阅读说明：本技术 一种用于人miR-34a基因甲基化快速检测的特异性引物组合、试剂盒及方法 (Specific primer combination, kit and method for quickly detecting methylation of human miR-34a gene ) 是由 刘枭男 张佳幸 华恺 崔亚丽 于 2020-12-30 设计创作，主要内容包括：本发明公开一种用于人miR-34a基因甲基化快速检测的特异性引物组合、试剂盒及方法。该方法采集的样本经过转化使其DNA甲基化的差异转化为DNA的序列差异；基于LAMP核酸扩增以及引物错配对扩增阻滞的原理,LAMP反应过程中实现对DNA序列差异的识别；利用扩增片段上标记的地高辛和金磁纳米微粒表面标记的地高辛单抗相互识别偶联,结合侧向流层析技术,实现特定基因甲基化的快速检测。本发明以LAMP技术执行核酸的扩增,极大地缩短了核酸扩增的时间并且降低了对于仪器设备的依赖,提高了灵敏度；并使扩增产物的检测彻底避免了引物二聚体造成的假阳性现象,提高了特异性；省去核酸扩增产物鉴定而导致的高成本、费时、费力等问题,使检测方法更易普及。(The invention discloses a specific primer combination, a kit and a method for quickly detecting human miR-34a gene methylation. The method comprises the steps of converting DNA methylation differences of samples collected by the method into DNA sequence differences; based on the principle of LAMP nucleic acid amplification and primer mismatching amplification block, the identification of DNA sequence difference is realized in the LAMP reaction process; the rapid detection of the methylation of the specific gene is realized by utilizing mutual recognition and coupling of the digoxin marked on the amplified fragment and the digoxin monoclonal antibody marked on the surface of the gold magnetic nanoparticle and combining a lateral flow chromatography technology. The invention uses LAMP technology to amplify nucleic acid, greatly shortens the time of nucleic acid amplification, reduces the dependence on instrument and equipment, and improves the sensitivity; the detection of the amplification product thoroughly avoids the false positive phenomenon caused by primer dimer, and improves the specificity; the problems of high cost, time consumption, labor consumption and the like caused by the identification of nucleic acid amplification products are saved, so that the detection method is easier to popularize.)

1. A specific primer combination for quickly detecting human miR-34a gene methylation is characterized in that: comprises a methylation specific primer group and an unmethylated specific primer group,

the nucleotide sequences of the methylation specific primer sets are as follows:

the nucleotide sequence of the upstream inner primer FIP in the methylation specific primer group is as follows:

5'-biotin-CACGTGAAATTTATTGCGTTGGAGGTCCGTTAGTGCGATGTACCC

the nucleotide sequence of the downstream inner primer BIP in the methylation specific primer group is as follows:

5'-biotin-ATTGCAAAAATCCAAGAAGCAGCGAGATGTGCCAATCACATAA

the nucleotide sequence of the upstream outer primer F3 in the methylation specific primer group is as follows:

GGAGAAAGTGTTGGAGGGTGAA

the nucleotide sequence of the downstream outer primer B3 in the methylation specific primer group is as follows:

ACTCAAACTAACCATATCACAACA；

the nucleotide sequence of the upstream inner primer FIP in the unmethylated specific primer group is as follows:

5'-biotin-ACATTAAAACATTACATCAATCGAGGGTTACCGATTAATCCTAGAATGTAAG

the nucleotide sequence of the downstream inner primer BIP in the unmethylated specific primer group is as follows:

5'-biotin-AAATAACCAAACTGGAATTGAACTTATGTGTTAGAAATGTCAAATGTGGTT

the nucleotide sequence of the upstream outer primer F3 in the unmethylated specific primer group is as follows: GGTGGAGAGAATTTGAGGAGGA

The nucleotide sequence of the downstream outer primer B3 in the unmethylated specific primer group is as follows: CTAAACCCAACTAACATACATCAA are provided.

2. A kit for quickly detecting methylation of human miR-34a genes is characterized in that: comprising the specific primer combination and the test strip of claim 1.

3. The kit for rapidly detecting methylation of human miR-34a genes according to claim 2, wherein the kit comprises: the quality control line of the test strip is sprayed with goat anti-rabbit IgG for quality control, the detection line is sprayed with streptavidin for capture, and the binding pad is sprayed with digoxin monoclonal antibody modified gold magnetic particles.

4. The kit for rapidly detecting methylation of human miR-34a genes according to claim 3, wherein the kit comprises: the digoxin monoclonal antibody modified gold magnetic particle is a superparamagnetic composite particle with a core-shell structure, the core of the digoxin monoclonal antibody modified gold magnetic particle is a nano Fe3O4 magnetic particle, the surface of the gold magnetic particle is a gold shell layer, and the surface of the gold magnetic particle is modified with digoxin monoclonal antibody to form the digoxin monoclonal antibody modified gold magnetic particle.

5. The kit for rapidly detecting methylation of human miR-34a genes according to claim 4, wherein the kit comprises: the base of digoxin label in the digoxin monoclonal antibody modified on the gold surface is dATP, dTTP, dCTP, dDTP or dUTP.

6. A rapid detection method for human miR-34a gene methylation is characterized by comprising the following steps:

1, obtaining a tissue genome DNA and/or an episome DNA sample;

2, modifying the DNA sample obtained in the step 1 to convert unmethylated cytosine into uracil, wherein the methylated cytosine is not changed;

performing two LAMP reactions in parallel on any one of the samples treated in step 2, wherein the methylation specific primer set of claim 1 is used in one reaction, and the unmethylated specific primer set of claim 1 is used in the other reaction;

adding bases marked by digoxin into two LAMP reaction systems of the same sample to mark amplification products, wherein the two LAMP reaction systems have the same reaction conditions and are simultaneously placed under the constant temperature condition to obtain the amplification products;

and 5, rapidly detecting the amplification product obtained by the treatment in the step 4 by using the test strip in the kit for rapidly detecting the methylation of the human miR-34a gene according to any one of claims 2 to 5, and judging the methylation mode of the sample.

7. The method for rapidly detecting methylation of human miR-34a genes according to claim 6, wherein in the step 1 ], the free DNA sample is peripheral blood free DNA, urine free DNA or other tissue fluid free DNA.

8. The method for rapidly detecting methylation of human miR-34a genes according to claim 6, wherein the modifying solution in the step 2) is 5mM-5M inorganic salt solution or organic solution.

9. The method for rapidly detecting methylation of human miR-34a genes according to claim 8, wherein the inorganic salt solution or the organic solution is a KOH solution, a NaOH solution, a Ca (OH)2 solution, a NaHSO3 solution, a NaHSO4 solution, a Na2SO3 solution or a C6H6O2 solution.

10. The method for rapidly detecting methylation of human miR-34a genes according to claim 8, wherein the step 5 specifically comprises:

dropping the two tubes of amplification products completing the same sample in the step 4 onto sample pads of two test strips respectively for detection, wherein the obtained amplification products are labeled with biotin and digoxin simultaneously, after dropping the amplification products onto the sample pads, the digoxin on the amplification products is specifically combined with the digoxin monoclonal antibody modified gold magnetic particles, and moving up to the detection line in a chromatography manner, the biotin on the amplification products is specifically combined with streptavidin on the detection line for color development, while the reaction solution amplified by the non-methylated specific primers is not developed on the detection line of the test strips, so that complete methylation is indicated; otherwise, the methylation is not carried out; if the detection lines of the two test strips are colored, partial methylation is indicated.

Technical Field

The invention belongs to the technical field of gene methylation detection, and particularly relates to a specific primer combination, a kit and a method for quickly detecting human miR-34a gene methylation.

Background

Cancer poses a serious threat to human health on a global scale and is the leading cause of human disease death. The main bases for cancer diagnosis include clinical symptoms, imaging detection, histopathological examination, etc., and the clinical symptoms of many cancer patients appear later, and most early cancer patients do not have any obvious clinical symptoms. For early screening of cancer, many methods are currently included, such as low-dose CT diagnosis, tissue-cell-level cancer diagnosis, and gene-level cancer diagnosis. However, these approaches have a number of disadvantages: the low-dose CT detection has the problem of false positive and high detection cost; tissue-level cancer diagnosis can cause great pain to the patient in terms of sampling and may lead to cancer cell spreading or metastasis; the detection of tumor specific molecular markers at the gene level can be used as a means for early diagnosis of cancer, but the technology is not mature.

Currently, methods for genetic detection of diseases are receiving wide attention. DNA methylation is an important component of epigenetics, not only maintaining normal cellular function, but also playing an important role in carcinogenesis, and alteration of methylation status is an important factor in causing cancer. The DNA methylation is adopted as a marker for disease diagnosis, and the advantages are mainly as follows: firstly, in the process of tumor formation, the occurrence frequency of promoter hypermethylation is very high, even higher than that of gene mutation, and important genes related to tumor formation are not lacked; secondly, methylation is an important event in the early stages of tumorigenesis; thirdly, DNA methylation exists stably, and detection can be carried out through a gene amplification effect. Therefore, methylation detection has potential application value in early diagnosis of tumors.

Although the prior art has found a plurality of cancer-related methylated genes, in early cancers, the methylation frequency of different genes and the hypermethylation sites of different genes are different, and the applicable detection method needs further research. In peripheral blood, DNA with abnormal methylation only accounts for about 0.1% -1% of the total DNA, and the unmethylated DNA and methylated DNA have only slight difference, so that trace abnormal methylated DNA needs to be detected from a highly complex interference background, and the detection method is highly required. In addition, methylation detection at the laboratory level is highly dependent on sophisticated instrumentation and tedious procedures of professionals, severely hampering the spread of individualized medicine in the context of precision medicine. Therefore, it is necessary to provide a rapid, sensitive and convenient means for detecting gene methylation, which is of great significance in early screening and popularization of cancer.

Disclosure of Invention

The invention provides a specific primer combination, a kit and a method for quickly detecting human miR-34a gene methylation, and mainly solves the problems that the existing method can only be limited to laboratory application, the detection cost is high, and quick and sensitive detection cannot be realized.

The specific technical solution of the invention is as follows:

the specific primer combination for quickly detecting the methylation of the human miR-34a gene comprises a methylation specific primer group and an unmethylated specific primer group,

the nucleotide sequences of the methylation specific primer sets are as follows:

the nucleotide sequence of the upstream inner primer FIP in the methylation specific primer group is as follows:

5'-biotin-CACGTGAAATTTATTGCGTTGGAGGTCCGTTAGTGCGATGTACCC

the nucleotide sequence of the downstream inner primer BIP in the methylation specific primer group is as follows:

5'-biotin-ATTGCAAAAATCCAAGAAGCAGCGAGATGTGCCAATCACATAA

the nucleotide sequence of the upstream outer primer F3 in the methylation specific primer group is as follows:

GGAGAAAGTGTTGGAGGGTGAA

the nucleotide sequence of the downstream outer primer B3 in the methylation specific primer group is as follows:

ACTCAAACTAACCATATCACAACA；

the nucleotide sequence of the upstream inner primer FIP in the unmethylated specific primer group is as follows:

5'-biotin-ACATTAAAACATTACATCAATCGAGGGTTACCGATTAATCCTAGAATGTAAG

the nucleotide sequence of the downstream inner primer BIP in the unmethylated specific primer group is as follows:

5'-biotin-AAATAACCAAACTGGAATTGAACTTATGTGTTAGAAATGTCAAATGTGGTT

the nucleotide sequence of the upstream outer primer F3 in the unmethylated specific primer group is as follows: GGTGGAGAGAATTTGAGGAGGA

The nucleotide sequence of the downstream outer primer B3 in the unmethylated specific primer group is as follows: CTAAACCCAACTAACATACATCAA are provided.

The kit for quickly detecting the methylation of the human miR-34a gene by applying the specific primer group comprises a specific primer combination and a test strip.

Furthermore, a quality control line of the test strip is sprayed with goat anti-rabbit IgG for quality control, a detection line is sprayed with streptavidin for capture, and a binding pad is sprayed with digoxin monoclonal antibody modified gold magnetic particles.

Further, the digoxin monoclonal antibody modified gold magnetic particles are superparamagnetic composite particles with a core-shell structure, the core of the digoxin monoclonal antibody modified gold magnetic particles is nano Fe3O4 magnetic particles, the surface of the core is a gold shell layer, and the surface of the gold is modified with digoxin monoclonal antibody to form digoxin monoclonal antibody modified gold magnetic particles.

Furthermore, the base of digoxin label in digoxin monoclonal antibody modified on the surface of the gold is dATP, dTTP, dCTP, dDTP or dUTP.

A rapid detection method for human miR-34a gene methylation is characterized by comprising the following steps:

1, obtaining a tissue genome DNA and/or an episome DNA sample;

2, modifying the DNA sample obtained in the step 1 to convert unmethylated cytosine into uracil, wherein the methylated cytosine is not changed;

performing two LAMP reactions in parallel on any sample treated in the step 2, wherein the methylation specific primer group is used in one reaction, and the unmethylated specific primer is used in the other reaction;

adding bases marked by digoxin into two LAMP reaction systems of the same sample to mark amplification products, wherein the two LAMP reaction systems have the same reaction conditions and are simultaneously placed under the constant temperature condition to obtain the amplification products;

and 5, rapidly detecting the amplification product obtained by the treatment in the step 4 by using the test strip in the kit for rapidly detecting the human miR-34a gene methylation, and judging the methylation mode of the sample.

Further, in the step 1), the free DNA sample is free DNA of peripheral blood, free DNA of urine or free DNA of other tissue fluids.

Further, in the step 2), the modification liquid is an inorganic salt solution or an organic solution with the concentration of 5 mM-5M.

Further, the inorganic salt solution or the organic solution is a KOH solution, a NaOH solution, a Ca (OH)2 solution, a NaHSO3 solution, a NaHSO4 solution, a Na2SO3 solution or a C6H6O2 solution.

Further, the step 5 specifically includes:

dropping the two tubes of amplification products completing the same sample in the step 4 onto sample pads of two test strips respectively for detection, wherein the obtained amplification products are labeled with biotin and digoxin simultaneously, after dropping the amplification products onto the sample pads, the digoxin on the amplification products is specifically combined with the digoxin monoclonal antibody modified gold magnetic particles, and moving up to the detection line in a chromatography manner, the biotin on the amplification products is specifically combined with streptavidin on the detection line for color development, while the reaction solution amplified by the non-methylated specific primers is not developed on the detection line of the test strips, so that complete methylation is indicated; otherwise, the methylation is not carried out; if the detection lines of the two test strips are colored, partial methylation is indicated.

The beneficial effects of the invention are embodied in the following aspects:

(1) reduce the detection cost and is convenient for popularization and use

The existing detection technologies for DNA methylation, such as fluorescence quantitative PCR, gene chip hybridization, PCR combined with gel electrophoresis, PCR combined with capillary electrophoresis, PCR combined with enzymatic cleavage, DNA direct sequencing, etc., usually require some special instruments, such as gene sequencer, chip spotting instrument, fluorescence quantitative PCR, electrophoresis and imaging equipment, etc., or require complicated enzyme treatment of amplification products, which requires high cost to complete detection. The invention not only saves the cost of the DNA purification reagent, but also does not need special instruments and equipment, and only visualizes the interpretation result through the test paper strip, thereby greatly reducing the whole detection cost and being convenient for popularization and use in all levels of medical institutions.

(2) Improving the detection sensitivity

Compared with the existing methylation detection method based on PCR, the method adopts LAMP nucleic acid amplification technology, obtains high sensitivity while ensuring accuracy and operation simplicity by optimizing conditions such as primers and an LAMP reaction system, and can still accurately detect trace target sequences when the proportion of the target sequences in the total detection template is 0.1%.

(3) Improving detection specificity

Existing methods for detecting DNA methylation based on nucleic acid amplification lead to serious false positives due to primer dimer factors when the primer concentration is increased in order to increase the sensitivity of detection. When the DNA methylation detection kit is used for DNA methylation detection, only one biomarker oligonucleotide participates in amplification reaction, and even if primer dimer is generated, color development on a chromatographic test strip cannot be caused to further cause misjudgment. The design of the invention enables the detection method to have high sensitivity and simultaneously maintain high specificity.

(4) Improve the detection efficiency

Most of the existing techniques based on nucleic acid amplification rely on PCR methods, but the PCR-dependent methods require thermal cycling and the whole nucleic acid amplification process takes up to 90 min. The LAMP nucleic acid amplification technology is adopted, so that the nucleic acid amplification process can be finished in an isothermal environment for 30min without thermal cycle, and the detection efficiency is greatly improved.

Drawings

FIG. 1 is a schematic diagram showing the color development result of a test strip when the methylation mode of the present invention is complete methylation;

FIG. 2 is a schematic diagram showing the color development of the test strip when the methylation pattern is unmethylated in accordance with the present invention;

FIG. 3 is a schematic diagram showing the color development of the test strip when the methylation pattern is partial methylation according to the present invention;

Detailed Description

The present invention is further described in detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description text.

The invention provides a rapid detection method for human miR-34a gene methylation, which comprises the following steps:

1 ] obtaining a sample: the sample type may be tissue genomic DNA, peripheral blood free DNA, urine free DNA, or other tissue fluid free DNA.

Processing of the sample: the DNA obtained in the first step needs to be treated by a set of modification solution so that unmethylated cytosine is converted into uracil and methylated cytosine is not changed.

Performing two LAMP reactions in parallel aiming at each sample, wherein one reaction uses a methylation specific primer, and the other reaction uses an unmethylated specific primer; the specific primers are mainly designed aiming at a promoter and a first exon region of a miR-34a gene to be detected, and a group of methylation specific primers and a group of non-methylation specific primers are respectively designed for recognition and amplification; 5' -end labeled biotin is arranged on both FIP primers and BIP primers in the two groups of specific primers;

adding bases marked by digoxin into two LAMP reaction systems to mark amplification products, wherein the two LAMP reaction systems have the same reaction conditions and are simultaneously placed under the constant temperature condition to obtain the amplification products, and the amplification reaction systems are as follows:

and 5, rapidly detecting the amplification product obtained in the step 5 by using a chromatographic test strip and judging the methylation mode of the sample by using the specific visual characteristic of the gold magnetic particles, the action of digoxin and digoxin monoclonal antibody and the action of biotin and streptavidin by using a chromatographic technique. Dropwise adding a reaction solution amplified by the methylation specific primer to develop color on a detection line of the test strip, and indicating that the reaction solution amplified by the unmethylated specific primer does not develop color on the detection line of the test strip, so that complete methylation is indicated; otherwise, the methylation is not carried out; if the detection lines of the two test strips are colored, partial methylation is indicated.

Wherein, the sample type in the step one is tissue genome DNA, peripheral blood free DNA, urine free DNA or other tissue fluid free DNA.

The modification solution in step 2 is inorganic salt solution or organic solution of 5mM-5M, which may be KOH solution, NaOH solution, Ca (OH)₂Solution, NaHSO₃Solution, NaHSO₄Solution, Na₂SO₃Solutions or C₆H₆O₂And (3) solution.

The digoxin labeled base described in step 4 is dATP, dTTP, dCTP, dDTP, or dUTP.

The test paper strip adopted by the chromatography in the step 5 meets the following conditions:

a. the quality control line of the test strip is sprayed with goat anti-rabbit IgG for quality control;

b. streptavidin for capture is sprayed on the detection line of the test strip;

c. the combination pad of the test strip is sprayed with digoxin monoclonal antibody modified gold magnetic particles;

and 5, performing parting detection, namely dripping the two amplification products finished in the fifth step on sample pads of the two test strips respectively for detection. And marking biotin and digoxin on the obtained amplification product, after the obtained amplification product is dripped on a sample pad, specifically binding the digoxin and the digoxin monoclonal antibody coupled gold magnetic particles, moving the sample up to a detection line through chromatography, specifically binding the biotin on the amplification product and streptavidin on the detection line for color development, and judging the methylation mode of the sample according to the result.

The digoxin monoclonal antibody modified gold magnetic particle in the sixth step is a superparamagnetic composite particle with a core-shell structure, and the core of the superparamagnetic composite particle is nano Fe₃O₄The surface of the magnetic particle is a gold shell layer, and the surface of the gold is modified with digoxin monoclonal antibody to form digoxin monoclonal antibody modified gold magnetic particles.

The following examples illustrate the implementation process of the rapid detection method for human miR-34a gene methylation provided by the invention.

Example 1 design and optimization of primers

Designing primers aiming at a miR-34a gene promoter region and a first exon region: the gene sequence of miR-34a is confirmed by NCBI database, and synthetic primers are designed according to the gene sequence. Aiming at miR-34a gene to be detected, a group of methylation specific primers and a group of non-methylation specific primers are respectively designed for identification and amplification, and each group of primers is complementary with a corresponding template base. In order to facilitate the subsequent chromatographic detection technology, the designed primers and bases are subjected to biological labeling. Wherein FIP and BIP are labeled with biotin and bases are labeled with digoxigenin. In order to increase the amplification specificity and the detection resolution, a plurality of groups of primers are designed for experimental screening.

The optimal primers were screened according to the following targets: (1) the amplification specificity is high: the completely methylated and unmethylated specific primers can only amplify specific target bands for corresponding templates. (2) The amplification sensitivity is high, and the target fragment can be effectively amplified under the condition that as low as 0.1 percent of the target sequence exists in a sample. (3) The kit has high adaptability to sample types, is suitable for common genome DNA samples, and can effectively and specifically amplify free DNA of peripheral blood, free DNA of urine or free DNA of other tissue fluids.

Wherein, for the amplification accuracy and sensitivity, the screening is carried out according to the following steps: gradient dilutions were performed using well-defined fully methylated and unmethylated human genomic DNA as template, so that the ratio of sequence of interest to total template was: 50%, 25%, 10%, 5%, 1%, 0.5%, 0.1%. And amplifying by using the templates of the designed multiple sets of alternative primers in each proportion, and setting positive quality control and negative quality control simultaneously. Dividing the amplified product into 2 parts, respectively carrying out agarose gel electrophoresis detection and test strip chromatography system detection, and determining the accuracy and sensitivity of the designed primer.

Experimental results show that after LAMP amplification is carried out on a sample by the optimized primers, the mutation with the proportion as low as 0.1 percent can be detected by using agarose gel electrophoresis and a test strip chromatography system. In addition, the detection signal intensity of the chromatographic test strip for 0.1 percent of the template is judged by naked eyes to be obviously higher than that of agarose gel electrophoresis, and no false positive appears, which is consistent with the expected result.

Example 2 detection of genomic DNA samples

Taking 10 cases of paraffin embedded tissue samples, purifying the genomic DNA, and then treating and converting the genomic DNA by using a modification solution for later use; a, B the following were added to each of the two tubes (methylation specific primer set was added to tube A and unmethylated specific primer set was added to tube B):

the A, B tubes were placed in a metal bath and incubated at 65 ℃ for 30 min.

And (3) respectively dripping the amplification products of the A, B two tubes onto the two test strips, and judging whether mutation is carried out according to the following principle: if the result is shown in FIG. 1, the T line of the test strip corresponding to the A tube methylation specific primer group is developed, and the T line of the test strip corresponding to the B tube non-methylation specific primer group is not developed, the site is completely methylated. As shown in FIG. 2, the test strip corresponding to the A-tube methylation specific primer set shows no color at the T line, and the test strip corresponding to the B-tube methylation specific primer set shows no methylation at the site when the test strip shows color at the T line, and as shown in FIG. 3, the test strips corresponding to the two tubes show color at both the T lines, which shows that the site is partially methylated.

Example 3 detection of free DNA in peripheral blood

Taking 10 fresh samples of peripheral blood, purifying free DNA of the peripheral blood, and treating and converting the peripheral blood with a modifying solution for later use; a, B the following were added to each of the two tubes (methylation specific primer set was added to tube A and unmethylated specific primer set was added to tube B):

the A, B tubes were placed in a metal bath and incubated at 65 ℃ for 30 min.

And (3) respectively dripping the amplification products of the A, B two tubes onto the two test strips, and judging whether mutation is carried out according to the following principle: if the result is shown in FIG. 1, the T line of the test strip corresponding to the A tube methylation specific primer group is developed, and the T line of the test strip corresponding to the B tube non-methylation specific primer group is not developed, the site is completely methylated. As shown in FIG. 2, the test strip corresponding to the A-tube methylation specific primer set shows no color at the T line, and the test strip corresponding to the B-tube methylation specific primer set shows no methylation at the site when the test strip shows color at the T line, and as shown in FIG. 3, the test strips corresponding to the two tubes show color at both the T lines, which shows that the site is partially methylated.

Example 4 detection of free DNA samples from urine

Taking 10 fresh urine samples, purifying free DNA of urine, and treating and converting the urine samples by using a modifying solution for later use; a, B the following were added to each of the two tubes (methylation specific primer set was added to tube A and unmethylated specific primer set was added to tube B):

the A, B tubes were placed in a metal bath and incubated at 65 ℃ for 30 min.

And (3) respectively dripping the amplification products of the A, B two tubes onto the two test strips, and judging whether mutation is carried out according to the following principle: if the result is shown in FIG. 1, the T line of the test strip corresponding to the A tube methylation specific primer group is developed, and the T line of the test strip corresponding to the B tube non-methylation specific primer group is not developed, the site is completely methylated. As shown in FIG. 2, the test strip corresponding to the A-tube methylation specific primer set shows no color at the T line, and the test strip corresponding to the B-tube methylation specific primer set shows no methylation at the site when the test strip shows color at the T line, and as shown in FIG. 3, the test strips corresponding to the two tubes show color at both the T lines, which shows that the site is partially methylated.

While embodiments of the invention have been disclosed above, it is not intended to be limited to the uses set forth in the specification and examples. It can be applied to all kinds of fields suitable for the present invention. Additional modifications will readily occur to those skilled in the art. It is therefore intended that the invention not be limited to the exact details and illustrations described and illustrated herein, but fall within the scope of the appended claims and equivalents thereof.

<110> Xian jin magnetic nanometer biotechnology, Inc

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