CN113604552A

CN113604552A - AHRR gene methylation detection primer and kit based on pyrosequencing technology

Info

Publication number: CN113604552A
Application number: CN202110834883.6A
Authority: CN
Inventors: 钟诗龙; 刘玉玲; 汪静; 汤雅男; 吴圆圆; 郑淑芬
Original assignee: Xinkaiyuan Jingrui Guangzhou Biomedical Technology Co ltd; Guangdong General Hospital
Current assignee: Xinkaiyuan Jingrui Guangzhou Biomedical Technology Co ltd; Guangdong General Hospital
Priority date: 2021-07-23
Filing date: 2021-07-23
Publication date: 2021-11-05

Abstract

The invention belongs to the technical field of methylation detection, and particularly relates to an AHRR gene methylation detection primer and a kit based on a pyrosequencing technology. The AHRR gene methylation detection primer based on the pyrosequencing technology can amplify CpG sites which contain methylation and are obviously related to the AHRR gene expression level; the detection primer is prepared into an AHRR gene methylation kit based on a pyrosequencing technology, and the methylation of the AHRR gene can be detected simply, conveniently, quickly and at low cost, so that the accurate evaluation of the methylation level of the AHRR gene is realized. The kit can effectively detect methylation of cg05575921 site of smoking related AHRR gene, thereby obtaining methylation degree of related gene of sample to be detected, is beneficial to judging occurrence risk degree of cardiovascular and lung cancer related diseases, and has wide application prospect.

Description

AHRR gene methylation detection primer and kit based on pyrosequencing technology

Technical Field

The invention belongs to the technical field of methylation detection, and particularly relates to an AHRR gene methylation detection primer and a kit based on a pyrosequencing technology.

Background

According to statistics, nearly 600 million people die of health damage caused by smoking every year in the world, and smokers have higher probability of suffering from coronary artery diseases, peripheral vascular diseases, chronic obstructive pulmonary diseases, stroke and other diseases. Either active or passive smoking can easily lead to cardiovascular events. Tobacco smoke has been identified with up to 7000 more chemicals, of which 69 are known human carcinogens (including polycyclic aromatic hydrocarbons). Smoking affects all stages of atherosclerosis, from endothelial dysfunction to acute clinical events mainly of thrombosis can be affected by smoking; smoking also causes increased platelet and macrophage adhesion, which stimulates the formation of procoagulant and inflammatory conditions. Therefore, by decoding the molecular mechanisms by which smoking drives the development of cardiovascular disease, insightful understanding of the possible mechanisms underlying the risk of smoking is of great significance to the development of new strategies for the prevention and treatment of relevant health impairments.

DNA methylation is an important modification mode in epigenetic regulation, and is closely related to gene regulation, biological development and disease occurrence. DNA methylation mainly refers to the process of covalent substitution of 5' cytosine by methyl within CpG (cytosine-phosphate-guanine) dinucleotide sequences catalyzed by DNA methyltransferases (DNMTs). DNA methylation is a process in which DNA methyltransferases (DNMTs) and DNA demethylases participate together in regulation and control, and can be inherited by progeny following cell division, playing important roles in regulation of gene expression, chromosome stabilization, and parentage. DNA methylation can inhibit transcription of a gene by altering chromatin structure and inhibiting the binding of transcription factors and cofactors to the corresponding target sites, thereby reducing the expression level of the gene. The DNA modification mode regulates and controls the expression of genes on the premise of not changing a DNA sequence, and plays an important role in maintaining the normal function of cells, the development and growth of embryos and the occurrence of human tumors.

Epigenetic modifications can also be used as biomarkers that can both reflect environmental exposure and predict the risk of disease onset. The research on the relevance of the whole genome reveals that the relation between smoking and the change of the DNA methylation level of an Aryl Hydrocarbon Receptor Repressor (AHRR) is most remarkable, and the change is only partially reversible even after smoking cessation, so that the DNA methylation level can be used as an index for judging whether smoking occurs or not. In addition, smoking is associated with elevated expression levels of AHRR (mrna) in human lung and lymphocytes, and a number of studies have indicated that methylation of AHRR is also associated with lung cancer. The AhR signal transduction pathway plays an important role in the metabolism of a plurality of compounds and environmental pollutants, and aromatic hydrocarbon receptor (AhR) inhibiting factors coded by AHRR can cause abnormal expression of genes such as cytochrome (such as CYP1A1/CYP1B1) and the like, thereby preventing the biotransformation process of exogenous harmful substances in organisms. The level of methylation of AHRR is low in the lung tissues of smokers (p <0.001) and the level of gene expression is high (p ═ 0.0047) compared to non-smokers. Thus, alterations in DNA methylation may reflect not only the exposure effects of smoking, but also one of the underlying mechanisms by which smoking causes related diseases.

Therefore, the degree of AHRR methylation can provide valuable information beyond the existing risk prediction model, and the method can judge the prognostic index of the lung cancer patient caused by smoking and the occurrence risk of related diseases by detecting the DNA methylation level of the AHRR gene, and has important significance for guiding clinical rational medication, realizing individual medication and early diagnosis of lung cancer. Therefore, the development of a simple, rapid and low-cost AHRR gene methylation detection method has important application value.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides an AHRR gene methylation detection primer based on a pyrosequencing technology, and an AHRR gene methylation kit prepared from the detection primer has the advantages of simplicity, convenience, rapidness, low cost, strong pertinence, high detection accuracy and the like in the detection process of AHRR gene methylation.

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

the invention provides an AHRR gene methylation detection primer based on pyrosequencing technology, which comprises specific amplification primers AHRR-F and AHRR-R for amplifying a sequencing fragment required by an AHRR gene and a sequencing primer AHRR-S matched with the sequencing fragment required by the AHRR gene, wherein the nucleotide sequences of the AHRR-F, AHRR-R and AHRR-S are respectively shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3.

Preferably, the AHRR gene is a human AHRR gene. Specifically, the AHRR gene is a nucleotide fragment on chromosome 5 of the human NCBI release 37 genome sequence (chr5: 321714-438285).

Preferably, the CpG site detected by the methylation detection primer is cg05575921 site of AHRR gene (chr5: 373378).

The invention also provides application of the AHRR gene methylation detection primer based on the pyrosequencing technology in preparation of an AHRR gene methylation detection product.

The invention also provides an AHRR gene methylation detection kit based on the pyrosequencing technology, and the kit comprises the AHRR gene methylation detection primer based on the pyrosequencing technology.

According to the AHRR gene methylation detection kit based on the pyrosequencing technology, firstly, a bisulfite conversion mode is utilized to convert unmethylated cytosine (C) into uracil (U), methylated cytosine (C) is kept unchanged, and an amplification product with a C/T single base difference is generated after amplification of an amplification primer, so that the aim of distinguishing different methylation conditions of cytosine (C) at the same site is fulfilled. And then quantitative detection of the target site is realized by utilizing a pyrosequencing technology, and the methylation rate of the original unit site is obtained according to the C/T ratio of the detected result, so that the method has the advantages of simplicity, convenience, rapidness, low cost and the like.

The method has the advantages of convenient detection, strong pertinence and high detection accuracy, and can be used for detecting the AHRR gene methylation degree of cardiovascular diseases, lung cancer and other diseases related to smoking. The methylation level of the CpG sites of the AHRR gene is negatively related to smoking, has influence on the prognosis of patients with coronary heart disease, is also negatively related to early lung cancer onset, and can assist in early diagnosis of lung cancer and cardiovascular diseases by measuring the methylation level of sample DNA. The kit can conveniently and quickly provide effective detection means and basis for evaluating the potential possibility of cardiovascular diseases, lung cancer and other smoking-related diseases on the molecular level, and has wide application prospect.

Preferably, the kit further comprises a reagent for amplifying an AHRR gene sequencing fragment and a pyrosequencing reagent.

Further, the reagent for amplifying the AHRR gene sequencing fragment comprises a premix 2 x mix, wherein the 2 x mix contains magnesium chloride, a deoxyribonucleotide triphosphate mixture and DNA polymerase.

Further, the pyrosequencing reagent comprises magnetic beads, pyrosequencing binding buffer, pyrosequencing annealing buffer, pyrosequencing denaturation buffer, pyrosequencing elution buffer, pyrosequencing substrate, pyrosequencing enzyme system and four dNTPs.

Preferably, the PCR reaction system of the kit is 25 μ L, and comprises 2 × mix 12.5 μ L, AHRR-F2 μ L, AHRR-R2 μ L and DNA50-100 ng.

Specifically, the concentration of AHRR-F and AHRR-R was 5. mu.M.

Preferably, the reaction procedure of the kit is: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 30sec, annealing at 60 ℃ for 50sec, and extension at 72 ℃ for 15sec for 50 cycles; extension at 72 ℃ for 5 min.

The invention also provides a use method of the AHRR gene methylation detection kit based on the pyrosequencing technology in detection of AHRR gene methylation, which comprises the following steps:

1) extracting DNA in a sample;

2) performing bisulfite conversion on the extracted DNA;

3) taking the sample DNA treated in the step 2) as a template, and performing PCR amplification by using AHRR gene specific amplification primers AHRR-F and AHRR-R;

4) pyrosequencing the amplification product obtained in the step 3) by using an AHRR gene methylation sequencing primer AHRR-S, thereby obtaining the methylation state of CpG sites in an AHRR gene sequencing fragment.

Preferably, the sample is selected from human blood, cell or tissue samples.

Compared with the prior art, the invention has the beneficial effects that:

the invention provides an AHRR gene methylation detection primer based on pyrosequencing technology, which can amplify CpG sites which contain methylation and are obviously related to the AHRR gene expression level; the detection primer is prepared into an AHRR gene methylation kit based on a pyrosequencing technology, and the methylation of the AHRR gene can be detected simply, conveniently, quickly and at low cost, so that the accurate evaluation of the methylation level of the AHRR gene is realized. The kit can effectively detect methylation of cg05575921 site of smoking related AHRR gene, thereby obtaining methylation degree of related gene of sample to be detected, and is beneficial to making judgment on occurrence risk degree of cardiovascular and lung cancer and other smoking related diseases. The invention has convenient detection, strong pertinence and high detection accuracy, can conveniently and quickly provide effective detection means and basis for evaluating the potential possibility of cardiovascular and lung cancer and other smoking-related diseases on the molecular level, and has wide application prospect.

Drawings

FIG. 1 is an agarose gel electrophoresis of the amplified fragment of AHRR gene [ M is Marker (Thermo Fisher Scientific, SM0332),

lanes

1, 2, 3 are the amplified samples, lane 4 is a blank control without DNA addition ];

FIG. 2 shows pyrosequencing results of AHRR gene methylation.

In FIG. 2, the peak value at each site is the fluorescence value of the site, the target site is the 4 th site in the sequencing sequence, and since the first two sites of the site are T, the result of the non-methylation at the site is superposed with the result of the T at the first two sites, and the methylation at the site is calculated as C/[ (C + T + T)/3 ].

Detailed Description

The following further describes the embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

The experimental procedures in the following examples were carried out by conventional methods unless otherwise specified, and the test materials used in the following examples were commercially available by conventional methods unless otherwise specified.

Example 1 establishment of AHRR Gene methylation detection kit based on pyrosequencing technology

(1) The kit comprises the following components: mainly comprises the following components:

1) specific primers AHRR-F and AHRR-R of the amplified AHRR gene are used, and a sequencing fragment to be amplified is the g05575921 site of the AHRR gene; detecting AHRR gene methylation sequencing primer AHRR-S, and matching with the sequencing fragment required by AHRR gene.

The amplification primer and the sequencing primer are designed according to g05575921 target site of AHRR gene by adopting Pyromark Assay Design 2.0 software (QIAGEN), and the sequences are as follows:

an amplification primer:

AHRR-F: 5 ' -Biotin-GGATTGTTTATTTTTGAGAGGGTAGT-3 ' (Biotin Biotin marker is carried at the 5 ' end);

AHRR-R：5’-AAAAAAACCCTACCAAAACCACTC-3’。

sequencing primer:

AHRR-S：5’-GGTTTTGGTTTTGTTTTGTA-3’。

2) reagent for amplifying target region of AHRR gene (g05575921 site): 2 × mix (containing magnesium chloride, deoxyribonucleotide triphosphate mixture, DNA polymerase, etc.) of premix required for mediating the amplification reaction.

3) Reagent for detecting methylation of target site of AHRR gene: magnetic beads, pyrosequencing binding buffer, pyrosequencing annealing buffer, pyrosequencing denaturation buffer, pyrosequencing elution buffer, pyrosequencing substrate, pyrosequencing enzyme system and four dNTPs.

(2) Optimization of amplification system and procedure:

1) optimization of an amplification system: the PCR reaction system of the kit was 25. mu.L, wherein 12.5. mu.L of 2 XMASTER mix (Thermo Fisher Scientific, NO.4371355) was added, the amounts of amplification primers (5. mu.M in concentration) were set in a gradient, specifically 0.5. mu.L, 1. mu.L, 1.5. mu.L, 2. mu.L, and 2.5. mu.L, and the amount of DNA was 50-100ng, and the whole system was made up to 25. mu.L with enzyme-free water. The optimal input amount of the amplification primers is finally determined to be 2 mu L according to the size of the amplified product strip with none or brightness.

2) Optimization of the amplification procedure: the process is mainly optimized for annealing temperature and extension time. According to the Tim value given by software during the design of the amplification primers, the annealing temperature in the PCR process is optimized in a gradient way and is respectively 54 ℃, 56 ℃, 58 ℃, 60 ℃ and 62 ℃ (the extension time is fixed). After that, the extension times were designed to be 11sec, 13sec, 15sec, 17sec, 19sec, respectively. After optimization of a plurality of experiments, the optimal annealing temperature is finally determined to be 60 ℃ and the extension time is 15 sec.

Finally, the amplification program was determined as: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 30 sec; annealing at 60 ℃ for 50 sec; extension at 72 ℃ for 15 sec; a total of 50 cycles; extension at 72 ℃ for 5 min.

Example 2 application of AHRR gene methylation detection kit based on pyrosequencing technology

(1) Blood cell DNA extraction (TIANAmp Genomic DNA Kit, DP304)

1) 200 μ L of fresh or frozen human blood samples (provided by volunteers diagnosed with coronary heart disease in Min Hospital, Guangdong province) were taken, and less than 200 μ L of the sample was made up with buffer GA.

2) Add 20. mu.L of protease K solution and mix well.

3) Adding 200 μ L buffer GB, mixing thoroughly, standing at 70 deg.C for 10min to clear the solution, and centrifuging briefly to remove water droplets on the inner wall of the tube.

4) Add 200. mu.L of absolute ethanol, mix well for 15sec with shaking, at which time a flocculent precipitate may appear, and centrifuge briefly to remove water droplets on the inner wall of the tube cover.

5) The solution and flocculent precipitate obtained in the previous step are added into an adsorption column, centrifuged at 12,000rpm for 30sec, the waste liquid is decanted, and the adsorption column is returned to the collection tube.

6) Add 500. mu.L of buffer GD to the adsorption column, centrifuge at 12,000rpm for 30sec, discard the waste, and place the adsorption column back into the collection tube.

7) 600. mu.L of the rinsing solution PW was added to the adsorption column, centrifuged at 12,000rpm for 30sec, the waste liquid was discarded, and the adsorption column was returned to the collection tube.

8) Operation 7 is repeated.

9) The column was returned to the collection tube, centrifuged at 12,000rpm for 2min and the waste liquid was decanted. The adsorption column was left at room temperature for several minutes to completely dry the residual rinse solution in the adsorption material.

10) Transferring the adsorption column into a clean centrifuge tube, suspending and dropwise adding 50-200 μ L of elution buffer TE to the middle part of the adsorption membrane, standing at room temperature for 2-5min, centrifuging at 12,000rpm for 2min, collecting the solution in the centrifuge tube, and extracting to obtain DNA.

And (3) measuring the concentration and purity of the extracted DNA, and taking the following components in mass: the DNA sample with OD260/280 being more than or equal to 1.8 and less than 2.0 and OD260/230 being more than 2.0 is qualified DNA for further use.

(2) DNA bisulfite conversion (EZ DNA Methylation-Lightning Kit, D5030T)

Performing bisulfite conversion by taking the DNA which is extracted in the step (1) and has qualified quality after detection as a sample, wherein the specific method comprises the following steps:

1) a20. mu.L sample was taken (the total amount of DNA contained therein was made to be 1. mu.g, and the remaining volume was made up with water).

2) 130 μ L of Lighting Conversion Reagent was added.

3) The system is placed in a PCR instrument, and the setting conditions are as follows: at 98 deg.C for 8 min; 54 ℃ for 60 min; 4 ℃ for 10 min.

4) In a dedicated reaction column, 600. mu. L M-Binding Buffer was added, and the sample was taken out from the PCR apparatus, transferred to the reaction column, mixed by inverting upside down, centrifuged in a centrifuge (12000r/s, 4 ℃, 30sec) and the liquid in the lower tube was removed.

5) 100 μ L M-Wash Buffer was added to the column and centrifuged (12000r/s, 4 ℃ C., 30 sec).

6) Then, 200. mu. L L-depletion Buffer was added thereto, and the mixture was left at room temperature for 20min and centrifuged (12000r/s, 4 ℃ C., 30 sec).

7) 200 μ L M-Wash Buffer was added and centrifuged (12000r/s, 4 ℃ C., 30sec), and this step was repeated once.

8) The lower tube was discarded, and the upper tube was placed in a 1.5mL low-adsorption centrifuge tube, and the transformation product in the lower tube was collected after Elution of DNA by centrifugation (12000r/s, 4 ℃ C., 30sec) with 10. mu. L M-elute Buffer.

(3) PCR amplification

1) The PCR reaction (25. mu.L) was configured as follows:

2) amplification of

After the reaction system is prepared, PCR amplification is carried out according to the following amplification procedure:

95℃、15min；50×(95℃、30sec；60℃、50sec；72℃、15sec)；72℃、5min。

3) agarose gel electrophoresis

2.5g of agarose was weighed and dissolved thoroughly by heating with 0.5X TBE buffer 100 mL. And (3) after the solution is cooled to room temperature, adding 10 mu L of special Gel Green dye, shaking and uniformly mixing, pouring the mixture into a prepared Gel making groove, inserting a comb, and demolding and placing the Gel into an electrophoresis device after the Gel is completely condensed and formed. The electrophoresis solution was subjected to 0.5X TBE buffer under 130V for 50 min. After electrophoresis is finished, the gel is placed under an ultraviolet projector to observe an electrophoresis result.

As shown in FIG. 1, after the sample DNA was amplified, there were distinct product bands between 140bp and 150bp (

lanes

1, 2 and 3), and the blank group used as a control had no corresponding product band (lane 4).

(4) AHRR gene methylated pyrophosphate sequencing

The reagent matched with the pyrosequencing comprises the magnetic beads, pyrosequencing combined buffer solution, pyrosequencing modified buffer solution, pyrosequencing elution buffer solution, pyrosequencing annealing buffer solution, pyrosequencing substrate, pyrosequencing enzyme system and four dNTPs (dATP alpha S, dTTP, dGTP and dCTP), and is purchased from Xinkayuan Feisite precision medical science and technology Limited company (Xiangchan instruments 20190409) in Hunan.

1) Single strand preparation

First, the corresponding buffer is diluted:

preparing a binding buffer solution: 38 μ L, beads: 2 mu L of the solution;

dilution of denaturation buffer (8 ×): denatured liquid (8X: 1V, ultrapure water: 7V, and (3) adding water;

dilution wash buffer (10 ×): wash buffer (10 ×):1V, ultrapure water: 9V.

Adding 40 mu L of binding buffer solution (containing micro-beads) into a clean EP tube, adding 20 mu L of PCR product into the clean EP tube, placing the mixture on a table-type oscillator, and oscillating the mixture for 15min at 1100 rpm;

③ 7,000 Xg, centrifuging for 1 min;

adding 150 mu L of diluted washing buffer into an EP tube, and centrifuging for 1min at 7,000 Xg (the step is operated for 3 times in total);

fifthly, taking a new clean EP tube, adding 6 mu L of annealing buffer solution into the EP tube, and then adding 1 mu L of sequencing primer AHRR-S;

sixthly, taking out the purification column, inserting the purification column into an EP tube, adding 22 mu L of diluted working solution of the denaturation buffer solution into the purification column, standing for 5min, centrifuging for 1min at 7,000 Xg, collecting a single-chain product by the EP tube, and removing the purification column.

2) Sequencing on computer [ real-time quantitative pyrophosphate sequence analyzer (QIAGEN GmbH, SFD (I)20113404004) ]

Dissolving sequencing enzyme and sequencing substrate: taking out vials of sequencing enzyme and sequencing substrate dry powder, respectively adding 620 mu L of ultrapure water, standing at room temperature for 5-10min, slightly rotating and shaking the vials during the standing period until the vials are completely dissolved, and subpackaging the vials into PCR tubes for later use, wherein each tube is divided into 53 mu L;

transferring the single-chain product in the EP tube to a sequencing tube, and adding 3 mu L of sequencing enzyme and 3 mu L of sequencing substrate into each sequencing tube;

③ taking a dNTP calandria, and adding 20 uL dATP alpha S, 20 uL dTTP, 20 uL dGTP and 20 uL dCTP from the round end to the flat end in sequence. Lightly knocking the bottom of the calandria against the tabletop to enable the bases to be flatly paved at the bottom of the calandria;

preparing a cleaning water tank: adding pure water into a cleaning water tank until the pure water is submerged by a brush, and placing absorbent paper at a specified position of the cleaning water tank;

starting the computer and waiting for the Windows desktop to appear;

sixthly, turning on a power switch of the pyrosequencing instrument and waiting for one minute;

and seventhly, operating iLight sequencing software by double clicking and clicking a tray in and out button. And (5) after the tray is moved out, putting the cleaning water tank, the dNTP discharging pipe and the sequencing pipe into a specified position. Clicking the 'tray in and out' button again to withdraw the tray;

clicking a 'new' button. Selecting a project to be sequenced from an A-I pull-down menu, and inputting the corresponding sample number;

the ninthly clicks the "start sequencing" button. After checking whether the preparation work is finished or not carefully by contrasting the prompt dialog box, clicking a 'confirm' button to start sequencing;

after sequencing on the r, the software pops up an "end" dialog box and clicks the "ok" button. Clicking the 'save' button can save the sequencing result file. Clicking the "output report" button can output the result (excel format).

As shown in fig. 2, the methylation rate of the blood sample at the cg05575921 site is: 12/[ (12+72+0)/3] ═ 42.9%; methylation was successfully detected in the blood sample.

The embodiments of the present invention have been described in detail, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, and the scope of protection is still within the scope of the invention.

Sequence listing

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Claims

1. An AHRR gene methylation detection primer based on pyrosequencing technology is characterized by comprising specific amplification primers AHRR-F and AHRR-R for amplifying a sequencing fragment required by an AHRR gene and a sequencing primer AHRR-S matched with the sequencing fragment required by the AHRR gene, wherein the nucleotide sequences of the AHRR-F, AHRR-R and AHRR-S are respectively shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3.

2. The primer for detecting AHRR gene methylation based on pyrosequencing technology of claim 1, wherein said AHRR gene is a human AHRR gene.

3. The AHRR gene methylation detection primer based on pyrosequencing technology of claim 1, wherein the CpG site detected by the methylation detection primer is cg05575921 site of the AHRR gene.

4. Use of the AHRR gene methylation detection primers of any one of claims 1-3 based on pyrosequencing technology in the preparation of an AHRR gene methylation detection product.

5. A kit for detecting AHRR gene methylation based on pyrosequencing technology, which comprises the AHRR gene methylation detection primer based on pyrosequencing technology of any one of claims 1-3.

6. The AHRR gene methylation detection kit based on pyrophosphate sequencing technology as claimed in claim 5, wherein said kit further comprises a reagent for amplifying AHRR gene sequencing fragment and a pyrophosphate sequencing reagent.

7. The AHRR gene methylation detection kit based on pyrophosphate sequencing technology as claimed in claim 6, wherein the reagent for amplifying the AHRR gene sequencing fragment comprises a premixed solution 2 x mix, and the 2 x mix contains magnesium chloride, a deoxyribonucleotide triphosphate mixture and DNA polymerase.

8. The AHRR gene methylation detection kit based on pyrosequencing technology of claim 6, wherein pyrosequencing reagents comprise magnetic beads, pyrosequencing binding buffer, pyrosequencing annealing buffer, pyrosequencing denaturation buffer, pyrosequencing elution buffer, pyrosequencing substrates, pyrosequencing enzyme system, and four dNTPs.

9. The AHRR gene methylation detection kit based on pyrosequencing technology as claimed in claim 5, wherein the PCR reaction system of the kit is 25 μ L, comprising 2 x mix 12.5 μ L, AHRR-F2 μ L, AHRR-R2 μ L, and DNA50-100 ng.

10. The AHRR gene methylation detection kit based on pyrosequencing technology according to claim 5, wherein the reaction procedure of the kit is as follows: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 30sec, annealing at 60 ℃ for 50sec, and extension at 72 ℃ for 15sec for 50 cycles; extension at 72 ℃ for 5 min.