Compositions and methods for detecting Epstein-Barr virus (EBV)

Abstract

A method for rapidly detecting the presence or absence of Epstein-Barr virus (EBV) in a biological or non-biological sample is disclosed. The method may include performing amplification, hybridization, and detection steps. Additionally, primers and probes targeting EBV and kits designed to detect target regions of EBV are provided. Kits, reaction mixtures, and oligonucleotides (e.g., primers and probes) for amplifying and detecting EBV are also disclosed. Primers and probes detecting different regions of EBV are also disclosed, and may be used in a dual-target assay to simultaneously detect two different, non-overlapping target regions of EBV.

Description

[Technical field]

[0001] The present disclosure relates to the field of in vitro diagnostics. In this field, the present invention relates to the amplification and detection of target nucleic acids that may be present in a sample, in particular the amplification, detection and / or quantification of target nucleic acids containing sequence variants and / or individual variants of Epstein-Barr Virus (EBV) using primers and probes. The present invention further provides reaction mixtures and kits containing primers and probes for the amplification and detection of EBV. [Background technology]

[0002] Epstein-Barr virus (EBV) is a DNA virus that is a member of the herpesvirus group (also known as human herpesvirus-4 or HHV-4). EBV infection is common, with an estimated prevalence of 90% in adults, and is often asymptomatic. EBV infection can cause mononucleosis in an early lytic phase before entering one or more of several latent stages. EBV is highly associated with life-threatening diseases in immunocompromised patients and several forms of cancer (Burkitt's lymphoma (BL), Hodgkin's lymphoma (HL), nasal-type NK / T-cell lymphoma (NKTL), lymphoepithelioma-like carcinoma (LELC), nasopharyngeal carcinoma (NPC), and gastric carcinoma (GC). Organ transplant patients with suppressed immune responses are particularly at risk as uncontrolled proliferation of latently EBV-infected B cells can lead to post-transplant lymphoproliferative disorder (PTLD), a serious complication. Transplantation involves a balance between the risk of rejection due to immunosuppression and the risk of disease. EBV is one of several common (90%), often asymptomatic viruses It is an infectious disease, but it poses a threat to immunosuppressed patients because it cannot eliminate the virus. PTLD is mainly a proliferation of B lymphocytes associated with EBV positivity (especially in the early onset cases). In fact, EBV is the second major post-transplant surveillance marker (after cytomegalovirus (CMV) infection), and EBV-associated PTLD is one of the most common malignancies after solid organ or hematopoietic stem cell transplantation. Up to 5% of transplant patients develop PTLD, which in the worst case can lead to lymphoma. The highest risk groups include patients in the first year after transplantation, organ transplants from EBV-positive donors to EBV-negative recipients, stem cell transplant patients and pediatric patients.

[0003] EBV may be detected in the bloodstream by viral particles and fragmented DNA in plasma, and by intracellular episomal DNA viral genomes and mRNA. EBV has a genome size of approximately 172 kb or more (depending on the number of internal repeats and deletions) and can exist in either linear or circular (episomal) structures. EBV genetic diversity is generally reported to include two types, which are listed in the literature, but characterization of the actual diversity of the virus is more complex and may depend on which part of the genome is sampled. EBNA2 and EBNA3A, B, and C gene sequences drive the classification of EBV strains into the traditional "type 1" and "type 2" categories. In general, type 1 is prevalent in most regions, but in Africa, both types 1 and 2 are highly prevalent. Outside of these genes, type 1 / type 2 differences are less relevant, and there may be different phylogenetic patterns for selected genes. Geographic variation has been described from differences between East Asian lineages of EBV and European and African lineages, but full characterization of EBV genomic diversity may require sampling of more than one gene region. Recombination appears to be a frequent event in the history of the virus, indicating that reinfection or co-infection may occur in single cells. There remains a need in the art for reliable, sensitive and reproducible means for detecting EBV.

[0004] Measurement of EBV viral load is becoming a routine test for managing transplant patients and diagnosing and tracking EBV-associated diseases. In patients undergoing EBV viral monitoring, serial DNA measurements can be used to indicate the need for potential treatment changes and evaluate viral response to treatment. Given the widespread prevalence of EBV infections worldwide and the frequent recombination that occurs in EBV, there is a need in the art for rapid, reliable, specific and sensitive methods for detecting and / or quantifying the presence of EBV in a sample. Summary of the Invention

[0005] In the field of molecular diagnostics, the amplification and detection of nucleic acids is of considerable importance. Such methods can be used to detect any number of microorganisms, such as viruses and bacteria. The most prominent and widely used amplification technique is the polymerase chain reaction (PCR). Other amplification techniques include ligase chain reaction, polymerase ligase chain reaction, Gap-LCR, repair chain reaction, 3SR, NASBA, strand displacement amplification (SDA), transcription-mediated amplification (TMA) and Qβ amplification. Automated systems for PCR-based analysis often utilize real-time detection of product amplification during the PCR process in the same reaction vessel. The key to such methods is the use of modified oligonucleotides bearing reporter groups or labels.

[0006] The present invention relates to reliable, sensitive and reproducible amplification and detection of EBV. EBV is a DNA virus with multiple well-conserved target regions for assay design, but the possibility of unencountered substitutions or deletions means that a dual targeting approach is prudent. Primer and probe design used available public sequence information to maximally include EBV and exclude other herpes viruses.

[0007] The present invention consists of a novel oligonucleotide design designed to maximally contain EBV and prevent cross-reactivity with other templates. This EBV assay can be used on cobas® 6800 / 8800. The primers and probes of the present invention can be used as a dual-target assay where both targets use the same dye as a countermeasure to viral sequence heterogeneity. The design strategy was to select conserved sequence regions from the EBV genome and evaluate several primer and probe combinations for each target. The five regions selected for the assay design were IR-1 repeats, LMP2A, EBNA-1 (BKRF-1), BMRF-2 and reductase / BORF-2. Sequences included both EBV types 1 and 2, and sequences from geographic locations around the world including Asia, Africa, North America, South America, Europe and Australia. After a screening and optimization process, two candidates were identified from just above the final exon of EBNA-1 and within the coding sequence of BMRF-2. These candidates can be used individually or together in dual-target assays. When used as a dual-target assay, two sets of primers and probes are used, with each set detecting either EBNA-1 or BMRF-2.

[0008] Certain embodiments of the present disclosure relate to methods for rapid detection of the presence or absence of EBV in biological or non-biological samples, for example, multiplex detection of EBV by real-time polymerase chain reaction (PCR) in a single test tube or container. The embodiments include a method for detection of EBV comprising performing at least one cycling step, which may include an amplification step and a hybridization step. Additionally, the embodiments include primers, probes and kits designed for detection of EBV in a single test tube or container.

[0009] One aspect of the invention is a method for detecting one or more target nucleic acids of Epstein-Barr Virus (EBV) in a sample, comprising: (a) providing a sample; (b) performing an amplification step, if said one or more target nucleic acids of EBV are present in said sample, comprising contacting said sample with one or more sets of primers to produce an amplification product; (c) performing a hybridization step, if said one or more target nucleic acids of EBV are present in said sample, comprising contacting said amplification product with one or more probes; and (d) performing a detection step, comprising detecting the presence or absence of said amplification product, wherein the presence of said amplification product indicates the presence or absence of said one or more target nucleic acids of EBV in said sample. and the absence of said amplification product indicates the presence of said one or more target nucleic acids of EBV in said sample, wherein said one or more sets of primers and said one or more probes comprise: (i) a set of primers comprising a first primer comprising a nucleic acid sequence of SEQ ID NO: 1 and a second primer comprising a nucleic acid sequence of SEQ ID NO: 3 or a complement thereof, and a probe comprising a nucleic acid sequence of SEQ ID NO: 2 or a complement thereof, and / or (ii) a set of primers comprising a first primer comprising a nucleic acid sequence of SEQ ID NO: 4 and a second primer comprising a nucleic acid sequence of SEQ ID NO: 6 or a complement thereof, and a probe comprising a nucleic acid sequence of SEQ ID NO: 3 or a complement thereof.

[0010] In a related embodiment, the sample is a biological sample. In a related embodiment, the biological sample is plasma. In a related embodiment, the biological sample is blood. In another embodiment, the method is for detecting a first target nucleic acid of EBV and a second target nucleic acid of EBV in a sample, wherein the set of one or more primers and the one or more probes for detecting the first target nucleic acid of EBV comprise (i) a set of primers comprising the nucleic acid sequences of SEQ ID NO: 1 and 3 or their complements, and a probe comprising the nucleic acid sequence of SEQ ID NO: 2 or its complement, and the set of one or more primers and the one or more probes for detecting the second target nucleic acid of EBV comprise (ii) a set of primers comprising the nucleic acid sequences of SEQ ID NO: 4 and 6 or their complements, and a probe comprising the nucleic acid sequence of SEQ ID NO: 3 or its complement. In another embodiment, the first target nucleic acid of EBV and the second target nucleic acid of EBV are different. In another embodiment, the first target nucleic acid of EBV and the second target nucleic acid of EBV are non-overlapping.

[0011] Another embodiment is a method for detecting a first target nucleic acid of EBV and a second target nucleic acid of EBV in a sample, comprising: (a) providing a sample; (b) performing an amplification step, if said one or more target nucleic acids of EBV are present in said sample, comprising contacting said sample with one or more sets of primers to produce an amplification product; (c) performing a hybridization step, if said one or more target nucleic acids of EBV are present in said sample, comprising contacting said amplification product with one or more probes; and (d) performing a detection step, comprising detecting the presence or absence of said amplification product, wherein the presence of said amplification product indicates the presence of said one or more target nucleic acids of EBV in said sample. and performing a detection step, wherein the presence of an amplification product indicates the presence of a target nucleic acid and the absence of the amplification product indicates the absence of the one or more target nucleic acids of EBV in the sample, wherein the one or more sets of primers and the one or more probes comprise (i) a set of primers comprising the nucleic acid sequences of SEQ ID NO:1 and 3 or a complement thereof, and a probe comprising the nucleic acid sequence of SEQ ID NO:2 or a complement thereof, and the one or more sets of primers and the one or more probes for detecting the second target nucleic acid of EBV comprise (ii) a set of primers comprising the nucleic acid sequences of SEQ ID NO:4 and 6 or a complement thereof, and a probe comprising the nucleic acid sequence of SEQ ID NO:3 or a complement thereof.

[0012] In another embodiment, the first target nucleic acid of EBV and the second target nucleic acid of EBV are different. In another embodiment, the first target nucleic acid of EBV and the second target nucleic acid of EBV do not overlap. In another embodiment, the sample is a biological sample. In another embodiment, the biological sample is plasma. In another embodiment, the biological sample is blood.

[0013] Another aspect of the invention relates to a kit for detecting one or more target nucleic acids of EBV possibly present in a sample, the kit comprising an amplification reagent comprising (a) a DNA polymerase, (b) nucleotide monomers, (c) one or more sets of primers, and (d) one or more probes, the one or more sets of primers and the one or more probes comprising (i) a set of primers comprising a first primer comprising a nucleic acid sequence of SEQ ID NO: 1 and a second primer comprising a nucleic acid sequence of SEQ ID NO: 3 or a complement thereof, and a probe comprising a nucleic acid sequence of SEQ ID NO: 2 or a complement thereof, and / or (ii) a set of primers comprising a first primer comprising a nucleic acid sequence of SEQ ID NO: 4 and a second primer comprising a nucleic acid sequence of SEQ ID NO: 6 or a complement thereof, and a probe comprising a nucleic acid sequence of SEQ ID NO: 3 or a complement thereof. In another embodiment, the one or more probes are labeled with a donor fluorescent moiety and a corresponding acceptor moiety. In another embodiment, the kit is for detecting a first target nucleic acid of EBV and a second target nucleic acid of EBV in a sample, and the one or more sets of primers and the one or more probes for detecting the first target nucleic acid of EBV include (i) a set of primers comprising the nucleic acid sequence of SEQ ID NO: 1 and 3 or a complement thereof, and a probe comprising the nucleic acid sequence of SEQ ID NO: 2 or a complement thereof, and the one or more sets of primers and the one or more probes for detecting the second target nucleic acid of EBV include (ii) a set of primers comprising a first primer comprising the nucleic acid sequence of SEQ ID NO: 4 and a second primer comprising the nucleic acid sequence of SEQ ID NO: 6 or a complement thereof, and a probe comprising the nucleic acid sequence of SEQ ID NO: 3 or a complement thereof. In another embodiment, the first target nucleic acid of EBV and the second target nucleic acid of EBV are different. In another embodiment, the first target nucleic acid of EBV and the second target nucleic acid of EBV do not overlap.

[0014] Another aspect of the present invention relates to a kit for detecting a first target nucleic acid of EBV and a second target nucleic acid of EBV in a sample, the kit comprising an amplification reagent comprising (a) a DNA polymerase, (b) a nucleotide monomer, (c) a first set of primers and one probe for detecting the first target nucleic acid of EBV, and (d) a second set of primers and one probe for detecting the second target nucleic acid of EBV, wherein the first set of primers and one probe for detecting the first target nucleic acid of EBV comprises (i) a set of primers comprising the nucleic acid sequences of SEQ ID NO: 1 and 3 or their complements, and a probe comprising the nucleic acid sequence of SEQ ID NO: 2 or its complement, and the second set of primers and one probe for detecting the second target nucleic acid of EBV comprises (ii) a set of primers comprising the nucleic acid sequences of SEQ ID NO: 4 and 6 or their complements, and a probe comprising the nucleic acid sequence of SEQ ID NO: 3 or its complement. In another embodiment, the probes are labeled with a donor fluorescent moiety and a corresponding acceptor moiety. In another embodiment, the first target nucleic acid of EBV and the second target nucleic acid of EBV are different. In another embodiment, said first target nucleic acid of EBV and said second target nucleic acid of EBV do not overlap.

[0015] Other aspects provide oligonucleotides comprising or consisting of a sequence of nucleotides selected from SEQ ID NOs: 1-6 or a complement thereof, having 100 or less nucleotides. In another embodiment, the disclosure provides oligonucleotides comprising a nucleic acid having at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90% or 95%) to one of SEQ ID NOs: 1-6 or a complement thereof, the oligonucleotide having 100 or less nucleotides. Generally, these oligonucleotides can be primer nucleic acids, probe nucleic acids, etc. in these embodiments. In certain of these embodiments, the oligonucleotide has 40 or less nucleotides (e.g., 35 or less nucleotides, 30 or less nucleotides, 25 or less nucleotides, 20 or less nucleotides, 15 or less nucleotides, etc.). In some embodiments, the oligonucleotide comprises at least one modified nucleotide, e.g., to alter nucleic acid hybridization stability compared to unmodified nucleotides. Optionally, the oligonucleotide comprises at least one label, and at least one quencher moiety. In some embodiments, the oligonucleotide comprises at least one conservatively modified mutation. "Conservatively modified variations" or simply "conservative variations" of a particular nucleic acid sequence refer to nucleic acids that encode identical or essentially identical amino acid sequences, or, where the nucleic acid does not encode an amino acid sequence, essentially identical sequences. Those of skill in the art will recognize that individual substitutions, deletions, or additions that alter, add, or delete a single amino acid or a small number of amino acids (usually less than 5%, more usually less than 4%, 2%, or 1%) in an encoded sequence are "conservatively modified variations" in which the modification results in the deletion of an amino acid, the addition of an amino acid, or the replacement of an amino acid with a chemically similar amino acid.

[0016] In one embodiment, the amplification may use a polymerase enzyme with 5' to 3' nuclease activity. Thus, the donor fluorescent moiety and the acceptor moiety, e.g., a quencher, may be within 5 to 20 nucleotides (e.g., 7 or 10 nucleotides) of each other along the length of the probe. In another embodiment, the probe includes a nucleic acid sequence that allows for the formation of a secondary structure. The formation of such a secondary structure may result in spatial proximity between the first fluorescent moiety and the second fluorescent moiety. According to the method, the second fluorescent moiety on the probe may be a quencher.

[0017] The present disclosure also provides methods for detecting the presence or absence of EBV or EBV nucleic acid in a biological sample from an individual. These methods may be used, for example, to detect the presence or absence of EBV nucleic acid in plasma for use in blood screening and diagnostic testing. In addition, the same tests may be used by those skilled in the art to evaluate urine and other types of samples to detect and / or quantify EBV nucleic acid. Such methods generally include performing at least one cycling step, including an amplification step and a dye binding step. Typically, the amplification step includes contacting the sample with a plurality of oligonucleotide primer pairs to generate one or more amplification products if nucleic acid molecules are present in the sample, and the dye binding step includes contacting the amplification products with a double-stranded DNA binding dye. Such methods also include detecting the presence or absence of binding of the double-stranded DNA binding dye to the amplification products, the presence of binding indicating the presence of EBV nucleic acid in the sample and the absence of binding indicating the absence of EBV in the sample. A representative double-stranded DNA binding dye is ethidium bromide. Other nucleic acid binding dyes include DAPI, Hoechst dyes, PicoGreen®, RiboGreen®, OliGreen®, and cyanine dyes such as YO-YO® and SYBR® Green. Additionally, such methods may also include determining the melting temperature between the amplification product and a double-stranded DNA binding dye, which confirms the presence or absence of EBV nucleic acid.

[0018] In a further embodiment, a kit for detecting and / or quantifying one or more nucleic acids of EBV is provided. The kit may include one or more sets of primers specific for amplifying a gene target; and one or more detectable oligonucleotide probes specific for detecting the amplification product.

[0019] In one embodiment, the kit may include a donor moiety and a corresponding acceptor moiety, e.g., a probe that is already labeled with another fluorescent moiety or a dark quencher, or may include a fluorophore moiety for labeling the probe. The kit may also further include nucleoside triphosphates, a nucleic acid polymerase, and buffers necessary for the function of the nucleic acid polymerase. The kit may also include a package insert and instructions for using the primers, probes, and fluorophore moieties to detect the presence or absence of EBV nucleic acid in a sample.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.Methods and materials similar or equivalent to those described herein can be used in the practice or testing of this subject, but suitable methods and materials are described below.In addition, materials, methods, and examples are only illustrative and are not intended to be limiting.All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.In case of conflict, the present specification, including definitions, will control.

[0021] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the drawings and detailed description, and from the claims. [Brief description of the drawings]

[0022] [Figure 1]Figure 1 shows the genomic structure of EBV. Figure 1A shows a schematic of the linear EBV genome (lytic form). Figure 1B shows a schematic of the circular EBV genome (latent form). EBV is a double-stranded DNA virus of approximately 172 kb or more. It has: (i) a mostly unique region, (ii) internal repeat regions (IR1-IR4), (iii) terminal repeats (TR), (iv) Ori P, the origin of latent EBV episomal replication with plasmid maintenance and DNA replication activity, (v) exons of latent EBV-induced membrane and nucleoproteins. Figure 1 highlights the EBV targets BMRF2 and EBNA1 that can be used as dual target assays. [Diagram 2] FIG. 2 shows the EBV target and EBNA1 oligonucleotide sets containing a 96 base pair amplicon generated by the primers and detected by the probe. [Diagram 3] FIG. 3 shows oligonucleotide sets for EBV targets and BMRF2, including a 98 base pair amplicon generated by the primers and detected by the probe. [Figure 4A] FIG. 4A shows the PCR growth curves of a dilution series of the Qnostics EBV Analytic Panel (Qnostics Catalog No. EBVAQP03-C) assay performance against the WHO criteria for EBV (NIBSC code 09 / 260) at 8,000 IU / ml (FIG. 4A). [Figure 4B] FIG. 4B shows the PCR growth curves of a dilution series of the Qnostics EBV Analytic Panel (Qnostics Catalog No. EBVAQP03-C) assay performance against the WHO criteria for EBV (NIBSC code 09 / 260) at 80 IU / ml (FIG. 4B). [Figure 4C]Figure 4C shows the PCR growth curves of a dilution series of the Qnostics EBV Analytic Panel (Qnostics Catalog No. EBVAQP03-C) assay performance against the WHO criteria for EBV (NIBSC code 09 / 260) at 8 IU / ml (Figure 4C). Other EBV sample types such as extracted EBV DNA spiked into EBV-negative plasma and Raji cell line DNA were also tested (data not shown). [Figure 5A] FIG. 5A shows the PCR growth curves of a dilution series of a multiplex assay run against a control template (1E9 IU / mL) (FIG. 5A). [Figure 5B] FIG. 5B shows the PCR growth curves of a dilution series of a multiplex assay run against the control template (1E8 IU / mL) (FIG. 5B). [Figure 5C] FIG. 5C shows the PCR growth curves of a dilution series of a multiplex assay run against a control template (1E7 IU / mL) (FIG. 5C). [Figure 5D] FIG. 5D shows the PCR growth curves of a dilution series of a multiplex assay run against the control template (1E6 IU / mL) (FIG. 5D). [Figure 5E] FIG. 5E shows the PCR growth curves of a dilution series of a multiplex assay run against the control template (1E5 IU / mL) (FIG. 5E). [Figure 5F] FIG. 5F shows the PCR growth curves of a dilution series of a multiplex assay run against the control template (1E4 IU / mL) (FIG. 5F). [Figure 5G] FIG. 5G shows the PCR growth curves of a dilution series of a multiplex assay run against the control template (1E4 IU / mL) (FIG. 5G). [Figure 5H] Figure 5H shows the PCR growth curves of a dilution series of a multiplexed assay run on cell culture derived material (1E3 IU / mL) (Figure 5H). [Figure 5I] Figure 5I shows the PCR growth curves of a dilution series of a multiplexed assay run on cell culture derived material (1E3 IU / mL) (Figure 5I). [Figure 5J]Figure 5J shows the PCR growth curves of a dilution series of a multiplexed assay run on cell culture derived material (1E2 IU / mL) (Figure 5J). [Figure 5K] FIG. 5K shows the PCR growth curves of a dilution series of a multiplexed assay run on cell culture derived material (40 IU / mL) (FIG. 5K). [Figure 5L] Figure 5L shows the PCR growth curves of a dilution series of a multiplexed assay run on cell culture-derived material (20 IU / mL) (Figure 5L). Channel 2 shows the dual target EBV assay and channel 5 is the internal control reaction. [Figure 6A] Figure 6A shows PCR growth curves of separate reactions for EBNA1 (Figure 6A) on plasmid templates containing individual targets (E1c, B2c) or both targets (dC), and on B95-8 cell line extracts (containing copies of EBV DNA). [Figure 6B] Figure 6B shows PCR growth curves of separate reactions for BMRF2 (Figure 6B) in plasmid templates containing individual targets (E1c, B2c) or both targets (dC), and in B95-8 cell line extracts (containing copies of EBV DNA). These studies demonstrate that the dual target EBNA1 and BMRF2 EBV assay is efficient and specifically identifies EBNA1 and BMRF2 EBV nucleic acid. [Figure 7] FIG. 7 shows linearity data for the dual target assay against genotype 1 cell culture-derived material and control templates. [Figure 8] FIG. 8 shows linearity data of the dual target assay against genotype 2 EBV-containing cell culture-derived material. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] Diagnosis of EBV infection by nucleic acid amplification provides a method for rapid, accurate, reliable, specific and sensitive detection and / or quantification of EBV infection. Disclosed herein is a real-time PCR assay for detecting and / or quantifying EBV nucleic acid, including DNA and / or RNA, in non-biological or biological samples. Primers and probes for detecting and / or quantifying EBV are provided, as are articles of manufacture or kits containing such primers and probes. The increased specificity and sensitivity of real-time PCR for the detection of EBV compared to other methods, and the improved features of real-time PCR, including sample containment and real-time detection of amplified products, make the implementation of this technology feasible for routine diagnosis of EBV infection in clinical laboratories. In addition, this technology may be used for blood screening and prognosis. This EBV detection assay may also be multiplexed in parallel with other assays for the detection of other nucleic acids, such as other bacteria and / or viruses.

[0024] The disclosure includes oligonucleotide primers and fluorescently labeled hydrolysis probes that hybridize to the EBV genome to specifically identify EBV, for example, using TaqMan® amplification and detection technology.

[0025] The disclosed method may include performing at least one cycling step that includes amplifying one or more portions of a nucleic acid molecular gene target from a sample using one or more primer pairs. As used herein, "EBV primer(s)" refers to an oligonucleotide primer that specifically anneals to a nucleic acid sequence found in EBV and initiates DNA synthesis therefrom under appropriate conditions to produce a respective amplification product. Examples of nucleic acid sequences found in the EBV genome include nucleic acids within the viral capsid protein region of the EBV genome, such as the VP2 region. Each of the contemplated EBV primers anneals to a target such that at least a portion of each amplification product contains a nucleic acid sequence corresponding to the target. If one or more nucleic acids are present in the sample, one or more amplification products are generated, and thus the presence of one or more amplification products indicates the presence of EBV in the sample. The amplification product must contain a nucleic acid sequence complementary to one or more detectable probes for EBV. As used herein, "EBV probe(s)" refers to an oligonucleotide probe that specifically anneals to a nucleic acid found in the EBV genome. Each cycling step includes an amplification step, a hybridization step and a detection step in which the sample is contacted with one or more detectable EBV probes to detect the presence or absence of EBV in the sample.

[0026] As used herein, the term "amplifying" refers to the process of synthesizing a nucleic acid molecule complementary to one or both strands of a template nucleic acid molecule (e.g., a nucleic acid molecule from the EBV genome). Amplifying a nucleic acid molecule typically involves denaturing the template nucleic acid, annealing a primer to the template nucleic acid at a temperature below the melting temperature of the primer, and enzymatically extending from the primer to generate an amplification product. Amplification typically requires the presence of deoxyribonucleoside triphosphates, a DNA polymerase enzyme (e.g., Platinum® Taq) and an appropriate buffer and / or cofactors (e.g., MgCl2 and / or KCl) for optimal activity of the polymerase enzyme.

[0027] The term "primer" as used herein is known to those skilled in the art and refers to oligomeric compounds, primarily oligonucleotides, but also modified oligonucleotides that are capable of "priming" DNA synthesis by a template-dependent DNA polymerase, i.e., for example, the 3' end of the oligonucleotide provides a free 3'-OH group to which a further "nucleotide" can be attached by a template-dependent DNA polymerase that establishes a 3' to 5' phosphodiester bond, thereby using a deoxynucleoside triphosphate, thereby releasing pyrophosphate. The term "hybridizing" refers to the annealing of one or more probes to an amplification product. Hybridization conditions typically include a temperature below the melting temperature of the probe, but which avoids non-specific hybridization of the probe. The term "5' to 3' nuclease activity" refers to the activity of a nucleic acid polymerase typically associated with nucleic acid chain synthesis whereby nucleotides are removed from the 5' end of a nucleic acid chain.

[0028] The term "thermostable polymerase" refers to a polymerase enzyme that is thermostable, i.e., that catalyzes the formation of primer extension products complementary to a template and does not irreversibly denature when exposed to elevated temperatures for the time required to effect denaturation of the double-stranded template nucleic acid. Generally, synthesis is initiated at the 3' end of each primer and proceeds in the 5' to 3' direction along the template strand. Thermostable polymerases have been isolated from Thermus flavus, T.ruber, T.thermophilus, T.aquaticus, T.lacteus, T.rubens, Bacillus stearothermophilus, and Methanothermus fervidus. Nevertheless, non-thermostable polymerases can also be used in PCR assays if the enzyme is supplemented as needed.

[0029] The term "complement thereof" refers to a nucleic acid that is the same length as and exactly complementary to a given nucleic acid.

[0030] The term "extension" or "lengthening" when used in reference to a nucleic acid refers to when additional nucleotides (or other similar molecules) are incorporated into a nucleic acid. For example, a nucleic acid is optionally extended by a nucleotide incorporating biocatalyst, such as a polymerase, which typically adds nucleotides to the 3' end of the nucleic acid.

[0031] The terms "identical" or percent "identity" in the context of two or more nucleic acid sequences refer to two or more sequences or subsequences that have the same or a specified percentage of identical nucleotides when compared and aligned for maximum correspondence as determined, for example, using one of the sequence comparison algorithms available to those of skill in the art or by visual inspection. Exemplary algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST programs, see, e.g., Altschul et al. (1990) "Basic local alignment search tool" J. Mol. Biol. 215:403-410, Gish et al. (1993) "Identification of protein coding regions by database similarity search" Nature Genet. 3:266-272, Madden et al. (1996) "Applications of network BLAST server" Meth. Enzymol. 266:131-141, Altschul et al. (1997) "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs" Nucleic Acids Res. 25:3389-3402, and Zhang et al. (1997) "PowerBLAST: A new network BLAST application for interactive or automated sequence analysis and annotation" Genome Genet. Res. 7:649-656, which are incorporated herein by reference.

[0032] "Modified nucleotide" in the context of oligonucleotide refers to a change in which at least one nucleotide of an oligonucleotide sequence is replaced by a different nucleotide that provides the oligonucleotide with desired properties.Exemplary modified nucleotides that can be substituted in the oligonucleotides described herein include, for example, t-butylbenzyl, C5-methyl-dC, C5-ethyl-dC, C5-methyl-dU, C5-ethyl-dU, 2,6-diaminopurine, C5-propynyl-dC, C5-propynyl-dU, C7-propynyl-dA, C7-propynyl-dG, C5-propargylamino-dC, C5-propargylamino-dU ... Examples of modified nucleotides include 7-propargylamino-dA, C7-propargylamino-dG, 7-deaza-2-deoxy-xanthosine, pyrazolopyrimidine analogs, pseudo-dU, nitropyrrole, nitroindole, 2'-0-methylribo-U, 2'-0-methylribo-C, N4-ethyl-dC, N6-methyl-dA, 5-propynyl-dU, 5-propynyl-dC, 7-deaza-deoxyguanosine (deazaG(u-deaza)), and the like. Many other modified nucleotides that can be substituted in an oligonucleotide are mentioned herein or known in the art. In certain embodiments, the modified nucleotide substitution modifies the melting temperature (Tm) of the oligonucleotide compared to the melting temperature of the corresponding unmodified oligonucleotide. To further illustrate, certain modified nucleotide substitutions can, in some embodiments, reduce non-specific nucleic acid amplification (e.g., minimize primer dimer formation, etc.), increase the yield of the intended target amplicon, and the like. Examples of these types of nucleic acid modifications are described, for example, in U.S. Patent No. 6,001,611, which is incorporated herein by reference. Other modified nucleotide substitutions may alter the stability of the oligonucleotide or provide other desirable characteristics.

[0033] Detection / quantification of EBV target nucleic acid The present disclosure provides methods for detecting EBV, for example, by amplifying a portion of an EBV nucleic acid sequence. Specifically, primers and probes for amplifying, detecting and / or quantifying EBV nucleic acid molecular targets are provided by embodiments of the present disclosure.

[0034] For the detection and / or quantification of EBV, primers and probes are provided for amplifying and detecting / quantifying EBV. EBV nucleic acids other than those exemplified herein can also be used to detect EBV in a sample. For example, functional variants can be evaluated for specificity and / or sensitivity by those skilled in the art using routine methods. Representative functional variants can include, for example, one or more deletions, insertions and / or substitutions in the EBV nucleic acids disclosed herein.

[0035] More specifically, each of the oligonucleotide embodiments includes a nucleic acid having a sequence selected from SEQ ID NOs: 1-6, a substantially identical variant thereof having at least, e.g., 80%, 90%, or 95% sequence identity to one of SEQ ID NOs: 1-6, or a complement or variant of SEQ ID NOs: 1-6. [Table 1]

[0036] In one embodiment, the primer sets and probes described above are used to provide for detection of EBV in biological samples suspected of containing EBV (Table 1). The primer sets and probes may comprise or consist of primers and probes specific for EBV nucleic acid sequences comprising or consisting of the nucleic acid sequences of SEQ ID NOs: 1-6. In another embodiment, the primers and probes for EBV targets comprise or consist of functionally active variants of any of the primers and probes of SEQ ID NOs: 1-6.

[0037] Functionally active variants of any of the primers and / or probes of SEQ ID NOs: 1-6 can be identified by using the primers and / or probes in the disclosed methods. Functionally active variants of any of the primers and / or probes of SEQ ID NOs: 1-6 relate to primers and / or probes that provide similar or higher specificity and sensitivity in the described methods or kits compared to the respective sequences of SEQ ID NOs: 1-6.

[0038] A variant may vary from the sequence of SEQ ID NO: 1-6 by the addition, deletion or substitution of one or more nucleotides, for example, the addition, deletion or substitution of one or more nucleotides at the 5'-end and / or 3'-end of each of the sequences of SEQ ID NO: 1-6. As detailed above, the primers and / or probes may be chemically modified, i.e., the primers and / or probes may contain modified nucleotides or non-nucleotide compounds. Thus, the probes (or primers) are modified oligonucleotides. A "modified nucleotide" (or "nucleotide analog") differs from a natural "nucleotide" by some modification, but still consists of a base or base-like compound, a pentofuranosyl sugar or pentofuranosyl sugar-like compound, a phosphate moiety or a phosphate-like moiety, or a combination thereof. For example, a "label" may be attached to the base portion of a "nucleotide" to obtain a "modified nucleotide". A natural base in a "nucleotide" may be replaced, for example, with a 7-deazapurine, thus also obtaining a "modified nucleotide". The terms "modified nucleotide" or "nucleotide analog" are used interchangeably in this application. A "modified nucleoside" (or "nucleoside analog") differs from a naturally occurring nucleoside by some modification, as outlined above for "modified nucleotides" (or "nucleotide analogs").

[0039] Oligonucleotides, including modified oligonucleotides and oligonucleotide analogs, that amplify nucleic acid molecules encoding EBV targets, nucleic acids encoding other sites of EBV, can be designed using computer programs such as, for example, OLIGO (Molecular Biology Insights Inc. Cascade, Colo.). Important features in designing oligonucleotides to be used as amplification primers include, but are not limited to, an appropriate size of the amplification product to facilitate detection (e.g., by electrophoresis), similar melting temperatures for the members of the pair of primers, and the length of each primer (i.e., primers must be long enough to anneal with sequence specificity and initiate synthesis, but not so long that fidelity is reduced during oligonucleotide synthesis). Typically, oligonucleotide primers are 8-50 nucleotides in length (e.g., 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50 nucleotides in length).

[0040] In addition to a set of primers, the method may use one or more probes to detect the presence or absence of EBV. The term "probe" refers to a synthetic or biologically produced nucleic acid (DNA or RNA), which, by design or selection, contains a specific nucleotide sequence that allows it to specifically (i.e., preferentially) hybridize to "target nucleic acid" under a defined, predetermined stringency when EBV (target) nucleic acid is present. "Probe" may be referred to as "detection probe", which means to detect target nucleic acid.

[0041] In some embodiments, the disclosed probe can be labeled with at least one fluorescent label.In one embodiment, the EBV probe can be labeled with a donor fluorescent moiety, such as a fluorescent dye, and a corresponding acceptor moiety, such as a quencher.In one embodiment, the probe comprises or consists of a fluorescent moiety, and the nucleic acid sequence comprises or consists of SEQ ID NO: 2 and / or 5.

[0042] The design of oligonucleotides used as probes may be performed in a similar manner to the design of primers. Embodiments may use a single probe or a pair of probes for detection of the amplification product. Depending on the embodiment, the probe(s) used may include at least one label and / or at least one quencher moiety. As with primers, the probes usually have similar melting temperatures, and the length of each probe must be sufficient for sequence-specific hybridization to occur, but not so long that fidelity is compromised during synthesis. Oligonucleotide probes are generally 15-40 (e.g., 16, 18, 20, 21, 22, 23, 24, or 25) nucleotides in length.

[0043] The constructs may each comprise one of the EBV primer and probe nucleic acid molecules (e.g., SEQ ID NOs: 1, 2, 3, 4 and 5). The constructs may be used, for example, as a control template nucleic acid molecule. Suitable vectors for use are commercially available and / or produced by recombinant nucleic acid technology methods routine in the art. The EBV nucleic acid molecule may be obtained, for example, by chemical synthesis, direct cloning from EBV, or by nucleic acid amplification.

[0044] Constructs suitable for use in the present methods typically include, in addition to an EBV nucleic acid molecule (e.g., a nucleic acid molecule comprising one or more of the sequences of SEQ ID NOs: 1-6), a sequence encoding a selection marker (e.g., an antibiotic resistance gene) for selecting the desired construct and / or transformant, and an origin of replication. The choice of vector system usually depends on several factors, including, but not limited to, the choice of host cell, replication efficiency, selectability, inducibility and ease of recovery.

[0045] Constructs containing EBV nucleic acid molecules can be propagated in host cells. The term host cell as used herein is meant to include prokaryotes and eukaryotes, such as yeast, plant cells and animal cells. Prokaryotic hosts can include E. coli, Salmonella typhimurium, Serratia marcescens and Bacillus subtilis. Eukaryotic hosts include yeasts such as S. cerevisiae, S. pombe, Pichia pastoris, mammalian cells such as COS cells or Chinese hamster ovary (CHO) cells, insect cells, and plant cells such as Arabidopsis thaliana and Nicotiana tabacum. The construct can be introduced into the host cell using any of the techniques commonly known to those skilled in the art. For example, calcium phosphate precipitation, electroporation, heat shock, lipofection, microinjection and virus-mediated nucleic acid transfer are common methods for introducing nucleic acid into the host cell. In addition, naked DNA can be delivered directly into the cell (see, for example, U.S. Patent Nos. 5,580,859 and 5,589,466).

[0046] Polymerase chain reaction (PCR) U.S. Patent Nos. 4,683,202, 4,683,195, 4,800,159 and 4,965,188 disclose conventional PCR techniques. PCR typically uses two oligonucleotide primers that bind to a selected nucleic acid template (e.g., DNA or RNA). In some embodiments, useful primers include oligonucleotides (e.g., SEQ ID NOs: 1, 3, 4 and 6) that can act as initiation points for nucleic acid synthesis within the described EBV nucleic acid sequence. Primers can be purified from restriction digests by conventional methods or can be produced synthetically. Primers are preferably single-stranded for maximum efficiency in amplification, but primers can also be double-stranded. Double-stranded primers are first denatured, i.e., treated to separate the strands. One method of denaturing double-stranded nucleic acids is by heating.

[0047] If the template nucleic acid is double-stranded, it is necessary to separate the two strands before it can be used as a template in PCR. Strand separation can be achieved by any suitable denaturing method, including physical, chemical, or enzymatic means. One method of separating the nucleic acid strands involves heating until the nucleic acid is predominantly denatured (e.g., greater than 50%, 60%, 70%, 80%, 90%, or 95% denatured). The heating conditions required to denature the template nucleic acid depend, for example, on the buffer salt concentration, and the length and nucleotide composition of the nucleic acid to be denatured, but typically range from about 90°C to about 105°C, depending on the characteristics of the reaction, such as temperature and nucleic acid length. Denaturation is typically performed for about 30 seconds to 4 minutes (e.g., 1 minute to 2 minutes and 30 seconds, or 1.5 minutes).

[0048] When the double-stranded template nucleic acid has been denatured by heat, the reaction mixture is cooled to a temperature that promotes annealing of each primer to its target sequence. The annealing temperature is usually about 35°C to about 65°C (e.g., about 40°C to about 60°C, about 45°C to about 50°C). The annealing time can be about 10 seconds to about 1 minute (e.g., about 20 seconds to about 50 seconds, about 30 seconds to about 40 seconds). The reaction mixture is then adjusted to a temperature that promotes or optimizes the activity of the polymerase, i.e., a temperature sufficient to cause extension from the annealed primers to generate products complementary to the template nucleic acid. The temperature must be sufficient to synthesize an extension product from each primer annealed to the nucleic acid template, but not so high as to denature the extension product from its complementary template (e.g., temperatures for extension generally range from about 40°C to about 80°C (e.g., about 50°C to about 70°C, about 60°C)). Extension times can be from about 10 seconds to about 5 minutes (e.g., from about 30 seconds to about 4 minutes, from about 1 minute to about 3 minutes, from about 1 minute 30 seconds to about 2 minutes).

[0049] The genome of retroviruses or RNA viruses, or the mRNA produced by DNA viruses such as EBV, consists of ribonucleic acid, i.e., RNA. In such cases, the template nucleic acid, RNA, must first be transcribed into complementary DNA (cDNA) through the action of the enzyme reverse transcriptase. Using the RNA template and a short primer complementary to the 3' end of the RNA, reverse transcriptase directs the synthesis of first strand cDNA, which can then be used directly as a template for the polymerase chain reaction.

[0050] PCR assays may use nucleic acids such as RNA or DNA (cDNA). Template nucleic acids do not need to be purified and may be a minor fraction of a complex mixture such as EBV nucleic acids contained in human cells. EBV nucleic acid molecules may be extracted from biological samples by routine techniques such as those described in Diagnostic Molecular Microbiology: Principles and Applications (Persing et al. (eds), 1993, American Society for Microbiology, Washington DC). Nucleic acids may be obtained from any number of sources, including plasmids or natural sources, including bacteria, yeast, viruses, organelles, or higher organisms such as plants or animals.

[0051] Oligonucleotide primers (e.g., SEQ ID NOs: 1, 2, 4, and 5) are combined with PCR reagents under reaction conditions conducive to primer extension. For example, a chain extension reaction typically contains 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 15 mM MgCl2, 0.001% (w / v) gelatin, 0.5-1.0 μg of denatured template DNA, 50 pmol of each oligonucleotide primer, 2.5 U of Taq polymerase, and 10% DMSO. Reactions typically contain 150-320 μM each of dATP, dCTP, dTTP, dGTP, or one or more analogs thereof.

[0052] The newly synthesized strands form double-stranded molecules that can be used in subsequent steps of the reaction. The steps of strand separation, annealing, and extension can be repeated as many times as necessary to generate the desired amount of amplification product corresponding to the target EBV nucleic acid molecule. The limiting factors of the reaction are the amount of primers, thermostable enzyme, and nucleoside triphosphates present in the reaction. The cycling steps (i.e., denaturation, annealing, and extension) are preferably repeated at least once. For detection use, the number of cycling steps depends, for example, on the nature of the sample. If the sample is a complex mixture of nucleic acids, more cycling steps will be required to amplify the target sequence enough for detection. Generally, the cycling steps are repeated at least about 20 times, but may be repeated 40, 60, or 100 times.

[0053] Fluorescence Resonance Energy Transfer (FRET) FRET technology (see, e.g., U.S. Pat. Nos. 4,996,143, 5,565,322, 5,849,489, and 6,162,603) is based on the concept that when a donor fluorescent moiety and a corresponding acceptor fluorescent moiety are positioned within a certain distance of each other, energy transfer occurs between the two fluorescent moieties, which can be visualized or otherwise detected and / or quantified. Typically, the donor transfers energy to the acceptor when excited by light radiation of a suitable wavelength. Typically, the acceptor re-emits the transferred energy in the form of light radiation of a different wavelength. In certain systems, non-fluorescent energy can be transferred between the donor and acceptor moieties via a biomolecule that includes a substantially non-fluorescent donor moiety (see, e.g., U.S. Pat. No. 7,741,467).

[0054] In one example, an oligonucleotide probe can contain a donor fluorescent moiety or dye (e.g., HEX or FAM) and a corresponding quencher (e.g., BlackHole Quencher™ (BHQ) (BHQ-2, etc.)) that may or may not be fluorescent and dissipates the transferred energy in a form other than light. When the probe is intact, energy transfer typically occurs between the donor and acceptor moieties such that the fluorescent emission from the donor fluorescent moiety is quenched by the acceptor moiety. During the extension step of the polymerase chain reaction, the probe bound to the amplification product is cleaved, for example, by the 5' to 3' nuclease activity of Taq polymerase such that the fluorescent emission of the donor fluorescent moiety is no longer quenched. Exemplary probes for this purpose are described, for example, in U.S. Patent Nos. 5,210,015, 5,994,056, and 6,171,785. Commonly used donor-acceptor pairs include the FAM-TAMRA pair. Commonly used quenchers are DABCYL and TAMRA. Commonly used dark quenchers include BlackHole Quencher™ (BHQ) (such as BHQ2), (Biosearch Technologies, Inc., Novato, Calif.), Iowa Black™ (Integrated DNA Tech., Inc., Coralville, Iowa), BlackBerry™ Quencher 650 (BBQ-650) (Berry & Assoc, Dexta, Michigan).

[0055] In another example, two oligonucleotide probes, each containing a fluorescent moiety, can hybridize to the amplification product at a specific position determined by the complementarity of the oligonucleotide probe to the target nucleic acid sequence. When the oligonucleotide probe hybridizes to the amplification product nucleic acid at the appropriate position, a FRET signal is generated. The hybridization temperature can range from about 35°C to about 65°C for about 10 seconds to about 1 minute.

[0056] Fluorescence analysis may be performed, for example, using a photon-counting epifluorescence microscope system (equipped with appropriate dichroic mirrors and filters to monitor a particular range of fluorescence emission), a photon-counting photomultiplier system, or a fluorometer. Excitation to initiate energy transfer or to allow direct detection of the fluorophore may be performed using an argon ion laser, a high-intensity mercury (Hg) arc lamp, a xenon lamp, a fiber optic light source, or other high-intensity light source appropriately filtered for excitation of the desired range.

[0057] As used herein with respect to a donor moiety and a corresponding acceptor moiety, "corresponding" refers to an acceptor fluorescent moiety or dark quencher that has an absorbance spectrum that overlaps with the emission spectrum of the donor fluorescent moiety. The wavelength maximum of the emission spectrum of the acceptor fluorescent moiety must be at least 100 nm greater than the wavelength maximum of the excitation spectrum of the donor fluorescent moiety. Thus, there can be efficient non-radiative energy transfer between them.

[0058] Fluorescent donor moieties and corresponding acceptor moieties are generally selected for (a) highly efficient Förster energy transfer, (b) a large final Stokes shift (>100 nm), (c) a shift in emission as far as possible into the red portion of the visible spectrum (>600 nm); and (d) a shift in emission to wavelengths higher than the Raman water fluorescence emission produced by excitation at the donor excitation wavelength. For example, a donor fluorescent moiety can be selected that has its maximum excitation wavelength near a laser line (e.g., helium-cadmium 442 nm or argon 488 nm), a high extinction coefficient, a high quantum yield, and good overlap of its fluorescence emission with the excitation spectrum of the corresponding acceptor fluorescent moiety. A corresponding acceptor fluorescent moiety can be selected that has a high extinction coefficient, a high quantum yield, good overlap of its excitation with the emission of the donor fluorescent moiety, and emission in the red portion of the visible spectrum (>600 nm).

[0059] Representative donor fluorescent moieties that can be used with various acceptor fluorescent moieties in FRET technology include fluorescein, lucifer yellow, B-phycoerythrin, 9-acridine isothiocyanate, lucifer yellow VS, 4-acetamido-4'-isothio-cyanatostilbene-2,2'-disulfonic acid, 7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin, succinimidyl 1-pyrenebutyrate, and 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid derivatives. Representative acceptor fluorescent moieties include LC Red 640, LC Red 705, Cy5, Cy5.5, Lissamine rhodamine B sulfonyl chloride, tetramethylrhodamine isothiocyanate, rhodamine x isothiocyanate, erythrosine isothiocyanate, fluorescein, diethylenetriamine pentaacetate, or other chelates of lanthanide ions (e.g., europium, or terbium), depending on the donor fluorescent moiety used. Donor and acceptor fluorescent moieties can be obtained, for example, from Molecular Probes (Junction City, OR) or Sigma Chemical Co. (St. Louis, MO).

[0060] The donor and acceptor fluorescent moieties can be attached to the appropriate probe oligonucleotide via a linker arm. The length of each linker arm is important since it affects the distance between the donor and acceptor fluorescent moieties. The length of the linker arm can be the distance in angstroms (Å) from the nucleotide base to the fluorescent moiety. Generally, the linker arm is from about 10 A to about 25 A. The linker arm can be of the type described in WO 84 / 03285. WO 84 / 03285 also discloses methods of attaching the linker arm to a particular nucleotide base and attaching the fluorescent moiety to the linker arm.

[0061] Acceptor fluorescent moieties such as LC Red 640 can be combined with oligonucleotides containing amino linkers (e.g., C6-amino phosphoramidites available from ABI (Foster City, Calif.) or Glen Research (Sterling, Va.)) to produce, for example, LC Red 640-labeled oligonucleotides. Linkers commonly used to attach donor fluorescent moieties such as fluorescein to oligonucleotides include thiourea linkers (derived from FITC, e.g., Fluorescein-CPG from Glen Research or ChemGene (Ashland, Mass.)), amide-linkers (derived from Fluorescein-NHS-esters, such as CX-Fluorescein-CPG from BioGenex (San Ramon, Calif.)), or 3'-amino-CPG, which requires attachment of a Fluorescein-NHS-ester after oligonucleotide synthesis.

[0062] The present disclosure provides a method for detecting the presence or absence of EBV in a biological or non-biological sample. The provided method avoids the problems of sample contamination, false negatives, and false positives. The method includes carrying out at least one cycling step, which includes using one or more primer pairs to amplify a portion of a target nucleic acid molecule from a sample, and a FRET detection step. The multiple cycling steps are preferably carried out in a thermocycler. The method can be carried out using primers and probes to detect the presence of EBV, and the detection of the target EBV gene indicates the presence of EBV in the sample. As described herein, the amplification product can be detected using a labeled hybridization probe that utilizes FRET technology. One FRET format utilizes TaqMan® technology to detect the presence or absence of the amplification product, and thus the presence or absence of EBV. TaqMan® technology utilizes a single-stranded hybridization probe that is labeled with, for example, one fluorescent moiety or dye (e.g., HEX or FAM) and one quencher (e.g., BHQ-2), which may or may not be fluorescent. When the first fluorescent moiety is excited with light of an appropriate wavelength, the absorbed energy is transferred to a second fluorescent moiety or dark quencher according to the principles of FRET. The second fluorescent moiety is generally a quencher molecule. During the annealing step of the PCR reaction, the labeled hybridization probe binds to the target DNA (i.e., the amplification product) and is degraded during the subsequent extension step, for example, by the 5' to 3' nuclease activity of Taq polymerase. As a result, the fluorescent moiety and the quencher moiety are spatially separated from each other. As a result, upon excitation of the first fluorescent moiety in the absence of the quencher, fluorescent emission from the first fluorescent moiety can be detected. By way of example, the ABI PRISM® 7700 Sequence Detection System (Applied Biosystems) uses TaqMan® technology and is suitable for carrying out the methods described herein for detecting the presence or absence of EBV in a sample.

[0063] Molecular beacons in combination with FRET can also be used to detect the presence of amplification products using real-time PCR. Molecular beacon technology uses a hybridization probe labeled with a first fluorescent moiety and a second fluorescent moiety. The second fluorescent moiety is generally a quencher, and a fluorescent label is typically placed at each end of the probe. Molecular beacon technology uses a probe oligonucleotide with a sequence that allows for secondary structure formation (e.g., a hairpin). The formation of a secondary structure within the probe results in both fluorescent moieties being in spatial proximity when the probe is in solution. After hybridization to the target nucleic acid (i.e., the amplification product), the secondary structure of the probe is disrupted and the fluorescent moieties are separated from each other, so that the emission of the first fluorescent moiety can be detected after excitation with light of an appropriate wavelength.

[0064] Another common form of FRET technology utilizes two hybridization probes. Each probe can be labeled with a different fluorescent moiety and is generally designed to hybridize in close proximity to each other within a target DNA molecule (e.g., an amplification product). A donor fluorescent moiety, e.g., fluorescein, is excited at 470 nm by the light source of the LightCycler® Instrument. During FRET, fluorescein transfers its energy to an acceptor fluorescent moiety, e.g., LightCycler®-Red 640 (LC Red 640) or LightCycler®-Red 705 (LC Red 705). The acceptor fluorescent moiety then emits light of a longer wavelength, which is detected by the optical detection system of the LightCycler® Instrument. Efficient FRET can only occur when the fluorescent moieties are in direct local proximity and the emission spectrum of the donor fluorescent moiety overlaps with the absorption spectrum of the acceptor fluorescent moiety. The intensity of the emitted signal can be correlated with the number of original target DNA molecules (e.g., the number of EBV genomes). When amplification of the target nucleic acid occurs and an amplification product is produced, the hybridizing step results in a detectable signal based on FRET between the members of the probe pair.

[0065] Generally, the presence of FRET indicates the presence of EBV in the sample, and the absence of FRET indicates the absence of EBV in the sample. However, inadequate specimen collection, shipping delays, improper shipping conditions, or the use of certain collection swabs (calcium alginate or aluminum shafts) are all conditions that can affect the success and / or accuracy of the test results.

[0066] Representative biological samples that can be used to carry out the method include, but are not limited to, whole blood, respiratory specimens, urine, fecal specimens, blood specimens, plasma, skin swabs, nasal swabs, wound swabs, blood cultures, skin and soft tissue infections. Methods for collecting and storing biological samples are known to those skilled in the art. The biological sample can be processed (e.g., by nucleic acid extraction methods and / or kits known in the art) to release EBV nucleic acid, or in some cases, the biological sample can be directly contacted with PCR reaction components and appropriate oligonucleotides. In some examples, the biological sample is whole blood. When whole blood is typically collected, it is often collected in a container that contains an anticoagulant, such as heparin, citrate, or EDTA, which allows the whole blood to be stored at an appropriate temperature. However, under such conditions, the nucleic acid in whole blood undergoes a significant amount of degradation. Therefore, it may be advantageous to collect the blood in a reagent that dissolves, denatures, and stabilizes whole blood components, including nucleic acid, such as a nucleic acid stabilizing solution. In such cases, the nucleic acid can be better preserved and stabilized for subsequent isolation and analysis, such as by nucleic acid testing, such as PCR. Such nucleic acid stabilizing solutions are well known in the art and include, but are not limited to, cobas PCR medium containing 4.2 M guanidinium salt (GuHCl) and 50 mM Tris at pH 7.5.

[0067] The sample can be collected by any method or device designed to adequately hold and preserve the sample before analysis. Such methods and devices are well known in the art. When the sample is a biological sample such as whole blood, the method or device can include a blood collection tube. Such blood collection tubes are well known in the art and can include, for example, blood collection tubes. In many cases, it may be advantageous to use a blood collection tube, where the blood collection tube is under pressure in the space intended for sample intake, such as a blood tube with a vacuum chamber, such as a vacutainer blood collection tube. Such blood collection tubes with vacuum chambers, such as vacutainer blood collection tubes, are well known in the art. It may be even more advantageous to collect blood into a blood collection tube with or without a vacuum chamber, such that the whole blood drawn into the blood collection tube immediately comes into contact with the nucleic acid stabilizing solution in the blood collection tube, such as a solution that dissolves, denatures, and stabilizes whole blood components, including nucleic acids, such as a nucleic acid stabilizing solution, which dissolves, denatures, and stabilizes whole blood components, including nucleic acids.

[0068] Melting curve analysis is an additional step that can be included in the cycle profile. Melting curve analysis is based on the fact that DNA melts at a characteristic temperature called the melting temperature (Tm), which is defined as the temperature at which one half of a DNA double strand separates into single strands. The melting temperature of DNA depends mainly on its nucleotide composition. Thus, DNA molecules rich in G and C nucleotides have a higher Tm than DNA molecules rich in A and T nucleotides. By detecting the temperature at which the signal is lost, the melting temperature of the probe can be determined. Similarly, by detecting the temperature at which the signal is generated, the annealing temperature of the probe can be determined. The melting temperature(s) of the probe from the amplification product may confirm the presence or absence of EBV in the sample.

[0069] During each thermocycler run, control samples can be cycled as well. A positive control sample can amplify a target nucleic acid control template (other than the amplification product of the described target gene), for example, using control primers and a control probe. A positive control sample can also amplify, for example, a plasmid construct containing a target nucleic acid molecule. Such a plasmid control can be amplified internally (e.g., in the sample) or in a separate sample run alongside the patient sample, using the same primers and probes used to detect the intended target. Such controls are indicators of the success or failure of the amplification, hybridization and / or FRET reaction. Each thermocycler run can also include a negative control, for example, lacking target template DNA. The negative control can measure contamination. This ensures that the system and reagents do not produce false positive signals. Thus, the control reaction can easily determine, for example, the ability of primers to anneal with sequence specificity and initiate extension, and the ability of probes to hybridize with sequence specificity and FRET to occur.

[0070] In one embodiment, the method includes a step to avoid contamination. For example, an enzymatic method utilizing uracil-DNA glycosylase is described in U.S. Patent Nos. 5,035,996, 5,683,896, and 5,945,313 to reduce or eliminate contamination between one thermocycling run and the next. This method can be carried out using conventional PCR combined with FRET technology. In one embodiment, the LightCycler® instrument is used. The following patent applications: WO 97 / 46707, WO 97 / 46714 and WO 97 / 46712 disclose real-time PCR used with LightCycler® technology.

[0071] Signals from the samples are obtained as the machine sequentially positions the capillaries on the optical unit. The software can display the fluorescence signal in real time immediately after each measurement. Fluorescence acquisition times range from 10 to 100 milliseconds (msec). After each cycling step, a quantitative display of fluorescence versus cycle number can be continuously updated for all samples. The data generated can be stored for further analysis.

[0072] The LightCycler® 480 II Real-Time PCR System may also be operated using a PC workstation. The instrument has a thermal block cycler, with heating and cooling achieved using Peltier elements. Fluorescent signals from samples are obtained from 96-well plates using high-intensity xenon lamps that emit light over a broad spectrum. Flexible combinations of built-in filters for specific excitation and emission allow the use of a variety of fluorescent dyes and detection formats. The software may display the fluorescent signals and calculate CT values, and the data generated may be stored for further analysis.

[0073] As an alternative to FRET, double-stranded DNA binding dyes such as fluorescent DNA binding dyes (e.g., SYBR® Green or SYBR® Gold (Molecular Probes)) can be used to detect the amplification products. Upon interaction with double-stranded nucleic acids, such fluorescent DNA binding dyes emit a fluorescent signal after excitation with light of a suitable wavelength. Double-stranded DNA binding dyes such as nucleic acid intercalating dyes can also be used. When using double-stranded DNA binding dyes, a melting curve analysis is usually performed to confirm the presence of the amplification product.

[0074] Those skilled in the art will understand that other nucleic acid or signal amplification methods can also be used. Examples of such methods include, but are not limited to, branched DNA signal amplification, loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), self-sustained sequence replication (3 SR), strand displacement amplification (SDA), or smart amplification process version 2 (SMAP 2).

[0075] It is understood that embodiments of the present disclosure are not limited by the configuration of one or more commercially available devices.

[0076] Manufactured products / kits The present disclosure further provides an article of manufacture, composition or kit for detecting EBV. The article of manufacture may include primers and probes used to detect target EBV genes, along with suitable packaging materials. Representative primers and probes for detecting EBV may hybridize to target nucleic acid molecules. In addition, the kit may also include appropriately packaged reagents and materials necessary for DNA immobilization, hybridization and detection, such as solid supports, buffers, enzymes and DNA standards. Methods for designing primers and probes are disclosed herein, and representative examples of primers and probes that amplify and hybridize to target nucleic acid molecules are provided.

[0077] The article of manufacture may also include one or more fluorescent moieties for labeling the probe, or may label the probe provided with the kit. For example, the article of manufacture may include donor and / or acceptor fluorescent moieties for labeling the probe. Examples of suitable FRET donor fluorescent moieties and corresponding acceptor fluorescent moieties are provided above.

[0078] The article of manufacture may also include a package insert or packaging label with instructions for using the primers and probes to detect EBV in a sample. The article of manufacture may further include reagents (e.g., buffers, polymerase enzymes, cofactors, or agents to prevent contamination) for carrying out the methods disclosed herein. Such reagents may be specific to one of the commercially available instruments described herein.

[0079] Embodiments of the present disclosure also provide a set of primers and one or more detectable probes for preferentially and / or selectively detecting and / or quantifying EBV in a sample.

[0080] Embodiments of the present disclosure are further described in the following examples, which do not limit the scope of the claimed invention. EXAMPLES

[0081] The following examples and figures are provided to aid the understanding of the subject matter, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.

[0082] In this study, fully automated sample preparation (nucleic acid extraction and purification) was followed by PCR amplification and detection. The system used was a cobas® 6800 / 8800 system, consisting of a sample delivery module, a transfer module, a processing module, and an analysis module. Automated data management was performed by the cobas® 6800 / 8800 system.

[0083] The master mix contained detection probes specific for EBV and control nucleic acids. The specific EBV detection probe and control detection probe were each labeled with one of two unique fluorescent dyes acting as reporters. Each probe also had a second dye acting as a quencher. The reporter dyes were measured at defined wavelengths, thereby allowing detection and discrimination of the amplified EBV targets and controls. The fluorescent signal of the intact probe was suppressed by the quencher dye. During the PCR amplification step, hybridization of the probe to the specific single-stranded DNA template led to cleavage by the 5' to 3' nuclease activity of DNA polymerase, separating the reporter and quencher dyes and producing a fluorescent signal. With each PCR cycle, the amount of cleaved probe increased, and the cumulative signal of the reporter dye increased simultaneously. The two specific reporter dyes were measured at fixed wavelengths, allowing simultaneous detection and discrimination of the amplified EBV targets and controls.

[0084] Primers and probes for EBV testing were designed by seeding the primers and probes according to the genome in the most conserved regions based on the alignment. One set of oligonucleotides (SEQ ID NO: 1-3) was designed to detect the BMRF2 region of the EBV genome. Another set of oligonucleotides (SEQ ID NO: 4-6) was designed to detect the EBNA1 region of the EBV genome. Each set of oligonucleotides (or primers / probes) may be used in singleplex in its own reaction to amplify and detect a specific target region of interest (i.e., BMRF2 and EBNA1). However, the sets of oligonucleotides may also be used together in a dual-targeting assay, whereby both BMRF2 and EBNA1 are amplified and detected in a sample in a single real-time PCR reaction since the reaction mixture contains both oligonucleotide sets (SEQ ID NO: 1-3 and SEQ ID NO: 4-6). For detection of the BMRF2 region, the forward primer corresponds to the nucleic acid sequence of SEQ ID NO: 1, the reverse primer corresponds to the nucleic acid sequence of SEQ ID NO: 3, and the probe corresponds to the nucleic acid sequence of SEQ ID NO: 2. For detection of the EBNA1 region, the forward primer corresponds to the nucleic acid sequence of SEQ ID NO: 4, the reverse primer corresponds to the nucleic acid sequence of SEQ ID NO: 6, and the probe corresponds to the nucleic acid sequence of SEQ ID NO: 5. These oligonucleotides may be used in separate assays for detection of the BMRF2 region of EBV (using SEQ ID NOs: 1-3) and for detection of the EBNA1 region of EBV (using SEQ ID NOs: 4-6). Alternatively, these oligonucleotides may be used in a dual target assay in which the oligonucleotides (SEQ ID NOs: 1-6) simultaneously detect the BMRF2 region and the EBNA1 region of EBV in the same sample. Dual target assays have certain advantages in that if one or more of the target regions contains any kind of mismatch or error, or if one of the reactions experiences an error, there is another target and another reaction that can occur.

[0085] Example 1: Primer and probe design for EBV detection by real-time PCR The EBV nucleic acid test was designed with two targets in mind. The two targets selected were BMRF2 and EBNA1, and are shown in FIG. 1. These candidates can be used individually or together in a dual target assay. When used as a dual target assay, two sets of primers and probes are used (each set detects either EBNA1 or BMRF2). As shown in Table 1 above, the primers for the BMRF2 target have the nucleic acid sequences of SEQ ID NO: 1 and 3, and the probe for the BMRF2 target has the nucleic acid sequence of SEQ ID NO: 2. Also as shown in Table 1 above, the primers for the EBNA1 target have the nucleic acid sequences of SEQ ID NO: 4 and 6, and the probe for the EBNA1 target has the nucleic acid sequence of SEQ ID NO: 5. The amplicon generated by the primers targeting BMRF2 is a 96 base pair long amplicon, and is shown in FIG. 2 (with the positions where the primers and probe overlap with the amplicon). The amplicon generated by the primers targeting EBNA1 is a 98 base pair long amplicon, and is shown in FIG. 3 (with the positions where the primers and probe overlap with the amplicon).

[0086] Example 2: EBV primers and probes detect EBV BMRF2 and EBNA-1 in a real-time PCR assay. EBV nucleic acid testing was performed using primers / probes to detect BMRF2 (SEQ ID NOs: 1-3) and EBNA1 (SEQ ID NOs: 4-6). A full EBV assay was performed. Four types of EBV-containing samples were used: (1) EBV-infected Raji cell extract spiked into EBV-negative plasma; (2) extracted EBV DNA (extracted from B95-8 cell line from Advanced Biotechnologies (catalog no. 17-926-500)); (3) Qnostics EBV analysis panel (EBV1604009C); and (4) EBV-specific ... 第1WHO International Standard. Reagents used include cobas® 6800 / 8800 Universal PCR Master Mix, profiles and conditions for use with cobas® 6800 / 8800 and TaqMan® amplification and detection technology. The final concentrations of oligonucleotides in the master mix were 0.3 μM for primers and 0.1 μM for probes. The cobas® 6800 / 8800 PCR profile used is shown in Table 2 below: [Table 2]

[0087] The results are shown in Table 3 below. [Table 3]

[0088] The first WHO International Standard for EBV stock was at a concentration of 5x106 IU / ml, while the Qnostics EBV Analytic Panel has samples at a range of different concentrations. Serial dilutions of the WHO material and further dilutions of some of the Qnostics panel were performed. The Qnostics EBV Analytic Panel was analyzed at 8,000, 800, 80 and 8 IU / ml, whereas the first WHO International Standard for EBV was analyzed at 800,000, 80,000, 8,000, 800, 80 and 8 IU / ml. Results for the Qnostics EBV Analytic Panel and the first WHO International Standard for EBV are shown in Figures 4A-4C. Another full process test of the dual target assay was performed with both cell culture derived virus material (accurate diagnostic supernatant) as shown in Figures 5H-L, and with control plasmid material containing both target regions as shown in Figures 5A-G. These two materials were diluted respectively and the estimated titers overlapped at 1E4 IU / mL and 1E3 IU / mL for both materials that could be tested. The resulting growth curves are shown in Figure 5A-L. These data and results demonstrate that the primers and probes (SEQ ID NOs: 1-6) amplify and detect the presence of EBV in various types of samples, including commercial and international standards.

[0089] The EBV dual-target and single-target plasmids were tested using the single-target EBV primers and probes. The results are shown in Figures 6A and 6B. This demonstrates that both the BMRF2 and EBNA1 positive control templates are amplified and detected by the respective EBV primers / probes for BMRF2 (SEQ ID NOs: 1-3) and EBNA1 (SEQ ID NOs: 4-6), but each single-target control plasmid does not cross-react with the other target, whereas the dual-target control plasmid and viral genomic DNA extract are amplified by both oligo sets.

[0090] Linearity testing of the dual target assay was performed on EBV genotype 1 and EBV genotype 2 material. For genotype 1, both cell culture derived viral material (accurate diagnosis) and control plasmid material containing both target regions were used, with overlapping estimated titers. For genotype 2, cell culture derived viral material (ATCC strain P3) was tested. The results are shown in Figures 7 and 8. These data and results indicate that the assay can be used to detect and quantify both EBV genotype 1 and genotype 2.

[0091] Although the foregoing invention has been described in some detail for clarity and understanding, it will be apparent to those skilled in the art upon reading this disclosure that various changes in form and detail may be made without departing from the true scope of the invention. For example, all of the techniques and devices described above may be used in various combinations. All publications, patents, patent applications, and / or other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, and / or other document was individually indicated to be incorporated by reference for all purposes.

Claims

1. A method for detecting one or more target nucleic acids of Epstein-Barr Virus (EBV) in a collected sample, comprising: (a) providing a nucleic acid sample from said sample; (b) if said one or more target nucleic acids of EBV are present in said sample, performing an amplification step comprising contacting said nucleic acid sample with one or more sets of primers to produce an amplification product; (c) if said one or more target nucleic acids of EBV are present in said sample, performing a hybridization step comprising contacting said amplification products with one or more probes; and (d) performing a detection step comprising detecting the presence or absence of said amplification product, the presence of said amplification product indicating the presence of said one or more target nucleic acids of EBV in said sample and the absence of said amplification product indicating the absence of said one or more target nucleic acids of EBV in said sample; the one or more sets of primers and the one or more probes (i) a set of primers comprising a first primer comprising the nucleic acid sequence of SEQ ID NO:1 and a second primer comprising the nucleic acid sequence of SEQ ID NO:3, and a probe comprising the nucleic acid sequence of SEQ ID NO:2 or its complement; and / or (ii) a set of primers comprising a first primer comprising the nucleic acid sequence of SEQ ID NO: 4 and a second primer comprising the nucleic acid sequence of SEQ ID NO: 6, and a probe comprising the nucleic acid sequence of SEQ ID NO: 5 or a complement thereof; method.

2. The method of claim 1 , wherein the sample is a biological sample, and the biological sample is plasma or blood.

3. A method for detecting a first target nucleic acid of EBV and a second target nucleic acid of EBV in the sample, comprising: the one or more sets of primers and the one or more probes for detecting the first target nucleic acid of EBV comprise: (i) a set of primers comprising a first primer comprising a nucleic acid sequence of SEQ ID NO:1 and a second primer comprising a nucleic acid sequence of SEQ ID NO:3, and a probe comprising a nucleic acid sequence of SEQ ID NO:2 or a complement thereof; The method of claim 1 or 2, wherein the one or more sets of primers and the one or more probes for detecting the second target nucleic acid of EBV comprise: (ii) a set of primers comprising a first primer comprising a nucleic acid sequence of SEQ ID NO: 4 and a second primer comprising a nucleic acid sequence of SEQ ID NO: 6, and a probe comprising a nucleic acid sequence of SEQ ID NO: 5 or its complement.

4. the first target nucleic acid of EBV and the second target nucleic acid of EBV are different; and The method of claim 3 , wherein the first target nucleic acid of EBV and the second target nucleic acid of EBV are non-overlapping.

5. A method for detecting a first target nucleic acid of EBV and a second target nucleic acid of EBV in a collected sample, comprising: (a) providing a nucleic acid sample from said sample; (b) if one or more of the target nucleic acids of EBV are present in the nucleic acid sample, performing an amplification step comprising contacting the nucleic acid sample with at least two sets of primers to produce an amplification product; (c) if one or more of said target nucleic acids of EBV are present in said sample, performing a hybridization step comprising contacting said amplification products with at least two probes; and (d) performing a detection step comprising detecting the presence or absence of said amplification product, the presence of said amplification product indicating the presence of one or more of said target nucleic acids of EBV in said sample and the absence of said amplification product indicating the absence of one or more of said target nucleic acids of EBV in said sample; one of the at least two sets of primers and one of the at least two probes for detecting the first target nucleic acid of EBV; (i) a set of primers comprising a first primer comprising the nucleic acid sequence of SEQ ID NO:1 and a second primer comprising the nucleic acid sequence of SEQ ID NO:3, and a probe comprising the nucleic acid sequence of SEQ ID NO:2 or its complement; one of the one or more sets of primers and one of the at least two probes for detecting the second target nucleic acid of EBV; (ii) a set of primers comprising a first primer comprising the nucleic acid sequence of SEQ ID NO: 4 and a second primer comprising the nucleic acid sequence of SEQ ID NO: 6, and a probe comprising the nucleic acid sequence of SEQ ID NO: 5 or a complement thereof; method.

6. 6. The method of claim 5, wherein the first target nucleic acid of EBV and the second target nucleic acid of EBV are different, and the first target nucleic acid of EBV and the second target nucleic acid of EBV do not overlap.

7. 7. The method of claim 5 or 6, wherein the sample is a biological sample, the biological sample being plasma or blood.

8. 1. A kit for detecting one or more target nucleic acids of EBV possibly present in a sample, said kit comprising: (a) a DNA polymerase; (b) a nucleotide monomer; (c) one or more sets of primers, and (d) one or more probes an amplification reagent comprising the one or more sets of primers and the one or more probes (i) a set of primers comprising a first primer comprising the nucleic acid sequence of SEQ ID NO:1 and a second primer comprising the nucleic acid sequence of SEQ ID NO:3, and a probe comprising the nucleic acid sequence of SEQ ID NO:2 or its complement; and / or (ii) a set of primers comprising a first primer comprising the nucleic acid sequence of SEQ ID NO: 4 and a second primer comprising the nucleic acid sequence of SEQ ID NO: 6, and a probe comprising the nucleic acid sequence of SEQ ID NO: 5 or a complement thereof; kit.

9. The kit of claim 8 , wherein the one or more probes are labeled with a donor fluorescent moiety and a corresponding acceptor moiety.

10. 10. A kit according to claim 8 or 9 for detecting a first target nucleic acid of EBV and a second target nucleic acid of EBV in a sample, comprising: The one or more sets of primers and the one or more probes for detecting the first target nucleic acid of EBV, (i) a set of primers comprising a first primer comprising the nucleic acid sequence of SEQ ID NO:1 and a second primer comprising the nucleic acid sequence of SEQ ID NO:3, and a probe comprising the nucleic acid sequence of SEQ ID NO:2 or its complement; The one or more sets of primers and the one or more probes for detecting the second target nucleic acid of EBV are (ii) a set of primers comprising a first primer comprising the nucleic acid sequence of SEQ ID NO: 4 and a second primer comprising the nucleic acid sequence of SEQ ID NO: 6, and a probe comprising the nucleic acid sequence of SEQ ID NO: 5 or a complement thereof; kit.

11. The kit of claim 10, wherein the first target nucleic acid of EBV and the second target nucleic acid of EBV are different, and the first target nucleic acid of EBV and the second target nucleic acid of EBV do not overlap.

12. 1. A kit for detecting an EBV first target nucleic acid and an EBV second target nucleic acid in a sample, the kit comprising: (a) a DNA polymerase; (b) a nucleotide monomer; (c) a first set of primers and one probe for detecting a first target nucleic acid of EBV; and (d) a second set of primers and one probe for detecting a second target nucleic acid of EBV; an amplification reagent comprising the first set of primers and one probe are for detecting the first target nucleic acid of EBV, and comprise a first primer comprising a nucleic acid sequence of SEQ ID NO:1, a second primer comprising a nucleic acid sequence of SEQ ID NO:3, and a probe comprising SEQ ID NO:2 or a complement thereof; the second set of primers and one probe is for detecting the second target nucleic acid of EBV, and comprises a first primer comprising a nucleic acid sequence of SEQ ID NO: 4, a second primer comprising a nucleic acid sequence of SEQ ID NO: 6, and a probe comprising a nucleic acid sequence of SEQ ID NO: 5 or its complement; kit.

13. The kit of claim 12 , wherein the probe is labeled with a donor fluorescent moiety and a corresponding acceptor moiety.

14. The kit of claim 12 or 13, wherein the first target nucleic acid of EBV and the second target nucleic acid of EBV are different, and the first target nucleic acid of EBV and the second target nucleic acid of EBV do not overlap.

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