CN115976202A - Primer probe combination and kit for detecting methylation of cervical cancer related genes - Google Patents
Primer probe combination and kit for detecting methylation of cervical cancer related genes Download PDFInfo
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Abstract
The invention discloses a primer probe combination and a kit for detecting methylation of genes related to cervical cancer. The invention designs a primer probe combination for detecting the methylation of the cervical cancer related gene based on the cervical cancer related gene RAB3C, ZNF and PAX1, and provides a kit for detecting the methylation of the cervical cancer related gene on the basis. The primer probe combination and the kit for methylation of the cervical cancer related gene can be used for detecting the cervical cancer and the high-grade precancerous lesion thereof so as to evaluate the cervical cancer risk, have the advantages of good detection specificity, high detection sensitivity, accurate detection result and the like, are simple to operate, have no wound, are suitable for preliminary screening of the cervical cancer, and provide reference for early diagnosis of the cervical cancer.
Description
Technical Field
The invention belongs to the technical field of gene methylation detection. More particularly, relates to a primer probe combination and a kit for detecting methylation of cervical cancer related genes.
Background
Cervical cancer is a common gynecological malignant tumor, the incidence rate of which is the second place among female malignant tumors in China, and the physical health of women is seriously threatened. Cervical Intraepithelial Neoplasia (CIN), a generic term for precancerous lesions closely related to cervical invasive carcinoma, occurs in association with infection with Human Papillomavirus (HPV). Because of the precancerous lesion of cervical cancer and no obvious symptom in early stage of cervical cancer, cervical cancer screening is considered as the most effective measure for reducing incidence rate and death rate of cervical cancer.
Cervical cancer screening is typically performed by primary screening through cervical cytology and confirmed by cervical biopsy histopathology. Cervical cytology screening is mainly to dye exfoliated cervical cells and observe morphological changes of the cells so as to judge whether cervical cells have canceration or not; the method has high specificity but low sensitivity, cytological result judgment needs a professional pathologist to judge under a microscope, is influenced by subjective factors, cells are easy to accumulate together, and therefore missed diagnosis and misdiagnosis are caused, and meanwhile, due to shortage of the pathologist, low-resource areas are difficult to popularize. Therefore, the kit and the method for simply and effectively detecting the cervical cancer are provided, the risk evaluation of the cervical cancer is carried out, the early diagnosis of the cervical cancer is facilitated, the timely and effective treatment is carried out, and the incidence rate and the death rate of the cervical cancer are reduced.
DNA methylation is an important epigenetic modification that plays an important regulatory role in the pattern of gene expression and stability of the genome. By detecting the methylation of the gene related to the cervical cancer, the cervical cancer and the precancerous lesion of the cervical cancer can be sensitively and specifically detected, and the risk evaluation of the cervical cancer can be carried out. For example, there are patents that the detection of the cervical cancer development process is realized by detecting the methylation of genes PAX1, FAM19A4 and miR124 2 related to the cervical cancer, but the detection sensitivity of the method still has a space for further improving.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a primer probe combination and a kit for detecting methylation of genes related to cervical cancer.
The first purpose of the invention is to provide the application of the reagent for detecting the methylation of RAB3C and/or ZNF93 genes in preparing a product for detecting the risk of cervical cancer.
The second purpose of the invention is to provide a primer probe combination for detecting methylation of cervical cancer related genes.
The third purpose of the invention is to provide a kit for detecting methylation of cervical cancer related genes.
The fourth purpose of the invention is to provide the application of the primer probe combination or the kit in the preparation of products for detecting the risk of cervical cancer.
The fifth purpose of the invention is to provide a kit for detecting the risk of cervical cancer.
The above purpose of the invention is realized by the following technical scheme:
the invention detects and discovers cervical cancer and samples of high-grade precancerous lesions of the cervical cancer (cervical intraepithelial neoplasia (CIN) stage 2 and 3) by using designed primers and probes for detecting RAB3C and ZNF93 gene methylation, can detect the cervical cancer and the high-grade precancerous lesions of the cervical cancer by detecting the RAB3C and ZNF93 gene methylation, and is used for evaluating the risk of the cervical cancer. Therefore, the invention discloses application of an agent for detecting methylation of RAB3C and/or ZNF93 genes in preparing a product for detecting the risk of cervical cancer.
As an alternative embodiment, the reagent for detecting the methylation of the RAB3C gene is a primer pair and a probe for detecting the methylation of the RAB3C gene, the sequences of the primer pair are shown in SEQ ID NO. 1-2, and the sequence of the probe is shown in SEQ ID NO. 3.
As an alternative embodiment, the reagent for detecting the methylation of the ZNF93 gene is a primer pair and a probe for detecting the methylation of the ZNF93 gene, the sequences of the primer pair are shown as SEQ ID NO. 4-5, and the sequence of the probe is shown as SEQ ID NO. 6.
The invention also designs a methylation detection primer probe combination with good detection specificity and high sensitivity based on the promoter sequences of the cervical cancer related genes RAB3C, ZNF and PAX1, wherein the primer probe combination comprises the following components:
(1) A primer pair and a probe for detecting the methylation of the RAB3C gene, wherein the sequence of the primer pair is shown as SEQ ID NO. 1-2, and the sequence of the probe is shown as SEQ ID NO. 3;
(2) A primer pair and a probe for detecting ZNF93 gene methylation, wherein the sequence of the primer pair is shown as SEQ ID NO. 4-5, and the sequence of the probe is shown as SEQ ID NO. 6;
(3) A primer pair and a probe for detecting PAX1 gene methylation, wherein the sequence of the primer pair is shown as SEQ ID NO. 7-8, and the sequence of the probe is shown as SEQ ID NO. 9.
The invention also designs a primer pair and a probe for detecting the reference gene ACTB, wherein the sequence of the primer pair is shown as SEQ ID NO. 10-11, and the sequence of the probe is shown as SEQ ID NO. 12.
The 5 'end of the probe is marked with a report fluorescent group, and the 3' end of the probe is marked with a quenching fluorescent group; the reporter fluorescent group is selected from one of FAM, JOE, VIC, HEX, ROX, CY3 and CY5, and the quenching fluorescent group is selected from one of BHQ, TAMRA and MGB.
Specifically, the 5 'end of the probe for detecting the methylation of the RAB3C gene is marked with a report fluorescent group FAM, and the 3' end is marked with a quenching fluorescent group BHQ; the 5 'end of the probe for detecting ZNF93 gene methylation is marked with a report fluorescent group ROX, and the 3' end is marked with a quenching fluorescent group BHQ; the 5 'end of the probe for detecting the methylation of the PAX1 gene is marked with a reporter fluorophore CY5, and the 3' end is marked with a quenching fluorophore BHQ; the 5 'end of the probe for detecting ACTB gene methylation is marked with a report fluorescent group JOE, and the 3' end is marked with a quenching fluorescent group BHQ.
The invention also provides application of the primer-probe combination, a primer pair for detecting the reference gene ACTB and the probe in detecting methylation of the genes related to the cervical cancer or preparing a kit for detecting methylation of the genes related to the cervical cancer.
Specifically, the cervical cancer related genes are RAB3C, ZNF and PAX1 genes.
The invention also provides a kit for detecting methylation of genes related to cervical cancer, and the kit comprises the primer and probe combination.
Specifically, the kit comprises a primer pair shown in SEQ ID NO. 1-12 and a probe.
Specifically, the cervical cancer related genes are RAB3C, ZNF and PAX1 genes.
The kit for detecting the methylation of the cervical cancer related gene also comprises a reagent required by a fluorescence PCR reaction, a positive quality control product and a negative quality control product; the positive quality control substance is human methylated genomic DNA, and the negative quality control substance is human non-methylated genomic DNA.
The primer probe combination and the kit for detecting the methylation of the cervical cancer related gene can be used for detecting the cervical cancer risk and evaluating the cervical cancer risk, and have the advantages of good detection specificity, high sensitivity and accurate detection result. Therefore, the invention also applies to protect the application of the methylation detection primer probe combination of the cervical cancer related genes or the methylation detection kit of the cervical cancer related genes in the preparation of products for detecting the risk of cervical cancer.
The invention also provides a kit for detecting the cervical cancer risk, and the kit contains the primer probe combination for detecting the cervical cancer related gene methylation.
Specifically, the primer probe combination or the kit for detecting the methylation of the cervical cancer related gene can be used for detecting Cervical Intraepithelial Neoplasia (CIN) stage 2, cervical intraepithelial neoplasia stage 3 and cervical cancer and detecting the risk of the cervical cancer; although it can not be distinguished specifically which stage of cervical intraepithelial neoplasia the tested sample is in or whether the tested sample has developed into cervical cancer, the primer probe combination or the kit has the advantages of simple, non-invasive, good specificity, high sensitivity, accurate result and the like when being used for detection, can be used for preliminary screening of cervical cancer, and can be further used for diagnosis confirmation through methods such as cervical biopsy histopathology and the like if the detection result is positive, thereby avoiding over-screening.
Specifically, the detection steps of the cervical cancer risk are as follows:
s1, extracting DNA of a sample to be detected;
s2, carrying out sulfite conversion on the DNA obtained in the step S1;
s3, carrying out real-time fluorescent quantitative PCR amplification reaction on the DNA sample converted by the sulfite by using a primer pair and a probe shown in SEQ ID NO. 1-12;
and S4, detecting a fluorescence signal and judging a result.
Specifically, the sample in step S1 is human cervical exfoliated cells, vaginal secretions, cervical tissue or a combination thereof; preferably human cervical exfoliated cells.
Specifically, the step S1 of extracting the DNA of the sample to be tested includes sample lysis, DNA binding, DNA washing and elution.
Specifically, the sulfite conversion in step S2 includes sulfite conversion, binding, first washing, desulfonation, second washing, third washing, drying, and elution.
Specifically, the reaction system of the real-time fluorescent quantitative PCR amplification reaction in step S3 includes: PCR buffer, dNTP, mgCl 2 Taq DNA polymerase and ddH 2 O, the final concentration of each primer in the reaction system is 200nM, the final concentration of each probe is 100nM, and other reagents are added according to the conventional method and dosage.
Specifically, the conditions of the reaction body of the real-time fluorescent quantitative PCR amplification reaction in step S3 are as follows: denaturation at 95 deg.C for 5min; denaturation at 95 ℃ for 5s, annealing at 55 ℃ and extension for 35s,45 cycles.
After the real-time fluorescent quantitative PCR amplification reaction is finished, analyzing the PCR amplification result by using a fluorescent PCR instrument matched analysis software, calculating the difference value between the Ct value of the fluorescence curve obtained by amplification and the Ct values of the ACTB detection primers and the probe of the internal reference gene, namely the delta Ct value, and judging the result by using the delta Ct value.
Specifically, the method for judging the cervical cancer risk result comprises the following steps: calculating the difference value of the Ct value of the gene with the amplification curve and the Ct value of the reference gene ACTB, namely the delta Ct value; when the delta Ct is less than or equal to 9, the detection result is positive, which indicates that the cervical cancer risk is high; when the delta Ct is more than 9 or is not detected, the detection result is negative, which indicates that the cervical cancer risk is low; if there is only one Δ Ct value, the result determination is performed using the Δ Ct value, and if there are a plurality of Δ Ct values, the result determination is performed using the smallest Δ Ct value.
The invention has the following beneficial effects:
the invention designs a primer probe combination for detecting the methylation of the cervical cancer related gene based on the cervical cancer related gene RAB3C, ZNF and PAX1, and provides a kit for detecting the methylation of the cervical cancer related gene on the basis. The primer probe combination and the kit for methylation of the cervical cancer related gene can be used for detecting the cervical cancer and the high-grade precancerous lesion thereof so as to evaluate the cervical cancer risk, have the advantages of good detection specificity, high detection sensitivity, accurate detection result and the like, are simple to operate, have no wound, are suitable for preliminary screening of the cervical cancer, and provide reference for early diagnosis of the cervical cancer.
Drawings
FIG. 1 is an amplification plot of the methylation detection of the RAB3C gene.
FIG. 2 is a graph of detection amplification of ZNF93 gene methylation.
FIG. 3 is a graph showing the detection and amplification of the methylation of the PAX1 gene.
FIG. 4 is a graph of the detection amplification of mixed methylation of RAB3C, ZNF and the PAX1 gene.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
EXAMPLE 1 design of primers and probes
Based on promoter sequences of cervical cancer related genes RAB3C, ZNF and PAX1, a plurality of pairs of primer pairs and probes are respectively designed according to a primer design principle, and then a primer probe combination for detecting methylation of the cervical cancer related genes with good detection specificity and high sensitivity is obtained; the invention also designs a primer pair and a probe for detecting the reference gene ACTB, and the sequences of the designed partial primers and probes are shown in the table 1:
TABLE 1 primer and Probe sequence Listing
Wherein, the 5 'end of the probe for detecting the methylation of the RAB3C gene is marked with a report fluorescent group FAM, and the 3' end is marked with a quenching fluorescent group BHQ; the 5 'end of the probe for detecting ZNF93 gene methylation is marked with a report fluorescent group ROX, and the 3' end is marked with a quenching fluorescent group BHQ; the 5 'end of the probe for detecting the PAX1 gene methylation is marked with a report fluorescent group CY5, and the 3' end is marked with a quenching fluorescent group BHQ; the 5 'end of the probe for detecting ACTB gene methylation is marked with a report fluorescent group JOE, and the 3' end is marked with a quenching fluorescent group BHQ.
The invention combines the primers and the probes and respectively detects the sensitivity and the specificity, and the results are shown in table 2:
TABLE 2 detection results of sensitivity and specificity of primer and probe combinations
According to the invention, a primer pair shown in SEQ ID NO. 1-11 and a probe are finally selected to be combined for preparing a kit for detecting methylation of genes related to cervical cancer by synthesizing all detection results.
Example 2 kit for detecting methylation of cervical cancer-related genes
The invention provides a kit for detecting methylation of cervical cancer related genes (RAB 3C, ZNF and PAX 1) based on the combination of the primer pair and the probe obtained in the embodiment 1, wherein the kit contains the primer pair and the probe shown in SEQ ID No. 1-12, and also contains a reagent, a positive quality control product and a negative quality control product required by a fluorescent PCR reaction; the positive quality control substance is human methylated genomic DNA, and the negative quality control substance is human non-methylated genomic DNA.
Example 3
The detection method for detecting methylation of the cervical cancer related gene by using the primer probe combination for detecting methylation of the cervical cancer related gene disclosed in embodiment 1 or the kit disclosed in embodiment 2 comprises the following steps:
1. materials, reagents, apparatus
The DNA extraction kit used in the invention is purchased from QIAGEN company; sulfite conversion kit was purchased from QIAGEN; PCR buffer, dNTP, taqDNA polymerase were purchased from Takara; mgCl 2 Purchased from Sigma company; the fluorescent quantitative PCR instrument is ABI7500.
2. Sample preparation
Cervical exfoliated cells of a cervical cancer patient are taken as a detection sample, cervical exfoliated cells of normal and healthy people are taken as negative control, meanwhile, human methylated genomic DNA is taken as a positive quality control product, and human non-methylated genomic DNA is taken as a negative quality control product.
3. DNA extraction
1. Adding 0.2mL of sample, 0.5mL of nucleic acid lysis adsorption solution and 10 mu L of magnetic beads into a 1.5mL centrifuge tube, uniformly mixing by vortex, and placing the centrifuge tube at 56 ℃ for 10 minutes;
2. placing the centrifuge tube in a magnetic frame for magnetic attraction for 2min, sucking away all the waste liquid, adding 0.5mL of washing liquid A, and uniformly mixing to ensure that the magnetic beads are thoroughly resuspended;
3. placing the centrifugal tube in a magnetic rack for magnetic attraction for 2min, sucking away all the waste liquid, removing the residual liquid as much as possible by using a 10-100 mu L gun head, transferring the centrifugal tube to a nonmagnetic test tube rack, opening a tube cover, and drying at room temperature for 5min;
4. adding 40 mu L of eluent, covering a tube cover, whirling, uniformly mixing and suspending magnetic beads, and incubating the centrifuge tube at 56 ℃ for 5 minutes;
5. place the centrifuge tube in a magnetic rack for magnetic attraction for 2min and move all eluents to a new 0.2mL PCR tube.
4. Sulfite conversion
1. Adding 40 mu L of DNA obtained by extraction into a 0.2mL PCR tube, then adding 110 mu L of sulfite solution, covering a centrifuge tube, performing vortex mixing, performing short-time centrifugation, placing the centrifuge tube in a common PCR instrument for reaction, wherein the reaction conditions are as follows: 5 minutes at 95 ℃, 10 minutes at 60 ℃,5 minutes at 95 ℃ and 10 minutes at 60 ℃;
2. transferring the DNA solution after the reaction to a new 1.5mL centrifuge tube, adding 600 mu L of binding solution and 10 mu L of magnetic beads, uniformly mixing by vortex, and standing for 5 minutes at room temperature;
3. placing the centrifuge tube in a magnetic rack for magnetic attraction for 2min, sucking away all the waste liquid, adding 500 μ L of washing liquid, and performing vortex mixing to ensure that the magnetic beads are thoroughly resuspended;
4. placing the centrifugal tube in a magnetic frame for magnetic attraction for 2min, sucking away all the waste liquid, adding 500 mu L of desulfonation liquid, vortex and uniformly mixing to ensure that the magnetic beads are thoroughly resuspended, and standing at room temperature for 15 min;
5. placing the centrifuge tube in a magnetic frame for magnetic attraction for 2min, sucking away all the waste liquid, adding 500 μ L of washing liquid, and vortex mixing to ensure thorough resuspension of the magnetic beads;
6. placing the centrifuge tube in a magnetic frame for magnetic attraction for 2min, sucking away all the waste liquid, adding 500 μ L of washing liquid, and vortex mixing to ensure thorough resuspension of the magnetic beads;
7. placing the centrifugal tube in a magnetic rack for magnetic attraction for 2min, sucking away all the waste liquid, removing the residual liquid as much as possible by using a 10-100 mu L gun head, transferring the centrifugal tube to a nonmagnetic test tube rack, opening a tube cover, and drying at room temperature for 2 min;
8. adding 50 mu L of eluent, covering a tube cover, whirling, uniformly mixing and resuspending magnetic beads, and incubating the centrifuge tube for 5 minutes at 56 ℃;
9. placing the centrifuge tube in a magnetic frame for magnetic attraction for 2min, and transferring all the eluent into a new centrifuge tube for later use.
5. PCR reaction
Preparation of PCR reaction solution
The invention respectively utilizes the detection primers and probes of single genes and the combination of the primers and the probes to carry out the fluorescent quantitative PCR reaction, and the prepared PCR reaction liquid systems are respectively shown in tables 3-6:
TABLE 3 detection reaction solution for methylation of RAB3C gene
TABLE 4 ZNF93 Gene methylation assay reaction solutions
| Components of a Single PCR reaction | Amount of Individual PCR reactions |
| Primer shown as SEQ ID NO.4 (10. Mu.M) | 0.5μL |
| Primer shown as SEQ ID NO.5 (10. Mu.M) | 0.5μL |
| Probe shown as SEQ ID NO.6 (10. Mu.M) | 0.25μL |
| Primer shown as SEQ ID NO.10 (10. Mu.M) | 0.5μL |
| Primer shown as SEQ ID NO.11 (10. Mu.M) | 0.5μL |
| Probe shown as SEQ ID NO.12 (10. Mu.M) | 0.25μL |
| TaqDNA polymerase (5 μm/μ L) | 0.5μL |
| dNTP(10mM) | 0.5μL |
| MgCl 2 (25mM) | 4μL |
| PCR buffer (10X) | 5μL |
| ddH 2 O | 7.5μL |
| Total | 20μL |
TABLE 5 methylation detection reaction solution for PAX1 Gene
TABLE 6 RAB3C ZNFC + PAX1 methylation detection reaction solution
2. Sample application
Respectively adding 20 mu L of prepared PCR reaction solution and 5 mu L of sample DNA into a prepared PCR reaction tube, covering a tube cover tightly, and performing instantaneous low-speed centrifugation; the sample adding of the negative quality control and the positive quality control is the same as that of the sample.
3. Fluorescent quantitative PCR detection
1) Fluorescence channel selection: the fluorescence channel is selected according to the fluorophore carried by the probe, and each sample in this example is selected from FAM, JOE, ROX and CY5, 4 channels in total. The Reference fluorescence (Passive Reference) is set to none;
2) The reaction conditions were set as follows (reaction volume was set to 25. Mu.L):
6. analysis of results
Analysis of PCR results
And automatically storing the result after the reaction is finished, automatically analyzing the result by using instrument matched software, and if any one of three genes of RAB3C, ZNF and PAX1 has an amplification curve in PCR amplification, calculating a delta Ct value of the result, wherein the delta Ct value is the difference value between the Ct value of any one of three genes of RAB3C, ZNF and PAX1 and the Ct value of the ACTB gene, and the size of the difference value reflects the relative quantification between the detected gene and the reference gene.
2. Determination of detection result
The minimum delta Ct value in three delta Ct values of RAB3C, ZNF and PAX1 is used as a judgment standard, and when the delta Ct is less than or equal to 9, the result is positive, which indicates that the cervical cancer has high risk; a negative result when Δ Ct >9 or n.d. is indicative of a low risk of having cervical cancer, wherein n.d. is an abbreviation for "Not Detected" meaning "Not Detected".
3. The result of the detection
The methylation of the RAB3C, ZNF and the PAX1 gene is not detected in a normal sample, the result is negative, and the result is positive when the methylation of the cervical cancer is detected in a cervical cancer sample. The amplification curve diagrams of RAB3C, ZNF, PAX1 and 3 gene mixed detection are sequentially shown in FIGS. 1-4, and the results shown in FIGS. 1-4 show that the combination of the primer and the probe can successfully detect the methylated RAB3C, ZNF and the PAX1 gene. The mixed detection Ct values and the judgment results of the RAB3C, ZNF, the PAX1 and the 3 genes are shown in tables 7-10 in sequence:
TABLE 7 RAB3C methylation assay results
TABLE 8 ZNF93 methylation assay results
TABLE 9 PAX1 methylation assay results
TABLE 10 RAB3C + ZNF93+ PAX1 methylation detection results
As can be seen from the results shown in tables 7 to 10, the methylation detection primers and probes for RAB3C, ZNF and PAX1 genes of the present invention can be used for detecting cervical cancer.
EXAMPLE 4 actual sample testing
In this embodiment, the kit of embodiment 2 of the present invention is used, and the detection method shown in embodiment 3 is used to detect clinical samples of cervical cancer, CIN iii, CIN ii and CIN I, so as to detect the detection sensitivity and specificity of the primers and probes for detecting methylation of genes related to cervical cancer of the present invention to cervical cancer and high-grade precancerous lesions thereof, and the experimental results are shown as follows:
TABLE 11 RAB3C test results
TABLE 12 ZNF93 test results
TABLE 13 PAX1 test results
TABLE 14 RAB3C ZNFC + PAX1 test results
From the results shown in tables 11 to 14, it is clear that: compared with the single detection of 1 gene, the detection sensitivity of the kit to cervical cancer, CIN III and CIN II is higher when 3 genes are jointly detected.
The embodiments show that the methylation detection primer probe combination and the kit for the cervical cancer related genes can be used for detecting the risk of the cervical cancer and provide reference for early diagnosis of the cervical cancer. Compared with the conventional cervical cancer diagnosis method, the method has the advantages of high sensitivity and high specificity when the primer probe combination or the kit is used for detecting the cervical cancer, is simple to operate, and can be used for carrying out early noninvasive diagnosis on the cervical cancer.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. The application of the reagent for detecting the methylation of the RAB3C and/or ZNF93 gene in preparing a product for detecting the risk of the cervical cancer.
2. The use according to claim 1, wherein the reagent for detecting the methylation of the RAB3C gene comprises a primer pair and a probe for detecting the methylation of the RAB3C gene, the sequences of the primer pair are shown as SEQ ID No. 1-2, and the sequence of the probe is shown as SEQ ID No. 3.
3. The use as claimed in claim 1 wherein the reagent for detecting methylation of ZNF93 gene is a primer pair for detecting methylation of ZNF93 gene and a probe, the sequence of the primer pair is shown as SEQ ID No. 4-5, and the sequence of the probe is shown as SEQ ID No. 6.
4. A primer probe combination for detecting methylation of cervical cancer related genes is characterized by comprising the following components:
(1) A primer pair and a probe for detecting the methylation of the RAB3C gene, wherein the sequence of the primer pair is shown as SEQ ID NO. 1-2, and the sequence of the probe is shown as SEQ ID NO. 3;
(2) A primer pair and a probe for detecting ZNF93 gene methylation, wherein the sequence of the primer pair is shown as SEQ ID NO. 4-5, and the sequence of the probe is shown as SEQ ID NO. 6;
(3) A primer pair and a probe for detecting PAX1 gene methylation, wherein the sequence of the primer pair is shown as SEQ ID NO. 7-8, and the sequence of the probe is shown as SEQ ID NO. 9.
5. The primer probe combination of claim 4, further comprising a primer pair and a probe for detecting the reference gene ACTB, wherein the sequences of the primer pair are shown as SEQ ID NO. 10-11, and the sequence of the probe is shown as SEQ ID NO. 12.
6. The use of claim 2 or 3 or the primer probe combination of claim 4 or 5, wherein the probe is labeled with a reporter fluorescent group at the 5 'end and a quencher fluorescent group at the 3' end; the reporter fluorescent group is selected from one of FAM, JOE, VIC, HEX, ROX, CY3 and CY5, and the quenching fluorescent group is selected from one of BHQ, TAMRA and MGB.
7. A kit for detecting methylation of genes related to cervical cancer is characterized in that the genes related to cervical cancer are RAB3C, ZNF and PAX1 genes; the kit comprises the primer probe combination of claim 4 or 5.
8. The kit of claim 7, further comprising reagents required for a fluorescent PCR reaction, a positive quality control material and a negative quality control material; the positive quality control substance is human methylated genomic DNA, and the negative quality control substance is human non-methylated genomic DNA.
9. Use of the primer probe combination of claim 4 or 5 or the kit of claim 7 or 8 for the preparation of a product for detecting the risk of cervical cancer.
10. A kit for detecting the risk of cervical cancer, which comprises the primer probe combination of claim 4 or 5.
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| CN117265096A (en) * | 2023-09-07 | 2023-12-22 | 北京美康基因科学股份有限公司 | Kit for cervical high-grade lesions and early detection of cervical cancer |
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| CN117265096A (en) * | 2023-09-07 | 2023-12-22 | 北京美康基因科学股份有限公司 | Kit for cervical high-grade lesions and early detection of cervical cancer |
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