CN117701718B - Gene methylation marker for diagnosing cervical cancer, primer pair and application thereof - Google Patents
Gene methylation marker for diagnosing cervical cancer, primer pair and application thereof Download PDFInfo
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Abstract
The invention provides a gene methylation marker for diagnosing cervical cancer, a primer pair and application thereof, and relates to the technical field of detection of cervical cancer; the gene methylation marker is selected from methylated nucleotide sequences in at least one target region in target genes PAX1 and ZCAN 1; specifically selected from the 500bp-1000bp sequence before the start codon and 200bp-500bp sequence after the start codon of each gene of PAX1 gene and ZCAN 1 gene. The invention uses methylation sensitive restriction enzyme to cut specific sites, because the methylation sensitive restriction enzyme is sensitive to CpG methylation, the methylation of CpG of specific sites affects the cutting of the restriction enzyme, and the specific sites which are not methylated are cut into two sections by the restriction enzyme, thus the amplification can not be completed by a complete template chain to distinguish the specific sites which are methylated and unmethylated.
Description
Technical Field
The invention relates to the technical field of detection of cervical cancer, in particular to a gene methylation marker for diagnosing cervical cancer, a primer pair and application thereof.
Background
In recent years, gene methylation has been widely used to detect cervical cancer and precancerous lesions. However, the methylation detection of cervical cancer is currently commercially available as a sulfite conversion method. Bisulfite treatment of DNA can change unmethylated cytosine (C) to thymine (T), and methylated C can distinguish methylated C from unmethylated C by adding a methyl group to the 5' carbon atom of methylated C under the action of methyltransferase, and protecting C from reaction during bisulfite treatment. Thus, various cervical cancer methylation detection methods have been developed, such as pyrosequencing, bisulfite sequencing, and real-time methylation-specific PCR (QMSP), among others. However, the method can break the DNA double chain, damage the integrity of the DNA to influence the subsequent detection, and the bisulfite treatment has complicated operation, and the nucleic acid is required to be recovered after the treatment, so that the loss of the DNA is caused; in addition, since the bisulfite treatment is carried out at a relatively high temperature, the reaction is carried out in a PCR instrument, and the treatment time is long, generally two hours are required to ensure the conversion efficiency of methyl group. Therefore, a cervical cancer methylation detection method with high specificity, high speed and convenient operation needs to be found so as to meet the market demand of cervical cancer detection.
The methylation sensitive restriction enzyme can identify and cut specific sites, many restriction enzymes are sensitive to CpG methylation, namely, if the CpG is methylated at the specific sites, the restriction enzyme cannot cut the specific sites, and sites which are not methylated at the specific sites can be cut under the action of the restriction enzyme, so that primer probe design is carried out near the restriction enzyme, fragments of the specific restriction enzyme where the CpG is methylated are not cut, and can be used as templates for smooth amplification, fragments of the specific restriction enzyme where the CpG is not methylated are cut into two fragments by the restriction enzyme, and template strand incompleteness cannot be amplified, so that methylation and unmethylation are distinguished.
The invention selects genes (PAX 1 and ZSCAN 1) related to cervical cancer and precancerous lesions, determines methylation conditions of the genes by sequencing, analyzes enzyme cutting sites of the genes, and selects specific sites and corresponding methylation-sensitive restriction enzymes according to the methylation conditions. The methylation level of specific sites of PAX1 gene and ZSSCAN 1 gene has good sensitivity and specificity to cervical cancer and precancerous high lesions.
Disclosure of Invention
The invention aims to select genes (PAX 1 and ZSCAN 1) related to cervical cancer and precancerous lesions, determine methylation conditions of the genes by sequencing, analyze enzyme cleavage sites of the genes, and select specific sites and corresponding methylation-sensitive restriction enzymes according to the methylation conditions. The methylation level of specific sites of PAX1 gene and ZSSCAN 1 gene has good sensitivity and specificity to cervical cancer and precancerous high lesions.
The invention provides a gene methylation marker for diagnosing cervical cancer, which is selected from a nucleotide sequence methylated in at least one target region in target genes PAX1 and ZSSCAN 1; wherein the target region is selected from a 500bp-1000bp sequence before the start codon and a 200bp-500bp sequence after the start codon of each gene of the PAX1 gene and the ZCAN 1 gene.
Alternatively, the target region is selected from the group consisting of a 700bp before start codon sequence and a 300bp after start codon sequence for each of the PAX1 gene and the ZCAN 1 gene.
Alternatively, the amplified fragment of the target gene comprises one or more of the cleavage sites of methylation sensitive restriction enzymes Bsh, 1236I, eco 72I, and HhaI, and the amplified fragment of the reference gene does not comprise the cleavage site.
The invention also provides a primer pair for diagnosing cervical cancer gene methylation, which comprises a primer sequence and a probe sequence, wherein the primer sequence and the probe sequence correspond to any pair of biomarkers as methylation markers, and the primer sequence is specifically expressed as follows:
The pre-primer of PAX1 gene: SEQ ID NO.1;
post primer of PAX1 gene: SEQ ID NO.2;
Probes for the PAX1 gene: SEQ ID NO.3;
the ZSSAN 1 gene pre-primer: SEQ ID NO.4;
Post primer of ZSSAN 1 gene: SEQ ID NO.5;
probes for ZSCAN1 gene: SEQ ID NO.6.
Optionally, the primer pair for diagnosing cervical cancer gene methylation further comprises a pair of primer sequences corresponding to a reference marker COL2A1 gene, and the primer sequences are specifically expressed as follows:
the pre-primer of COL2A1 gene: SEQ ID NO.7;
post-primer of COL2A1 gene: SEQ ID NO.8;
Probes for the COL2A1 gene: SEQ ID NO.9.
Alternatively, the fluorescent group label used for the probe is set to be either one of FAM, VIC, HEX, TET, CY, ROX, JOE, CY 3.
Alternatively, the quenching group is set to any one of BHQ1, BHQ2, BHQ3, TAMRA, DABCYL, MGB.
Furthermore, the primer for diagnosing cervical cancer gene methylation is applied to preparing a cervical cancer detection kit.
The invention also provides a kit for diagnosing cervical cancer gene methylation, which comprises the primer pair, an enzyme digestion amplification system and a primer probe mixed solution; wherein,
The buffer solution of the enzyme digestion amplification system is prepared from the following components in percentage by weight:
；
the preparation ratio of the primer probe mixed solution is as follows:
。
optionally, the kit for diagnosing cervical cancer gene methylation described above is applied to detection of cervical cancer gene methylation markers.
Alternatively, in the detection of biomarkers of cervical cancer using the kit, the methylation level of a specific gene target region is reflected with Δcp values, wherein Δcp values are expressed as follows:
ΔCp(PAX1)= Cp(PAX1)- Cp(COL2A1)
ΔCp(ZSCAN1)= Cp(ZSCAN1)- Cp(COL2A1)。
compared with the prior art, the invention has the following beneficial effects:
The invention uses methylation sensitive restriction enzyme to cut specific sites, because the methylation sensitive restriction enzyme is sensitive to CpG methylation, the methylation of CpG of specific sites affects the cutting of the restriction enzyme, and the specific sites which are not methylated are cut into two sections by the restriction enzyme, thus the amplification can not be completed by a complete template chain, and the specific sites which are methylated and unmethylated are distinguished. The technology does not need to carry out bisulfite treatment on the nucleic acid, can carry out the subsequent PCR reaction only by carrying out short enzyme digestion at 37 ℃, has mild enzyme digestion reaction conditions and short reaction time, is easy to operate, does not need to recycle the digested nucleic acid, and can directly carry out the methylation detection of the next step after enzyme digestion, thereby avoiding the risk of nucleic acid loss. The invention well solves the problems of long reaction time, serious nucleic acid loss and the like of the bisulfite conversion method.
In addition to the objects, features and advantages described above, the present invention has other objects, features and advantages. The present invention will be described in further detail with reference to the drawings.
Drawings
The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain the application. In the drawings:
FIG. 1 is a schematic diagram showing the amplification of PAX1 and ZCAN 1 genes by using Bsh 1236I restriction enzyme in the experimental example of the present invention;
FIG. 2 is a schematic diagram showing the amplification of PAX1 and ZCAN 1 genes by enzyme digestion with Eco 72I restriction enzyme in the experimental example of the present invention;
FIG. 3 is a schematic diagram showing the digestion and amplification curves of PAX1 and ZCAN 1 genes using HhaI restriction enzyme in the experimental example of the invention;
FIG. 4 is a schematic diagram showing the amplification of PAX1 and ZCAN 1 genes by using Bsh 1236I and Eco 72I restriction enzymes in the experimental example of the present invention;
FIG. 5 is a schematic diagram showing the amplification curves of PAX1 and ZCAN 1 genes by enzyme digestion with Eco 72I and HhaI restriction enzymes in the experimental example of the invention;
FIG. 6 is a schematic diagram showing the amplification curves of PAX1 and ZCAN 1 genes by using Bsh 1236I and HhaI restriction enzymes in the experimental example of the present invention;
FIG. 7 is a schematic diagram showing the amplification curves of PAX1 and ZCAN 1 genes by using Bsh 1236I, eco 72I and HhaI restriction enzymes in the experimental example of the present invention;
FIG. 8 is a schematic diagram showing a test subject who has been diagnosed with cervical cancer by performing amplification of PAX1 gene using Bsh 1236I, eco 72I, and HhaI restriction enzymes in the experimental example of the present invention;
FIG. 9 is a schematic diagram showing the experimental results of the amplification of ZSCAN1 gene by enzyme digestion using Bsh 1236I, eco 72I, hha I restriction enzymes for diagnosing cervical cancer in the experimental examples of the present invention.
Detailed Description
The following are specific embodiments of the present invention and the technical solutions of the present invention will be further described with reference to the accompanying drawings, but the present invention is not limited to these embodiments.
Unless defined otherwise, 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. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
Example 1
The gene methylation marker for cervical cancer provided by the invention is selected from a sequence about 700bp before a start codon and a sequence about 300bp after the start codon of each gene in target genes PAX1, ZSCAN1 and internal reference genes COL2A1, and the methylation condition of a promoter region is determined by sequencing. And primer design was performed on the target gene fragment as described above, and the primer design is shown in Table 1. Wherein the target gene amplified fragment contains one or more of the cleavage sites in methylation sensitive restriction enzymes Bsh 1236I, eco 72I and HhaI, and the reference gene amplified fragment does not contain the cleavage sites; further preferred, bsh 1236I-specific cleavage CG/CG, eco 72I-specific cleavage CAC/GTG, hhaI-specific cleavage GCG/C; still more preferably, methylation sensitive restriction enzymes Bsh 1236I, eco 72I, and HhaI are all available commercially.
TABLE 1 detection of target Gene and primer probe nucleic acid sequences
Wherein the probe sequences shown in the table are not fluorescently labeled and quenching group labeled. The fluorescent group label used by the probe is set to be any one of FAM, VIC, HEX, TET, CY and ROX, JOE, CY 3; the quenching group is set to any one of BHQ1, BHQ2, BHQ3, TAMRA, DABCYL, MGB.
Specifically, the target fragment of the target gene PAX1 is as follows:
ATTCTGTGGTTTAACAGAGACTTCCTGAAGCTGCAGGTGCTGGGAGAAAGACGAGGCCAGGCCACCTGGGATAGAGTGGTGCGAGATTCCACCGCCAGGGAGAAAGGAACTTGTCCTTCAGACCTAGAGCTGGAGCTATGCATTTGGCCTCCCGCCGGTCGCGCTTGGGGACAGGAGGGCGCCGGATCTATGCGCCCCTTAGCAGCAGATCGGGATCCTTTTGCCTCCTGCCCCTTCTGTCACTGCTTGGAGAGGGATGAGTTCTGGTGGCTGGGCCTGGCTGTAGGAGACAGGATTTGGACCGTGCCCCTCTCGCATCACCGAAATCACCCCCACTATTCCAAGAGTGGTTG
specifically, the target fragment of the target gene ZSCAN1 is as follows:
AGAACAAGGAAGTAAGGAGCCGAGGGGCATCCTGAGTACAGAGGGCCACCATCTGATGTGACCCCTCTGTCCCTGCCCCTCTCTGCAGGCCCCTGATTGCTGATTCTGTCCGCCTGCCACACCGGCTCTGTCCGGAGCCACTGGGACTCGGGATCCAGTCCACACACACCCCTCAGAGGGGCACTGTGGCCAGAGAAATGCTTCCACGGCCCAAAGCCCCTGCCTCCCCCAGACGCCCCCAGACCCCAACCCCGAGTGAGCAGGACGCAGACCCTGGGCCAGCAAGCCCCAGGGACACCGAAGCCCAGCGTCTGCGCTTCCGGCAGTTCCAGTACCACGTGGCGAGCGGGCCGCACCTCGCGCTGGGCCAGCTCTGGACGCTGTGCCGCCAGTGGCTGAGGCCCGAGGCGCGCTCCAAGGAACGCGTCGATCG
Specifically, the target fragment of the reference gene COL2A1 is as follows:
CTATGAATTAGCAGACATGGAGTAAGAAGGACTCTACAGTGCACAAAATGCATAGCTTTATATAACATCAATTCTGGCTATTTAGTGGTTTAGTTCCCCTTCTTTTCAGTTTCTTGGGTATGGTATGGATAGTGAGATTGTTGGACGATCAATTTCCAAATCAGTGATTTGCATATGTGTGCCACATATAGGTCTAATTCCAGTTTGAGTCACAAATTAGAGGTTTTTAATTAGTTGTTTGGAGTCACAGATGGGTAAAATATTAATGTTGGTGTTCAATTGGCTTTAGAATTTTAATAATTTCCATAGTTTATTTTGGCTTTGTCAATTTAAATTTTAAATTTTATTTTTCTTATGTCAATTCCCGTTGCTGGTGAAAAGAAATTTTCCCACAGTGGGTGGAGAGCTTAACTTGTACTCATAGATGGACCATGATTGCTGGAAGGAATCCAAAGCTCTTATATGTAGACACAGTCTATTGGCAAAGATGAATCACAGAGCTAGAGGATGAAATTAAGAACAA
specifically, for better application of the enzyme digestion system and the subsequent amplification system of three endonucleases, an enzyme digestion amplification buffer system is prepared according to table 2; preparing a mixed solution of primer probes according to Table 3;
TABLE 2 enzyme digestion and amplification buffers
TABLE 3 primer probe mixture
Example 2
The kit provided by the invention is used for detecting and testing the methylation of the PAX1 gene and the methylation of the ZSCAN1 gene in early screening diagnosis of cervical cancer, and comprises the following steps:
1. sample DNA extraction
And extracting DNA of the cervical exfoliated cell sample by adopting a magnetic bead method, and monitoring the quality of the extracted DNA, wherein the total amount of the DNA is between 0.05 mug and 1 mug, so that the requirement of the next experimental nucleic acid can be met.
2. Enzyme-cleaved fluorescent quantitative PCR amplification
2.1 Enzyme digestion amplification system
The enzyme-digested amplification system was prepared according to Table 4, and the amount of DNA used was 0.2. Mu.g, the lowest was not less than 0.05. Mu.g, and the highest was not more than 1. Mu.g. The probes of PAX1, ZSCAN1 and COL2A1 genes adopt different fluorescent groups, and can finish detection of three target genes and one reference gene in the same reaction system.
TABLE 4 preparation of enzyme-cleaved amplification System
2.2 Enzyme digestion amplification procedure
The invention firstly carries out enzyme digestion reaction at the low temperature of 37 ℃, and three restriction enzymes can not interfere with each other in an enzyme digestion amplification system. Then, the real-Time fluorescent quantitative PCR amplification was performed, and methylation detection was performed on an ABI 7500 Real-Time PCR apparatus according to the enzyme digestion amplification procedure of Table 5.
TABLE 5 parameters of the enzyme digestion and amplification procedure
The enzyme-cutting amplification positive quality control substance P and the enzyme-cutting amplification negative quality control substance N are added in each reaction, and a blank control NTC is used for enzyme-cutting amplification. Wherein P is a synthesized methylation positive plasmid fragment, N is normal human DNA of which the target genes are not methylated through sequencing verification, and NTC is nuclease-free water.
3. Gene methylation result interpretation
3.1 Quality control is qualified: the internal control gene COL2A1 is amplified in an S type, and Cp is less than or equal to 33 and is in control.
3.2 The Δcp value is used to reflect the methylation level of a particular gene target region.
ΔCp(PAX1)= Cp(PAX1)- Cp(COL2A1)
ΔCp(ZSCAN1)= Cp(ZSCAN1)- Cp(COL2A1)
3.3 Cervical lesions prediction interpretation
The level of methylation of the gene is the Δcp value, the smaller the Δcp value the higher the degree of methylation and vice versa. The invention uses three restriction enzyme amplification systems, uses high-grade squamous intraepithelial lesions as tangent points according to clinical sample data, and uses the principle of maximum about dengue index for distinguishing lesions, and positive judgment standards of PAX1 and ZSCAN1 are shown in Table 6.
TABLE 6 Gene methylation results interpretation criteria
Experimental example 1:
204 cervical exfoliated cells with well-known pathological consequences were selected, 92 patients with cervical normal or chronic inflammation, 47 patients with low-grade squamous intraepithelial lesions, 55 patients with high-grade squamous intraepithelial lesions, and 10 patients with cervical cancer.
According to example 2, DNA from cervical exfoliated cell samples was extracted, digested with methylation restriction enzymes, and the methylation status of PAX1 and ZSCAN1 genes was detected by real-time quantitative PCR, to obtain Δcp values for the methylation results of PAX1 and ZSCAN1 genes. Subject work curve analysis was performed on the PAX1 and ZCAN 1 genes (see FIG. 1 to FIG. 9), and positive judgment values of the two genes were determined on the principle of about maximum Density index with cervical high-grade lesions as tangent points, and the results are shown in Table 7.
TABLE 7 PAX1, ZSCAN1 Gene subject working Curve analysis
And combining the actual results, determining that the positive judgment value of the PAX1 gene is 10.50, and the positive judgment value of the ZSCAN1 gene is 8.80. The performance of the two genes in combined diagnosis of cervical high-grade lesions was analyzed according to the positive judgment value determined, and statistics of each pathological group are shown in the following table 8.
TABLE 8 analysis of PAX1, ZCAN 1 Gene methylation and disease State correlation
The performance of the single gene and dual gene combination diagnosis of cervical high grade lesions was analyzed and the results are shown in Table 9.
TABLE 9 Performance analysis of double Gene methylation in combination with diagnosis of cervical high grade lesions
It can be seen from Table 9 that although the methylation detection of the PAX1 and ZCAN 1 single genes has better detection performance, the methylation detection of the combination of the double genes has better detection performance on cervical high-grade lesions than that of the single genes. When any gene in the double genes is positive and judged to be positive, the detection sensitivity and the negative judgment value are the highest and are 93.85% and 96.95% respectively; when both genes are positive, the detection specificity and the positive predictive value are highest, namely 96.40% and 90.38%, respectively, but the detection sensitivity is lowest and only 72.31%. And the detection sensitivity, the detection specificity, the positive predictive value and the negative predictive value are synthesized, and the positive is judged to be the best when the methylation detection result of any one gene of the PAX1 and ZSCAN1 genes is positive.
The advantages of the present invention over the prior art include:
1. Compared with the common bisulfite treatment, the methylation sensitive restriction enzyme digestion amplification method has the advantages that the detection time is short, three hours are needed for the bisulfite treatment, the restriction enzyme digestion reaction only needs fifteen minutes, the recovery of nucleic acid is not needed after enzyme digestion, and the loss of nucleic acid can be reduced; 2. compared with HPV detection, the gene methylation detection specificity is higher, and misdiagnosis can be effectively reduced; 3. compared with cytological examination, the invention has higher sensitivity and can effectively reduce missed diagnosis.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (6)
1. A primer probe set for diagnosing methylation of cervical cancer genes, comprising a primer sequence and a probe sequence for detecting a gene methylation marker consisting of a nucleotide sequence methylated in one target region in a target gene PAX1 and a nucleotide sequence methylated in one target region in a target gene ZSCAN1, wherein the primer sequence and the probe sequence are specifically expressed as follows:
The pre-primer of PAX1 gene: SEQ ID NO.1;
post primer of PAX1 gene: SEQ ID NO.2;
Probes for the PAX1 gene: SEQ ID NO.3;
the ZSSAN 1 gene pre-primer: SEQ ID NO.4;
Post primer of ZSSAN 1 gene: SEQ ID NO.5;
probes for ZSCAN1 gene: SEQ ID NO.6.
2. The primer probe set according to claim 1, further comprising a set of primer sequences and probe sequences corresponding to a set of reference markers COL2A1 gene, specifically expressed as follows:
the pre-primer of COL2A1 gene: SEQ ID NO.7;
post-primer of COL2A1 gene: SEQ ID NO.8;
Probes for the COL2A1 gene: SEQ ID NO.9.
3. The primer probe set according to claim 1 or 2, wherein the fluorescent group label for the probe is set to be either one of FAM, VIC, HEX, TET, CY and ROX, JOE, CY 3;
the quenching group is set to any one of BHQ1, BHQ2, BHQ3, TAMRA, DABCYL, MGB.
4. The use of the primer probe set for diagnosing methylation of cervical cancer genes according to claim 3 for preparing a cervical cancer detection kit.
5. A kit for diagnosing cervical cancer gene methylation, which is characterized by comprising the primer probe set, the enzyme digestion amplification system and a primer probe mixed solution according to claim 3; wherein,
The buffer solution of the enzyme digestion amplification system is prepared from the following components in percentage by weight:
Tris-HCl:15mmol/L; naCl:50mmol/L; mgCl2:10mmol/L; triton X-100:0.1% -1%; DMSO:0.1% -1%; BSA:0.1mg/mL; mercaptoethanol: 1mmol/L; dNTPs:1mmol/L;
Wherein, the unit of each reagent is concentration;
the preparation ratio of the primer probe mixed solution is as follows:
PAX 1-pre-primer: 10 mu mol/L; PAX 1-post primer: 10 mu mol/L; PAX 1-probe: 20 mu mol/L: ZSSAN 1-pre-primer: 10 mu mol/L; ZSSAN 1-post primer: 10 mu mol/L; ZSSCAN 1-probe: 20 mu mol/L; COL2A 1-pre-primer: 10 mu mol/L; COL2A 1-post primer: 10 mu mol/L; COL2 A1-probe: 20 mu mol/L;
wherein, each primer unit is working concentration.
6. The kit according to claim 5, wherein the methylation level of the target region of the specific gene is reflected in a Δcp value in the detection of the biomarker of cervical cancer, wherein the Δcp value is expressed as follows:
ΔCp(PAX1)= Cp(PAX1)- Cp(COL2A1)；
ΔCp(ZSCAN1)= Cp(ZSCAN1)- Cp(COL2A1)。
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