阅读说明：本技术 一种检测系统性红斑狼疮基因突变位点的方法 (Method for detecting mutation sites of systemic lupus erythematosus genes ) 是由 尹志华 叶志中 曾惠琼 陈新鹏 戴莉萍 于 2020-04-22 设计创作，主要内容包括：本发明属于分子生物学技术领域,公开了一种检测系统性红斑狼疮基因突变位点的方法,包括：针对待测序样本,制备系统性红斑狼疮基因测序反应体系；利用单管多重PCR扩增反应得到测序引物片段,利用单碱基延伸技术进行双向延伸,得到待检测的系统性红斑狼疮基因位点；通过核酸质谱分析待检测基因位点,精确测定目标DNA测序引物序列；将目标DNA测序引物序列锚定在待测模板链上,对待测模板链上的系统性红斑狼疮基因突变位点进行检测。本发明通过对测序反应体系的制备能够获得均匀分布的待测模板链和测序引物链,能够提高后续测序过程中的测序引物序列的锚定效率,进而提高测序效率和准确性,为个人临床用药提供安全指导的作用。(The invention belongs to the technical field of molecular biology, and discloses a method for detecting a mutation site of a systemic lupus erythematosus gene, which comprises the following steps: aiming at a sample to be sequenced, preparing a systemic lupus erythematosus gene sequencing reaction system; obtaining a sequencing primer fragment by using a single-tube multiplex PCR amplification reaction, and performing bidirectional extension by using a single-base extension technology to obtain a systemic lupus erythematosus gene locus to be detected; analyzing the gene locus to be detected by nucleic acid mass spectrometry, and accurately determining a target DNA sequencing primer sequence; anchoring a target DNA sequencing primer sequence on a template chain to be detected, and detecting the systemic lupus erythematosus gene mutation site on the template chain to be detected. According to the invention, the template chain to be detected and the sequencing primer chain which are uniformly distributed can be obtained by preparing the sequencing reaction system, the anchoring efficiency of the sequencing primer sequence in the subsequent sequencing process can be improved, the sequencing efficiency and accuracy are further improved, and the safety guidance effect is provided for the personal clinical medication.)

1. A method for detecting a mutation site of a systemic lupus erythematosus gene comprises the following steps:

extracting a sample to be sequenced, and preparing a systemic lupus erythematosus gene sequencing reaction system aiming at the extracted sample to be sequenced; the sequencing reaction system comprises a template chain to be tested and a sequencing primer chain, wherein one end of the template chain and one end of the sequencing primer chain are connected to the same solid phase carrier;

secondly, obtaining sequencing primer fragments by utilizing a single-tube multiplex PCR amplification reaction through a designed sequencing primer chain, performing bidirectional extension on the sequencing primer fragments by utilizing a single-base extension technology, adding a magnetic particle suspension into an extension product according to a certain proportion range, uniformly mixing the extension product and the magnetic particle suspension, and incubating for the first time;

placing the product of the first incubation on a magnetic frame, removing supernatant after the magnetic beads are completely adsorbed, cleaning the magnetic beads by using HPLC-aqueous clear liquid, resuspending the magnetic beads in a biotin coating solution for the second incubation, performing short-time centrifugation, and performing desalination and purification to obtain the gene locus of the systemic lupus erythematosus to be detected;

analyzing the systemic lupus erythematosus gene locus to be detected through nucleic acid mass spectrometry, and accurately determining a target DNA sequencing primer sequence;

anchoring the target DNA sequencing primer sequence on a template chain to be tested, and detecting the systemic lupus erythematosus gene mutation site to be sequenced on the template chain to be tested.

2. The method of claim 1, wherein in step one, the method for preparing the systemic lupus erythematosus gene sequencing reaction system comprises:

firstly, searching a DNA sequence of an exon of a systemic lupus erythematosus gene on Genebank, and looking up related literature data to determine a sequence position containing a mutation site of the systemic lupus erythematosus gene;

secondly, uploading a target gene sequence by using online Primer design software Primer Explorer V4, and designing a sequencing Primer chain of the systemic lupus erythematosus gene;

finally, designing a primer and a closed probe capable of forming a hairpin structure aiming at the mutation site.

3. The method of claim 1, wherein in step one, the system lupus erythematosus gene sequencing reaction system further comprises a probe;

the sequencing primer chain comprises a sequencing primer sequence and a sequencing connection sequence; the sequencing connection sequence is positioned at the fixed end of the sequencing primer chain, and the sequencing primer sequence is positioned at the free end of the sequencing primer chain and is used as a sequencing primer of the template chain to be detected; the sequencing primer comprises a wild-type primer and a mutation primer, and the probe comprises a wild-type probe and at least one mutation probe.

4. The method for detecting the mutation sites of the systemic lupus erythematosus gene as in claim 3, wherein the 5 'end of the probe sequence is provided with FAM fluorescent label, and the 3' end is provided with MGB or BHQ fluorescent label; the 5 'end of the wild type detection probe sequence is provided with a HEX fluorescent label, and the 3' end is provided with a MGB or BHQ fluorescent label.

5. The method according to claim 1, wherein in the second step, the reaction procedure of the single-tube multiplex PCR amplification comprises: 100-120 seconds at 90-95 ℃, 30-35 seconds at 90-92 ℃, 20-30 seconds at 50-56 ℃, 60-65 seconds at 70-72 ℃, 45-50 cycles, 5-7 minutes at 70-72 ℃ and 2-4 ℃ storage.

6. The method of claim 1, wherein in step two, the single base extension technique is performed by using a single base extension primer set to perform bidirectional extension on the target fragment.

7. The method for detecting the mutation site of the systemic lupus erythematosus gene according to claim 1, wherein the desalting and purifying method comprises the following steps:

1) adding a proper amount of Buffer DE-A solution into a centrifuge tube, turning upside down, mixing uniformly, and then putting into a water bath kettle at 80 ℃ for heating to fully melt the gel;

2) after the gel is completely melted, adding a Buffer DE-B solution with the volume 0.5 times that of the Buffer DE-A solution, and turning upside down and uniformly mixing to eliminate the generated floccules and ensure that a bright yellow solution is formed;

3) transferring the yellow solution obtained in the step 2) into a DNA preparation tube by using a pipette, placing the DNA preparation tube into a 2m1 centrifuge tube prepared in advance, centrifuging the solution at a high speed of 12000 Xg for 1 minute, and removing the filtrate;

4) the DNA preparation tube is put back into a 2m1 centrifuge tube, then 500 microliters of BufferW1 solution is added, the mixture is kept stand for 2 minutes at room temperature, and is centrifuged at 12000 Xg at a high speed for 1 minute, and the filtrate is discarded;

5) the DNA preparation tube is put back into a 2m1 centrifuge tube, 700 microliters of BufferW2 solution is added, high-speed centrifugation is carried out for 1 minute at 12000 Xg, and the filtrate is discarded;

6) repeat step 5) again to ensure complete desalination; and is centrifuged at 12000 Xg at high speed for 2 minutes;

7) the DNA preparation tube was placed in a new 1.5m1 centrifuge tube, 30. mu.l of Eluent Eluent heated in advance in a water bath was added to the center of the filtration membrane of the preparation tube, and the tube was left standing at room temperature for 10 minutes, and then centrifuged at 12000 Xg at high speed for 2 minutes to elute the DNA.

8. The method for detecting the mutation site of the systemic lupus erythematosus gene of claim 1, wherein in the fourth step, the nucleic acid mass spectrometry method comprises:

(1) spreading the resin for later use on a resin plate, and air-drying for 5-10 minutes;

(2) adding a sample to be detected into a reaction hole of a sample plate, supplementing 25ul ddHO, sealing by using a sealing film, and performing instant centrifugation at 3000 rpm;

(3) removing the sealing film, reversely buckling the sample plate on the resin plate obtained in the step (1) for fixing, then turning over the sample plate and the resin plate together to enable the air-dried resin to fall into the reaction hole of the sample plate, sealing by using the sealing film, and instantly centrifuging at 3000 r/min;

(4) the sample plate is placed on a rotator to rotate at a slow speed for 20 minutes, then is centrifuged at 3000 rpm for 5 minutes, and then is spotted on a machine to be detected by a nucleic acid mass spectrometer.

9. The method of claim 1, wherein in step four, the target DNA sequencing primer is a primer pair capable of specifically amplifying DNA fragments containing 3 sites of SNPs against 3 sites of Single Nucleotide Polymorphisms (SNPs) of A49G of CTLA4 gene and C677T of T-1772C, MTHFR gene.

10. The method of claim 1, wherein in step five, the template chain to be tested is designed for 3 SNPs of A49G of CTLA4 gene and C677T of T-1772C, MTHFR gene, and DNA sequencing primer capable of detecting the genotype of the 3 SNPs specifically is used.

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to a method for detecting a mutation site of a systemic lupus erythematosus gene.

Background

Currently, Systemic Lupus Erythematosus (SLE) is a common autoimmune disease, which is characterized by the production of multiple autoantibodies and the formation of immune complexes, eventually causing damage to almost all systems of the whole body such as lesions of the circulatory system, urinary system, digestive system and central nervous system due to immune response against itself. The etiology and pathogenesis of SLE are not completely understood, and the SLE is considered to belong to polygenic genetic diseases at present, and genetic factors and environmental factors are jointly involved in the occurrence and development processes of the SLE. SLE is well developed in women, especially women of childbearing age, and affects many important organs of the whole body, such as heart, brain, kidney, etc. It is estimated that there are currently over 100 million people in our country with SLE patients, with the average prevalence being second worldwide. A plurality of researches find that the disease has higher genetic tendency, and the risk of SLE of a subject can be evaluated in advance by detecting genetic loci closely related to SLE occurrence through molecular diagnosis, so as to prevent and treat SLE in advance.

Currently, two commonly used PCR-based gene mutation detection methods are ARMS-PCR and digital PCR. The ARMS-PCR technology is established on the basis of allele specific extension reaction, the extension reaction can be carried out only when the 3' terminal base of a certain allele specific primer is complementary with the base at the mutation site, and the upstream primer which is not completely matched with the template can not anneal and can not generate a PCR product. The biggest limitation of clinical application of ARMS-PCR is that the sensitivity is low, the ARMS-PCR sensitivity is about 1% generally, and the detection sensitivity of mutation lower than 1%, especially blood samples is insufficient, so that the judgment of a clinician on the curative effect of a targeted drug is limited.

Microdroplet digital PCR technology is the "single-molecule template PCR amplification" by distributing a standard PCR reaction into a large number of tiny reaction units, with or without one or more copies of the target molecule (DNA template) in each reaction unit. After amplification is finished, the initial copy number or concentration of the target molecule is obtained according to the Poisson distribution principle and the number and proportion of the positive microdroplets, and the absolute quantification technology of the nucleic acid molecule is realized. However, ddPCR is often used for gene mutation requiring deep optimization of probe sequences. Since the difference in Tm between the mutant and wild-type probes is to be highlighted, the sequence design is usually relatively short, and specially modified probes, such as MGBs or Locked Nucleic Acids (LNAs), are generally used. Moreover, the position of the mutation site in the probe sequence is not fixed, a large amount of experiment optimization is needed, the optimization time is long, the cost is high, the dynamic range of detection is small, and false positive is easy to generate. Therefore, the application of ARMS-PCR and digital PCR detection methods to the detection of the mutation sites of the systemic lupus erythematosus gene has great limitations.

Clinical research data at home and abroad show that the CTLA-4 gene-1722T/C site polymorphism is closely related to the occurrence of SLE, and can be used as an independent risk factor for the occurrence of various diseases such as leucoderma, breast cancer and the like. Meanwhile, homozygous variation of MTHFR gene 677TT is related to the pathogenesis of SLE, and is a risk factor of SLE. 3 SNPs of A49G on CTLA4 gene and C677T on T-1772C, MTHFR gene can be used as independent risk factors of SLE for gene detection. However, in the prior art, no method has been reported for gene detection using 3 SNPs of A49G in CTLA4 gene and C677T in T-1772C, MTHFR gene as independent risk factors for SLE.

Through the above analysis, the problems and defects of the prior art are as follows:

(1) the biggest limitation of clinical application of ARMS-PCR is that the sensitivity is low, the ARMS-PCR sensitivity is about 1% generally, and the detection sensitivity of mutation lower than 1%, especially blood samples is insufficient, so that the judgment of a clinician on the curative effect of a targeted drug is limited.

(2) While ddPCR is often used for gene mutation to deeply optimize probe sequences, the sequence design is usually relatively short due to the difference in Tm between the mutant and wild-type probes, and typically a specially modified probe such as MGB or Locked Nucleic Acid (LNA) is used. Moreover, the position of the mutation site in the probe sequence is not fixed, a large amount of experiment optimization is needed, the optimization time is long, the cost is high, the dynamic range of detection is small, and false positive is easy to generate.

(3) In the prior art, no method for gene detection by using 3 SNPs of A49G on CTLA4 gene and C677T on T-1772C, MTHFR gene as independent risk factors of SLE has been reported.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a method for detecting the mutation sites of systemic lupus erythematosus genes.

The invention is realized in such a way that the method for detecting the systemic lupus erythematosus gene mutation site comprises the following steps:

extracting a sample to be sequenced, and preparing a systemic lupus erythematosus gene sequencing reaction system aiming at the extracted sample to be sequenced; the sequencing reaction system comprises a template chain to be detected and a sequencing primer chain, wherein one end of the template chain and one end of the sequencing primer chain are connected to the same solid phase carrier.

Secondly, obtaining sequencing primer fragments by utilizing a single-tube multiplex PCR amplification reaction through a designed sequencing primer chain, performing bidirectional extension on the sequencing primer fragments by utilizing a single-base extension technology, adding a magnetic particle suspension into an extension product according to a certain proportion range, uniformly mixing the extension product and the magnetic particle suspension, and incubating for the first time;

and step three, placing the product of the first incubation on a magnetic frame, removing the supernatant after the magnetic beads are completely adsorbed, cleaning the magnetic beads by using HPLC-aqueous clear liquid, resuspending the magnetic beads in a biotin coating solution for the second incubation, performing short-time centrifugation, and performing desalination and purification to obtain the systemic lupus erythematosus gene locus to be detected.

Analyzing the systemic lupus erythematosus gene locus to be detected through nucleic acid mass spectrometry, and accurately determining a target DNA sequencing primer sequence.

Anchoring the target DNA sequencing primer sequence on a template chain to be tested, and detecting the systemic lupus erythematosus gene mutation site to be sequenced on the template chain to be tested.

Further, in the first step, the preparation method of the systemic lupus erythematosus gene sequencing reaction system comprises the following steps:

firstly, searching a DNA sequence of an exon of a systemic lupus erythematosus gene on Genebank, and looking up related literature data to determine a sequence position containing a mutation site of the systemic lupus erythematosus gene;

secondly, uploading a target gene sequence by using online Primer design software Primer Explorer V4, and designing a sequencing Primer chain of the systemic lupus erythematosus gene;

finally, designing a primer and a closed probe capable of forming a hairpin structure aiming at the mutation site.

Further, in the first step, the systemic lupus erythematosus gene sequencing reaction system further comprises a probe;

the sequencing primer chain comprises a sequencing primer sequence and a sequencing connection sequence; the sequencing connection sequence is positioned at the fixed end of the sequencing primer chain, and the sequencing primer sequence is positioned at the free end of the sequencing primer chain and is used as a sequencing primer of the template chain to be detected; the sequencing primer comprises a wild-type primer and a mutation primer, and the probe comprises a wild-type probe and at least one mutation probe.

Furthermore, the 5 'end of the probe sequence is provided with a FAM fluorescent label, and the 3' end is provided with a MGB or BHQ fluorescent label; the 5 'end of the wild type detection probe sequence is provided with a HEX fluorescent label, and the 3' end is provided with a MGB or BHQ fluorescent label.

Further, in the second step, the reaction procedure of the single-tube multiplex PCR amplification is as follows: 100-120 seconds at 90-95 ℃, 30-35 seconds at 90-92 ℃, 20-30 seconds at 50-56 ℃, 60-65 seconds at 70-72 ℃, 45-50 cycles, 5-7 minutes at 70-72 ℃ and 2-4 ℃ storage.

Further, in the second step, the single-base extension technology is to use a single-base extension primer set to perform bidirectional extension on the target fragment.

Further, in step three, the desalination purification method comprises:

1) adding a proper amount of Buffer DE-A solution into a centrifuge tube, turning upside down, mixing uniformly, and then putting into a water bath kettle at 80 ℃ for heating to fully melt the gel;

2) after the gel is completely melted, adding a Buffer DE-B solution with the volume 0.5 times that of the Buffer DE-A solution, and turning upside down and uniformly mixing to eliminate the generated floccules and ensure that a bright yellow solution is formed;

3) transferring the yellow solution obtained in the step 2) into a DNA preparation tube by using a pipette, placing the DNA preparation tube into a 2m1 centrifuge tube prepared in advance, centrifuging the solution at a high speed of 12000 Xg for 1 minute, and removing the filtrate;

4) the DNA preparation tube is put back into a 2m1 centrifuge tube, then 500 microliters of BufferW1 solution is added, the mixture is kept stand for 2 minutes at room temperature, and is centrifuged at 12000 Xg at a high speed for 1 minute, and the filtrate is discarded;

5) the DNA preparation tube is put back into a 2m1 centrifuge tube, 700 microliters of BufferW2 solution is added, high-speed centrifugation is carried out for 1 minute at 12000 Xg, and the filtrate is discarded;

6) repeat step 5) again to ensure complete desalination; and is centrifuged at 12000 Xg at high speed for 2 minutes;

7) the DNA preparation tube was placed in a new 1.5m1 centrifuge tube, 30. mu.l of Eluent Eluent heated in advance in a water bath was added to the center of the filtration membrane of the preparation tube, and the tube was left standing at room temperature for 10 minutes, and then centrifuged at 12000 Xg at high speed for 2 minutes to elute the DNA.

Further, in step four, the method for mass spectrometry of nucleic acid comprises:

(1) spreading the resin for later use on a resin plate, and air-drying for 5-10 minutes;

(2) adding a sample to be detected into a reaction hole of a sample plate, supplementing 25ul ddHO, sealing by using a sealing film, and performing instant centrifugation at 3000 rpm;

(3) removing the sealing film, reversely buckling the sample plate on the resin plate obtained in the step (1) for fixing, then turning over the sample plate and the resin plate together to enable the air-dried resin to fall into the reaction hole of the sample plate, sealing by using the sealing film, and instantly centrifuging at 3000 r/min;

(4) the sample plate is placed on a rotator to rotate at a slow speed for 20 minutes, then is centrifuged at 3000 rpm for 5 minutes, and then is spotted on a machine to be detected by a nucleic acid mass spectrometer.

Furthermore, in the fourth step, the target DNA sequencing primer is a primer pair which can specifically amplify DNA fragments containing 3 Single Nucleotide Polymorphisms (SNPs) sites of a49G on the CTLA4 gene and C677T on the T-1772C, MTHFR gene.

Further, in the fifth step, the template chain to be detected is designed aiming at 3 SNPs loci of A49G on CTLA4 gene and C677T on T-1772C, MTHFR gene, and DNA sequencing primer capable of specifically detecting the genotypes of the 3 SNPs loci by DNA sequencing technology.

By combining all the technical schemes, the invention has the advantages and positive effects that: according to the invention, the template chain to be tested and the sequencing primer sequence are fixed on the same solid phase carrier, and the template chain to be tested and the sequencing primer chain which are uniformly distributed can be obtained by preparing the sequencing reaction system, so that the anchoring efficiency of the sequencing primer sequence in the subsequent sequencing process is improved, and the sequencing efficiency is further improved. When a plurality of samples to be sequenced are sequenced in the same system, additional sequencing primers are not required to be added in the sequencing step, so that mutual interference caused by different sequencing primers is avoided, and the sequencing efficiency and accuracy are improved.

According to the invention, a sequencing primer fragment is obtained by utilizing a single-tube multiplex PCR amplification reaction through a designed sequencing primer chain, then a gene site to be detected is obtained by utilizing a single-base extension technology through bidirectional extension, and finally a target DNA sequence is accurately determined through nucleic acid mass spectrometry; the mutation site in the target DNA sequence can be accurately positioned, and the safety guidance effect is provided for the personal clinical medication.

Drawings

FIG. 1 is a flowchart of a method for detecting mutation sites of systemic lupus erythematosus genes according to an embodiment of the present invention.

FIG. 2 is a schematic diagram of an amplification curve for PCR amplification at different concentrations according to an embodiment of the present invention.

FIG. 3 is a schematic diagram of elution peaks of an amplification product obtained by nucleic acid mass spectrometry provided in an embodiment of the present invention.

FIG. 4 is a flowchart of a method for sequencing systemic lupus erythematosus.

FIG. 5 is a flow chart of a desalination and purification method provided by an embodiment of the invention.

FIG. 6 is a flow chart of a method for mass spectrometry of nucleic acids according to an embodiment of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Aiming at the problems in the prior art, the invention provides a method for detecting the mutation sites of the systemic lupus erythematosus gene, and the invention is described in detail with reference to the attached drawings.

As shown in fig. 1, the method for detecting mutation sites of systemic lupus erythematosus provided by the embodiment of the present invention includes the following steps:

s101, extracting a sample to be sequenced, and preparing a systemic lupus erythematosus gene sequencing reaction system aiming at the extracted sample to be sequenced; the sequencing reaction system comprises a template chain to be detected and a sequencing primer chain, wherein one end of the template chain and one end of the sequencing primer chain are connected to the same solid phase carrier.

S102, obtaining a sequencing primer fragment by utilizing a single-tube multiplex PCR amplification reaction through a designed sequencing primer chain, performing bidirectional extension on the sequencing primer fragment by utilizing a single-base extension technology, adding a magnetic particle suspension into an extension product according to a certain proportion range, uniformly mixing the extension product and the magnetic particle suspension, and incubating for the first time.

S103, placing the product of the first incubation on a magnetic frame, removing supernatant after the magnetic beads are completely adsorbed, cleaning the magnetic beads by using HPLC-water clear liquid, resuspending the magnetic beads in biotin coating liquid for the second incubation, performing short-time centrifugation, and performing desalination and purification to obtain the systemic lupus erythematosus gene locus to be detected.

S104, analyzing the systemic lupus erythematosus gene locus to be detected through nucleic acid mass spectrometry, and accurately determining a target DNA sequencing primer sequence.

S105, anchoring the target DNA sequencing primer sequence on a template chain to be tested, and detecting the systemic lupus erythematosus gene mutation site to be sequenced on the template chain to be tested.

As shown in fig. 4, in step S101, the method for preparing a systemic lupus erythematosus gene sequencing reaction system provided by the embodiment of the present invention includes:

s201, searching DNA sequences of exons of the systemic lupus erythematosus gene on Genebank, and determining sequence positions of mutation sites containing the systemic lupus erythematosus gene by referring to related literature data;

s202, uploading a target gene sequence by using online Primer design software Primer Explorer V4, and designing a sequencing Primer chain of the systemic lupus erythematosus gene;

s203, designing a primer and a blocking probe capable of forming a hairpin structure aiming at the mutation site.

In step S101, the system lupus erythematosus gene sequencing reaction system provided in the embodiment of the present invention further includes a probe; the sequencing primer chain comprises a sequencing primer sequence and a sequencing connection sequence; the sequencing connection sequence is positioned at the fixed end of the sequencing primer chain, and the sequencing primer sequence is positioned at the free end of the sequencing primer chain and is used as a sequencing primer of the template chain to be detected; the sequencing primer comprises a wild-type primer and a mutation primer, and the probe comprises a wild-type probe and at least one mutation probe.

The 5 'end of the probe sequence provided by the embodiment of the invention is provided with a FAM fluorescent label, and the 3' end is provided with a MGB or BHQ fluorescent label; the 5 'end of the wild type detection probe sequence is provided with a HEX fluorescent label, and the 3' end is provided with a MGB or BHQ fluorescent label.

In step S102, the reaction procedure of single-tube multiplex PCR amplification provided in the embodiment of the present invention is: 100-120 seconds at 90-95 ℃, 30-35 seconds at 90-92 ℃, 20-30 seconds at 50-56 ℃, 60-65 seconds at 70-72 ℃, 45-50 cycles, 5-7 minutes at 70-72 ℃ and 2-4 ℃ storage.

In step S102, the single-base extension technology provided by the embodiment of the present invention is to perform bidirectional extension on the target fragment by using a single-base extension primer set.

As shown in fig. 5, in step S103, the desalination and purification method provided by the embodiment of the present invention includes:

s301, adding a proper amount of Buffer DE-A solution into a centrifuge tube, turning upside down, mixing uniformly, and then putting into a water bath kettle at 80 ℃ for heating to fully melt the gel;

s302, after the gel is completely melted, adding a Buffer DE-B solution with the volume 0.5 times that of the Buffer DE-A solution, and turning upside down and uniformly mixing to eliminate the generated floccules so as to ensure that a transparent yellow solution is formed;

s303, transferring the yellow solution obtained in the step S302 into a DNA preparation tube by using a pipette, placing the DNA preparation tube into a 2m1 centrifuge tube prepared in advance, centrifuging the solution at a high speed of 12000 Xg for 1 minute, and removing the filtrate;

s304, putting the DNA preparation tube back into a 2m1 centrifugal tube, adding 500 microliters of BufferW1 solution, standing for 2 minutes at room temperature, centrifuging for 1 minute at a high speed of 12000 Xg, and removing the filtrate;

s305, putting the DNA preparation tube back into a 2m1 centrifuge tube, adding 700 microliters of BufferW2 solution, centrifuging at 12000 Xg at a high speed for 1 minute, and removing the filtrate;

s306, repeating the step S305 again to ensure complete desalination; and is centrifuged at 12000 Xg at high speed for 2 minutes;

s307, the DNA preparation tube is placed in a new 1.5m1 centrifuge tube, 30 microliters of Eluent Eluent which is heated in advance in a water bath is added to the center of a filter membrane of the preparation tube, the mixture is kept stand at room temperature for 10 minutes, and is subjected to high-speed centrifugation at 12000 Xg for 2 minutes to elute DNA.

As shown in fig. 6, in step S104, the method for analyzing nucleic acid by mass spectrometry according to the embodiment of the present invention includes:

s401, paving the resin for standby on a resin plate, and air-drying for 5-10 minutes;

s402, adding a sample to be detected into a reaction hole of a sample plate, supplementing 25ul ddHO, sealing by using a sealing film, and performing instant centrifugation at 3000 r/min;

s403, removing the sealing film, reversely buckling the sample plate on the resin plate obtained in the step S401, fixing, turning over the sample plate and the resin plate together, enabling the air-dried resin to fall into the reaction hole of the sample plate, sealing by using the sealing film, and instantly centrifuging at 3000 r/min;

s404, placing the sample plate on a rotator, rotating the sample plate for 20 minutes at a low speed, centrifuging the sample plate for 5 minutes at 3000 rpm, then spotting the sample plate on a machine, and detecting the sample plate by using a nucleic acid mass spectrometer.

In step S104, the target DNA sequencing primer provided in the embodiment of the present invention is a primer pair capable of specifically amplifying DNA fragments containing 3 Single Nucleotide Polymorphisms (SNPs) of a49G on the CTLA4 gene and a C677T on the T-1772C, MTHFR gene.

In step S105, the template strand to be tested provided in the embodiment of the present invention is designed for 3 SNPs sites of a C677T on a CTLA4 gene a49G and a T-1772C, MTHFR gene, and is a DNA sequencing primer capable of specifically detecting genotypes of the 3 SNPs sites by a DNA sequencing technology.

The above description is only for the purpose of illustrating the present invention and the appended claims are not to be construed as limiting the scope of the invention, which is intended to cover all modifications, equivalents and improvements that are within the spirit and scope of the invention as defined by the appended claims.