JP-7854681-B2 - Methods for testing the disease activity of bovine lymphoma, primers, and test kits for testing the disease activity of bovine lymphoma.

Inventors

• 佐藤 賢文
• 松尾 美沙希
• 小林 朋子

Assignees

• 国立大学法人 熊本大学
• 学校法人東京農業大学

Dates

Publication Date

20260507

Application Date

20210730

Claims (2)

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 .

Description

This invention relates to a method for testing the disease activity of bovine lymphoma, a primer, and a test kit for testing the disease activity of bovine lymphoma. Cattle are an important food resource, providing 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 account for an extremely large share of the livestock industry. Bovine lymphoma virus (Bovine Leukemia Virus: BLV) is a virus that infects B lymphocytes in cattle. BLV is pathogenic and can cause lymphoma in some infected cattle. 60-70% of infected cattle are asymptomatic and do not develop the disease, but 30% develop persistent lymphocytosis, and a few percent develop lymphoma. In recent years, both the infection rate of BLV and the number of cases of infectious lymphoma have been increasing in Japan, highlighting the growing need for countermeasures. In Japan, cattle infected with BLV but without developing lymphoma are sold as meat. However, if infectious lymphoma is detected during slaughter inspection, the entire animal is discarded according to the provisions of the Act on the Prevention of Infectious Diseases of Domestic Animals. Therefore, the occurrence of infectious lymphoma results in significant economic losses for cattle farmers. Accurate diagnosis is necessary to detect the onset of infectious lymphoma. However, despite its importance, the current diagnosis of infectious lymphoma relies on veterinarians visually or palpating the presence or absence of lymph node enlargement. Therefore, the diagnostic accuracy is not high. 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. 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. Japanese Patent Publication No. 2019-180351 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. This diagram illustrates the state of infected cells in cattle that develop infectious lymphoma and those that do not.This is a schematic diagram illustrating the principle of the inspection method according to one embodiment.This is a schematic diagram showing an example of step (b) of the inspection method of one embodiment.This figure shows the results of evaluating the homogeneity of host DNA in the genomic boundary region of cattle that do not develop infectious lymphoma, cattle with lymphocytosis, and cattle that develop infectious lymphoma. A shows the spectral data from Sanger sequencing. B shows the results of analyzing the spectral data from Sanger sequencing using EditR.This figure shows the results of evaluating the homogeneity of host DNA in the genomic boundary region of cattle that do not develop infectious lymphoma, cattle with lymphocytosis, and cattle that develop infectious lymphoma. A shows the results of analyzing Sanger sequencing spectral data with EditR. B shows the results of evaluating the homogeneity of infected cells by Next Generation Sequencer (NGS) analysis.This figure shows the results of evaluating the uniformity of host DNA in the genomic boundary region of BLV-infected cattle over time. "NGS analysis" shows the results of evaluating the uniformity of infected cells by NGS analysis. "PVL (%)" shows the quantitative result of the proviral load by real-time PCR. "IS" shows the number of insertion sites of the BLV provirus.This figure shows the results of evaluating the uniformity of host DNA in the genomic boundary region of BLV-infected cattle over time. This is a different case from Figure 6.This figure shows the results of evaluating the uniformity of host DNA in the genomic boundary region of BLV-infected cattle over time. This is a different case from Figures 6 and 7.This figure shows the results of evaluating the uniformity of host DNA in the genomic boundary region of BLV-infected cattle over time. This is a different case from Figures 6-8.This figure shows the results of evaluating the homogeneity of host DNA in the genomic boundary region in 12 cases of cattle that developed infectious lymphoma and 11 cases of cattle that did not develop infectious lymphoma or had lymphocytosis. The diversity evaluation results from NGS analysis and the clonal index values are shown together.This graph shows a comparison of clonal index values between cows that developed infectious lymphoma and cows that did not.This graph shows a comparison of BLV provi