CN116814789B - System for cervical cancer high-grade lesion or cervical cancer diagnosis and related gene methylation qPCR detection kit - Google Patents

Abstract

The invention discloses a system for diagnosing cervical cancer high-grade lesions or cervical cancer and a related gene methylation qPCR detection kit. The combination of the specific markers of the kit realizes high-sensitivity and specificity cervical high-level lesion detection, so that cervical cancer can be discovered and intervened in early stage. In addition, the kit adopts enzymatic methylation detection to avoid the defect of severe reaction of the bisulfite, and greatly improves the sensitivity and specificity of cervical high-level lesion detection by optimizing conditions and interpretation rules of results.

Description

System for cervical cancer high-grade lesion or cervical cancer diagnosis and related gene methylation qPCR detection kit

The application is a divisional application of Chinese patent application 202211645332.6, the application date of the original application is 2022, 12 and 15, and the invention is named as a kit for detecting methylation qPCR of cervical high-grade lesion related genes and a using method and a system thereof, and the whole content of the kit is incorporated by reference.

Technical Field

The invention relates to the cervical lesion detection field, in particular to a system for diagnosing cervical cancer high-grade lesions or cervical cancer and a related gene methylation qPCR detection kit.

Background

Cervical cancer is one of the common female malignancies, which has a long history of progression from precancerous lesions to invasive cancers, with a latency period of 5-10 years. The standardized physical examination can prevent more than 90% of cervical cancer and kill the sprouting state of 'precancerous lesions'. The cervical cancer screening method commonly used in clinic at present comprises visual inspection of acetic acid staining, cytology inspection and HPV DNA detection. The sensitivity of HPV DNA detection is higher, but most HPVs are general infection, and the HPV does not develop into cervical precancerous lesions or cervical cancer, so that unnecessary panic is caused for women, and medical resources are wasted. Thus, there is a need for a biological marker that can identify truly high risk.

The research shows that the methylation level of host genes increases with the increase of cervical lesion severity, and can be used as a biological marker for screening cervical cancer and precancerous lesions. Methods for diagnosing cervical cancer-related diseases based on methylation levels have been disclosed. For example, chinese patent publication CN111893183a discloses a DNA methylation qPCR kit for cervical cancer detection and methods of use. The kit screens and diagnoses cervical cancer by detecting or measuring the methylation state or level of one or more specific genes in the DNA of a sample to be tested, and can diagnose human cervical cancer in time in early stage, so that early and accurate diagnosis of cervical cancer is possible.

For another example, chinese patent publication CN107653322a discloses a kit and method for detecting cervical lesions and cervical cancer related gene methylation, the kit comprises reagents including a PCR reaction solution, a positive control, a negative control, and a cell lysate for extracting sample DNA; the detection method comprises the steps of extracting sample DNA, processing and purifying the sample DNA, preparing a PCR reaction system and adding samples, carrying out PCR amplification reaction, collecting fluorescent signals, obtaining an analysis amplification curve and a melting curve, obtaining a Ct value and a Tm value of each marker through the analysis amplification curve and the melting curve, preparing a positive judgment method based on a weighting algorithm, carrying out positive judgment and integration on each marker, and finally explaining a sample result. The kit has the characteristics of high sensitivity, strong specificity, no wound and the like.

However, the above methods are all based on methylation detection of bisulfite conversion, and since the number of unmethylated C in the gene is large, there is base imbalance in the gene obtained based on bisulfite conversion, which affects the PCR reaction and detection result, and the bisulfite method is a chemical method, and has a large damage to DNA.

The information in the background section is only for the purpose of illustrating the general background of the invention and is not to be construed as an admission or any form of suggestion that such information forms the prior art that is well known to those of ordinary skill in the art.

Disclosure of Invention

In order to solve at least part of the technical problems in the prior art, the invention provides an enzymatic-based cervical lesion and cervical cancer related gene methylation detection test, which has milder reaction conditions and greatly improves the sensitivity and specificity of enzymatic detection through optimization. Specifically, the present invention includes the following.

In a first aspect of the invention, there is provided a cervical high-grade lesion-associated gene methylation qPCR assay kit comprising a reagent strip, a container 1 and a container 2; wherein the reagent strip is provided with at least one small tube, a methylation specific primer pair containing mismatched bases is fixed on the inner wall of the small tube, the container 1 contains buffer solution containing 3-morpholinopropane sulfonic acid, alpha-KG, naCl, dithiothreitol and ascorbic acid, and the container 2 contains 2-methylpyridine-borane. The mismatched base herein refers to a base corresponding to a wild type, and generally corresponds to a methylation site of the wild type, particularly a methylated C base. The mismatched base at this time is referred to as a T base. The small tube can be fixedly connected with the reagent strip or can be detachably connected with the reagent strip. The mismatched base is preferably located at the 3' end of the primer.

In certain embodiments, the cervical high-grade lesion-associated gene methylation qPCR detection kit according to the present invention, wherein the kit is provided with at least 6 vials, and the inner wall of each vial is immobilized with a primer pair corresponding to one methylation site or gene, respectively.

In certain embodiments, the cervical high grade lesion related gene methylation qPCR detection kit according to the present invention, wherein the methylation site or gene is selected from ZNF671, ASTN1, DLX1, ITGA4, RXFP3, SOX17.

In certain embodiments, the cervical high grade lesion related gene methylation qPCR detection kit according to the present invention, wherein the primer pair is selected from the sequences set forth in SEQ ID NOs 1-12.

In certain embodiments, the cervical high grade lesion related gene methylation qPCR assay kit according to the present invention further comprises a container 3 containing a TET enzyme and β -glucosyltransferase complex enzyme.

In certain embodiments, the cervical high grade lesion related gene methylation qPCR detection kit according to the present invention further comprises a container 4 containing a ferrous ammonium sulfate solution.

In certain embodiments, the cervical high-grade lesion-associated gene methylation qPCR detection kit according to the present invention, further comprises a container 5 containing a PCR reaction solution.

In a second aspect of the present invention, there is provided a reaction system for methylation qPCR detection of cervical high-grade lesions related genes, comprising at least one of reaction solution 1, reaction solution 2 and reaction solution 3, wherein:

the reaction solution 1 contains 40-60mM 3-morpholinopropane sulfonic acid, 0.5-1.5mM alpha-KG, 40-60mM NaCl, 0.5-1.5mM dithiothreitol and 1.5-2.5mM ascorbic acid, 0.1-1mM TET enzyme, 0.1-1mM beta-glucosyltransferase and 90-150mM ferrous ammonium sulfate. The concentration of 3-morpholinopropionic acid is preferably 45 to 55mM, more preferably 50 to 55mM; the concentration of α -KG is preferably 0.8 to 1.3mM, more preferably 1 to 1.2mM; the concentration of NaCl is preferably 45 to 55mM, more preferably 50 to 55mM; the concentration of dithiothreitol is preferably 0.6 to 1.2mM, more preferably 0.8 to 1mM; the concentration of ascorbic acid is preferably 1.2-2mM, more preferably 1-1.5mM; the concentration of TET enzyme is preferably 0.2-0.8mM, more preferably 0.4-0.6mM; the concentration of beta-glycosyltransferase is preferably 0.2-0.8mM, more preferably 0.4-0.6mM; the concentration of ferrous ammonium sulphate is preferably 95-120mM, more preferably 100-110mM. The TET enzyme and the beta-glucosyltransferase constitute a complex enzyme, and the molar ratio of the two enzymes is generally (0.8-1.2): 1, such as 1:1.

The reaction liquid 2 contains 1-10M 2-methylpyridine-borane and 1-5M sodium acetate;

the reaction solution 3 contains dNTPs, taq enzyme, dye and solvent, preferably further contains magnesium ions, and the concentration of the magnesium ions in the reaction solution 3 in the present application is higher than that of the conventional qPCR under the same reaction conditions.

In a third aspect of the invention, there is provided a method of using the kit of the first aspect, comprising the steps of:

constructing a reaction solution 1, a reaction solution 2 and a reaction solution 3, wherein the reaction solution 1 comprises 40-60mM 3-morpholinopropane sulfonic acid, 0.5-1.5mM alpha-KG, 40-60mM NaCl, 0.5-1.5mM dithiothreitol and 1.5-2.5mM ascorbic acid, 0.1-1mM TET enzyme, 0.1-1mM beta-glucosyltransferase and 90-150mM ferrous ammonium sulfate, the reaction solution 2 comprises 1-10M 2-picoline-borane and 1-5M sodium acetate, and the reaction solution 3 comprises dNTPs, taq enzyme, dye and solvent;

adding the extracted DNA into the reaction solution 1, reacting for 1h-3h at 25-40 ℃, then adding proteinase K, reacting for 0.5h-1h at 35-45 ℃, and purifying the DNA;

adding the purified DNA into the reaction solution 2 to react for 10 to 20 hours at the temperature of between 25 and 40 ℃ and further purifying to obtain the treated DNA;

the reaction solution 3 and the treated DNA are added into a small tube of a reagent strip respectively or simultaneously, and amplified by fluorescence quantitative PCR after sealing.

In a fourth aspect of the present invention, there is provided a system for diagnosis of cervical cancer high-grade lesions or cervical cancer, comprising:

a. a data acquisition unit for acquiring data of an analytical amplification curve and a melting curve obtained by using the kit according to any one of claims 1 to 7 or Ct value and Tm value further obtained therefrom;

b. a data storage unit for storing at least the acquired Ct value and Tm value data, and a positive determination range of the Ct value and Tm value of the marker gene and a rule for giving the marker gene;

c. the data processing unit is used for retrieving the data acquired by the data acquisition unit, carrying out positive judgment according to the Ct value and the Tm value of the corresponding marker genes, respectively giving out the corresponding scores of the marker genes, and calculating the sum of the scores of the marker genes; if the sum of the scores is smaller than the sample positive judgment value, the sample detection result is negative; if the sum of the scores is greater than or equal to the sample positive judgment value, the sample detection result is positive, and the cervical cancer transformation risk is prompted to be high;

d. and an output unit for outputting the result of the data processing unit.

The kit adopts the combination of specific markers, can detect cervical high-grade lesions (CIN III) with high sensitivity and specificity, and can discover and intervene cervical cancer treatment in an early stage. The kit adopts an enzyme method to avoid severe chemical reaction conditions of the bisulfite. The harsh reaction conditions lead to degradation of the template DNA, which severely affects the detection amount of the sample and the detection sensitivity of qPCR, which is very disadvantageous for detection of fewer samples. Second, the bisulfite method relies on the complete conversion of unmodified cytosines to thymines, which account for about 95% of the total cytosines in the human genome, and converting all of these positions to thymines severely reduces sequence complexity, affecting the choice of sites for primer design. In addition, the bisulfite method is also prone to false detection of 5mC and 5 hmC. The enzymatic methylation detection of the present invention can overcome the above-described problems. Although the conversion efficiency of the enzymatic method to methylated C base is lower than that of the bisulfite method, the application greatly improves the sensitivity and specificity of cervical high-level lesion detection by optimizing conditions and result interpretation rules.

Drawings

FIG. 1ASTN1 sequence and primer positions.

FIG. 2DLX1 sequence and primer correspondence.

FIG. 3ITGA4 sequence and primer corresponding positions.

FIG. 4RXFP3 sequence and primer corresponding positions.

FIG. 5SOX17 sequence and primer correspondence.

FIG. 6ZNF671 sequence and primer corresponding positions.

FIG. 7QC sequence and primer correspondence.

FIG. 8QM sequence and primer correspondence.

Detailed Description

Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in the present invention, it is understood that the upper and lower limits of the ranges and each intermediate value therebetween are specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control. Unless otherwise indicated, "%" is percent by weight.

Example 1

The embodiment is an exemplary cervical high-grade lesion related gene methylation Qpcr detection kit, which comprises an octant tube and various solutions or reagents: TET enzyme buffer (pH 8.0), reducing agent solution, complex enzyme, ferrous ammonium sulfate solution and PCR reaction solution, which are independent of each other.

In this embodiment, the eight-connected pipes are 12 eight-connected pipes, and include 12 corresponding pipe covers. Wherein, 10 green mark eight allies oneself with the pipe for sample detection, 1 red mark eight allies oneself with the pipe and is used for detecting positive quality control, and 1 yellow mark eight allies oneself with the pipe and is used for detecting negative quality control. The eight-connected tubes are respectively provided with 8 fixed small tubes, which are respectively numbered 1-8, and different primers are fixed in the eight-connected tubes. A primer set for ASTN1 (star actin 1), a primer set for DLX1 (homologous transcription factor 1), a primer set for ITGA4 (integrin. Alpha.4), a primer set for RXFP3 (relaxin family receptor 3), a primer set for SOX17 (SRY-Box 17), a primer set for ZNF671 (zinc finger protein 671), a primer set for QC (TET enzyme treatment quality control) for 7, and a primer set for QM (methylation quality control) for 8. See in particular the following table. Quality control QC and QM were used as internal parameters for PCR amplification.

TABLE 1 primer pair information in eight-tube tubules

TET enzyme buffer (pH 8.0, 1X) contained 50mM 3-morpholinopropane sulfonic acid, 1mM alpha-KG, 50mM NaCl, 1mM dithiothreitol, and 2mM ascorbic acid.

The kit may further comprise a reagent bottle for containing a complex enzyme including TET enzyme and beta-glucosyltransferase.

The kit may further comprise a reagent bottle for containing a reducing agent, wherein the reducing agent comprises a 2-methylpyridine-borane solution having a concentration of 8M 2-methylpyridine-borane. Optionally, a sodium acetate solution is also included, at a concentration of 3M.

The kit can also comprise a reagent bottle for containing the ferrous ammonium sulfate solution, wherein the concentration of the ferrous ammonium sulfate is 200Mm.

The kit can also comprise a PCR reaction liquid, and compared with the common PCR reaction liquid (such as 200 mu m Dntp, 1 mu m magnesium ion and a proper amount of dye), the PCR reaction liquid contains magnesium ion with higher concentration.

In addition to the above description, other reagents in this embodiment are conventional reagents or amounts, and the kit may further include conventional reagents known in the art, which are not specifically described herein.

Example 2

1. DNA extraction

DNA was extracted using cervical exfoliated cells or tissues as a sample. The specific method comprises the following steps:

(1-1) vortexing the samples at maximum speed for 5+ -1 seconds, and immediately transferring 1Ml of sample into a 1.5Ml EP tube;

(1-2) 10000 Xg, centrifuging for 5 minutes;

(1-3) carefully remove supernatant, removing up to 980 ml;

(1-4) adding 40 ml of cell lysate to the sample precipitate and vortexing at maximum speed for 3+ -1 seconds;

(1-5) placing the centrifuge tube with the mixture in a constant temperature shaking device, and incubating at 60 ℃ and 1000rpm for 30 minutes to obtain sample DNA;

2. TET enzyme treatment and reduction of DNA

(2-1) the DNA obtained in step 1 was added to the reaction solution 1 of the EP tube, reacted at 37℃for 1-2 hours, followed by addition of proteinase K, and further incubation at 40℃for 1 hour.

TABLE 2 composition of reaction solution 1

(2-2) adding purified magnetic beads (MagMAX), mixing thoroughly, incubating at room temperature for 20min, taking EP tube on a magnetic rack, and removing supernatant.

(2-3) washing with ethanol, removing ethanol by suction, drying at room temperature, and eluting DNA by adding 50. Mu.l of DDW.

(2-4) 35. Mu.l of the DNA of step (2-3) was taken, 5. Mu.l of 2-methylpyridine-borane solution and 10. Mu.l of sodium acetate solution were added, and incubated at room temperature for 20 hours under stirring, and the DNA was isolated and purified by centrifugation.

Step 2 in this application allows the complete operation to be accomplished in one tube without the need to replace the small tube.

3. Methylation detection of cervical high-grade lesion related genes

(3-1) preparing a PCR reaction system and adding samples:

70. Mu.L of PCR water was added to each DNA sample, the DNA samples were vortexed at maximum speed for 3.+ -.1 sec, centrifuged, the PCR reaction solution (appropriate amount of magnesium ions was added to a concentration of 2 mM) and PCR octants were taken out from the kit, and the PCR octants were placed on a PCR tube rack, wherein a green label octant was used for detecting the samples, a red label octant was used for positive quality control, and a yellow label octant was used for negative quality control. Note that: in the requirement of the arrangement direction of the PCR eight-connection tube, the protruding part on the tube cover is required to be on the left side, in addition, the reaction tubes of the PCR eight-connection tube are provided with numbers 1-8, the reaction tube 1 is required to be on the A row of the PCR tube frame, and the reaction tube 8 is positioned on the H row of the PCR tube frame.

The PCR reaction solution was vortexed for 3.+ -.1 sec, centrifuged briefly, and the tube caps of all PCR octants were opened and discarded. 10. Mu.L of each PCR reaction solution was added to each of the reaction tubes of the eight PCR tubes. Note that: because the reaction tube of each PCR octant contains the primer, the gun head must be replaced every time the liquid is added.

mu.L of DNA was added to each of the PCR octant reaction tubes. Note that: because the reaction tube of each PCR octant already contains the primer, the gun head must be changed every time the sample is added.

After the sample is added, all the PCR eight-joint tubes are directly covered with a flat top transparent cover and sealed. Mix all PCR octants 3±1 seconds with maximum speed vortex and briefly centrifuge. Note that: the tube cap is removed and capped without touching the inside of the tube cap and is checked from the side to ensure the tube cap is closed.

(3-2) qPCR amplification

Before the PCR eight-joint tube is put into a fluorescent quantitative PCR instrument, the PCR reaction software and parameter setting are finished. Note that: the configured reaction system may be stored in the refrigerator for no more than 15 minutes during the PCR system set-up. The following settings were made for the instrument according to the instructions provided by the fluorescent quantitative PCR instrument manufacturer: reference fluorescence (Reference Dye): ROX; 20 μl per well reaction volume; collecting signals: "stage 2" (30 second hold) and "stage 3" (1% heat up rate).

Using a fluorescent quantitative PCR instrument, PCR amplification was performed on the PCR reaction system to which DNA was added according to the PCR amplification procedure shown in the following table:

TABLE 3PCR amplification procedure

* The fluorescence signal was collected during the incubation period of 30 seconds at 67℃in stage 2 and during the heating period of 30 seconds before the reaction at 97℃in stage 3, and thus an analytical amplification curve and a melting curve were obtained.

4. Interpretation of test results

On a fluorescent quantitative PCR apparatus, an analysis threshold suitable for a sample and a cycle starting and ending baseline are set. Main pair is stored in STM ^TM The samples in (1) are tested and analysis threshold values, start and end cycle reaction baselines applicable to the samples are determined.

The Ct value and the Tm value of markers DLX1, ITGA4, RXFP3, SOX17, ZNF671, ASTN1, QC and QM are respectively obtained by detecting the embedded fluorescent dye and defining the peak value of a melting curve and analyzing an amplification curve and the melting curve; then, different positive judgment methods based on a weighting algorithm are prepared:

TABLE 4 Table 4

Marker(s)	Ct value	Tm value range (. Degree. C.)
			ASTN1	<42	57.40-57.96
DLX1	<42	58.27-67.73
			ITGA4	<42	60.92-65.36
RXFP3	<42	53.41-62.13
			SOX17	<42	53.41-68.12
ZNF671	<42	57.55-67.42
			Quality control QC	≤36	63.95-65.66
Quality control QM	<42	65.77-68.72

Corresponding scores are assigned to DLX1, ITGA4, RXFP3, SOX17, ZNF671, ASTN1, respectively, according to the following table:

TABLE 5

Marker(s)	Score of
		ASTN1	1
DLX1	1
		ITGA4	1
RXFP3	1
		SOX17	1
ZNF671	3

The Ct values and Tm values of 6 cervical lesion markers DLX1, ITGA4, RXFP3, SOX17, ZNF671, ASTN1 were analyzed using the positive determination ranges of the Ct values and Tm values of the respective markers given in table 4. If the Ct value and the Tm value of a certain marker are within the positive determination range, the marker is defined as positive, and the corresponding score given by the marker in Table 5 is obtained. If the Ct value and the Tm value of a certain marker are out of the positive determination range, the marker is defined as negative, and the obtained score is zero. From this, the total score of 6 marker combinations from DLX1, ITGA4, RXFP3, SOX17, ZNF671, ASTN1 can be calculated. If the sum of the scores is less than 3, the sample detection result is negative; if the sum of the scores is more than or equal to 3, the sample detection result is positive, the risk of suffering from high-level cervical lesions is high, and the tissue pathology detection result needs to be further checked and combined.

5. Result verification

The present application is a kit for detecting methylation based on a TET enzymatic method, which enables efficient detection of methylation without affecting unmodified cytosines by using mild enzymatic and chemical reactions, as compared to the bisulfite-based kits developed by the applicant.

In addition, the inventors compared the detection results of the kit of the present application with the bisulfite-based kit. By optimizing conditions and result interpretation rules, the detection sensitivity of the kit is equivalent to that of the bisulfite kit, and the specificity is obviously higher than that of the bisulfite kit.

TABLE 6 comparison of the detection results for different kits

As shown in Table 6, the TET enzymatic method of the present application has excellent detection sensitivity for CIN II and above cervical lesions, while the detection rate for CIN I is lower, and the difference between the detection rate and CIN II and above cervical lesions is obvious. The detection result is equivalent to that of the bisulfite method, and the specificity of the TET enzymatic method is higher.

While the invention has been described with reference to exemplary embodiments, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. Various modifications or changes may be made to the exemplary embodiments of the present disclosure without departing from the scope or spirit of the invention. The scope of the claims is to be accorded the broadest interpretation so as to encompass all modifications and equivalent structures and functions.

Claims (5)

1. A system for cervical cancer lesions or diagnosis of cervical cancer above CIN II, comprising:

a. a data acquisition unit for acquiring data of Ct value and Tm value obtained by using a quantitative polymerase chain reaction (qPCR) detection kit for cervical cancer lesion related genes of CIN II or more, wherein the kit comprises a reagent strip, a container 1, a container 2 and a container 3, the reagent strip is provided with at least one small tube, a methylation specific primer pair containing mismatched bases is fixed on the inner wall of the small tube, the container 1 contains 40-60mM of TET enzyme buffer containing 3-morpholinopropane sulfonic acid, 0.5-1.5mM of alpha-KG, 0.5-60 mM of NaCl, 0.5-1.5mM of dithiothreitol and 1.5-2.5mM of ascorbic acid, the container 2 contains 2-methylpyridine-borane, the container 3 contains TET enzyme and beta-glucosyltransferase complex enzyme, genes of which methylation sites are ZNF671, ASTN1, X1, RXGA 4, FP3 and SOX17, and the primer pair shown in SEQ ID 12;

b. a data storage unit for storing at least the acquired Ct value and Tm value data, and a positive determination range of the Ct value and Tm value of the marker gene and a rule for giving the marker gene;

c. the data processing unit is used for retrieving the data acquired by the data acquisition unit, carrying out positive judgment according to the Ct value and the Tm value of the corresponding marker genes, respectively giving out the corresponding scores of the marker genes, and calculating the sum of the scores of the marker genes; if the sum of the scores is smaller than the sample positive judgment value, the sample detection result is negative; if the sum of the scores is greater than or equal to the sample positive judgment value, the sample detection result is positive, the cervical cancer transformation risk is high, and the positive judgment ranges of the Ct value and the Tm value are as follows:

，

the rules for the assignment of marker genes are: the score values of DLX1, ITGA4, RXFP3, SOX17 and ASTN1 are 1, respectively, and ZNF671 is 3;

d. and an output unit for outputting the result of the data processing unit.

2. A methylation qPCR kit for detecting cervical lesions above CIN II or cervical cancer, characterized in that the kit comprises a reagent strip, a container 1, a container 2 and a container 3; wherein the reagent strip is provided with at least one small tube, the inner wall of the small tube is fixed with a methylation specific primer pair containing mismatched bases, the container 1 contains 40-60mM TET enzyme buffer containing 3-morpholinopropane sulfonic acid, 0.5-1.5mM alpha-KG, 40-60mM NaCl, 0.5-1.5mM dithiothreitol and 1.5-2.5mM ascorbic acid, the container 2 contains 2-methylpyridine-borane, the container 3 contains TET enzyme and beta-glucosyltransferase complex with the molar ratio of (0.8-1.2) 1, genes of the methylation sites are ZNF671, ASTN1, DLX1, ITGA4, RXFP3 and SOX17, and the sequences of the primer pair are shown as SEQ ID NO: 1-12.

3. The kit according to claim 2, wherein the reagent strip is provided with at least 6 vials, and each vial has a primer pair corresponding to one methylation site immobilized on an inner wall thereof.

4. The kit of claim 2, further comprising a container 4 containing a ferrous ammonium sulfate solution.

5. The kit according to claim 3, further comprising a container 5 for containing the qPCR reaction solution.