Methods for testing the disease activity of bovine lymphoma, primers, and test kits for testing the disease activity of bovine lymphoma.

Abstract

To provide methods for examining the disease progression of bovine infectious lymphoma, which can evaluate the disease progression of bovine infectious lymphoma easily and rapidly, to provide primers that can be used in the examination method, and to provide examination kits.SOLUTION: Provided is a method for examining the disease progression of bovine infectious lymphoma, comprising the steps of: (a) preparing an infected cell sample of a host animal infected with bovine infectious lymphoma virus; (b) using the genomic DNA of the infected cell as a template to amplify a DNA fragment of the genome border region containing both the proviral DNA of bovine infectious lymphoma virus and the genomic DNA of the host animal; (c) performing Sanger sequencing of the DNA fragment and obtaining spectral data by Sanger sequencing; (d) based on the spectral data, identifying the region derived from the proviral DNA and the region derived from the genomic DNA of the host animal in the DNA fragment, and determining the clonality of the region derived from the genomic DNA of the host animal; and the like.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for examining the disease state of bovine infectious lymphoma, a primer, and a kit for examining the disease state of bovine infectious lymphoma.

Background Art

[0002] Cattle are important food resources such as milk and meat. In recent years, the high quality of Japanese beef cattle and dairy products has been globally evaluated, and the demand for them is increasing annually not only in Japan but also overseas. Globally, dairy farming and beef cattle production occupy a very large share in the livestock industry.

[0003] Bovine leukemia virus (BLV) is a virus that infects bovine B lymphocytes. BLV has pathogenicity that causes lymphoma in some infected cattle. 60 - 70% of infected cattle do not show symptoms and do not develop the disease, but 30% of infected cattle develop persistent lymphocytosis, and a few percent of infected cattle develop lymphoma.

[0004] In recent years in Japan, both the infection rate of BLV and the number of cases of infectious lymphoma have been on the increase, and the need for countermeasures has been growing. In Japan, cattle that have not developed lymphoma only due to BLV infection are distributed in the market as meat. However, if the development of infectious lymphoma is recognized during livestock inspection, all of them will be discarded according to the provisions of the Livestock Infectious Diseases Prevention Law. Therefore, the development of infectious lymphoma causes a great economic loss to the producing farmers.

[0005] In order to detect the development of infectious lymphoma, accurate diagnosis is necessary. However, despite this important problem, the diagnosis of infectious lymphoma is currently carried out by veterinarians by visually or palpating to confirm the presence or absence of swollen lymph nodes. Therefore, the diagnostic accuracy is not high.

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

[0007] Furthermore, a method for evaluating the clonality of BLV-infected cells using next-generation sequencing analysis has been proposed (Non-Patent Literature 1). However, next-generation sequencers are expensive and have limited versatility. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] Japanese Patent Publication No. 2019-180351 [Non-patent literature]

[0009] [Non-Patent Document 1] 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. [Overview of the Initiative] [Problems that the invention aims to solve]

[0010] As described above, methods for quantifying BLV provirus cannot quantify the number of tumorigeneized cells and therefore cannot assess the disease activity of bovine lymphoma. Furthermore, methods using next-generation sequencing are costly and difficult to use practically. Therefore, there is a need for a diagnostic technique that can easily and rapidly assess the disease activity of bovine lymphoma.

[0011] Therefore, the object of the present invention is to provide a method for testing the disease activity of bovine infectious lymphoma, a primer usable in the said testing method, and a test kit that can evaluate the disease activity of bovine infectious lymphoma simply and rapidly. [Means for solving the problem]

[0012] The present invention includes the following embodiments. [1] A method for testing the disease activity of bovine infectious lymphoma, comprising: (a) preparing an infected cell sample of a host animal infected with bovine leukemia disease bovine lymphoma virus; (b) amplifying a DNA fragment of the genomic boundary region containing both the proviral DNA of the bovine leukemia disease bovine lymphoma virus and the genomic DNA of the host animal, using the genomic DNA of the infected cells as a template; (c) performing Sanger sequencing of the DNA fragment and obtaining spectral data by Sanger sequencing; (d) identifying, based on the spectral data, a region in the DNA fragment derived from the proviral DNA and a region derived from the genomic DNA of the host animal, and determining the uniformity of the region derived from the genomic DNA of the host animal; and (e) determining the disease activity of bovine infectious lymphoma in the host animal based on the uniformity of the region derived from the genomic DNA of the host animal. [2] A method for testing the disease activity of bovine infectious lymphoma according to [1], wherein in step (b) above, at least one primer selected from the group consisting of (i) to (iv) below is used: (i) a primer having the nucleotide sequence described in SEQ ID NO: 1, 2, or 3; (ii) a primer that specifically anneals to the region in the genomic DNA of bovine leukemia bovine infectious lymphoma virus in which the primer containing the nucleotide sequence described in SEQ ID NO: 1, 2, or 3 specifically anneals; (iii) a primer having a nucleotide sequence in which one or more nucleotides are deleted, added, or substituted in the nucleotide sequence described in SEQ ID NO: 1, 2, or 3, and that specifically anneals to the genomic DNA of bovine leukemia bovine infectious lymphoma virus; and (iv) a primer having a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence described in SEQ ID NO: 1, 2, or 3, and that specifically anneals to the genomic DNA of bovine leukemia bovine infectious lymphoma virus. [3] The step in (b) is to (b1) perform a DNA extension reaction using the genomic DNA of the infected cell as a template and a first primer that specifically anneals to the proviral DNA; (b2) purify the first DNA fragment produced in step (b1); (b3) attach a first adapter to the first DNA fragment; and (b4) use the first DNA fragment with the first adapter attached as a template to specifically anneal to the first adapter and to the second adapter A method for testing the disease activity of bovine infectious lymphoma according to [1] or [2], comprising: (b) a step of performing a DNA extension reaction using a second primer containing; (b5) a step of purifying the second DNA fragment produced in step (b4); and (b6) a step of performing a nucleic acid amplification reaction using the second DNA fragment purified in step (b5) as a template, using a third primer that specifically anneals to the proviral DNA contained in the second DNA fragment and a fourth primer that specifically anneals to the second adapter. [4] The method for testing the disease activity of bovine infectious lymphoma according to [3], wherein step (b) is followed by (b6), and (b7) a nucleic acid amplification reaction is performed using the third DNA fragment obtained in step (b6) as a template, with a fifth primer that specifically anneals to the proviral DNA contained in the third DNA fragment and a sixth primer that specifically anneals to the second adapter. [5] At least one primer selected from the group consisting of (i) to (iv) below: (i) a primer having the nucleotide sequence described in SEQ ID NO: 1, 2, or 3; (ii) a primer that specifically anneals to the region in the proviral DNA of bovine leukemia disease bovine infectious lymphoma virus in which a primer containing the nucleotide sequence described in SEQ ID NO: 1, 2, or 3 specifically anneals; (iii) a primer having a nucleotide sequence in which one or more nucleotides are deleted, added, or substituted in the nucleotide sequence described in SEQ ID NO: 1, 2, or 3, and that specifically anneals to the proviral DNA; and (iv) a primer having a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence described in SEQ ID NO: 1, 2, or 3, and that specifically anneals to the proviral DNA. A disease activity test kit for bovine lymphoma, including the primers described in [6][5]. [Effects of the Invention]

[0013] The present invention provides a method for testing the disease activity of bovine infectious lymphoma, a primer usable in the testing method, and a testing kit, all of which enable a simple and rapid evaluation of the disease activity of bovine infectious lymphoma. [Brief explanation of the drawing]

[0014] [Figure 1] This diagram illustrates the state of infected cells in cattle that develop infectious lymphoma and those that do not. [Figure 2] This is a schematic diagram illustrating the principle of the inspection method according to one embodiment. [Figure 3]It is a schematic diagram showing an example of step (b) of the inspection method of the embodiment. [Figure 4] It is a figure showing the results of evaluating the homogeneity of the host-side DNA in the genomic boundary region in non-developing infectious lymphoma cattle, lymphocytosis cattle, and developing infectious lymphoma cattle. A shows the spectral data of Sanger sequencing. B shows the results of analyzing the spectral data of Sanger sequencing with EditR. [Figure 5] It is a figure showing the results of evaluating the homogeneity of the host-side DNA in the genomic boundary region in non-developing infectious lymphoma cattle, lymphocytosis cattle, and developing infectious lymphoma cattle. A shows the results of analyzing the spectral data of Sanger sequencing with EditR. B shows the results of evaluating the homogeneity of infected cells by next-generation sequencer (NGS) analysis. [Figure 6] It is a figure showing the results of evaluating the homogeneity of the host-side DNA in the genomic boundary region of BLV-infected cattle over time. "NGS analysis" shows the results of evaluating the homogeneity of infected cells by NGS analysis. "PVL (%)" shows the quantification results of provirus amounts by real-time PCR. "IS" shows the number of provirus insertion sites of BLV. [Figure 7] It is a figure showing the results of evaluating the homogeneity of the host-side DNA in the genomic boundary region of BLV-infected cattle over time. It is a different case from FIG. 6. [Figure 8] [[ID=1...]]It is a figure showing the results of evaluating the homogeneity of the host-side DNA in the genomic boundary region of BLV-infected cattle over time. It is a different case from FIGS. 6 and 7. [Figure 9] It is a figure showing the results of evaluating the homogeneity of the host-side DNA in the genomic boundary region of BLV-infected cattle over time. It is a different case from FIGS. 6 to 8. [Figure 10] It is a figure showing the results of evaluating the homogeneity of the host-side DNA in the genomic boundary region in 12 cases of developing infectious lymphoma cattle and 11 cases of non-developing infectious lymphoma cattle or lymphocytosis cattle. The diversity evaluation results by NGS analysis and the clone index values are shown together. [Figure 11] A graph showing a comparison of the clone index values between cattle with infectious lymphoma and cattle without infectious lymphoma is shown. [Figure 12] A graph showing a comparison of the amount of BLV provirus between cattle with infectious lymphoma and cattle without infectious lymphoma is shown. [Mode for Carrying Out the Invention]

[0015] [Definitions] The "host animal" refers to an animal that is a target for BLV infection. The host animal of BLV is not particularly limited as long as it can be infected with BLV. The host animal is a non-human animal, and examples include, but are not limited to, cattle, water buffalo, capybara, sheep, etc. The "infected cell" refers to a cell of the host animal in which the BLV provirus has been integrated into the genomic DNA of the host animal. The "disease state of bovine infectious lymphoma" refers to the symptomatic state of bovine infectious lymphoma. Typically, when bovine infectious lymphoma develops, lymphomas occur. The "examination of the disease state of bovine infectious lymphoma" includes determining the symptomatic state of bovine infectious lymphoma and determining the risk of developing bovine infectious lymphoma.

[0016] The "genomic boundary region" refers to the region in the genomic DNA of the infected cell where the DNA of the BLV provirus integrated into the host animal genomic DNA and the host animal genomic DNA are linked. In the genomic boundary region, both the DNA of the BLV provirus and the host animal genomic DNA are present. The DNA of the BLV provirus in the genomic boundary region may be referred to as the "provirus-side DNA" or "provirus-side sequence". The DNA of the host animal genome in the genomic boundary region may be referred to as the "host-side DNA" or "host-side sequence".

[0017] Sanger sequencing refers to sequence analysis based on the principle of the Sanger method (Sanger, F., et al. (1975)). The Sanger method utilizes the termination of the DNA chain elongation reaction by ddNTPs (2',3'-dideoxynucleotides). DNA polymerase elongates the DNA chain in the presence of four types of nucleotides (dNTPs: dATP, dTTP, dGTP, dCTP). When the DNA chain elongation reaction is carried out in the presence of ddNTPs (2',3'-dideoxynucleotides; ddATP, ddTTP, ddGTP, ddCTP), the DNA elongation reaction stops where ddNTPs are incorporated. This yields DNA fragments of various chain lengths, with each base having a different length. ddNTPs are incorporated into the 3' end of these DNA fragments. By labeling ddATP, ddTTP, ddGTP, and ddCTP with different fluorescent dyes, the ddNTPs incorporated into the 3' end of the aforementioned DNA fragments can be identified by distinguishing the fluorescent dyes. By reading the type of ddNTP incorporated into the 3' end, sequence analysis can be performed.

[0018] "Sanger sequencing" refers to a DNA elongation reaction performed in the presence of four types of dNTPs and four types of ddNTPs labeled with fluorescent dyes. A "Sanger sequencing sequencer" is a sequencer equipped with the function of acquiring sequence data from the reaction product obtained by the Sanger sequencing reaction. In a Sanger sequencing sequencer, DNA fragments obtained by the Sanger sequencing reaction are separated by chain length, and sequence data is acquired by identifying the ddNTPs present at the 3' end of each DNA fragment of each chain length using a fluorescent dye.

[0019] "Spectral data obtained by Sanger sequencing" (hereinafter also simply referred to as "spectral data") refers to fluorescence spectral data obtained by analyzing the reactants of a Sanger sequencing reaction with a Sanger sequencing sequencer. A Sanger sequencing sequencer identifies a fluorescent label for each DNA strand length and outputs fluorescence spectral data with the fluorescent signals arranged in order from shortest to longest strand length. When Sanger sequencing is performed on a single-sequence DNA fragment, the ddNTPs incorporated into the 3' end of the same strand length are identical, and the same fluorescent label is detected. Therefore, the waveform of the spectral data is almost uniform. On the other hand, when Sanger sequencing is performed on DNA fragments with some sequence differences, the spectral data will have a waveform with multiple peaks at the positions where the sequences differ.

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

[0021] Sequence identity between nucleotide sequences is determined by juxtaposing the two nucleotide sequences, inserting gaps in the regions corresponding to insertions and deletions so that the most corresponding nucleotides match. The resulting alignment is then calculated as the proportion of matching nucleotides relative to the entire nucleotide sequence excluding the gaps. 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 obtained by calculation based on the alignment obtained using the known homology search software BLASTN.

[0022] [Methods for assessing the disease activity of bovine lymphoma] A first aspect of this disclosure is a method for testing the disease activity of bovine lymphoma. In one embodiment, the testing method of this disclosure includes the following steps (a) to (e). (a) A step of preparing an infected cell sample from a host animal infected with bovine lymphoma virus. (b) A step of amplifying a DNA fragment of the genomic boundary region, which includes both the proviral DNA of the bovine lymphoma virus and the genomic DNA of the host animal, using the genomic DNA of the infected cell as a template. (c) A step of performing Sanger sequencing on the DNA fragment and obtaining spectral data obtained by Sanger sequencing. (d) A step of identifying, based on the spectral data, the region in the DNA fragment that originates from the proviral DNA and the region that originates from the host animal's genomic DNA, and determining the uniformity of the region that originates from the host animal's genomic DNA. (e) A step of determining the disease activity of bovine lymphoma in the host animal based on the uniformity of the region derived from the genomic DNA of the host animal.

[0023] Bovine lymphoma virus (BLV) is classified as a delta-retrovirus in the retroviridae family and is the virus that causes infectious bovine lymphoma. When BLV infects a host animal, the BLV provirus is incorporated into the genomic DNA of the host animal's cells. BLV primarily infects B lymphocytes. The site where the BLV provirus is incorporated into the host animal's genomic DNA is not particularly limited. Therefore, the insertion site of the BLV provirus may differ depending on the infected cell.

[0024] Figure 1 is a schematic diagram showing the state of BLV-infected cells in a host animal. In the infected cell population shown in Figure 1, the proportion of infected cells is 100% in both animals that do not develop infectious lymphoma and animals that develop infectious lymphoma. On the other hand, in cases where the disease progression of infectious lymphoma is not advanced (non-infectious lymphoma), there is a diverse range of infected cells with different BLV provirus insertion sites. However, when infectious lymphoma develops and specific infected cells proliferate neoplastically, the diversity of infected cells is lost. Therefore, in animals that develop infectious lymphoma, the cells become almost entirely occupied by specific infected cells. Figure 1 shows a typical example of the results of evaluating the diversity of infected cells by NGS analysis. Typical NGS analysis results show that animals that do not develop infectious lymphoma have high diversity of infected cells, while animals that develop infectious lymphoma are dominated by infected cells of a single clone.

[0025] Figure 2 illustrates the principle of the testing method according to one embodiment. In animals that do not develop infectious lymphoma, there are diverse infected cells with different BLV provirus insertion sites. Since the BLV provirus sequence is known, the insertion site of the BLV provirus in the host animal genome can be identified from the BLV provirus sequence. At the genomic boundary region between BLV proviral DNA and host animal genomic DNA, the proviral DNA is common to all infected cells. On the other hand, the host DNA differs depending on the insertion site of the provirus. In animals that do not develop infectious lymphoma, the host DNA at the genome boundary region exhibits diversity due to the high diversity of infected cells. Therefore, spectral data obtained by Sanger sequencing in the host DNA shows a waveform in which four different fluorescent dye peaks corresponding to each base are mixed at each nucleotide position. In animals that develop infectious lymphoma, the host DNA at the genome boundary lacks diversity due to the high clonality of infected cells. Therefore, spectral data obtained by Sanger sequencing shows a waveform in which fluorescent dye peaks corresponding to approximately a single base are formed at each nucleotide position, even in the host DNA.

[0026] Therefore, by obtaining spectral data from the genomic boundary region, the diversity of infected cells can be evaluated. Furthermore, the disease activity of infectious lymphoma can be assessed from the diversity of infected cells. In this specification, the region of the DNA fragment obtained in step (b) that has the nucleotide sequence of the host animal's genomic DNA may be referred to as the host animal's genomic DNA or host DNA. The region of the DNA fragment obtained in step (b) that has the nucleotide sequence of the BLV provirus DNA may be referred to as the BLV provirus DNA or provirus DNA.

[0027] <Process (a)> In step (a), an infected cell sample is prepared from a host animal infected with bovine lymphoma virus (BLV).

[0028] Whether or not a host animal is infected with BLV can be confirmed by known methods. For example, a method of amplifying BLV provirus DNA using primers that specifically anneal to the BLV provirus; or a method using antibodies that specifically bind to BLV proteins. Examples include methods for detecting BLV (blende leukemia).

[0029] Since BLV primarily infects B cells, infected cell samples can usually be obtained by collecting blood from a host animal. The blood may also be separated into mononuclear cell components using methods such as density gradient centrifugation.

[0030] After preparing the infected cell sample, genomic DNA may be extracted from the infected cells. Genomic DNA extraction can be performed by known methods. Alternatively, a cell lysate obtained by disrupting the infected cells may be prepared and used in step (b).

[0031] <Process (b)> In step (b), the genomic DNA of the infected cell is used as a template to amplify a DNA fragment from the genomic boundary region that contains both the proviral DNA of bovine lymphoma virus (BLV) and the genomic DNA of the host animal.

[0032] In the genome boundary region, the host DNA is usually unknown. On the other hand, the BLV provirus DNA is known. Therefore, by using primers that specifically anneal to the provirus DNA in the genome boundary region, DNA fragments in the genome boundary region can be amplified.

[0033] The genome boundary region may be set at the 3' end or the 5' end of the BLV provirus. In one embodiment, the genome boundary region is set at the 3' end of the BLV provirus. By setting the genome boundary region at the 3' end of the BLV provirus, a DNA extension reaction can be performed in the direction of the host DNA using a forward primer that specifically anneals to the provirus DNA. An adapter may be added to the 3' end of the DNA strand obtained by the DNA extension reaction. By adding an adapter to the 3' end, a nucleic acid amplification reaction of the genome boundary region can be performed using a reverse primer that specifically anneals to the adapter and a forward primer that specifically anneals to the provirus DNA. The adapter sequence is not particularly limited, and any sequence can be used. To specifically amplify DNA fragments in the genome boundary region, it is preferable that the adapter has a sequence not present in the provirus and host animal genomes. The DNA fragment may be purified before or after adding the adapter sequence. In this case, the adapter sequence may be a sequence present in the provirus and host animal genomes. Examples of adapter sequences include poly(A) sequences.

[0034] (Primer) The primers used in step (b) can be designed based on the proviral sequence of the genome boundary region. The entire BLV genome has been sequenced and is registered in sequence databases such as NCBI. Therefore, the BLV genome sequence and proviral sequence can be obtained from these sequence databases.

[0035] Examples of forward primers that specifically anneal to the proviral DNA in the genome boundary region include the following primers (i) to (iv). (i) A primer having the nucleotide sequence described in SEQ ID NO: 1, 2, or 3. (ii) A primer that specifically anneals to a region in the proviral DNA of bovine infectious lymphoma virus in which a primer containing the nucleotide sequence described in SEQ ID NO: 1, 2, or 3 specifically anneals. (iii) A primer having a nucleotide sequence in which one or more nucleotides are deleted, added, or substituted in the nucleotide sequence described in SEQ ID NO: 1, 2, or 3, and which specifically anneals to the proviral DNA. (iv) A primer having a nucleotide sequence that has 90% or more sequence identity with the nucleotide sequence described in Sequence ID No. 1, 2, or 3, and that specifically anneals to the proviral DNA.

[0036] The primers described in (i) to (iv) above are primers that can specifically anneal to the 3'LTR or the region immediately preceding it of the BLV provirus. The primers described in (i) to (iv) can specifically anneal to sequences that are commonly present in known BLVs. Therefore, they can be applied to a wide range of BLVs.

[0037] In (iii) above, "multiple items" can refer to, for example, 2 to 6 items, 2 to 5 items, 2 to 3 items, or 2 items. In (iv) above, examples of sequence identity include 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.

[0038] The length of the primer is not particularly limited and can be the length commonly used in nucleic acid elongation reactions. Examples of lower limits for primer length include 15 mer or longer, 16 mer or longer, 17 mer or longer, 18 mer or longer, or 19 mer or longer. Examples of upper limits for primer length include 30 mer or shorter, 29 mer or shorter, 28 mer or shorter, 27 mer or shorter, 26 mer or shorter, or 25 mer or shorter. These upper and lower limits can be combined in any way.

[0039] (Example of process (b)) Step (b) may be carried out by combining known methods such as DNA extension reaction, purification of DNA fragments, and nested PCR. Figure 3 is a schematic diagram illustrating an example of process (b). In the method shown in Figure 3, process (b) includes the following steps (b1) to (b7). (b1) A step of performing a DNA extension reaction using the genomic DNA of the infected cell as a template and a first primer that specifically anneals to the proviral DNA. (b2) A step of purifying the first DNA fragment produced in step (b1). (b3) A step of adding the first adapter to the first DNA fragment. (b4) A step of performing a DNA extension reaction using the first DNA fragment to which the first adapter has been attached as a template, specifically annealing to the first adapter, and using a second primer containing the second adapter. (b5) A step of purifying the second DNA fragment produced in step (b4). (b6) A step of performing a nucleic acid amplification reaction using the second DNA fragment purified in step (b5) as a template, a third primer that specifically anneals to the proviral DNA contained in the second DNA fragment, and a fourth primer that specifically anneals to the second adapter. (b7) A step of performing a nucleic acid amplification reaction using the third DNA fragment obtained in step (b6) as a template, with a fifth primer that specifically anneals to the proviral DNA contained in the third DNA fragment and a sixth primer that specifically anneals to the second adapter.

[0040] Process (b1): In step (b1), a DNA extension reaction is performed using the genomic DNA of the infected cell as a template and a first primer that specifically anneals to the proviral DNA, thereby obtaining a single-stranded DNA fragment.

[0041] In Figure 3, the first primer is represented by F1. The first primer F1 is a forward primer that specifically anneals to the proviral DNA in the genome boundary region. The first primer F1 anneals to the proviral DNA and serves as the starting point for the DNA extension reaction to the host DNA. In the example in Figure 3, the first primer F1 anneals to the region immediately adjacent to the 3'LTR of the proviral DNA. Examples of the first primer include the primer having the nucleotide sequence described in Sequence ID No. 1, and modified primers thereof (e.g., (ii) to (iv) above).

[0042] The first primer F1 may be labeled with the first element of a binding pair for the purpose of purifying the DNA fragment in a later step. A "binding pair" refers to two molecules (a pair of molecules) that bind to each other. The "first element" of a binding pair refers to one of the molecules that make up the binding pair. The "second element" of a binding pair refers to the other molecule that makes up the binding pair. Examples of binding pairs include, for example, biotin or its derivatives and avidin or its derivatives (streptavidin, neutraavidin, etc.); antigens and antibodies or antibody fragments; ligands and receptors, etc. In one embodiment, the first primer F1 is biotin-labeled.

[0043] The DNA extension reaction can be carried out by known methods. Genomic DNA may be cut. The cut may be performed using restriction enzymes. The reaction solution used for the DNA extension reaction may contain dNTPs, DNA polymerase, a first primer, genomic DNA from infected cells, and magnesium salts. The DNA extension reaction can be carried out by repeating a cycle of pre-denaturation (e.g., 94-98°C, 1-5 minutes), denaturation (e.g., 94-98°C, 5-20 seconds), and extension (e.g., 70-75°C, 30-100 seconds). As a result of the DNA extension reaction, a first DNA fragment containing proviral DNA and host DNA is produced. The first DNA fragment may be obtained as single-stranded DNA by denaturing it after the DNA extension reaction.

[0044] Process (b2): In step (b2), the first DNA fragment produced in step (b1) is purified.

[0045] The first DNA fragment can be purified by known methods. For example, a commercially available DNA purification column can be used. By purifying the first DNA fragment, unreacted first primers and genomic DNA from infected cells can be removed.

[0046] Process (b3): In step (b3), the first adapter is attached to the first DNA fragment.

[0047] The first adapter can be any type. The first adapter can be attached to the first DNA fragment by known methods. In one embodiment, the first adapter is typically attached to the 3' end of the first DNA fragment. An example of a first adapter is a poly(A) sequence. This poly(A) sequence, as the first adapter, can be added to the 3' end of a first DNA fragment using, for example, TdT (Terminal Deoxynucleotidyl Transferase) and dATP. TdT catalyzes the addition of a deoxynucleotide to the 3'-OH end of single-stranded or double-stranded DNA. The reaction solution for the poly(A) addition reaction to the 3' end of the first DNA fragment may include, for example, dATP, TdT, the first DNA fragment, and a cobalt salt. The first DNA fragment used in the poly(A) addition reaction may be single-stranded DNA. The reaction temperature for the poly(A) addition reaction can be, for example, 30-40°C or 35-40°C. As an example, the reaction temperature is 37°C. The reaction time is not particularly limited, but can be, for example, 10-60 minutes, 15-50 minutes, or 20-40 minutes. In the reaction solution, by using dTTP, dCTP, or dGTP instead of dATP, a poly-T sequence, poly-C sequence, or poly-G sequence can be added to the 3' end of the first DNA fragment as a first adapter.

[0048] Process (b4): In step (b4), a DNA extension reaction is performed using a first DNA fragment to which the first adapter has been attached as a template, specifically annealing to the first adapter, and a second primer containing the second adapter.

[0049] In Figure 3, the second primer is represented by R1. The second primer R1 is a reverse primer that specifically anneals to the first adapter. The second primer R1 contains a sequence that specifically anneals to the first adapter (hereinafter referred to as the "first adapter annealing sequence"). For example, the second primer contains a complementary sequence to the first adapter. If the first adapter is a poly-A sequence, the second primer contains a poly-T sequence.

[0050] The second primer R1 includes a second adapter in addition to the first adapter annealing sequence. In the second primer R1, the second adapter is located at the 5' end of the first adapter annealing sequence. The second adapter is not particularly limited, and any adapter can be used. To prevent nonspecific amplification during the nucleic acid amplification reaction, it is preferable that the second adapter does not contain sequences located in the genome boundary region. The second adapter may include the adapter sequence ADP1 used in the nucleic acid amplification reaction of step (b6) described below, and the adapter sequence ADP2 used in the nucleic acid amplification reaction of step (b7).

[0051] The DNA extension reaction can be carried out by known methods. The DNA extension reaction can be carried out by repeating a cycle of pre-denaturation (e.g., 94-98°C, 1-5 minutes), denaturation (e.g., 94-98°C, 5-20 seconds), annealing (e.g., 40-60°C, 40-100 seconds), and extension (e.g., 70-75°C, 30-100 seconds). The number of cycles may be one. As a result of the DNA extension reaction, a second DNA fragment is produced containing proviral DNA, host DNA, a first adapter sequence, and a second adapter sequence.

[0052] Process (b5): In step (b5), the second DNA fragment produced in step (b4) is purified.

[0053] The second DNA fragment can be purified by any method. If a primer labeled with the first element of a binding pair is used as the first primer, the second DNA can be purified by utilizing the binding action with the second element of the binding pair. For example, if a biotin-labeled primer is used as the first primer, the second DNA fragment can be purified using magnetic beads or the like surface-modified with avidin or its derivatives (streptavidin, neutraavidin, etc.).

[0054] Process (b6): In step (b6), the second DNA fragment purified in step (b5) is used as a template, and a nucleic acid amplification reaction is performed using a third primer that specifically anneals to the proviral DNA contained in the second DNA fragment and a fourth primer that specifically anneals to the second adapter.

[0055] In Figure 3, the third primer is represented by F2. The third primer F2 is a forward primer that specifically anneals to the proviral DNA contained in the second DNA fragment. The third primer F2 may be the same as or different from the first primer F1. From the viewpoint of eliminating DNA fragments generated by nonspecific amplification, it is preferable that the third primer F2 is different from the first primer F1. If the third primer F1 is different from the first primer F1, it is preferable that the third primer F2 specifically anneals to a region 3' to the annealing site of the first primer F1. Examples of third primers include primers having the nucleotide sequence described in Sequence ID No. 2, and modified primers thereof (e.g., (ii) to (iv) above).

[0056] In Figure 3, the fourth primer is represented by R2. The fourth primer R2 is a reverse primer that specifically anneals to the adapter sequence ADP1 of the second adapter. In one embodiment, the fourth primer R2 contains a complementary sequence to the adapter sequence ADP1.

[0057] The nucleic acid amplification reaction can be carried out by known methods. The reaction solution used in the nucleic acid amplification reaction may include dNTPs, DNA polymerase, a third primer F2, a fourth primer R2, a second DNA fragment purified in step (b5), and a magnesium salt, etc.

[0058] The nucleic acid amplification reaction can be carried out by known methods. The nucleic acid amplification reaction can be carried out by repeating a cycle of pre-denaturation (e.g., 94-98°C, 1-5 minutes), denaturation (e.g., 94-98°C, 5-20 seconds), annealing (e.g., 40-60°C, 40-100 seconds), and extension (e.g., 70-75°C, 30-100 seconds). Alternatively, the nucleic acid amplification reaction may be carried out by repeating a cycle of denaturation (e.g., 94-98°C, 5-20 seconds) and extension (e.g., 70-75°C, 30-100 seconds). The number of cycles can be, for example, 10-50. As a result of the nucleic acid amplification reaction, a third DNA fragment containing proviral DNA, host DNA, and a second adapter is produced.

[0059] Process (b7): In step (b7), the third DNA fragment obtained in step (b6) is used as a template, and a nucleic acid amplification reaction is performed using a fifth primer that specifically anneals to the proviral DNA contained in the third DNA fragment and a sixth primer that specifically anneals to the second adapter.

[0060] In Figure 3, the fifth primer is represented by F3. The fifth primer F3 is a forward primer that specifically anneals to the proviral DNA sequence contained in the third DNA fragment. Examples of the fifth primer include the primer having the nucleotide sequence described in Sequence ID No. 3, and modified primers thereof (e.g., (ii) to (iv) above).

[0061] In Figure 3, the sixth primer is represented by R3. The sixth primer R3 is a reverse primer that specifically anneals to the adapter sequence ADP2 of the second adapter. In the second adapter, the adapter sequence ADP2 is located 3' to the side of the adapter sequence ADP1. In one embodiment, the fourth primer R3 contains the complementary sequence of the adapter sequence ADP2.

[0062] Nucleic acid amplification reactions can be carried out by known methods. The reaction solution used in the nucleic acid amplification reaction may contain dNTPs, DNA polymerase, a fifth primer F3, a sixth primer R3, a third DNA fragment, and a magnesium salt, etc.

[0063] The nucleic acid amplification reaction can be carried out by known methods. The nucleic acid amplification reaction can be carried out by repeating a cycle of pre-denaturation (e.g., 94-98°C, 1-5 minutes), denaturation (e.g., 94-98°C, 5-20 seconds), annealing (e.g., 40-60°C, 40-100 seconds), and extension (e.g., 70-75°C, 30-100 seconds). Alternatively, the nucleic acid amplification reaction may be carried out by repeating a cycle of denaturation (e.g., 94-98°C, 5-20 seconds) and extension (e.g., 70-75°C, 30-100 seconds). The number of cycles can be, for example, 10-50. As a result of the nucleic acid amplification reaction, a fourth DNA fragment containing proviral DNA, host DNA, and adapter ADP2 is produced.

[0064] Steps (b1) to (b7) may be partially omitted. Examples of steps that can be omitted include steps (b2), (b5), (b6), and (b7).

[0065] <Process (c)> In step (c), Sanger sequencing is performed on the DNA fragment obtained in step (b), and spectral data is obtained by the Sanger sequencing.

[0066] Sanger sequencing of DNA fragments can be performed by known methods. For example, Sanger sequencing can be performed according to the instructions provided with the Sanger sequencing sequencer. For example, Sanger sequencing can be performed by carrying out the Sanger sequencing reaction of the DNA fragment obtained in step (b) and analyzing the reaction solution with a Sanger sequencing sequencer. Sanger sequencing allows for the acquisition of spectral data of the ddNTP labeling fluorescent dye (spectral data obtained by Sanger sequencing). The Sanger sequencing reaction can be performed using, for example, the BigDye Terminator v3.1 Cycyle Sequencing Kit (ThermoFisher Scientific). Examples of Sanger sequencing sequencers include the Applied Biosystems 3130 series genetic analyzer, 3500 series genetic analyzer, and 3730 series genetic analyzer (ThermoFisher Scientific).

[0067] <Process (d)> In step (d), based on the spectral data obtained in step (c), the region of the DNA fragment derived from proviral DNA (proviral DNA) and the region derived from the host animal's genomic DNA are identified, and the uniformity of the region derived from the host animal's genomic DNA (host DNA) is determined.

[0068] From the spectral data obtained in step (c), the sequence of the DNA fragment in the genome boundary region can be determined. Since the sequence of the proviral DNA is known, the boundary between the proviral DNA and the host DNA can be determined based on the sequence of the proviral DNA.

[0069] The sequence of the host DNA varies depending on the insertion site of the provirus into the host animal genome. When the host DNA is highly homogeneous, the spectral data will be a waveform in which peaks formed by approximately a single fluorescent dye (the base corresponding to the fluorescent dye) are continuous. On the other hand, when the host DNA is not highly homogeneous, the spectral data will be a waveform in which peaks of multiple fluorescent dyes (the bases corresponding to the fluorescent dyes) are mixed at each nucleotide position (see Figure 2). Therefore, the homogeneity of the host DNA can be determined from the waveform of the spectral data of the host DNA.

[0070] The uniformity of the host DNA may be determined based on numerical values ​​calculated from the waveforms of each fluorescent dye (the base corresponding to the fluorescent dye). For example, the ratio of the total area of ​​other peaks to the area of ​​the peak with the largest peak area at each nucleotide position may be calculated and used to determine the uniformity of the host DNA. The uniformity of the host DNA may be quantified using a program that processes spectral data. Examples of such programs include EditR (Mitchell G. Kluesner et al., CRISPR J. 2018 Jun 1; 1(3): 239-250). EditR calculates a "clone index value" from the spectral data. This clone index value can be used to determine the uniformity of the host DNA. A higher clone index value indicates greater DNA uniformity.

[0071] <Process (e)> In step (e), the disease activity of bovine lymphoma in the host animal is determined based on the homogeneity of the region derived from the host animal's genomic DNA (host DNA).

[0072] High uniformity of host DNA in the genome boundary region indicates that the disease progression of bovine lymphoma is advanced in the host animal. Conversely, low uniformity of host DNA in the genome boundary region indicates that the disease progression of bovine lymphoma is not advanced in the host animal.

[0073] For example, if the clonal index value of the host DNA obtained by EditR is 0.5 or higher, 0.6 or higher, 0.7 or higher, 0.8 or higher, 0.85 or higher, 0.9 or higher, 0.95 or higher, or 1.0 or higher, the host animal may be judged to have developed bovine lymphoma. Furthermore, for example, if the clonal index value of the host DNA is 0.05 or higher, 0.1 or higher, 0.15 or higher, 0.2 or higher, 0.25 or higher, or 0.3 or higher, the host animal may be judged to have an increased risk of developing bovine lymphoma.

[0074] The testing method disclosed herein utilizes spectral data obtained by Sanger sequencing of the genome boundary region to determine the disease activity of bovine infectious lymphoma. Therefore, compared to NGS analysis, it is possible to determine the disease activity of bovine infectious lymphoma in a low-cost, simple, and rapid manner. By performing step (b) using primers selected from the group consisting of (i) to (iv) above, it is possible to apply this to almost all currently known BLVs. Furthermore, it is possible to accurately determine the disease activity of bovine lymphoma.

[0075] [primer] A second aspect of this disclosure is at least one primer selected from the group consisting of (i) to (iv) below. (i) A primer having the nucleotide sequence described in SEQ ID NO: 1, 2, or 3. (ii) A primer that specifically anneals to a region in the proviral DNA of bovine infectious lymphoma virus in which a primer containing the nucleotide sequence described in SEQ ID NO: 1, 2, or 3 specifically anneals. (iii) A primer having a nucleotide sequence in which one or more nucleotides are deleted, added, or substituted in the nucleotide sequence described in SEQ ID NO: 1, 2, or 3, and which specifically anneals to the proviral DNA. (iv) A primer having a nucleotide sequence that has 90% or more sequence identity with the nucleotide sequence described in Sequence ID No. 1, 2, or 3, and that specifically anneals to the proviral DNA.

[0076] The primers in (i) to (iv) are the same as those exemplified in step (b) above. The primers can be synthesized by known solid-phase synthesis methods such as the phosphoramidate method.

[0077] [Test kit] A third aspect of this disclosure is a disease activity testing kit for bovine lymphoma, comprising the primers of the second aspect.

[0078] The test kit may contain only one primer selected from the group consisting of (i) to (iv) above, or it may contain two or more primers. The following are possible combinations of primers included in the test kit:

[0079] (A) A primer having the nucleotide sequence described in SEQ ID NO: 1; a primer that specifically anneals to the region in the proviral DNA of bovine infectious lymphoma virus in which a primer containing the nucleotide sequence described in SEQ ID NO: 1 specifically anneals; a primer having a nucleotide sequence in which one or more nucleotides are deleted, added, or substituted in the nucleotide sequence described in SEQ ID NO: 1, and that specifically anneals to the proviral DNA; or a primer having a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence described in SEQ ID NO: 1, and that specifically anneals to the proviral DNA. (B) A primer having the nucleotide sequence described in SEQ ID NO: 2; a primer that specifically anneals to the region in the proviral DNA of bovine infectious lymphoma virus in which a primer containing the nucleotide sequence described in SEQ ID NO: 2 specifically anneals; a primer having a nucleotide sequence in which one or more nucleotides are deleted, added, or substituted in the nucleotide sequence described in SEQ ID NO: 2, and that specifically anneals to the proviral DNA; or a primer having a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence described in SEQ ID NO: 2, and that specifically anneals to the proviral DNA. A combination.

[0080] The primer (A) and, (C) A primer having the nucleotide sequence described in SEQ ID NO: 3; a primer that specifically anneals to the region in the proviral DNA of bovine infectious lymphoma virus in which a primer containing the nucleotide sequence described in SEQ ID NO: 3 specifically anneals; a primer having a nucleotide sequence in which one or more nucleotides are deleted, added, or substituted in the nucleotide sequence described in SEQ ID NO: 3, and that specifically anneals to the proviral DNA; or a primer having a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence described in SEQ ID NO: 3, and that specifically anneals to the proviral DNA. A combination.

[0081] A combination of the primer (B) and the primer (C). A combination of primer (A), primer (B), and primer (C).

[0082] The test kit may include other components in addition to the primers. Examples of other components include an adapter, a primer that specifically anneals to the adapter, DNA polymerase, dNTPs, TdT, reaction buffer, magnesium salt, and instructions for use.

[0083] The test kit of this disclosure can be used in the test method of the first embodiment. [Examples]

[0084] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0085] [Testing Method] <Primer Design> The primer was designed based on the following principles: (1) It is necessary to design primers that anneal to the DNA sequence in the terminal region of the BLV genome. Since the BLV genome has LTRs (Long Terminal Repeats) at both ends, it is necessary to distinguish between them. Therefore, we designed a primer to be placed immediately adjacent to the 3'LTR and amplified it from the 3'LTR to the host genome junction, thereby distinguishing it from the 5'LTR.

[0086] (2) In order to determine the uniformity of the host sequence in the genome boundary region from spectral data obtained by Sanger sequencing, nonspecific amplification should be avoided. Therefore, we decided to perform nested PCR to increase specificity. To that end, we designed multiple primer sets.

[0087] (3) In order to apply the primers to BLV in general, rather than to specific BLV strains, it is necessary to design primers that can be used in common with the genomic DNA of a wide range of BLV strains. Therefore, we obtained a total of 58 BLV genome sequences from sequence databases such as NCBI. We compared and analyzed these genome sequences and selected sequences common to these genome sequences from the 3'LTR sequences, including the most recent ones.

[0088] (4) The primers needed to be capable of amplification by PCR, but the 3'LTR sequence, including the immediate vicinity, was very GC-rich. Therefore, primer candidate sequences with a GC% ratio of about 40 to 60% were selected from the common sequences selected in (3).

[0089] As a result of the above, three types of primers, "Biotinylated-BLV-F1", "BLV-F2", and "BLV-F3" shown in Table 1 were designed.

[0090] <Preparation of DNA Sample> The preparation of the DNA sample was carried out using the QIAamp DNA Blood Mini kit (QIAGEN) from bovine blood according to the manufacturer's instructions. Specifically, it was carried out as follows. a) 20 μL of QIAGEN Protenase was added to 200 μL of the blood sample. b) After stirring each tube well, 200 μL of Buffer AL was added. c) After reacting at 56°C for 10 minutes, 200 μL of ethanol was added to each sample and vortexed for 15 seconds. Then, each tube was spun down to collect the solution at the bottom. d) 300 μL was taken from each tube and transferred to a labeled QIAquick column. e) Centrifuged at 8000 rpm for 1 minute. f) The supernatant was removed. g) 500 μL of Buffer AW1 was added to the QIAamp Mini Spin column. h) Centrifuged at 8000 rpm for 1 minute. i) The supernatant was removed. j) 500 μL of Buffer AW2 was added to the QIAamp Mini Spin column. k) Centrifuged at 14000 rpm for 3 minutes. l) The QIAamp Mini Spin column was placed in a new 1.5 mL tube. m) 100 μL of Elution Buffer (Buffer AE) was added to the center of the QIAamp Mini Spin column, centrifuged for 1 minute, and then stored at -20°C.

[0091] <Preparation of samples for Sanger sequencing> The primers used are shown in Table 1.

[0092] [Table 1]

[0093] (1) Synthesis of single-stranded DNA (ssDNA) The reaction solutions shown in Table 2 were prepared, and the DNA extension reaction was carried out under the conditions shown in Table 3. Next, the temperature of the thermal cycler was reduced to 4°C, and the tubes were removed from the thermal cycler and subjected to short-term centrifugation.

[0094] [Table 2]

[0095] [Table 3]

[0096] (2) Column purification ssDNA purification was performed using the Monarch PCR & DNA Cleanup Kit (NEW England biolabs) according to the manufacturer's instructions. Specifically, the procedure was as follows:

[0097] a) 250 μL of Buffer PB was added to 50 μL of ssDNA sample (volume ratio 5:1). b) Each tube was thoroughly mixed by pipetting. c) Labels were attached to the QIAquick column (QIAGEN) and 2 mL blood collection tubes. d) 300 μL was collected from each tube, transferred to a labeled QIAquick column, and placed in a 2 mL blood collection tube. e) Centrifuged at 12000 rpm for 1 minute. f) The supernatant was removed. g) 730 μL of Buffer PE was added to the QIAquick column. h) The cells were centrifuged at 12,000 rpm for 1 minute. i) Remove the supernatant and return the QIAquick column to the same blood collection tube. j) The centrifugal force was removed at 12,000 rpm for 1 minute. k) Remove the QIAquick column from the blood collection tube and place it in a new 1.5 mL low-bind Eppendorf tube. l) 30 μL of Elution Buffer (Buffer EB) was added to the center of the QIAquick membrane and centrifuged for 1 minute. m) Stored at 20°C.

[0098] (3) Poly A-tailing PolyA-tailing was performed using the reaction mixture shown in Table 4, followed by incubation at 37°C for 30 minutes.

[0099] [Table 4]

[0100] (4) Synthesis of double-stranded DNA The reaction solutions shown in Table 5 were prepared, and the nucleic acid amplification reaction was carried out under the conditions shown in Table 6. Next, the temperature of the thermal cycler was lowered to 4°C, and the tubes were removed from the thermal cycler and subjected to short-term centrifugation.

[0101] [Table 5]

[0102] [Table 6]

[0103] (5) Avidin bead purification The purification of double-stranded DNA is performed using Magnosphere. TMThe procedure was performed using (registered trademark) MS300 / Streptavidin (JSR Life Science) in accordance with the manufacturer's instructions.

[0104] Magnosphere TM The reagents and equipment used for fixing biotinylated DNA to MS300 / streptavidin are listed below. a)Binding buffer(2x):20mM Tris-HCl(pH7.4) with 1mM EDTA,2M NaCl,0.1% Tween20 b) Equipment: Magnetic separator, vortex tube mixer, tube rotor

[0105] Specifically, we did the following: i) Labels were attached to the microtubes. ii) Magnosphere TM MS300 / streptavidin was suspended using a vortex mixer, and a 30 μm suspension (i.e., 1 mg beads) was placed in a microcentrifuge tube. iii) Place the tubes on the magnetic tube stand for at least one minute and carefully remove the supernatant. iv) Add 200 μL of 1x Binding buffer and suspend the beads using a vortex mixer. Then, remove the supernatant in the same manner as in iii). v) Biotinylated DNA solution and an equal volume of 2x Binding buffer were added to a microtube, and the beads were suspended using a vortex mixer. vi) The tube was rotated using a tube rotor at room temperature for 10 minutes. The supernatant was removed in the same manner as in vii)iii). viii) The beads were washed with 200 μL of 1x Binding buffer and then suspended in a vortex mixer. The supernatant was removed in the same manner as in ix) and iii). Steps x)viii) and ix) were repeated a total of three times. xi) The beads were suspended in 50 μL of distilled water and stored at 2-8°C until use.

[0106] (6) First PCR The reaction solutions shown in Table 7 were prepared, and the nucleic acid amplification reaction was carried out under the conditions shown in Table 8. Next, the temperature of the thermal cycler was lowered to 4°C, and the tubes were removed from the thermal cycler and subjected to short-term centrifugation.

[0107] [Table 7]

[0108] [Table 8]

[0109] (7) Second PCR The reaction solutions shown in Table 9 were prepared, and the nucleic acid amplification reaction was carried out under the conditions shown in Table 10. Next, the temperature of the thermal cycler was lowered to 4°C, and then the tubes were removed from the thermal cycler and stored at 20°C.

[0110] [Table 9]

[0111] [Table 10]

[0112] <Sanger Sequencing> Sanger sequencing was outsourced to FASMAC Co., Ltd. The Applied Biosystems 3130xl Genetic Analyzer or Applied Biosystems 3730xl DNA Analyzer was used as the sequencer for Sanger sequencing. The Sanger sequencing reaction was performed using the Applied Biosystems Big Dye Terminator v3.1 (ThermoFisher Scientific).

[0113] <Example 1> Blood samples were collected from cattle that did not develop infectious lymphoma, cattle with lymphocytosis (not affected by infectious lymphoma), and cattle that developed infectious lymphoma. DNA samples were prepared from the collected blood samples, and spectral data of fluorescent dyes corresponding to each base were obtained according to the [Testing Method] described above. The spectral data was analyzed using EditR (Mitchell G. Kluesner et al., CRISPR J. 2018 Jun 1; 1(3): 239-250.) to obtain a clonal index.

[0114] The results are shown in Figure 4. In Figure 4, A shows the spectral data obtained by Sanger sequencing. B shows the results of the spectral data analysis by EditR and the clone index values. In B, "signal" shows the peak area of ​​the signal peak (the peak with the largest area), and "noise" shows the peak area of ​​peaks other than the signal peak.

[0115] As shown in Figure 4, cows without infectious lymphoma had low clonal index values, indicating that cloning of infected cells had not progressed. In cows with lymphocytosis, compared to cows without infectious lymphoma, the clonal index values ​​were higher, indicating that cloning of infected cells had progressed. In cows with infectious lymphoma, the clonal index value was greater than 1, suggesting that the infected cells were almost entirely single clones.

[0116] <Example 2> Next-generation sequencing (NGS) analysis was performed using the DNA samples prepared in Example 1. NGS analysis was used to analyze the diversity of the host animal's genomic DNA sequence at the genomic boundary region between BLV proviral DNA and the host animal's genomic DNA.

[0117] The results are shown in Figure 5. In Figure 5, A shows the EditR analysis results and clonal index values ​​obtained in Example 1. B shows the diversity of genomic DNA sequences in the host animal at the genome boundary region obtained by NGS analysis.

[0118] As shown in Figure 5, the NGS analysis results showed that cloning of infected cells progressed in the following order: cattle without infectious lymphoma, cattle with lymphocytosis, and cattle with infectious lymphoma. In cattle with infectious lymphoma, the host animal DNA in the genomic boundary region was 100% identical. These results were consistent with the clonal index values ​​obtained in Example 1.

[0119] <Example 3> Blood samples were collected from cattle infected with BLV in 2015 and 2017. Tissue samples were also collected from two tumors at the time of infectious lymphoma onset (2018). DNA samples were prepared from the collected blood or tissue samples, and spectral data was obtained by Sanger sequencing according to the [Testing Method] described above. The spectral data was analyzed using EditR to obtain a clonal index. Furthermore, NGS analysis was performed using the same DNA samples. The proviral load of BLV was also quantified by real-time PCR using the same DNA samples. Similar analyses were performed for four cases of BLV-infected cattle.

[0120] The results for each case are shown in Figures 6-9. In Figures 6-9, "PVL (%)" indicates the amount of BLV provirus obtained by real-time PCR. "IS" indicates the number of insertion sites of the BLV provirus.

[0121] In all cases shown in Figures 6-9, cloning was more advanced in 2017 than in 2015. Tumors (Tumor(1), Tumor(2)) showed even more advanced cloning in 2017 than in 2015. In the cases shown in Figures 8 and 9, the tumors were composed of almost identical infected cells. The clonal index values ​​showed a similar trend to the NGS analysis results. Proviral load (PVL(%)) did not necessarily correlate with disease activity.

[0122] <Example 4> Tissue samples were collected from tumors of 12 cattle that developed infectious lymphoma. Blood samples were also collected from 11 cattle that did not develop infectious lymphoma or cattle with lymphocytosis. DNA samples were prepared from the tissue or blood samples, and spectral data was obtained by Sanger sequencing according to the [Testing Method] described above. The spectral data was analyzed using EditR to obtain a clone index. Furthermore, NGS analysis was performed using the same DNA samples.

[0123] The results are shown in Figure 10. In Figure 10, each bar represents the NGS analysis result for each case. Above the bar representing the NGS analysis result, the clonal index value obtained from that case is shown. As shown in Figure 10, the clonal index value and the NGS analysis result showed similar trends. Cloning was advanced in cattle that developed infectious lymphoma. Diversity was high in cattle that did not develop infectious lymphoma or in cattle with lymphocytosis.

[0124] <Example 5> Figure 11 shows the results of plotting clonal index values ​​obtained from multiple cattle that developed infectious lymphoma and those that did not. A significant difference in clonal index values ​​(p ≤ 0.00083, Wilcoxon rank-sum test) was observed between cattle that developed infectious lymphoma and those that did not.

[0125] Figure 12 shows the results of real-time PCR quantification of BLV proviral levels in blood samples from multiple cattle with and without infectious lymphoma. Proviral levels tended to be higher in cattle without infectious lymphoma, and did not correlate with disease severity.

[0126] Based on these results, it was confirmed that the results obtained by this testing method are consistent with the disease activity of bovine infectious lymphoma. Therefore, it was demonstrated that this testing method can be used to assess the disease activity of bovine infectious lymphoma. [Industrial applicability]

[0127] The present invention provides a method for testing the disease activity of bovine infectious lymphoma, a primer usable in the testing method, and a testing kit, all of which enable a simple and rapid evaluation of the disease activity of bovine infectious lymphoma.

Claims

1. (a) A step of preparing an infected cell sample from a host animal infected with bovine lymphoma virus, (b) A step of amplifying a DNA fragment of the genomic boundary region, which includes both the proviral DNA of the bovine lymphoma virus and the genomic DNA of the host animal, using the genomic DNA of the infected cell as a template; (c) A step of performing Sanger sequencing on the DNA fragment and obtaining spectral data by Sanger sequencing, (d) A step of identifying, based on the spectral data, the region in the DNA fragment that originates from the proviral DNA and the region that originates from the host animal's genomic DNA, and determining the uniformity of the region that originates from the host animal's genomic DNA, (e) A step of determining the disease activity of bovine infectious lymphoma in the host animal based on the uniformity of the region derived from the genomic DNA of the host animal, Includes, The above step (b) is (b1) A step of performing a DNA extension reaction using the genomic DNA of the infected cell as a template and a first primer that specifically anneals to the proviral DNA, (b2) A step of purifying the first DNA fragment produced in step (b1), (b3) A step of attaching a first adapter to the first DNA fragment, (b4) A step of performing a DNA extension reaction using the first DNA fragment to which the first adapter is attached as a template, specifically annealing to the first adapter, and using a second primer containing the second adapter, (b5) A step of purifying the second DNA fragment produced in step (b4), (b6) A step of performing a nucleic acid amplification reaction using the second DNA fragment purified in step (b5) as a template, a third primer that specifically anneals to the proviral DNA contained in the second DNA fragment, and a fourth primer that specifically anneals to the second adapter, (b7) Using the third DNA fragment obtained in step (b6) as a template, a nucleic acid amplification reaction is performed using a fifth primer that specifically anneals to the proviral DNA contained in the third DNA fragment and a sixth primer that specifically anneals to the second adapter. Includes, The first primer is a primer consisting of the nucleotide sequence described in Sequence ID No. 1, The third primer is a primer consisting of the nucleotide sequence described in Sequence ID No. 2, The fifth primer is a primer consisting of the nucleotide sequence described in Sequence ID No.

3. Methods for testing the disease activity of bovine lymphoma.

2. A test kit for detecting the disease activity of bovine infectious lymphoma, comprising a primer consisting of the nucleotide sequence described in SEQ ID NO: 1, a primer consisting of the nucleotide sequence described in SEQ ID NO: 2, and a primer consisting of the nucleotide sequence described in SEQ ID NO: 3.

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