

The differential detection probe used in the present invention can be labeled with a known label. As the labeled substance, for example, radioactive substances, enzymes, fluorescent substances, luminescent substances, antigens, haptens, enzyme substrates, insoluble carriers and the like can be used. The position of the label may be an end label or may be in the middle of the sequence. Further, any label may be attached to the sugar, phosphate group and base moiety. Detection is achieved by using a detection method that depends on the respective label.

As the detection method for separating the amplification products generated as a result of the amplification reaction, various known methods can be used using the HHV separation detection probe of the present invention. In one form, the amplification product is immobilized on a solid phase carrier such as a membrane, plate or bead and hybridized with a labeled probe for differential detection of HHV (dot blot hybridization, Southern hybridization). Blotting, hybridization, etc.). In another form, a probe for capturing an amplification product may be immobilized on a solid phase to perform a sandwich assay (sandwich hybridization).
In the sandwich assay, if one of the probes for detection / capture is the probe of the present invention, specific detection is possible with this probe, and there is no problem even if the other probe has a slightly low specificity.

[0032]

EXAMPLES The effects of the present invention will be further clarified by exemplifying the examples of the present invention. Example 1 Preparation of Various Oligonucleotides Using ABI DNA Synthesizer Model 391, various oligonucleotides having the sequences shown in the Sequence Listing and SEQ ID NOs: 1 to 36 were synthesized by the phosphoamidite method. The oligonucleotides shown in the sequence listing and SEQ ID NOS: 1 to 36 are respectively oligonucleotides (1) to (36)
Call. The method is 0.2 μM according to the ABI company manual.
Performed on a scale. Deprotection of various oligonucleotides was carried out with ammonia water at 55 ° C. overnight. Purification was carried out by FPLC manufactured by Pharmacia on a reverse phase column. If necessary, the synthesized oligonucleotide can be prepared according to the method of Jablonski et al. (Nucl. Acid Res. 14: 6115-6128, 1986).
Alkaline phosphatase (Alp) was attached.

Example 2 HHV Amplification and Detection (1) Preparation of Viral Nucleic Acid The following strains were used as HHV standard strains. -HSV-1: Seivert strain-HSV-2: UW268 strain-VZV: Kawaguchi strain-EBV: B95-8 strain-CMV: AD169 strain-HHV-6A: U1102 strain-HHV-6B: Hashimoto strain-HHV-7: RK strain The viral nucleic acid was prepared by a conventional method.

(2) Amplification of HHV Nucleic Acid-Part 1 The above nucleic acid solution or human placenta DNA was added to 94 μl of the reaction solution.
5 μl of solution, oligonucleotides (1), (3) of Example 1
, (4), (5), (8), (9), (12), (15), (17), (1
1 μl of the mixed solution of 8), (19) and (20) was added as a primer to amplify a human herpesvirus-derived nucleic acid.
The composition of the reaction solution is as follows. Reaction solution composition KCl 50 mM Tris-HCl (pH 8.3) 10 mM MgCl ₂ 1.5 mM Gelatin 0.01% dATP, dCTP, dGTP, dTTP 0.2 mM Tth DNA polymerase 40 units / ml The reaction conditions are as follows. Thermal denaturation: 94 ° C., 1 minute Annealing: 55 ° C., 2 minutes Polymerization reaction: 75 ° C., 1.5 minutes 30 times. These operations are Perkin-Elmer /
It was performed using a DNA thermal cycler manufactured by Cetus (Perkin-Elmer / Cetus).

(3) Fractional Detection of HHV by Dot Blot Hybridization Dot blot hybridization was performed to identify which human herpesvirus the resulting amplification product was derived from as a result of the above amplification reaction. . Examples of the probe include (22), (24), (26), (27), (29), and (3) of Example 1.
The oligonucleotides of 1), (33) and (35) were labeled with Alp and used. To 1 μl of the above PCR product, add 100 μl of 0.3 N NaOH and denature for 15 minutes at room temperature.
It was blotted on a nylon membrane (High Bond N Plus, manufactured by Amersham) wetted with 5 × SSC using a dot blotter (manufactured by BRL). SSC means 15 mM trisodium citrate and 15 mM sodium chloride solution,
5 × SSC means a 5 times concentrated solution of SSC. This film is 8
Nucleic acid-immobilized by heat treatment at 0 ° C for 30 minutes 8
One sheet was prepared and hybridization was performed using each Alp-labeled probe.

First, the dried film (1 × 8 cm) is treated with 5 × S.
Immerse in SC for 5 minutes. Next, the membrane was transferred to a hybridization bag (manufactured by BRL), and 5 ml of a hybridization buffer (5 × SSC, 0.5% bovine serum albumin, 0.5% polyvinylpyrrolidone, 1% sodium dodecylsulfate) was added to the policyr. Seal with
Prehybridization was performed at 50 ° C. for 15 minutes. Next, hybridization was carried out at 55 ° C. for 15 minutes with 5 ml of a hybridization buffer containing 5 μl of any of the above probes. The membrane was taken out from the polybag, and washed with Shake Solution-1 (1 × SSC, 1% sodium dodecyl sulfate) at 55 ° C. for 5 minutes with shaking twice. Further, the membrane was dried after washing with Washing solution-2 (1 × SSC) twice at room temperature for 5 minutes with shaking.

Each 1 ml of color developing solution (200 mM Tri
s-HCl (pH 8.5), 0.58% NaCl,
1% MgCl ₂ , 0.00136% ZnCl ₂ , 0.0
2% NaN ₃ , 75 mg / ml NBT (nitro blue tet
razolium), 50 mg / ml BCIP (5-bromo-4-chlo
ro-3-indolyl phosphate)) was added, and the color reaction was carried out at 37 ° C. for 30 minutes to detect human herpesvirus. As a result, the oligonucleotide probe specific to each virus specifically reacted only with the amplification product derived from each virus, and no reaction with the amplification products derived from other viruses or human placenta DNA was observed. (Table 1).

[0038]

[Table 1]

(4) Amplification of HHV nucleic acid-Part 2 The above nucleic acid solution or human placental DNA was added to 94 μl of the reaction solution.
5 μl of solution, oligonucleotides (1), (2) of Example 1
, (6), (7), (10), (11), (15) and (20) were added as a primer to amplify human nucleic acid derived from human herpesvirus. The composition of the reaction solution conforms to the experiment of (2) above.

(5) Differential detection of HHV by sandwich hybridization Sandwich hybridization was performed to identify which human herpesvirus the amplification product generated in the experiment of (4) originated. As the solid phase, a polystyrene microtiter plate (Microlite 2, Dynatech) is used, and the oligonucleotides (21), (23), (25), (28), (30), (of Example 1 are used. 32), (34) and (36) were fixed by physical adsorption. 2 μl of each HHV-derived amplification product denatured on each capture probe-immobilized plate
And hybridization buffer (5 x SSC, 0.5
% Polyvinylpyrrolidone K-30, 1% sodium laurylsarcosine) 100 μl was added, and prehybridization was performed at 50 ° C. for 60 minutes. After removing the liquid, (22), (24), (26), (27), (29), (31), (33), (3 of Example 1
Each of the oligonucleotides of 5) labeled with Alp was added to a corresponding plate, and hybridization was carried out at 50 ° C. for 60 minutes. After removing the probe solution, 200 μl of washing solution- (1) (2 × SSSC, 1.0
% Sodium lauryl sulfate), and washing solution- (2) (1
X SSC). Next, a 1,2-dioxetane compound, which is a luminescent substrate of Alp, (Lumiphos48
0, Wako Pure Chemical Industries, Ltd.) was added, and a luminescence reaction was performed at 37 ° C. for 15 minutes. Micro light 10
00 (Dynatech). As a result, all HHVs could be separately detected without being confused with each other (Table 2).

[0041]

[Table 2]

[0042]

EFFECTS OF THE INVENTION According to the present invention, it becomes possible to detect HHV, which has conventionally required a long time for culturing, in a specific manner and with high sensitivity in a rapid and simple manner. By using the differential detection method of the present invention, all the HHV amplification reactions can be performed simultaneously without using a single tube, so that the time, cost, and labor required for the test can be significantly reduced.

[0043]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 25 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..25 Method for determining features : S Other features: Sequence common to HSV. Array GTTTGTGTGT TGTTCGGTCA TAAGC 25

SEQ ID NO: 2 Sequence length: 22 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..22 Features Method determined: S Other features: Sequence common to HSV. Array GCGTAGTACA CCGTGATCGG GA 22

SEQ ID NO: 3 Sequence length: 24 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..24 Method determined: S Other features: HSV-1 specific sequence. Sequence TCGGCCATCT TGAGAGAGGC ATCC 24

SEQ ID NO: 4 Sequence length: 24 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..24 Method determined: S Other features: HSV-2 specific sequence. Sequence GGCTGAATGT GGTAAACACG CTTC 24

SEQ ID NO: 5 Sequence length: 23 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1.23 Method determined: S Other features: VZV specific sequence. Sequence GTTTCTCCCA CTGGTACGTC AAG 23

SEQ ID NO: 6 Sequence length: 24 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..24 Method determined: S Other features: VZV specific sequence. Sequence GGGCGAAATG TAGGATATAA AGGA 24

SEQ ID NO: 7 Sequence length: 24 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..24 Features Method determined: S Other features: VZV specific sequence. Sequence ATCGCGGCTT GTTGTTTGTC TAAT 24

SEQ ID NO: 8 Sequence length: 23 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..23 Features Method determined: S Other features: VZV specific sequence. Sequence CAGTGGCCGC TACGTATAAA CAT 23

SEQ ID NO: 9 Sequence length: 18 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..18 Features Method determined: S Other features: Sequence common to EBV and CMV. Sequence TTTGGCCACC ACCAAGGT 18

SEQ ID NO: 10 Sequence length: 23 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1.23 Method determined: S Other features: Sequence common to EBV and CMV. Sequence GGTTTGATCC TTATCGACAT GTA 23

SEQ ID NO: 11 Sequence length: 19 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..19 Features Method determined: S Other features: Sequence common to EBV and CMV. Sequence AGAAGACACG GATCTGCTG 19

SEQ ID NO: 12 Sequence length: 19 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..19 Determine features Method: S Other features: Sequence common to EBV and CMV. Sequence AGTGCTTGGG CAGGATAAA 19

SEQ ID NO: 13 Sequence length: 18 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..18 Features Method determined: S Other features: EBV-specific sequence. Sequence GAGATTCCTC GCCTCTTT 18

SEQ ID NO: 14 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..20 Method determined: S Other features: CMV specific sequence. Sequence ATGCATGGCC AAGACTAACT 20

SEQ ID NO: 15 Sequence length: 22 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..22 Features Method determined: S Other features: Sequence common to HHV-6A, HHV-6B, HHV-7. Array ATAATTGGCA ATGAACACCG TT 22

SEQ ID NO: 16 Sequence length: 24 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..24 Method determined: S Other features: Sequence common to HHV-6A, HHV-6B, HHV-7. Sequence TTTTGATATG GGTTTAGGTT TTAG 24

SEQ ID NO: 17 Sequence length: 22 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..22 Method determined: S Other features: HHV-6A specific sequence. Sequence GTGCCTATTA TACAGTTCCA GA 22

SEQ ID NO: 18 Sequence length: 24 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..24 Features determined Method: S Other features: Sequence specific for HHV-6B. Array GTCAACAAGC TATCTGCGAA GTCG 24

SEQ ID NO: 19 Sequence length: 24 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..24 Method determined: S Other features: HHV-7 specific sequence. Array TACACCAACC CTACTGTAAA TAGT 24

SEQ ID NO: 20 Sequence length: 23 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence characteristics Location: 1..23 Determined method: S Other characteristics: HHV-6A, HHV-6B, HHV-7C
Sequence common to MV. Sequence GATCCTTTTT GAGATGCCCA AGG 23

SEQ ID NO: 21 Sequence length: 25 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..25 Features Method determined: S Other features: HSV-1 specific sequence. Array AGCGCGAACG ACCAACTACC CCGAT 25

SEQ ID NO: 22 Sequence length: 25 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..25 Features Method determined: S Other features: HSV-1 specific sequence. Sequence TCAGTTATCC TTAAGGTCTC TTTTG 25

SEQ ID NO: 23 Sequence length: 25 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..25 Features Method determined: S Other features: HSV-2 specific sequence. Sequence TCCCAATCGA TTTCGCGGGA AGAAC 25

SEQ ID NO: 24 Sequence length: 25 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..25 Features Method determined: S Other features: HSV-2 specific sequence. Sequence TTCCGGTTTT GGACCAGCTG ACCGA 25

SEQ ID NO: 25 Sequence length: 25 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..25 Features Method determined: S Other features: VZV specific sequence. Sequence TTTACCACGC TAACGTTAAA TTTTG 25

SEQ ID NO: 26 Sequence length: 22 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..22 Features Method determined: S Other features: VZV specific sequence. Sequence TATGCCACCT TTACAGTTGG AG 22

SEQ ID NO: 27 Sequence length: 24 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..24 Method determined: S Other features: EBV-specific sequence. Sequence GCTAAACCAT TTCGTGATCC ACGT 24

SEQ ID NO: 28 Sequence length: 24 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..24 Method determined: S Other features: EBV-specific sequence. Sequence AGAGATTGCC AAGATCGCTC ACAT 24

SEQ ID NO: 29 Sequence length: 24 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..24 Method determined: S Other features: CMV specific sequence. Sequence ATTCCGTTGC GGCGTGTCAT CTTT 24

SEQ ID NO: 30 Sequence length: 26 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..26 Method determined: S Other features: CMV specific sequence. Array GTTCAACACC ATTAATTTTC ACTACG 26

SEQ ID NO: 31 Sequence length: 23 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..23 Method determined: S Other features: HHV-6A specific sequence. Array GTTAATGTTT GTCGTAACCG GGG 23

SEQ ID NO: 32 Sequence length: 22 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..22 Features Method determined: S Other features: HHV-6A specific sequence. Array CTCTTGGGTT GTGGGGCGAT TT 22

SEQ ID NO: 33 Sequence length: 23 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence characteristics Location: 1..23 Method determined: S Other features: HHV-6B specific sequence. Array GTTAATATTT GTCGTGACCG GAG 23

SEQ ID NO: 34 Sequence length: 22 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..22 Features Method determined: S Other features: HHV-6B specific sequence. Sequence CTCTGGGGTT ATGGGGCAAT TT 22

SEQ ID NO: 35 Sequence length: 31 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..31 Features Method determined: S Other features: HHV-7 specific sequence. Sequence TCATTACTCC AGTGACTTCC GATATTAATT T 31

SEQ ID NO: 36 Sequence length: 30 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..30 Method determined: S Other features: HHV-7 specific sequence. Array CTTTCGGTAA GATAAATAAG CAATCACAAT 30

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Yutaka Takarada 1-1-1, Katata, Otsu City, Shiga Prefecture Toyobo Co., Ltd.

Claims (23)

[Claims] 1. Fractionation of human herpesvirus, characterized in that at least two kinds of human herpesvirus nucleic acids in a sample are previously amplified using at least four kinds of amplification primers, and then differentially detected using a specific detection probe. Detection method. 2. Differential detection of human herpesvirus, which comprises amplifying all human herpesvirus nucleic acids in a sample in advance using at least four types of amplification primers, and then differentially detecting with a specific detection probe. Method. 3. At least four kinds of amplification primers are sequence group A (SEQ ID NOS: 1-8) and sequence group B (SEQ ID NOS: 9-1).
4) and sequence group C (SEQ ID NOS: 15 to 20), comprising an oligonucleotide having 70% or more homology with a nucleic acid sequence selected from at least two sequence groups selected from the group consisting of: The method for differential detection of human herpesvirus according to claim 1 or claim 2. 4. The specific detection probes are sequence group A ′ (SEQ ID NOS: 21 to 26) and sequence group B ′ (SEQ ID NOS: 27 to 30).
And an oligonucleotide having a homology of 70% or more with a nucleic acid sequence selected from at least two sequence groups selected from the group consisting of sequence group C ′ (SEQ ID NOS: 31 to 36),
Alternatively, the method for differentially detecting human herpesvirus according to claim 1 or 2, which is an oligonucleotide having a nucleic acid sequence complementary thereto. 5. The method for differential detection of human herpesvirus according to claim 1 or 2, wherein the specific detection probe has a label. 6. The human herpesvirus is HSV-1,
Fractionation of human herpesvirus according to claim 1 or 2, which is HSV-2, VZV, EBV, CMV, HHV-6 (type A), HHV-6 (type B) or HHV-7. Detection method. 7. The sequence group A (SEQ ID NO: 1 to
8), sequence group B (SEQ ID NOS: 9 to 14) and sequence group C
After amplification using at least four kinds of amplification primers containing an oligonucleotide having a homology of 70% or more with a nucleic acid sequence selected from at least two sequence groups selected from the group consisting of (SEQ ID NOS: 15 to 20) , Sequence group A '
(SEQ ID NOS: 21 to 26), sequence group B '(SEQ ID NOS: 27 to
30) and a sequence group C ′ (SEQ ID NOS: 31 to 36), an oligonucleotide having a homology of 70% or more with a nucleic acid sequence selected from at least two sequence groups selected from the group consisting of SEQ ID NOS: 31 to 36, or a complementary nucleic acid sequence thereof. The method for differential detection of human herpesvirus according to claim 1, wherein the specific detection probe which is an oligonucleotide has differential detection. 8. The sequence groups A (SEQ ID NOS: 1 to 8) and the sequence group B are preliminarily analyzed for all kinds of human herpesvirus nucleic acids in a sample.
(SEQ ID NOS: 9-14) and sequence group C (SEQ ID NOS: 15-
20) Sequence group A ′ after amplification using at least 4 kinds of amplification primers containing an oligonucleotide having 70% or more homology with a nucleic acid sequence selected from at least two sequence groups selected from the group consisting of (SEQ ID NOs: 21 to
26), a nucleic acid sequence selected from at least two sequence groups selected from the group consisting of sequence group B ′ (SEQ ID NOS: 27 to 30) and sequence group C ′ (SEQ ID NOS: 31 to 36), and
3. The method for differential detection of human herpesvirus according to claim 2, wherein the specific detection probe is an oligonucleotide having a homology of not less than% or an oligonucleotide having a nucleic acid sequence complementary thereto. 9. A nucleic acid sequence selected from the sequence group A (SEQ ID NOs: 1 to 8) and 70 using at least two human herpesvirus nucleic acids in a sample as amplification primers in advance.
An oligonucleotide having a homology of at least% and a nucleic acid sequence selected from the sequence group B (SEQ ID NOS: 9 to 14);
After carrying out an amplification reaction using a mixture of oligonucleotides having a homology of 0% or more, as a specific detection probe, a sequence group A ′ (SEQ ID NOS: 21 to 26) and a sequence group B ′ (SEQ ID NOS: 27 to 30) are used. HSV- in a sample characterized by differential detection using an oligonucleotide having a homology of 70% or more with a nucleic acid sequence selected from the above, or an oligonucleotide having a complementary nucleic acid sequence thereof
1, HSV-2, VZV, EBV or CMV classification detection method. 10. A nucleic acid sequence selected from the sequence group A (SEQ ID NOS: 1 to 8) with at least two human herpesvirus nucleic acids in a sample as amplification primers in advance and 7
After carrying out an amplification reaction by using an oligonucleotide having 0% or more homology and an oligonucleotide having 70% or more homology with a nucleic acid sequence selected from sequence group C (SEQ ID NOS: 15 to 20) , As a specific detection probe, an oligonucleotide having 70% or more homology with a nucleic acid sequence selected from the sequence group A ′ (SEQ ID NOS: 21 to 26) and the sequence group C ′ (SEQ ID NOS: 31 to 36), or its complement In a sample characterized by differential detection using an oligonucleotide having a specific nucleic acid sequence
1, HSV-2, VZV, HHV-6 (Type A), H
A method for differential detection of HV-6 (type B) or HHV-7. 11. An oligonucleotide having a homology of 70% or more with a nucleic acid sequence selected from Sequence Group B (SEQ ID NOS: 9 to 14) using at least two human herpesvirus nucleic acids in a sample as amplification primers in advance, and After performing an amplification reaction by mixing and using an oligonucleotide having a homology of 70% or more with the nucleic acid sequence selected from the sequence group C (SEQ ID NOS: 15 to 20), the sequence group B ′ ( Fractionation using an oligonucleotide having 70% or more homology with a nucleic acid sequence selected from SEQ ID NOs: 27 to 30 and sequence group C ′ (SEQ ID NOs: 31 to 36), or an oligonucleotide having a complementary nucleic acid sequence thereof EB in a sample characterized by detecting
A method for differential detection of V, CMV, HHV-6 (type A), HHV-6 (type B) or HHV-7. 12. A nucleic acid sequence selected from the sequence group A (SEQ ID NOS: 1 to 8) with at least two human herpesvirus nucleic acids in a sample as amplification primers in advance and 7
An oligonucleotide having a homology of 0% or more, a nucleic acid sequence selected from the sequence group B (SEQ ID NOS: 9 to 14) and 70
After performing an amplification reaction using a mixture of an oligonucleotide having a homology of not less than% and a nucleic acid sequence selected from the sequence group C (SEQ ID NOS: 15 to 20) and an oligonucleotide having a homology of not less than 70%, Sequence group A ′ (SEQ ID NOS: 21 to 26) and sequence group B ′ as specific detection probes
(SEQ ID NOs: 27 to 30) and sequence group C ′ (SEQ ID NO: 3
HSV-1 in a sample characterized by differential detection using an oligonucleotide having 70% or more homology with a nucleic acid sequence selected from 1 to 36) or an oligonucleotide having a complementary nucleic acid sequence thereof, HSV-2,
VZV, EBV, CMV, HHV-6 (Type A), H
A method for differential detection of HV-6 (type B) or HHV-7. 13. An oligonucleotide having a nucleic acid sequence selected from sequence group A (SEQ ID NOS: 1 to 8) and sequence group B (SEQ ID NO: 9) using at least two kinds of human herpesvirus nucleic acids in a sample as amplification primers in advance. ~
After performing an amplification reaction by mixing and using an oligonucleotide having a nucleic acid sequence selected from 14), as a specific detection probe, a sequence group A ′ (SEQ ID NOS: 21 to 26) and a sequence group B ′ (SEQ ID NO: 27) are used. To 30), HSV-1, HSV-2, VZV, EBV or C in a sample characterized by differential detection using an oligonucleotide having a nucleic acid sequence selected from
MV differential detection method. 14. An oligonucleotide having a nucleic acid sequence selected from sequence group A (SEQ ID NOS: 1 to 8) and sequence group C (SEQ ID NO: 15) using at least two kinds of human herpesvirus nucleic acids in a sample as amplification primers in advance.
To 20), an amplification reaction is performed using a mixture of oligonucleotides having a nucleic acid sequence selected from the group consisting of sequence group A ′ (SEQ ID NOS: 21 to 26) and sequence group C ′ (SEQ ID NO :). 31-36), and HSV-1, HSV-2, VZV, HHV-6 in a sample, characterized by differential detection using an oligonucleotide having a nucleic acid sequence selected from the above or a complementary nucleic acid sequence thereof.
(Type A), HHV-6 (Type B) or HHV-
7. Discrimination detection method of 7. 15. An oligonucleotide having a nucleic acid sequence selected from sequence group B (SEQ ID NOS: 9 to 14) and sequence group C (SEQ ID NO: 1) using at least two kinds of human herpesvirus nucleic acids in a sample as amplification primers in advance.
5 to 20), an amplification reaction is carried out using a mixture of oligonucleotides having a nucleic acid sequence selected from the group consisting of sequence group B '(SEQ ID NOS: 27 to 30).
And human herpesvirus, EBV, CMV in a sample characterized by differential detection using an oligonucleotide having a nucleic acid sequence selected from Sequence Group C ′ (SEQ ID NOS: 31 to 36) or a complementary nucleic acid sequence thereof , HH
V-6 (Type A), HHV-6 (Type B) or H
HV-7 differential detection method. 16. An oligonucleotide having a nucleic acid sequence selected from sequence group A (SEQ ID NOS: 1 to 8) and sequence group B (SEQ ID NO: 9) using at least two human herpesvirus nucleic acids in a sample as amplification primers in advance. ~ 1
4) An oligonucleotide having a nucleic acid sequence selected from 4) and an oligonucleotide having a nucleic acid sequence selected from sequence group C (SEQ ID NOS: 15 to 20) are mixed and used to perform an amplification reaction, and then used as a specific detection probe. , Sequence group A '(SEQ ID NOS: 21-26), sequence group B' (SEQ ID NOS: 27-30) and sequence group C '(SEQ ID NOS: 31-36)
HSV-1, HSV-2, VZ in a sample characterized by differential detection using an oligonucleotide having a nucleic acid sequence selected from the following or a complementary nucleic acid sequence thereof
V, EBV, CMV, HHV-6 (Type A), HHV
-6 (Type B) or HHV-7 differential detection method. 17. The method for differential detection of human herpesvirus according to claim 1, wherein the detection method is a method by sandwich hybridization. 18. The method for differential detection of human herpesvirus according to claim 1, wherein the detection method is a method by Southern blot hybridization. 19. The method for differential detection of human herpesvirus according to claim 1, wherein the detection method is a detection method by dot blot hybridization. (1) At least two selected from the group consisting of sequence group A (sequence numbers 1 to 8), sequence group B (sequence numbers 9 to 14) and sequence group C (sequence numbers 15 to 20) At least four kinds of amplification primers containing an oligonucleotide having a homology of 70% or more with a nucleic acid sequence selected from a sequence group, (2) amplification enzyme, (3) amplification substrate, (4) amplification buffer, (5) Sequence group A ′ (SEQ ID NOs: 21 to 26),
Sequence group B ′ (SEQ ID NOS: 27 to 30) and sequence group C ′
Specific detection which is an oligonucleotide having 70% or more homology with a nucleic acid sequence selected from at least two sequence groups selected from the group consisting of (SEQ ID NOS: 31 to 36), or an oligonucleotide having a complementary nucleic acid sequence thereof Probe, (6) Detection Buffer, (7) Detection Wash Solution, (8)
A reagent for differential detection of human herpesvirus, which comprises a detection substrate. 21. (2) The amplification enzyme is a nucleic acid synthase selected from the group consisting of DNA polymerase, RNA polymerase and reverse transcriptase, and (3) the amplification substrate is dN.
21. The reagent for differential detection of human herpesvirus according to claim 20, which is TP and / or rNTP. 22. (5) The specific detection probe as a label,
3. A radioactive substance, an enzyme, a fluorescent substance, an antigen, a hapten, an enzyme substrate or an insoluble carrier.
The reagent for differential detection of human herpesvirus according to 0. 23. (5) The specific detection probe as a label,
21. The reagent for the differential detection of human herpesvirus according to claim 20, which has alkaline phosphatase and (8) the detection substrate is a luminescent 1,2-dioxetane compound.