CN116536415A

CN116536415A - Material and kit for detecting EB virus genome differential methylation genes of CAEBV and IM and application of material and kit

Info

Publication number: CN116536415A
Application number: CN202310523521.4A
Authority: CN
Inventors: 谢正德; 王然; 张林琳; 艾军红; 张萌
Original assignee: Beijing Childrens Hospital
Current assignee: Beijing Childrens Hospital
Priority date: 2023-05-10
Filing date: 2023-05-10
Publication date: 2023-08-04

Abstract

The invention discloses application of a substance for detecting methylation levels of EBNA-LP, EBNA-3A, A73, BALF5 and BNRF1 loci in a genome of a sample to be detected in preparation of a product for identifying or assisting in identifying chronic active EB virus infection and infectious mononucleosis. The invention can detect the methylation level of the gene locus by using a detection substance so as to perform early identification of CAEBV and IM.

Description

Material and kit for detecting EB virus genome differential methylation genes of CAEBV and IM and application of material and kit

Technical Field

The invention relates to a primer group and a kit for identifying methylation differences, in particular to a primer group and a kit for identifying EBV (Epstein-Barr Virus) differential methylation genes in chronic active EB virus infection (CAEBV) and Infectious Mononucleosis (IM) and application thereof, belonging to the field of biotechnology.

Background

Epstein-Barr virus (EBV) is a member of the subfamily gamma herpes virus of the family herpesviridae, a human lymphotropic virus, and is widely distributed throughout the world, with more than about 90% of healthy adults carrying the virus worldwide. Epstein barr virus proliferates in oropharyngeal epithelial cells and then infects B lymphocytes, which enter the blood circulation in large amounts to cause systemic infection, and can be hidden in human lymphoid tissues for a long period of time. EBV infection can be manifested as proliferative and latent infection. Different infection states express different antigens, the antigen expressed in the proliferative infection stage is EBV early antigen, EBV capsid protein and EBV membrane antigen, and the antigen expressed in the latent infection stage is EBV nuclear antigen and latent membrane protein. EBV is related to the development of various human lymphocytic and epithelial tumors, such as hodgkin's lymphoma, nasopharyngeal carcinoma, and EBV-related gastric cancer, and is a well-known DNA tumor virus.

EBV is the causative agent of infectious mononucleosis (infectious mononucleosis, abbreviated IM) and chronic active EBV infection (chronic active EBV infection, abbreviated CAEBV). IM is an acute lymphoproliferative disease, which is most commonly developed after EBV infection in adolescence, and is manifested by fever, pharyngitis, lymphadenitis, splenomegaly, abnormal liver function, and a large number of mononuclear cells and atypical lymphocytes in peripheral blood. CAEBV is a lymphoproliferative disease caused by EBV infection of T lymphocytes, NK cells or B lymphocytes. CAEBV usually occurs after primary EBV infection and is characterized mainly by fever, lymphadenopathy and splenomegaly. Laboratory examination can find high copy number EBV-DNA and/or abnormal EBV related antibodies, pathological examination can detect EBV-encoded RNA and various virus related proteins in damaged tissues, and research finds that allogeneic hematopoietic stem cell transplantation for patients as early as possible is an effective method for curing the disease.

It was found that epigenetic modifications, in particular DNA methylation modifications, play an important role in EBV infection-related diseases. After EBV infection of host cells, EBV viral genes may be involved in the EBV pathogenic process by controlling the level of DNA methylation of the host genes by methyltransferases. The methylation profile of the EBV genome varies among EBV-associated diseases, and thus the corresponding disease can be identified and diagnosed by detecting the differential DNA methylation genes.

At present, the potential role played by alterations in EBV whole genome DNA methylation in the occurrence and progression of chronic active EBV infection (CAEBV) and Infectious Mononucleosis (IM) has not yet been investigated. Comparing the differential methylation genes between CAEBV and IM in the whole EBV genome, thereby screening DNA methylation biological marker for clinical diagnosis and prognosis, further revealing the mechanism of methylation in the occurrence and development of the disease, and having important significance as the basis of early detection and diagnosis of the disease.

Disclosure of Invention

The invention aims to provide a novel application of a substance for detecting methylation level of relevant sites in EB virus genome of a sample to be detected in identifying or assisting in identifying chronic active EB virus infection and infectious mononucleosis.

Another technical problem to be solved by the present invention is to provide a detection primer set for detecting the above-mentioned related sites.

In order to achieve the technical purpose, the invention adopts the following technical scheme:

the application of substances for detecting the methylation levels of EBNA-LP, EBNA-3A, A, BALF5 and BNRF1 sites in EB virus genome of a sample to be detected in the preparation and identification or auxiliary identification of chronic active EB virus infection and infectious mononucleosis products.

Wherein preferably the substance is a primer set.

Wherein preferably the primer set is selected from the group consisting of the primer pair of SEQ ID No.1 and SEQ ID No.2, the primer pair of SEQ ID No.3 and SEQ ID No.4, the primer pair of SEQ ID No.5 and SEQ ID No.6, the primer pair of SEQ ID No.7 and SEQ ID No.8, and the primer pair of SEQ ID No.9 and SEQ ID No. 10.

A primer set for detecting differential methylation genes in the epstein barr virus genome of chronic active epstein barr virus infection and infectious mononucleosis, wherein the primer set is used for detecting the methylation level of five sites of EBNA-LP, EBNA-3A, A, BALF5 and BNRF1 of the epstein barr virus genome.

The use of the primer set in a detection reagent or a detection kit for detecting or assisting in detecting differential methylation levels in the EB virus genome of chronic active EB virus infection and infectious mononucleosis.

A kit for detecting differential methylation genes in the genome of epstein barr virus (epstein barr) for chronic active epstein barr virus infection and infectious mononucleosis, comprising the primer set.

Compared with the prior art, the invention has the following technical effects:

(1) The invention obtains the differential methylation site between CAEBV and IM with strong stability and obvious specificity through optimizing and improving the screening method. The present invention proposes for the first time a method for identifying CAEBV and IM by detecting the methylation level of the EBV gene of interest.

(2) The present invention provides 5 methylation sites located on the EB virus genome, and the methylation site changes caused by EBV infection are related to CAEBV, and early diagnosis of CAEBV can be performed by detecting the methylation site changes of the EB virus genome.

(3) The five differential methylation sites between CAEBV and IM provided by the invention have good specificity and excellent stability, and can be used for further detecting the change of the methylation sites at the positions of EBNA-LP (30069 bp), EBNA-3A (53968 bp), A73 (157064 bp), BALF5 (154371 bp) and BNRF1 (2422 bp) of an EB virus genome to perform early identification of the CAEBV and the IM.

(4) The invention provides a detection primer of methylation sites, which can detect the methylation level of corresponding genes by carrying out PCR amplification and pyrosequencing detection on the sequences of the primers, thereby carrying out early identification of CAEBV and IM.

Drawings

FIG. 1 is a graph of methylation levels of the complete genome of CAEBV and IM group EBV;

FIG. 2 is a graph of EBV DNA methylation level analysis of PBMC samples from CAEBV and IM cases;

FIG. 3 is an analysis of the differential methylation genes in EBV and IM genomes detected according to the first screening criteria;

FIG. 4 is a graph showing analysis of the differential methylation genes in EBV and IM genomes detected according to the second screening criteria;

FIG. 5A is a graph showing methylation levels of five genes EBNA-LP, EBNA-3A, A73, BALF5 and BNRF1 screened for significant methylation differences between CAEBV and IM by the second screening criteria after optimization;

FIG. 5B is a graph of methylation level analysis of five genes BcRF1, EBNA1, BPLF1, LMP-2A and BSLF1 screened for significant methylation differences between CAEBV and IM according to the first screening criteria.

Detailed Description

The technical contents of the present invention will be described in detail with reference to the accompanying drawings and specific examples.

Example 1 screening and analysis methods for differential methylation sites of CAEBV and IM

1. Sample source

36 Peripheral Blood Mononuclear Cell (PBMC) samples, 19 from chronically active EBV infected infants (CAEBV) and 17 from Infectious Mononucleosis (IM).

2. Extraction of Total DNA

The extraction of PBMC sample DNA was performed using the magnetic bead method universal genomic DNA extraction kit (Tiangen). After passing the test, the sample DNA was subjected to a Bisulfofield treatment (EZ DNA Methylation Gold Kit, zymo Research), and after the treatment, unmethylated C was changed to U (changed to T after PCR amplification), while methylated C was kept unchanged.

EBV methylation primer Panel custom platform development

The EBV (NC_ 007605.1) genome is used as an EBV reference sequence to complete 98 pairs of BS-PCR primer design and synthesis. And (5) quality control and comparison of the data after sequencing.

4. BSP amplification of Bisulfite treated templates by high fidelity U base resistant DNA polymerase

The 1/20 eluted product was used as a template for 35 PCR amplifications using a KAPA HiFi HotStart Uracil +Readymix PCR Kit (KAPA Biosystems, wilmington, mass., USA).

5. Library construction

Mixing BSP amplification products from the same sample, carrying out label primer amplification, carrying out illumine sequencing joint on the mixture, finally obtaining sequencing libraries with different labels of each sample, and carrying out purification, quantification, mixing of various libraries, quality inspection and sequencing on a machine.

6. Data quality control and comparison (Excel)

Qualified data is the basis of information analysis, so quality control (quality control) of sequenced data is the primary content of data analysis, and mainly comprises:

(1) Performing basic statistics on the output and quality of data (raw reads) after the machine is started;

(2) Sequencing adaptors and low quality bases and reads were removed using software trimmatic-0.36, using a sliding window approach, 4 bases were used as a window from which reads were truncated if the average base matrix value of the window was below 15, and parameters were selected: SLIDINGWINDOW 4:15. Clear reads were obtained.

(3) Next, the clean data was aligned with the amplified target sequence, and the clean reads and target sequence were aligned using methylation sequencing data alignment software (bsmap, version v 2.73), the alignment model was map to 2forward strands,i.e.BSW (++) and BSC (- +).

7. Methylation site detection and analysis

After alignment, the methylation level of CG bases on each target sequence of each sample was calculated using the BSMAP self-contained calculation methylation python program, and the methylation level was calculated as follows:

methylation level of C site = number of reads supporting methylation/(number of reads supporting methylation + number of reads supporting unmethylation).

The methylation level of each site in each sample was counted, where if the depth was less than 30X, it would be denoted NA. Finally, according to the methylation levels calculated above, statistical and visual display was performed using the R language, and group-to-group variance analysis was performed using the two-tailed t-test, and when the test result p-value was less than 0.05, and the absolute value of the variance of the mean value of the methylation levels between groups was greater than 0.1, the variance was considered significant. Thus obtaining effective methylation information of single sites on the whole genome.

As shown in FIG. 3, the current screening method has poor site specificity and cannot effectively screen specific differential methylation gene sites for identifying CAEBV and IM. In order to further screen for effective methylation sites, the inventors have performed the following optimization and improvement of the screening method.

8. Screening for significantly different methylation sites

(1) To screen for specific iso-methylated gene loci that can effectively distinguish CAEBV from IM, we further optimized the screening protocol. And taking 500bp as a sliding window, scanning methylation information of each section of the whole genome, screening a site with the largest absolute value difference of the inter-group methylation level mean value in each section, and counting effective methylation information of each section.

(2) Increasing the screening threshold of the inter-group differential methylation sites for screening again, wherein the screening conditions are as follows:

1) For the sites where the methylation level of the EBV genome is different between the CAEBV group and the IM group, selecting the sites where the absolute value of the difference between the methylation level average of the CAEBV group and the mean of the IM group is more than 0.3;

2) Stability within the differential methylation site group was evaluated, i.e. the coefficient of difference (coefficient of variation, CV) was calculated, the formula being cv=s/M x 100% (S is the standard deviation of the samples, M is the average number of samples), and sites with a CV of less than 20% were considered to be stable. Sites with a CV of 20% or less in the CAEBV group were selected as significant differential methylation sites.

9. Through repeated test screening and verification, the primer with high amplification efficiency and good specificity and capable of detecting the methylation gene level with obvious difference between CAEBV and IM samples is obtained.

Example 2 screening and analysis of differential methylation sites for CAEBV and IM

1. Methylation sites were detected for 19 CAEBV and 17 IM samples, and a total of 1019 differential methylation sites were detected.

Based on the 1019 differential methylation sites, the methylation levels of the CAEBV and IM samples are visualized, the methylation level of each site in each sample is graphically displayed based on the absolute coordinates of the detected gene region, different colors are marked according to biological groups, the average methylation level of each site in the group is displayed in the form of a dot diagram, the positions, the methylation levels, the significance of the differences and the like of the differential methylation on the genes are visually displayed, the visualization results are shown in figure 1, and the DNA methylation levels of the CpG sites of the whole EBV genome in sequencing data of the CAEBV and IM case PBMC samples are shown in figure 1. Each dot in the figure represents the methylation level of the corresponding CpG site. The CAEBV methylation level is shown in figure 1 to be higher than in the IM group.

Differential methylation analysis between CAEBV and IM

The difference at each site of each amplicon of each comparative group was detected by t-test, the average methylation level of each amplicon was counted, the methylation profile of each region between groups was shown by violin, and the group difference analysis was performed, as shown in fig. 2, and the EBV DNA methylation levels of CAEBV and IM case PBMC samples were shown in fig. 2. Each point in the graph represents the average methylation level of one sample. The CAEBV group EBV genome methylation level was 78.4%, the IM group EBV genome methylation level was 66.7%, and the CAEBV group EBV genome methylation level was higher than that of the IM group (P < 0.0001). The methylation sites of the differences between groups were screened against the first screening criteria (using the two-tailed t-test for group difference analysis when the p-value of the test results was less than 0.05 and the absolute value of the difference in the mean value of the methylation levels between groups was greater than 0.1), and the methylation of each gene between groups was shown by a heat map, and the results are shown in FIG. 3. FIG. 3 is a thermal chart of EBV genome differential methylation gene cluster analysis in PBMC samples of CAEBV and IM cases. Each column represents one sample. Each row represents the average methylation level of each EBV encoding gene in the viral genome in all samples. The methylation level corresponding to each region of all samples is indicated in a different color. The change in the degree of methylation of the gene from low to high is indicated by a light to dark color gradient. CAEBV: chronic active EBV infection; IM: infectious mononucleosis.

3. Screening for differential methylation sites specific between CAEBV and IM

Because the specificity of the 1019 differential methylation sites detected preliminarily is poor, in order to further screen out differential methylation sites capable of specifically distinguishing CAEBV from IM, the inventor optimizes the screening conditions, and the specific optimization method is as follows:

(1) Selecting sites with absolute value of methylation level mean difference greater than 0.3 between two groups, detecting 171 EBV differential methylation sites, and displaying methylation condition of each gene between groups by using a heat map, wherein the result is shown in figure 4; FIG. 4 is a thermal graph of EBV genome differential methylation gene cluster analysis in PBMC samples from the second screening criteria CAEBV and IM cases. Each column in the figure represents a sample. Each row represents the average methylation level of each EBV encoding gene in the viral genome in all samples. The methylation level corresponding to each region of all samples is indicated in a different color. The color gradation from blue to red indicates a change in the degree of methylation of the gene from low to high. CAEBV: chronic active EBV infection; IM: infectious mononucleosis.

(2) The stability in groups of 171 differential methylation sites was further assessed, i.e. the coefficient of difference (coefficient of variation, CV) was calculated, the formula being cv=s/M x 100% (S being the standard deviation of the samples, M being the average number of samples), with sites with a CV of less than 20% being considered to be stable.

After optimization of the method, five genes EBNA-LP (30069 bp), EBNA-3A (53968 bp), A73 (157064 bp), BALF5 (154371 bp) and BNRF1 (2422 bp) with obvious methylation differences between CAEBV and IM were screened out, and methylation levels and CV values of the genes are shown in the following Table and in FIG. 5A and FIG. 5B, which are representative differential methylation genes in the EBV and IM genomes. Wherein FIG. 5A shows the methylation levels of five genes EBNA-LP, EBNA-3A, A73, BALF5 and BNRF1 screened according to the optimized second screening criteria for significant methylation differences between CAEBV and IM; FIG. 5B shows the methylation levels of five genes BcRF1, EBNA1, BPLF1, LMP-2A and BSLF1 screened for significant methylation differences between CAEBV and IM according to the first screening criteria. CAEBV: chronic active EBV infection; IM: infectious mononucleosis.

Table 1 lists a portion of the first screening site data, table 2 shows the second optimization screening data, and the methylation level of the second screening methylation site can be directly judged to be high by diff values, and the specificity of the second screening site can be judged to be far higher than that of other sites by CV values.

TABLE 1

TABLE 2

Example 2 primer for detecting level of significantly different methylation genes of EB virus genome in CAEBV and IM samples

1. Primer set

The inventor designs primer groups with high amplification efficiency and good specificity aiming at the differential methylation loci, and can detect the gene level of the obvious differential methylation loci in CAEBV and IM samples, and the sequences of the primer groups are shown in the following table.

TABLE 3 Table 3

2. Basic methods for detection of methylation levels of interest in CAEBV and IM samples using the above primers:

pyrophosphate sequencing PCR detection was performed on the methylation levels of the genes of interest in CAEBV and IM samples using the primers described above.

(1) DNA sample preparation: extracting by using a magnetic bead method universal genome DNA extraction kit (Tiangen);

(2) Sulfite conversion: sodium bisulphite modification of the extracted DNA samples was performed using the EZ DNA Methylation-Gold Kit D5005 Kit (ZYMO RESEARCH) to convert unmethylated cytosines to uracil, while methylated cytosines remained unchanged.

(3) PCR amplification reaction: and enriching and amplifying the gene fragments to be detected through PCR reaction, wherein the product length is between 200 and 700 bp.

a. The sample is subjected to sulfite conversion and low-speed short-time centrifugation for standby.

b. The PCR reaction mixture was prepared as shown in the following table (the reaction system preparation process was completed under low temperature conditions, preventing deactivation by long-term exposure to high temperature).

TABLE 4 methylation detection amplification PCR reaction Components

c. Amplification PCR reaction procedure

The procedure was set as follows: 95 ℃,5 min- & gt (94 ℃,30 s- & gt 60 ℃,45 s- & gt 72 ℃ 45 s) 40 times of circulation- & gt 72 ℃,10 min- & gt 10min.

d. After the reaction is finished, 10 mu L of PCR product is taken, electrophoresis is carried out for 45min by 2% agarose gel, and after the electrophoresis is finished, ultraviolet photographing is carried out, so as to verify the amplification effect of the BSP reaction product.

(4) Pyrophosphate sequencing:

a. separating single-stranded DNA from the PCR product by adopting a single-stranded purification device matched with a pyroMark Q96 ID pyrosequencing instrument;

b. the sequencing reaction plate is placed in a sequencer for sequencing.

c. Data analysis: methylation analysis software matched with a pyrophosphoric acid sequencer is adopted to analyze methylation results.

Claims

1. The application of substances for detecting the methylation levels of EBNA-LP, EBNA-3A, A73, BALF5 and BNRF1 loci in the genome of a sample to be detected in the preparation and identification or auxiliary identification of products for chronic active EB virus infection and infectious mononucleosis.

2. The use according to claim 1, wherein:

the substance is a primer group.

3. The use according to claim 2, wherein:

the primer set is selected from the primer set consisting of the primer pair of SEQ ID No.1 and SEQ ID No.2, the primer pair of SEQ ID No.3 and SEQ ID No.4, the primer pair of SEQ ID No.5 and SEQ ID No.6, the primer pair of SEQ ID No.7 and SEQ ID No.8 and the primer pair of SEQ ID No.9 and SEQ ID No. 10.

4. A primer set for detecting host differential methylation genes for chronic active epstein barr virus infection and infectious mononucleosis, characterized in that:

the primer group is used for detecting methylation levels of EBNA-LP, EBNA-3A, A73, BALF5 and BNRF1 loci of the human genome.

5. The primer set of claim 4, wherein:

the primer group consists of a primer pair of SEQ ID No.1 and SEQ ID No.2, a primer pair of SEQ ID No.3 and SEQ ID No.4, a primer pair of SEQ ID No.5 and SEQ ID No.6, a primer pair of SEQ ID No.7 and SEQ ID No.8 and a primer pair of SEQ ID No.9 and SEQ ID No. 10.

6. Use of the primer set of claim 4 or 5 in a detection reagent or detection kit for detecting or aiding in the detection of host differential methylation levels of chronic active epstein barr virus infection and infectious mononucleosis.

7. A kit for detecting host differential methylation genes for chronic active epstein barr virus infection and infectious mononucleosis, comprising:

the kit comprises the primer set according to claim 4 or 5.