CN117904303B - Application of SORCS gene methylation and PAX1 gene methylation combined diagnosis detection primer probe group in preparation of cervical cancer diagnosis product - Google Patents
Application of SORCS gene methylation and PAX1 gene methylation combined diagnosis detection primer probe group in preparation of cervical cancer diagnosis product Download PDFInfo
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- 238000007069 methylation reaction Methods 0.000 title claims abstract description 93
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- 206010008342 Cervix carcinoma Diseases 0.000 title claims abstract description 30
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Abstract
The invention provides an application of a SORCS gene methylation and PAX1 gene methylation combined diagnosis detection primer probe group in preparation of cervical cancer diagnosis products, wherein a SORCS gene methylation and PAX1 gene methylation primer sequence is specifically expressed as follows: the pre-primer pair of SORCS gene: SEQ ID NO.1; post primer pair for SORCS gene: SEQ ID NO.2; front primer pair of PAX1 gene: SEQ ID NO.4; rear primer pair of PAX1 gene: SEQ ID NO.5. The invention selects the genes related to cervical cancer and precancerous lesions, and scientifically selects a methylation interval by detecting the specific methylation sites of the genes, thereby well predicting the cervical lesion degree. The 2 selected genes form functional complementation, and have the characteristics of good sensitivity and specificity for cervical cancer and cervical cancer front high-grade lesions.
Description
Technical Field
The invention relates to the technical field of Gong Gengliang malignant lesions detection, in particular to application of a detection primer probe group for combined diagnosis of SORCS gene methylation and PAX1 gene methylation in preparation of cervical cancer diagnosis products.
Background
Cervical cancer is the fourth most common cancer in women, accounting for 7.5% of female cancer deaths. At present, the diagnosis and treatment process of cervical cancer is high-risk human papilloma virus (hrHPV) primary screening, and in women with positive hrHPV primary screening, cytology is adopted to carry out shunt management. Wherein:
The cytological detection is based on subjective interpretation, a large number of doctors need to be trained specifically, the human interference factor is large, the inconsistent proportion of results is high, and a large number of ambiguous results (ASCUS) are also caused, so that the clinical doctor is bothered. And while hrHPV detection is very sensitive to detection of cervical lesions, about 90% self-clearance in two years is less than ideal in view of most infections, resulting in female cancer panic and even excessive medical treatment for personal reasons.
(II) colposcopy is an interpretation of the image after enlargement of the cervical region, the sensitivity is between 40% and 80%, hidden lesions of the cervical canal are difficult to find, the sampling of colpossessor also affects the final pathological result, and the doctor can not accurately judge the most serious part of the cervical lesion, so that missed diagnosis can be caused.
(Iii) the slip is a gold standard for diagnosing cervical cancer and precancerous lesions, but it is invasive, hrHPV more easily enters basement membrane cells through the wound, increasing the risk of cervical cancer. In addition, the pathological junction is mainly based on morphology, and the potential risk of the pathological junction cannot be judged.
Disclosure of Invention
The invention aims to provide a gene methylation biomarker which is used as a specimen for detecting the shunt of hrHPV positive patients so as to identify a very small number of hrHPV positive women suffering from cervical high-grade diseases, thereby achieving the aim of achieving clinical required effects while eliminating the need of professional technology by relying on molecular automatic detection rather than detection relying on morphological characteristics (such as cytology and histopathology).
The invention provides an application of a detection primer probe group for SORCS gene methylation and PAX1 gene methylation joint diagnosis in preparation of cervical cancer diagnosis products, wherein a primer sequence for SORCS gene methylation and PAX1 gene methylation is specifically expressed as follows:
The pre-primer pair of SORCS gene: SEQ ID NO.1;
post primer pair for SORCS gene: SEQ ID NO.2;
Front primer pair of PAX1 gene: SEQ ID NO.4;
rear primer pair of PAX1 gene: SEQ ID NO.5.
Further, the kit also comprises a probe sequence for detecting SORCS gene and a probe sequence for detecting PAX1 gene, wherein the probe sequences are specifically expressed as follows:
Probe of SORCS Gene: SEQ ID NO.3;
Probes for the PAX1 gene: SEQ ID NO.6.
Further, the kit also comprises a pair of primer sequences corresponding to the reference marker COL2A1 genes, and the primer sequences are specifically expressed as follows:
the pre-primer pair of COL2A1 gene: SEQ ID NO.7;
rear primer pair of COL2A1 gene: SEQ ID NO.8;
probes for the COL2A1 gene: SEQ ID NO.9.
Further, in the detection process of the Gong Gengliang malignant lesion biomarker, the result of diagnosing cervical cancer by gene methylation is interpreted as follows:
The methylation level of a target region of a particular gene is reflected in a Δcp value of:
ΔCp(SORCS1)= Cp(SORCS1)- Cp(COL2A1)，ΔCp(PAX1)= Cp(PAX1)- Cp(COL2A1)。
wherein COL2A1 is an internal control gene.
Further, in the detection of the biomarkers of Gong Gengliang malignant lesions, SORCS methylation and PAX1 methylation were ranked as follows, with the principle of maximum about sign index to distinguish lesions:
。
Further, in the detection process of the biological marker of Gong Gengliang malignant lesions, the prediction and interpretation of Gong Gengliang malignant lesions are as follows:
。
Compared with the prior art, the invention has the following beneficial effects:
The invention selects the genes (SORCS gene and PAX1 gene) related to cervical cancer and precancerous lesions, and scientifically selects a methylation interval by detecting the specific methylation site of the genes, thereby well predicting the cervical lesion degree. The 2 selected genes form functional complementation, and have the characteristics of good sensitivity and specificity for cervical cancer (squamous carcinoma and adenocarcinoma) and cervical cancer anterior altitude lesions.
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 (A) is a schematic diagram showing the distribution of ORCS 1-methylated ΔCp in a sample of different lesions in an example of the present invention;
FIG. 1 (B) is a schematic diagram showing the distribution of PAX1 methylation ΔCp in different lesions in a sample according to an embodiment of the present invention;
FIG. 2 (A) is a graphical representation of the percent predictions for each grade after grading SORCS methylation levels in an example of the present invention;
FIG. 2 (B) is a graphical representation of the percent predictions for each grade after grading the methylation level of PAX1 in the examples of the present invention;
FIG. 3 (A) is a graphical representation of the Area (AUC) of the working curve ROC of a subject identified by the SORCS.sup.1 methylation test in an example of the present invention to cervical intraepithelial neoplasia CIN2+;
FIG. 3 (B) is a graphical representation of the Area (AUC) of the working curve ROC of a subject identified by the SORCS.sup.1 methylation test in an example of the present invention;
FIG. 3 (C) is a graphical representation of the Area (AUC) of the working curve ROC of a subject identified by the PAX1 methylation test for cervical intraepithelial neoplasia CIN2+ in an embodiment of the present invention;
fig. 3 (D) is a graphical representation of the Area (AUC) of the working curve ROC of subjects identified as cervical intraepithelial neoplasia cin3+ by the PAX1 methylation test in an example of the present invention.
Wherein,Is critical value,/>For sensitivity,/>Is specificity.
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 biomarker for early screening diagnosis of cervical cancer (particularly comprises screening diagnosis of cervical cancer and precancerous high lesions CIN2/CIN 3), which is provided by the invention, at least comprises nucleotide sequences methylated in at least one target region of SORCS genes and PAX1 genes and at least comprises nucleotide sequences methylated in one target region of internal control genes COL2A 1. Among them, the chromosome positions in ORCS gene, PAX1 gene, internal control gene COL2A1 are shown in Table 1.
TABLE 1 chromosomal location in SORCS1 Gene, PAX1 Gene, internal control Gene COL2A1
Specifically, a primer probe for PCR amplification of the target gene is designed:
The reagent for detecting the target gene is divided into a first reagent for detecting SORCS gene, a second reagent for detecting PAX1 gene, and a third reagent for detecting internal control gene COL2A 1; wherein:
The first reagent is a reagent suitable for detecting SORCS gene methylation by adopting a PCR method, and a target area detected by the first reagent is a partial area selected from chr10: 108924300-108924805;
The second reagent is a reagent suitable for detecting PAX1 gene methylation by adopting a PCR method, and the target region detected by the second reagent is selected from partial regions of chr20: 21694472-21694870;
the third reagent is a reagent suitable for detecting methylation of the COL2A1 gene by a PCR method, and the target region detected by the third reagent is selected from partial regions of chr12: 48366748-48398285.
The first reagent comprises a first primer pair and a first probe, and the sequences of the first primer pair and the first probe for detecting the target area are shown in the table 2; the second reagent comprises a second primer pair and a second probe, and the sequences of the second primer pair and the second probe for detecting the target area are shown in the table 3; the third reagent comprises a third primer pair and a third probe, and the sequences of the third primer pair and the third probe for detecting the target region are shown in Table 4.
TABLE 2 nucleotide sequences of first primer pair and first probe for detecting SORCS Gene methylation
Note that: probe sequences shown in the table were not fluorophore labelled and quencher labelled.
Specifically, SORCS1 (chr10: 108924300-108924805) is selected from the DNA strand in UCSC Genome Browser on Human (GRCh 37/hg 19), > hhg19_dnarange=chr10: 108924300-108924805 trand = +:
CAGCGGAGAGAGGGGTCCCAGAACGAAGGTGGCGGCACGAGCTCTGCGCTGGCGGCTGTGGGGGGCCGGCGCTCAGGACCCCAACTCCATCCAAGTTGCGCCGCGGTGGGGGCGGGCGGAGGCGGCGCCGGGCAGGTGGCGGCCGCTTGCCCGGGCTGGGCTTGTGCCGAGCGCGGGTCCGGACGGAGCGAGCCGCCGCCGCGCGGGGGTGGAGCTGAGCTGAAGTCACTGGCGGAGCGGGCGCGGGCGCTGGCGGGCGACGCGGGAGGGAGGGCAGGCGCTGCCGGGACTCCGGGCGCCGCGTCCCGCGGCCGAGCTGCGCTGCGCACCGGGTCTCCGGGCGCTGGAGGCGCCGCCGGCGACCTCCCCTCGGCCTCGCAGGCGCTGCCGCCGCCTCTCCCCGCCGCCCACTCTGCGCCCCCGCGGCCGCGGGTCCCGGCGCTGAGTCCGCGCACCCTGCCCGCGGGGGCGGTCGACCCCGGGCTCCCGGAGCAGTCGATGTGTCG
the DNA sequence was subjected to bisulfite conversion (unmethylated C to T, methylated C remained unchanged) to give converted DNA strands:
TAGCGGAGAGAGGGGTTTTAGAACGAAGGTGGCGGTACGAGTTTTGCGTTGGCGGTTGTGGGGGGTCGGCGTTTAGGATTTTAATTTTATTTAAGTTGCGTCGCGGTGGGGGCGGGCGGAGGCGGCGTCGGGTAGGTGGCGGTCGTTTGTTCGGGTTGGGTTTGTGTCGAGCGCGGGTTCGGACGGAGCGAGTCGTCGTCGCGCGGGGGTGGAGTTGAGTTGAAGTTATTGGCGGAGCGGGCGCGGGCGTTGGCGGGCGACGCGGGAGGGAGGGTAGGCGTTGTCGGGATTTCGGGCGTCGCGTTTCGCGGTCGAGTTGCGTTGCGTATCGGGTTTTCGGGCGTTGGAGGCGTCGTCGGCGATTTTTTTTCGGTTTCGTAGGCGTTGTCGTCGTTTTTTTTCGTCGTTTATTTTGCGTTTTCGCGGTCGCGGGTTTCGGCGTTGAGTTCGCGTATTTTGTTCGCGGGGGCGGTCGATTTCGGGTTTTCGGAGTAGTCGATGTGTCG
the underlined part of the converted DNA strand sequence is the position of the primer probe sequence of SORCS (chr 10: 108924300-108924805) finally obtained, and 10 methylation sites are detected.
TABLE 3 nucleotide sequences of a second primer pair and a second probe for detecting methylation of PAX1 gene
Note that: probe sequences shown in the table were not fluorophore labelled and quencher labelled.
Specifically, PAX1 (chr 20: 21694472-21694870) is selected from the DNA strand in UCSC Genome Browser on Human (GRCh 37/hg 19), > hr19_dnarange=chr20: 21694472-21694870 strand = +:
GCGCATCTGGATCCCCGGACCTCGTGGGCACAACGTAGTCTGCCGCTCACTTAAAGCGCAACTGCCTCCTACGTCGGAGGAATCCAGGCCGCTCCAGGGAAATCTCTGGTGGCTCATTTCCCGCAAGATTTCCCCCTGCCCACAGCGAGCTCCGAGCCGCGGGTGTCAGCGGACTCGACGCATTGGCTGCTCTTCGCACTTGTGCGGGCGCCTGGGGTTTGGAAAGGGTGAGGGTCCGCGCAACGTTAGAGTCCTGTTCCCTTCTGCGGAAGTCGCCATCAGACCTGAAACCTTCCCAGCCCTCTGCGCCCTTCACTTCCTTTACTGAGCAGGTGGTGAGGGGGTTGGACGGCAACGGGAATTGCACCTGGCCAGCGGTCAGTGTTTGCAAATGGCCTT
the DNA sequence was subjected to bisulfite conversion (unmethylated C to T, methylated C remained unchanged) to give converted DNA strands:
GCGTATTTGGATTTTCGGATTTCGTGGGTATAACGTAGTTTGTCGTTTATTTAAAGCGTAATTGTTTTTTACGTCGGAGGAATTTAGGTCGTTTTAGGGAAATTTTTGGTGGTTTATTTTTCGTAAGATTTTTTTTTGTTTATAGCGAGTTTCGAGTCGCGGGTGTTAGCGGATTCGACGTATTGGTTGTTTTTCGTATTTGTGCGGGCGTTTGGGGTTTGGAAAGGGTGAGGGTTCGCGTAACGTTAGAGTTTTGTTTTTTTTTGCGGAAGTCGTTATTAGATTTGAAATTTTTTTAGTTTTTTGCGTTTTTTATTTTTTTTATTGAGTAGGTGGTGAGGGGGTTGGACGGTAACGGGAATTGTATTTGGTTAGCGGTTAGTGTTTGTAAATGGTTTT
The underlined part of the converted DNA chain sequence is the position of the primer probe sequence of the PAX1 (chr 20: 21694472-21694870) finally obtained, and 7 methylation sites are detected.
TABLE 4 nucleotide sequences of third primer pair and third probe for detecting methylation of internal control gene COL2A1
Note that: probe sequences shown in the table were not fluorophore labelled and quencher labelled.
Example 2:
the kit provided by the invention is used for a detection test method for early screening diagnosis ORCS genes, PAX1 genes and internal control genes COL2A1 of cervical cancer, and comprises the following steps:
1. sample DNA extraction
The DNA of the cervical exfoliated cell sample is extracted by adopting a manual magnetic bead method, the quality of the extracted DNA is monitored, the total amount of the DNA is between 1ug and 10ug, the OD260/280 is between 1.9 and 2.0, and the DNA electrophoresis gel has better prompt yield and purity and can be used for the next test.
2. DNA bisulfite conversion
The method comprises the steps of performing bisulfite conversion on extracted DNA by using a bisulfite conversion kit, namely a methylation detection sample pretreatment kit (Hunan Hongya Gene technology Co., ltd., hunan Long mechanical equipment 20200131) as a DNA bisulfite conversion reagent, wherein unmethylated cytosine (C) in the DNA is converted into uracil (U), and methylated cytosine (C) is unchanged, so that DNA (B-DNA) after the bisulfite conversion is obtained.
3. Methylation specific fluorescent quantitative PCR amplification
3.1, Preparing a fluorescence quantitative PCR reaction system, and table 5. The SORCS, PAX1 and COL2A1 probes adopt different fluorophores, so that detection can be performed in the same reaction system, and separate detection can be performed.
Table 5 PCR reaction system (25 ul/person)
Note that: the DNA is sample DNA after bisulfite conversion, or negative/positive quality control.
3.2 PCR reaction
The invention skillfully designs SORCS, PAX1 and COL2A1 primer probes, the optimal temperature of the PCR reaction is quite close, and the primers do not interfere with each other in the same reaction system. PCR reactions were performed in an LC482 II fluorescent quantitative PCR instrument according to the program parameters of Table 6.
Table 6 PCR machine program parameters
4. Gene methylation result interpretation
4.1, Qualified quality control: the internal control gene COL2A1 is amplified in an S type, and Cp is less than or equal to q which is tested by LC480 is under control.
4.2, The methylation level of a target region of a specific gene is reflected in the ΔCp value.
ΔCp(SORCS1)= Cp(SORCS1)- Cp(COL2A1)；
ΔCp(PAX1)= Cp(PAX1)- Cp(COL2A1)；
4.3 Prediction interpretation of cervical lesions
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. According to clinical sample data, the method takes the principle of maximum about dengue index for distinguishing lesions as SORCS grades of methylation of PAX1, and the method comprises the following steps of: 1. slightly methylated, 2. Hypomethylated, 3. Moderately methylated, 4. Hypermethylated, 5. Hypermethylated. The range of values for each grade is shown in Table 7.
TABLE 7 methylation level ranking of genes
4.4 Interpretation of Gene methylation results
The methylation level of the gene is distributed differently in the shedding cells of patients with normal pathology, CIN1, CIN2, CIN3 and cervical cancer, and the aim of clinically detecting CIN2+ is to be achieved, so that the invention selects the optimal critical value for SORCS.PAX 1 methylation by taking the principle that the clinical sensitivity and specificity are equally important and detecting as much CIN2+ as possible. The range of the critical value is shown in Table 8.
TABLE 8 interpretation of methylation results of genes
Experimental example 1:
291 samples of cervical exfoliated cells of patients with known pathological results were selected, of which 149 were normal, 67 were CIN1, 40 were CIN2, 23 were CIN3, and 12 were cervical cancer. 8 of cervical cancers, 4 of them, squamous carcinoma. Sample DNA extraction, DNA bisulfite conversion, and methylation specific fluorescent quantitative PCR amplification were performed according to example 2, resulting in Δcp values for SORCS1 and PAX1 methylation results. The distribution of SORCS a and Δcp of PAX1 in the sample for different lesions is shown in fig. 1 (a) and 1 (B), NS: p is more than 0.05, and no obvious difference exists; * : p is more than 0.01 and less than 0.05; * *: p is more than 0.001 and less than 0.01; * **: p < 0.001.
For SORCS1 methylation ranking, a1=14.77, a2=10.26, a3=6.42, a4=4.66 was taken and divided into 5 classes: SORCS1 slight methylation (ΔCp > 14.77), 2.SORCS1 hypomethylation (10.26 < ΔCp. Ltoreq.14.77), 3. SORCS1 moderate methylation (6.42 < ΔCp. Ltoreq.10.26), 4. SORCS1 hypermethylation (4.66 < ΔCp. Ltoreq.6.42), 5. SORCS1 hypermethylation (ΔCp. Ltoreq.4.66). Following the SORCS1 methylation level ranking, the number and percentage of lesions predicted for each grade are shown in Table 9 and FIG. 2 (A). The possibility of cervical lesions can be predicted according to the interval in which Δcp is located. SORCS1 methylation at 6.42 < ΔCp.ltoreq.10.26, the probability of 68.9% in the sample of this experimental example was CIN2.SORCS1 methylation at ΔCp > 14.77, the patient was most likely normal, with a 1.6% probability of CIN1.
For the grading of PAX1 methylation, b1=16.96, a2=11.99, a3=8.98, a4=4.57 was taken and divided into 5 grades: PAX1 is slightly methylated (Δcp > 16.96), 2.pax 1 is hypomethylated (11.99 < Δcp.ltoreq.16.96), 3.pax 1 is hypomethylated (8.98 < Δcp.ltoreq.11.99), 4.pax 1 is hypermethylated (4.57 < Δcp.ltoreq.8.98), 5.pax 1 is hypermethylated (Δcp.ltoreq.4.57). After grading the PAX1 methylation levels, each grade predicts the number and percentage of lesions, see table 10 and fig. 2 (B). When PAX1 methylation ΔCp > 16.96, 100% of the samples in this experimental example were normal.
TABLE 9 number and ratio of cervical lesions in SORCS methylation intervals
TABLE 10 number and ratio of cervical lesions in the PAX1 methylation interval
The ability of the primer probe reagents of the invention to identify CIN2+ and CIN3+ is shown in FIGS. 3 (A) -3 (D) (where AUC is expressed as the area under the subject's working curve) and is analyzed as follows:
SORCS1 (chr 10: 108924300-108924805) methylation test the Area (AUC) of the working curve ROC of the subject identifying cervical intraepithelial neoplasia CIN2+ was 0.944.
SORCS1 (chr 10: 108924300-108924805) methylation test the Area (AUC) of the working curve ROC of the subject identifying cervical intraepithelial neoplasia CIN3+ was 0.904.
PAX1 (chr 20: 21694472-21694870) methylation test the Area (AUC) of the working curve ROC of subjects identified as cervical intraepithelial neoplasia CIN2+ was 0.968.
PAX1 (chr 20: 21694472-21694870) methylation test identifies the Area (AUC) of the subject working curve ROC of cervical intraepithelial neoplasia CIN3+ as 0.974.
In this experimental example, the critical values of SORCS and PAX1 methylation were taken as k1=10.26 and k2=11.84, respectively. Clinical performance against CIN2+ (CIN2, CIN3 and cervical cancer) and CIN3+ (CIN3 and cervical cancer) are presented in Table 11.SORCS1 methylation detection CIN2+ sensitivity of 89.3%, specificity of 92.6%, positive predictive value of 80.7% and negative predictive value of 96.2%; sensitivity of detection of CIN2+ by PAX1 methylation is 96.0%, specificity is 94.4%, positive predictive value is 85.7%, and negative predictive value is 98.6%; SORCS1 methylation detection CIN3+ sensitivity of 82.9%, specificity of 78.9%, positive predictive value of 34.9% and negative predictive value of 97.1%; the sensitivity of the PAX1 methylation detection CIN3+ is 91.7%, the specificity is 80.5%, the positive predictive value is 40.5%, and the negative predictive value is 99.5%.
TABLE 11 clinical performance of SORCS1 and PAX1 methylation diagnosis of CIN2+ and CIN3+
SORCS1 and PAX1 methylation can be diagnosed in combination, see table 12 for clinical performance against cin2+ and cin3+a. When SORCS and PAX1 methylation are positive, the sensitivity to CIN2+ is improved to 97.3%, and particularly the sensitivity to CIN3+ detection reaches 100%. When SORCS and PAX1 methylation genes were positive, the specificity was improved, especially 94.9% for CIN2+ detection.
Table 12 clinical performance of PAX1 and SORCS1 methylation in combination diagnosis of CIN2+ and CIN3+
In this experimental example, 8 squamous cell carcinomas were detected, and 5 cases were detected for all 8 squamous cell carcinomas with PAX1 methylation and SORCS methylation. In this experimental example, 4 cases of adenocarcinoma were detected by SORCS methylation, and 3 cases of adenocarcinoma were detected by PAX1 methylation. The combination of SORCS and PAX1 gene methyl groups, functionally complementary, detected cervical cancer at maximum efficiency, see Table 13.
TABLE 13 distribution and interpretation of PAX1 and SORCS1 methylation in cervical squamous carcinoma and adenocarcinoma
The advantages of the present invention over the prior art include:
The SROCS and PAX1 gene methylation markers highly related to Gong Gengliang malignant lesions are screened out, the methylation levels of the SROCS and PAX1 genes are classified into grades by adopting a scientific method, and the SROCS and PAX1 gene methylation interpretation standards are set for detecting cervical lesions CIN2+. SROCS1 and PAX1 genes can well predict cervical lesion degree. According to the standard of SROCS and PAX1 gene methylation, the method has accuracy superior to the existing means on CIN2, CIN3 and cervical cancer, and the two genes are combined to form functional complementation, so that the detection rate of squamous carcinoma and adenocarcinoma of cervical cancer is improved. The invention provides an automatic detection for screening and diagnosing cervical cancer and can give objective result reflecting Gong Gengliang malignant lesions.
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. The application of a SORCS gene methylation and PAX1 gene methylation combined diagnosis detection primer probe group in the preparation of cervical cancer diagnosis products is characterized in that the primer sequences of SORCS gene methylation and PAX1 gene methylation are specifically expressed as follows:
The pre-primer pair of SORCS gene: SEQ ID NO.1;
post primer pair for SORCS gene: SEQ ID NO.2;
Front primer pair of PAX1 gene: SEQ ID NO.4;
rear primer pair of PAX1 gene: SEQ ID NO.5.
2. The use according to claim 1, wherein the primer-probe set further comprises a probe sequence for detecting SORCS gene and a probe sequence for detecting PAX1 gene, said probe sequences being specifically expressed as follows:
Probe of SORCS Gene: SEQ ID NO.3;
Probes for the PAX1 gene: SEQ ID NO.6.
3. The use according to claim 2, wherein the primer probe set further comprises a set of primer sequences corresponding to a pair of reference markers COL2A1 gene, specifically expressed as follows:
the pre-primer pair of COL2A1 gene: SEQ ID NO.7;
rear primer pair of COL2A1 gene: SEQ ID NO.8;
probes for the COL2A1 gene: SEQ ID NO.9.
4. The use according to claim 3, wherein in the detection of the biomarkers of Gong Gengliang malignant lesions, the result of diagnosing cervical cancer by gene methylation is interpreted as follows:
The methylation level of a target region of a particular gene is reflected in a Δcp value of:
ΔCp(SORCS1)= Cp(SORCS1)- Cp(COL2A1)，ΔCp(PAX1)= Cp(PAX1)- Cp(COL2A1)；
wherein COL2A1 is an internal control gene.
5. The use according to claim 4, wherein in the detection of biomarkers of Gong Gengliang malignant lesions, SORCS and PAX1 methylation are graded on the principle of maximum about index to distinguish lesions:
。
6. The use according to claim 5, wherein in the detection of the biomarker for Gong Gengliang malignant lesions, the predictive interpretation of Gong Gengliang malignant lesions is performed as follows:
。
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