CN106367515B

CN106367515B - Kit for detecting DNA methylation degree based on gold nanoparticle probe and detection method and application thereof

Info

Publication number: CN106367515B
Application number: CN201610856439.3A
Authority: CN
Inventors: 黄金; 谭志荣; 俞竟; 周宏灏
Original assignee: Individual
Current assignee: Individual
Priority date: 2016-09-27
Filing date: 2016-09-27
Publication date: 2019-12-17
Anticipated expiration: 2036-09-27
Also published as: CN106367515A

Abstract

the invention relates to a kit for detecting DNA methylation degree based on a gold nanoparticle probe, and a detection method and application thereof, wherein the kit comprises the gold nanoparticle probe, the gold nanoparticle probe comprises gold nanoparticles and an upstream primer for carrying out methylation specific polymerase chain reaction, a sulfydryl group is modified at the 5' end of the upstream primer, the upstream primer is coupled with the gold nanoparticles through the sulfydryl group, and the particle size of the gold nanoparticles is 10-15 nm. According to the invention, the standard colorimetric card is designed, the methylation degree of the DNA to be detected can be detected in real time, quickly, simply and economically according to the color of the system, a standard curve of ultraviolet absorbance and DNA methylation percentage is designed, and the accurate DNA methylation percentage can be obtained by substituting related data into the standard curve through testing the ultraviolet visible light absorption spectrum of the system, so that a valuable instrument is not required to be used, and the detection method is simpler, more convenient and more economic and has higher practical value.

Description

Kit for detecting DNA methylation degree based on gold nanoparticle probe and detection method and application thereof

Technical Field

the invention relates to the fields of biological detection and medicine, in particular to a kit for detecting DNA methylation degree based on a gold nanoparticle probe, and a detection method and application thereof.

Background

DNA methylation is one of the major forms of epigenetics, and the development of cancer is closely related. DNA methylation is realized by selectively adding a methyl group to the fifth C atom of cytosine in CpG dinucleotide in a genome under the catalysis of methyltransferase to form a 5-methylcytosine structure which is commonly seen in a 5'-CG-3' sequence of a gene. DNA methylation does not alter the base sequence of a gene, but can affect gene expression. Research shows that the occurrence and development of cancer are closely related to abnormal methylation of cancer suppressor gene DNA. In normal cells, oncogenes regulate cell growth, repair DNA, and control apoptosis. And the over-methylation of the cancer suppressor gene promoter can reduce or inactivate the expression of the cancer suppressor gene, so that the growth of cancer cells is out of control, and the apoptosis of the cells is blocked, thereby causing the occurrence of tumors. In recent years, DNA methylation has become one of the research hotspots of the tumorigenic mechanism.

At present, pyrosequencing, BSP and methylation-specific PCR are mainly used as methods for DNA methylation detection, but the existing DNA methylation detection method needs a complex and precise instrument and is difficult to obtain a detection result in real time and quickly.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to provide a kit for detecting the DNA methylation degree based on a gold nanoparticle probe, and a detection method and application thereof, so as to detect the DNA methylation degree in real time, quickly, economically and practically.

In a first aspect, the kit for detecting DNA methylation degree provided by the invention comprises a gold nanoparticle probe, wherein the gold nanoparticle probe comprises gold nanoparticles and an upstream primer for performing methylation specific polymerase chain reaction, a sulfydryl group is modified at the 5' end of the upstream primer, the upstream primer is coupled with the gold nanoparticles through the sulfydryl group, the particle size of the gold nanoparticles is 10-15 nm, and the average particle size is 13 nm.

Preferably, the DNA is PAX1 gene, and the upstream primer corresponds to PAX1 gene promoter region; i.e., the extent to which DNA methylation of the promoter region of the tumor suppressor gene PAX1 occurs.

Preferably, the upstream primer is SH-5 'CGGTTAGACGAATTTTTTTTAATCGGATGA-3' (SEQ ID NO. 1).

Preferably, the kit further comprises a downstream primer, the sequence of the downstream primer is 5'-CCCGCGACCCCAAAC-3' (SEQ ID NO. 2).

preferably, the preparation method of the gold nanoparticle probe comprises the following steps:

1) incubating the upstream primer and the gold nanoparticles at room temperature to obtain a first mixture;

2) adding a Tris-hydrochloric acid buffer solution and a NaCl solution into the first mixture, and then incubating under a dark condition to obtain a second mixture;

3) And centrifuging the second mixture, and taking the precipitate to obtain the gold nanoparticle probe.

Preferably, in the step 1), the molar ratio of the upstream primer to the gold nanoparticles is 95-105: 1, and the incubation time is 15-20 h; in the step 2), the incubation time is 20-30 h. The method specifically comprises the following steps: in the step 1), the molar ratio of the upstream primer to the gold nanoparticles is 100:1, and the incubation time is 16 h; in step 2), 10. mu.L of Tris-hydrochloric acid buffer solution having a concentration of 100mM and a pH of 8.2 and 20. mu.L of 1M NaCl solution were added dropwise to the first mixture, and the resulting system was incubated for 24 hours in the absence of light.

Optionally, the kit further comprises gold nanoparticles or gold nanoparticle sols (colloidal solutions).

Optionally, the kit further comprises NaCl powder or an aqueous solution.

optionally, the kit further comprises other reagents required for Polymerase Chain Reaction (PCR).

in a second aspect, the invention provides the use of the kit or gold nanoparticle probes in the kit as described above in the preparation of a system for detecting the degree of DNA methylation, said system further comprising other reagents required for performing a methylation specific polymerase chain reaction.

In a third aspect, the present invention provides a method for detecting the degree of DNA methylation, comprising the steps of:

a) Carrying out methylation specific polymerase chain reaction on DNA to be detected after bisulfite treatment in a system containing the gold nanoparticle probe in the kit; the method specifically comprises the following steps: configuring a 25 μ L PCR amplification system comprising: 100ng of DNA template to be detected after bisulfite treatment, 0.8 mu M gold nanoparticle probe, 0.4 mu M downstream primer, 5 mu L of 10 xEpiTaq PCRbuffer, 0.3mM dNTPs and 1.25U of DNA polymerase; the reaction conditions are that the reaction is carried out for 4min at 95 ℃, then the reaction is carried out for 30s at 95 ℃, 30s at 55 ℃ and 1min at 72 ℃ for 16 cycles, and finally the reaction is carried out for 7min at 72 ℃, and the reaction is finished after the temperature is reduced to 4 ℃;

b) adding a product obtained after methylation specific polymerase chain reaction into a colloidal solution of gold nanoparticles, standing and incubating to obtain a third mixture, wherein the particle size of the gold nanoparticles is 10-15 nm, and the average particle size is 13 nm; optionally, purifying a product obtained after methylation specific polymerase chain reaction by using a PCR purification kit, and then adding the purified product into a colloidal solution of gold nanoparticles;

c) adding a NaCl solution into the third mixture, and reacting for at least 4 minutes to obtain a fourth mixture, wherein the reaction time is preferably 6 minutes;

d) The fourth mixture is tested.

Further, the method for detecting the methylation degree of the DNA further comprises the following steps: comparing the color of the fourth mixture with a standard color comparison card, and detecting the methylation degree of the DNA to be detected; the preparation method of the standard colorimetric card comprises the following steps:

Preparing a series of standard DNAs with different methylation degrees by adopting standard methylated DNAs and standard unmethylated DNAs; alternatively, the standard unmethylated DNA is peripheral blood gDNA of a healthy human, and the standard methylated DNA is obtained by treating the standard unmethylated DNA with CpG methyltransferase;

Respectively carrying out the operations in the steps a) to c) on a series of standard DNAs with different methylation degrees to obtain a series of fourth mixtures with different colors;

A series of fourth mixtures of different colors are arranged in ascending or descending order of the corresponding degree of methylation.

Further, the method for detecting the methylation degree of the DNA further comprises the following steps: detecting the fourth mixture by adopting an ultraviolet-visible light spectrophotometry;

the method for detecting the fourth mixture by using ultraviolet-visible spectrophotometry specifically comprises the following steps:

S1) establishing a standard curve:

Preparing a series of standard DNAs with different methylation degrees by adopting standard methylated DNAs and standard unmethylated DNAs; respectively carrying out the operations in the steps a) to c) on the series of standard DNAs with different methylation degrees to obtain corresponding fourth mixtures; alternatively, the standard unmethylated DNA is peripheral blood gDNA of a healthy human, and the standard methylated DNA is obtained by treating the standard unmethylated DNA with CpG methyltransferase;

Testing the ultraviolet visible light absorption spectrum of a fourth mixture obtained by adopting standard DNA with the methylation degree of 0%, and recording the wavelength X of the maximum absorption peak, wherein the X is 650nm specifically;

Adopting standard DNA with the methylation degree of 100 percent to carry out the operation in the steps a) to b), testing the ultraviolet visible light absorption spectrum of the obtained system, and recording the wavelength Y of the maximum absorption peak, wherein the Y is 520nm specifically;

Testing the corresponding fourth mixture for UV-visible absorption spectrum, and recording the absorbance values A at X and Y_XAnd A_YTo obtain A_X/A_YA ratio;

A is to be_X/A_Yfitting the ratio with the corresponding methylation degree percentage to prepare a standard curve;

S2) testing the ultraviolet-visible light absorption spectrum of the fourth mixture obtained by using the DNA to be tested, and recording the absorbance values A at the wavelengths X and Y_XAnd A_YTo obtain A_X/A_YAnd (4) substituting the ratio into the standard curve to obtain the methylation degree percentage of the DNA to be detected.

The principle for detecting the DNA methylation degree provided by the invention is as follows: after the DNA to be detected is treated by bisulfite, the methylated C basic group is reserved, the unmethylated C basic group is converted into U, a methylation specific polymerase chain reaction (MSP) primer is designed, when MSP amplification is carried out, if methylation occurs at a detection site, a fragment product can be obtained through PCR amplification, and if methylation does not occur at the detection site, no amplification product exists. The invention is based on the target detection sequence of the gene DNA to be detected, such as the promoter region of the cancer suppressor gene PAX1,Designing and synthesizing a gold nanoparticle probe, wherein the gold nanoparticle probe is a coupling body of a gold nanoparticle and an MSP (mixed protein) upstream primer modified with a sulfydryl at the 5' end, and the upstream primer is coupled with the gold nanoparticle through the sulfydryl. The gold nanoparticle probe is used as an upstream primer of an MSP amplification system, and if an amplified fragment exists, the amplified product is connected to the surface of the gold nanoparticle through sulfydryl. The inventor researches the influence of the sulfydryl modified MSP primer on MSP amplification efficiency, the MSP amplification efficiency is divided into two groups of samples, the 5' end of one group of upstream primers is modified with sulfydryl, the other group of upstream primers is not modified with sulfydryl, MSP amplification experiments are carried out under the same experiment conditions, gel electrophoresis experiments show that the results are consistent, and the primers modified with sulfydryl do not influence the MSP amplification efficiency. After salt is added into a colloidal solution of gold nanoparticles with uniformly dispersed particles, if NaCl is added, the gold nanoparticles can be agglomerated, and further, the color of the system and the ultraviolet visible light absorption spectrum can be changed to a certain extent. In the gold nanoparticle colloidal solution with the MSP amplification product on the surface of the gold nanoparticles, the gold nanoparticles can still be kept stable under the addition of salt ions, the agglomeration is avoided, and the color of a corresponding system and the change of an ultraviolet visible light absorption spectrum are very small and basically kept unchanged; in a system without amplification product generation, because the surface of the gold nanoparticles is not protected by macromolecules, the gold nanoparticles can be subjected to an agglomeration phenomenon under the action of salt ions, the color of a colloidal solution is changed from wine red to gray and then tends to be stable, and the change of the color and the ultraviolet absorbance of the system is related to the DNA methylation degree. As shown in FIGS. 4 and 5, after a salt solution (NaCl solution) is added into the system according to the detection method provided by the invention, because the gold nanoparticles are modified by the amplification product, the color of the system has no obvious change, the system still presents red as shown in an inset (right) in FIG. 5 after 6 minutes, and the ultraviolet-visible spectrum A is recorded every second within 0-6 minutes after the NaCl is added₆₅₀/A₅₂₀The ratio, the result corresponds to PAX1 of FIG. 5^m+The curve shows that the ratio is almost unchanged, namely the system of the positive sample is kept unchanged, and the transmission electron microscope image in FIG. 4 shows that the gold nanoparticles are still uniformly dispersed; standard unmethylated DNA with a degree of methylation of 0% was usedAccording to the detection method provided by the invention, after the salt solution (NaCl solution) is added into the system, the color of the system gradually changes from wine red to gray within 0-4 min, the color tends to be stable after 4min, and the change of the ultraviolet visible light absorption spectrum of the system within 0-4 min is as follows: the position of the maximum absorption peak of the ultraviolet visible spectrum is redshifted from 520nm to 650nm, the peak shape is gradually widened, the gold nanoparticles in the system can be seen to generate a serious agglomeration phenomenon from figure 4 (right), and the A of the ultraviolet visible spectrum of the system is recorded within 0-6 min per second after NaCl is added₆₅₀/A₅₂₀Ratio, corresponding to PAX1 of FIG. 5^m-Curve, negative sample system A₆₅₀/A₅₂₀The ratio is increased with time within 0-4 min from 0.3 to 1.4, and the ratio is stably maintained after 4 min.

According to the invention, the standard colorimetric card is designed, the methylation degree of the DNA to be detected can be detected in real time, quickly, simply and economically according to the color of the system, a standard curve of ultraviolet absorbance and DNA methylation percentage is designed, and accurate DNA methylation percentage can be obtained by substituting related data into the standard curve through testing the ultraviolet visible light absorption spectrum of the system, so that a valuable instrument is not required to be used, and the detection method is simpler, more convenient, more economic and more practical.

additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

drawings

In order to more clearly illustrate the technical solution of the embodiments of the present invention, the drawings used in the embodiments will be briefly described below.

fig. 1 shows a transmission electron micrograph (a) and an ultraviolet-visible light absorption spectrum (B) of a gold nanoparticle sol of example 1 of the present invention;

FIG. 2 shows a photograph of a standard color chart provided in example 1 of the present invention;

FIG. 3 shows a standard curve provided in example 1 of the present invention;

fig. 4 shows a transmission electron micrograph: adding a salt solution into the standard methylated DNA system to react for 4min (left) and adding a salt solution into the standard unmethylated DNA system to react for 4min (right);

FIG. 5 shows the UV-VIS absorption spectrum A of a reaction system of standard methylated DNA with standard unmethylated DNA₆₅₀/A₅₂₀the values change in a curve within 0-6 min after the salt solution is added, and the color of two reaction systems is shown in an inset of 6 min.

Detailed Description

The present invention will be described in detail below with reference to the accompanying drawings and examples. The following examples are illustrative and are intended to more clearly illustrate the technical solutions of the present invention and should not be construed as limiting the present invention.

Example 1

the kit for detecting the DNA methylation degree provided by the embodiment is used for detecting the methylation degree of the DNA of the PAX1 gene, the PAX1 is an anti-cancer gene, hypermethylation of a promoter region of the PAX1 is often used for diagnosis and screening of some cancers, particularly in cervical cancer, and research shows that the PAX1 gene methylation detection and HPV detection are combined clinically, so that the misdiagnosis rate of the cervical cancer can be reduced, the diagnosis level of the cervical cancer is greatly improved, and the PAX1 gene is selected. The kit comprises a gold nanoparticle probe, gold nanoparticle sol and a downstream primer.

The preparation method of the gold nanoparticle sol comprises the following steps: soaking the used glassware in aqua regia overnight; during preparation, 100mL of deionized water and 4.8mL of chloroauric acid aqueous solution with the mass fraction of 1.0% are added into a 250mL three-neck flask, the flask is placed on a magnetic stirrer, a condensing reflux pipe is added on the flask, the flask is heated to boiling, 10mL of freshly prepared trisodium citrate aqueous solution with the mass fraction of 1.0% is rapidly added under vigorous stirring, the solution is kept boiling for reaction for 5min, heating is stopped, the solution is changed into wine red, the solution is continuously stirred until the solution is cooled to room temperature, and the gold nanoparticle sol is prepared and stored at 4 ℃. Wherein trisodium citrate is available from the national pharmaceutical group and chloroauric acid is available from Shanghai Sigma Aldrich.

Fig. 1 is a transmission electron microscope (a) and an ultraviolet-visible light absorption spectrum (B) of the gold nanoparticle sol, as shown in fig. 1(a), the prepared gold nanoparticles have uniform particle size of 10-15 nm, an average particle size of 13nm, and good dispersibility, and the ultraviolet-visible light absorption spectrum of the gold nanoparticle sol is as shown in fig. 1(B), and the maximum absorption peak is 520 nm.

the preparation method of the gold nanoparticle probe comprises the following steps: designing and synthesizing a PAX1 gene MSP primer, wherein the 5' end of an upstream primer is modified with sulfydryl, and the sequence of the upstream primer is as follows:

an upstream primer F: SH-5 'CGGTTAGACGAATTTTTTTTAATCGGATGA-3' (SEQ ID NO. 1);

Taking 20 mu L of 100 mu M upstream primer and 200 mu L of 100nM gold nanoparticle sol, placing the mixture at room temperature (about 20 ℃) and incubating the mixture for 16h with gentle shaking to obtain a first mixture; adding 10 mu L (100mM, pH 8.2) of Tris-hydrochloric acid buffer solution and 20 mu L of 1M NaCl solution into the first mixture dropwise, incubating the system for 24h in a dark place to obtain a second mixture, placing the second mixture in a low-temperature centrifuge 8000g, centrifuging for 15min, and taking the precipitate to obtain the gold nanoparticle probe.

a downstream primer R: 5'-CCCGCGACCCCAAAC-3' (SEQ ID NO.2), and the forward primer and the reverse primer were synthesized from Shanghai Boshang Biotechnology Ltd.

The method for detecting the DNA methylation degree of the PAX1 gene by using the kit provided by the embodiment 1 comprises the following steps:

Step 1: extracting DNA to be detected, standard methylated DNA and standard unmethylated DNA; the DNA to be detected is extracted from cervical exfoliated cells of normal persons, the standard unmethylated DNA is taken from peripheral blood gDNA of healthy volunteers, and the standard methylated DNA is obtained after the standard unmethylated DNA is treated by CpG methyltransferase.

Step 2: preparing a series of standard DNAs with different methylation degrees; standard methylated DNA and standard unmethylated DNA are adopted, standard DNA with the mass concentration of the standard methylated DNA being 0%, 20%, 40%, 60%, 80% and 100% respectively is prepared, the mass concentration is the percentage of methylation degree, and standard DNA with the mass concentration of the standard methylated DNA being 0% is the standard unmethylated DNA.

And step 3: and (3) treating the DNA to be detected and a series of standard DNAs by using bisulfite by using a conventional treatment method.

And 4, step 4: carrying out methylation specific polymerase chain reaction on the DNA to be detected after bisulfite treatment and a series of standard DNAs, specifically: preparing a 25 mu L PCR amplification system, wherein the PCR amplification system comprises 100ng of DNA template (DNA to be detected or standard DNA) treated by bisulfite, 0.8 mu M gold nanoparticle probe, 0.4 mu M downstream primer, 5 mu L of 10 multiplied by EpiTaq PCRbuffer, 0.3mM dNTPs and 1.25U DNA polymerase; the reaction conditions are that the reaction is carried out for 4min at 95 ℃, then for 30s at 95 ℃, for 30s at 55 ℃ and for 1min at 72 ℃ for 16 cycles, and finally for 7min at 72 ℃, and the reaction is finished after the temperature is reduced to 4 ℃. Methylation specific polymerase chain reaction (MSP) products were purified using PCR purification kits, following the instructions for product use. Among them, dNTP was purchased from Shanghai bioengineering Co., Ltd, DNA polymerase (dedicated to DNA after bisulfite conversion) and 10 × EpiTaq PCR buffer were purchased from Baojie bioengineering (Dalian) Co., Ltd, and PCR purification kit was purchased from Kajie biotech (Shanghai) Co., Ltd.

And 5: establishing a standard colorimetric card; respectively taking 5 mu L of MSP products of a series of purified standard DNAs, adding the MSP products into 20 mu L of gold nanoparticle sol in the kit, incubating for 30min, and then adding 2 mu L of NaCl solution with the concentration of 5 mol/L. Starting timing, reacting for 6 minutes to obtain a series of systems with different colors, sequencing the systems with different colors according to the corresponding methylation degrees (namely the mass concentration of the standard methylated DNA) in an ascending order, as shown in FIG. 2, wherein the methylation degrees are respectively 0%, 20%, 40%, 60%, 80% and 100% from left to right, and the colors of the corresponding systems are respectively gray, purple gray, bluish purple, deep red and wine red to obtain the standard colorimetric card. Wherein NaCl was purchased from the national pharmaceutical group.

Step 6: detecting the methylation degree of the DNA to be detected by a colorimetric method; and adding 5 mu L of the MSP product of the purified DNA to be detected into 20 mu L of gold nanoparticle sol in the kit, incubating for 30min, and then adding 2 mu L of NaCl solution with the concentration of 5 mol/L. And starting timing, after reacting for 6 minutes, recording the color of the reaction system, carrying out color comparison with a standard color comparison card, judging the interval where the color is located, and estimating the approximate range of the methylation degree. The color of the system obtained in this example changed from wine red to purple gray after the NaCl solution was added, the color was stable after 6 minutes, and compared with the color comparison card, the color of the system was very close to the color with the methylation degree of 20% in the color comparison card, and the methylation percentage of the DNA to be detected was determined to be about 20%.

further, step 5 also includes establishing a standard curve; adding a purified MSP amplification product obtained by adopting standard DNA (namely standard methylated DNA) with the methylation degree of 100% into the gold nanoparticle sol, incubating for 30min, testing the ultraviolet-visible light absorption spectrum of the system, and recording the wavelength of the maximum absorption peak as 520 nm; adding a purified MSP amplification product obtained by adopting standard DNA (namely standard non-methylated DNA) with the methylation degree of 0% into gold nanoparticle sol, incubating for 30min, adding 2 mu L of NaCl solution with the concentration of 5mol/L, reacting for 6min, testing the ultraviolet visible light absorption spectrum of the obtained system, and recording the wavelength of the maximum absorption peak, wherein the wavelength is 650 nm. Testing the obtained ultraviolet visible light absorption spectra of a series of systems with different colors, and recording the absorbance values at the 520nm and 650nm wavelengths to obtain a series of A₆₅₀/A₅₂₀Ratio, A series of₆₅₀/A₅₂₀The ratios were fitted to the corresponding percent methylation to generate a standard curve, as shown in FIG. 3. A. the₆₅₀/A₅₂₀The relationship between the ratio and the percentage of DNA methylation of the PAX1 gene is

Y＝1.45-0.03×X-1.22×X² (1)；

wherein X represents the percentage of DNA methylation and Y represents A of the system after 6 minutes of reaction₆₅₀/A₅₂₀a ratio. The established standard curve can be used for detecting the proportion of methylation in a DNA sample to be detected with unknown methylation degree, and only needs to detect the A of the system after NaCl is added for 6min₆₅₀/A₅₂₀ratio, inverse function substituted into equation (1)

M＝0.82×(1.769-1.22×A)^1/2-0.0123 (2)；

The percent methylation of the DNA to be tested can be calculated, where M is the percent methylation of the sample to be tested, and A is the detected A_650/A₅₂₀a ratio.

Further, step 6 includes passing violetDetecting the methylation percentage of the DNA to be detected by an external visible light spectrophotometry, which specifically comprises the following steps: and adding 5 mu L of the MSP product of the purified DNA to be detected into 20 mu L of gold nanoparticle sol in the kit, incubating for 30min, and then adding 2 mu L of NaCl solution with the concentration of 5 mol/L. Starting timing, reacting for 6 minutes, testing the ultraviolet visible light absorption spectrum of the obtained system, and calculating A₆₅₀/A₅₂₀The value, counted as A, is substituted into a standard curve chart, and the methylation percentage of the DNA to be detected is calculated to be 20.98% through equation (2), which is very close to the result of colorimetric detection.

example 2

The kit and the method for detecting the degree of DNA methylation provided in this example are the same as those in example 1, except that the DNA to be detected used in this example is obtained from exfoliated cervical cells of a patient with cervical cancer. Through colorimetric detection, the obtained system has unobvious color change after the NaCl solution is added, is still wine-red after 6 minutes, is very close to the color with the methylation percentage of 100 percent in a colorimetric card, and judges that the methylation degree of the DNA to be detected is close to 100 percent. And detecting the methylation percentage of the DNA to be detected by an ultraviolet-visible spectrophotometry, and calculating to obtain the methylation percentage of 85.23%.

Comparative example

The methylation percentage of the DNA to be detected in the embodiment 1 and the methylation percentage of the DNA to be detected in the embodiment 2 are respectively detected by a qMSP method by adopting a PAX1 methylation kit of Rixiang life science Limited company, the methylation percentage of the DNA to be detected in the embodiment 1 is 19.67 percent, the methylation percentage of the DNA to be detected in the embodiment 2 is 88.47 percent, the consistency of the detection result of the invention and the detection result of the existing method is higher by adopting the existing method, and the error is within an acceptable range, so that the feasibility and the accuracy of the method for detecting the DNA methylation degree provided by the invention are proved.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; such modifications and substitutions do not depart from the spirit and scope of the present invention, and they should be construed as being included in the following claims and description.

Claims

1. A kit for detecting DNA methylation degree based on a gold nanoparticle probe is characterized by comprising the gold nanoparticle probe, wherein the gold nanoparticle probe comprises gold nanoparticles and an upstream primer for carrying out methylation specific polymerase chain reaction, a sulfydryl group is modified at the 5' end of the upstream primer, the upstream primer is coupled with the gold nanoparticles through the sulfydryl group, and the particle size of the gold nanoparticles is 10-15 nm; the DNA is PAX1 gene, and the upstream primer corresponds to a promoter region of PAX1 gene; the upstream primer is SH-5 'CGGTTAGACGAATTTTTTTTAATCGGATGA-3'; the kit also comprises a downstream primer, and the sequence of the downstream primer is 5'-CCCGCGACCCCAAAC-3';

The preparation method of the gold nanoparticle probe comprises the following steps:

1) incubating the upstream primer and the gold nanoparticles at room temperature to obtain a first mixture; the molar ratio of the upstream primer to the gold nanoparticles is 95-105: 1, and the incubation time is 15-20 h;

2) adding a Tris-hydrochloric acid buffer solution and a NaCl solution into the first mixture, and then incubating under a dark condition to obtain a second mixture; wherein the incubation time is 20-30 h;

3) And centrifuging the second mixture to obtain the gold nanoparticle probe.

2. Use of the kit of claim 1 or the gold nanoparticle probe of claim 1 for the preparation of a system for detecting the degree of DNA methylation.