A method and primer pair for quantitatively detecting different levels of classical swine fever antibodies

Abstract

The invention discloses a method and primer pair for quantitatively detecting different levels of classical swine fever antibodies. The invention uses Oligo 6.0 software and MethyPrimer online software to design self-designed primers, uses Bisulfite sequencing, and combines with a classical bisulfite sequencing method for verification, so that different classical swine fever antibody levels can be quickly, accurately and efficiently determined. The primer pair provided by the invention is a primer pair composed of DNAs shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10 in a sequence table. The method provided by the invention is to perform PCR amplification on genomic DNA of a pig to be tested treated with bisulfite by using the primer pair, and then judge whether a CD4 gene promoter region of the pig to be tested is highly methylated or moderately methylated according to a pyrophosphate sequencing result, and the classical swine fever antibody level of a highly methylated pig is higher than that of a moderately methylated pig. The classical swine fever antibody level is detected by using a test paper. The method provided by the invention is convenient to use, accurate and highly sensitive, and can be used for detecting the level of classical swine fever antibodies in Landrace and Large White pigs.

Description

Technical Field

[0001] The invention relates to the fields of epigenetics and molecular immunology, and in particular to a method and a primer pair for quantitatively detecting different levels of swine fever antibodies. Background Art

[0002] my country is one of the countries with the largest number of pigs in the world, and pig farming occupies a pivotal position in my country's animal husbandry. However, in actual production, Classic Swine Fever Virus can infect pigs by single infection or mixed infection with multiple viruses such as PRRSV, SIV and PRV. These viruses usually target lymphocytes, macrophages, endothelial cells and some tissue epithelial cells with immune functions, destroying the immune function of host cells. Because viruses such as Classic Swine Fever usually cause immunosuppression in the body, it is manifested as inducing apoptosis of tissue intrinsic cells and lymphocytes, inhibiting the production of type I interferon, inducing leukopenia, and ultimately causing immunosuppression in infected pigs. In this case, on the one hand, pigs are in a sub-healthy state, grow slowly or even become ill, and eventually lead to the collapse of the immune system; on the other hand, on the basis of tissue degeneration caused by infection, the exudate includes natural killer cells (NK cells), monocytes and specific T cells, which form a network system of signals and effects to comprehensively inhibit the damage caused by viral infection. The fundamental reason is that viral infection causes a series of changes in host cell gene expression. Some of these changes are conducive to the reproduction of pathogens, while others are the host's initiation of pathogen elimination.

[0003] How to quantitatively detect whether swine fever is successfully immunized will become the top priority in this research field. The most researched immunity to swine fever is related to immune genes such as cytokines and cell surface receptors, interferon response factors that are essential for innate resistance to infection, and genes related to T cell differentiation. The pathogenesis of swine fever was studied using expression profiling methods and was related to the host's cellular response, and some gene expression levels were found to be related to viral stimulation. However, gene expression is controlled by complex gene networks and environmental factors, including viral virulence, feeding conditions, stress, etc., which brings great interference and difficulty to the monitoring of antibody levels after swine fever immunization. Therefore, studying complex immune mechanisms only from a genetic perspective is subject to certain limitations. Scientists are expected to find swine fever immunity-related genes through the study of the virus-body-environment interaction mechanism, and stably distinguish the antibody levels after swine fever immunization through DNA methylation and molecular markers, thereby laying a theoretical foundation for epigenetic treatment and prevention of swine fever.

[0004] The sequencing technology of DNA methylation mainly includes Bisulfite sequencing, Bsulfite clone sequencing and pyrophosphate sequencing. Bisulfite sequencing is combined with clone sequencing to analyze DNA methylation differential sites. Through a pre-designed reaction system, one or more methylation sites can be quickly and accurately quantified in one test, and the Bisulfite sequencing results can reflect the methylation status of individual cells. The results of Bisulfite sequencing are presented in the form of sequences, and the trend is relatively consistent with the results of clone sequencing. It is currently a relatively fast and convenient research method for detecting DNA methylation of target genes. In addition, this technology can also detect SNP mutations at methylation sites. In humans, more than 10% of the common SNPs of different alleles overlap with differentially methylated regions. Therefore, DNA methylation differences may be affected by genetic differences between animals. Since DNA methylation is cell-specific, special attention should be paid to cell populations when studying immune responses. For example, there are certain differences in the DNA methylation status of whole blood cells and lymphocytes. The use of blood can reflect the immune status of the entire group of immune cells. It is more convenient and reliable to use whole blood to study the immunity of pigs in animal husbandry production. The stable, reliable and easy-to-operate Bisulfite sequencing method is used to quantify DNA methylation levels for distinguishing antibody levels for classical swine fever immunity, which is of great significance for the research on classical swine fever immunity in production.

[0005] The porcine CD4 gene is located on chromosome 5 (Genebank accession number: NC_010447; NCBI: Gene ID_404704), with a total length of about 26Kb (including 11 exons and 10 introns), encoding the leukocyte surface antigen CD4 protein. Leukocytes are a type of nucleated cells. Common leukocytes in normal pig blood mainly include neutrophils, lymphocytes and monocytes. The initial resistance encountered by viruses invading the body comes from leukocytes, so leukocytes are the body's defense cells. Lymphocytes are a type of cell with specific immune functions, which can be further divided into T lymphocytes and B lymphocytes, which perform cellular immune functions and humoral immune functions, respectively. Mature T lymphocytes are divided into CD4+ helper T cells (including CD4 differentiation antigens) and CD8+ anti-toxic T cells (including CD8 differentiation antigens) according to their surface antigen markers and functions. The main function of CD4+T cells is to enhance and expand the immune response process, activate macrophages, activate humoral immunity and promote humoral immunity through the secretion of lymphokines. CD4 differentiation antigens exist on the surface of mature CD4+T cells and participate in immune responses. Pigs infected with classical swine fever virus show a dramatic change in the ratio of CD4+T cells. In addition, the ratio of CD4+ / CD8+ is much higher in uninfected pigs than in infected pigs. In recent years, it has been found that epigenetic modification of the CD4 gene has a significant effect on the expression of T cells in the immune process. For example, epigenetic inhibition of the CD4 gene affects the immune response. Our previous studies have found that the CD4 gene is an important candidate gene related to viral response, but there is currently no report on the effect of the DNA methylation level of the pig CD4 gene on the level of classical swine fever immune antibodies. Summary of the invention

[0006] The purpose of the present invention is to provide a method and primer pair for quantitatively detecting different levels of classical swine fever antibodies. By successfully marking DNA methylation molecules, the effect of the DNA methylation level of the pig CD4 gene on the level of classical swine fever immune antibodies can be studied, which is of great significance. At the same time, the method provided by the present invention is easy to use and has high sensitivity, and can be used for the detection of classical swine fever antibody levels in large white and landrace pigs and the identification of immune effects. The present invention provides a new method for detecting classical swine fever antibody levels, which has extremely significant application value and social benefits.

[0007] A primer pair for quantitatively detecting different levels of classical swine fever antibodies, wherein a reagent for quantitatively detecting pigs with different classical swine fever antibody levels is prepared by the primer pair: the reagent comprises a primer pair consisting of a DNA shown in SEQ ID N0.5 and a DNA shown in SEQ ID N0.6, a primer pair consisting of a DNA shown in SEQ ID N0.7 and a DNA shown in SEQ ID N0.8, and a quantitative detection primer pair consisting of a DNA shown in SEQ ID N0.9 and a cDNA shown in SEQ ID N0.10.

[0008] The primer pairs composed of the DNA shown in SEQ ID N0.5 and the DNA shown in SEQ ID N0.6, the primer pairs composed of the DNA shown in SEQ ID N0.7 and the DNA shown in SEQ ID N0.8, and the quantitative detection primer pairs composed of the DNA shown in SEQ ID N0.9 and the cDNA shown in SEQ ID N0.10 can be used to prepare a kit for quantitatively detecting pigs with different levels of classical swine fever antibodies.

[0009] The primer pairs consisting of the DNA shown in SEQ ID N0.5 and the DNA shown in SEQ ID N0.6, the primer pairs consisting of the DNA shown in SEQ ID N0.7 and the DNA shown in SEQ ID N0.8, and the quantitative detection primer pairs consisting of the DNA shown in SEQ ID N0.9 and the cDNA shown in SEQ ID N0.10 can be used to quantitatively detect the DNA methylation levels of different classical swine fever antibodies.

[0010] A method for quantitatively detecting different levels of classical swine fever antibodies comprises the following steps: detecting the methylation degree of the CpG (and Y) position in the DNA shown in sequence 2 in the sequence table in chromosome 5 of the tested pig, determining whether the methylation level of the tested pig is high methylation or moderate methylation, and the classical swine fever antibody level of the pig with low methylation level is lower than the classical swine fever antibody level of the pig with high methylation level.

[0011] It is clear to those skilled in the art that there are many analytical methods that can be used to detect whether DNA methylation modification exists in the CD4 gene promoter region sequence. These techniques include (but are not limited to): DNA sequencing, DNA chip, methylase-specific PCR, PCR-RFLP, quantitative PCR, etc.;

[0012] The step of determining whether the CD4 gene promoter region in the peripheral blood of the pig to be tested is highly methylated or moderately methylated and the degree of methylation may specifically include the following steps:

[0013] (1) Extracting genomic DNA from the blood and lymphocytes of the pig to be tested;

[0014] (2) bisulfite treatment of genomic DNA;

[0015] (3) using the genomic DNA as a template, performing PCR amplification using a primer pair consisting of the DNA shown in SEQ ID No. 3 and the DNA shown in SEQ ID No. 8 to obtain a PCR amplification product;

[0016] (4) using the bisulfite-treated genomic DNA as a template, performing hot-start PCR amplification using a primer pair consisting of the DNA shown in SEQ ID No. 2 and the DNA shown in SEQ ID No. 6 in the sequence listing to obtain a PCR amplification product;

[0017] (5) Quantitative detection of DNA methylation. The hot-start PCR amplification product was cloned and sequenced using an ABI 377 automatic sequencer. SEQ ID No. 6 can quantitatively detect the methylation degree of 9 CpG loci (1st to 9th CpG), and SEQ ID No. 8 can detect the SNP genotype at the 2nd CpG locus;

[0018] (6) extracting RNA from the blood of the pig to be tested and reverse transcribing it into cDNA;

[0019] (7) Using cDNA as a template, fluorescent quantitative PCR amplification was performed using a primer pair consisting of the cDNA shown in SEQ ID No. 4 and the cDNA shown in SEQ ID No. 10 to obtain the Ct value of the PCR product.

[0020] Preferably, the pigs are Large White pigs and Landrace pigs.

[0021] Preferably, the pigs to be tested may be pigs with high DNA methylation-modified CD4 genes and pigs with moderate DNA methylation-modified CD4 genes.

[0022] Preferably, the method for detecting the level of classical swine fever antibodies in the present invention is to use pigs immunized with low levels of classical swine fever antibodies of CD4 genes with moderate DNA methylation modification identified by the method described above.

[0023] Preferably, SEQ ID NO.2 shows a DNA sequence after bisulfite conversion.

[0024] The DNA shown in SEQ ID NO.2 is used to prepare a kit for quantitatively detecting different levels of classical swine fever antibodies.

[0025] The beneficial effects of the present invention are as follows: the present invention targets 9 CpG loci in the CD4 gene promoter region of peripheral blood and lymphocytes PBMCs in Chinese Large White and Landrace pigs, and for the first time finds that the second site G / A SNP mutation and its DNA methylation level are significantly different between high classical swine fever antibody immunity level and low classical swine fever antibody immunity level and extremely significantly affect the gene expression (p<0.01), wherein Large White pigs with low classical swine fever antibody levels show significant CD4 hypomethylation and high expression, and appear as GA heterozygotes. The method provided by the present invention is easy to use and highly sensitive, and can be used for Large White and Landrace pigs to detect classical swine fever antibody levels and identify immune effects. The present invention provides a new method for detecting classical swine fever antibody levels, which has extremely significant application value and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Extraction of partial pig DNA

[0027] A DNA extraction from anticoagulated blood; DNA extraction from B lymphocytes and PBMCs; C RNA extraction from anticoagulated blood. M: DL2000 molecular weight marker; 1-7: test samples 1-7.

[0028] Figure 2 Effect of PCR Amplification of Porcine CD4 Gene

[0029] A: 365 bp PCR fragment used for re-pressed sulfate cloning sequencing; B: 224 bp PCR fragment used for SNP sequencing; M: DL600 molecular weight marker; 1-7: test samples No. 1-7; 8: negative control.

[0030] Figure 3 Bisulfite individual sequencing and SNP sequencing results of Large White pig CD4 gene.

[0031] A Peripheral blood CD4 methylation sequencing; B PBMCs CD4 methylation sequencing; C Peripheral blood CD4 SNP sequencing results; DPBMCs CD4 SNP sequencing results.

[0032] Figure 4 PCR electrophoresis identification of recombinant plasmid

[0033] A circles represent randomly picked clones; B recombinant plasmid PCR.

[0034] Figure 5 Methylation clone sequencing of GA heterozygotes in Large White pigs.

[0035] A. DNA methylation level of peripheral blood CD4 after sequencing; B. DNA methylation level of PBMCs CD4 after sequencing.

[0036] Figure 6 Comparison of CD4 DNA methylation levels in Large White and Landrace pigs with different levels of classical swine fever antibody immunization.

[0037] A. Methylation level of low classical swine fever antibody in Large White pigs; B. Methylation level of high classical swine fever antibody in Large White pigs; C. Methylation level of low classical swine fever antibody in Landrace pigs; D. Methylation level of high classical swine fever antibody in Landrace pigs.

[0038] Figure 7 Detection of CD4 gene expression in peripheral blood at different levels of classical swine fever antibodies. DETAILED DESCRIPTION

[0039] The following examples are provided for a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified. The % in the following examples are by mass percentage unless otherwise specified. The quantitative tests in the following examples were repeated three times, and the results were averaged.

[0040] Example 1: Optimization of DNA extraction from blood and PBMCs

[0041] 1. DNA Extraction from Anticoagulated Blood

[0042] The Wizard Genomic DNA Purification Kit (Promega, Beijing agent) was used to optimize the method for extracting anticoagulated blood genomic DNA. The blood sample was mixed, 300 μL of whole blood was taken, 900 μL of cell lysis solution was added, and the mixture was mixed by inversion. The red blood cells were lysed at room temperature for 10 min, and the vortex was vortexed for 5-10 s, 14000 rpm, and centrifuged for 20 s. The supernatant was removed and the white precipitate was retained (if it is a frozen blood sample, the above steps were repeated until the precipitate turns white). The white precipitate was resuspended by vortexing for 10-15 s, 300 μL of nuclear lysis solution was added to the resuspended cells, and the cells were lysed by pipetting with a pipette. The solution became viscous (if cell clumps were visible after mixing, incubate at 37°C for 1 h), 1.5 μL of RNase solution was added to the nuclear lysis solution to remove RNA contamination, incubate at 37°C for 15 min, and cool to room temperature. Detection was performed by 1% agarose gel electrophoresis.

[0043] 2. Isolation of porcine anticoagulant lymphocytes PBMCs

[0044] 1ml fresh blood (K3EDTA anticoagulation), add 1640 serum-free culture medium or 1×PBS reagent in a 1:1 ratio; add porcine lymphocyte separation solution (Ficoll separation solution, TBD company) in advance in a 15ml / 50ml centrifuge tube. Centrifuge at room temperature, 2000rpm, use a flat-bottom centrifuge for 25min. The cells are divided into four layers, from top to bottom: plasma and tissue homogenate layer, ring milky white lymphocyte layer, transparent separation liquid layer, red blood cell layer) Carefully remove the white blood cell layer, resuspend in 4ml PBS, mix well, 1500rpm, centrifuge for 10min, repeat the previous step of washing 2-3 times, and collect the cells for DNA extraction.

[0045] 3. DNA extraction from porcine PBMCs

[0046] The Wizard Genomic DNA Purification Kit (Promega, Beijing agent) was used to optimize the method for extracting genomic DNA from PBMCs. The cells were washed with 200 μL PBS, centrifuged at 16,000 rpm for 10 min, and 600 μL nuclear lysis solution was added. The cells were repeatedly pipetted until the cell clumps disappeared. 1.5 μL RNase was incubated at 37°C for 15 min, and then 200 μL protein precipitation solution was added. The cells were shaken vigorously for 20 s, cooled on ice for 5 min, and centrifuged at 16,000 rpm for 4 min to form a white dense protein precipitate. Take a clean 1.5ml centrifuge tube, add 600μL isopropanol, carefully take the supernatant and add isopropanol, mix by inversion until white linear DNA forms a precipitate, centrifuge at 16000rpm for 1min, remove the supernatant, add 600μL 70% ethanol, invert several times to wash the DNA precipitate, centrifuge at 16000rpm for 1min, gently aspirate the ethanol (avoid aspirating the precipitate), dry naturally for 10-15min, dissolve the DNA with 100μL DNAhydration, incubate at 65℃ for 1h or 4℃ overnight. Detect by 1% agarose gel electrophoresis.

[0047] Example 2: Optimizing PCR reaction

[0048] 1. Bisulfite treatment of genomic DNA

[0049] The quantified DNA was treated with bisulfite using an optimized method using the EZ DNA Methylation-Gold Kit (Zymo research, Beijing Tianmo Technology Co., Ltd.). 130μL CT Conversion Reagent and 20μL DNA (1μg) were mixed in a PCR tube. After mixing and PCR thermal cycling, 600μL M-Binding buffer was added, and the tube was inverted to mix. The tube was centrifuged at 12000rpm for 30s and the waste liquid was discarded. 100μL M-Washbuffer was added to the column. The tube was centrifuged at 12000rpm for 30s and the waste liquid was discarded. 200μL M-Desulphonationbuffer was added to the column, and the tube was left at room temperature for 30min. The tube was centrifuged at 12000rpm for 30s and the waste liquid was discarded. 200μL M-Washbuffer was added to the column. The tube was centrifuged at 12000rpm for 30s and the waste liquid was discarded. 200μL M-Elutionbuffer was added to the column and transferred to a new centrifuge tube. The tube was left at room temperature for 20min. The tube was centrifuged at 12000rpm for 30s and the converted DNA was collected, diluted 1:5 and stored at -20℃.

[0050] 2. Hot-start PCR based on bisulfite sequencing

[0051] The PCR 50μL amplification system was: 2×Reaction Buffer 25μL, dNTP Mix 0.25μL, upstream and downstream primers 1μL each (10μmol / L), bisulfite-treated DNA template 6μL, Zymo Taq enzyme 0.4μL (Zymo research, Beijing Tianmo Technology Agent), DNase / RNase free water to 50μL. The reaction conditions were: 95℃ denaturation 10min; 94℃ 30s, 50-60℃ 40s, 72℃ 45s, 40 cycles; 72℃ extension 7min, 4℃ storage.

[0052] 3. PCR based on SNP sequencing

[0053] The PCR 50μL amplification system was: 2×TaqPCR MasterMix 25μL (Beijing Zhongke Tairui, RTC3104-03), upstream primer 1μL (10μmol / L), downstream primer 1μL (10μmol / L), genomic DNA template 4μL, DNase / RNasefree water to 50μL. The reaction conditions were: 94℃ denaturation for 5min; 94℃ 30s, 50-60℃ 30s, 72℃ 45s, 35 cycles; 72℃ extension for 5min, -20℃ storage in the dark.

[0054] IV. PCR amplification result detection

[0055] After amplification, 2 μL of PCR product was detected on a 2% agarose gel. A single band with a strong signal should be obtained without primer dimers or other non-specific bands.

[0056] Example 3: Bisulfite direct sequencing

[0057] 1. Bisulfite pooled sequencing and individual direct sequencing

[0058] The bisulfite-treated DNA diluted 1:5 was mixed in equal amounts, and the pool DNA was used as a template for temperature gradient hot-start PCR amplification to obtain the PCR product at the appropriate temperature. After the PCR product was tested by gel electrophoresis, it was tested by ABI 377 direct sequencing. Individual sequencing is to directly sequence the individual DNA after the appropriate temperature amplification in the previous step (Beijing Tianyi Huiyuan).

[0059] 2. Bisulfite cloning sequencing

[0060] (1) Gel excision and recovery of amplified DNA

[0061] (2) Connection

[0062] Add the recovered target fragment and pGM-T vector into the reaction tube and mix well, then place in a 15°C water bath for overnight connection;

[0063] (3) Transformation of recombinant plasmid

[0064] Under completely sterile conditions, take out a tube of 100μL fresh DH5α E. coli competent bacteria from the -80℃ refrigerator and melt it by hand; add 5μL of the linked product to each competent cell, mix gently, and let it stand on ice for 30min; heat activate in a 42℃ water bath for 90s; quickly transfer the EP tube to ice to quickly cool the cells for 1-2min; on a sterile operating table, add 600μL LB liquid culture medium (without AMP) to the EP tube to revive the bacteria (200rpm, 50min, 37℃); take 100μL of the revived bacterial solution and plate it on the LA plate culture medium. Culture it in a 37℃ incubator overnight; the next day, observe the growth of the culture plate colonies, and randomly pick 8 to 10 colonies to streak culture on another LA solid culture medium (8 to 12h).

[0065] (4) Identification of recombinant plasmid

[0066] Bacterial PCR, the original PCR amplification reaction is the same except that the DNA is replaced with bacterial solution;

[0067] After PCR, the DNA was sent to Beijing Tianyi Huiyuan Company for sequencing.

[0068] Example 4: Determination of RNA expression in pig anticoagulant blood

[0069] 1. RNA extraction from anticoagulated blood

[0070] Fresh anticoagulated blood was mixed with cell lysis solution (RL) at a ratio of 1:3 to fully lyse the cells; 0.2 ml of chloroform was added to every 1 ml of RL, and the cells were shaken vigorously for 15 seconds and incubated at room temperature for 3 minutes. Centrifuge at 4°C, 12,000 rpm for 10 minutes, and the sample was separated into 3 layers; the upper aqueous phase was taken, and the volume was 60% of the RL, that is, 0.6 ml was taken into a new centrifuge tube, and 1 volume of 70% ethanol was added, and the mixture was mixed by inversion, and precipitation may occur; the solution and precipitation were transferred to an adsorption column (Beijing Biotech Biotechnology Co., Ltd., RP1202), and centrifuged at 10,000 rpm for 45 seconds, and the waste liquid was discarded. Add 50 μL of deproteinizing solution RE (Biotech), centrifuge at 12000rpm for 45s, and discard the waste liquid; add 700 μL of rinsing solution RW (DEPC water with 70% alcohol), centrifuge at 12000rpm for 60s, and discard the waste liquid; add 500 μL of rinsing solution RW, centrifuge at 12000rpm for 60s, and discard the waste liquid; centrifuge at 12000rpm for 2min, and idle; place the adsorption column in an RNase-free centrifuge tube, add 30 μL of RNase-free water, incubate at 65℃ for 10min, leave at room temperature for 2min, and centrifuge at 12000rpm for 1min to collect blood RNA.

[0071] 2. Anticoagulated RNA reverse transcription into cDNA

[0072] Use oligo (dT) to initiate the synthesis of the first-strand cDNA. Starting template amount: 2 μg total RNA, 1 μL control RNA (50 ng / μl). Before use, shake each component in the kit and centrifuge briefly; add the following components to a 0.2 ml sterile PCR tube: Prepare RNA / primer mixture, as shown in Table 1:

[0073] Table 1 RNA / primer mixed reaction

[0074] RNA / primer mix composition Volume (μL) 10mM dNTP Mix 1 Oligo(dT)12-18 1 DEPC water 1 RNA n <![CDATA[Make up to ddH2O]]> 10

[0075] Incubate the above mixture at 65℃ for 5min and place on ice for at least 1min. Prepare 2× reaction mixture in a new sterile PCR tube as shown in Table 2:

[0076] Table 2 2× reaction mixed reaction

[0077] 2× Reaction Mixture Composition Volume (μL) 10×RT buffer 2 <![CDATA[25mM MgCl2]]> 4 0.1M DTT 2 Recombinant RNase inhibitor (40U / μL) 1 Total volume 9

[0078] Add 9 μl of 2× reaction mixture to the mixture in the previous step, mix gently and centrifuge briefly, incubate at 42℃ for 2 min; add 1 μl of GoldScript RT to each tube, add 1 μl of DEPC water to the no RT control, incubate at 42℃ for 50 min; incubate at 70℃ for 15 min, terminate the reaction, cool on ice; centrifuge briefly to collect the reaction; add 1 μl of RNaseH to each tube, incubate at 37℃ for 20 min; store at -20℃ or perform fluorescence quantitative PCR reaction. Dilute cDNA 1:50 before fluorescence quantitative PCR.

[0079] 3. Reverse transcription PCR of cDNA

[0080] The PCR 50μL amplification system was: 2×Taq PCR Master Mix 25μL (Beijing Zhongke Tairui, RTC3104-03), upstream primer 1μL (10μmol / L), downstream primer 1μL (10μmol / L), genomic DNA template 4μL, DNase / RNasefree water to 50μL. The reaction conditions were: 94℃ denaturation for 5min; 94℃ 30s, 60℃ 30s, 72℃ 45s, 35 cycles; 72℃ extension for 5min, -20℃ storage in the dark.

[0081] IV. CD4 Real-time PCR Primer Design and Real-time PCR Reaction Conditions

[0082] Oligo6.0 software was used to design primers for target genes and housekeeping genes. The sequences were synthesized by Shanghai Yingjun Company. The sequences of the primers are shown in Table 3. Each q-PCR reaction used the same procedure: incubation at 95°C for 10 minutes, amplification for 45 cycles (95°C for 10s, 60°C for 10s and 72°C for 10s), and high temperature reduction and cooling were used for the melting curve. The intersection point (Cp value) was obtained using the secondary derivative maximum analysis tool in Roche 480 software. RT-PCR technology combined with the 2-ΔΔCt method was used to calculate the relative expression of the CD4 gene. The reaction conditions are shown in Table 3:

[0083] Table 3 Real time PCR reaction system

[0084] Real time PCR reaction system Volume (μL) Master Mix 10 Forward Primer 1 Reverse Primer: 1 Diluted cDNA: 2 <![CDATA[Make up to ddH2O]]> 20

[0085] Example 5: Classical Swine Fever Antibody

[0086] Detection of CD4 DNA methylation

[0087] 1. Gel electrophoresis detection of DNA extracted from porcine K3EDTA anticoagulated blood

[0088] from Figure 1A shows that 10-20 μg DNA was extracted from anticoagulated blood. The OD A260 / A280 values ​​were between 1.80 and 2.0, and the OD A260 / A230 values ​​were between 1.50 and 1.8, respectively, indicating that the protein removal effect was very good, and there was no residual RNA and organic solvents such as ethanol. This provides a basis for subsequent research on DNA methylation. Figure 1 B shows that the DNA extracted from lymphocyte PBMCs has a high concentration and is free of exogenous RNA contamination.

[0089] from Figure 1 C shows that the concentration of RNA extracted from anticoagulated blood is qualified at 200-300 ng / μL, the OD A260 / A280 values ​​are between 1.80-2.0, and the OD A260 / A230 values ​​are between 1.30-1.5, indicating that there is no residual DNA and organic solvents such as ethanol. RNA is basically not degraded, providing a basis for subsequent research on RNA expression.

[0090] 2. Agarose gel detection of porcine CD4 gene PCR products for bisulfite sequencing and clone sequencing

[0091] Figure 2 The CD4 gene is shown in the amplification results for bisulfite sequencing and pyrophosphate sequencing, and the electrophoresis bands are bright and neat, with no non-specific bands, no primer dimers, and no contamination, indicating that the amplification effect is good and methylation sequencing analysis can be performed. The concentration of the PCR product used for methylation sequencing is lower than that of the PCR product used for SNP sequencing under the amplification conditions of 40 cycles.

[0092] 3. Results of bisulfite sequencing and SNP sequencing of porcine CD4 gene

[0093] In order to preliminarily detect whether the CpG loci in blood and lymphocytes are methylated, the present invention first performs hybrid sequencing on the PCR products. Figure 3 Shown is bisulfite sequencing of the porcine CD4 gene in blood and lymphocytes from one individual ( Figure 3 A, 3B) and SNP sequencing of the same locus ( Figure 3 C, 3D) Results: A total of 9 CpG loci were detected. Figure 3 A and 3B show two CpG sequencing loci, and the red and blue double peaks represent differentially methylated loci. The black arrow shows the methylation degree of the first CpG locus, which is 58.1% (peripheral blood) and 79.8% (lymphocytes) on average; the red arrow shows the SNP locus, and the green and black double peaks represent heterozygotes of the G / A mutation. Figure 3 C. The red color in 3D shows that this individual is confirmed to be a heterozygote based on genomic DNA sequencing.

[0094] IV. Clone selection and PCR results of recombinant plasmids

[0095] The purified PCR product was cloned and transformed into a recombinant plasmid, and then PCR electrophoresis was performed to detect the positive bands. The results were as follows: Figure 4 shown. Figure 4 A, 4B black circles represent 8 clones after randomly picking CD4 gene clones from peripheral blood and PBMCs for recombinant plasmid transformation and sequencing; Figure 4 C represents the recombinant plasmid for PCR electrophoresis identification, among which 7 bands, 1, 3, 7, 8, 9, 10, and 11, are the target bands for subsequent cloning and sequencing analysis.

[0096] 5. Methylation clone sequencing of pig GA heterozygotes

[0097] Cloning and sequencing of bisulfite PCR products is a traditional method for detecting DNA methylation. We further used this method to determine whether DNA methylation modification had indeed occurred at the above CpG loci. Figure 5 The results of bisulfite cloning sequencing of CD4 gene in pig peripheral blood and PBMCs are shown. A total of 9 CpG loci were detected. The average DNA methylation levels of the 9 YG loci in the two cells were 91.7% and 94.4%, respectively. The results showed that the average levels of differentially methylated loci in the promoter region of CD4 gene in the two cells were relatively consistent. However, there was a significant difference in the second CpG locus ( Figure 5 As indicated by the red arrows, the average is 15% and 31.5%). Therefore, peripheral blood whole blood cells will be selected to analyze the methylation degree of the second differentially methylated locus.

[0098] VI. Comparison of CD4 DNA methylation levels in large white and landrace pigs with different levels of classical swine fever antibodies

[0099] In order to analyze the differences in CD4 gene promoter methylation and SNP differences between Large White pigs and Landrace pigs at different levels of classical swine fever antibodies, 5 Large White pigs GA heterozygous individuals and 5 Landrace pigs GG homozygous individuals were selected to analyze the differences in CD4 methylation. It was observed in both breeds that the CD4 methylation level in the high antibody group was higher than that in the low antibody group. The overall methylation level of the CD4 gene in Landrace pigs was higher than that in Large White pigs. In the high antibody group, the methylation level of this CpG locus in Landrace pigs (97.3%) was approximately 20% higher than that in Large White pigs (72.4%). In the low antibody group, the CD4 methylation level at the same locus in Landrace pigs (82.1%) was higher than that in Large White pigs (67.9%) ( Figure 6 ), the differential methylation level was approximately 15%.

[0100] VII. Detection of CD4 gene expression in peripheral blood at different levels of swine fever antibodies

[0101] We continued to analyze the CD4 DNA methylation level in the peripheral blood of Large White and Landrace pigs, and initially found that the methylation level of a CpG locus in Landrace pigs was slightly higher than that of Large White pigs by 20%, and found a G / A mutation at this YG locus in the Landrace pig breed, which may be one of the important reasons for the downregulation of CD4 gene expression. The DNA methylation regulation mechanism of the weakened resistance of Landrace pigs was further confirmed before and after immunization with attenuated swine fever vaccine. Figure 7 The results show that the expression level of CD4 in the high antibody group of Large White pigs was reduced, and the difference was extremely significant compared with the low antibody group, P = 1.5402E-07 ( Figure 7 ). In the high antibody group, the DNA methylation level of GG large white pigs increased by about 15% compared with the low antibody group. This preliminarily indicates that the CD4 promoter and SNP affect the expression of the CD4 gene. The attenuated swine fever virus may have caused the body's own immunosuppression, resulting in a transient inhibition of CD4 gene expression, which in turn caused the inactivation of CD4+T cell function. Sequence Listing <110> Inner Mongolia Agricultural University Vocational and Technical College <120> A method and primer pair for quantitatively detecting different levels of classical swine fever antibodies <130> 2020 <160> 3 <170> SIPOSequenceListing 1.0 <210> 1 <211> 361 <212> DNA <213> Pig <400> 1 tatggaggtt cccaggctag gggtctgatt ggagctatag atgctggctt acaccagagc 60 cacagcaaag ccagatctga gctgcgtctt caacctgcac cggttcactg caacgccgga 120 tccttaaccc actgagcgag ggcagggatc gaacccgaaa cctcatggtt cctaggtgga 180 ttcgttaatc actgagccac aacgggaact cccaaaaaaa aattttttaa ggggtttgtt 240 cagctcctaa ctccaactat gaacacaagt gtttctgttg aggcaaccat tatactttat 300 ttgtagccaa agtgctttat gctacttctc atttcataac agagtgtact caagagttga 360 g 361 <210> 2 <211> 361 <212> DNA <213> CD4 gene sequence after bisulfite transformation <400> 2 tatggaggtt tttaggttag gggtttgatt ggagttatag atgttggttt atattagagt 60 tatagtaaag ttagatttga gttgygtttt taatttgtat yggtttattg taaygtygga 120 tttttaattt attgagygag ggtagggaty gaattygaaa ttttatggtt tttaggtgga 180 ttygttaatt attgagttat aaygggaatt tttaaaaaaa aattttttaa ggggtttgtt 240 tagtttttaa ttttaattat gaatataagt gtttttgttg aggtaattat tatattttat 300 ttgtagttaa agtgttttat gttatttttt attttataat agagtgtatt taagagttga 360 g 361 <210> 3 <211> 224 <212> DNA <213> SNP sequencing sequence <400> 3 cttacaccag agccacagca aagccagatc tgagctgcgt cttcaacctg caccgcggtt 60 cactgcaacg ccggatcctt aacccactga gcgagggcag ggatcgaacc cgaaacctca 120 tggttcctag gtggattcgt taatcactga gccacaacgg gaactcccaa aaaaaatttt 180 tttaaggggt ttgttcagct cctaactcca actatgaaca caag 224

Claims

1. A method for quantitatively detecting different levels of classical swine fever antibodies, characterized in that: Determining whether the CD4 gene promoter region in the peripheral blood of the pig to be tested is highly methylated or moderately methylated and the degree of methylation may specifically include the following steps: (1) extracting genomic DNA from the blood of the pig to be tested; (2) bisulfite treatment of genomic DNA; (3) using the genomic DNA as a template, performing PCR amplification using a primer pair consisting of the DNA shown in SEQ ID No. 3 and the DNA shown in SEQ ID No. 8 to obtain a PCR amplification product; (4) using the bisulfite-treated genomic DNA as a template, performing hot-start PCR amplification using a primer pair consisting of the DNA shown in SEQ ID No. 2 and the DNA shown in SEQ ID No. 6 to obtain a PCR amplification product; (5) Quantitative detection of DNA methylation: The hot-start PCR amplification product was cloned and sequenced using an ABI 377 automatic sequencer. SEQ ID No. 6 can quantitatively detect the methylation degree of 9 CpG loci, and SEQ ID No. 8 can detect the SNP genotype at the second CpG locus; (6) extracting RNA from the blood of the pig to be tested and reverse transcribing it into cDNA; (7) Using cDNA as a template, performing fluorescent quantitative PCR amplification with a primer pair consisting of the cDNA shown in SEQ ID No. 4 and the cDNA shown in SEQ ID No. 10 to obtain the Ct value of the PCR product; SEQ ID NO.8 5'-CTTACACCAGAGCCACAGC-3' 5'-CTTGTGTTCATAGTTGGAGTT-3' SEQ ID NO.6 5'-TATGGAGGTTTTTAGGTTAGG-3' 5'-CTCAACTCTTAAATACACTCT-3' SEQ ID NO.10 5'-CCTTCTGTTTCTCGCTGGG-3' 5′-GGCAGGTCTTCTTCTCACTA-3′.

2. A method for quantitatively detecting different levels of classical swine fever antibodies according to claim 1, characterized in that: The pigs are specifically Large White pigs and Landrace pigs.

3. A method for quantitatively detecting different levels of classical swine fever antibodies according to claim 1, characterized in that: The pigs with moderate DNA methylation modification of CD4 gene identified by the above method are distinguished.

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