EB virus detection technology based on capture sequencing

An Epstein-Barr virus and sequencing technology, which is applied in recombinant DNA technology, microbial measurement/inspection, DNA/RNA fragments, etc., can solve problems such as poor sample quality, large impact on test results, and inability to combine

Pending Publication Date: 2021-01-05
3D BIOMEDICINE SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Although conventional PCR can directly detect Epstein-Barr virus DNA, the quality of the sample is not good, and if the DNA fragmentation is severe, the primers cannot amplify the target fragment.
Immunohistochemistry is highly dependent on the quality of antibodies, and antibodies with poor production quality or storage conditions have a greater impact on the test results
In addition, the judgment of the immunohistochemical te

Method used

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  • EB virus detection technology based on capture sequencing
  • EB virus detection technology based on capture sequencing
  • EB virus detection technology based on capture sequencing

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0052]Example 1: Proof of specificity of EB virus probe design

[0053]● Use bedtools2 (the same below v 2.25.0) software to extract the area of ​​the probe design (that is, the area covered by the probe of the present invention) and integrate it to form a bed file;

[0054]● Use bedtools software to extract the Epstein-Barr virus reference genome sequence (GenBank ID: NC_007605.1) according to the generated bed file to form a fasta file;

[0055]● Use the internal python script to split each sequence in the fasta file to form a fastq file. The split method is: use 1bp as the step size and 100bp as the window, slide on each record, and finally generate a fastq file with a length of 100bp ;

[0056]● Use bwa v0.7.12 software to compare the split fastq file to the human reference genome (GRCH37) to generate a comparison file (bam format);

[0057]● Use the python script to view the comparison result information to verify whether the Epstein-Barr virus and the human genome have homologous regions.

[00...

Example Embodiment

[0061]Example 2: Proof of specificity of EBV-1 and EBV-2 probe design

[0062]● Use bedtools2 (the same below v 2.25.0) software to extract the corresponding ranges of the two types of EB virus in the probe design area (that is, the area covered by the probe of the present invention) and integrate them to form a bed file;

[0063]● Use bedtools software to extract the Epstein-Barr virus reference genome sequence according to their respective bed files, and form fasta files respectively;

[0064]● Use the internal python script to split each sequence in the fasta file to form a fastq file. The split method is: use 1bp as the step size and 100bp as the window, slide on each record, and finally generate a fastq file with a length of 100bp ;

[0065]● Use bwa v0.7.12 software to compare the split fastq files to the reference genome of the Epstein-Barr virus, and generate comparison files (bam format) respectively;

[0066]● Use python script to view the comparison result information.

[0067]● The final ...

Example Embodiment

[0072]Example 3: Library building, capturing and sequencing of samples

[0073]After obtaining the tissue sample, first use a mature kit on the market to extract the DNA. The DNA from tissues needs to be interrupted by ultrasonic technology, DNA digestion technology or transposase technology to form a double-stranded DNA fragment with a length of about 200 bases. Then use the corresponding kits to construct the library for DNA from different sources. After the library is built, the libraries of one to four samples are mixed, and then the Epstein-Barr virus capture probe is used to capture the Epstein-Barr virus genome fragments. The EB virus capture probe can be mixed with other probes to capture together, and the capture efficiency of the EB virus probe will not be affected. The captured library is finally sequenced by a high-throughput sequencer.

[0074]Operation steps of wet experiment:

[0075]Step 1: Build a library:

[0076]1. Reagents and consumables

[0077]a) KAPA Hyper Library Preparati...

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Abstract

The invention relates to an EB virus detection technology based on capture sequencing. Specifically, the invention relates to a probe group for detecting EB virus based on capture sequencing. The probe group is characterized by comprising probes which are specific to EB virus genes LMP1, LMP2, EBNA-1, EBNA-2, EBNA-3 and BZLF1 respectively, preferably the probes which are specific to the EBNA-2, and/ or the probes which are specific to the EBNA-3, wherein the probes which are specific to the EBNA-2 comprises a probe which is specific to EBNA-2 of an EB virus genotype 1 and a probe which is specific to EBNA-2 of an EB virus genotype 2; and the probes which are specific to the EBNA-3 comprises a probe which is specific to EBNA-3 of the EB virus genotype 1 and a probe which is specific to EBNA-3 of the EB virus genotype 2.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the detection of Epstein-Barr virus. Background technique [0002] Epstein-Barr virus (EBV) is a double-stranded DNA virus belonging to the herpesvirus gamma subfamily. Epstein-Barr virus mainly infects nasopharyngeal epithelial cells and B lymphocytes, and the main diseases it can cause include nasopharyngeal carcinoma, gastric cancer, and some benign / malignant lymphoproliferative diseases. The detection of Epstein-Barr virus plays a good reference role in the diagnosis, staging, treatment and prognosis of related diseases or tumors. Currently in clinical practice, the detection of Epstein-Barr virus is mainly through: 1. Conventional PCR amplification, the target fragment of the main amplification is EBNA-1 , EBNA-2 , EXLF-1 Gene and Bam HI-w region; 2. Immunohistochemical method, such as serum IgA, IgG antibody and LMP-1 antibody detection; 3. Epstein-bar virus-encoded RNA i...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6874C12N15/11
CPCC12Q1/705C12Q1/6874C12Q2600/166C12Q2535/122C12Q2545/113C12Q2545/101C12Q2565/501
Inventor 王磊刘士毅梁雷郭浩
Owner 3D BIOMEDICINE SCI & TECH CO LTD
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