JPH05276998A - Method for detecting virus of aujeszki disease virus - Google Patents

Abstract

PURPOSE:To obtain a new oligonucleotide pair useful as a primer for detecting Aujeszki disease virus(ADV) in a specimen. CONSTITUTION:The objective method for detecting ADV is to amplify a genetic region specific for the ADV with at least one of oligonucleotide pairs of formulas I to VII as a primer according to a polymerase chain reactional(PCR) method and thereby enable the detection of the amplified genetic region by hybridization. Attention is paid to thymidine kinase gene and an initial expression protein gene and a primer DNA pair for the PCR is synthesized by a DNA synthesizer (model 391, manufactured by Applied Biosystems, Inc.) and purified by an 18C reversed phase column.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention relates to Aujeszky's disease (hereinafter referred to as
Method for specifically detecting ADV using a specific gene region of Aujeszky's disease virus (ADV), which is the causative virus of this disease), specific DNA used therefor, and AD
It relates to a detection kit for V detection.

[0002]

ADV belongs to the herpesviridae family and is a pathogenic virus for domestic animals such as pigs, cattle, sheep and goats, dogs and cats, as well as experimental animals such as rats and mice, and many wild animals. .. When animals other than pigs are infected, most animals die due to acute encephalomyelitis, but the incidence is low in animals other than pigs, and the damage of this disease is concentrated in pigs. When pigs are infected with ADV, a high rate of death occurs in young pigs, and about half of pregnant pigs abort. In fattening pigs and adult pigs, it often results in subclinical infection, but it is recently becoming clear that it causes viral pneumonia or worsens the disease due to mixed infection with pathogenic bacteria such as Pasteurella. .. In addition, since subclinical infected pigs and overdeveloped pigs continue to carry the virus in their lifespans, they become a new source of infection for this disease. This disease occurs in most of the countries where pig farming is active, such as European countries, the United States, Latin America, North Africa, Iran, Malaysia, Thailand and Taiwan. It first occurred in Yamagata in Japan in 1981, and since then, it has become localized habitually in the Kanto region and a part of the surrounding region, but in recent years the region has shown a marked expansion trend, and it has also been reported in the Tohoku and Kyushu regions. Came to be. Called Aujeszky's disease in Japan and Europe, but pseudorabies in the United States P
It is called seudorabies (PR). Although the transmission route of this disease is often unknown, it is thought that the transmission to remote areas depends on the movement of latently infected pigs. When this disease occurs, isolation of the place of occurrence and taking preventive measures in the surrounding area, etc.
Prompt action is required to prevent the spread of infection,
For that purpose, it is necessary to detect and eliminate this disease by a quick and reliable diagnosis.

For diagnosing this disease, cell culture method, serological diagnosis method, histopathological diagnosis method and the like are used. Serological diagnosis is performed by antibody detection methods such as neutralization test, ELISA and latex test. The antibody detection method has high specificity and is useful for detecting latently infected pigs, but it has a problem of lack of reliability because it does not directly indicate the presence of the virus. Therefore, the most reliable method of diagnosing ADV infection at present is isolation of virus by cell culture method. However, isolation and confirmation require skill and a long time, and sampling after onset is required. There is a problem that the result is affected by the storage condition of the sample up to. In order to solve these problems, it is possible to apply the gene diagnosis technology which has been showing rapid progress in recent years. However, conventional methods such as Southern hybridization have poor sensitivity and are not practical. Because ADV
In swine (animals) infected with A. cinerea, live virus is detected in various tissues such as nervous tissue and various excrements such as secretions and nasal discharge. Is so low that the gene cannot be detected by conventional methods.

In recent years, the PCR method has been used as a highly sensitive gene diagnostic method.
(Polymerase chain reaction method) was developed (Saiki et al., Science, Volume 230, 1350-13).
54 (1985)). According to this method, even one virus can theoretically be detected. Further, since it is completed in a short time without requiring skill, it is far easier than virus isolation by the cell culture method. There have been some reports on the detection of ADV by the PCR method (Maes et al., Veterinary Microbiology, Vol. 24,
281-295; others), all have problems in reproducibility, and some are not detected in field virus strains in the field, so they are difficult to use for actual diagnosis. This is considered to be mainly due to the following two reasons. (1) ADV has an extremely high GC content of about 80%, and therefore has a strong double-stranded DNA binding strength and is difficult to amplify under normal PCR reaction conditions. (2) In most of the previous reports, the viral coat protein gene group is targeted by the PCR method, but these genes are easily mutated and there is a high possibility that the nucleotide sequences differ depending on the strain.

[0005]

DISCLOSURE OF THE INVENTION An object of the present invention is to provide a method for specifically detecting ADV using a specific gene region of Aujeszky's disease virus (ADV), which is the causative virus of Aujeszky's disease. DNA and A
It is to provide a detection kit for DV detection.

[0006]

The present invention provides an oligonucleotide pair used as a primer for selectively detecting Aujeszky's disease virus (ADV) in a sample, which comprises a thymidine kinase (TK) gene of ADV. Alternatively, it provides a synthetic oligonucleotide pair selected from the group consisting of the sequence pairs (I) to (VII) shown below, which targets the early expressed protein (IE) gene. 5 ′ TCTAC GCCAT GCTGG TCAAC (I) a SEQ ID NO: 1 5 ′ AGGTC GACGG TGGAG ACGC (I) b SEQ ID NO: 2 5 ′ CCCGT CTTCA TCCCG GAGAT (II) a SEQ ID NO: 3 5 ′ CACGC TGCCG GTGAT GAAGG (II) b sequence SEQ ID NO: 4 5'GAGGC CATGT CCTGG CTGC (III) a SEQ ID NO: 5 5'GTAGG CCCGG CGGAG GCT (III) b SEQ ID NO: 6 5'GCCGC CTGGA TGCGG CAG (IV) a SEQ ID NO: 7 5'CCAGT TCCCG GCCCA GGC (IV) b SEQ ID NO: 8 5 ′ GCCCA GGGGG TGCTG CTG (V) a SEQ ID NO: 9 5 ′ CCAGC GCACG GCGCA CTG (V) b SEQ ID NO: 10 5 ′ TTCGC CCGCG TGGAG GCC (VI) a SEQ ID NO: 11 5 ′ GCGCC GAGCC CCCAG CG ( VI) b SEQ ID NO: 12 5'TCTAC GCCAT GCTGG TCAAC (VII) a SEQ ID NO: 1 5'TTGGC CACGA GCGCG TCCAT (VII) b SEQ ID NO: 13

The present invention also relates to an oligonucleotide pair used as a primer for selectively detecting ADV in a sample, which comprises the above formulas (I), (II), (III), (IV),
(V), (VI) or (VII) of the pair consisting of at least 10 consecutive nucleotide sequences in one row (a) of the pair and at least 10 consecutive rows in the other row (b) The present invention provides a synthetic oligonucleotide pair consisting of an oligonucleotide consisting of a base sequence. The present invention further uses at least one of the above-mentioned oligonucleotide pairs as a primer to bind an ADV-specific gene region to P
The present invention provides a method for selectively detecting ADV in a sample, which comprises amplifying by a CR method and detecting the amplified gene region by hybridization. The present invention also includes the above oligonucleotide pair,
Or A in a sample, which contains the above-mentioned oligonucleotide pair and a DNA probe, or contains the above-mentioned oligonucleotide pair, a DNA probe and a DNA replication enzyme.
The present invention provides a detection kit for selectively detecting DV.

The present invention will be described in detail below. In order to achieve the above-mentioned subject, the inventors proceeded with the research in the following procedure. (1) Of the ADV genes, thymidine kinase (TK)
Genes and early expressed proteins (Immediately Expresse
Focusing on the d Protein (IE) gene, we estimated highly conserved regions between strains, and used this as a primer DNA for PCR.
Combine pairs. (2) Search for conditions under which the PCR reaction proceeds with high sensitivity and reproducibility even in DNA with a high GC content. (3) Further, the method (procedure) obtained in (1) and (2) is tested with various herpesvirus strains closely related to ADV strains.

The present invention will be described more specifically in the following order. TK has the activity of adding phosphate to thymidine, which is one of the constituents of the gene, and the gene is
Herpes viruses have in common. It is not always essential for growth, but is said to be strongly involved in pathogenicity. IE also has in common with herpesviruses, and there are two genes, one in each of the two repeat sequences on the 3'side of the genome. It is presumed that IE is expressed early after the herpes virus infection and before the initiation of DNA transcription, and functions as an activator of transcription.
It is an essential factor for virus growth. However, even though the herpesviruses exist in common, their nucleotide sequences are different and it is possible to specifically detect individual virus species. For example, both TK and IE genes have ADV
And the herpes simplex virus (HSV) have already been determined for their nucleotide sequences (Prieto et al., J. Gen. Virol., 72: 1435-1439 (1991), Cheung.
Et al., Nucleic Acid Research, Volume 17, 4637-4.
646 (1989)), it has been elucidated that the sequence is divided into several common regions and species-specific regions.

The inventors of the present invention compared these sequences and estimated a region that seems to be highly conserved. A candidate primer group was selected and synthesized in these regions, subjected to PCR, and screened for pairs in which a PCR product was detected in 20 or more types of ADV field strains. As a result, for example, a primer DNA pair represented by the above formula (I), (II), (III), (IV), (V), (VI) or (VII) is suitable for the above purpose. The inventors have found that and completed the present invention. Using at least one of the above oligonucleotide pairs as a primer, a gene region specific for ADV
ADV in the sample can be selectively detected by amplifying by the R method and detecting the amplified gene region by agarose gel electrophoresis or dot blot hybridization. As a DNA probe for detecting the amplified gene region, any DNA sequence complementary to part or all of the amplified gene region can be used, but preferably at least 1
It is a DNA sequence of 0 bases, more preferably 15 bases or more. As specific examples thereof, the following DNA sequences and complementary sequences thereof, and at least one contiguous sequence of these sequences:
A sequence including a 0 base sequence can be mentioned.

(A) AGTCG CGCGT GGTCT GGTCG SEQ ID NO: 14 (B) CCCAC GCGTG CACCT CGAGC SEQ ID NO: 15 (C) GGCGT GCAGA GGCGG TGGGC G SEQ ID NO: 16 The present inventors further investigated the reaction conditions of PCR. .. In PCR, heat denaturation (Denaturing) in the presence of a thermostable DNA replication enzyme → association of a primer with a denatured DNA
(annealing) → 3 steps of extension reaction (Extension) are repeated. The biggest problem is Denaturing
Is the temperature of. Usually done at 95 ° C, but ADV D
In the case of NA, denaturation does not occur well at this temperature, and a higher temperature (98 ° C or higher) is required for sufficient denaturation. However, when a DNA replication enzyme conventionally used in PCR (for example, Ampitaq manufactured by Cetus) is used at such a high temperature, the activity is lost and the reaction does not proceed. Therefore, in order to amplify ADV DNA by the PCR method, it is necessary to use an enzyme having higher thermostability. Therefore, as a result of examining several enzymes, Ve
It has been found that an enzyme such as nt polymerase (manufactured by NEB) or Pfu polymerase (manufactured by Stratagene) that can remain for a long time even at 100 ° C. is suitable for this.

[0012] ADV DNA can be prepared according to a known method from a culture cell line infected with ADV, an organ of an infected animal, excrement or the like. The PCR method may be performed by preparing a gene amplification reaction solution containing a DNA replication enzyme such as Vent polymerase or Pfu polymerase and using an automatic gene amplification device (Perkin Elmer Cetus). For detection of the amplified DNA, for example, agarose gel electrophoresis or dot blot hybridization can be used. In dot blot hybridization, for example, when a primer pair represented by (I) is used, an ADV-specific sequence in the amplified gene region, for example, a DNA sequence represented by formula (A) is used as a probe. , The amplified gene can be detected. Labeling of the primer for detection can be carried out by a known method such as radioisotope or enzyme. Regarding the detection sensitivity, for example, by serially diluting a virus solution prepared from a sample, extracting viral DNA, performing PCR, and comparing the results obtained using various techniques with the results obtained by the cell culture method, , Can be determined. In fact, it was possible to detect a virus using the method according to the present invention even in a sample determined to have a virus number of 1 or less by the cell culture method.

[0013]

【Example】

Example 1 Amplification and detection of ADV TK gene by PCR method 1-1. Synthesis and purification of DNA for primers and probes The oligonucleotides represented by the formulas (I), (VI), (A), (B) and (C) were transferred to a DNA synthesizer (Applied Biosystems, Model 391). Used to purify using a C18 reverse phase column. 1-2. Preparation of various ADV strains and closely related herpesvirus liquids and measurement of virus number (titer) by the culture cell method After inoculating various virus into susceptible cell subculture cells such as CPK and culturing at 37 ° C. for 24 to 36 hours The culture supernatant was collected and centrifuged at about 1500 xg for 30 minutes, and the supernatant was used as a virus solution. Separate each virus solution at -80 ℃
Saved in. Viral titer measurement (titration)
Was carried out by the microplate method using 96-well microplate culture cells, and the virus titer was calculated by the Behrens-Kaerber method and expressed as TCID _50/50 μl. The number of virus particles (titer) was calculated from the virus titer obtained by titration as 1 TCID ₅₀ = 0.69 virus particles. That is, 10 ^5.0 TCID _50/50 μl
It is considered that 6.9 × 10 ⁴ active virus particles are present in 50 µl of the virus solution having a titer of.

1-3. Preparation of DNA from virus solution Add 0.1 to 1 ml of virus solution to Proteinase K (Merck
Company) at 100 μg / ml and treated at 70 ° C for 30 minutes, then add an equal volume of 1: 1 mixture of phenol / chloroform and stir well, and centrifuge at about 10,000 xg for 5 minutes. , 1/10 volume of 3M sodium acetate (p
H5.2), 2.5 volumes of ethanol was added and stirred,-
After standing at 70 ° C. for 15 minutes or more, centrifugation was performed at about 10,000 × g for 5 minutes to recover the precipitate. This was washed with 70% ethanol, dried, and dissolved in distilled water. 1-4. Amplification of target DNA by PCR method Take a certain amount of DNA solution prepared from virus solution, and use a reaction solution (30 μl) having the following composition, and an automatic gene amplification device (Perkin Elmer Cetus, GeneAmp System 9600)
Was used to perform PCR.

[0015]

[Table 1] Table 1 Composition of PCR reaction solution ────────────────────────── Tris-HCl, pH 8.8 20 mM Potassium chloride 10 mM Ammonium sulfate 10 mM Sulfuric acid Magnesium 2 mM Sodium edetate 0.5 mM Bovine serum albumin 100 μg / ml Triton X-100 0.1% Substrate (dATP, dGTP, dCTP, dTTP) 100 μM each primer pair 0.4 μg Vent polymerase 0.4 unit each ──── ────────────────────── The composition shown in Table 1 was used as a detection kit. PC
The reaction conditions for R are 98 ° C., 30 seconds → 55 ° C., 1 minute → 76
One cycle was performed at 1 ° C. for 1 minute, and this was repeated 35 times. After the reaction, the reaction solution was directly subjected to electrophoresis or dot blot hybridization.

1-5. Detection of target DNA amplified by PCR method A. Detection by Electrophoresis After the completion of the PCR reaction, 5 to 20 μl of the reaction solution was taken and subjected to electrophoresis using 3% Nusieve GTG agarose (FMC) gel to detect the amplified DNA fragment by the ethidium bromide staining method. B. Detection by Dot Blot Hybridization The oligonucleotide for probe (purified) represented by the formula (A) was labeled with ³² P at the 5'end using a megalabel kit (Takara Shuzo). The sequence of formula (A) is present in the wild strain, but is missing in the commercial vaccine strain which is a vaccine strain. Hybridization was performed as follows. PCR was carried out on the ADV YS-81 strain and the commercial vaccine 1 strain DNA using the primer pair (I),
The reaction solution was aliquoted and made up to 100 μl with 20 × SSC (0.3M trisodium citrate, 3M sodium chloride).
A nylon membrane (Nytran 13 N, manufactured by Schleicher & Schuell) equilibrated with 20 × SSC was set on a dot blot apparatus (DP-96, manufactured by Toyo Advantech Co., Ltd.), a sample reaction solution was applied, and 100 μl of 0.
Add 2N NaOH, let stand for 3 minutes, and after suction, add 100 μl of 20
Wash with xSSC. The filter was removed from the device, air-dried and then irradiated with ultraviolet rays (120 mJ / cm ² ) to fix the test DNA. Pre-hybridization buffer (5x Denhardt's solution, 5xSSC, 0.1% SD)
S, 50 μg / ml yeast tRNA), shaken at 60 ° C. for 1 hour, and a fixed amount of ³² P-labeled probe was added.
Shake at 0 ° C. for 2 hours. Then, the filter was washed with a washing solution (5 × SSC, 0.2% SDS) at 60 ° C. for 10 minutes (three times in total), air-dried, and then autoradiography was performed to determine spots produced. The results are shown in Table 2 to Table 4.
Shown in.

[0017]

[Table 2] Table 2 Assay for detection sensitivity of PCR method <Comparison with titer by cultured cell method> ──────────────────────────── ───── Titer ^(*) Primer pair (I) Primer pair (VI) ──────────────────────────────── ─ 4.4 x 10 ³ ＋ ＋ 4.4 x 10 ² ＋ ＋ 4.4 x 10 ¹ ＋ ＋ 4.4 x 10 ⁰ ＋ ＋ 4.4 x 10 ^-1 ＋ ＋ 4.4 x 10 ^-2 − − ──────────── ───────────────────── For ADV, YS-81 strain was used. The detection was performed by agarose electrophoresis. + Is detected and-is not detected (the same applies hereinafter). * 4.4 × 10 ³ (pieces / 50 μl) is the value obtained by serially diluting 10 times.

[0018]

[Table 3] Table 3 Detection results of various ADVs (including vaccine strains) and related viruses ─────────────────────────────── ── Virus strain Primer pair (I) Primer pair (VI) ──────────────────────────────── ADV YS-81 + + ZK + + ZK (TK deficiency) + + ZH + + ZH (TK deficiency) + + Tochi 85 + + Tochi 86 + + Tochi 88-1 + + Tochi 88-2 + + Tochi 89-1 + + Tochi 89 -2 + + Tochi 89-3 + + Tochi 89-4 + + Tochi 90 + + Tochi 91 + + Group 88-1 + + Group 88-2 + + Group 88-3 + + Group 88-4 + + Group 89 -1 + + group 89-2 + + group 89-3 + + group 89-4 + + group 90-1 + + group 90-2 + + group 90-3 + + group 90-4 + + Group 91-1 + + group 91-2 + + group 91-3 + + group 91-4 + + commercial vaccine 1 + (*) + commercial vaccine 2 ++ commercial vaccine 3 ++ related virus ERV − − HSV -1 --- HSV-2--IBRV ------ ────────────────────────────

As ADV, YS-81 strain was used. The detection was performed by agarose electrophoresis.・ + Is detected and-is not detected.・ *: Fragment of about 130 base pairs, unmarked is about 280 base pairs ・ ERV: Equine rhinitis virus ・ HSV: Herpes simplex virus ・ IBRV: Bovine infectious rhinotracheitis virus ・ Commercial vaccine 1, commercial vaccine 2, commercial vaccine 3 are vaccine strains, respectively. Of these, the commercial vaccine 1
Is an attenuated virus produced by gene recombination,
The region amplified by the primer pair (I) lacks approximately 150 base pairs. Therefore, in commercial vaccine 1, 280-15
A DNA fragment of 0, that is, about 130 bases is detected.・ The titer of all virus strains is measured, and the titer of each virus strain used in Table 3 is 10
⁴ cells / μl or more.

[0020]

[Table 4] Table 4 Detection results using hybridization method ──────────────────────────────── Virus strain electrophoresis Hybridization (size of DNA fragment) ──────────────────────────────── Probe (A) YS-81 ++ (About 280 base pairs) Commercial vaccine 1 +-(About 130 base pairs) ERV --- HSV-1--HSV-2--IBRV --- Probe (B) YS-81 ++ (About 280 base pairs) Commercial vaccine 1 + + (about 130 base pairs) ERV − − HSV-1 − − HSV-2- − IBRV − − probe (C) YS-81 + + (about 200 base pairs) Commercial vaccine 1 ++ (about 200 base pairs) ) ERV − − HSV-1 − − HSV-2 − − IBR − − ──────────────────────────────── PCR uses the sequence (A) or (B) as a probe. Was carried out using the primer pair (I) and the primer pair (VI) when the sequence (C) was used as a probe. As shown in this, it was found that the DNA fragment amplified by the primer pair was detected when the probe sequence was contained, and was not detected when the DNA sequence was not contained as in the commercial vaccine 1. This result means that highly specific determination can be performed by detecting the PCR reaction product by the hybridization method.

Example 2. Detection of ADV from nasal discharge and organs of experimentally infected pigs 2-1. Viral fluid and virus DN from organs and nasal discharge
Preparation of A Each organ of a pig experimentally infected with the ADV YS81 strain was removed (in the case of nasal discharge, wiped with a cotton swab), and a 9-fold volume of ME was used.
The suspension was suspended in M medium and made into an emulsion using a Dounce glass homogenizer. After centrifugation at 1,500 xg for 30 minutes,
The supernatant was taken and used as a virus solution. For long-term storage of the virus solution, fetal calf serum was added to 5% and stored at -80 ° C. The titer was measured and the DNA was prepared from the virus solution according to the method described in Example 1. 2-2. Amplification and detection by PCR method According to the method described in Example 1, the titer was measured from the virus extract, and the extracted DNA was subjected to PCR using the detection kit described in Example 1, and the amplified DNA fragment was electrophoresed. Detected. The results are shown in Table 5.

[0022]

[Table 5] Table 5 ─────────────────────────────── Specimens (organs, nasal discharge) Titer PCR method (electrophoresis) (TCID ₅₀ / 50μl) ─────────────────────────────── cerebrum 10 ^1.75 + cerebellum 10 ^2.0 + tonsils 10 ^4.5 ＋ trigeminal Nerve 10 ^3.5 + Lung 10 ^4.25 + Nasal discharge 10 ^5.0 + Uninfected pig cerebrum 0 − Uninfected pig nasal discharge 0 − ─────────────────────────── ─────

[0023]

INDUSTRIAL APPLICABILITY In the present invention, a primer pair selected from a gene region specific to ADV DNA is used, and PCR conditions are devised so as to be compatible with amplification of ADV DNA. It can be detected with high sensitivity. In addition, by forming a kit, ADV can be easily detected in a short time, and highly reliable results can be obtained. Sequence Listing SEQ ID NO: 1 Sequence Length: 20 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid (Synthetic DNA for PCR) Sequence TCTACGCCAT GCTGGTCAAC SEQ ID NO: 2 Sequence Length: 19 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acids (synthetic DNA for PCR) Sequence AGGTCGACGG TGGAGACGC SEQ ID NO: 3 Sequence length: 20 Sequence Type: Nucleic acid Number of strands: 1 Strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA for PCR) Sequence CCCGTCTTCA TCCCGGAGAT SEQ ID NO: 4 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA for PCR) Sequence CACGCTGCCG GTGATGAAGG SEQ ID NO: 5 Sequence length: 19 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Type of sequence: other nucleic acid (PC Synthetic DNA for R) Sequence GAGGCCATGT CCTGGCTGC SEQ ID NO: 6 Sequence length: 18 Sequence type: Nucleic acid Number of strands: 1 strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA for PCR) Sequence GTAGGCCCGG CGGAGGCT SEQ ID NO: 7 Sequence length: 18 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA for PCR) Sequence GCCGCCTGGA TGCGGCAG SEQ ID NO: 8 Sequence Length: 18 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA for PCR) Sequence CCAGTTCCCG GCCCAGGC SEQ ID NO: 9 Sequence length: 18 Sequence type : Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA for PCR) Sequence GCCCAGGGGG TGCTGCTG SEQ ID NO: 10 Sequence length: 18 Sequence type: Nucleic acid Number of strands: 1 Main chain Topology: Linear Sequence type: Other nucleic acid (synthetic DNA for PCR) Sequence CCAGCGCACG GCGCACTG SEQ ID NO: 11 Sequence length: 18 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (Synthetic DNA for PCR) Sequence TTCGCCCGCG TGGAGGCC SEQ ID NO: 12 Sequence length: 17 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (Synthetic DNA for PCR) Sequence GCGCCGAGCC CCCAGCG SEQ ID NO: 13 Sequence length: 20 Sequence type: Nucleic acid Number of strands: 1 strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA for PCR) Sequence TTGGCCACGA GCGCGTCCAT SEQ ID NO: 14 Sequence Length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (hybridization probe DNA) Sequence AGTCGCGCGT GGTCTGGTCG SEQ ID NO: 15 Sequence length : 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (hybridization probe DNA) Sequence CCCACGCGTG CACCTCGAGC SEQ ID NO: 16 Sequence length: 21 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (hybridization probe DNA) Sequence GGCGTGCAGA GGCGGTGGGC G

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Reference number within the agency FI technical indication location // (C12Q 1/70 C12R 1:92) (72) Inventor Mika 1 Ebina-shi, Kanagawa Prefecture Central −10-1-403 (72) Shizuo Mise 4-25-8 Morinosato, Atsugi City, Kanagawa Prefecture (72) Isao Shibata 2-14-17 Osakidai, Sakura City, Chiba Prefecture

Claims (5)

[Claims] 1. Aujeszky's disease virus (AD) in a sample
An oligonucleotide pair used as a primer for selectively detecting V), which comprises a thymidine kinase (TK) gene of ADV or an early expressed protein (I).
E) Sequence pairs (I) to
A synthetic oligonucleotide pair selected from the group consisting of (VII). a 5'TCTAC GCCAT GCTGG TCAAC b 5'AGGTC GACGG TGGAG ACGC (I) a 5'CCCGT CTTCA TCCCG GAGAT b 5'CACGC TGCCG GTGAT GAAGG (II) a 5'GAGGC CATGT CCTGG CTGC b 5'GTAGG CCCGG CGGAG GCT (III ) a 5 'GCCGC CTGGA TGCGG CAG b 5' CCAGT TCCCG GCCCA GGC (IV) a 5 'GCCCA GGGGG TGCTG CTG b 5' CCAGC GCACG GCGCA CTG (V) a 5 'TTCGC CCGCG TGGAG GCC b 5' GCGCC GAGCC CCCAG CG ( VI) a 5'TCTAC GCCAT GCTGG TCAAC b 5'TTGGC CACGA GCGCG TCCAT (VII) 2. Aujeszky's disease virus (AD) in a sample
A pair of oligonucleotides for use as a primer for selectively detecting V), the formula of claim 1
Consists of at least 10 consecutive nucleotide sequences in one row (a) of the pair represented by (I), (II), (III), (IV), (V), (VI) or (VII) A synthetic oligonucleotide pair consisting of an oligonucleotide and an oligonucleotide consisting of at least 10 consecutive base sequences in the other row (b). 3. An ADV-specific gene region is amplified by PCR using at least one pair of the oligonucleotide pairs according to claim 1 or 2 as a primer, and the amplified gene region is detected by hybridization. A method for selectively detecting Aujeszky's disease virus (ADV) in a sample, which comprises: 4. A detection kit for selectively detecting Aujeszky's disease virus (ADV) in a sample, which comprises the oligonucleotide pair according to claim 1 or 2 and a DNA replication enzyme. 5. An Aujeszky's disease virus (AD) in a sample, which comprises the oligonucleotide pair according to claim 1 or 2, a DNA replication enzyme and a DNA probe.
A detection kit for selectively detecting V).