CN119372296A - A kit for human B lymphocyte counting based on gene methylation detection - Google Patents

Abstract

The invention provides a kit for realizing human B lymphocyte counting based on gene methylation detection, which is a kit for detecting poly ADP-ribose polymerase 1 (PARP 1) gene methylation in a sample. The invention provides a biomarker which can be used for identifying B lymphocytes and is prepared from an unmethylated gene region of PARP1 genes for the first time. The invention quantifies the copy numbers of the PARP1 target gene and the reference gene by the PCR technology, and can conveniently and rapidly conduct relative quantitative analysis on B lymphocytes in a small amount of blood or dry blood. The target detected by the invention is the methylation level of the leucocyte DNA in the sample, can be suitable for various samples, including fresh or frozen blood, isolated PBMC cells, dry blood spots and the like, and greatly reduces the requirements on the samples.

Description

Kit for realizing human B lymphocyte counting based on gene methylation detection

Technical Field

The invention relates to the field of molecular biology, in particular to a kit for realizing human B lymphocyte counting based on gene methylation detection.

Background

Lymphocytes are important cellular components of the human body that resist disease, provoke immune response functions, and express differentiation antigens, i.e., CD molecules, during the process of differentiation and maturation. Depending on the CD molecule expressed, lymphocyte subsets mainly include T lymphocytes, B lymphocytes and NK cells. B lymphocytes are traditionally defined as "cell populations expressing a clonal diversity of cell surface immunoglobulin (Ig) receptors, recognizing specific epitopes", derived from hematopoietic stem cells in bone marrow, and feature markers include CD19, CD20, and B lymphocyte antigen receptors (BCR). B lymphocytes can be divided into two lineages, T cell independent B1 cells and T cell dependent B2 cells. The B lymphocyte developed and mature in the bone marrow migrates to peripheral lymphoid organs through blood, and after being stimulated by antigen, the B lymphocyte is differentiated and proliferated into plasma cells to synthesize antibodies, plays a role in humoral immunity and plays an important role in pathogenesis of various human diseases.

The proportion, the number and the functions of different lymphocyte subsets directly influence the immune state of an organism, and the detection of the lymphocyte subset is an important index for evaluating the current immune function and the balance state of the organism, and is an indispensable diagnostic item especially for patients with immune function deficiency. Normally, the lymphocyte subsets maintain a proportion and quantity, cooperate, and maintain the body's steady state. During the course of the disease, the proportion and number of immune cells changes pathologically, resulting in immune dysfunction. The application of lymphocyte counting is wide, and mainly comprises early screening of abnormal total lymphocyte subpopulations of patients with infection and malignant tumors, verification after primary immunodeficiency screening, auxiliary diagnosis of Chronic Lymphocytic Leukemia (CLL), detection monitoring after treatment of HIV positive patients, immune and immune reconstitution monitoring, such as evaluation of immune reconstitution after immunosuppressive treatment of transplantation, autoimmunity and other immune conditions, hematopoietic stem cell transplantation and the like.

Currently, flow Cytometry (FCM) is a standard method for lymphocyte subpopulations or B lymphocyte counts, which uses different monoclonal antibodies to bind CD antigens on lymphocyte surfaces, and then cooperates with multicolor fluorescent dyes to detect surface antigens of several lymphocytes simultaneously, so as to separate B lymphocytes from white blood cells, thereby obtaining the number and relative proportion of B lymphocytes. The flow cytometry has the limitations that firstly, the requirement on a sample is high, the cells to be detected are required to be complete, the blood sample is required to be detected in 8 hours, secondly, the requirement on the sample is high due to the fact that the flow B lymphocytes have high requirements on the sample, the blood sampling problem of certain application scenes such as infants is high, thirdly, the flow cytometry is used for detecting fluorescent signals of fluorescent labeled antibodies combined with antigen on the cell surface and then the fluorescent signals of complexes between yin and yang are required to be determined, but for surface antigens of different cell subsets, the expression of the surface antigens is not a process or is not a process, but is low, medium and high in different expression levels, so that the sub-populations of the B lymphocytes can be inaccurate in count due to the fact that the threshold is determined, fourthly, the difference of detection results of each laboratory is large due to the differences of the sample, the immunological detection technology and the operation process, and the standardization of the flow B lymphocyte count is still a challenge to be established.

During the differentiation development of B lymphocytes, DNA methylation modification plays an important role by regulating gene expression and shut down, thus providing specific methylation markers for B lymphocytes, such as certain gene promoter regions involved in the differentiation development of B lymphocytes, which are completely unmethylated, and which are highly methylated in cell subsets other than B lymphocytes, such as T lymphocytes, NK cells, and non-lymphocytes such as granulocytes and monocytes. These unmethylated CpG sites can be used as specific Differential Methylation Regions (DMR), i.e. as biomarkers, for identifying B lymphocytes and for quantification of B lymphocytes in peripheral blood. Such B lymphocytes are characterized not by cell morphology or surface markers, but by epigenetic means, hopefully overcoming the limitations of the flow detection methods described above.

Currently, methods based on epigenetic DNA methylation biomarkers as lymphocyte counts have been reported. Patent CN 108026578A reports that identification and quantification of B lymphocytes is achieved based on the demethylation or lack of methylation status of at least one CpG position in the gene region of low density lipoprotein receptor-related protein 5 (LRP 5) and that it is distinguished from all other cells in complex samples such as other blood cells or immune cells. However, in this patent, the validation of LRP5 as a B lymphocyte specific marker is insufficient, and cell type specificity is demonstrated only by plasmid DNA. Furthermore, the description and data of the whole blood and/or tissue not digested with trypsin to carry out the method are ambiguous.

Disclosure of Invention

In order to overcome the problems, the invention provides a kit for realizing human B lymphocyte count based on gene methylation detection, wherein the kit is used for detecting PARP1 gene methylation in a sample.

In one embodiment, the kit is a kit for detecting a fragment of the differential methylation region hg38 chr1: 226372321-226371196 on the PARP1 gene.

In one embodiment, the kit is a kit for detecting the differential methylation region hg38 chr1: 226371363-226371254 on the PARP1 gene.

In one embodiment, the kit is a kit for detecting a gene region comprising at least one of the differential methylation sites hg38 chr1: 226371196,hg38 chr1: 226371228,hg38 chr1: 226371279 and hg38 chr1: 226371333 on the PARP1 gene.

In one embodiment, the kit calculates the proportion of human B lymphocytes to human white blood cells by detecting the copy numbers of the reference gene and the PARP1 gene.

In one embodiment, the reference gene is the RPP30 gene.

In one embodiment, the kit is a sequencing kit, a quantitative PCR kit, or a digital PCR kit.

In one embodiment, the kit includes primers and probes for detecting the methylation status of CpG sites in the region of hg38 chr1: 226371363-226371254 and primers and probes for detecting the copy number of the reference gene.

In one embodiment, the kit comprises an upstream primer SEQ ID No. 13 GGAAGTGTTGGATATGTAGAAATGG, a downstream primer SEQ ID No. 14: CCCACACTCTATATCCTAAAACATCA, and a probe SEQ ID No. 15: AGGTGTAGTTATGGGTT.

In one embodiment, the sample is selected from a peripheral blood sample, a capillary blood sample, or venous blood.

Based on the above state of the art, in order to overcome the technical limitations of the current cytological-based B lymphocyte counting and expand its applicability, the present invention first proposes that the unmethylated gene region of the PARP1 gene can be used as a biomarker for B lymphocyte identification.

The beta value of the CpG sites in the methylation chip represents the methylation degree, and the difference value (delta beta value) of the beta of the corresponding CpG sites among the groups of samples can be compared to screen the differential methylation sites, so that the larger the absolute value of the delta beta value is, the larger the difference of the methylation degree of the sites is, and the stronger the specificity of the sites is. When B lymphocyte differential CpG sites are screened according to the principle, the invention creatively sets another screening condition that delta beta values of adjacent 4-5 CpG sites in a methylation chip are all as large as possible, and the sites are continuous and as dense as possible, so that the gene fragments formed by the 4-5 methylation sites have the maximum specificity, thereby meeting the requirement of potential biological markers. According to this screening condition, the present invention finally determines 4 differential methylation sites of relatively dense distribution with larger Δβ values on the PARP1 gene, cg08247449 (hg 38 chr1: 226371196), cg00278472 (hg 38 chr1: 226371228), cg09201889 (hg 38 chr1: 226371279) and cg06184361 (hg 38 chr1: 226371333).

In view of the regulatory effect on the gene or the fact that a gene sequence is used for methylation detection, a differential methylation region is determined from the screened differential methylation sites and used as a specific marker for distinguishing B lymphocytes. Meanwhile, cpG sites covered by probes in the methylation chip are also discontinuous, the invention takes 4 screened differential methylation sites as base points, and verifies the methylation state of 22 CpG sites contained in sequences of about 1130bp at the upstream and downstream of a gene through Sanger sequencing after bisulfite conversion and PCR amplification, wherein the CpG sites are in an unmethylated state in B lymphocytes, and the CpG sites are in a methylation state in other subgroups and completely coincide with the methylation state of the 4 screened differential methylation sites, so that a differential methylation region (hg 38 chr1: 226372321-226371196) of about 1130bp consisting of 22 methylation sites is determined and used as a specific unmethylated region of the B lymphocytes.

In the above verified differential methylation regions of about 1130bp, a plurality of amplicon detection fragments (hg 38 chr1: 226371363-226371254) with relatively concentrated CpG sites and simultaneously covering different methylation sites are selected, primers and probes of the corresponding methylation transformed fragments are designed, and the higher the delta Ct value (Ct _{Negative of} -Ct _{Positive and negative} ) of the candidate amplicon is compared by the result of fluorescence quantitative PCR (qPCR), the better the specificity of the candidate amplicon is indicated, so that the 110bp differential methylation region (hg 38 chr1: 226371363-226371254) of the corresponding amplicon AMP1333 on the PARP1 gene is preferable, and the better specificity and sensitivity are achieved, and can be used as biomarkers for B lymphocyte identification.

Firstly, extracting gDNA of a sample to be detected, and using the extracted gDNA as a DNA template after bisulfite conversion. Then, double qPCR amplification was performed using the amplicon-specific primer probe of the PARP1 target gene and the primer probe specific for the reference gene RPP 30. The amplification reaction is operated on a qPCR instrument, the amplification result is given in the form of a fluorescence curve, the purified B lymphocytes are selected as a reference system, and the relative content of the B lymphocytes in the white blood cells can be calculated according to the amplification curve and a method of 2 ^-ΔΔCt. In addition, a digital PCR detection platform can be adopted, and the relative content of B lymphocytes in white blood cells can be calculated by directly detecting the copy numbers of PARP1 target genes and RPP30 reference genes and according to the corresponding relation between the copy numbers of the reference genes and the cell numbers (the copy number of the RPP 30: the cell number=2:1) and using the formula that the relative content (%) of the B lymphocytes is=the copy number of the PARP1 target genes/the copy number of the reference genes multiplied by 100. The RPP30 reference gene detection fragment selected by the invention is stably expressed in all white blood cells, and cytosine in the sequence is in an unmethylated state.

The target of the detection is the methylation level of the leucocyte DNA in the sample, can be suitable for various samples, including fresh or frozen blood, isolated PBMC cells, dried blood spots and the like, has greatly reduced requirements on the sample and no requirements on the preservation state of the sample, secondly, the DNA amplification detection has small requirement on the sample, the trace DNA can meet the detection requirement, furthermore, detection signals obtained by PCR are digital, represent a positive or negative value of each cell and are not a positive threshold value which is arbitrarily defined as a flow cytometry method, and finally, the detection can be performed in an automatic and operator-independent mode based on epigenetic qPCR, and the sensitivity to reagent variability is reduced. The kit is not only a beneficial supplement to traditional flow cytometry detection, but also can be used for obviously distinguishing the infant suffering from X-linked agaropectinemia (X-linked agammaglobulinemia, XLA) from a normal newborn, and can be used for screening primary immunodeficiency diseases of the newborn.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments described in the present application, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a DMR sequence of PARP1 gene for identifying B lymphocytes;

FIG. 2 is a bisulphite converted DMR sequence of the PARP1 gene used to identify B lymphocytes;

FIG. 3 is a graph of the results of PCR amplification curves for verifying TPG specificity by plasmid DNA;

FIG. 4 is a graph showing the results of PCR amplification curves for verifying CPG specificity by plasmid DNA;

FIG. 5 is a graph of PCR amplification for methylation standards validating TPG specificity;

FIG. 6 is a plot of PCR amplification of methylation standards validating CPG specificity;

FIG. 7 is a bar graph of gradient methylation standard validation of PCR system specificity;

FIG. 8 is a linear graph of gradient methylation standard validation of PCR system specificity;

FIG. 9 is a graph of digital PCR results specific for purified cell markers.

Detailed Description

In order that those skilled in the art will better understand the present application, the following description will proceed with reference being made to illustrative embodiments, only being described in terms of a few, but not all embodiments of the present application. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, shall fall within the scope of the application. In the following examples, unless otherwise specified, conventional methods are used.

The experimental methods in the following examples, in which specific conditions are not noted, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. The various chemical reagents used in the examples are all commercially available products.

Example 1 differential Gene discovery and discovery of differential methylation regions

In order to find the specific DNA methylation region of B lymphocyte, the invention firstly analyzes 450K and 850K methylation chip data from Illumina, and obtains methylation beta value through probe filtration and other operations. And then setting the threshold value of beta to be 0.2 and 0.8, and after processing and analyzing the confirmed data set and the verified data set, obtaining differential sites by comparing the whole methylation levels of different lymphocyte subpopulations and performing data quality control and standardization operation, wherein the differential of the sites is measured by delta beta value. The larger absolute value of Δβ value indicates more significant difference between groups, and Q value is smaller than 0.01 (Q <0.01 represents significant difference) according to the absolute value of Δβ value being larger than 0.5. Accordingly, the present invention prefers the differential gene PARP1 with more differential sites (the results are shown in Table 1). ‌ PARP1 (poly ADP-ribose polymerase 1) ‌ is mainly involved in various biological processes in cells including DNA damage repair, gene transcription regulation, cell differentiation, proliferation, tumor transformation, etc. On the basis of obtaining differential genes, the invention sets a further screening condition that delta beta values of adjacent 4-5 CpG sites in a methylation chip are all as large as possible, and the sites are continuous and as dense as possible, so that the gene fragments formed by the 4-5 methylation sites can be ensured to have the maximum specificity, thereby meeting the requirement of potential biological markers. The invention screens 4 differential methylation sites with larger delta beta value and relatively dense distribution on the differential gene PARP1, namely, specific positions corresponding to the PARP1 gene are hg38 chr1: 226371196,hg38 chr1: 226371228,hg38 chr1: 226371279 and hg38 chr1: 226371333, namely, cg08247449, cg00278472, cg09201889 and cg 06184361.

Table 1 differential sites and differential Gene conditions screened

Next, the present invention experimentally confirmed the differential sites. Sanger sequencing and pyrosequencing verification were performed after qPCR amplification using purified B lymphocytes derived from fresh peripheral blood, bisulphite-converted gDNA as a positive template and non-B lymphocytes of the same origin, bisulphite-converted gDNA as a negative template. As shown in the results of the verification shown in Table 2, the 4 differential methylation sites (cg 08247449, cg00278472, cg09201889 and cg 06184361) on the PARP1 gene were indeed differential methylation sites.

In view of the regulatory effect on genes or the fact that a gene sequence is used for methylation detection, a DMR (DMR) is determined from the screened differential methylation sites and used as a specific marker for distinguishing B lymphocytes. At the same time, the CpG sites covered by the probes in the methylation chips are also discontinuous. The invention uses the above-determined 4 differential methylation sites as base points, expands the possible range of the differential sites in the sequence of about 900bp upstream of the gene (about 200bp downstream of the 4 differential sites has no CG points), and further searches for possible differential methylation regions DMR. gDNA derived from purified B lymphocytes was bisulfite converted and amplified by designed pairs of universal primers to target gene fragments at which multiple possible differential methylation sites were located, followed by Sanger sequencing. As can be seen from the sequencing results shown in Table 3, each site to be tested showed a "T" (unmethylated state) when the positive template was amplified, and a "C" (methylated state) when the negative template was amplified, which was completely consistent with the methylation state of the 4 differentially methylated sites screened. Accordingly, the present invention has determined DMR (hg 38 chr1: 226372321-226371196) consisting of the above 22 differential methylation sites, and the DMR is distributed in the promoter region of the PARP1 gene.

Table 2 Sanger sequencing and pyrosequencing of 4 different methylation sites on the PARP1 gene verify the results.

TABLE 3 Sanger sequencing results for each site tested

In particular, fig. 1 shows the DNA sequence of DMR for identifying PARP1 gene of B lymphocytes, cpG site analysis and specific gene localization of 4 differential methylation sites, the 4 differential methylation sites being bold and underlined in fig. 1.

Example 2 defined amplicon and specific primer probes

FIG. 2 shows the DNA sequence of the defined DMR region after bisulfite conversion. The horizontal box in figure 2 corresponds to (CPG 1, 2..22) in the PARP1 gene analyzed, indicated position (hg 38 chr1: 226372321, hg38 chr1: 226372269.... 226371196) corresponds to CPG1, 2. To further determine the optimal target gene amplicon sequence, 5 sets of primer probes were designed according to the MethyLight rules for 5 candidate amplicons AMP2269 (CpG 2-CpG5, specific genome located at hg38 chr1: 226372287-226372167), AMP2110 (CpG 6-CpG8, specific genome located at hg38 chr1: 226372134-226372009), AMP1948 (CpG 9-CpG11, specific genome located at hg38 chr1: 226371978-226371882), AMP1787 (CpG 13-CpG16, specific genome located at hg38 chr1: 226371814-226371711), AMP1333 (CpG 18-CpG20, specific genome located at hg38 chr1: 226371363-226371254), primers and probes were optimized for optimal specificity and sensitivity. As shown in Table 4, SEQ ID Nos. 1-21 show specific primer and probe sequences used according to the present invention. The invention uses positive samples (gDNA extracted and converted by B lymphocytes) and negative samples (gDNA extracted and converted by non-B lymphocytes) as templates of a TPG system of a qPCR detection platform, uses delta Ct (Ct _{Negative of} -Ct _{Positive and negative} )) to evaluate amplicon to identify the specificity of the B lymphocytes, and the higher the delta Ct value is, the better the specificity aiming at the B lymphocytes is indicated. The qPCR results are shown in Table 5, and the primer probe corresponding to AMP1333 shows the maximum delta Ct, which shows that the amplicon has better specificity and sensitivity and can be used as a biomarker for B lymphocyte identification.

Preferably, the underlined position (genomic position hg38 chr1: 226371363-226371254) in FIG. 2 shows CPG site analysis on the amplicon determined for the identification of the PARP1 target gene of B lymphocytes. The horizontal boxes correspond to CPG positions (CPG 18, 19, 20) in the amplicon analyzed, the indicated positions (hg 38 chr1: 226371333, hg38 chr1: 226371325, hg38 chr1: 226371279) corresponding to CPG18, 19, 20.

SEQ ID No. 22 and SEQ ID No. 23 show the DNA sequences of bisulfite converted target gene amplicons according to the qPCR and digital PCR detection systems of the invention. The methylated "CG" site still maintains the differential methylation site of "CG" and specific primers and probes for detecting the site. TPG system refers to the specific primer and probe for detecting the differential methylation site of unmethylated CG site converted into TG after sulfate conversion of DNA sequence of target gene amplicon.

Preferably, the system-specific amplicon (of SEQ ID No. 22) of CPG (for methylation site detection):

GGAAGTGTTGGATATGTAGAAATGGAAAGGCGTAGTTACGGTTTTTAGGGAGTTTAGAAGGGTTTATTGGTAAAGTTTTAGAGACGATGTTTTAGGATATAGAGTGTGG

Preferably, the TPG (of the unmethylated site) system-specific amplicon (SEQ ID No. 23):

GGAAGTGTTGGATATGTAGAAATGGAAAGGTGTAGTTATGGTTTTTAGGGAGTTTAGAAGGGTTTATTGGTAAAGTTTTAGAGATGATGTTTTAGGATATAGAGTGTGGG

TABLE 4 sequences of target gene PARP1 specific primer probes

Table 55 qPCR amplification results of primer probes

Example 3 TPG and verification of CPG-specific PCR System

The specificity of the PCR system was verified using a test template (plasmid DNA), two concentration gradients of templates were set, the sequence of the C plasmid was identical to SEQ ID No. 22, and the sequence of the T plasmid was identical to SEQ ID No. 23. The TPG and CPG systems were tested by PCR amplification, respectively, and the results are shown in FIGS. 3 and 4.

As can be seen from FIG. 3, qPCR amplification curves were typical S amplification curves when the added template was either high concentration of T plasmid or 100-fold dilution of high concentration of T plasmid, but the amplification curves were not shown when the template was either high or low concentration of C plasmid, consistent with NTC amplification. The results prove that the primer probe of the TPG system has good specificity.

As can be seen from FIG. 4, when the added template is 100-fold diluted with high concentration of T plasmid or high concentration of C plasmid, the qPCR amplification curve is a typical S amplification curve, but when the template is high or low concentration of T plasmid, the amplification curve is not out, consistent with the amplification of NTC. The result proves that the primer probe of the CPG system has good specificity.

In addition, the specificity of TPG and CPG systems was verified using a methylation standard with a methylation rate of 0% and a methylation standard with a methylation rate of 100%, the methylation standard being derived from QIAGEN. As can be seen from FIGS. 5 and 6, when 0% methylation standard was used as a template, the TPG system showed a typical amplification curve, whereas the CPG system showed no amplification curve. Likewise, CPG systems show typical amplification curves when 100% methylation standards are used as templates, whereas TPG systems do not. The results show that the primer probe of the TPG and CPG system has good specificity.

The specificity of the PCR system was further verified using gradient methylation standards taking into account the differences in methylation levels in the actual samples. For this purpose, a methylation standard with a methylation rate of 0% was mixed with a methylation standard with a methylation rate of 100% to simulate a methylation value in the range of 0% -100%. The ratio (%) of TPG template in these mixtures was then detected using the assay of the invention. As a result, as shown in fig. 7 and 8, when the methylation ratio was changed from 0% to 100%, the ratio calculated by the digital PCR test was equally changed, and a good linear relationship was exhibited (R ² =0.9982). The test results of the gradient methylation standard fully show that the specificity of the TPG and CPG systems is good, and different methylation levels can be distinguished.

Example 4 validation of PARP1 target Gene as B lymphocyte specific marker

The specificity of PARP1 as B lymphocyte marker was verified using purified cells. Therefore, the DNA obtained by separating and purifying lymphocytes (T lymphocytes, CD4+T lymphocytes, CD8+T lymphocytes, B lymphocytes and NK cells) with magnetic beads is used as a template, the copy numbers of the target gene PARP1 and the reference gene RPP30 are directly obtained by utilizing a digital PCR platform, and the relative content of each cell subset can be directly calculated by the formula that the relative content (%) of the cell subset is=the copy number of the PARP1 target gene/the copy number of the reference gene is multiplied by 100. As shown in fig. 9, the ratio of B lymphocytes detected by the TPG system is very high, whereas the ratio of T lymphocytes, cd4+ T lymphocytes, cd8+ T lymphocytes, and NK cells is <1.7%, and the detected results are consistent with the results of the flow cytometry detection. The above results also demonstrate that the PARP target gene can be used as a marker specific for B lymphocytes.

Example 5 determination of B lymphocyte content in actual sample

The PARP1 primer probe of the present invention was used to quantify the B lymphocyte content (%) in neonatal dry blood spot samples. XLA is one of the most serious diseases in B lymphocyte immunodeficiency, and XLA patients cannot produce mature B lymphocytes, thereby affecting humoral immunity and increasing the probability of serious, lethal bacterial infection. The copy numbers of the PARP1 gene and the reference gene are obtained through digital PCR detection, and the relative content of the B lymphocyte in the white blood cell can be calculated by using the formula of the ratio (%) of the B lymphocyte to the PARP1 target gene/the copy number of the reference gene multiplied by 100. As shown in Table 6, the target gene copy number of the XLA sample was 3-13 copies/. Mu.L, the ratio in the white blood cells was 0.2-1.6%, while the target gene copy number of the normal neonatal sample was 40-288 copies/. Mu.L, the ratio in the white blood cells was 3.9-8.8%. The results show that the method provided by the invention can be used for distinguishing the XLA infant from the normal newborn infant and can be used for screening the primary immunodeficiency diseases of the newborn infant.

TABLE 6 quantification of PARP1 Gene and B lymphocyte occupancy in Dry blood spot samples

It is to be understood that this invention is not limited to the particular methodology, protocols, and materials described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are also encompassed by the appended claims.

Claims (10)

1. A kit for realizing human B lymphocyte counting based on gene methylation detection, characterized in that the kit is a kit for detecting PARP1 gene methylation in a sample. 2. The kit according to claim 1, characterized in that the kit is used to detect the differentially methylated region hg38 chr1: 226372321-226371196 on the PARP1 gene. 3. The kit according to claim 2, characterized in that the kit is a kit for detecting the differentially methylated region hg38 chr1: 226371254-226371363 on the PARP1 gene. 4. The kit according to claim 1, characterized in that the kit is a kit for detecting a gene region comprising at least one of the differentially methylated sites hg38 chr1: 226371196, hg38 chr1: 226371228, hg38 chr1: 226371279 and hg38 chr1: 226371333 on the PARP1 gene. 5. The kit according to claim 1, characterized in that the kit calculates the proportion of human B lymphocytes to human white blood cells by detecting the copy number of the internal reference gene and the PARP1 gene. 6. The kit according to claim 5, wherein the internal reference gene is the RPP30 gene. 7. The kit according to claim 5, characterized in that the kit is a sequencing kit, a quantitative PCR kit or a digital PCR kit. 8. The kit according to claim 7, characterized in that the kit comprises: primers and probes for detecting the methylation status of CpG sites in the region of hg38 chr1:226371254-226371363 and primers and probes for detecting the copy number of an internal reference gene. 9. The kit according to claim 8, characterized in that the kit comprises: an upstream primer SEQ ID No. 13 GGAAGTGTTGGATATGTAGAAATGG, a downstream primer SEQ ID No. 14: CCCACACTCTATATCCTAAAACATCA, and a probe SEQ ID No. 15: AGGTGTAGTTATGGGTT. 10. The kit according to claim 1, characterized in that the sample is selected from a peripheral blood sample, a capillary blood sample or a venous blood.