JP2006223180A - Method for detecting herpes virus gene by using multiplex pcr - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a primer set for multiplex PCR capable of simultaneously and specifically detecting herpes virus (KSHV, EBV and HCMV) gene in high sensitivity and to provide a method for detecting herpes virus gene by using the primer set. <P>SOLUTION: The kit is used for detecting presence or expression of two or more kinds of herpes viruses in a sample and the kit comprises two or more primer sets selected from a group consisting of a primer set for detection of at least one Kaposi's sarcoma-related herpes virus (KSHV), a primer set for detection of at least one Epstein-Barr Virus (EBV) and a primer set for detection of at least one cytomegalovirus (CMV). <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a primer set for multiplex PCR for detecting a herpesvirus (KSHV, EBV, HCMV) gene and a method for detecting a herpesvirus gene using the primer set.

  Kaposi's Sarcoma-associated Herpesvirus (KSHV) is the causative virus of Kaposi's sarcoma. When patients using immunosuppressants are infected with KSHV, it is known to develop Kaposi's sarcoma and phosphorus tumor, but so far there is no effective drug therapy. Therefore, this virus is becoming a serious problem not only for opportunistic infections in AIDS patients but also for organ transplantation surgery using immunosuppressive drugs.

  Epstein-Barr virus (EBV) is already infected in 95% of Japanese and can cause carcinogenesis by latent infection. In addition, HCMV (human cytomegalovirus) infection can cause encephalopathy, but the presence of drug-resistant strains has become a serious problem in medical practice.

  Currently, in the clinical setting, methods such as antibody reaction using a patient's serum (Western blot) and immunohistological staining that stains pathological sections with antiviral protein antibodies are used as diagnostic methods for herpes virus infection. . However, these methods are complicated in operation and require techniques and experience for analysis, so there is a problem in that they are inaccurate and prone to false positives.

  In addition, as a method for detecting herpesvirus using PCR, a method for amplifying and detecting only a target herpesvirus gene using primers specific to each virus gene has been reported. Since the amplification reaction had to be performed in separate tubes, the operation was complicated and difficult to implement with a small amount of sample. Foldes-Papp et al. (Non-Patent Document 1) describes a method for detecting HSV-1, HSV-2, VZV, (EBV, CMV and HHV-6 using a DNA array, but nothing about KSHV. Not mentioned.

Pozo et al. (Non-Patent Document 4) describe a method for detecting six types of viruses of EBV, HCMV, HHV6-A, HHV6-B, HHV7, and HHV8 / KSHV using multiplex PCR. So-called nested PCR is used. Although this method has the advantage of high sensitivity, it takes time and labor to perform PCR twice, and the amount of virus DNA is biased by the first PCR, that is, the amount of virus is quantitatively detected. There was a drawback that could not be done.
Foldes-Papp, et al., Mol. Diagn. (2004) 8, 1-9 Druce et al., J. Clin. Microbiol. (2002) 40, 1728-1732. Markoulatos et al., J. Clin. Microbiol. (2001) 39, 4426-4432. Pozo et al., J. Virol.Methods. (1999) 79, 9-19.

  Therefore, a simple method that can simultaneously detect KSHV, EBV, and HCMV genes has been desired.

  The present invention provides a primer set for multiplex PCR capable of simultaneously and specifically detecting a herpesvirus (KSHV, EBV, HCMV) gene and a method for detecting a herpesvirus gene using the same. Objective.

The present invention is a kit for detecting the presence or expression of two or more herpesviruses in a sample, the following AC:
(A) Primer set KSHV1, which includes two oligonucleotides each consisting of a base sequence represented by SEQ ID NO: 1 and SEQ ID NO: 2, and two oligonucleotides each comprising a base sequence represented by SEQ ID NO: 3 and SEQ ID NO: 4 Primer set KSHV2, containing
At least one primer set for detecting Kaposi's sarcoma-associated herpesvirus (KSHV) selected from the group consisting of:
(B) Primer set EBV1, comprising two oligonucleotides each having the base sequence represented by SEQ ID NO: 5 and SEQ ID NO: 6
Primer set EBV2, which includes two oligonucleotides each consisting of a base sequence represented by SEQ ID NO: 7 and SEQ ID NO: 8, and a primer which includes two oligonucleotides each composed of a base sequence represented by SEQ ID NO: 9 and SEQ ID NO: 10 Set EBV3
At least one Epstein Barr virus (EBV) detection primer set selected from the group consisting of:
(C) Primer set CMV1, comprising two oligonucleotides each having the nucleotide sequence represented by SEQ ID NO: 11 and SEQ ID NO: 12
Primer set CMV2, which includes two oligonucleotides each consisting of a base sequence represented by SEQ ID NO: 13 and SEQ ID NO: 14, and a primer which includes two oligonucleotides each composed of a base sequence represented by SEQ ID NO: 15 and SEQ ID NO: 16 Set CMV3
At least one cytomegalovirus (CMV) detection primer set selected from the group consisting of:
A kit comprising two or more primer sets selected from the group consisting of:

  The target virus and the primer sequences according to the present invention are shown in Table 1. The kit of the present invention is useful for easily and simultaneously detecting two or three kinds of KSHV, EBV and CMV in a sample.

  As used herein, “consisting of” means that the primer included in the kit of the present invention is sufficient if it has the sequence specified by the SEQ ID NO to such an extent that the effects of the present invention are exhibited. , Does not imply that no additional sequences are included. A person skilled in the art can add any amount of sequence to the 5 ′ side and 3 ′ side in order to achieve a desired effect as a primer, that is, to be able to specifically amplify the target. Of course you can understand.

  Preferably, the kit of the present invention comprises a primer set KSHV1 comprising two oligonucleotides each consisting of a nucleotide sequence represented by SEQ ID NO: 1 and SEQ ID NO: 2, respectively, from a nucleotide sequence represented by SEQ ID NO: 5 and SEQ ID NO: 6, respectively. A primer set EBV1 including two oligonucleotides, and a primer set CMV1 including two oligonucleotides each having a base sequence represented by SEQ ID NO: 11 and SEQ ID NO: 12, respectively.

  Also preferably, the kit of the present invention is a primer set KSHV1 comprising two oligonucleotides each consisting of a nucleotide sequence represented by SEQ ID NO: 1 and SEQ ID NO: 2, respectively, and a nucleotide sequence represented by SEQ ID NO: 3 and SEQ ID NO: 4, respectively. Primer set KSHV2 comprising two oligonucleotides consisting of: primer set EBV2 comprising two oligonucleotides consisting of base sequences represented by SEQ ID NO: 7 and SEQ ID NO: 8, respectively, and represented by SEQ ID NO: 9 and SEQ ID NO: 10, respectively Primer set EBV3 containing two oligonucleotides consisting of base sequences, primer set CMV2 containing two oligonucleotides consisting of base sequences represented by SEQ ID NO: 13 and SEQ ID NO: 14 respectively, and SEQ ID NO: 15 and SEQ ID NO: 1 respectively In including a primer set CMV3 comprising two oligonucleotides consisting of the nucleotide sequence.

  In another aspect, a method for detecting the presence or expression of two or more herpesviruses in a sample is provided. This method uses a DNA obtained from the sample or a cDNA prepared by reverse transcription from mRNA obtained from the sample as a template, and performs a polymerase chain reaction using any of the kits of the present invention described above, and Detecting an amplified herpesvirus gene.

  In the method of the present invention, detection of the amplified herpesvirus gene is preferably performed using a DNA array or electrophoresis.

  In order to detect herpes virus using the kit of the present invention, for example, total DNA is prepared from a blood sample of a patient, and PCR is performed by mixing primers corresponding to a plurality of viruses to be tested. PCR products are detected by hybridization using a DNA array. Alternatively, after capillary electrophoresis, the PCR product is detected with high sensitivity using a dedicated reading analyzer. According to the method of the present invention, it becomes possible to simultaneously diagnose KSHV, EBV, and CMV infection from a small amount of blood sample in a single analysis, before administration of an immunosuppressant at the time of organ transplantation, and for patients with latent HIV infection. It is possible to predict opportunistic infections before the onset of AIDS.

  Each oligonucleotide of the primer set of the present invention can be chemically synthesized using, for example, a general-purpose DNA synthesizer (for example, Model 394 manufactured by Applied Biosystems). Oligonucleotides may be synthesized using any other method well known in the art.

  In order to detect the presence of a herpesvirus gene in a sample using the primer set of the present invention, DNA is extracted from the sample, and this is used as a template to perform a polymerase chain reaction using the primer set of the present invention. Amplify. As a sample, a blood sample, a tissue sample, a biopsy, a smear, etc. obtained from a subject suspected of being infected with herpes virus can be used.

  Methods for extracting DNA from a sample are well known in the art, and for example, extraction with phenol / chloroform can be used. Alternatively, a commercially available DNA extraction reagent may be used.

  Furthermore, since the primer set of the present invention targets genes expressed during the herpesvirus gene latency and lytic infection, detection of herpesvirus gene expression in the sample allows detection of latent infection and reactivation of the virus. Can be used to diagnose. In order to detect the expression of the herpesvirus gene, RNA is extracted from the sample, template cDNA is prepared, and this is used as a template to perform polymerase chain reaction using the primer set of the present invention and expressed in the sample. Amplifies the herpesvirus gene.

  Methods for extracting RNA from a sample are well known in the art, and for example, guanidine-isothiocyanate and phenol / chloroform extraction can be used. Alternatively, a commercially available RNA extraction reagent may be used.

  A method for synthesizing cDNA using RNA as a template is also well known in the art. A commercially available random primer can be used as a primer, and cDNA can be synthesized using reverse transcriptase in the presence of dNTPs. For example, SuperScript II (Invitrogen) can be used as the reverse transcriptase.

  Next, polymerase chain reaction (PCR) is performed using the DNA or cDNA thus prepared as a template and the primer set mixture of the present invention as a primer. As the polymerase, for example, Ex Taq (registered trademark) (Takara Bio) can be used. Methods for selecting appropriate PCR reaction conditions based on the Tm of the primer are well known in the art, eg, denaturation at 94 ° C. for 15 minutes, then 94 ° C. for 30 seconds, 60 ° C. for 1.5 minutes. It can be carried out by extending 35 minutes at 72 ° C for 1 minute and then at 72 ° C for 3 minutes. In order to facilitate the detection of PCR products, PCR can be performed using labeled nucleotides. As such a labeling substance, a radioisotope, a fluorescent substance, a chemiluminescent substance, biotin and the like well known in the art can be used.

  Detection of the amplification product can be performed by hybridization using a DNA array or electrophoresis.

  The DNA array for detection is a support on which a DNA fragment having a sequence capable of hybridizing with the sequence of the herpesvirus gene to be detected is immobilized. The support is not limited, and a nylon film, a nitrocellulose film, glass, a silicon chip, and the like can be used. The DNA fragment is immobilized by dropping an aqueous solution of the DNA fragment onto a support by an automatic machine or manually and drying it. Further, the DNA fragment may be fixed on the support by the UV cross-linking method.

  The DNA fragments immobilized on the detection array are selected so as to hybridize only with the corresponding type of herpesvirus gene and not with other types of herpesvirus genes. Most preferably, the detection array used in the present invention is a DNA fragment obtained by PCR amplification of viral DNA using a primer set used for detection. Hybridization and detection can be easily performed by methods well known in the art.

  When PCR amplification products are detected by electrophoresis, a primer set is designed so that the length of the amplification product varies depending on the type of virus to be detected, and the amplification product is subjected to conventional electrophoresis such as gel electrophoresis or capillary electrophoresis. Separate by electrophoresis. Based on the length of the obtained PCR amplification product, the type of virus present in the sample can be determined.

  In addition, two or more genes can be amplified for one target virus. When a DNA array is used for detection, DNA fragments corresponding to each amplified gene are immobilized on the array. When electrophoresis is used for detection, primers are designed so that PCR amplification products having different lengths can be obtained for each gene. This makes it easier to determine false positives, and virus detection can be performed more reliably.

  The kit of the present invention is characterized in that each herpesvirus gene can be specifically amplified when multiplex PCR is performed using a mixture of primer sets corresponding to two or three types of herpesviruses. . Therefore, it is possible to examine the presence or absence of 2-3 herpesviruses in one test tube at the same time, so compared with the prior art method in which PCR is performed in separate test tubes using type-specific primers. Thus, the assay is simple and has the advantages that the time, cost and sample required for the assay are small.

  EXAMPLES The present invention will be described below in more detail with reference to examples, but the present invention is not limited to these examples.

Primer design The gene sequence of the herpesvirus was obtained from a public database (GenBank accession numbers AF360120 and AF148805 for KSHV, V01555, AJ3157572 and M12553 for EBV, AY315197, M21295, AY442286 for HCMV).

  Based on the sequence of each herpesvirus gene, candidate primers were designed so that the gene expressed in the early stage of virus infection was amplified and the length of the amplified product was in the range of 300-600 bp. Next, sequences that had significant homology with other types of herpesviruses, sequences that existed in multiple copies in the human genome sequence, and sequences that could form secondary structures in the molecule were excluded. For the combination of several tens of kinds of primers selected in this way, a PCR reaction was actually performed with a sample mixed with human genomic genes and each viral DNA, and it was confirmed whether amplification efficiency and non-specific amplification were exhibited. As a result, several primer sets shown in Table 1 were selected as the most preferable primer sets.

Detection of herpesviruses using multiplex PCR and DNA macroarray (1) Extraction of viral DNA For KSHV and EBV, 2 × 10 ⁵ KSHV ⁺ B cells (BC2) or EBV + B cells (BC2) ( Raji) was cultured and extracted with a commercially available DNA extraction kit (GenElute Plasmid midiprep Kit, Sigma). For HCMV, 1 × 10 ⁶ human fibroblasts HEL were seeded on a 10 cm plate. Cells were infected with HCMV (Town strain) at MOI = 10. Seven days later, the cells were collected and extracted with a commercially available DNA extraction kit (GenElute Plasmid midiprep Kit, Sigma).

(2) Preparation of cDNA Macroarray A nylon microarray was prepared by spotting DNA from three types of viral genomic DNA clones shown in Table 2, two types of external control control clones, and two types of internal control clones at seven locations. All clones were amplified by PCR using the primers shown in Table 2. The external control (E1, E2) gene was cloned from Burevundimonas diminuta.

ＬＡＮＡ，潜在的核抗原；ＥＢＥＲ，ＥＢＶによりコードされるＲＮＡ；ＩＥ，前初期遺蛋白質遺伝子；ＡＬＡＳ１，アミノレブリン酸シンターゼ；ＧＡＰＤＨ，グリセルアルデヒド−３−リン酸デヒドロゲナーゼ
／Ｆ，フォワードプライマーの配列；／Ｒ，リバースプライマーの配列

LANA, potential nuclear antigen; RNA encoded by EBER, EBV; IE, immediate early protein gene; ALAS1, aminolevulinate synthase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase / F, forward primer sequence; R, reverse primer sequence

  The amplified product was then purified by QIAQUICK 8 PCR Purification Kit (QIAGEN), resuspended in 100 μl distilled water and 10% (v / v) xylene cyanol was added. Purified 0.2 μl (4 ng) DNA was manually spotted onto a nylon membrane Hybond-N + (Amersham Biosciences) and then the DNA was cross-linked to the membrane in GSGENELINKER UV CHAMBER (BIORAD).

(3) Preparation of label target by multiplex PCR Multiplex PCR labeling was performed by mixing external standard-specific primers and all virus primers. PCR was performed in each 25 μl reaction mixture containing 1 × PCR buffer, 1 unit ExTaq (TAKARA BIO INC.), 2 mM dNTPs and 2 mM biotinylated dUTP and 1 pmol of specific primer pairs. Alternatively, 10 ⁴ copies of each plasmid was mixed with the PCR reaction solution. Specific primer pairs are shown in Table 2 above.

As a template, 10 ³ copies of each viral genome plasmid or external standard plasmid (external standard 2) were used. As a control for hybridization efficiency, an external standard plasmid subjected to biotinylated PCR was used (external standard 1). As external standard clones, a plurality of clones were preliminarily examined for non-specific amplification and detection with the human genome and each virus genome clone, and clones that were not confirmed to have non-specific detection were selected.

  PCR is performed at 95 ° C. for 5 minutes, followed by a reaction cycle consisting of 30 cycles of amplification at 95 ° C. for 30 seconds, 55 ° C. for 30 seconds, 72 ° C. for 30 seconds, and extension step at 72 ° C. for 5 minutes. It was. The PCR product was ethanol precipitated, dried, dissolved in DDW and used as a label target.

(4) cDNA macroarray hybridization The specificity of multiplex PCR labeling and hybridization was verified. The PCR amplification product denatured at 100 ° C. for 5 minutes was added to the prehybridized macroarray membrane, and hybridization was performed at 68 ° C. for 10 hours. After hybridization, the membrane was washed 3 times with 2 × SSC, 0.1% SDS at 68 ° C. for 5 minutes each and then 3 times with 0.1 × SSC, 0.1% SDS at 68 ° C. for 5 minutes each. Washed. Nucleic acids were detected from the resulting membranes using a Phototope-Star detection kit (New England Biolabs) according to the manufacturer's guidelines. Detection was performed by chemiluminescence image analysis, and an image was recorded by a Fluor-SMAX2 multi-imaging system (BIORAD).

  The results are shown in FIG. In the figure, KSHV (A); EBV (B); CMV (C); Columns E1 and E2: External control; Column 1: KSHV; Column 2: EBV; Column 3: CMV; Columns I1 and I2: Internal control; Indicates a viral signal. It was confirmed that a signal specific to the corresponding spot was detected only when a viral plasmid was present. Since no internal standard primer was added, no signal was detected.

(5) Detection sensitivity of each virus Next, the detection sensitivity was analyzed. The primer mix was prepared by mixing the virus-specific primer, the external standard, and the internal standard primer in equal amounts. The plasmids of the viral genomic clone, external standard clone, and internal standard clone cloned in the plasmid were mixed together so as to have an equal amount. 10-fold dilution series of the cloned viral DNA (10 ³ to 10 ⁰ copies / reaction tube) were mixed and used as template for multiplex PCR labeling. Multiplex PCR labeling was performed using these primer mix and template mix, and the detection sensitivity of each virus was analyzed.

The results are shown in FIG. In the figure, 10 ³ (A), 10 ² (B), 10 ¹ (C), 10 ⁰ (D) copy / reaction tubes; each column is the same as shown in FIG. As a result, EBV is but 10 ² and the sensitivity is low, with respect to KSHV and CMV was detectable up to 10 ⁰ copies.

(6) Detection of virus from virus-infected cells Next, total DNA was prepared from the virus-infected cells and the array was analyzed. As virus-infected cells, BC2 (KSHV, EBV double positive), BC3 (KSHV positive), Raji (EBV positive), Akata (EBV positive) and HEL # 1, # 2 (CMV positive) were used. DNA was prepared from about 1 × 10 ⁶ cells using GenElute Mammalian Genomic DNA Miniprep Kit (Sigma). The external standard plasmid used 10 ² copies. PCR and macroarray hybridization were performed in the same manner as described above using a primer mix in which virus-specific primers, external standards, and internal standard primers were mixed in equal amounts.

  The results are shown in FIG. In the figure, KSHV, EBV double positive BC2 (A), KSHV positive BC3 (B), EBV positive Raji (C), EBV positive Akata (D), CMV positive HEL # 1, # 2 (E, F); each column Is the same as shown in FIG. 1; the arrow indicates the viral signal. As is clear from the figure, it was possible to detect the virus specifically for any virus using the multiplex PCR and macroarray of the present invention.

Detection of herpes virus using multiplex PCR and electrophoresis (1) Multiplex PCR
Multiplex PCR was performed by using plasmids cloned from each virus as templates and mixing corresponding primers. Table 3 shows the target genes for multiplex PCR and the sequences of the primer sets used. ALAS1 (aminolevulinate delta synthase 1) was amplified as an internal control for human genome, and an unknown DNA fragment derived from Brevundimonas dimita was amplified as an external control. Specific amplification using each primer was confirmed in advance.

ｖＦＬＩＰ：ウイルスＦＬＩＣＥ阻害蛋白質；ＬＡＮＡ：潜在的核抗原；ＥＢＮＡ１：エプステインバーウイルス核抗原１，ＬＭＰ１：潜在的膜蛋白質１；ＩＥ：前初期蛋白質遺伝子；ＡＬＡＳ１：アミノレブリン酸シンターゼ
／Ｆ，フォワードプライマーの配列；／Ｒ，リバースプライマーの配列

vFLIP: viral FLICE inhibitor protein; LANA: potential nuclear antigen; EBNA1: Epstein-Barr virus nuclear antigen 1, LMP1: potential membrane protein 1; IE: immediate early protein gene; ALAS1: aminolevulinate synthase / F, forward primer Sequence; / R, reverse primer sequence

The template plasmid was serially diluted to 10 ⁸ to 10 ⁰ copy number was determined detectable sensitivity. Two clones targeted for each virus were selected from a plurality of clones after confirming by a preliminary experiment that no cross reaction occurred. For the internal control, ALAS1 (aminolevulinate synthase), which was confirmed to be a single copy on the database, was used, and the copy number of the internal control was adjusted to be the same as that of the virus clone.

PCR is performed in a master mix (multiplex PCR Kit, QIAGEN), 25 μl of each reaction mixture containing 3.125 pmol of each specific primer pair, and the extracted sample DNA or 10 ⁴ copies of each plasmid is mixed with the PCR reaction solution. did. PCR consists of a denaturation step at 94 ° C for 15 minutes, followed by 35 cycles of amplification at 94 ° C for 30 seconds, 60 ° C for 1.5 minutes, 72 ° C for 1 minute, and extension step at 72 ° C for 3 minutes. Done in a cycle.

(2) Detection of PCR products and electrophoretic analysis The obtained PCR products were separated and analyzed using 2100 Bioanalyzer and DNA 500 LabChip kit (Agilent). High resolution and high sensitivity analysis was performed using a 2100 bioanalyzer. The pseudo image of the gel was processed and analyzed using Photoshop 6.0 (Adobe Systems). Finally, the type of virus was identified by the size of the PCR product.

As a result, the detection sensitivity, KSHV 10 ⁰ copies, EBV · CMV was 10 ¹ copies (Fig. 4). The arrow indicates the human genome internal control.

(3) Detection of virus from virus-infected cells Next, multiplex PCR was performed using genomic DNA extracted and purified from virus-infected cultured cells as a template, and corresponding primers and external / internal standard primers were mixed. As an external standard, B.I. A plasmid into which a clone derived from the dimita genome was introduced was used after adjusting to 10 ⁴ copies. The cell lines used for the templates are BC2 (KSHV, EBV double positive), BC3 (KSHV positive), Raji (EBV positive), Akata (EBV positive), HEL # 1, # 2 (CMV positive) and SiHa (negative control). ). PCR and electrophoresis of amplification products were performed under the same conditions as described above.

  The results are shown in FIG. In the figure, KSHV, EBV double positive BC2 (lane 1), KSHV-positive BC3 (lane 2), EBV-positive Raji (lane 3), EBV-positive Akata (lane 4), CMV-positive HEL # 1, # 2 (Lanes 5 and 6) and SiHa (negative control) (lane 7); arrows indicate external control (top) and human genome internal control (bottom).

  As is clear from the figure, a band derived from the target virus genome was specifically detected from virus-infected cells. It was also confirmed that two types of viruses could be detected simultaneously.

  According to the method of the present invention, it becomes possible to simultaneously diagnose KSHV, EBV, and CMV infection from a small amount of blood sample in a single analysis, before administration of an immunosuppressant at the time of organ transplantation, and for patients with latent HIV infection. It is possible to predict opportunistic infections before the onset of AIDS.

FIG. 1 shows detection of viral DNA using multiplex PCR and macroarray. FIG. 2 shows the detection sensitivity of viral DNA using multiplex PCR and macroarray. FIG. 3 shows the detection of each viral DNA in virus-infected cell lines using multiplex PCR and macroarray. FIG. 4 shows the detection sensitivity of viral genes using multiplex PCR and electrophoresis. FIG. 5 shows the detection of each viral DNA in a virus-infected cell line using multiplex PCR and electrophoresis.

Claims (6)

A kit for detecting the presence or expression of two or more herpesviruses in a sample, comprising the following AC:
(A) Primer set KSHV1, which includes two oligonucleotides each consisting of a base sequence represented by SEQ ID NO: 1 and SEQ ID NO: 2, and two oligonucleotides each comprising a base sequence represented by SEQ ID NO: 3 and SEQ ID NO: 4 Primer set KSHV2, containing
At least one primer set for detecting Kaposi's sarcoma-associated herpesvirus (KSHV) selected from the group consisting of:
(B) Primer set EBV1, comprising two oligonucleotides each having the base sequence represented by SEQ ID NO: 5 and SEQ ID NO: 6
Primer set EBV2, which includes two oligonucleotides each consisting of a base sequence represented by SEQ ID NO: 7 and SEQ ID NO: 8, and a primer which includes two oligonucleotides each composed of a base sequence represented by SEQ ID NO: 9 and SEQ ID NO: 10 Set EBV3
At least one Epstein Barr virus (EBV) detection primer set selected from the group consisting of:
(C) Primer set CMV1, comprising two oligonucleotides each having the nucleotide sequence represented by SEQ ID NO: 11 and SEQ ID NO: 12
Primer set CMV2, which includes two oligonucleotides each consisting of a base sequence represented by SEQ ID NO: 13 and SEQ ID NO: 14, and a primer which includes two oligonucleotides each composed of a base sequence represented by SEQ ID NO: 15 and SEQ ID NO: 16 Set CMV3
At least one cytomegalovirus (CMV) detection primer set selected from the group consisting of:
A kit comprising two or more primer sets selected from the group consisting of: Primer set KSHV1, comprising two oligonucleotides each consisting of a base sequence represented by SEQ ID NO: 1 and SEQ ID NO: 2
Primer set EBV1, which includes two oligonucleotides each consisting of a base sequence represented by SEQ ID NO: 5 and SEQ ID NO: 6, and a primer which includes two oligonucleotides each composed of a base sequence represented by SEQ ID NO: 11 and SEQ ID NO: 12 Set CMV1,
The kit according to claim 1, comprising: Primer set KSHV1, comprising two oligonucleotides each consisting of a base sequence represented by SEQ ID NO: 1 and SEQ ID NO: 2
Primer set KSHV2, comprising two oligonucleotides each consisting of the base sequence represented by SEQ ID NO: 3 and SEQ ID NO: 4
Primer set EBV2, comprising two oligonucleotides each consisting of a base sequence represented by SEQ ID NO: 7 and SEQ ID NO: 8
Primer set EBV3 comprising two oligonucleotides each consisting of a base sequence represented by SEQ ID NO: 9 and SEQ ID NO: 10
Primer set CMV2, which includes two oligonucleotides each consisting of a base sequence represented by SEQ ID NO: 13 and SEQ ID NO: 14, and a primer which includes two oligonucleotides each composed of a base sequence represented by SEQ ID NO: 15 and SEQ ID NO: 16 Set CMV3
The kit according to claim 1, comprising: A method for detecting the presence or expression of two or more herpesviruses in a sample, wherein DNA obtained from the sample or cDNA prepared by reverse transcription from mRNA obtained from the sample is used as a template. A method comprising performing a polymerase chain reaction using the kit according to any one of 3 and detecting an amplified herpesvirus gene. The method according to claim 4, wherein detection of the amplified herpesvirus gene is performed using a DNA array. The method according to claim 4, wherein detection of the amplified herpesvirus gene is performed using electrophoresis.

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