JP7582674B2 - Kit for detecting bovine leukemia virus - Google Patents

Description

This invention relates to a kit comprising a primer set for detecting bovine leukemia virus.

Bovine leukemia is a disease whose main symptoms are an increase in white blood cells and systemic malignant lymphosarcoma, and its causes are broadly divided into viral and non-viral. Non-viral bovine leukemia is called sporadic and has an unknown cause, and is divided into three types based on symptoms: calf type, thymic type, and cutaneous type. Viral bovine leukemia is called endemic, is caused by the bovine leukemia virus (BLV), and accounts for the majority of bovine leukemia cases.

Most BLV-infected cattle are asymptomatic, but 20-30% of infected cattle develop persistent lymphocytosis, which is characterized by abnormal proliferation of polyclonal B cells. Furthermore, 2-3% of infected cattle develop endemic bovine leukemia, a type of B-cell leukemia, which leads to the formation of malignant lymphosarcoma in the lymph nodes and leads to death with a poor prognosis.

In addition to the economic losses caused by the onset of bovine leukemia, BLV often causes economic losses due to reduced milk production. A survey by the USDA's National Animal Health Monitoring System found that for every 10% increase in BLV-infected cows in a herd, 95 kg of milk is lost per cow per year.

BLV belongs to the family Retroviridae, subfamily Orthoretrovirinae, and subfamily Deltaretrovirus. The genome consists of 8714 nucleotides and is flanked by two identical long terminal repeats (LTRs) (N Sagata, T Yasunaga, J Tsuzuku-Kawamura, K Ohishi, Y Ogawa, Y Ikawa. (1985). Complete nucleotide sequence of the genome of bovine leukemia virus: its evolutionary relationship to other retroviruses. Proceedings of the National Academy of Sciences of the United States of America. 82: 677-681). The genes gag, pro, pol, and env, which code for structural proteins and enzymes, are essential for the viral life cycle, infectivity, and infectious virion production (Sagata et al., etc.).

A schematic diagram of the BLV genome structure is shown in FIG.
BLV infects various peripheral mononuclear cells and is integrated into the host genome as a provirus, and the BLV provirus is present in the host genome at all stages of the disease progression. ELISA for serological diagnosis and real-time PCR for provirus detection are used for BLV testing (Schoepf KC, Kapaga AM, Msami HM, Hyera JM. (1997). Serological evidence of the occurrence of enzootic bovine leukosis (EBL) virus infection in cattle in Tanzania. Tropical Animal Health and Production. 29:15-9, Y Somura, E Sugiyama, H Fujikawa, K Murakami. (2014). Comparison of the copy numbers of bovine leukemia virus in the lymph nodes of cattle with enzootic bovine leukosis and cattle with latent infection. Archives of virology. 159:2693-7).

The advantages of diagnosis using real-time PCR include the ability to use a variety of samples, high sensitivity, rapidity, and the ability to quantify provirus. Disadvantages include complicated procedures, the need for expensive equipment, and the need to design new primers for mutant strains. A large number of mutant strains of BLV have been registered with NCBI, and it is expected that mutant strains with unknown sequences will continue to appear in the future, so it is extremely important to develop a PCR method that targets sequences with few mutations.

Schoepf KC, Kapaga AM, Msami HM, Hyera JM. (1997). Serological evidence of the occurrence of enzootic bovine leukosis (EBL) virus infection in cattle in Tanzania. Tropical Animal Health and Production. 29:15-9 Y Somura, E Sugiyama, H Fujikawa, K Murakami. (2014). Comparison of the copy numbers of bovine leukemia virus in the lymph nodes of cattle with enzootic bovine leukosis and cattle with latent infection. Archives of virology. 159:2693－7

There was a demand for a kit for detecting bovine leukemia virus that contained a primer set that could detect with high sensitivity even subjects infected with the bovine leukemia virus, which had previously been undetectable.

The above problem is based on the finding from examples that by using a kit that employs a certain primer set, subjects infected with bovine leukemia virus that was previously undetectable can be detected with high sensitivity.

One of the inventions described in this specification relates to a kit for detecting bovine leukemia virus, which includes a primer set. The kit may be any kit as long as it includes the primer set.
The primer set includes a forward primer and a reverse primer.
The forward primer is an oligonucleotide consisting of the base sequence shown in SEQ ID NO:1 or SEQ ID NO:4.
The reverse primer is an oligonucleotide consisting of the base sequence shown in SEQ ID NO:2 or SEQ ID NO:5.
SEQ ID NO: 1: ACCATGGACTTTCACTTCTGG
SEQ ID NO: 2: GGCCAAGACAAGGGGAAC
SEQ ID NO: 4: CTCCCCAACTTCCCCTTTC
SEQ ID NO: 5: ACGGGTCTCAGGAGAAG
The bases constituting the primer may be appropriately modified.

A preferred example of the above kit further comprises a probe which is an oligonucleotide consisting of the base sequence shown in SEQ ID NO:3 or 6.
SEQ ID NO: 3: CCGGGTGGACATGAGGTGGG
SEQ ID NO: 6: TCGAGTTAGCGGCACCAGAAGC
The bases constituting the probe may be appropriately modified. Specifically, for example, they may be modified with a fluorescent dye, a quencher, a quenching agent, or a dark quencher. In particular, the 5'-end or 3'-end may have these modifications. They may also be modified with an internal quencher.
In particular, those in which the 5'-terminus and 3'-terminus are modified with a quencher and a dark quencher are preferred, and further, those in which the base between the 5'-terminus and the 3'-terminus is modified with an internal quencher may also be used.
An example of a probe having modifications is, for SEQ ID NO: 3,
56-FAM/CCGGGTGGACATGAGGTGGG/3IABkFQ, or 56-FAM/CCGGGTGGA/ZEN/CATGAGGTGGG/3IABkFQ.
An example of a probe having modifications is, for SEQ ID NO: 6,
56-FAM/TCGAGTTAGCGGCACCAGAAGC/3IABkFQ, or 56-FAM/TCGAGTTAG/ZEM/CGGCACCAGAAGC/3IABkFQ.
56-FAM is one of the isomers of fluorescein and is a commonly used fluorescent dye.
ZEN means ZEN quencher, which is an internal quencher that is usually located between 9 and 10 bases from the fluorescent dye at the 5' end and cooperates with the quencher at the 3' end to perform quenching.
3IABkFQ is a dark quencher. 3IABkFQ can also be modified at the 5' end.

An example of a primer set is one in which the forward primer is an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 1, and the reverse primer is an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 2. In this example, the preferred probe is an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 3.

Another example of a primer set is an oligonucleotide in which the forward primer consists of the base sequence shown in SEQ ID NO: 4, and the reverse primer consists of the base sequence shown in SEQ ID NO: 5. In this example, the preferred probe is an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 6.

This specification also provides a PCR method using the above primer set or kit, and a method for detecting a subject infected with the bovine leukemia virus. A sample such as blood is obtained from the subject, and the target nucleic acid is amplified and detected using the above primer set or kit. In this way, it is possible to detect with higher sensitivity than ever before whether or not a subject is infected with the bovine leukemia virus.

The invention described in this specification makes it possible to detect with high sensitivity subjects infected with bovine leukemia virus, which was previously undetectable.

FIG. 1 is a schematic diagram of the genome structure of bovine leukemia virus (BLV). FIG. 2 is a diagram showing the location of the storage area. FIG. 3 is a graph and a table in place of a drawing showing the results of real-time PCR of synthetic DNA. FIG. 4 is a graph and a table in place of a drawing showing the results of real-time PCR of synthetic DNA. FIG. 5-1 is a graph and table in place of a drawing showing the results of real-time PCR of DNA extracted from BLV-negative bovine whole blood. FIG. 5-2 is a graph and table in place of a drawing showing the results of real-time PCR of DNA extracted from BLV-negative bovine whole blood. FIG. 6 is a photograph, instead of a drawing, showing the results of electrophoresis. FIG. 7 is a graph and a table, instead of a drawing, showing the results of the spike test. FIG. 8 is a graph and a table, instead of a drawing, showing the results of the spike test.

Hereinafter, the embodiments for carrying out the present invention will be described with reference to the drawings. The present invention is not limited to the embodiments described below, but also includes appropriate modifications of the embodiments described below within the scope obvious to those skilled in the art.

As used herein, "amplification," "amplification method," or "amplification reaction" are used interchangeably herein and refer to a method or process for increasing the representation of a population of specific nucleic acid (any type of DNA or RNA) sequences (e.g., target sequences or target nucleic acids) in a test sample. Specific examples of amplification methods that may be used in the present invention include PCR, real-time PCR, RT-PCR, competitive RT-PCR, LCR, gap LCR, SDA, NASBA, and TMA.

As used herein, the term "amplification conditions" refers to conditions that promote annealing and/or extension of primer sequences. Such conditions are well known to those of skill in the art and depend on the amplification method selected. For example, PCR amplification conditions generally include thermal cycling, e.g., cycling the reaction mixture between two or more temperatures. In isothermal amplification reactions, an initial temperature increase may be required to initiate the reaction, but amplification occurs without thermal cycling. Amplification conditions include all reaction conditions, including, but not limited to, temperature and temperature cycling, buffers, salts, ionic strength, pH, etc.

As used herein, the term "amplification reagents" refers to reagents used in an amplification reaction and may include, but are not limited to, buffers, reagents, enzymes having reverse transcription and/or polymerase or exonuclease activity; enzyme cofactors, such as magnesium or manganese, salts; and deoxynucleotide triphosphates (dNTPs), such as deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP), deoxythymidine triphosphate (dTTP), and deoxyuridine triphosphate (dUTP). Amplification reagents can be readily selected by one of skill in the art depending on the amplification method used.

The term "directly detectable" as used herein with respect to a detectable label or detectable moiety means that the detectable label or detectable moiety does not require further reaction or manipulation in order to be detectable. For example, a fluorescent moiety is directly detectable by fluorescence spectroscopy. In contrast, the term "indirectly detectable" as used herein with respect to a detectable label or detectable moiety means that the detectable label or detectable moiety becomes detectable following further reaction or manipulation. For example, a hapten becomes detectable following reaction with an appropriate antibody conjugated to a reporter such as a fluorescent dye.

The terms "fluorophore," "fluorescent moiety," "fluorescent label," and "fluorescent dye" are used interchangeably herein to refer to a molecule that absorbs a quantum of electromagnetic radiation of one wavelength and in response emits one or more photons of a different, typically longer, wavelength. Numerous fluorescent dyes of widely varying structures and characteristics are suitable for use in the practice of the present invention. Methods and materials for fluorescently labeling nucleic acid molecules are known (see R.P. Haugland, "Molecular Probes: Handbook of Fluorescent Probes and Research Chemicals 1992-1994", 5th Ed., 1994, Molecular Probes, Inc.). Preferably, the fluorescent label or moiety absorbs and emits light with high efficiency (e.g., exhibits a high molar absorption coefficient and a high fluorescence quantum yield at the excitation wavelength used) and is photostable (e.g., does not undergo significant decomposition upon photoexcitation within the time required to perform the assay). Instead of being directly detectable themselves, some fluorescent dyes transfer energy to another fluorescent dye in a process called fluorescence resonance energy transfer (FRET), which second dye generates a detectable signal. Such FRET fluorescent dye pairs are also included in the term "fluorescent moiety". Physically linked fluorescent reporter/quencher moieties are also within the scope of the present invention. In these embodiments, when the fluorescent reporter and quencher moieties are kept in close proximity, for example at the end of a probe, the quencher moiety prevents detection of the fluorescent signal from the reporter moiety. When the two moieties are physically separated, for example after cleavage by DNA polymerase, the fluorescent signal from the reporter moiety becomes detectable.

As used herein, the term "hybridization" refers to the formation of a complex between nucleic acid sequences that are sufficiently complementary to form a complex by Watson-Crick base pairing or non-canonical base pairing. For example, when a primer "hybridizes" to a target sequence (template), such a complex (or hybrid) is stable enough to perform the priming function required, for example, by a DNA polymerase to initiate DNA synthesis. It will be understood by those of skill in the art that hybridizing sequences need not have perfect complementarity to form a stable hybrid. In many cases, stable hybrids will result when less than about 10% of the bases are mismatched. Thus, the term "complementary" as used herein refers to an oligonucleotide that forms a stable duplex with its corresponding complement under assay conditions, and generally refers to about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% or greater homology. Those of skill in the art understand how to estimate and adjust the stringency of hybridization conditions such that sequences with at least a desired level of complementarity will stably hybridize, while sequences with less complementarity will not stably hybridize. Specific examples of hybridization conditions and parameters can be found, for example, in Sambrook et al., "Molecular Cloning: A Laboratory Manual", 1989, Second Edition, Cold Spring Harbor Press: Plainview, NY; F. M. Ausubel, "Current Protocols in Molecular Biology", 1994, John Wiley & Sons: Secaucus, NJ.

As used herein, the terms "labeled" and "labeled with a detectable label (or substance or moiety)" are used interchangeably herein and mean that an entity (e.g., a primer or probe) can be visualized, for example, after binding to another entity (e.g., an amplification product or amplicon). Preferably, the detectable label is selected such that it generates a signal that can be measured and whose intensity is related (e.g., proportional) to the amount of bound entity. A wide variety of systems for labeling and/or detecting nucleic acid molecules (e.g., primers and probes) are well known in the art. Labeled nucleic acids can be prepared by incorporating or binding to a label that is directly or indirectly detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, chemical or other means. Suitable detectable substances include, but are not limited to, radionuclides, fluorophores, chemiluminescent substances, microparticles, enzymes, colorimetric labels, magnetic labels, haptens, molecular beacons, aptamer beacons, and the like.

The term "primer" refers to an oligonucleotide that can act as a point of initiation for the synthesis of a primer extension product that is a complementary strand of a nucleic acid (any type of DNA or RNA) when placed under appropriate amplification conditions (e.g., buffer, salt, temperature and pH) in the presence of nucleotides and an agent for nucleic acid polymerization (e.g., DNA- or RNA-dependent polymerase). The primer can be single-stranded or double-stranded. If double-stranded, the primer can first be treated (e.g., denatured) to allow separation of its strands before being used to prepare an extension product. Such a denaturation step is typically performed using heat, but can alternatively be performed by the use of alkali followed by neutralization. The primers of the present invention have a length of about 15 to 50 nucleotides, preferably about 20 to about 40 nucleotides, and most preferably about 22 to 30 nucleotides. The primers of the present invention can contain additional nucleotides in addition to those described in more detail herein. For example, primers used in SDA can contain a restriction endonuclease recognition site 5' to the target binding sequence (see U.S. Patent Nos. 5,270,184 and 5,455,166), and NASBA and TMA primers can contain an RNA polymerase promoter linked to the target binding sequence of the primer. Methods for linking such specialized sequences to the target binding sequence used in the selected amplification reaction are well known to those of skill in the art. Also, in some cases, the primers can be labeled with a detectable label.

The term "forward primer" refers to a primer that hybridizes (or anneals) to a target sequence (e.g., a template strand). The term "reverse primer" refers to a primer that hybridizes (or anneals) to the complementary strand of a target sequence. The forward primer hybridizes to the target sequence 5' to the reverse primer.

As used herein, the term "set" or "primer set" refers to two or more primers that together can prime the amplification of a target sequence or nucleic acid of interest (e.g., a target sequence in bovine leukemia virus). In certain embodiments, the term "primer set" refers to a pair of primers that includes a 5' (upstream) primer (or forward primer) that hybridizes to the 5' end of the target sequence or nucleic acid to be amplified and a 3' (downstream) primer (or reverse primer) that hybridizes to the complement of the target sequence or nucleic acid to be amplified. Such primer sets or primer pairs are particularly useful in PCR amplification reactions.

As used herein, the term "probe" refers to an oligonucleotide that can selectively hybridize to at least a portion of a target sequence (e.g., a portion of an amplified target sequence) under appropriate amplification conditions. In general, probe sequences are recognized to be either "complementary" (i.e., complementary to the coding or sense strand (+)) or "reverse complementary" (i.e., complementary to the antisense strand (-)). In some cases, probes may be labeled with a detectable label.

As used herein, the term "primer and probe set" refers to a combination that includes two or more primers that together can prime the amplification of a target sequence or nucleic acid, and at least one probe that can detect the target sequence or nucleic acid. The probe typically hybridizes to a strand of an amplification product to form an amplification product/probe hybrid that can be detected using conventional techniques known to those of skill in the art.

The terms "target sequence" and "target nucleic acid" are used interchangeably herein to mean something whose presence or absence is desired to be detected. In the context of the present invention, a target sequence preferably includes a nucleic acid sequence to which one or more primers form a complex. The target sequence may also include a region that hybridizes to a probe, where the probe forms a stable hybrid under appropriate amplification conditions. As will be appreciated by those of skill in the art, a target sequence may be single-stranded or double-stranded.

As used herein, the term "test sample" generally refers to a biological material suspected of containing a target nucleic acid of interest and/or being tested.

The primers and probes of the present invention can be produced by any of a variety of methods known in the art (see, for example, Sambrook et al., "Molecular Cloning. A Laboratory Manual", 1989, 2. Supp. Ed., Cold Spring Harbor Laboratory Press: New York, NY; "PCR Protocols. A Guide to Methods and Applications", 1990, M.A. Innis (ed.), Academic Press: New York, NY; P. Tijssen, "Hybridization with Nucleic Acids", 1990, M.A. Innis (ed.), Academic Press: New York, NY; See, "Probes-Laboratory Techniques in Biochemistry and Molecular Biology (Parts I and II)", 1993, Elsevier Science; "PCR Strategies", 1995, M. A. Innis (ed.), Academic Press: New York, NY; and "Short Protocols in Molecular Biology", 2002, F. M. Ausubel (ed.), 5. Supp. Ed., John Wiley & Sons: Secaucus, NJ. For example, the primers and probes described herein can be prepared by chemical synthesis and template-based polymerization (e.g., as described in Narang et al., Meth. Enzymol., 1979, 68:90-98; Brown et al., Meth. Enzymol., 1979, 68:109-151 and Belousov et al., Nucleic Acids Res., 1997, 25:3440-3444).

The sequences of the primers and probes may be confirmed using any suitable sequencing method known in the art, including, but not limited to, chemical degradation (see Maxam et al., Methods of Enzymology, 1980, 65:499-560), matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (see Pieles et al., Nucleic Acids Res., 1993, 21:3191-3196), combined alkaline phosphatase and exonuclease digestion followed by mass spectrometry (Wu et al., Anal. Biochem., 2001, 290:347-352), and the like.

As already described above, modified primers and probes can be prepared by any of several means known in the art. Non-limiting examples of such modifications include methylation, "caps," substitution of one or more of the natural nucleotides with analogs, and internucleotide modifications, such as those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.) or charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.). Primers and probes can contain one or more additional covalently linked moieties, such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalators (e.g., acridine, psoralen, etc.), chelators (e.g., for chelating metals, radioactive metals, oxidative metals, etc.), and alkylators. Primers and probes can be derivatized by formation of methyl or ethyl phosphotriester or alkyl phosphoramidate linkages. Additionally, the primers, probes or primers and probes of the present invention may also be modified with a detectable label.

As mentioned above, in certain embodiments of the invention, the primers, probes, or both the primers and probes may be labeled with a detectable label or moiety prior to use in the amplification/detection method. Preferably, when used in the methods described herein, one or more probes are labeled with a detectable label or moiety. The role of the detectable label is to allow visualization and detection of the amplified target sequence. Preferably, the detectable label is selected such that it generates a signal that can be measured and whose intensity is related (e.g., proportional) to the amount of amplification product in the test sample being analyzed.

The bond between one or more probes and the detectable label can be covalent or non-covalent. Labeled probes can be prepared by incorporation of or conjugation to a detectable moiety. The label can be attached directly or indirectly (e.g., via a linker) to the nucleic acid sequence. Linkers or spacer arms of various lengths are known in the art and commercially available, and can be selected to reduce steric hindrance or to impart other useful or desired properties to the resulting labeled molecule (see, e.g., Mansfield et al., Mol. Cell. Probes, 1995, 9:145-156).

Methods for labeling oligonucleotides such as probes are known to those of skill in the art. Reviews of labeling protocols and label detection techniques can be found, for example, in L. J. Kricka, Ann. Clin. Biochem., 2002, 39:114-129; van Gijlswijk et al., Expert Rev. Mol. Diagn., 2001, 1:81-91; and Joos et al., J Biotechnol, 1994, 35:135-153. Standard nucleic acid labeling methods include incorporation of radioactive materials, direct attachment of fluorescent dyes (see Smith et al., Nucl. Acids Res., 1985, 13:2399-2412) or enzymes (see Connoly et al., Nucl. Acids Res., 1985, 13:4485-4502); chemical modification of the nucleic acid molecule to render it detectable immunochemically or by other affinity reactions (see Broker et al., Nucl. Acids Res., 1978, 5:363-384; Bayer et al., Methods of Biochem. Analysis, 1980, 26:1-45; Langer et al., Proc. Natl. Acad. Sci., 1999, 26:1-45; Langer et al., Proc. Natl. Acad. Sci., 1999, 26:1-45). USA, 1981, 78:6633-6637; Richardson et al., Nucl. Acids Res., 1983, 11:6167-6184; Brigati et al., Virol., 1983, 126:32-50; Tchen et al., Proc. Natl. Acad. Sci. USA, 1984, 81:3466-3470; Landegent et al., Exp. Cell Res., 1984, 15:61-72; and A. H. Hopman et al., Exp. Cell Res. Res., 1987, 169:357-368); enzyme-mediated labeling methods, such as random priming, nick translation, PCR, and terminal transferase tailing (for a review on enzyme labeling, see, e.g., Temsamani et al., Mol. Biotechnol., 1996, 5:223-232).

Any of a wide variety of detectable labels may be used in the present invention. Suitable detectable labels include, but are not limited to, various ligands, radionuclides (e.g., 32P, 35S, 3H, 14C, 125I, 131I, etc.); fluorescent dyes; chemiluminescent materials (e.g., acridinium esters, stabilized dioxetanes, etc.); spectrally resolvable inorganic fluorescent semiconductor nanocrystals (e.g., quantum dots), metal nanoparticles (e.g., gold, silver, copper, and platinum) or nanoclusters; enzymes (e.g., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase); colorimetric labels (e.g., dyes, colloidal gold, etc.); magnetic labels (e.g., Dynabeads™); and biotin, dioxygenin, or other haptens, proteins for which antisera or monoclonal antibodies are available.

In certain embodiments, the detection probes of the invention are fluorescently labeled. Numerous known fluorescent labeling moieties of widely varying chemical structures and physical properties are suitable for use in the practice of the invention. Suitable fluorescent dyes include the Quasar® dyes available from Biosearch Technologies, Novato, CA, fluorescein and fluorescein dyes such as fluorescein isothiocyanine or FITC, naphthofluorescein, 4',5'-dichloro-2',7'-dimethoxy-fluorescein, 6-carboxyfluorescein (e.g., FAM), VIC, NED, carbocyanine, merocyanine, cytyryl dyes, oxonol dyes, fucoerythrin, erythrosine, eosin, rhodamine dyes (e.g., carboxytetramethylrhodamine or are TAMRA, carboxyrhodamine 6G, carboxy-X-rhodamine (ROX), lissamine rhodamine B, rhodamine 6G, rhodamine green, rhodamine red, tetramethylrhodamine or TMR), coumarins and coumarin dyes (e.g., methoxycoumarin, dialkylaminocoumarin, hydroxycoumarin, and aminomethylcoumarin or AMCA), Oregon Green dyes (e.g., Oregon Green 488, Oregon Green 500, Oregon Green 514), Texas Red, Texas Red-X, Spectrum Red™, Spectrum Green™, cyanine dyes (e.g., Cy-3™, Cy-5™, Cy-3.5™, Cy-5.5™), Alexa Fluor dyes (e.g., Alexa Fluor 350, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660, and Alexa Fluor 680), BODIPY dyes (e.g., BODIPY FL, BODIPY 594, Alexa Fluor 633, Alexa Fluor 660, and Alexa Fluor 680), Examples of suitable fluoropolymers include, but are not limited to, BODIPY R6G, BODIPY TMR, BODIPY TR, BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 630/650, BODIPY 650/665), IRDyes (e.g., IRD 40, IRD 700, IRD 800), and the like. Other suitable fluorescent dyes that may be used, and specific examples of methods for linking or incorporating fluorescent dyes into oligonucleotides such as probes, can be found in RP Haugland, "The Handbook of Fluorescent Probes and Research Chemicals", Publisher, Molecular Probes, Inc., Eugene, Oreg. (June 1992). Fluorescent dyes and labeling kits are available, for example, from Amersham Biosciences, Inc. (Piscataway, N.J.), Molecular Probes Inc. (Eugene, Oreg.) and New England Biolabs Inc. (Beverly, MA).

Some fluorescent groups (donors), instead of being directly detectable themselves, transfer energy to another fluorescent group (acceptor) in a process of fluorescence resonance energy transfer (FRET), which second group generates a detectable fluorescent signal. In these embodiments, the probe may be detectable, for example, when hybridized to an amplified target sequence. Examples of FRET acceptor/donor pairs suitable for use in the present invention include, but are not limited to, fluorescein/tetramethylrhodamine, IAEDANS/FITC, IAEDANS/5-(iodoacetomido)fluorescein, B-phycoerythrin/Cy-5, and EDANS/Dabcyl.

The use of physically linked fluorescent reporter/quencher molecules is also within the scope of the invention, either in TaqMan® assays (e.g., as described in U.S. Pat. Nos. 5,210,015, 5,804,375, 5,487,792, and 6,214,979) or as Molecular Beacons (e.g., Tyagi et al., Nature Biotechnol., 1996, 14:303-308; Tyagi et al., Nature Biotechnol., 1998, 16:49-53; Kostrikis et al., Science, 1998, 279:1228-1229; Sokol et al., Proc. Natl. Acad. Sci., 1999, 279:1228-1229). USA, 1998, 95:11538-11543; Marras et al., Genet. Anal, 1999, 14:151-156; and U.S. Patent Nos. 5,846,726, 5,925,517, 6,277,581, and 6,235,504) the use of such systems is well known to those of skill in the art. In the TaqMan® assay format, the product of the amplification reaction can be detected as it is formed in a "real-time" manner. As a result, amplification product/probe hybrids are formed and detected while the reaction mixture is under amplification conditions.

In some embodiments of the invention, the PCR detection probe is a TaqMan®-like probe labeled with a fluorescent moiety at the 5' end and a quencher moiety at the 3' end. Suitable fluorophores and quenchers for use in TaqMan®-like probes are disclosed in U.S. Patent Nos. 5,210,015, 5,804,375, 5,487,792 and 6,214,979, and WO 01/86001, each of which is incorporated herein by reference. Specific examples of quenchers include DABCYL (e.g., 4-(4'-dimethylaminophenylazo)benzoic acid), succinimidyl ester, diarylrhodamine carboxylic acid, succinimidyl ester (or QSY-7), and 4',5'-dinitrofluorescein carboxylic acid, succinimidyl ester (or QSY-33), all of which are available from Molecular Probes, which is part of Invitrogen, Carlsbad, Calif.; Quencher 1 (Q1; available from Epoch Biosciences, Bothell, Wash.); or "Black hole quenchers" BHQ-1, BHQ-2, and BHQ-3 (BioSearch, Inc.). Examples of suitable PCR detection probes include, but are not limited to, TaqMan®-like probes labeled with FAM at the 5' end and Black Hole Quencher® or Black Hole Quencher® plus at the 3' end (both available from Biosearch Technologies, Novato, Calif.).

Alternatively, a "tail" of normal or modified nucleotides can be added to the probe for detection purposes. A second hybridization with a nucleic acid complementary to the tail and containing one or more detectable labels (e.g., fluorophores, enzymes, or radiolabeled bases) allows visualization of the amplicon/probe hybrids.

The choice of a particular labeling technique will be situational and will depend on several factors, such as the ease and cost of the labeling method, the spectral spacing between the different detectable labels used, the quality of sample labeling desired, the effect of the detectable moiety on the hybridization reaction (e.g., on the speed and/or efficiency of the hybridization process), the nature of the amplification method used, the nature of the detection system, and the nature and strength of the signal generated by the detectable label.

The use of the primers or primer sets of the present invention is not limited to any particular nucleic acid amplification technique or any particular variant thereof. Indeed, the primers and primer sets of the present invention can be used in a variety of nucleic acid amplification methods known in the art (e.g., Kimmel et al., Methods Enzymol., 1987, 152:307-316; Sambrook et al., "Molecular Cloning. A Laboratory Manual", 1989, 2. Supp. Ed., Cold Spring Harbor Laboratory Press: New York, NY; "Short Protocols in Molecular Biology", F.M. Ausubel (ed.), 2002, 5. Supp. Ed., John Wiley & Sons, 2003, 114:111-115, 1997; Sons: Secaucus, NJ).

Such nucleic acid amplification methods include, but are not limited to, the polymerase chain reaction (PCR), which is described in "PCR Protocols: A Guide to Methods and Applications", M. A. Innis (ed.), 1990, Academic Press: New York; "PCR Strategies", M. A. Innis (ed.), 1995, Academic Press: New York; "Polymerase chain reaction: basic principles and automation in PCR. A Practical Approach", McPherson et al. (eds.), 1991, IRL Press: Oxford; Saiki et al., Nature, 1986, 324:163; and U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,889,818, each of which is incorporated by reference in its entirety. Also included are variations on PCR, including TaqMan®-based assays (see Holland et al., Proc. Natl. Acad. Sci, 1991, 88:7276-7280) and reverse transcription polymerase chain reaction (or RT-PCR; e.g., as described in U.S. Pat. Nos. 5,322,770 and 5,310,652, each of which is incorporated herein by reference).

Generally, in PCR, a pair of primers are added in excess to (and thus contacted with) a test sample from a subject and hybridized to complementary strands of the target nucleic acid. Each of the primers is extended by a DNA polymerase using the target sequence as a template. The extension product, after dissociation (denaturation) from the original target strand, becomes the target itself. New primers are then hybridized and extended by the polymerase, and the cycle is repeated to exponentially increase the number of copies of the amplicon. Specific examples of DNA polymerases capable of producing primer extension products in a PCR reaction include E. coli DNA polymerase I, Klenow fragment of DNA polymerase I, T4 DNA polymerase, thermostable DNA polymerase isolated from Thermus aquaticus (Taq), available from a variety of sources (e.g., Perkin Elmer, Waltham, Mass.), Thermus thermophilus (USB Corporation, Cleveland, Ohio), Bacillus stereothermophilus (Bio-Rad Laboratories, Hercules, Calif.), AmpliTaq Examples of suitable polymerases include, but are not limited to, recombinant Thermus thermophilus (rTth) DNA polymerase (Applied Biosystems, Foster City, CA), or Thermococcus litoralis ("Vent" polymerase, New England Biolabs, Ipswich, MA). RNA target sequences can be amplified by reverse transcribing (RT) the mRNA into cDNA followed by PCR (RT-PCR) (as described above). Alternatively, a single enzyme can be used for both steps, as described in US Pat. No. 5,322,770, which is incorporated herein by reference.

Strand displacement amplification combines the ability of a restriction endonuclease to nick the unmodified strand of its target DNA with the action of an exonuclease-deficient DNA polymerase to extend the 3' end at the nick and remove the downstream DNA strand at a constant temperature (Walker et al., Proc.
. Natl. Acad. Sci. USA, 1992.) Primers used in SDA contain a restriction endonuclease recognition site at a site 5' to the target binding sequence (see U.S. Pat. Nos. 5,270,184 and 5,344,166, each of which is incorporated herein by reference).

Nucleic Acid Sequence Based Amplification (NASBA) utilizes three enzymes (e.g., RNase H, avian myeloblastosis virus (AMV) reverse transcriptase, and T7 RNA polymerase) that act cooperatively at low isotherms, typically 41°C (see Compton, Nature, 1991, 350:91-92; Chan et al., Rev. Med. Microbiol, 1999, 10:185-196). The product of the NASBA reaction is primarily single-stranded RNA.

In certain embodiments of the invention, the probes described herein are used to detect the amplification products generated by the amplification reaction. The probes described herein can be used using a variety of well-known homogeneous or heterogeneous methods.

Homogeneous detection methods include FRET labels that are attached to the probe and emit a signal in the presence of the target sequence, Molecular Beacons (Tyagi et al., Nature Biotechnol., 1996, 14:303-308; Tyagi et al., Nature Biotechnol., 1998, 16:49-53; Kostrikis et al., Science, 1998, 279:1228-1229; Sokol et al., Proc. Natl. Acad. Sci., 1998, 279:1228-1229). USA, 1998, 95:11538-11543; Marras et al., Genet. Anal., 1999, 14:151-156; and U.S. Pat. Nos. 5,846,726, 5,925,517, 6,277,581, and 6,235,504.) and TaqMan® assays (U.S. Pat. Nos. 5,210,015, 5,804,375, 5,
See, for example, US Pat. Nos. 4,877,792 and 6,214,979, and WO 01/86001. Using these detection techniques, the product of the amplification reaction can be detected as it is formed, i.e., in a real-time manner. As a result, amplification product/probe hybrids are formed and detected while the reaction mixture is under amplification conditions.

In one embodiment, the probes of the invention are used in a TaqMan® assay. In a TaqMan® assay, analysis is performed in conjunction with heating cycling by monitoring the generation of a fluorescent signal. The assay system has the ability to generate quantitative data that allows for the determination of target copy number. For example, a standard curve can be generated using serial dilutions of a pre-quantified suspension of one or more bovine leukemia virus types or human beta globin sequences, against which unknown samples can be compared. A TaqMan® assay is conveniently performed using, for example, AmpliTaqGold™ DNA polymerase, which has intrinsic 5' nuclease activity, to digest a probe labeled with both a fluorescent reporter dye and a quenching moiety, as described above. The assay result is obtained by measuring the change in fluorescence that occurs during the amplification cycle as the probe is digested, separating the fluorescent and quenching moieties, and causing an increase in the fluorescent signal that is proportional to the amplification of the target sequence.

Other examples of homogeneous detection methods include hybridization protection assays (HPA). In such assays, the probe is labeled with a highly chemiluminescent molecule, acridinium ester (AE) (see Weeks et al., Clin. Chem., 1983, 29:1474-1479; Berry et al., Clin. Chem., 1988, 34:2087-2090), using non-nucleotide-based linker arm chemistry (see U.S. Pat. Nos. 5,585,481 and 5,185,439). Chemiluminescence is induced by hydrolysis of AE with alkaline hydrogen peroxide. In this case, the hydrolysis gives excited N-methylacridone, which is then inactivated with the emission of light. In the absence of target sequence, hydrolysis of AE is rapid. However, when the probe is bound to the target sequence, the rate of hydrolysis of AE is significantly reduced. Thus, hybridized and unhybridized AE-labeled probes are directly detectable in solution without the need for physical separation.

Heterogeneous detection systems are also well known in the art and generally use a capture material to separate the amplified sequences from other materials in the reaction mixture. The capture material typically comprises a solid support material (e.g., a microtiter well, a bead, a chip, etc.) coated with one or more specific binding sequences. The binding sequence may be complementary to a tail sequence added to an oligonucleotide probe of the invention. Alternatively, the binding sequence may be complementary to a sequence of a capture oligonucleotide, which itself may comprise a sequence complementary to the tail sequence of the probe. After separating the amplification product/probe hybrid bound to the capture material from the remainder of the reaction mixture, the amplification product/probe hybrid may be detected using any detection method, such as those described herein.

Typically, the method of the present invention first involves obtaining a test sample from a subject. The subject from which the test sample can be obtained is any mammal. Preferably, the mammal includes a bovine. Examples of bovines are Wagyu, Angus, and Holstein.

After a test sample is obtained from a subject, the test sample is contacted with primers (and optionally one or more probes) from at least one of the primer sets or primer and probe sets disclosed herein to form a reaction mixture. The reaction mixture is then placed under amplification conditions. The primers hybridize to any target nucleic acid in the test sample. The target nucleic acid present in the sample is amplified to obtain at least one amplification product (i.e., at least one target sequence).

At least one amplification product is detected by detecting hybridization between at least one amplification product and at least one of the probes of the invention (e.g., one or more probes from the primer and probe sets described herein).

Depending on the method, the kit may further include one or more of wash buffers, hybridization buffers, labeling buffers, detection means, and other reagents. The buffers and/or reagents are preferably optimized for the particular amplification/detection technique for which the kit is intended. Protocols for using these buffers and reagents to perform the various steps of the method may also be included in the kit.

Additionally, the kit may include internal controls as a test standard for amplification efficiency, to prevent false negative test results due to failure of said amplification, to verify cell validity, sample extraction, etc. An optimal internal control sequence is selected such that it does not compete with the target nucleic acid sequence in the amplification reaction.

The kit may also contain reagents for isolation of nucleic acid from the test sample prior to amplification prior to nucleic acid extraction.

The reagents may be supplied in solid (e.g., lyophilized) or liquid form. The kits of the invention may optionally include various containers (e.g., vials, ampoules, test tubes, flasks or bottles) for each individual buffer and/or reagent. Each component will generally be preferably aliquoted in its respective container or provided in concentrated form. Other containers suitable for carrying out certain steps of the amplification/detection assay may also be provided. The individual containers are preferably maintained in close confinement for commercial sale.

The kits may also include instructions for using the amplification reagents and primer sets or primers and probes described herein, instructions for processing test samples, extracting nucleic acid molecules, and/or performing tests, and instructions for interpreting the results obtained, as well as notes in a form prescribed by a government agency. Such instructions may, in some cases, be in printed form or on a CD, DVD, or other format of a recording medium.

By way of non-limiting example, specific examples of the present disclosure are provided below.

[Test Example]
Experimental Materials and Methods
Alignment of BLV genome and design and preparation of primers and probes
195 types of BLV nucleotide sequences registered in NCBI were selected and aligned using CLC Genomic Workbench 12.0. Five conserved regions were selected from the alignment results. The nucleotide sequences of the selected conserved regions are shown in Table 1, and an overview is shown in Figure 2. Primers and TaqMan probes were designed based on the selected conserved regions using IDT's primer design tool. The designed primers and probes are shown in Table 2.

Design and preparation of synthetic DNA A 500 bp synthetic DNA containing the sequence of the selected conserved region was designed and purchased from IDT (Integrated DNA Technologies, Inc., Coralville, IA). The sequence of the designed synthetic DNA is shown in Table 3. In this case, the sequence of the conserved region was designed to be located exactly in the center of the synthetic DNA. The purchased synthetic DNA was dissolved in TE according to the IDT instructions and left to stand overnight at 4°C. After leaving it to stand overnight, it was diluted with TE to a concentration of 5 x ¹⁰⁹ {copy number/μl} and stored at -30°C until use.

DNA extraction from bovine whole blood
DNA was extracted from 200 μl of bovine whole blood using the High Pure Viral Nucleic Acid Kit (Roche) (final volume: 50 μl). The amount of DNA in the extract was measured using Qubit The DNA was measured using a 2.0 Fluorometer (Invitrogen) and the extracted DNA was stored at -30°C until use.

Real-time PCR
In this study, real-time PCR was performed to detect BLV using a LightCycler Nano (Roche). The reaction mixture consisted of 10 μl of Premix Ex Taq (Perfect Real Time) (Takara Bio), 2 μl of PrimeTime qPCR Assay (IDT), 6 μl of Nuclease-Free Water, and 2 μl of sample, totaling 20 μl. The reaction conditions were 45°C for 5 min, 95°C for 30 s, followed by 40 cycles of 95°C for 10 s, 55°C for 20 s, and 72°C for 20 s. A standard curve was drawn with eight points of synthetic DNA concentrations: 10 ⁶ , 10 ⁵ , 10 ⁴ , 10 ³ , 10 ² , 10 ¹ , 10 ⁰ , and 0, and the copy number contained in the sample was quantified.

In addition to the above conditions, real-time PCR was performed to detect BLV using the Applied Biosystems 7300 Real-Time PCR System (Applied Biosystems). The reaction mixture consisted of 10 μl of Premix Ex Taq (Perfect Real Time) (Takara Bio), 0.4 μl of ROX Reference Dye (Takara Bio), 2 μl of PrimeTime qPCR Assay (IDT), 5.6 μl of Nuclease-Free Water, and 2 μl of sample, for a total of 20 μl. The reaction conditions were 45°C for 5 minutes, 95°C for 30 seconds, followed by 40 cycles of 95°C for 10 seconds, 55°C for 30 seconds, and 72°C for 20 seconds.

Spike test: The synthetic DNA was diluted with extracted DNA from a BLV-negative cow to concentrations of 10, ^{10 , 10 , 10} , ¹⁰ , ¹⁰ , and 0 (copy number/2 μl), and real-time PCR was performed using a LightCycler Nano (Roche) and an Applied Biosystems 7300 Real-Time PCR System (Applied Biosystems).

Agarose gel electrophoresis
The PCR products obtained by performing real-time PCR using a LightCycler Nano (Roche) were electrophoresed for 30 minutes using a 4% agarose gel and Tris-borate-EDTA buffer, stained with ethidium bromide, and then confirmed for the presence or absence of PCR products of the desired size and non-specific amplified products using a UV transilluminator.

BLV detection by real-time PCR using the designed primers and probes. The real-time PCR designed for this test was performed on a total of 29 samples, including 24 samples of DNA extracted from bovine whole blood that were determined to be positive by real-time PCR provided by University A and 5 samples that were determined to be negative, using a LightCycler Nano (Roche).
In addition, a total of 40 samples were provided by University B: 30 real-time PCR-negative and ELISA-positive samples, 5 real-time PCR-negative and ELISA-negative samples, and 5 real-time PCR-positive samples. The real-time PCR designed for this test was performed using a LightCycler Nano (Roche).

Results Real-time PCR of synthetic DNA
The results using a LightCycler Nano (Roche) are shown in Figure 3. Amplification of synthetic DNA was observed with all five primer sets. Real-time PCR was performed using synthetic DNA diluted 10-fold from ¹⁰⁶ to ¹⁰⁰ copies, and detection was possible up to ¹⁰⁰ copies. This experiment was performed twice, and detection was possible up to ¹⁰⁰ copies both times, but the Cq value was less stable for samples with lower copy numbers, and primers 1 ^{, 2} , 3, and 4 showed similar Cq values at 101 copies and ¹⁰⁰ copies.
The results of real-time PCR of the synthetic DNA using the Applied Biosystems 7300 Real-Time PCR System are shown in Figure 4. Primer sets 1, 3, and 5 were used, and all three were able to detect up to ¹⁰ copies.

Real-time PCR of DNA extracted from BLV-negative whole blood of cattle
Real-time PCR was performed using the LightCycler Nano with two types of DNA extracted from cattle that were determined to be BLV negative as samples to check for false positives. The results are shown in Figure 5 (Figure 5-1 and Figure 5-2). Because a positive result was obtained in real-time PCR using primers 2 and 4, real-time PCR was performed again using these primer sets. No false positives were found in the second real-time PCR, but agarose gel electrophoresis confirmed that the real-time PCR product using primer 4 had a different length from the target region. These results are shown in Figure 6.

Spike Test
The results of the spike test using the LightCycler Nano are shown in Figure 7. Primer sets 1, 3, and 5 were able to detect up to ¹⁰ copies.
The results of the spike test using the Applied Biosystems 7300 Real-Time PCR System are shown in Figure 8. Primer sets 1, 3, and 5 were able to detect up to ¹⁰ copies.

BLV detection using samples from University A Real-time PCR was performed using a LightCycler Nano with samples of DNA extracted from bovine whole blood provided by University A, and the results are shown in Table 4 (Table 4-1 to Table 4-3). With primer sets 1, 3, and 5, all 24 samples that had tested positive in the previous test were positive, and all 5 samples that had tested negative in the previous test were negative.

Detection of BLV using samples from University B Real-time PCR was performed using a LightCycler Nano with samples of DNA extracted from bovine whole blood provided by University B, and the results are shown in Table 5 (Tables 5-1 to 5-3). Of the 30 samples that were ELISA positive and real-time PCR negative in the preliminary test, 12 were positive with primer set 1, 6 with primer set 3, and 13 with primer set 5. For the five samples that were ELISA negative and real-time PCR negative in the preliminary test, all were negative with primer sets 1 and 5 (not performed with primer set 3). For the five samples that were real-time PCR positive in the preliminary test, all were positive with primer sets 1 and 5 (not performed with primer set 3).

Discussion In this study, we aimed to select highly conserved sequences from existing BLV sequences and demonstrate the usefulness of real-time PCR designed for those sequences. First, in real-time PCR of synthetic DNA performed with the LightCycler Nano, all primer sets were able to detect up to 10 ⁰ copies, but the Cq values for the samples with 10 ¹ and 10 ⁰ copies were very close, and the quantitation between these copy numbers is thought to be low. However, if the lower limit is 10 ¹ copies, it is thought that this is a sufficiently reliable diagnostic system. Next, in real-time PCR using DNA extracted from BLV-negative cows, primer sets 2 and 4 were suspected of producing false positives, so they were excluded from the candidates and the study was terminated. In the spike test using the LightCycler Nano, up to 10 ¹ copies could be detected, and it was determined that the detection sensitivity in the presence of cow-derived DNA was sufficient. In both real-time PCR of synthetic DNA and the spike test using the Applied Biosystems 7300 Real-Time PCR System, it was possible to detect up to 10 ¹ copies. There were no problems with the calibration curve, and it was determined that this detection system also had sufficient sensitivity. The Applied Biosystems 7300 Real-Time PCR System is said to have a slightly lower sensitivity than the LightCycler Nano, but it can handle multiple samples at once. Since it is expected that such equipment will be used in the field, a different real-time PCR device was used in this study. In real-time PCR using samples provided by University A, it was confirmed that the detection sensitivity of the system developed in this study was not inferior to that of the existing real-time PCR detection system. In addition, all samples that were negative in the preliminary diagnosis were negative, and it was confirmed that the specificity was high. In real-time PCR using samples provided by University B, we attempted to detect BLV that could not be detected with the existing real-time PCR detection system. As a result, we succeeded in detecting BLV that could not be detected by the existing real-time PCR, although not all of them. At this time, primer set 3 was determined to have a low sensitivity compared to primer sets 1 and 5, so it was removed from the candidates and was not used in subsequent studies. Five samples that were positive in real-time PCR in the preliminary diagnosis were positive without any problems, and all five samples that were negative in both ELISA and real-time PCR in the preliminary diagnosis were negative, and there were no problems with specificity. Based on these results, we concluded that primer sets 1 and 5 developed in this study have higher sensitivity than existing real-time PCR detection systems and have no problems with specificity, making them useful as new detection systems.

Conventional real-time PCR for BLV includes TaKaRa cycleave PCR targeting the Tax gene and BLV-CoCoMo-qPCR targeting the LTR [M Jimba et al. 2012]. In contrast, the primers finally adopted in this test target the gag gene and LTR. Since there are two LTRs in the BLV genome, two copies are detected per provirus. Therefore, it has been reported that the detection sensitivity is high [M Jimba et al. 2010]. Since BLV-CoCoMo-qPCR uses degenerate primers, it has the advantage that in principle it can detect any base substitution in the target sequence, but it has the disadvantage that the concentration of each primer is low. Since the primers used in this test are not degenerate primers, the primer concentration can be set high, which may allow for the detection of smaller amounts of BLV. In addition, the discovery of a conserved region in the gag gene other than the genes that were previously targeted and the practical application of this method are of great significance for the field where BLV testing will continue in the future.

Conclusion: The novel real-time PCR detection system developed in this study has higher detection sensitivity and no specificity problems compared to conventional real-time PCR, and is therefore considered to be useful for diagnosing BLV.

The invention described in this specification may be used in veterinary medicine and animal husbandry.

Free text in sequence listing SEQ ID NO: 1: primer SEQ ID NO: 2: primer SEQ ID NO: 3: probe SEQ ID NO: 4: primer SEQ ID NO: 5: primer SEQ ID NO: 6: probe

Claims (4)

A primer set in which a forward primer is an oligonucleotide having a base sequence shown in SEQ ID NO: 1 or 4 , and a reverse primer is an oligonucleotide having a base sequence shown in SEQ ID NO: 2 or 5 ,
A kit for detecting bovine leukemia virus.
SEQ ID NO: 1: ACCATGGACTTTCACTTCTGG
SEQ ID NO: 2: GGCCAAGACAAGGGGAAC
SEQ ID NO: 4: CTCCCCAACTTCCCCTTTC
SEQ ID NO: 5: ACGGGTCTCAGGAGAAG 2. The kit of claim 1 ,
A kit further comprising a probe which is an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 3 or 6.
SEQ ID NO: 3: CCGGGTGGACATGAGGTGGG
SEQ ID NO: 6: TCGAGTTAGCGGCACCAGAAGC 2. The kit of claim 1 ,
A primer set in which a forward primer is an oligonucleotide having a base sequence shown in SEQ ID NO: 1 and a reverse primer is an oligonucleotide having a base sequence shown in SEQ ID NO: 2 ,
A kit for detecting bovine leukemia virus. 2. The kit of claim 1 ,
A primer set in which a forward primer is an oligonucleotide having a base sequence shown in SEQ ID NO: 4 and a reverse primer is an oligonucleotide having a base sequence shown in SEQ ID NO: 5 ,
A kit for detecting bovine leukemia virus.

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