Primer probe composition for thyroid cancer methylation level detection and application thereof

Abstract

The invention provides a primer probe composition for thyroid cancer methylation level detection and application thereof, and relates to the technical field of thyroid cancer detection. The primer probe composition comprises at least any one group of biomarkers as a primer probe sequence corresponding to a methylation marker, the methylation marker is composed of a nucleotide sequence of a PLEKHA4 gene and a nucleotide sequence of an REC8 gene, and primer probe design is carried out on the PLEKHA4 gene and the REC8 gene respectively. According to the invention, a specific methylation gene marker of thyroid cancer is selected, primer and probe design is carried out on the screened gene, and the gene methylation state is detected by using a fluorescent quantitative PCR technology; and multi-gene multi-target region combination and functional complementation are adopted, so that the diagnosis performance is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of thyroid cancer detection, and in particular to a primer-probe composition for detecting methylation level of thyroid cancer and application thereof. Background Art

[0002] Thyroid cancer is the most common malignant tumor of the endocrine system and head and neck, mainly including papillary carcinoma (PTC), follicular carcinoma (FTC), Hürthle cell carcinoma, medullary carcinoma (MTC) and anaplastic carcinoma (ATC). Among them, papillary carcinoma is the most common type, accounting for 79% to 94% of thyroid cancer.

[0003] Existing methods for thyroid cancer examination include neck palpation, ultrasound examination, blood test, fine needle aspiration biopsy, etc. Among them: the method of using neck palpation for detection is non-invasive, simple, and low-cost, and can quickly detect thyroid abnormalities (such as nodules or enlargement); its disadvantage is that it relies on the doctor's experience, is highly subjective, and cannot distinguish between benign and malignant. The method of using ultrasound examination is non-invasive and radiation-free, and can show the shape, size, boundary and internal echo of thyroid nodules, but it cannot determine the nature of nodules. The existing methods of using blood tests include: measuring the levels of thyroid stimulating hormone (TSH) and thyroid hormone (T3 and T4) in the blood; these detection methods can help determine whether the thyroid function is normal, but cannot directly detect thyroid nodules; and for early thyroid cancer, thyroid function may be normal, so blood tests cannot be used as a screening method alone. When thyroid nodules are found by ultrasound examination, fine needle aspiration biopsy can be used to obtain cell samples for pathological examination to determine whether the nodules are malignant. Fine needle aspiration biopsy can obtain nodule tissue samples for pathological examination, with a high diagnosis rate. Relatively safe and less traumatic. However, there is a certain risk of pain and bleeding. For cystic or smaller nodules, it may be difficult to obtain sufficient tissue samples, and there is a possibility of false negatives or false positives.

[0004] The detection of gene methylation levels in thyroid cancer is a molecular biological detection method that diagnoses or predicts thyroid cancer by analyzing DNA methylation patterns. DNA methylation refers to the process of adding methyl groups to the cytosine bases of DNA molecules. This modification can affect gene expression. During the development of cancer, certain tumor suppressor genes may be hypermethylated in tumor cells, causing them to lose their tumor suppressor function. Gene methylation can detect abnormalities in gene methylation before cell or tissue morphology changes, which helps to detect thyroid cancer at an early stage. Gene methylation can be detected through cytological samples obtained from blood or fine needle aspiration biopsy, which is relatively non-invasive. Gene methylation patterns are highly specific in thyroid cancer and help improve the accuracy of diagnosis. Summary of the invention

[0005] The present invention provides a primer-probe composition for detecting the methylation level of thyroid cancer, comprising a biomarker composed of methylated nucleotide sequences in the following target regions of PLEKHA4 gene and REC8 gene as primer-probe sequences corresponding to the methylation marker, wherein the target region of the PLEKHA4 gene is selected from chr19:49340488-49340774, and the REC8 gene comprises a REC8-1 gene whose target region is selected from chr14:24641053-24641452 and a REC8-2 gene whose target region is selected from chr14:24641637-24642033; The primer probe sequences are specifically shown as follows: The forward primer sequence of PLEKHA4 gene: CGGTTTTGTTATAAGACGAGGG, the rear primer sequence of PLEKHA4 gene: ACGATACCTCCAAACCACGT, the probe sequence of PLEKHA4 gene: CGCTCCGATTAACTACCGCTTTTAAACGAA; The forward primer sequence of REC8-1 gene: CGTTATTTAATGAGGAGCGAGGT, the rear primer sequence of REC8-1 gene: ACCTTACACTAACTTAAACGACCG, the probe sequence of REC8-1 gene: ATATAACCCCGAACCGCCTACTACAAAACT; The forward primer sequence of REC8-2 gene: CGGGTAGCGTTCGTTATCG, the rear primer sequence of REC8-2 gene: AAACAATATTTCTACTATCCCAACGT, the probe sequence of REC8-2 gene: CTTCAACGCCACACCGACTACTTTACCAC.

[0006] Furthermore, the primer-probe combination also includes an internal reference gene, and the internal reference gene is selected from any one of the Col2A1 gene, the ACTB gene or the GAPDH gene.

[0007] Furthermore, the present invention also provides a group of uses of the primer-probe combination as described above in the preparation of a kit for detecting the methylation level of thyroid cancer.

[0008] Further, when the PLEKHA4 gene, REC8-1 gene and REC8-2 gene combination are used as the detection primer probe combination, the P value is calculated using the logistic regression prediction probability formula; the logistic regression prediction probability formula is expressed as: ; in, Expressed as natural logarithm, Expressed as a constant, Expressed as PLEKHA4 The methylation coefficient, Expressed as REC8_1 The methylation coefficient, Expressed as REC8_2 Methylation coefficient.

[0009] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention selects specific methylated gene markers for thyroid cancer, designs primers and probes for the selected genes, and uses fluorescent quantitative PCR technology to detect the gene methylation status; then, multiple genes and multiple target regions are combined to complement each other's functions and improve diagnostic performance.

[0010] (2) The biomarkers screened out by the present invention for detecting thyroid cancer-specific methylation levels have abnormal gene methylation that precedes morphological changes, which is beneficial for the early detection of thyroid cancer. At the same time, the non-invasive collection of peripheral blood samples for gene methylation increases the patient's compliance with testing. In addition, by using multiple genes and multiple target regions to jointly detect thyroid cancer-specific methylation levels, the detection has high sensitivity and can effectively reduce the missed diagnosis of thyroid cancer.

[0011] In addition to the above-described purposes, features and advantages, the present invention has other purposes, features and advantages. The present invention will be further described in detail with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The drawings constituting a part of this application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings: FIG. 1 (a) is a schematic diagram showing that the methylation value of the PLEKHA4 gene Ct in the experimental example of the present invention is different between benign thyroid cancer and thyroid cancer; FIG1 (b) is a schematic diagram showing that the methylation value of the REC8_1 gene ΔCt in the experimental example of the present invention is different between benign thyroid cancer and thyroid cancer; FIG2 (a) is a schematic diagram of a receiver operating characteristic curve of the ability of using the PLEKHA4 gene to diagnose the gene methylation level of thyroid cancer in the experimental example of the present invention; FIG2 (b) is a schematic diagram of a receiver operating characteristic curve of the ability of using the REC8_1 gene to diagnose the gene methylation level of thyroid cancer in the experimental example of the present invention; FIG2 (c) is a schematic diagram of a receiver operating characteristic curve of the ability of using the REC8_2 gene to diagnose the gene methylation level of thyroid cancer in the experimental example of the present invention; FIG2 (d) is a schematic diagram of a receiver operating characteristic curve of the ability of the combined methylation of PLEKHA4 gene, REC8_1 gene and REC8_2 gene to diagnose the gene methylation level of thyroid cancer in the experimental example of the present invention; Figure 3 It is a schematic diagram of the prediction probability curve for diagnosing thyroid cancer using the gene methylation prediction formula in the experimental example of the present invention. DETAILED DESCRIPTION

[0013] The following are specific embodiments of the present invention and the accompanying drawings to further describe the technical solution of the present invention, but the present invention is not limited to these embodiments.

[0014] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the present invention specification herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention. Example

[0015] 1. Screening of gene methylation markers for thyroid cancer methylation level detection The screened gene methylation markers and their target regions for thyroid cancer methylation level detection are specifically shown in Table 1.

[0016] Table 1: Methylated genes used for methylation level detection in thyroid cancer

[0017] 2. Design of primer and probe set sequences Query and download the DNA sequence of the target region chr19:49340488-49340774 of the gene PLEKHA4 in the UCSC data, and use PCR design software to design primers and probes; The DNA sequences of two CpG islands chr14:24641053-24641452 (REC8_1) and chr14:24641637-24642033 (REC8_2) in the target region of gene REC8 were queried and downloaded from UCSC data, and primers and probes were designed for them respectively using PCR design software; The primer and probe sequences for PCR amplification of methylated genes for detecting methylation levels of thyroid cancer as shown in Table 2 were obtained.

[0018] Furthermore, in the process of designing primer probes for methylation gene PCR amplification for thyroid cancer methylation level detection, an internal reference gene is also selected, and the internal reference gene is preferably set to any one of Col2A1 gene, ACTB gene or GAPDH gene. Specifically, in the field of gene methylation level detection technology, the internal reference gene Col2A1 gene, ACTB gene or GAPDH gene is selected as a common technical means, and those skilled in the art can also obtain the corresponding primer probe sequence according to the prior art, and the core technical point of the present application is to combine the internal reference gene Col2A1 gene, ACTB gene or GAPDH gene with the methylation gene screened for thyroid cancer methylation level detection to detect the gene methylation level of thyroid cancer, therefore, the primer probe of the internal reference gene Col2A1 gene, ACTB gene or GAPDH gene is no longer disclosed in the present application.

[0019] Table 2: Target regions, primer probe sequences and melting temperatures of methylated genes for thyroid cancer methylation level detection

[0020] Specifically, the methylation region sequence of the PLEKHA4 gene is selected from the DNA strand in hg19_dna range=chr19:49340488-49340774 5'pad=0 3'pad=0 strand=-: CGGTGTGCCGCTGCCTCCTTCGGACCCCACGCGCCAGGAGACCCCTCCCCCCAGATCTCCCCCGGTGGCTAATTCGGGTTCCACGGGGTTCTCTCGCCGAGGGAGTGGGCGTGGAGGAGGTCCCACCCCCTGGGGGCCCGCGT GGGATGCCGGGATCGCCCCTCCGGTCCTGCCACAAGACGAGGGGGCATGGCCTCTGCGAGTCACTCTGCTACAATCCAGCTTCTAATCCGCCCAAAAGCGGCAGCCAATCGGAGCGCGAGGACGTGGTCTGGAGGTACCGCCGG All C in CG in this DNA sequence is methylated and bisulfite converted (unmethylated C is converted to T, and methylated C remains unchanged) to obtain the converted DNA chain (B-DNA): CGGTGTGTCGTTGTTTTTTTCGGATTTTACGCGTTAGGAGATTTTTTTTTTTAGATTTTTTTCGGTGGTTAATTCGGGTTTTACGGGGTTTTTCGTCGAGGGAGTGGGCGTGGAGGAGGTTTTATTTTTTGGGGGTTCGCGT GGGATGTCGGGATCGTTTTTCGGTTTGTTATAAGACGAGGGGGTATGGTTTTTGCGAGTTATTTTGTTATAATTTAGTTTTTAATTCGTTTAAAAGCGGTAGTTAATCGGAGCGCGAGGACGTGGTTTGGAGGTATCGTCGG Based on the converted DNA chain sequence, the methylation primer probe sequence of PLEKHA4 (chr19:49340488-49340774) was obtained.

[0021] Specifically, the methylation region sequence of the REC8_1 gene is selected from the DNA strand in hg19_dna range=chr14:24641053-24641452 5'pad=0 3'pad=0 strand=+: GCGCCTGTTACTAGAGGCGAGAACCGGAGCCCATTGGTCGGAACACCTCACAATGGACCCCAGCGGCGCGCAAAATCCTTATGATTGGTTTGCTGGCTGCCTCGGGAGACCCTGTTGCCAGGATACTTGGCGTTCCCGACCCGACCCCCGTTCCCCATTGGCTGTCAGGGCAAAAGCCGCCATCTAATGAGGAGCGAGGT GCGGTGCCCCGAAGCGCTCGCTTCCCGCGGTGCGATCTAGTCCTGCAGTAGGCGGCCCGGGGCCACACCGCGGCCGCCCAAGCCAGTGCAAGGCCCAGGGGCCTGACATCGCTCCCAGGCTCTCGAGGACCGAGGCCTGCTGTGGAGGACACCGTGCTCCCTCGGGACCTGCTCTGGATTCCGGCCCGGACGTCCCCTTGG All C in CG in this DNA sequence is methylated and bisulfite converted (unmethylated C is converted to T, and methylated C remains unchanged) to obtain the converted DNA chain (B-DNA): GCGTTTGTTATTAGAGGCGAGAATCGGAGTTTATTGGTCGGAATATTTTATAATGGATTTTAGCGGCGCGTAAAATTTTTATGATTGGTTTGTTGGTTGTTTCGGGAGATTTTGTTGTTAGGATATTTGGCGTTTTCGATTCGATTTTCGTTTTTTATTGGTTGTTAGGGTAAAAGTCGTTATTTAATGAGGAGCGAGGTGCGGTGTTTCGAAGCGTTCGTTTTTCGCGGTGCGATTTAGTTTTGTAGTAGGCGGTTCGGGGTTATATCGCGGTCGTTTAAGTTAGTGTAAGGTTTAGGGGTTTGATATCGTTTTTAGCGTTCGAGGATCGAGGTTTGTTGTGGAGGATATCGTGTTTTTTCGGGATTTGTTTTGGATTTCGGTTCGGACGTTTTTTTGG Based on the converted DNA strand sequence, the methylation primer probe sequence of REC8 (chr14:24641053 - 24641452) is obtained.

[0022] Specifically, the methylation region sequence of the REC8_2 gene is selected from the DNA strand in hg19_dna range = chr14:24641637 - 24642033 5'pad = 0 3'pad = 0 strand = -: TCACCCCCCGCACCAGGCCCTCCTTCCCTTTTCTGGGCCTTACCAGGTTTTCACCACATTCACCCTCAGGTATTCGCGCTTCACCAACCGGCTGCCGCGAGTCGCCGCCAGCCTGAAGTGGGTGGGGAAGGTGGGAGAATGGGGATGGAGGGGGAGCTGTCAGGCTGGGATCCCGGGCAGCGTCCGCCATCGCGCGCCAACGGGCCCCGCCCTTACCAGATGGTGGCAAAGCAGCCGGTGTGGCGCTGAAGCACGTTGGGATAGTAGAACATTGTCCCTCTGGTCTTTCACCCTCGATTCCACCAGATCCCCGTTCGGATCTGAGTCGGAATTCTTTAGGGCACAGAATTCCCAACACCTTGGAGAGAAGTCAGGATTTGGACTTCTCTGGTGGCAA All C in CG in this DNA sequence is methylated and bisulfite converted (unmethylated C is converted to T, and methylated C remains unchanged) to obtain the converted DNA chain (B-DNA): TTATTTTTCGTATTAGGTTTTTTTTTTTTTTGGGTTTTATTAGGTTTTTATTATTTATTTTTAGGTATTCGCGTTTTATTAATCGGTTGTCGCGAGTCGTCGTTAGTTTGAAGTGGGTGGGGAAGGTGGGAGAATGGGGATGGAGGGGGAGTTGTTAGGTTGGGATTTCGGGTAGCGTTCGTTATCGCGCGTT AACGGGTTTCGTTTTTATTAGATGGTGGTAAAGTAGTCGGTGTGGCGTTGAAGTACGTTGGGATAGTAGAATATTGTTTTTTTGGTTTTTTATTTTCGATTTTATTAGATTTTCGTTCGGATTTGAGTCGGAATTTTTAGGGTATAGAATTTTTAATATTTTGGAGAGAAGTTAGGATTTGGATTTTTTTGGTGGTAA Based on the converted DNA chain sequence, the methylation primer probe sequence of REC8 (chr14:24641637-24642033) was obtained.

[0023] 3. Construction of a PCR system for thyroid cancer gene methylation detection S3.1. Pre-test treatment of nucleic acid DNA samples Peripheral blood free DNA extraction reagent was used to extract DNA from plasma or serum, and the DNA quality (yield, purity, and integrity) was monitored at the same time, requiring the total DNA amount to be between 1ug-10ug and OD260 / 280 to be between 1.9-2.0. A commercially available bisulfite conversion kit (Methylation Detection Sample Pretreatment Kit (Hunan Hongya Gene Technology Co., Ltd. Xiangchang Equipment No. 20200131)) was used as a DNA bisulfite conversion reagent to perform bisulfite conversion on the extracted DNA, and the unmethylated cytosine (C) in the DNA was converted to uracil (U), while the methylated cytosine (C) remained unchanged, obtaining bisulfite-converted DNA (B-DNA).

[0024] S3.2. Construction of PCR reaction system and PCR reaction procedure The components of the PCR reaction system for thyroid cancer gene methylation detection are configured in a certain proportion, and there is no requirement for the total reaction volume, which can be 10ul-50ul.

[0025] Taking the common 25ul reaction system as an example, its specific components are: (i) 12.5ul PCR reaction mixture (Mix), (ii) 1.5ul of gene methylation primer-probe mixture, including primers and probes of target gene and internal reference gene, (iii) 3ul of sample B-DNA after bisulfite conversion, (iv) Make up the volume to 25ul with double distilled water.

[0026] In addition, external quality control was set up, using positive quality control products and negative quality control products to replace sample B-DNA.

[0027] Thyroid cancer gene methylation was amplified and detected by fluorescent quantitative PCR. The PCR reaction procedure was as follows: (1) Pre-denaturation, 94℃-98℃ for 5-10 minutes; (2) Denaturation, 92℃-96℃ for 5-10 seconds; (3) Annealing, 56℃-60℃ for 5 seconds; extension, 57℃-72℃ for 35 seconds; (4) Repeat steps (2) to (4) 45 to 50 times; (5) Store at 4℃-10℃ for 10 minutes.

[0028] 4. Establishing standards for clinical interpretation of gene methylation When the internal reference gene (IC) is qualified, the results of the methylation level detection of the PLEKHA4 gene, REC8_1 gene and REC8_2 gene are obtained respectively. Among them, the result of the methylation level detection of the PLEKHA4 gene is expressed by the Ct value, and the result of the methylation level detection of the REC8_1 gene and REC8_2 gene is expressed by ΔCt, ΔCt methylated gene = Ct methylated gene - Ct internal reference gene.

[0029] S4.1. Criteria for judging gene methylation The larger the Ct value of the result of the PLEKHA4 gene methylation level detection, the lower the methylation level, and vice versa.

[0030] The larger the ΔCt value of the REC8 gene methylation level detection result, the lower the methylation level, and vice versa.

[0031] When the Ct value of the internal reference gene (IC) is less than 38, the sample tested is qualified and the PCR reaction is successful.

[0032] The criteria for judging the methylation results of target genes are shown in Table 3: Table 3: Interpretation of single gene methylation results in thyroid cancer

[0033] S4.2. Interpretation criteria for the results of dual-gene methylation combined detection In peripheral blood testing for gene methylation markers of thyroid cancer, the two genes are combined to improve sensitivity in an OR format, that is, any one gene is positive and is interpreted as positive, and both genes are negative and are interpreted as negative.

[0034] Table 4: Interpretation of combined detection of dual gene methylation in thyroid cancer

[0035] S4.3. Gene methylation prediction formula for three-gene methylation combined detection The three-gene methylation combined detection uses the logistic regression prediction probability formula to calculate the P value, which is convenient for selecting the appropriate critical value according to clinical needs: ;

[0036] in, Expressed as natural logarithm, Expressed as a constant, Expressed as PLEKHA4 The methylation coefficient, Expressed as REC8_1 The methylation coefficient, Expressed as REC8_2 Methylation coefficient.

[0037] Experimental example: A total of 40 paraffin sections of thyroid tissues were collected, including 15 known cases of thyroid malignant tumors, including 8 cases of papillary thyroid carcinoma, 4 cases of follicular thyroid carcinoma, 2 cases of medullary thyroid carcinoma, and 1 case of anaplastic thyroid carcinoma; and 25 cases of benign tissues, including 15 benign lesions, 6 cases of follicular adenomas, and 4 cases of papillary adenomas.

[0038] In addition, peripheral blood samples were collected from 318 patients, of whom 56 were known to have thyroid cancer and 262 were known to have benign lesions.

[0039] All collected specimens (including 40 paraffin sections of thyroid tissue and peripheral blood of 318 patients) were subjected to gene methylation testing to obtain methylation results.

[0040] 1. Distribution of gene methylation in thyroid tissue In 40 tissue paraffin sections, the methylation values ​​of PLEKHA4 (Ct), REC8_1 (ΔCt), and REC8_2 (ΔCt) were different in benign tissues (see Figure 1 (a)) and thyroid cancer (see Figure 1 (b)), and are potential markers for distinguishing thyroid cancer.

[0041] 2. Diagnostic performance of thyroid cancer gene methylation The methylation of PLEKHA4 (ΔCt), REC8_1 (ΔCt) and REC8_2 (ΔCt) was detected in 318 peripheral blood samples according to Example 1. The ability of PLEKHA4, REC8_1 and REC8_2 to diagnose thyroid cancer is shown in the receiver operating characteristic curves (ROC) shown in Figures 2 (a) to 2 (c), and the areas under the ROC curves (AUCs) were 0.640, 0.830 and 0.791, respectively.

[0042] According to the maximum Youden index principle of the ROC curve, the closest critical values ​​for diagnosing thyroid cancer were taken for PLEKHA4, REC8_1, and REC8_2. The methylation level of PLEKHA4 was set as PLEKHA4m, and the critical value of PLEKHA4m was Ct=40.52, Ct≤40.52, PLEKHA4m was positive, otherwise it was negative; the methylation level of REC8_1 was set as REC8_1m, and the critical value of REC8_1m was ΔCt=10.38, ΔCt≤10.38, REC8_1m was positive, otherwise it was negative; the methylation level of REC8_2 was set as REC8_2m, and the critical value of REC8_2m was ΔCt=5.73, ΔCt≤5.73, REC8_2m was positive, otherwise it was negative.

[0043] Table 5: Critical values ​​of gene methylation for diagnosing thyroid cancer

[0044] 3. Clinical diagnostic performance of single gene methylation After determining the critical values ​​of PLEKHA4m, REC8_1m and REC8_2m (the critical values ​​of PLEKHA4m, REC8_1m and REC8_2m gene methylation for diagnosing thyroid cancer are shown in Table 5), their clinical performance was calculated. The clinical sensitivities of PLEKHA4m, REC8_1m and REC8_2m were 76.8%, 66.1% and 67.9%, respectively, and the negative predictive values ​​were 92.9%, 92.1% and 92.6%, respectively (see Table 6).

[0045] Table 6: Clinical performance of single gene methylation detection in diagnosing thyroid cancer

[0046] 4. Performance of dual-gene methylation combined detection The clinical sensitivity of single gene methylation in diagnosing thyroid cancer is low, and the combined detection of double gene methylation adopts the form of "or" to improve the sensitivity. As shown in Table 7, the sensitivities of PLEKHA4m / REC8_1m, PLEKHA4m / REC8_2m and REC8_1m / REC8_2m are 91.1%, 96.4% and 91.1% respectively, and the negative predictive values ​​are all above 95%, which reduces the missed diagnosis of thyroid cancer.

[0047] Table 7: Clinical performance of dual gene methylation combined detection in diagnosing thyroid cancer

[0048] 5. Performance of gene methylation prediction formula detection Logistic regression analysis was performed on the methylation level of three genes, PLEKHA4, REC8_1 and REC8_2 (the receiver operating characteristic curve of the ability of the three-gene methylation combination to diagnose the gene methylation level of thyroid cancer is shown in Figure 2 (d)). , , , , the gene methylation prediction formula is: ; By selecting different critical values, fixed sensitivity and specificity performance can be obtained. Figure 3 As shown in the figure, when P>0.05 is judged as positive, the sensitivity of the gene methylation prediction formula for diagnosing thyroid cancer is 98% and the specificity is 55%. When P>0.10 is judged as positive, the sensitivity of the gene methylation prediction formula for diagnosing thyroid cancer is 91% and the specificity is 70%. The gene methylation prediction formula can adjust the critical value according to the clinical application scenario to achieve the effect required by the clinical situation.

[0049] Table 8: Clinical performance of gene methylation prediction formula in diagnosing thyroid cancer

[0050] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A primer-probe combination for detecting methylation level of thyroid cancer, characterized in that: A biomarker comprising a methylated nucleotide sequence in the following target region of the PLEKHA4 gene and the REC8 gene as a primer probe sequence corresponding to the methylation marker, wherein the target region of the PLEKHA4 gene is selected from chr19:49340488-49340774, and the REC8 gene comprises a REC8-1 gene whose target region is selected from chr14:24641053-24641452 and a REC8-2 gene whose target region is selected from chr14:24641637-24642033; The primer probe sequences are specifically shown as follows: The forward primer sequence of PLEKHA4 gene: CGGTTTTGTTATAAGACGAGGG, the rear primer sequence of PLEKHA4 gene: ACGATACCTCCAAACCACGT, the probe sequence of PLEKHA4 gene: CGCTCCGATTAACTACCGCTTTTAAACGAA; The forward primer sequence of REC8-1 gene: CGTTATTTAATGAGGAGCGAGGT, the rear primer sequence of REC8-1 gene: ACCTTACACTAACTTAAACGACCG, the probe sequence of REC8-1 gene: ATATAACCCCGAACCGCCTACTACAAAACT; The forward primer sequence of REC8-2 gene: CGGGTAGCGTTCGTTATCG, the rear primer sequence of REC8-2 gene: AAACAATATTTCTACTATCCCAACGT, the probe sequence of REC8-2 gene: CTTCAACGCCACACCGACTACTTTACCAC.

2. The primer-probe combination according to claim 1, characterized in that It also includes an internal reference gene, which is selected from any one of the Col2A1 gene, the ACTB gene or the GAPDH gene.

3. Use of a group of primer-probe combinations as described in claim 1 in the preparation of a kit for detecting methylation levels of thyroid cancer.

4. The use according to claim 3, characterized in that: When the PLEKHA4 gene, REC8-1 gene and REC8-2 gene combination are used as the detection primer probe combination, the P value is calculated using the logistic regression prediction probability formula; the logistic regression prediction probability formula is expressed as: ； in, Expressed as natural logarithm, Expressed as a constant, Expressed as PLEKHA4 The methylation coefficient, Expressed as REC8_1 The methylation coefficient, Expressed as REC8_2 Methylation coefficient.

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