Kit and system for methylation detection of cervical cancer related genes

Abstract

The invention belongs to the technical field of gene methylation detection, and particularly relates to a cervical cancer related gene methylation detection kit and system, which comprises primers and probes for detecting APC, CALCA, DAPK and ESR1 gene methylation, primers and probes for monitoring internal control genes, a PCR reaction liquid, a positive quality control product and a negative quality control product, the use method comprises the following steps: 1) performing nucleic acid extraction on a sample; (2) converting the extracted genome DNA by bisulfite; (3) premixing and subpackaging the gene methylation detection reagent to obtain a PCR reaction system; (4) adding the DNA obtained in the step (2) into the PCR reaction system in the step (3), uniformly mixing, and carrying out PCR amplification; 5, gene methylation interpretation is carried out after the reaction is ended, the method has the advantages of being high in sensitivity, low in cost, easy and convenient to operate, short in detection period, visual in interpretation and the like, and methylation of the cervical cancer related genes can be rapidly and accurately detected.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of gene methylation detection, and particularly relates to a kit and system for methylation detection of a cervical cancer related gene. BACKGROUND

[0002] Cervical cancer is one of the most common malignant tumors in women, and has always been ranked first in gynecological malignant tumors. More than 90% of cervical cancer is caused by HPV infection. Persistent high-risk HPV infection can cause cervical mucosa cells to mutate into cancer cells, forming cervical cancer. Once cervical cancer spreads, only 20% of women can survive for more than 5 years. Globally, there is no good solution to viral clearance for high-risk HPV infection for a long time, and the prognosis is poor. At present, HPV prevention and control still mainly relies on detection screening. According to the screening strategy of cervical cancer, there is a great opportunity to find the problem when it is still in the precancerous lesion, and then solve it as soon as possible. The cure rate of early cervical precancerous lesions is 100%. Active and effective development of cervical cancer screening, early detection, early diagnosis and early intervention can significantly reduce the incidence and disease burden of cervical cancer.

[0003] Cervical cytology and / or hrHPV detection is the main screening method. However, both cytology and hrHPV detection have certain limitations in diagnostic accuracy. DNA methylation is an epigenetic mechanism that can cause genetic silencing without changing its coding sequence. According to a number of research reports, more than 100 human (host) gene methylation is considered to be a biomarker of cervical cancer, and a part of them has been verified to be associated with the development of cervical cancer. At present, the existing technology discloses a gene methylation detection kit related to cervical cancer, which is focused on PAX1, JAM3, FAM19A4, SOX1, EPB41L3 and ZNF671, etc. such as CN201910898042, CN202110287291, CN202110489434, CN202210054100, CN202210107725, CN202310072061, CN202310390823, etc. Therefore, the inventors expect to find new molecular indicators closely related to precancerous lesions to make up for the deficiencies in the currently used cervical cancer screening methods in China. Finding and researching new methylation markers specific to diseases can provide new ideas for cervical cancer screening, diagnosis and treatment. SUMMARY

[0004] In order to solve the problems in the prior art, the present application aims at the clinical needs at the present stage and the deficiencies of the prior art, and provides a kit for detecting cervical cancer related gene methylation and a use method based on qMSP technology and TaqMan fluorescent probe technology.The kit has the characteristics of high sensitivity, low cost, simple operation, short detection period, intuitive judgment, etc., and can quickly and accurately detect cervical cancer related APC, CALCA, DAPK and ESR1 gene methylation.

[0005] The present application solves the technical problems by adopting the following technical solutions:

[0006] The present application aims to provide a kit for detecting cervical cancer related gene methylation, which comprises primers and probes for detecting APC, CALCA, DAPK and ESR1 gene methylation.

[0007] Further, the primer and probe sequence for detecting APC gene methylation are as follows: the forward primer sequence is as shown in SEQ ID NO. 6, the reverse primer sequence is as shown in SEQ ID NO. 7, and the nucleic acid sequence of the fluorescent probe is as shown in SEQ ID NO. 8.

[0008] The primer and probe sequence for detecting CALCA gene methylation are as follows: the forward primer sequence is as shown in SEQ ID NO. 9, the reverse primer sequence is as shown in SEQ ID NO. 10, and the nucleic acid sequence of the fluorescent probe is as shown in SEQ ID NO. 11.

[0009] The primer and probe sequence for detecting DAPK gene methylation are as follows: the forward primer sequence is as shown in SEQ ID NO. 12, the reverse primer sequence is as shown in SEQ ID NO. 13, and the nucleic acid sequence of the fluorescent probe is as shown in SEQ ID NO. 14.

[0010] The primer and probe sequence for detecting ESR1 gene methylation are as follows: the forward primer sequence is as shown in SEQ ID NO. 15, the reverse primer sequence is as shown in SEQ ID NO. 16, and the nucleic acid sequence of the fluorescent probe is as shown in SEQ ID NO. 17.

[0011] Further, the 5' end fluorescent group of the probe for APC, CALCA, DAPK and ESR1 gene methylation is one of FAM, VIC and CY5, and the 3' end quenching group is MGB or BHQ2.

[0012] Further, the kit further comprises primers and probes for monitoring the internal control gene GAPDH, and the primer and probe sequence for detecting the internal control gene GAPDH are as follows: the forward primer sequence is as shown in SEQ ID NO. 18, the reverse primer sequence is as shown in SEQ ID NO. 19, and the nucleic acid sequence of the fluorescent probe is as shown in SEQ ID NO. 20.

[0013] Further, the kit further comprises a PCR reaction solution, a positive quality control and a negative quality control, the PCR reaction solution comprising Taq enzyme, UNG enzyme, buffer, magnesium ions and dNTP substances.

[0014] The method for obtaining the positive quality control is as follows: according to the sequence information of the APC, CALCA, DAPK and ESR1 genes and the promoter region sequence information published by the NCBI database, a plasmid is constructed to synthesize a sequence gene fragment after bisulfite treatment, the fragment is then inserted into a T vector, and the plasmid is transformed by using an Escherichia coli DH5α strain and extracted, and the plasmids are mixed in equal proportions to obtain the positive quality control.

[0015] The negative quality control is DEPC-treated deionized water.

[0016] A system for detecting cervical cancer-related gene methylation comprises the kit, a PCR amplification system and a genotyping interpretation system.

[0017] Further, the reagents in the kit are pre-mixed and divided to obtain PCR reaction solution 1 and PCR reaction solution 2 to form a PCR reaction system.

[0018] Further, the conditions for PCR amplification in the PCR amplification system are as follows: the conditions for UNG enzyme reaction are 25°C for 10 minutes; the conditions for UNG enzyme inactivation are 95°C for 2 minutes;

[0019] The conditions for system amplification are a two-step method: denaturation at 95°C for 5 seconds; annealing and extension at 58°C for 30 seconds, with fluorescence signal collection; and the number of cycles is set to 45.

[0020] Further, the genotyping interpretation system comprises: using the fluorescence detection result to determine the sample to be detected, when GAPDH Ct≤34, calculating the methylation index (MI) of each gene according to the ΔCt (Ct 靶基因 -Ct 内参基因 ) of the APC, CALCA, DAPK and ESR1 genes, and the assignment method is as follows:

[0021]

[0022] The MI values of the four genes are added to obtain a total MI value, and when the total MI value≥6, the sample is determined to be methylation positive for cervical cancer, and when the total MI value<6, the sample is determined to be methylation negative for cervical cancer; when GAPDH Ct>34, it is determined that the sample is insufficient or that there is an inhibitor, and the PCR reaction is invalid, and the experiment or sampling needs to be repeated.

[0023] A method for using a kit for detecting cervical cancer-related gene methylation comprises the following steps:

[0024] 1) Take the sample containing cervical exfoliative cells for nucleic acid extraction;

[0025] 2) Transform the genomic DNA into B-DNA using bisulfite;

[0026] 3) Pre-mix and sub-pack the gene detection reagent to obtain PCR reaction liquid 1 and PCR reaction liquid 2 respectively to form a PCR reaction system;

[0027] 4) Add the DNA obtained in step 2) into the PCR reaction system in step 3), mix well and then perform PCR amplification;

[0028] 5) Perform genotyping interpretation after the reaction is completed.

[0029] Compared with the prior art, the beneficial technical effects of the present application are:

[0030] (1) The four cervical cancer-related gene methylation detection reagents are sub-packed in a 2-reaction PCR reaction strip, each reaction strip includes 1 human detection reagent, pre-mixed and packaged, convenient for detection, realizing multi-gene methylation analysis of one reaction system, reducing the cost while improving the detection efficiency, detecting two methylation sites in the same tube, saving the sample.

[0031] (2) The present application proposes four new methylation sites related to cervical cancer, which is a supplement to the methylation detection scheme related to cervical cancer. The present application precisely detects the methylation of cervical cancer-related genes, has high sensitivity, low cost, simple operation, short detection period, intuitive interpretation and other characteristics.

[0032] The above description is only a summary of the technical scheme of the present application, in order to more clearly understand the technical means of the present application, which can be implemented according to the content of the specification, and in order to make the above content and purposes, characteristics and advantages of the present application more obvious and easy to understand, the following specific embodiments of the present application are described. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 is the prediction of the CpG island of the APC gene in the present application and the result of the methylation primer design. DETAILED DESCRIPTION

[0034] The technical scheme of the present application will be further described in detail below in combination with specific embodiments. It should be understood that the following embodiments are only exemplary to illustrate and explain the present application, and should not be interpreted as limiting the scope of protection of the present application. Any technology realized based on the above content of the present application is covered within the scope of protection intended by the present application.

[0035] In addition, unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present application can be purchased or prepared by existing methods.

[0036] The present application screens four genes closely related to cervical cancer by analyzing the methylation degree of genes related to cervical cancer samples in clinic, and prepares a primer probe composition for detecting the methylation of the four genes and a detection kit using the composition, and establishes an accurate detection system for detecting the methylation of genes related to cervical cancer, which is expected to realize early screening of cervical cancer.

[0037] Example 1

[0038] This example is directed to the design of primers for APC, CALCA, DAPK and ESR1 genes and their promoter regions, and each gene selects a highly methylation-differentiated region, and designs a GAPDH primer and probe for the internal reference gene. The target region sequence (Table 1) and the primer sequence (Table 2) are as follows:

[0039] Table 1. Sequence information of target region of each gene

[0040]

[0041] Table 2. Primer and probe sequence information for detecting methylation of each gene

[0042]

[0043]

[0044] Example 2: Establishment of kit detection system

[0045] First, the sequence information of the CpG island site of APC, CALCA, DAPK and ESR1 genes and their promoter regions on NCBI was sorted out using an online database, and the sequence after sodium bisulfite conversion was synthesized into a plasmid, and the four plasmids were diluted to 10 -4 ng / μL concentration to simulate methylation samples.

[0046] According to the sequence characteristics of the genes and their promoter CpG island regions, the methylation primer design tool MathPrimer was used to design the primers for sample detection, and the probes for methylation detection were designed according to the probe design principles, and the TaqMan probes, forward primers and reverse primers for the PCR detection system were obtained. The primers and probes were synthesized by Shengong Bioengineering (Shanghai) Co., Ltd.

[0047] Two multiplex PCR detection systems for detecting APC, CALCA, DAPK and ESR1 gene sites were prepared respectively, wherein the APC and CALCA gene systems share one tube (reaction system 1), and the DAPK and ESR1 gene systems share one tube (reaction system 2). The reaction system configuration is shown in Table 3:

[0048] Table 3. Kit detection system configuration list

[0049]

[0050]

[0051] The prepared detection system was subjected to PCR amplification, and the PCR reaction program was as follows:

[0052] First stage: 25℃, 10 minutes;

[0053] Second stage: 95℃, 2 minutes;

[0054] Third stage: 95℃, 5 seconds; 58℃, 30 seconds, cycle number setting 45; collect fluorescence at 58℃.

[0055] Experimental results: PCR was performed on the above primers and probes, and the CT value of the detection system was directly read, and the experimental results of each group are shown in Table 4:

[0056] Table 4

[0057] Gene site Ct value ΔCt GAPDH 32.42 - APC 32.70 0.28 CALCA 30.81 -1.61 DAPK 33.88 1.46 ESR1 32.16 -0.26 .

[0058] According to the ΔCt results, the system in this embodiment can effectively detect the simulated cervical cancer methylation sample.

[0059] Example 3: Use of gene detection kit

[0060] The present application provides a fluorescent quantitative PCR kit for detecting cervical cell methylation genes, and optimized primers and probes are designed for one or a combination of APC gene, CALCA gene, DAPK gene and ERS1 gene, which can simultaneously detect cervical cell methylation genes through a reaction system. The kit of the present application can quickly, sensitively and specifically detect cervical cell methylation genes. The typical use method of the kit is as follows:

[0061] 1. Sample collection and processing

[0062] Cervical exfoliated cells of the person to be tested were collected, and nucleic acid extraction was performed using the blood / cell / tissue genomic DNA extraction kit (DP304) of Tiangen Biochemical. The experimental steps are described in the instruction manual.

[0063] 2. DNA transformation

[0064] The genomic DNA in Step 1 was transformed into DNA template to be tested using the DNA Bisulfite Conversion Kit (Spin Column Type) (DP215-02) of Tiangen Biochemical. The specific experimental steps were referred to the instruction manual.

[0065] 3. Preparation of reaction system

[0066] The reaction system was prepared according to the PCR reaction liquid 1 and 2 in Example 2.

[0067] 4. PCR reaction

[0068] The DNA extracted from the sample was added to the prepared reaction system, and the amount of template added was 1-200 ng. When performing the PCR reaction, the sample to be tested, positive quality control and negative quality control were detected together in parallel, and two reaction liquids were added to each sample for reaction.

[0069] 5. Selection of instrument channel and reaction volume

[0070] ① Select FAM channel (Reporter: FAM, Quencher: MGB), VIC channel (Reporter: VIC, Quencher: MGB) and CY5 channel (Reporter: CY5, Quencher: BHQ2) to detect amplification.

[0071] ② The reaction volume (Sample Volume) was 25 μL.

[0072] ③ Reference fluorescence (Reference Dye): If an ABI series PCR instrument is used, please make sure to select "none" for passive reference; the specific detection channel settings can be referred to the instrument instruction manual.

[0073] 6. PCR amplification

[0074] First stage: 25°C, 10 minutes;

[0075] Second stage: 95°C, 2 minutes;

[0076] Third stage: 95°C, 5 seconds; 58°C, 30 seconds, cycle number set to 45; collect fluorescence at 58°C.

[0077] 7. Experimental results

[0078] After the reaction program was completed, the results were saved, the ΔCt corresponding to each point and the total MI value of the sample were calculated, and the results were interpreted.

[0079] The FAM, VIC and CY5 fluorescence detection signals in the positive quality control 1-2 reaction tube form logarithmic amplification "S" type curve; the negative quality control 1-2 tube has no amplification curve.

[0080] Example 4: Accuracy performance evaluation of the kit

[0081] 25 healthy people and 25 cervical cancer CIN II+ cervical cell samples were selected respectively, and were detected according to the detection method of Example 3, and the detection results were compared with the clinical diagnosis results for accuracy. The detection results are as follows in Table 5.

[0082] Table 5: Accuracy detection results

[0083]

[0084]

[0085] According to the detection results, the specificity of the kit for detecting healthy people was 92%, and the sensitivity for detecting cervical cancer samples was 88%; the total positive predictive value of the kit was 91.6%, and the negative predictive value was 88.5%. It is shown that the detection system of the kit of Example 2 has good accuracy.

[0086] The above-mentioned example numbers of the present application are only for description, and do not represent the advantages and disadvantages of the examples.

[0087] The embodiments of the present application are described above in combination with the drawings, but the present application is not limited to the above-mentioned specific embodiments, and the above-mentioned specific embodiments are only illustrative, but not limiting, and those skilled in the art can make many forms under the inspiration of the present application without departing from the purpose of the present application and the scope protected by the claims, which all belong to the protection of the present application.

Claims

1. A kit for detection of uterine cervix cancer related gene methylation, characterized by, The kit comprises primers and probes for detecting methylation of APC, CALCA, DAPK and ESR1 genes.

2. The kit for detecting methylation of cervical cancer-related genes as described in claim 1, characterized in that: The primer and probe sequence for detecting methylation of the APC gene is as follows: the forward primer sequence is as shown in SEQ ID NO. 6, the reverse primer sequence is as shown in SEQ ID NO. 7, and the nucleic acid sequence of the fluorescent probe is as shown in SEQ ID NO.

8. The primer and probe sequence for detecting methylation of the CALCA gene is as follows: the forward primer sequence is as shown in SEQ ID NO. 9, the reverse primer sequence is as shown in SEQ ID NO. 10, and the nucleic acid sequence of the fluorescent probe is as shown in SEQ ID NO.

11. The primer and probe sequence for detecting methylation of the DAPK gene is as follows: the forward primer sequence is as shown in SEQ ID NO. 12, the reverse primer sequence is as shown in SEQ ID NO. 13, and the nucleic acid sequence of the fluorescent probe is as shown in SEQ ID NO.

14. The primer and probe sequence for detecting methylation of the ESR1 gene is as follows: the forward primer sequence is as shown in SEQ ID NO. 15, the reverse primer sequence is as shown in SEQ ID NO. 16, and the nucleic acid sequence of the fluorescent probe is as shown in SEQ ID NO.

17.

3. The kit for detecting methylation of cervical cancer-related genes as described in claim 2, characterized in that: The 5' end fluorescent group of the probe for detecting methylation of the APC, CALCA, DAPK and ESR1 genes is one of FAM, VIC and CY5, and the 3' end quenching group is MGB or BHQ2.

4. The kit for detecting methylation of cervical cancer-related genes as described in claim 1, characterized in that: The kit further comprises primers and probes for monitoring the internal control gene GAPDH, and the primer and probe sequence for detecting the internal control gene GAPDH is as follows: the forward primer sequence is as shown in SEQ ID NO. 18, the reverse primer sequence is as shown in SEQ ID NO. 19, and the nucleic acid sequence of the fluorescent probe is as shown in SEQ ID NO.

20.

5. The kit for detecting cervical cancer related gene methylation according to claim 1, wherein: The kit further comprises PCR reaction solution, positive quality control and negative quality control, and the PCR reaction solution comprises Taq enzyme, UNG enzyme, buffer, magnesium ions and dNTP substances.

6. A system for detection of cervical cancer related gene methylation, characterized by: The kit, the PCR amplification system and the genotyping interpretation system of any one of claims 1-5 are included.

7. The system for detecting cervical cancer related gene methylation according to claim 6, wherein: The reagents in the kit are pre-mixed and divided to obtain PCR reaction solution 1 and PCR reaction solution 2, which constitute the PCR reaction system.

8. The system for detecting cervical cancer related gene methylation according to claim 6, wherein: The conditions for PCR amplification in the PCR amplification system are as follows: the conditions for UNG enzyme reaction are 25°C for 10 minutes; the conditions for UNG enzyme inactivation are 95°C for 2 minutes; The amplification conditions of the system are two-step method: denaturation: 95°C, 5 seconds; annealing and extension: 58°C, 30 seconds, with fluorescence signal collection; the cycle number is set to 45.

9. The system for detecting cervical cancer related gene methylation according to claim 6, wherein: The genotyping interpretation system includes: using the fluorescence detection results for determination, when GAPDH Ct≤34, according to the ΔCt(Ct 靶基因 -Ct 内参基因 ) of the APC, CALCA, DAPK and ESR1 genes, the methylation index (MI) of each gene is calculated, and the value assignment mode is as follows: The MI values of the four genes are added to obtain a total MI value, and when the total MI value is greater than or equal to 6, it is determined that the methylation of cervical cancer is positive, and when the total MI value is less than 6, it is determined that the methylation of cervical cancer is negative; when the GAPDH Ct is greater than 34, it is determined that there is an inhibitor, and the PCR reaction is invalid.

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