Method for examining disease progression of enzootic bovine leukosis, primer, cfdna quantitation kit, and kit for examining disease progression of enzootic bovine leukosis

Abstract

Provided is a method for examining disease progression of enzootic bovine leukosis, the method comprising a step (A) for quantifying cell-free DNA derived from proviral DNA of bovine leukemia virus, in a sample containing cell-free DNA of an animal of interest.

Description

Testing method for infectious bovine lymphoma progression, primers, cfDNA quantification kit, and test kit for infectious bovine lymphoma progression

[0001] The present invention relates to a method for testing the progression of infectious bovine lymphoma, primers, a cfDNA quantification kit, and a test kit for testing the progression of infectious bovine lymphoma. This application claims priority to Japanese Patent Application No. 2024-064965, filed April 12, 2024, the contents of which are incorporated herein by reference.

[0002] Cows are an important food resource, producing milk and meat. In recent years, the high quality of Japanese beef cattle and dairy products has been recognized worldwide, and demand is increasing year by year, not only in Japan but also overseas. Globally, dairy and beef cattle production accounts for an extremely large share of the livestock industry.

[0003] Bovine Leukemia Virus (BLV) is a virus that infects B lymphocytes in cattle. BLV is pathogenic enough to cause lymphoma in some infected cattle. While 60-70% of infected cattle are asymptomatic and do not develop the disease, 30% of infected cattle develop persistent lymphocytosis, and a small percentage of infected cattle develop lymphoma.

[0004] In recent years, the infection rate of BLV and the incidence of infectious bovine lymphoma (Enzootic Bovine Leukosis: EBL) have both been on the rise in Japan, increasing the need for countermeasures. In Japan, cattle infected only with BLV but not with EBL are distributed to the market as meat. However, if EBL is detected during slaughter inspection, the entire animal is discarded in accordance with the provisions of the Livestock Infectious Diseases Prevention Act. Therefore, the onset of EBL causes significant economic losses for producers.

[0005] Accurate diagnosis is necessary to detect the onset of EBL. However, despite the importance of this issue, EBL is currently diagnosed by veterinarians by visually or palpably checking for the presence or absence of lymph node enlargement. Therefore, the diagnostic accuracy is not high.

[0006] Nucleic acid testing methods for quantifying the amount of BLV provirus are used to test for BLV-infected cattle (see, for example, Patent Document 1). However, although the amount of BLV proviral DNA reflects the number of BLV-infected cells, it cannot evaluate the number of tumorigenic cells.

[0007] As a diagnostic method for EBL, a method of evaluating the clonality of BLV-infected cells by next-generation sequencing (NGS) analysis has been proposed (Non-Patent Document 1). However, next-generation sequencers are expensive and not very versatile.

[0008] Furthermore, a method for diagnosing EBL has been proposed in which the nucleotide sequence of the junction between the BVL provirus and the host bovine genome is analyzed by Sanger sequencing to evaluate the clonality of BLV-infected cells (Patent Document 2).

[0009] JP 2019-180351 A JP 2023-20700 A

[0010] Nicolas A. Gillet et al., Massive Depletion of Bovine Leukemia Virus Proviral Clones Located in Genomic Transcriptionally Active Sites during Primary Infection. PLoS Pathog. 2013;9(10):e1003687.

[0011] As described above, the method for quantifying BLV provirus cannot quantify the number of tumorigenic cells and cannot evaluate the progression of EBL. The method using NGS analysis is expensive and difficult to use in practice. The method using Sanger sequencing analysis is cheaper than NGS analysis, but requires sequence analysis, so it cannot be said to be simple and low-cost. Therefore, there is a need for a testing technology that can evaluate the progression of bovine infectious lymphoma more simply and at lower cost than conventional technologies.

[0012] Therefore, an objective of the present disclosure is to provide a method for testing the progression of infectious bovine lymphoma, which enables easy evaluation of the progression of the disease, as well as primers, a cell-free DNA quantification kit, and a testing kit that can be used in the testing method.

[0013] The present disclosure includes the following aspects: (1) A method for testing the progression of bovine infectious lymphoma, comprising step (A) of quantifying cell-free DNA derived from proviral DNA of bovine infectious lymphoma virus in a sample containing cell-free DNA from a target animal. (2) The method for testing the progression of bovine infectious lymphoma according to (1), wherein the cell-free DNA derived from proviral DNA comprises at least a portion of a region selected from the group consisting of the LTR and pol gene of the proviral DNA. (3) The method for testing the progression of bovine infectious lymphoma according to (1) or (2), wherein step (A) comprises a step of performing a nucleic acid amplification reaction using the cell-free DNA as a template and a primer capable of specifically annealing to the proviral DNA. (4) The method for testing the progression of bovine infectious lymphoma according to (3), wherein the primer is a primer capable of specifically annealing to at least one region selected from the group consisting of the LTR and pol gene of the proviral DNA. (5) A method for testing the progression of bovine infectious lymphoma according to (3), wherein the primer comprises at least one type selected from the group consisting of the following (i) to (iv): (i) a primer having a nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 6 and 19; (ii) a primer capable of specifically annealing to a region in the proviral DNA to which a primer comprising a nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 6 and 19 can specifically anneal; (iii) a primer having a nucleotide sequence in which one or several nucleotides have been deleted, added, or substituted in the nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 6 and 19, and capable of specifically annealing to the LTR or pol gene region of the proviral DNA; and (iv) a primer having a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 6 and 19, and capable of specifically annealing to the LTR or pol gene region of the proviral DNA.(6) A method for testing the progression of bovine infectious lymphoma according to (4), wherein the primer comprises at least one type selected from the group consisting of the following (i') to (iv'): (i') a primer having the nucleotide sequence set forth in SEQ ID NO: 1 or 2; (ii') a primer capable of specifically annealing to a region in the proviral DNA to which a primer comprising the nucleotide sequence set forth in SEQ ID NO: 1 or 2 can specifically anneal; (iii') a primer having a nucleotide sequence in which one or several nucleotides have been deleted, added, or substituted in the nucleotide sequence set forth in SEQ ID NO: 1 or 2, and capable of specifically annealing to the LTR of the proviral DNA; and (iv') a primer having a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence set forth in SEQ ID NO: 1 or 2, and capable of specifically annealing to the LTR of the proviral DNA. (7) A primer selected from the group consisting of (i) to (iv) below: (i) a primer having a nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 6 and 19; (ii) a primer capable of specifically annealing to a region in the proviral DNA of bovine infectious lymphoma virus to which a primer comprising a nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 6 can specifically anneal; (iii) a primer having a nucleotide sequence in which one or several nucleotides have been deleted, added, or substituted in the nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 6 and 19, and capable of specifically annealing to the LTR or pol gene region of the proviral DNA; and (iv) a primer having a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 6 and 19, and capable of specifically annealing to the LTR or pol gene region of the proviral DNA. (8) A kit for quantifying cell-free DNA derived from proviral DNA of bovine infectious lymphoma virus, comprising the primer described in (7), a probe capable of annealing to a nucleic acid fragment obtained by a nucleic acid amplification reaction using the primer described in (7), and a standard DNA containing the nucleotide sequence of the nucleic acid fragment obtained by a nucleic acid amplification reaction using the primer described in (7).(9) A test kit for the progression of bovine infectious lymphoma, comprising a primer capable of annealing to cell-free DNA derived from proviral DNA of bovine infectious lymphoma virus. (10) The test kit for the progression of bovine infectious lymphoma according to (9), further comprising a probe capable of annealing to a nucleic acid fragment obtained by a nucleic acid amplification reaction using the primer, and a standard DNA comprising the nucleotide sequence of the nucleic acid fragment obtained by a nucleic acid amplification reaction using the primer. (11) The test kit for the progression of bovine infectious lymphoma according to (9) or (10), further comprising a cell-free DNA extraction reagent. (12) The test kit for the progression of bovine infectious lymphoma according to any one of (9) to (11), wherein the primer comprises the primer according to (7).

[0014] According to the present invention, there are provided a method for testing the progression of infectious bovine lymphoma, which enables easy evaluation of the progression of the disease, as well as primers, a cell-free DNA quantification kit, and a testing kit that can be used in the testing method.

[0015] The genome structure of bovine infectious lymphoma virus (BLV) and the positions of the primers and probes designed in Experimental Example 1 are shown. The structure of the standard plasmid prepared in Experimental Example 1 is shown. ６ 1 shows the amplification curve of real-time PCR performed using a standard plasmid solution containing 10 copies / μL of the plasmid. ５1 shows the results of droplet digital PCR performed using a standard plasmid solution of 1 copy / μL. In Experimental Example 2, the results of confirming whether the nucleotide sequences of the primers (LTR_F, LTR_R) and probe (LTR_P) for region 1 (LTR U3) are conserved among BLV virus strains are shown. In Experimental Example 2, the results of confirming whether the target region of the primers (LTR_F, LTR_R) and probe (LTR_P) for region 1 (LTR U3) is present in the cfDNA of cattle affected with infectious bovine lymphoma are shown. In Experimental Example 3, the results of quantifying BVL-derived cfDNA by real-time PCR using cfDNA extracted from serum samples of cattle affected with infectious bovine lymphoma (EBL) and cattle not affected with infectious bovine lymphoma (Non-EBL) as templates are shown. Experimental Example 4 shows the results of quantifying BVL proviral DNA by real-time PCR using DNA extracted from blood cell samples of infectious bovine lymphoma-affected cattle (EBL) and non-infectious bovine lymphoma-affected cattle (non-EBL) as a template. Experimental Example 5 shows the results of evaluating the separability of infectious bovine lymphoma-affected cattle (EBL) and non-infectious bovine lymphoma-affected cattle (non-EBL) by combining the results of quantifying BLV-derived cfDNA in Experimental Example 3 and the results of quantifying intracellular BLV proviral DNA in Experimental Example 4. Experimental Example 6 shows the results of quantifying BVL provirus-derived DNA by real-time PCR using DNA extracted from whole blood samples of infectious bovine lymphoma-affected cattle (EBL) and non-infectious bovine lymphoma-affected cattle (non-EBL) as a template. LTR_F and LTR_R were used as primers, and LTR_P was used as a probe.

[0049] In Experimental Example 6, BVL provirus-derived DNA was quantified by real-time PCR using cfDNA extracted from plasma samples of infectious bovine lymphoma-affected cattle (EBL) and non-infectious bovine lymphoma-affected cattle (Non-EBL) as a template. LTR_F and LTR_R were used as primers, and LTR_P was used as a probe.

[0050] In Experimental Example 6, BVL provirus-derived DNA was quantified by real-time PCR using DNA extracted from whole blood samples of infectious bovine lymphoma-affected cattle (EBL) and non-infectious bovine lymphoma-affected cattle (Non-EBL) as a template.Pol_F1 and Pol_R3 were used as primers, and Pol_P3 was used as the probe. In Experimental Example 6, the results of real-time PCR quantification of BVL provirus-derived DNA using cfDNA extracted from plasma samples of infectious bovine lymphoma-affected cattle (EBL) and non-EBL cattle as templates are shown. Pol_F1 and Pol_R3 were used as primers, and Pol_P3 was used as the probe. The results of real-time PCR quantification of BVL provirus-derived DNA using cfDNA extracted from plasma samples of non-EBL cattle as templates are shown. In the figure, "86 bp" indicates the results when LTR_F and LTR_R were used as primers and LTR_P was used as the probe. "183 bp" indicates the results when CoCoMo was used as the primer and probe. ＴＭ The figures show the results when using the primers and probe contained in the BLV Primer / Probe (Nippon Gene). The figures show the results of quantifying BVL provirus-derived DNA by real-time PCR using cfDNA extracted from the plasma sample of cattle affected by infectious bovine lymphoma (EBL) as a template. In the figure, "86 bp" indicates the results when LTR_F and LTR_R were used as primers and LTR_P was used as a probe. "183 bp" indicates the results when CoCoMo LTR_F and LTR_R were used as primers and probe. ＴＭ The results shown are for the case where the primers and probe included in the BLV Primer / Probe (Nippon Gene) were used.

[0016] [Definitions] Numerical ranges expressed using "to" mean a range that includes the numerical values ​​before and after "to" as the lower and upper limits. Unless otherwise specified, "a," "an," and "the" are understood to include singular and plural and mean "one or more."

[0017] The terms "comprise" and "have" mean that components other than the target component may be included. The term "consist of" means that components other than the target component are not included. The term "consist essentially of" means that components other than the target component are not included in a form that performs a special function (such as a form that completely loses the effect of the invention). In this specification, when "comprise" or "have" is used, it includes both an embodiment that "consists of" and an embodiment that "consists essentially of."

[0018] "Host animal" refers to an animal that is the target of infection with BLV. The host animal for BLV is not particularly limited as long as it can be infected with BLV. Host animals are non-human animals, and examples include, but are not limited to, cattle, buffalo, capybaras, sheep, etc. "Infectious bovine lymphoma virus-infected cells" refer to host animal cells in which the BLV provirus has been integrated into the genomic DNA of the host animal. "Infectious bovine lymphoma disease progression" refers to the onset state of infectious bovine lymphoma. Typically, when infectious bovine lymphoma develops, lymphoma occurs. "Testing the onset state of infectious bovine lymphoma disease" includes determining the onset state of infectious bovine lymphoma, determining the possibility of the onset of infectious bovine lymphoma, and determining the risk of developing infectious bovine lymphoma.

[0019] "Cell-free DNA" (cfDNA) refers to extracellular DNA present in body fluids. cfDNA includes DNA released from cells that have died due to apoptosis or necrosis, DNA contained in extracellular vesicles such as exosomes, and the like. In one embodiment, cfDNA is DNA present in serum or plasma.

[0020] "Specifically annealing" means annealing to the nucleotide sequence to be annealed and not to other nucleotide sequences under annealing conditions typically used in nucleic acid amplification reactions. Examples of annealing conditions typically used in nucleic acid amplification reactions include a salt concentration of 1.5 to 2.5 mM and an annealing temperature of 50 to 70°C. The annealing temperature may be set based on the Tm value of the primer. For example, the annealing temperature may be set to a temperature that is about 2 to 3 degrees lower than the Tm value.

[0021] The sequence identity between nucleotide sequences is calculated by aligning two nucleotide sequences with gaps at the insertion and deletion sites so that the number of corresponding nucleotides is the greatest, and calculating the percentage of matched nucleotides relative to the entire nucleotide sequence excluding gaps in the resulting alignment. The sequence identity between nucleotide sequences can be determined using various homology search software known in the art. For example, the sequence identity value of nucleotide sequences can be calculated based on the alignment obtained by the known homology search software BLASTN.

[0022] [Method for testing the progression of infectious bovine lymphoma] A first aspect of the present disclosure is a method for testing the progression of infectious bovine lymphoma. In one embodiment, the testing method of the present disclosure comprises a step (A) of quantifying cell-free DNA derived from proviral DNA of bovine infectious lymphoma virus in a sample containing cell-free DNA from a target animal. The testing method of the present disclosure can be performed in vitro.

[0023] <Step (A)> Step (A) is a step of quantifying cell-free DNA derived from bovine infectious lymphoma virus proviral DNA in a sample containing cell-free DNA from a target animal. Step (A) can be performed in vitro.

[0024] The infectious bovine lymphoma virus (BLV) is a virus that belongs to the genus Deltaretrovirus in the family Retroviridae and causes infectious bovine lymphoma (EBL). When BLV infects a host animal, the BLV provirus is integrated into the genomic DNA of the host animal's cells. BLV primarily infects B lymphocytes.

[0025] A "subject animal" is an animal that is the subject of testing for the progression of EBL. The subject animal is not particularly limited as long as it is an animal that develops EBL due to infection with BLV. The subject animal is a non-human animal, and examples thereof include, but are not limited to, cattle, buffalo, capybara, sheep, etc. The subject animal is preferably a cattle.

[0026] A "sample containing cell-free DNA of a target animal" (hereinafter also referred to as "target animal sample") is a biological sample containing cell-free DNA collected from a target animal. In one embodiment, the target animal sample is a body fluid sample of the target animal. As a body fluid sample, a serum sample or a plasma sample is preferred. Serum samples and plasma samples can be prepared from blood collected from a target animal by known methods. Serum samples can be prepared, for example, by allowing the blood to stand, centrifuging it, and collecting the supernatant. Plasma samples can be prepared, for example, by adding an anticoagulant (EDTA, sodium citrate, heparin, etc.) to the blood, followed by centrifugation and collecting the supernatant.

[0027] "Cell-free DNA derived from bovine infectious lymphoma virus proviral DNA" refers to cfDNA containing at least a portion of BLV proviral DNA. cfDNA derived from BLV proviral DNA (hereinafter also referred to as "BLV cfDNA") is considered to be cfDNA derived from BLV-infected cells. BLV cfDNA contains at least a portion of the nucleotide sequence of BLV proviral DNA.

[0028] The BLV cfDNA to be quantified preferably contains a region that is highly conserved among BLV virus strains. Examples of highly conserved regions include the LTR, pol gene region, and pro gene region (see FIG. 1 ). The LTR (long terminal repeat) is a repetitive sequence located in the 5'- and 3'-terminal regions of the BLV genome. The pol gene is a reverse transcriptase gene. The pro gene is a protease gene. In one embodiment, the BLV cfDNA contains at least a portion of a region selected from the group consisting of the LTR and the pol gene. In one embodiment, the BLV cfDNA contains at least a portion of the LTR.

[0029] Step (A) may be performed by extracting cfDNA from a sample containing cfDNA from a subject animal (such as a plasma sample or serum sample) and quantifying the BLV cfDNA contained in the cfDNA. Extraction of cfDNA from a body fluid sample can be performed by a known method. Examples of cfDNA extraction methods include the CTAB method, silica membrane method, ion exchange column method, and magnetic bead method. Various cfDNA extraction kits are commercially available. Extraction of cfDNA from a sample may be performed using a commercially available cfDNA extraction kit.

[0030] The BLV cfDNA contained in the cfDNA extracted from the sample (hereinafter also referred to as a "cfDNA sample") can be quantified using known DNA quantification methods. Examples of such methods include real-time PCR, digital PCR, droplet digital PCR, DNA microarrays, and next-generation sequencing analysis. Real-time PCR is preferred as a quantification method because it is highly quantitative and allows for simple and low-cost quantification.

[0031] Quantification of BLV cfDNA can be performed using primers and / or probes capable of specifically binding to BLV cfDNA. "Specific binding" means that, under commonly used binding reaction conditions, the primers and / or probes bind to the target nucleotide sequence and do not or barely bind to other nucleotide sequences. The primers and / or probes preferably target regions highly conserved among BLV virus strains. Targeting highly conserved regions among BLV virus strains allows for application to a wide range of BLV virus strains. Examples of highly conserved regions include the LTR, pol gene region, and pro gene region (see FIG. 1). In one embodiment, the primers and / or probes target a region selected from the group consisting of the LTR and the pol gene. In one embodiment, the primers and / or probes target the LTR. Since two copies of the LTR exist in one provirus, targeting the LTR is particularly preferred in terms of high detection sensitivity.

[0032] In one embodiment, step (A) includes a step of performing a nucleic acid amplification reaction using cfDNA contained in a sample (plasma sample, serum sample, etc.) from a subject animal as a template and a primer capable of specifically annealing to BLV proviral DNA.

[0033] The nucleic acid amplification reaction is carried out using cfDNA contained in a sample (plasma sample, serum sample, etc.) from a target animal as a template. The cfDNA used as a template can be cfDNA extracted from the sample (cfDNA sample).

[0034] The method of the nucleic acid amplification reaction is not particularly limited as long as it is a method capable of quantifying the target DNA in a DNA sample. Examples of the nucleic acid amplification reaction include the PCR (Polymerase Chain Reaction) method, the LAMP (Loop-Mediated Isothermal Amplification) method, and the NEAR (Nicking Enzyme Amplification Reaction) method. The nucleic acid amplification reaction is preferably the PCR method. Examples of the PCR method capable of quantifying the target DNA include real-time PCR, digital PCR, and droplet digital PCR.

[0035] (Primer) A "primer capable of specifically annealing to BLV proviral DNA" is a primer capable of specifically annealing to any region of BLV proviral DNA. The primer is preferably capable of annealing to a region that is highly conserved among BLV virus strains. The primer is more preferably a primer capable of specifically annealing to at least one region selected from the group consisting of the LTR and pol gene of BLV proviral DNA, and even more preferably a primer capable of specifically annealing to the LTR of BLV proviral DNA. By targeting the LTR, the progression of bovine infectious lymphoma can be assessed with high accuracy.

[0036] The size of the nucleic acid fragment obtained by the nucleic acid amplification reaction using primers is preferably about 50 to 300 bp. cfDNA is expected to be shorter than genomic DNA present in cells. Therefore, it is preferable to use primers that can amplify short regions. The size of the nucleic acid fragment obtained by the nucleic acid amplification reaction targeting cfDNA is, for example, about 70 to 160 bp, preferably about 70 to 150 bp, and more preferably about 70 to 130 bp.

[0037] The primer preferably comprises at least one type selected from the group consisting of the following (i) to (iv): (i) a primer having a nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 6 and 9; (ii) a primer capable of specifically annealing to a region in the BLV proviral DNA to which a primer comprising a nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 6 and 9 can specifically anneal; (iii) a primer having a nucleotide sequence in which one or several nucleotides have been deleted, added, or substituted in the nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 6 and 9, and capable of specifically annealing to at least one region selected from the group consisting of the LTR and pol gene of the BLV proviral DNA; and (iv) a primer having a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 6 and 9, and capable of specifically annealing to at least one region selected from the group consisting of the LTR and pol gene of the BLV proviral DNA.

[0038] The primer preferably comprises at least one type selected from the group consisting of the following (i') to (iv'): (i') a primer having the nucleotide sequence set forth in SEQ ID NO: 1 or 2; (ii') a primer capable of specifically annealing to a region in the BLV proviral DNA to which a primer comprising the nucleotide sequence set forth in SEQ ID NO: 1 or 2 can specifically anneal; (iii') a primer having a nucleotide sequence in which one or several nucleotides have been deleted, added, or substituted in the nucleotide sequence set forth in SEQ ID NO: 1 or 2, and capable of specifically annealing to the LTR of the BLV proviral DNA; and (iv') a primer having a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence set forth in SEQ ID NO: 1 or 2, and capable of specifically annealing to the LTR of the BLV proviral DNA.

[0039] In (iii) and (iii') above, "several" includes, for example, 2 to 6, 2 to 5, 2 to 3, or 2. In (iv) and (iv') above, sequence identity includes, for example, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. In (i) to (iv) and (i') to (iv') above, the embodiment of "having a specific nucleotide sequence" encompasses the embodiment of "consisting of the specific nucleotide sequence".

[0040] The primers (i) to (iv) are primers capable of specifically annealing to the LTR or pol gene region of the BLV provirus. The primers (i') to (iv') are primers capable of specifically annealing to the LTR of the BLV provirus.

[0041] More specifically, a primer containing the nucleotide sequence set forth in SEQ ID NO: 1 or a mutant nucleotide sequence thereof can be used as a forward primer capable of annealing to the LTR of a BLV provirus. A primer containing the nucleotide sequence set forth in SEQ ID NO: 2 or a mutant nucleotide sequence thereof can be used as a reverse primer capable of annealing to the LTR of a BLV provirus. A partial region of the LTR contained in BLV cfDNA can be amplified by performing a nucleic acid amplification reaction using these primers as forward and reverse primers.

[0042] A primer containing the nucleotide sequence set forth in SEQ ID NO: 3 or a mutant nucleotide sequence thereof can be used as a forward primer capable of annealing to the pol gene region of a BLV provirus. A primer containing the nucleotide sequence set forth in SEQ ID NO: 4 or a mutant nucleotide sequence thereof can be used as a reverse primer capable of annealing to the pol gene region of a BLV provirus. By using these primers as forward and reverse primers in a nucleic acid amplification reaction, a portion of the 5' region of the pol gene contained in BLV cfDNA can be amplified.

[0043] A primer containing the nucleotide sequence set forth in SEQ ID NO:5 or a mutant nucleotide sequence thereof can be used as a forward primer capable of annealing to the pol gene region of a BLV provirus. A primer containing the nucleotide sequence set forth in SEQ ID NO:6 or a mutant nucleotide sequence thereof can be used as a reverse primer capable of annealing to the pol gene region of a BLV provirus. By using these primers as forward and reverse primers in a nucleic acid amplification reaction, a portion of the 3' region of the pol gene region contained in BLV cfDNA can be amplified.

[0044] A primer containing the nucleotide sequence of SEQ ID NO: 19 or a mutated nucleotide sequence thereof can be used as a reverse primer capable of annealing to the pol gene region of the BLV provirus. A nucleic acid amplification reaction can be performed using a primer containing the nucleotide sequence of SEQ ID NO: 19 or a mutated nucleotide sequence thereof as a reverse primer and a primer containing the nucleotide sequence of SEQ ID NO: 3 or a mutated nucleotide sequence thereof as a forward primer to amplify a portion of the 5' region of the pol gene region contained in BLV cfDNA.

[0045] The length of the primer can be a length commonly used in nucleic acid amplification reactions. Examples of the lower limit of the primer length include 15 mer or more, 16 mer or more, 17 mer or more, 18 mer or more, or 19 mer or more. Examples of the upper limit of the primer length include 30 mer or less, 29 mer or less, 28 mer or less, 27 mer or less, 26 mer or less, or 25 mer or less. These upper and lower limits can be arbitrarily combined. Examples of the primer length range include 15 mer or more and 30 mer or less, 16 mer or more and 30 mer or less, 17 mer or more and 30 mer or less, 18 mer or more and 30 mer or less, or 19 mer or more and 30 mer or less.

[0046] The primers preferably include a forward primer selected from the group consisting of the following (iF) to (ivF) and a reverse primer selected from the group consisting of the following (iR) to (ivR):

[0047] (iF) a forward primer having the nucleotide sequence set forth in SEQ ID NO: 1; (iiF) a forward primer capable of specifically annealing to a region in BLV proviral DNA to which a primer containing the nucleotide sequence set forth in SEQ ID NO: 1 can specifically anneal; (iiiF) a forward primer having a nucleotide sequence in which one or more nucleotides in the nucleotide sequence set forth in SEQ ID NO: 1 have been deleted, added, or substituted, and capable of specifically annealing to the LTR of BLV proviral DNA; and (ivF) a forward primer having a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence set forth in SEQ ID NO: 1, and capable of specifically annealing to the LTR of BLV proviral DNA.

[0048] (iR) A reverse primer having the nucleotide sequence set forth in SEQ ID NO: 2; (iiR) A reverse primer capable of specifically annealing to a region in the BLV proviral DNA to which a primer containing the nucleotide sequence set forth in SEQ ID NO: 2 can specifically anneal; (iiiR) A reverse primer having a nucleotide sequence in which one or more nucleotides have been deleted, added, or substituted in the nucleotide sequence set forth in SEQ ID NO: 2 and capable of specifically annealing to the LTR of the BLV proviral DNA; and (ivR) A reverse primer having a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence set forth in SEQ ID NO: 2 and capable of specifically annealing to the LTR of the BLV proviral DNA.

[0049] Examples of "several" in (iiiF) and (iiiR) above include the same as "several" in (iii) and (iii') above. Examples of "sequence identity" in (ivF) and (ivR) above include the same as "sequence identity" in (iv) and (iv') above. In the above (iF) to (ivF) and (iR) to (ivR), the embodiment of "having a specific nucleotide sequence" encompasses the embodiment of "consisting of the specific nucleotide sequence".

[0050] (Real-time PCR Probe) When performing real-time PCR, a probe capable of annealing to a nucleic acid fragment obtained by a nucleic acid amplification reaction using a primer may be used. The design of the probe used in real-time PCR can be performed by a known method. The Tm value of the probe is preferably about 3 to 10°C higher than the Tm value of the primer. Examples of the lower limit of the probe length include 15 mer or more, 16 mer or more, 17 mer or more, 18 mer or more, or 19 mer or more. Examples of the upper limit of the probe length include 30 mer or less, 29 mer or less, 28 mer or less, 27 mer or less, 26 mer or less, or 25 mer or less. These upper and lower limits can be combined arbitrarily. Examples of the probe length range include 15 mer or more and 30 mer or less, 16 mer or more and 30 mer or less, 17 mer or more and 30 mer or less, 18 mer or more and 30 mer or less, or 19 mer or more and 30 mer or less.

[0051] The probe contains a fluorescent substance and a quencher substance. For example, the 5'-end of the probe is modified with a fluorescent substance and the 3'-end is modified with a quencher substance. The probe may be a double quencher probe. A double quencher probe contains a quencher substance at one site in the middle region of the probe in addition to the 3'-end. Known fluorescent substances and quencher substances can be used without particular limitation. Examples of fluorescent substances include FAM. Examples of quencher substances include TAMRA, ZEN, and Iowa Black FQ.

[0052] The probes include the following probes (a) to (d): (a) a probe having a nucleotide sequence set forth in any one of SEQ ID NOs: 13 to 15 and 20; (b) a probe capable of specifically annealing to a region in the BLV proviral DNA to which a probe comprising a nucleotide sequence set forth in any one of SEQ ID NOs: 13 to 15 can specifically anneal; (c) a probe having a nucleotide sequence in which one or several nucleotides have been deleted, added, or substituted in the nucleotide sequence set forth in any one of SEQ ID NOs: 13 to 15 and 20, and capable of specifically annealing to the LTR or pol gene region of the BLV proviral DNA; and (d) a probe having a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence set forth in any one of SEQ ID NOs: 13 to 15 and 20, and capable of specifically annealing to the LTR or pol gene region of the BLV proviral DNA.

[0053] Examples of "several" in (c) above include the same as "several" in (iii) and (iii') above. Examples of "sequence identity" in (d) above include the same as "sequence identity" in (iv) and (iv') above. In the above (a) to (d), the embodiment of "having a specific nucleotide sequence" encompasses the embodiment of "consisting of the specific nucleotide sequence".

[0054] The probes (a) to (d) are probes capable of specifically annealing to the LTR or pol gene region of the BLV provirus.

[0055] More specifically, a probe containing the nucleotide sequence set forth in SEQ ID NO: 13 or a mutated nucleotide sequence thereof can be used as a probe capable of annealing to the LTR of a BLV provirus. These probes can anneal to a nucleic acid fragment obtained by a nucleic acid amplification reaction using a forward primer selected from the group consisting of (iF) to (ivF) and a reverse primer selected from the group consisting of (iR) to (ivR). These probes can be used in real-time PCR using a forward primer selected from the group consisting of (iF) to (ivF) and a reverse primer selected from the group consisting of (iR) to (ivR).

[0056] Probes containing the nucleotide sequence set forth in SEQ ID NO: 14 or a mutated nucleotide sequence thereof can be used as probes capable of annealing to the pol gene region of the BLV provirus. These probes are capable of annealing to nucleic acid fragments obtained by a nucleic acid amplification reaction using a forward primer containing the nucleotide sequence set forth in SEQ ID NO: 3 or a mutated nucleotide sequence thereof and a reverse primer containing the nucleotide sequence set forth in SEQ ID NO: 4 or a mutated nucleotide sequence thereof. These probes can be used in real-time PCR using the forward primer and the reverse primer.

[0057] Probes containing the nucleotide sequence set forth in SEQ ID NO: 15 or a mutated nucleotide sequence thereof can be used as probes capable of annealing to the pol gene region of the BLV provirus. These probes are capable of annealing to nucleic acid fragments obtained by a nucleic acid amplification reaction using a forward primer containing the nucleotide sequence set forth in SEQ ID NO: 5 or a mutated nucleotide sequence thereof and a reverse primer containing the nucleotide sequence set forth in SEQ ID NO: 6 or a mutated nucleotide sequence thereof. These probes can be used in real-time PCR using the forward primer and the reverse primer.

[0058] A probe containing the nucleotide sequence set forth in SEQ ID NO: 20 or a mutant nucleotide sequence thereof can be used as a probe capable of annealing to the pol gene region of the BLV provirus. These probes are capable of annealing to a nucleic acid fragment obtained by a nucleic acid amplification reaction using a forward primer containing the nucleotide sequence set forth in SEQ ID NO: 3 or a mutant nucleotide sequence thereof and a reverse primer containing the nucleotide sequence set forth in SEQ ID NO: 4 or 19 or a mutant nucleotide sequence thereof. These probes can be used in real-time PCR using the forward primer and the reverse primer.

[0059] The reaction solution for the nucleic acid amplification reaction can be prepared by known methods. In the case of real-time PCR, for example, the reaction solution can contain a cfDNA sample as template DNA, a forward primer and a reverse primer, a probe, a thermostable DNA polymerase (such as Taq DNA polymerase), and dNTPs (a mixture of dATP, dCTP, dGTP, and dTTP). The magnesium concentration in the reaction solution can be, for example, 1 to 10 mM, preferably 2 to 8 mM, and more preferably 3 to 6 mM. Magnesium can be added to the reaction solution, for example, as magnesium chloride or magnesium sulfate. A fluorescent passive reference dye (such as ROX) may also be added to the reaction solution.

[0060] The nucleic acid amplification reaction can be carried out by a known method. In the case of real-time PCR, for example, the nucleic acid amplification reaction can be carried out by performing pre-denaturation, followed by repeating cycles of denaturation, annealing, and extension.

[0061] Examples of pre-denaturation temperatures include 94 to 98°C, with 95°C typically being used. Examples of pre-denaturation times include 20 seconds to 3 minutes (e.g., 30 seconds). Examples of denaturation temperatures include 94 to 98°C, with 95°C typically being used. Examples of denaturation times include 5 to 30 seconds (e.g., 25 seconds). The annealing temperature can be set based on the Tm of the primers. The annealing temperature can be set, for example, 2 to 3 degrees lower than the Tm of the primers. Examples of annealing temperatures include 45 to 65°C. Examples of annealing times include 40 to 100 seconds (e.g., 30 seconds). Examples of extension temperatures include 70 to 75°C, with 70 to 72°C typically being used. The extension time can be set depending on the length of the nucleic acid fragment to be amplified. Examples of extension reaction times include 30 to 100 seconds. Annealing and extension may be performed simultaneously. In this case, the annealing / extension temperature is, for example, 55 to 65°C, and is usually 60°C. The annealing / extension time can be set depending on the length of the nucleic acid fragment to be amplified. The annealing / extension time is, for example, 30 to 100 seconds.

[0062] The number of cycles of denaturation, annealing, and extension can be, for example, 30 to 100, preferably 40 to 60. When cycles of denaturation and annealing / extension are used, the number of cycles can be the same as those described above.

[0063] In the case of real-time PCR, the concentration of BLV cfDNA can be calculated by comparing the Ct value of a nucleic acid amplification reaction using a cfDNA sample as a template with the Ct (Threshold Cycle) value of a calibration curve. The calibration curve can be created by performing a nucleic acid amplification reaction using a standard sample containing target region DNA at a known concentration. The target region DNA is DNA containing the target region to be amplified in the nucleic acid amplification reaction. For example, when the target region is the LTR of the BLV provirus, the target region DNA contains the LTR of the BLV provirus.

[0064] <Other Steps> In one embodiment, the method for testing the progression of EBL may include other steps in addition to the above step (A). Examples of the other steps include a step of preparing a cfDNA sample (a cfDNA sample preparation step), a step of assessing the progression of EBL (assessment step), etc.

[0065] (cfDNA sample preparation step) A cfDNA sample can be prepared from a subject animal sample by a known method. For example, cellular components are removed from the subject animal sample to obtain a body fluid sample (e.g., plasma, serum, etc.). Removal of cellular components from the sample can be performed, for example, by centrifugation. Next, a cfDNA sample can be prepared by extracting DNA from the body fluid sample. Extraction of cfDNA from a body fluid sample can be performed by a known method as described above. The cfDNA sample preparation step can be performed in vitro.

[0066] (Determination step) The progression of EBL can be determined based on the quantitative value of BLV cfDNA obtained in step (A). In step (A), the quantitative value of BLV cfDNA may be calculated, for example, as the concentration of BLV cfDNA in a sample from a subject animal or in the body fluid of the subject animal. The quantitative value of BLV cfDNA may be calculated, for example, as the number (copy number) of BLV cfDNA containing the target region contained in 1 mL of the body fluid of the subject animal.

[0067] For example, if the quantitative value of BLV cfDNA (e.g., concentration in a body fluid) is higher than a reference value, it can be determined that the subject animal is likely to have developed EBL. Alternatively, if the quantitative value of BLV cfDNA is lower than the reference value, it can be determined that the subject animal is likely not to have developed EBL.

[0068] The reference value may be, for example, a non-onset reference value calculated by performing statistical processing or the like from quantitative values ​​of BLV cfDNA obtained in a group of multiple EBL-non-onset animals. In this case, if the quantitative value of BLV cfDNA obtained in step (A) is higher than the non-onset reference value, it can be determined that the subject animal is highly likely to have developed EBL, or that the subject animal is low in likelihood of not having developed EBL. If the quantitative value of BLV cfDNA obtained in step (A) is lower than or similar to the non-onset reference value, it can be determined that the subject animal is highly likely not to have developed EBL, or that the subject animal is low in likelihood of having developed EBL. The reference value may be, for example, an onset reference value calculated by performing statistical processing or the like from quantitative values ​​of BLV cfDNA obtained in a group of any number of EBL-onset animals. In this case, if the quantitative value of BLV cfDNA obtained in step (A) is higher than or similar to the onset reference value, it can be determined that the subject animal is highly likely to have developed EBL, or that the subject animal is low in likelihood of not having developed EBL. If the quantitative value of BLV cfDNA obtained in step (A) is lower than the onset reference value, it can be determined that the subject animal is highly likely not to have developed EBL, or that the subject animal is low in likelihood of having developed EBL. The reference value may be a cutoff value calculated by performing statistical processing or the like on the quantitative values ​​of BLV cfDNA obtained in multiple EBL-nondeveloping animal groups and multiple EBL-developing animal groups. In this case, if the quantitative value of BLV cfDNA obtained in step (A) is higher than the cutoff value, it can be determined that the subject animal is highly likely to have developed EBL, or that the subject animal is low in likelihood of not having developed EBL. When the quantitative value of BLV cfDNA obtained in step (A) is lower than the cutoff value, it can be determined that the subject animal is highly likely not to have developed EBL, or that the subject animal is highly likely to have developed EBL.

[0069] In the assessment step, the progression of EBL may be assessed by combining data obtained by other methods with the quantitative value of BLV cfDNA. For example, real-time PCR may be performed using DNA extracted from cellular components in a blood sample as a template, and the obtained quantitative value of intracellular BLV proviral DNA may be combined. Alternatively, real-time PCR may be performed using DNA extracted from a whole blood sample as a template, and the obtained quantitative value of BLV proviral DNA in whole blood may be combined.

[0070] Alternatively, the method described in JP 2018-023329 A may be combined. The method described in JP 2018-023329 A uses cells that are infectable with BLV, into which a gene construct comprising: 1) an expression control sequence derived from a deltaretrovirus LTR, which has reduced or no U5 region activity but has U3 region promoter activity; and 2) a reporter gene linked to the expression control sequence in an expressible manner is introduced into the cells. The cells are contacted with a sample (e.g., a body fluid sample containing leukocytes) collected from a subject animal, and the progression of EBL is assessed based on the expression of the reporter gene. For example, if the expression level of the reporter gene is equal to or greater than a predetermined threshold, it can be determined that the subject animal is highly likely to have developed EBL.

[0071] When the target animal is a livestock such as a cow, the assessment step may be performed as an auxiliary step in the step of determining whether to discard all of the target animals. All cattle confirmed to have EBL at slaughter inspection are discarded. Raising EBL-affected cattle results in economic losses for livestock farmers. Therefore, the quantitative value of BLV cfDNA obtained in step (A) may be used as an auxiliary test when determining whether to discard all of the target animals. For example, if the target animal is determined to have a high probability of having EBL in the assessment step, further tests for assessing the severity of EBL may be performed. Examples of further tests for assessing the severity of EBL include clinical tests, necropsies, antibody tests, and histopathological tests. If the target animal is finally diagnosed as having EBL as a result of the further tests for assessing the severity of EBL, the target animal may be discarded. For example, the method for testing the progression of EBL may include, after the determination step, a step of conducting a disease progression assessment test on the target animal that has been determined to have developed EBL in the determination step, and a step of discarding the target animal that has been diagnosed as having developed EBL in the disease progression assessment test.

[0072] According to the method for testing the progression of infectious bovine lymphoma of this embodiment, the progression of infectious bovine lymphoma can be tested more simply and at lower cost than conventional methods such as NGS analysis. As will be shown in the Examples below, the testing method of this embodiment can diagnose the onset of EBL with high sensitivity and high specificity.

[0073] In the early stages of EBL, tumors develop in lymph nodes, heart, liver, spleen, uterus, etc. Therefore, it was unexpected that the amount of BLV cfDNA in body fluids (plasma, etc.) correlates with the onset of EBL. However, as shown in the Examples below, it has been confirmed that the onset of EBL can be determined based on the amount of BLV cfDNA in body fluids.

[0074] [Primer] A second aspect of the present disclosure is at least one primer selected from the group consisting of the following (i) to (iv): (i) a primer having a nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 6 and 19; (ii) a primer capable of specifically annealing to a region in BLV proviral DNA to which a primer comprising a nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 6 and 19 can specifically anneal; (iii) a primer having a nucleotide sequence in which one or several nucleotides have been deleted, added, or substituted in the nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 6 and 19, and capable of specifically annealing to the LTR or pol gene region of BLV proviral DNA; and (iv) a primer having a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 6 and 19, and capable of specifically annealing to the LTR or pol gene region of BLV proviral DNA.

[0075] The primers (i) to (iv) are the same as those described above in the "Method for testing the progression of bovine infectious lymphoma." The primers can be synthesized by known solid-phase synthesis methods such as the phosphoramidate method.

[0076] [Cell-Free DNA Quantification Kit] A third aspect of the present disclosure is a kit for quantifying cell-free DNA derived from bovine infectious lymphoma virus proviral DNA. In one embodiment, the kit includes the primer according to the second aspect, a probe capable of annealing to a nucleic acid fragment obtained by a nucleic acid amplification reaction using the primer according to the second aspect, and a standard DNA containing the nucleotide sequence of the nucleic acid fragment obtained by a nucleic acid amplification reaction using the primer according to the second aspect.

[0077] <Primers> The primers are those according to the second aspect. The primers are the same as those described above in the "Method for testing the progression of bovine infectious lymphoma." The primers preferably include at least one primer selected from the group consisting of (i) to (iv) above, or at least one primer selected from the group consisting of (i') to (iv') above. The primers preferably include a forward primer selected from the group consisting of (iF) to (ivF) above, and a reverse primer selected from the group consisting of (iR) to (ivR) above.

[0078] <Probe> The probe is a probe capable of annealing to a nucleic acid fragment obtained by a nucleic acid amplification reaction using the primers. Examples of the probe include the probes listed above in the "Method for testing the progression of bovine infectious lymphoma." Examples of the probe include the probes (a) to (d) listed above.

[0079] <Standard DNA> The standard DNA is DNA containing the nucleotide sequence of the nucleic acid fragment obtained by the nucleic acid amplification reaction using the above primers. The standard DNA is, for example, a plasmid.

[0080] Combinations of primers, probes, and standard DNA include the following. Examples of "several" and "90% or more sequence identity" below include the same as above. In the primers and probes below, an embodiment "having a specific nucleotide sequence" encompasses an embodiment "consisting of that specific nucleotide sequence."

[0081] (Combination targeting LTR) Primers: A forward primer selected from the group consisting of (iF) to (ivF) above, and a reverse primer selected from the group consisting of (iR) to (ivR). Probe: A probe selected from the group consisting of a probe having the nucleotide sequence set forth in SEQ ID NO: 13; a probe capable of specifically annealing to a region in BLV proviral DNA to which a probe comprising the nucleotide sequence set forth in SEQ ID NO: 13 can specifically anneal; a probe having a nucleotide sequence in which one or several nucleotides are deleted, added, or substituted in the nucleotide sequence set forth in SEQ ID NO: 13, and capable of specifically annealing to the LTR of BLV proviral DNA; and a probe having a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence set forth in SEQ ID NO: 13, and capable of specifically annealing to the LTR of BLV proviral DNA. Standard DNA: Standard DNA containing a target region for nucleic acid amplification reaction in the LTR (particularly U3) of the BLV provirus. The standard DNA may contain the entire U3, may contain the entire 5'LTR, or may contain the entire 3'LTR.

[0082] (Combination targeting the 5' side of the pol gene) Primers: A forward primer selected from the group consisting of: a forward primer having the nucleotide sequence set forth in SEQ ID NO: 3; a forward primer capable of specifically annealing to a region in the BLV proviral DNA to which a primer comprising the nucleotide sequence set forth in SEQ ID NO: 3 can specifically anneal; a forward primer having a nucleotide sequence in which one or several nucleotides have been deleted, added, or substituted in the nucleotide sequence set forth in SEQ ID NO: 3 and capable of specifically annealing to the pol gene region of the BLV proviral DNA; and a forward primer having a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence set forth in SEQ ID NO: 3 and capable of specifically annealing to the pol gene region of the BLV proviral DNA; A reverse primer selected from the group consisting of: a reverse primer having the nucleotide sequence set forth in SEQ ID NO: 4 or 19; a reverse primer capable of specifically annealing to a region in BLV proviral DNA to which a primer comprising the nucleotide sequence set forth in SEQ ID NO: 4 or 19 can specifically anneal; a reverse primer having a nucleotide sequence in which one or several nucleotides are deleted, added, or substituted in the nucleotide sequence set forth in SEQ ID NO: 4 or 19 and capable of specifically annealing to the pol gene region of BLV proviral DNA; and a reverse primer having a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence set forth in SEQ ID NO: 4 or 19 and capable of specifically annealing to the pol gene region of BLV proviral DNA.Probe: A probe selected from the group consisting of: a probe having the nucleotide sequence set forth in SEQ ID NO: 14 or 20; a probe capable of specifically annealing to a region in BLV proviral DNA to which a probe comprising the nucleotide sequence set forth in SEQ ID NO: 14 or 20 can specifically anneal; a probe having a nucleotide sequence in which one or several nucleotides are deleted, added, or substituted in the nucleotide sequence set forth in SEQ ID NO: 14 or 20 and capable of specifically annealing to the pol gene region of BLV proviral DNA; and a probe having a nucleotide sequence with 90% or more sequence identity to the nucleotide sequence set forth in SEQ ID NO: 14 or 20 and capable of specifically annealing to the pol gene region of BLV proviral DNA. Standard DNA: Standard DNA containing a target region in the pol gene of BLV provirus for nucleic acid amplification reactions. The standard DNA may contain the entire pol gene.

[0083] (Combination targeting the 3' side of the pol gene) Primers: A forward primer selected from the group consisting of: a forward primer having the nucleotide sequence set forth in SEQ ID NO: 5; a forward primer capable of specifically annealing to a region in the BLV proviral DNA to which a primer comprising the nucleotide sequence set forth in SEQ ID NO: 5 can specifically anneal; a forward primer having a nucleotide sequence in which one or several nucleotides have been deleted, added, or substituted in the nucleotide sequence set forth in SEQ ID NO: 5 and capable of specifically annealing to the pol gene region of the BLV proviral DNA; and a forward primer having a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence set forth in SEQ ID NO: 5 and capable of specifically annealing to the pol gene region of the BLV proviral DNA; A reverse primer selected from the group consisting of: a reverse primer having the nucleotide sequence set forth in SEQ ID NO: 6; a reverse primer capable of specifically annealing to a region in BLV proviral DNA to which a primer comprising the nucleotide sequence set forth in SEQ ID NO: 6 can specifically anneal; a reverse primer having a nucleotide sequence in which one or more nucleotides have been deleted, added, or substituted in the nucleotide sequence set forth in SEQ ID NO: 6 and capable of specifically annealing to the pol gene region of BLV proviral DNA; and a reverse primer having a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence set forth in SEQ ID NO: 6 and capable of specifically annealing to the pol gene region of BLV proviral DNA.Probe: A probe selected from the group consisting of a probe having the nucleotide sequence set forth in SEQ ID NO: 15; a probe capable of specifically annealing to a region in BLV proviral DNA to which a probe comprising the nucleotide sequence set forth in SEQ ID NO: 15 can specifically anneal; a probe having a nucleotide sequence in which one or several nucleotides are deleted, added, or substituted in the nucleotide sequence set forth in SEQ ID NO: 15 and capable of specifically annealing to the pol gene region of BLV proviral DNA; and a probe having a nucleotide sequence with 90% or more sequence identity to the nucleotide sequence set forth in SEQ ID NO: 15 and capable of specifically annealing to the pol gene region of BLV proviral DNA. Standard DNA: Standard DNA containing a target region in the pol gene of BLV provirus for nucleic acid amplification reactions. The standard DNA may contain the entire pol gene.

[0084] <Other Components> The quantification kit may include other components, such as reagents used in PCR reactions, including a thermostable DNA polymerase (such as Taq DNA polymerase), dNTPs, a PCR reaction buffer, and magnesium salts.

[0085] The quantification kit according to this embodiment can be used to quantify BLV cfDNA by real-time PCR.

[0086] [Test Kit] A fourth aspect of the present disclosure is a test kit for detecting the progression of bovine infectious lymphoma. In one embodiment, the test kit comprises a primer capable of annealing to cell-free DNA derived from proviral DNA of bovine infectious lymphoma virus.

[0087] Examples of the primer include the primers listed above in the section "Method for testing the progression of bovine infectious lymphoma." The primer is preferably the primer according to the second aspect.

[0088] The test kit may contain, in addition to the primers, a probe capable of annealing to a nucleic acid fragment obtained by a nucleic acid amplification reaction using the primers. Examples of the primer include the probes listed above in the "Testing method for the progression of bovine infectious lymphoma."

[0089] In addition to the primers or the primers and probes, the test kit may also include a standard DNA containing the nucleotide sequence of a nucleic acid fragment obtained by a nucleic acid amplification reaction using the primers. Examples of the standard DNA include the standard DNAs listed in the "Quantitation Kit" section above. Examples of combinations of primers, probes, and standard DNA include the combinations listed in the "Quantitation Kit" section above.

[0090] The test kit may include the quantitative kit according to the third aspect.

[0091] In addition to the above components, the test kit may further include a cfDNA extraction reagent. The cfDNA extraction reagent may be a known reagent. Examples of the cfDNA extraction reagent include, but are not limited to, a silica membrane column, an ion exchange resin column, and silicon-coated magnetic beads.

[0092] The test kit according to this embodiment can be used in the test method according to the first embodiment.

[0093] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0094] [Experimental Example 1] (Selection of target region) <Design of primers and probes for real-time PCR> In the BLV genome, regions 1 to 5 shown in Figure 1 were selected as candidate target regions for real-time PCR. Regions 1 to 5 are as follows: 1. U3 region of LTR 2. pro gene region (Pro) 3. pro-pol junction region (Pro-Pol) 4. 5' region of pol gene (Pol1) 5. 3' region of pol gene (Pol2)

[0095] Primers and probes for real-time PCR were designed for each candidate target region. The designed primers are shown in Table 1. The designed probes are shown in Table 2. Tables 1 and 2 also show primers and probes for the reference gene (actβ).

[0096]

[0097]

[0098] <Preparation of standard plasmid> A SalI-NotI fragment was prepared containing the reference gene actβ and regions 1 to 5 of BLV shown in Figure 1. pUC-L-Pr-PI (custom-prepared by VectorBuilder Japan) was cleaved with SalI and NotI, and the SalI-NotI fragment was ligated. This produced the standard plasmid (pL-pR-pL) shown in Figure 2.

[0099] <Preparation of dilution series of standard plasmid> A dilution series of the standard plasmid was prepared by the following procedure. (1) 45 μL of EB buffer (Qiagen) was dispensed into six microtubes. (2) 5 μL of the standard plasmid (pL-pR-pL) solution (1.0 × 10 ７ (3) 5 μL of the solution was taken from the No. 6 microtube and added to the No. 5 microtube. 5 μL of the solution was then transferred to the No. 4, No. 3, No. 2, and No. 1 microtubes in order, followed by serial dilutions.

[0100] <Real-time PCR> Real-time PCR was performed using each sample of a dilution series of the standard plasmid. Real-time PCR was performed using a StepOne Plus Real-Time PCR system (Applied Biosystems). Real-time PCR was performed for each of the candidate target regions shown in Figure 1 using the primers and probes shown in Tables 1 and 2 (see Tables 1 and 2).

[0101] The composition of the reaction solution for real-time PCR is shown in Table 3. The reaction cycle for real-time PCR is shown in Table 4.

[0102]

[0103]

[0104] In FIG. ６ Figure 3 shows the amplification curves of real-time PCR performed using a standard plasmid solution (copies / μL). From the results shown in Figure 3, region 1 (LTR U3), region 4 (Pol1), and region 5 (Pol2) were considered to be suitable target regions for real-time PCR.

[0105] Table 5 shows 10 ６ Copies / μL, 10 ４ Copies / μL, or 10 ２ The threshold cycle (Ct) values ​​of real-time PCR performed using standard plasmid solutions (copies / μL) are shown.

[0106]

[0107] The results shown in Table 5 also suggest that region 1 (LTR U3), region 4 (Pol1), and region 5 (Pol2) are suitable target regions for real-time PCR.

[0108] <Droplet Digital PCR (ddPCR)> 10 ５ A standard plasmid solution of copies / μL was prepared, and ddPCR was performed using region 1 (LTR U3), region 4 (Pol1), or region 5 (Pol2) as the target region. The primers used for each region are shown in Table 1. ddPCR was performed using a Digital PCR QX200 system (Bio-Rad).

[0109] The results of ddPCR are shown in Figure 4. From the results shown in Figure 4, it was considered that among regions 1 to 3, region 1 (LTR U3) had high amplification specificity.

[0110] From the above results, it was considered that region 1 (LTR U3) had the highest amplification efficiency and specificity in the nucleic acid amplification reaction, and therefore region 1 (LTR U3) was selected as the final candidate target region.

[0111] Experimental Example 2 (Evaluation of Primers and Probes) <Evaluation of Commonality Between BLV Virus Strains> The primers (LTR_F (SEQ ID NO: 1), LTR_R (SEQ ID NO: 2)) and probe (LTR_P (SEQ ID NO: 13)) for region 1 (LTR U3) shown in Tables 1 and 2 were evaluated for their common applicability to various known BLVs. Nucleotide sequences of 190 types of BLV were obtained from sequence databases such as NCBI. The nucleotide sequences of the primers (LTR_F, LTR_R) and probe (LTR_Probe) for region 1 (LTR U3) were confirmed to be commonly present in the genomes of 190 types of BLV.

[0112] A portion of the analysis results is shown in Figure 5. In Figure 5, dots (•) indicate nucleotide residues that are common among BLV virus strains.

[0113] <Evaluation of Applicability to cfDNA> It was investigated whether the primers (LTR_F, LTR_R) and probe (LTR_P) for region 1 (LTR U3) shown in Tables 1 and 2 could be applied to cfDNA from cattle with infectious bovine lymphoma.

[0114] Blood was collected from cattle with infectious bovine lymphoma. The collected blood was centrifuged (1,600 x g, 10 minutes) to obtain the supernatant, which was used as a serum sample. cfDNA was then extracted from the serum sample using a QIAamp Circulating Nucleic Acid kit (Qiagen). A DNA library was prepared from the obtained cfDNA (NEBNext UltraII DNA library preparation kit, NEB) and analyzed using a next-generation sequencer (NGS) (MiSeq, Illumia).

[0115] The results are shown in Figure 6. Figure 6 shows the results of NGS analysis of cfDNA extracted from serum samples from three cases of cattle with infectious bovine lymphoma. In Figure 6, the lower figure is an enlarged view of a portion of the upper figure. The results of the NGS analysis in Figure 6 confirmed that region 1 (LTR U3) was present in sufficient amounts in the cfDNA of cattle with infectious bovine lymphoma.

[0116] Experimental Example 3 (Evaluation of the correlation between BLV-derived cfDNA and the onset of infectious bovine lymphoma) Blood was collected from cattle with and without infectious bovine lymphoma, and serum samples were prepared and cfDNA was extracted in the same manner as described above. Real-time PCR was performed using the extracted cfDNA as a template and the primers (LTR_F, LTR_R) and probe (LTR_P) for region 1 (LTR U3) shown in Tables 1 and 2. Real-time PCR was performed in the same manner as in Experimental Example 1. A standard curve was prepared using a dilution series of the standard plasmid (pL-pR-pL).

[0117] The results are shown in Figure 7. Compared to cattle without infectious bovine lymphoma (Non-EBL), cattle with infectious bovine lymphoma (EBL) showed higher measured values ​​of cfDNA containing region 1 (LTR U3). The sensitivity of this method for diagnosing infectious bovine lymphoma was 90%, and the specificity was 90%. These results confirmed that the onset of infectious bovine lymphoma can be diagnosed by quantifying BLV-derived cfDNA.

[0118] Experimental Example 4 (Evaluation of the correlation between intracellular BLV proviral DNA and the onset of bovine infectious lymphoma) Blood was collected from cattle with and without bovine infectious lymphoma. The collected blood was centrifuged (3,000 rpm, 30 minutes) to obtain a precipitate, which was used as a blood cell sample. DNA was then extracted from the blood cell sample using a DNeasy Blood and Tissue kit (Qiagen). Real-time PCR was performed in the same manner as in Experimental Example 3, except that the DNA extracted from the blood cell sample was used as a template.

[0119] The results are shown in Figure 8. There was no significant difference in the measured values ​​of DNA containing region 1 (LTR U3) between cattle affected by infectious bovine lymphoma (EBL) and cattle not affected by infectious bovine lymphoma (Non-EBL). The sensitivity of this method for diagnosing infectious bovine lymphoma was 55%, and the specificity was 60%. These results confirmed that it is difficult to diagnose infectious bovine lymphoma by quantifying BLV proviral DNA in blood cells.

[0120] [Experimental Example 5] (Evaluation of correlation between combination of cfDNA and intracellular DNA and onset of infectious bovine lymphoma) An evaluation was conducted to determine whether combining the results of Experimental Example 3 and Experimental Example 4 would improve the ability to separate cattle that have developed infectious bovine lymphoma from cattle that have not developed infectious bovine lymphoma.

[0121] The results are shown in Figure 9. In Figure 9, the vertical axis shows the measured values ​​of BLV-derived cfDNA, and the horizontal axis shows the measured values ​​of BLV proviral DNA in blood cells. The results shown in Figure 9 confirmed that combining the measured values ​​of BLV-derived cfDNA and the measured values ​​of BLV proviral DNA in blood cells improves the ability to separate cattle that develop infectious bovine lymphoma from cattle that do not develop infectious bovine lymphoma.

[0122] These results confirmed that the results obtained by this test method are consistent with the progression of infectious bovine lymphoma, demonstrating that this test method can be used to detect the progression of infectious bovine lymphoma.

[0123] Experimental Example 6 (Evaluation of the correlation between the amount of BLV-derived DNA in whole blood and plasma and the onset of infectious bovine lymphoma) Blood was collected from cattle with and without infectious bovine lymphoma. Plasma was prepared from the blood. DNA was extracted from the whole blood and plasma using a DNeasy Blood and Tissue kit (Qiagen). The DNA extracted from the whole blood was used as a whole blood DNA sample. The DNA extracted from the plasma was used as a cfDNA sample. Real-time PCR was performed using the whole blood DNA and cfDNA as templates, respectively, and the primers and probes shown in Tables 6 and 7. Real-time PCR was performed using a LightCycler (registered trademark) 96 system (Roche Diagnostics) and a StepOne Plus Real-Time PCR system (Applied Biosystems). The composition of the PCR reaction solution was as shown in Table 3. The reaction cycle for real-time PCR was as shown in Table 4.

[0124]

[0125]

[0126] The results are shown in Figures 10 to 13. Figure 10 shows the results of real-time PCR using whole blood DNA as a template, LTR_F and LTR_R as primers, and LTR_P as a probe. Figure 11 shows the results of real-time PCR using cfDNA as a template, LTR_F and LTR_R as primers, and LTR_P as a probe. Figure 12 shows the results of real-time PCR using whole blood DNA as a template, Pol_F3 and Pol_R3 as primers, and Pol_P3 as a probe. Figure 13 shows the results of real-time PCR using cfDNA as a template, Pol_F3 and Pol_R3 as primers, and Pol_P3 as a probe.

[0127] In all cases, cattle with infectious bovine lymphoma (EBL) showed higher values ​​than cattle without infectious bovine lymphoma (Non-EBL). Regardless of the primers and probes used, the ability to separate cattle with infectious bovine lymphoma from those without was improved when cfDNA was used as a template compared to when whole blood DNA was used as a template.

[0128] [Experimental Example 7] (Study on the detection sensitivity of BLV provirus) cfDNA samples from cattle with infectious bovine lymphoma and cattle without infectious bovine lymphoma were prepared in the same manner as in Experimental Example 6. Using the cfDNA as a template, real-time PCR was performed in the same manner as in Experimental Example 6. The following primers and probes were used: 86 bp: Primers: LTR_F and LTR_R, Probe: LTR_P, PCR product size: 86 bp 183 bp: CoCoMo ＴＭ -BLV Primer / Probe (Nippon Gene) primer and probe, PCR product size: 183 bp

[0129] The results are shown in Figures 14 and 15. Figure 14 shows the results when cfDNA from cattle not affected by infectious bovine lymphoma (nonEBL) was used as a template. Figure 15 shows the results when cfDNA from cattle affected by infectious bovine lymphoma (EBL) was used as a template. The 86 bp primer set showed significantly higher sensitivity for detecting BLV DNA than the 183 bp primer set. From these results, it was concluded that the LTR_F and LTR_R primer sets were suitable for CoCoMo ＴＭ -It was confirmed that this primer set is superior to the BLV Primer / Probe primer set in terms of detecting BLV DNA in cfDNA.

[0130] According to the present invention, there are provided a method for testing the progression of infectious bovine lymphoma, which allows for a simple and rapid evaluation of the progression of the disease, primers that can be used in the testing method, and a testing kit. While preferred embodiments of the present invention have been described above, the present invention is not limited to these embodiments. Addition, omission, substitution, and other modifications of the components are possible within the scope of the present invention. The present invention is not limited by the above description, but is limited only by the scope of the appended claims.

Claims

1. A method for testing the progression of bovine infectious lymphoma, comprising the step (A) of quantifying cell-free DNA derived from the proviral DNA of bovine infectious lymphoma virus in a sample containing cell-free DNA from a target animal.

2. A method for testing the progression of bovine infectious lymphoma according to claim 1, wherein the cell-free DNA derived from the proviral DNA contains at least a portion of a region selected from the group consisting of the LTR and pol gene of the proviral DNA.

3. A method for testing the progression of bovine infectious lymphoma as described in claim 1 or 2, wherein step (A) comprises a step of carrying out a nucleic acid amplification reaction using the cell-free DNA as a template and a primer capable of specifically annealing to the proviral DNA.

4. A method for testing the progression of bovine infectious lymphoma as described in claim 3, wherein the primer is capable of specifically annealing to at least one region selected from the group consisting of the LTR and pol gene of the proviral DNA.

5. A method for testing the progression of bovine infectious lymphoma according to claim 3, wherein the primer comprises at least one type selected from the group consisting of the following (i) to (iv): (i) a primer having a nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 6 and 19; (ii) a primer capable of specifically annealing to a region in the proviral DNA to which a primer comprising a nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 6 and 19 can specifically anneal; (iii) a primer having a nucleotide sequence in which one or more nucleotides have been deleted, added or substituted in the nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 6 and 19, and capable of specifically annealing to the LTR or pol gene region of the proviral DNA; and (iv) a primer having a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 6 and 19, and capable of specifically annealing to the LTR or pol gene region of the proviral DNA.

6. A method for testing the progression of bovine infectious lymphoma according to claim 4, wherein the primers comprise at least one type selected from the group consisting of the following (i') to (iv'): (i') a primer having the nucleotide sequence set forth in SEQ ID NO: 1 or 2; (ii') a primer capable of specifically annealing to a region in the proviral DNA to which a primer comprising the nucleotide sequence set forth in SEQ ID NO: 1 or 2 can specifically anneal; (iii') a primer having a nucleotide sequence in which one or several nucleotides have been deleted, added, or substituted in the nucleotide sequence set forth in SEQ ID NO: 1 or 2, and capable of specifically annealing to the LTR of the proviral DNA; and (iv') a primer having a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence set forth in SEQ ID NO: 1 or 2, and capable of specifically annealing to the LTR of the proviral DNA.

7. A primer selected from the group consisting of (i) to (iv) below: (i) a primer having a nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 6 and 19; (ii) a primer capable of specifically annealing to a region in the proviral DNA of bovine infectious lymphoma virus to which a primer comprising a nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 6 and 19 can specifically anneal; (iii) a primer having a nucleotide sequence in which one or more nucleotides have been deleted, added, or substituted in the nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 6 and 19, and capable of specifically annealing to the LTR or pol gene region of the proviral DNA; and (iv) a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 6 and 19, and capable of specifically annealing to the LTR or pol gene region of the proviral DNA.

8. A kit for quantifying cell-free DNA derived from proviral DNA of bovine infectious lymphoma virus, comprising: the primer according to claim 7; a probe capable of annealing to a nucleic acid fragment obtained by a nucleic acid amplification reaction using the primer according to claim 7; and standard DNA containing the nucleotide sequence of the nucleic acid fragment obtained by a nucleic acid amplification reaction using the primer according to claim 7.

9. A test kit for detecting the progression of bovine infectious lymphoma, comprising a primer capable of annealing to cell-free DNA derived from the proviral DNA of bovine infectious lymphoma virus.

10. A test kit for detecting the progression of bovine infectious lymphoma according to claim 9, further comprising: a probe capable of annealing to a nucleic acid fragment obtained by a nucleic acid amplification reaction using the primers; and standard DNA containing the nucleotide sequence of the nucleic acid fragment obtained by a nucleic acid amplification reaction using the primers.

11. The test kit for detecting the progression of bovine infectious lymphoma according to claim 9 or 10, further comprising a cell-free DNA extraction reagent.

12. A test kit for detecting the progression of bovine infectious lymphoma according to claim 9 or 10, wherein the primers include the primers according to claim 7.

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