Patent Grant
11814690
This application claims priority to and the benefit of Korean Patent Application No. 10-2019-0139264, filed on Nov. 4, 2019, the disclosure of which is incorporated herein by reference in its entirety.
The instant application contains a Sequence Listing which has been submitted in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on Dec. 28, 2020, is named “ELIP111seq.txt” and is 58.5 kilobytes in size.
The present invention relates to a probe for detecting hepatitis B virus and a method for detecting an insertion site of hepatitis B virus at high efficiency based on the analysis method of next-generation sequencing using the probe.
Hepatitis B virus (HBV) is a disease which is the main cause of liver cancer, and approximately 300 million people worldwide are affected by HBV. Hepatitis B virus (hereinafter, referred to as ‘HBV’) is a virus belonging to the Hepadnaviridae family and infects only liver cells of humans specifically. Symptoms of hepatitis are fatigue for mild cases, and jaundice may appear in severe cases. In the late stage of the disease, complications of cirrhosis such as, ascites, edema, gastroesophageal variceal bleeding, hepatic encephalopathy, blood coagulation abnormality, and hepatorenal syndrome can appear.
In the case of patients who have been infected in childhood, the period of immune tolerance occurs continuously for 10 to 30 years in which the proliferation of virus occurs but no symptoms of hepatitis appear, but when these healthy carriers reach a certain period (15 to 30 years old), hepatocytes are damaged by the action of the immune system and develop into hepatitis. When e-antigen seroconversion (HBeAg seroconversion) occurs quickly, viral proliferation is suppressed and symptoms of hepatitis do not develop any further, but when the proliferation of virus is not effectively suppressed, and it develops into chronic hepatitis and liver cirrhosis, and in severe cases, it develops into liver cancer.
Hepatitis B virus can be inserted (integration) into the human genome during viral proliferation and life cycle, and although this step is not essential for viral replication, integration of the HBV DNA into a host genome contributes to the occurrence of liver cancer by inducing genomic instability and altering the expression of cancer-related genes. Until recently, the existence of this genomic insertion phenomenon has traditionally been discovered by polymerase chain reaction (PCR), but this method has a limitation in finding all of HBV-inserted molecules in the entire human genome because it biases detection of only the inserted virus localized in the human genome region designated by a specific primer. Therefore, a new method was necessary to investigate HBV insertion in the entire human genome.
Recently, with the introduction of next-generation sequencing (NGS) technology, it is possible to overcome the limitations of traditional PCR-based studies and to attempt non-biased detection of HBV insertion sites across the entire human genome. The present invention provides a method for analyzing HBV insertion sites at high efficiency based on NGS and a probe applied thereto.
The present invention provides a probe for detecting hepatitis B virus and a method for detecting an insertion site of hepatitis B virus at high efficiency based on the analysis method of next-generation sequencing using the probe.
The present invention provides a probe composition for detecting hepatitis B virus (HBV) consisting of sequences of SEQ ID NO: 1 to SEQ ID NO: 215.
In addition, the present invention may provide a kit for detecting hepatitis B virus (HBV) including the probe composition.
In addition, the present invention may provide a method for detecting hepatitis B virus (HBV), wherein the method is a method for detecting hepatitis B virus (HBV) through next-generation sequencing (NGS), the method including hybridizing a target sample with a probe composition for detecting hepatitis B virus (HBV) consisting of a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 215 to capture a target gene.
In addition, the present invention may provide a method for providing information for the diagnosis of liver cancer using the method.
According to the present invention, a probe may be provided that is capable of confirming an insertion site of HBV in the human genome with a possibility of developing into liver cancer. In addition, by applying the probe to the analysis method of next-generation sequencing, HBV insertion sites in the human genome can be analyzed at low cost and high efficiency.
The above and other objects, features and advantages of the present invention will become more apparent to those of ordinary skill in the art by describing in detail exemplary embodiments thereof with reference to the accompanying drawings, in which:
Hereinafter, the present invention will be described in detail.
The present invention may detect an insertion site of hepatitis B virus (HBV) located in the human genome at high efficiency based on next-generation sequencing (NGS). Specifically, in a DNA library constructed from a patient's liver tissue, an HBV sequence may be captured with a probe complementary to the self-constructed HBV. Based on this, HBV and breakpoints of the human genome may be detected (refer to
As used herein, the term “probe” refers to a nucleic acid fragment corresponding to several bases to several hundred bases for specific binding to DNA or RNA, and afterwards, the presence or absence of specific DNA or RNA may be confirmed by amplification, separation, and detection.
The present invention provides a probe for detecting hepatitis B virus (HBV) consisting of nucleotide sequences of SEQ ID NO: 1 to SEQ ID NO: 215.
The probe may detect an insertion site of hepatitis B virus in the human genome. More specifically, the probe may detect an insertion site of hepatitis B virus (HBV) using the analysis method of next-generation sequencing.
The probe may be applied to the detection of hepatitis B virus of Koreans, and more specifically, it may be applied to the detection of genotype hepatitis C virus.
The length of the probe is 120 nucleotides. When the length of the probe is too short or too long, false hybridization increases and the likelihood of a decrease in specificity increases. In the present invention, hybridization efficiency was maximized by optimizing the length of a probe as above.
In addition, the probe is based on the complete genome sequences of 8 prototypes of hepatitis B virus (HBV) of Koreans, and by allowing each HBV nucleotide sequence to overlap, it is designed to have almost 100% coverage for hepatitis B virus (HBV) of Koreans.
In addition, the present invention provides a composition for detecting hepatitis B virus (HBV), including the probe. The composition may include deoxynucleoside triphosphate (dNTP), heat-resistant polymerase, and a metal ion salt such as magnesium chloride and the like, in addition to the probe.
In addition, the present invention provides a kit for detecting hepatitis B virus (HBV), including the composition.
The kit may include a barcoding primer in which an adapter suitable for the NGS device to be used is combined with a barcode sequence.
In addition, the kit may further include a reagent commonly used in a method for detecting nucleic acid. For example, it may include deoxynucleoside triphosphate (dNTP), heat-resistant polymerase, and a metal ion salt such as magnesium chloride and the like that are required for PCR reaction, and may include dNTP, sequenase, and the like that are required for sequencing. In addition, the kit may take the form of a bottle, a tub, a sachet, an envelope, a tube, an ampoule, and the like, and these may be partially or entirely formed from plastic, glass, paper, foil, wax, and the like. The container may be equipped with a completely or partially removable plug, which is initially part of a container or may be attached to the container by mechanical, adhesive, or other means. The container may be equipped with a stopper that may allow access to the contents by an injection needle. The kit may include an external package, and the external package may include instructions for use of the components.
The present invention provides a method for detecting hepatitis B virus (HBV), wherein the method is a method for detecting hepatitis B virus (HBV) through next-generation sequencing (NGS), the method including hybridizing a target sample with a probe for detecting hepatitis B virus (HBV) composed of a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 215 to capture a target gene.
As used herein, the term “hybridization” means that complementary single-stranded nucleic acids form double-stranded nucleic acids. The degree of complementarity required for hybridization may vary depending on the hybridization conditions, and in particular, if it can be optimized at temperature, it may be preferably optimized to a temperature described in the protocol that can be specified by the probe manufacturer.
As used herein, the term “target gene” refers to a gene sequence to be detected, and it is hybridized with a probe under hybridization, annealing, or amplification conditions.
As used herein, the term “target gene” is not different from the terms used in the present specification such as “target gene”, “target gene sequence”, or “target sequence”, and these terms are used interchangeably in the present specification.
As used herein, a target sample refers to a sample including a gene region to be detected, and it may be collected from at least one selected from the group consisting of tissue, blood, serum, saliva, urine, semen, and body fluid, and specifically, it may be liver tissue derived from a patient.
In addition, the present invention provides a method for detecting hepatitis B virus (HBV), including (a) hybridizing a target sample including a target gene with a probe for detecting hepatitis B virus (HBV) composed of a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 215 to capture a target gene and amplifying to create a library; and (b) sequencing the library to map the produced nucleotide sequence in the human and HBV reference sequences for analysis to confirm an insertion site of hepatitis B virus (HBV) in the human genome.
The hybridizing may be performed at a temperature of 65° C. for 16 hours to 24 hours.
Since it is a temperature and time condition that optimizes the efficiency of probe hybridization, the hybridization efficiency may be lowered when an experiment outside this range is performed.
The target gene may be a hepatitis B virus (HBV) gene of Koreans.
In addition, the present invention may provide a method for providing information for the diagnosis of liver cancer, using the method.
Hereinafter, the present invention will be described in more detail through exemplary embodiments. Objects, features, and advantages of the present invention will be easily understood through the following exemplary embodiments. The present invention is not limited to the exemplary embodiment described herein, and may be embodied in other forms. The exemplary embodiments introduced herein are provided in order to sufficiently convey the spirit of the present invention to those of ordinary skill in the technical field to which the present invention pertains. Therefore, the present invention should not be limited by the following exemplary embodiments.
In order to perform next-generation sequencing analysis for the detection of an HBV insertion site, a probe for HBV capture was prepared based on the following complete genome sequences of 8 representative Korean HBV types. Complementary probes were prepared such that each HBV nucleotide sequence overlapped with each other. The probe was synthesized through the HPLC purification method, and the concentration and purity of the synthesized probe were confirmed using the BioAnalyzer device.
(Sequence Information)
The probe targets the following 8 viruses.
complete genome“,”AY641559.1 Hepatitis B virus isolate He53 (can be found at www.ncbi.nlm.nih.gov as AY641559.1)
complete genome“,”DQ683578.1 Hepatitis B virus from South Korea (can be found at www.ncbi.nlm.nih.gov as DQ683578.1)
complete genome“,”GQ872211.1 Hepatitis B virus (can be found at www.ncbi.nlm.nih.gov as GQ872210.1)
complete genome“,”JN315779.1 Hepatitis B virus genotype C2 (can be found at www.ncbi.nlm.nih.gov as JN315779)
complete genome“,”KR184660.1 Hepatitis B virus isolate SS 3 22 (can be found at www.ncbi.nlm.nih.gov as KR184660.1)
complete sequence, isolate 22Y04HCC″,“AB014381.1 Hepatitis B virus genomic DNA (can be found at www.ncbi.nlm.nih.gov as 3582357)
complete sequence, isolate 36Y18HCC”,“AB014395.1 Hepatitis B virus genomic DNA (can be found at www.ncbi.nlm.nih.gov as 3551389)
D23680.1 Hepatitis B virus (B4-HBVST1) complete genome sequence (can be found at www.ncbi.nlm.nih.gov as D23680.1)
Based on the above contents, it was prepared by Tilling density 1X, Boosting: balanced, probe group size: 25.595 kbp, Total probe: 215. The sequence information of each designed probe was shown in Table 2 below.
2-1. DNA shearing
2-3. Repairing the ends
2-4. Addition of A′ base to the 3′ end of DNA fragment
2-5. Adapter ligation to DNA fragment
2-6. Amplification of adapter-ligated library
2-8. Assessment of quality and quantity
In order to confirm whether the library size was made within the intended range to optimize the efficiency of hybridization and to confirm the concentration to check if the amount at which hybridization could be attempted was achieved, the size and concentration of a library were measured using Agilent 4200 Tape Station and D1000 Screen Tape, and the result was shown in
2-9. Hybridization
2-10. Preparation of magnetic beads
2-11. Hybridization capture selection with SureSelect
2-12. Addition of index tags by amplification after hybridization (post-hybridization)
2-13. Purification of sample using Agencourt AMPure XP beads
2-14. Confirmation of Library It is a library state after hybridization has been performed and only a target region has been amplified. In
2-15. Analysis of HBV Gene Insertion Site
The sequenced reads were mapped to the reference sequence (HBV+Human genome) to create a BAM file, which is a binary of the Sequence Alignment map (SAM) file. Among the mapped reads, the chimeric read that was split-mapped to HBV and the human genome was selected to identify break points. Next, for each point, a region that satisfied read count >10, average mapping quality (MQ)>20 was defined as an HBV-human integration site, and the location of HBV and the human genome was searched. Recurrently inserted human genes were collected, gene-annotation was performed and analyzed to discover the overall biological function of each gene, and the results were shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2019-0139264 | Nov 2019 | KR | national |
| Number | Name | Date | Kind |
|---|---|---|---|
| 10597736 | Storch | Mar 2020 | B2 |
| 20180223380 | Lin | Aug 2018 | A1 |
| Number | Date | Country |
|---|---|---|
| 10-2013-0113046 | Oct 2013 | KR |
| 10-2014-0022971 | Apr 2015 | KR |
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| Haslam et. al. Optimal probe length varies for targets with high sequence variation: Implications for probe library design for resequencing highly variable genes. Plos ONE. 3(6), 2008, 1-10. [retrieved on Jan. 31, 2023]. Retrieved from the Internet <DOI: 10.1371/journal.pone.0002500> (Year: 2008). |
| Weiner et. al. Kits and their unique role in molecular biology: a brief retrospective. BioTechniques. 44, 2008, 701-704. (Year: 2008). |
| Jang et al., “Detection of HBV Integration in the Human Genome Using High-Throughput Targeted Sequencing”, Division of Hepatology, Department of Internal Medicine, The Catholic University of Korea, 2019. |
| Jang, “Detection of HBV Integration in the Human Genome Using High-Throughput Targeted Sequencing”, Free Paper Session, The Catholic University of Korea, Jun. 21, 2019. |
| Jang et al, “Detection of HBV Integration in the Human Genome Using High-Throughput Targeted Sequencing”, Department of Internal Medicine, The Catholic University of Korea Liver Research Center, 2019. |
| Yang, L. et al., “Molecular Characterization of HBV DNA Integration in Patients with Hepatitis and Hepatocellular Carcinoma”, Journal of Cancer, 9(18), pp. 3225-3235, 2018. |
| Number | Date | Country | |
|---|---|---|---|
| 20210130916 A1 | May 2021 | US |
