Oral cancer methylation prognostic marker combination and oral cancer detection kit

Abstract

The invention discloses an oral cancer methylation prognostic marker combination and an oral cancer detection kit. The oral cancer methylation prognostic marker combination is composed of PAX1, PAX9, PRAMEF33 and CPLANE2, the oral cancer detection kit contains a PAX1 PCR reaction solution, a PAX9 PCR reaction solution, a PRAMEF33 PCR reaction solution and a CPLANE2 PCR reaction solution, and can specifically detect the methylation level of CpG dinucleotide sites in the full length of nucleotide sequences of four target genes PAX1, PAX9, PRAMEF33 and CPLANE2 in a biological sample or the nucleotide sequence of any partial region of the nucleotide sequences of the four target genes PAX1, PAX9, PRAMEF33 and CPLANE2. Through methylation joint detection of a plurality of oral cancer methylation prognosis markers, the accuracy of oral cancer prognosis detection is improved, and a new strategy and method are provided for early diagnosis, prognosis evaluation and individualized treatment of oral cancer.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a combination of oral cancer methylation prognosis markers and an oral cancer detection kit. Background Art

[0002] In the field of biotechnology, the early diagnosis and prognosis evaluation of oral cancer are crucial for improving the survival rate of patients. In recent years, with the in-depth study of epigenetics, DNA methylation, as a key epigenetic modification mechanism, has become a hot topic in oral cancer research. DNA methylation markers play an important role in the occurrence, development, and prognosis evaluation of oral cancer.

[0003] The methylation prognosis markers of oral cancer mainly involve changes in DNA methylation levels, which can be used as early indicators of tumor development. For example, the methylation inactivation of the tumor suppressor gene p16 has been proven to be able to be used for the early diagnosis of oral mucosal dysplasia carcinogenesis. The proportion of patients with oral mucosal dysplasia with p16 gene methylation progressing to oral cancer is significantly higher than that of patients without this gene methylation. In addition, by detecting methylation markers related to tumor development, it is possible to assist in the early diagnosis of cancer, evaluate the risk of progression, and evaluate minimal residual disease (MRD) and monitor recurrence after treatment or radical surgery.

[0004] Therefore, providing effective oral cancer methylation prognosis markers and developing corresponding detection reagents and detection methods will provide new strategies and methods for the early diagnosis, prognosis evaluation, and individualized treatment of oral cancer, which is also a topic for the technical personnel of this unit to research and overcome. Summary of the Invention

[0005] To solve the problems existing in the prior art, the object of the present invention is to provide a combination of oral cancer methylation prognosis markers and an oral cancer detection kit, so as to achieve the methylation combined detection of multiple oral cancer methylation prognosis markers, thereby improving the accuracy of oral cancer prognosis detection and providing new strategies and methods for the early diagnosis, prognosis evaluation, and individualized treatment of oral cancer.

[0006] To achieve the object of the invention, the technical solutions provided by the present invention are as follows: First, the present invention provides a combination of oral cancer methylation prognosis markers, which is characterized by being composed of four oral cancer DNA methylation prognosis markers, namely PAX1, PAX9, PRAMEF33, and CPLANE2. Among them, the nucleotide sequence of the methylation region to be detected of the marker PAX1 is as shown in SEQ ID No. 1; The nucleotide sequence of the methylation region to be detected of the marker PAX9 is as shown in SEQ ID No. 2; The nucleotide sequence of the methylation region to be detected for the biomarker PRAMEF33 is shown in SEQ ID No. 3; The nucleotide sequence of the methylation region to be detected for the biomarker CPLANE2 is shown in SEQ ID No. 4.

[0007] The NCBI database accession number for the PAX1 gene sequence involved in the present invention is NC_000020.11, the NCBI database accession number for the PAX9 gene sequence is NC_000014.9, the NCBI database accession number for the PRAMEF33 gene sequence is NC_000001.11, and the NCBI database accession number for the CPLANE2 gene sequence is NC_000001.11.

[0008] The nucleotide sequences of the methylation regions to be detected for the four target genes involved in the present invention are shown in Table 1 below.

[0009] Table 1: Nucleotide sequences of the methylation regions to be detected for the four target genes of the present invention Sequence Name Oligonucleotide Sequence 5'-3' Number Region to be detected for PAX1 methylation CGAAACACCTTTCCACGATAAACTTTTATTCTCTATCTTATTATTAATGGTGGCGGAAATAAAACTAAAACCAAAACGAAAACGAGTACTAGTACTAACACACTAATCAATTTGAGATGACTTCCCCCTCATTCCCAAAGCTAGAGGAGGAAGGGGGCTGAAAGGGGCTCAGAGCAGTGGAAGGTCCCAGGCCCAGCTGGGGTTGGGACGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTAGAGGGGTGGGTTCCGGGGGGGTGGT SEQ ID No.1 Region to be detected for PAX9 methylation GGGCCCAGCCGAAGCTTGGGGCCTAGAGGGCAGACGTCCGGCTCTGATCAGAGTACAGCGGGCTGGGCCTGGCAGCCTGCAGGCAACCTGGACCTTGCAATAAAGCTGCATTCAACATAACTCAGACCACAGTGGGAGCCCCCGAAGGGCTTCATGTGAGTCCCGAAACCAGAGTTTAGAGGCGCGGCCTGGTTCTCGTTTATCTTTTCCAGACAGCATTTAGGCCAATAAGGTCTTTGAAAGGAGGCAGCCCCAAAGCCCTCAGGCCGACACCTGA SEQ ID No.2 Region to be detected for PRAMEF33 methylation CAGATTTTAGTTTTCTGTCTAATATCTGGGAAAGATTAGACCCTTCCCTGCTTCAGACTCAGGACTATGCAGGTCACATATTAGTGAAATTCCATCAGTGTTTGTGAAGTTCACGAATGAATGAATTTTTTTTTTCCGACAAATTCTCCCTCTGTCACCCAGAGTGGAGTGCAATGGTGCAATCTTGGTTCACTGCAACCATTGCCTCTTGGGTTCAAGCGATTCTCCCACCTCAGCCTCCTGAGTAGCTGGATTACAGGCATGTGCCATCGTCCCTGCC SEQ ID No.3 Region to be detected for CPLANE2 methylation GTGCGGGTGGGCGTTTAAAGGGGCCTTCGGCACCCAGGTCGGTTCGCCGCCGGGACTGCTGACGGGGAGGGCTAGGTAGCCCTGGGAGTCGGGATCTAGAAGGTAGAGGAGAAGCGGGGCTGTCTGACCCCCGGAGGTGACAAGGGGAGAGGGGCTGTGTGAATACCCTCCATGGGCAGTAATGGGATGACTATCCCCAGGGAGCATTGCGAAGGAGAGAGGCGGCTGAGGGACTGCACTTTCTGGGGCCCCCCGGGAGAGTCAGAGGGCAGTTAAG SEQ IDNo.4 Second, the present invention provides an oral cancer detection kit, characterized in that the kit contains the following detection reagents: PAX1 PCR reaction solution, used to detect the methylation degree in at least one target region in the promoter region of the PAX1 gene, and the nucleotide sequence of the methylation region to be detected for PAX1 is shown in SEQ ID No. 1; PAX9 PCR reaction solution, used to detect the methylation degree in at least one target region in the promoter region of the PAX9 gene, and the nucleotide sequence of the methylation region to be detected for PAX9 is shown in SEQ ID No. 2; PRAMEF33 PCR reaction solution, used to detect the methylation degree in at least one target region in the promoter region of the PRAMEF33 gene, and the nucleotide sequence of the methylation region to be detected for PRAMEF33 is shown in SEQ ID No. 3; CPLANE2 PCR reaction solution, used to detect the methylation degree in at least one target region in the promoter region of the CPLANE2 gene, and the nucleotide sequence of the methylation region to be detected for the CPLANE2 gene is shown in SEQ ID No. 4; Positive control and negative control, wherein the positive control substance contained in the positive control is selected from the DNA of human oral cancer cell NCI-H1703; the negative control is sterile water.

[0010] The kit also includes droplet generation oil, and the droplet generation oil described in the present invention is purchased from Bio-rad, USA.

[0011] The promoter region is not strictly limited to the region within the promoter fragment, but rather a gene fragment in the region near the promoter.

[0012] The described PAX1 PCR reaction solution includes forward and reverse PAX1 primers, a PAX1 methylation probe, a PAX1 unmethylated probe, forward and reverse internal reference GAPDH primers, and a GAPDH probe; among them, the base sequence of the PAX1 forward primer is as shown in SEQ ID No. 5; the base sequence of the PAX1 reverse primer is as shown in SEQ ID No. 6; the base sequence of the PAX1 methylation probe is as shown in SEQ ID No. 7; the base sequence of the PAX1 unmethylated probe is as shown in SEQ ID No. 8; The described PAX9 PCR reaction solution includes forward and reverse PAX9 primers, a PAX9 methylation probe and an unmethylated probe, forward and reverse internal reference GAPDH primers, and a GAPDH probe; among them, the base sequence of the PAX9 forward primer is as shown in SEQ ID No. 9; the base sequence of the PAX9 reverse primer is as shown in SEQ ID No. 10; the base sequence of the PAX9 methylation probe is as shown in SEQ ID No. 11; the base sequence of the PAX9 unmethylated probe is as shown in SEQ ID No. 12; The described PRAMEF33 PCR reaction solution includes forward and reverse PRAMEF33 primers, a PRAMEF33 methylation probe and an unmethylated probe, forward and reverse internal reference GAPDH primers, and a GAPDH probe; among them, the base sequence of the PRAMEF33 forward primer is as shown in SEQ ID No. 13; the base sequence of the PRAMEF33 reverse primer is as shown in SEQ ID No. 14; the base sequence of the PRAMEF33 methylation probe is as shown in SEQ ID No. 15; the base sequence of the PRAMEF33 unmethylated probe is as shown in SEQ ID No. 16; The described CPLANE2 PCR reaction solution includes forward and reverse CPLANE2 primers, a CPLANE2 methylation probe and an unmethylated probe, forward and reverse internal reference GAPDH primers, and a GAPDH probe; among them, the base sequence of the CPLANE2 forward primer is as shown in SEQ ID No. 17; the base sequence of the CPLANE2 reverse primer is as shown in SEQ ID No. 18; the base sequence of the CPLANE2 methylation probe is as shown in SEQ ID No. 19; the base sequence of the CPLANE2 unmethylated probe is as shown in SEQ ID No. 20; The base sequences of the forward and reverse primers and the probe of the internal reference GAPDH in the PAX1 PCR reaction solution, PAX9 PCR reaction solution, PRAMEF33 PCR reaction solution, and CPLANE2 PCR reaction solution are as follows: The base sequence of the forward primer of the internal reference GAPDH is shown in SEQ ID No. 21; the base sequence of the reverse primer of the internal reference GAPDH is shown in SEQ ID No. 22; the base sequence of the probe of the internal reference GAPDH is shown in SEQ ID No. 23.

[0013] The above-mentioned PAX1 PCR reaction solution, PAX9 PCR reaction solution, PRAMEF33 PCR reaction solution, and CPLANE2 PCR reaction solution also all contain PCR buffer, MgCl2, dNTP, and Taq enzyme.

[0014] The Taq enzyme described above can be a commonly used Taq DNA polymerase in the art. The formulation amounts of the PCR buffer, MgC12, and dNTP in the present invention can be selected according to conventional methods in the art. One can select appropriate formulation amounts of individual reagents and mix them by oneself, or directly select a commercially available finished reaction system. The differences in these components have no obvious influence on the experimental results. The PCR buffer is a conventional buffer in the art, such as 30 - 60 mmol / L Tris and 10 - 50 mmol / L KC1, or 30 - 600 mmol / L Tris and 10 - 50 mmol / L KAc, or 30 - 60 mmol / L Tris and 10 - 50 mmol / L CsC1, etc. These differences have no significant influence on the reagents and detection results described in the present invention.

[0015] The nucleotide sequences of the target gene-specific primers, probes, internal reference gene primers, and probes involved in the detection reagents in the oral cancer detection kit of the present invention are shown in Table 2 below.

[0016] Table 2: Nucleotide sequences of target gene-specific primers, probes, internal reference gene primers, and probes Sequence name Oligonucleotide sequence 5'-3' Number PAX1 forward primer CGAAATATTTTTTTACGATAAATT SEQ ID No.5 PAX1 reverse primer ACCACCCCCCCGAAACCCACCCC SEQ ID No.6 PAX1 methylation probe GTTGAAAGGGGTTTAGAGTAGTGG SEQ ID No.7 PAX1 unmethylated probe GGGCTGAAAGGGGCTCAGAGCAGTGG SEQ ID No.8 PAX9 forward primer GGGTTTAGTCGAAGTTTGGGGTTTAG SEQ ID No.9 PAX9 reverse primer TCAAATATCGACCTAAAAACTTTAAAACTACC SEQ ID No.10 PAX9 methylation probe ATGTGAGTTTCGAAATTAGAGTTTAGA SEQ ID No.11 PAX9 unmethylated probe TGTGAGTCCCGAAACCAGAGTTTAGA SEQ ID No.12 PRAMEF33 forward primer TAGATTTTAGTTTTTTGTTTAATATT SEQ ID No.13 PRAMEF33 reverse primer AACAAAAACGATAACACATACCTAT SEQ ID No.14 PRAMEF33 methylation probe TTTAAGCGATTTTTTTATTTTAGTTTT SEQ ID No.15 PRAMEF33 unmethylated probe GTTCAAGCGATTCTCCCACCTCAGCCTC SEQ ID No.16 CPLANE2 forward primer GTGCGGGTGGGCGTTTAAAGGG SEQ ID No.17 CPLANE2 reverse primer CTTAACTACCCTCTAACTCTCCCGA SEQ ID No.18 CPLANE2 methylation probe CGAAGGAGAGAGGCGGTTGAGGGATTGTATT SEQ ID No.19 CPLANE2 non-methylated probe CGAAGGAGAGAGGCGGCTGAGGGACTGCACT SEQ ID No.20 Reference GAPDH forward primer TATCGATTATTTTTTATCGGGTTAATT SEQ ID No.21 Reference GAPDH reverse primer TTCTCCCCGCTTTTTTTTTTCCG SEQ ID No.22 Reference GAPDH probe TACGTTCGGTGCGTGTTTAGTTG SEQ ID No.23 Meanwhile, the nucleotide sequences involved in Table 1 and Table 2 of the present invention will be submitted to the National Intellectual Property Administration in the form of a sequence listing that conforms to the WIPO ST.26 standard.

[0017] Both ends of the described PAX1 methylation probe and non-methylation probe, PAX9 methylation probe and non-methylation probe, PRAMEF33 methylation probe and non-methylation probe, CPLANE2 methylation probe and non-methylation probe, and GAPDH probe are respectively labeled with a fluorescent reporter group and a fluorescent quenching group; the fluorescent reporter group is selected from FAM, HEX, ROX, JOE, VIC, TET, NED, FITC, CY3 or CY5; the fluorescent quenching group is selected from BHQ1, BHQ2, BHQ3, TAMRA, Eclipse, DABCYL.

[0018] The PAX1 PCR reaction solution, PAX9 PCR reaction solution, PRAMEF33 PCR reaction solution, and CPLANE2 PCR reaction solution of the detection reagent provided by the present invention contain specific primers and fluorescent probes for the target genes, and can rapidly detect the methylation of four target genes PAX1, PAX9, PRAMEF33, and CPLANE2 in the detection sample.

[0019] Furthermore, the present invention also provides a method for applying the oral cancer methylation prognosis marker combination and the oral cancer detection kit to oral cancer prediction, which specifically includes the following steps: S1. Extract the DNA of the detection sample, subject the DNA of the detection sample to bisulfite conversion treatment, deaminate the unmethylated cytosine in the nucleic acid of the target gene in the detection sample to uracil, and obtain a purified Bis-DNA sample; S2. Using the purified Bis-DNA sample as a template, respectively prepare a target gene PCR reaction system with the PAX1 PCR reaction solution, PAX9 PCR reaction solution, PRAMEF33 PCR reaction solution, and CPLANE2 PCR reaction solution in the oral cancer detection kit of the present invention, amplify the nucleic acid with the specific primers and fluorescent probes in the PAX1 PCR reaction solution, PAX9 PCR reaction solution, PRAMEF33 PCR reaction solution, and CPLANE2 PCR reaction solution, and use a droplet digital PCR instrument to detect the methylation levels of PAX1, PAX9, PRAMEF33, and CPLANE2 genes in the DNA of the detection sample; S3. Judgment of the detection result: If the total number of droplets in the detection sample is greater than 10,000, it is judged that this reaction is effective; if the sum of the methylated copy number and the non-methylated copy number of the detection sample is ≥100, then the detection result of this sample is effective; if it is less than 100, then the detection of this sample is invalid and needs to be retested; The methylation ratio of the test sample is calculated by the following formula: Methylation ratio = Methylated copy number / [(Methylated copy number + Unmethylated copy number)] × 100%. If the methylation ratio of the target gene ≥ 2%, the interpretation result is methylation positive; if the methylation ratio of the target gene < 2%, the interpretation result is methylation negative; When any one of the four target genes, PAX1, PAX9, PRAMEF33, and CPLANE2, is methylation positive, the test sample is interpreted as cancer positive. When all four target genes, PAX1, PAX9, PRAMEF33, and CPLANE2, are methylation negative, the test sample is interpreted as cancer negative.

[0020] The beneficial effects of the present invention are as follows: The present invention provides an oral cancer methylation prognosis marker combination and an oral cancer detection kit. The oral cancer methylation prognosis marker combination includes the following methylated genes: PAX1, PAX9, PRAMEF33, and CPLANE2 genes. The detection reagents provided by the present invention, the PAX1 PCR reaction solution, PAX9 PCR reaction solution, PRAMEF33 PCR reaction solution, and CPLANE2 PCR reaction solution, contain specific primers and fluorescent probes for the target genes. The PAX1, PAX9, PRAMEF33, and CPLANE2 genes involved in the present invention are all related to the prognosis of oral cancer patients. The methylation detection region of each target gene contains multiple CpGs. The methylation determination of the target gene is not limited to one or two bases, and the randomness of base changes is reduced compared with the detection of a single base. Therefore, the combined methylation detection of the four target genes, PAX1, PAX9, PRAMEF33, and CPLANE2, effectively compensates for the differences that may be caused by site selection and reaction systems, reduces the randomness of base changes, improves the accuracy of the prognosis detection of oral cancer patients, and provides new strategies and methods for the early diagnosis, prognosis evaluation, and individualized treatment of oral cancer. Brief Description of the Drawings

[0021] Figure 1 . is the amplification results of positive and negative methylation of the target gene in the application experiment of Example 3 of the present invention; Figure 2 is the receiver operating characteristic ROC curve established in the application experiment of Example 3 of the present invention; Figure 3 is the amplification results of positive and negative methylation of the target gene in the verification experiment of Example 4 of the present invention; Figure 4 is the comparison of the ROC curve of the methylation detection result and the 5-year survival rate of the patient in the verification experiment of Example 4 of the present invention; Figure 5This is a comparison table of the detection results and the 5-year survival status of patients in the verification experiment of Example 4 of the present invention. Detailed implementation mode

[0022] The present invention will be further described in detail below with reference to the drawings and embodiments. Embodiment 1

[0023] This embodiment is about the composition of the methylation prognostic marker combination for oral cancer and the oral cancer detection kit.

[0024] Referring to Table 1, the methylation prognostic marker combination for oral cancer provided by the present invention consists of four oral cancer DNA methylation prognostic markers, namely PAX1, PAX9, PRAMEF33, and CPLANE2. Among them, the nucleotide sequence of the methylation region to be detected of the marker PAX1 is shown in SEQ ID No. 1; The nucleotide sequence of the methylation region to be detected of the marker PAX9 is shown in SEQ ID No. 2; The nucleotide sequence of the methylation region to be detected of the marker PRAMEF33 is shown in SEQ ID No. 3; The nucleotide sequence of the methylation region to be detected of the marker CPLANE2 is shown in SEQ ID No. 4.

[0025] The PAX1, PAX9, PRAMEF33, and CPLANE2 genes involved in the present invention are all related to the prognosis of oral cancer patients, and the methylation detection region of each target gene contains multiple CpGs.

[0026] The present invention provides an oral cancer detection kit, and the composition of the kit is shown in Table 3 below.

[0027] Table 3: Composition of the kit Component Main component Specification PAX1 PCR reaction solution <![CDATA[Forward and reverse primers for PAX1, PAX1 methylation and unmethylation probes, forward and reverse primers for internal reference GAPDH, internal reference GAPDH probe, PCR buffer, dNTP, MgCl2 and Taq enzyme.]]> 400uL PAX9 PCR reaction solution <![CDATA[Forward and reverse primers for PAX9, PAX9 methylation and non-methylation probes, forward and reverse primers for internal reference GAPDH, internal reference GAPDH probe, PCR buffer, dNTP, MgCl2 and Taq enzyme.]]> 400uL PRAMEF33 PCR reaction solution <![CDATA[Forward and reverse primers for PRAMEF33, methylated and unmethylated probes for PRAMEF33, forward and reverse primers for internal reference GAPDH, probe for internal reference GAPDH, PCR buffer, dNTP, MgCl2 and Taq enzyme.]]> 400uL CPLANE2 PCR reaction solution <![CDATA[Forward and reverse primers of CPLANE2, methylated and unmethylated probes of CPLANE2, forward and reverse primers of internal reference GAPDH, probe of internal reference GAPDH, PCR buffer, dNTP, MgCl2 and Taq enzyme.]]> 400uL Droplet generation oil Droplet generation oil 150uL Positive control DNA of NCI-H1703 50uL Negative control Sterile water 50uL Referring to Table 1, Table 2, and Table 3, an oral cancer detection kit provided by the present invention contains the following detection reagents: PAX1 PCR reaction solution, used to detect the methylation degree in at least one target region in the promoter region of the PAX1 gene. The nucleotide sequence of the methylation region to be detected of PAX1 is shown in SEQ ID No. 1; PAX9 PCR reaction solution, used to detect the methylation degree in at least one target region in the promoter region of the PAX9 gene. The nucleotide sequence of the methylation region to be detected of PAX9 is shown in SEQ ID No. 2; The PRAMEF33 PCR reaction solution is used to detect the methylation degree in at least one target region in the promoter region of the PRAMEF33 gene. The nucleotide sequence of the region to be detected for PRAMEF33 methylation is shown in SEQ ID No. 3; The CPLANE2 PCR reaction solution is used to detect the methylation degree in at least one target region in the promoter region of the CPLANE2 gene. The nucleotide sequence of the region to be detected for CPLANE2 gene methylation is shown in SEQ ID No. 4; Positive control and negative control. The positive control substance contained in the positive control is selected from the DNA of human oral cancer cell line NCI-H1703; the negative control is sterile water.

[0028] The kit also includes droplet generation oil, and the droplet generation oil described in the present invention is purchased from Bio-rad, USA.

[0029] The promoter region is not strictly limited to the region within the promoter fragment, but rather the gene fragment in the region near the promoter.

[0030] The described PAX1 PCR reaction solution includes PAX1 forward and reverse primers, PAX1 methylation probe, PAX1 unmethylated probe, internal reference GAPDH forward and reverse primers, and internal reference GAPDH probe; among them, the base sequence of the PAX1 forward primer is shown in SEQ ID No. 5; the base sequence of the PAX1 reverse primer is shown in SEQ ID No. 6; the base sequence of the PAX1 methylation probe is shown in SEQ ID No. 7; the base sequence of the PAX1 unmethylated probe is shown in SEQ ID No. 8; The described PAX9 PCR reaction solution includes PAX9 forward and reverse primers, PAX9 methylation probe and unmethylated probe, internal reference GAPDH forward and reverse primers, and internal reference GAPDH probe; among them, the base sequence of the PAX9 forward primer is shown in SEQ ID No. 9; the base sequence of the PAX9 reverse primer is shown in SEQ ID No. 10; the base sequence of the PAX9 methylation probe is shown in SEQ ID No. 11; the base sequence of the PAX9 unmethylated probe is shown in SEQ ID No. 12; The described PRAMEF33 PCR reaction solution includes forward and reverse primers for PRAMEF33, methylated and unmethylated probes for PRAMEF33, forward and reverse primers for the internal reference GAPDH, and a probe for the internal reference GAPDH; among them, the base sequence of the PRAMEF33 forward primer is as shown in SEQ ID No. 13; the base sequence of the PRAMEF33 reverse primer is as shown in SEQ ID No. 14; the base sequence of the PRAMEF33 methylated probe is as shown in SEQ ID No. 15; the base sequence of the PRAMEF33 unmethylated probe is as shown in SEQ ID No. 16. The described CPLANE2 PCR reaction solution includes forward and reverse primers for CPLANE2, methylated and unmethylated probes for CPLANE2, forward and reverse primers for the internal reference GAPDH, and a probe for the internal reference GAPDH; among them, the base sequence of the CPLANE2 forward primer is as shown in SEQ ID No. 17; the base sequence of the CPLANE2 reverse primer is as shown in SEQ ID No. 18; the base sequence of the CPLANE2 methylated probe is as shown in SEQ ID No. 19; the base sequence of the CPLANE2 unmethylated probe is as shown in SEQ ID No. 20. The base sequences of the forward and reverse primers and the probe for the internal reference GAPDH in the above-mentioned PAX1 PCR reaction solution, PAX9 PCR reaction solution, PRAMEF33 PCR reaction solution, and CPLANE2 PCR reaction solution are as follows: the base sequence of the forward primer for the internal reference GAPDH is as shown in SEQ ID No. 21; the base sequence of the reverse primer for the internal reference GAPDH is as shown in SEQ ID No. 22; the base sequence of the probe for the internal reference GAPDH is as shown in SEQ ID No. 23.

[0031] The PAX1 PCR reaction solution, PAX9 PCR reaction solution, PRAMEF33 PCR reaction solution, and CPLANE2 PCR reaction solution of the present invention also all contain a PCR buffer, MgCl2, dNTP, and Taq enzyme.

[0032] The Taq enzyme described above can be a commonly used Taq DNA polymerase in the art. The formulation amounts of the PCR buffer, MgC12, and dNTP in the present invention can be selected according to conventional methods in the art. One can choose the appropriate formulation amounts of individual reagents and mix them by oneself, or directly select a commercially available finished product of the reaction system. The differences in these components have no obvious impact on the experimental results. Among them, the PCR buffer can be a conventional buffer in the art, such as 30 - 60 mmol / L Tris and 10 - 50 mmol / L KC1, or 30 - 600 mmol / L Tris and 10 - 50 mmol / L KAc, or 30 - 60 mmol / L Tris and 10 - 50 mmol / L CsC1, etc. These differences have no significant impact on the reagents and detection results described in the present invention.

[0033] Both ends of the described PAX1 methylation probe and non - methylation probe, PAX9 methylation probe and non - methylation probe, PRAMEF33 methylation probe and non - methylation probe, CPLANE2 methylation probe and non - methylation probe, and GAPDH probe are labeled with a fluorescent reporter group and a fluorescent quenching group respectively; the fluorescent reporter group is selected from FAM, HEX, ROX, JOE, VIC, TET, NED, FITC, CY3, or CY5; the fluorescent quenching group is selected from BHQ1, BHQ2, BHQ3, TAMRA, Eclipse, DABCYL.

[0034] As a preferred embodiment, the oral cancer detection kit provided by the present invention, the specific compositions of the four detection reagents, namely PAX1 PCR reaction solution, PAX9 PCR reaction solution, PRAMEF33 PCR reaction solution, and CPLANE2 PCR reaction solution, are as follows: The described PAX1 PCR reaction solution includes 0.2 μmol / L - 0.5 μmol / L of each of the forward and reverse primers of PAX1, 0.1 - 0.5 μmol / L of each of the PAX1 methylation probe and non - methylation probe, 0.2 μmol / L - 0.5 μmol / L of each of the forward and reverse primers of the internal reference GAPDH, 0.1 - 0.5 μmol / L of the internal reference GAPDH probe, PCR buffer, 2 - 8 mmol / L of MgCl2, 0.2 - 0.4 mmol / L of dNTP, and 200 U / ml - 1000 U / ml of Taq enzyme; The described PAX9 PCR reaction solution includes forward and reverse primers for PAX9 each at 0.2 μmol / L to 0.5 μmol / L, methylated and unmethylated probes for PAX9 each at 0.1 to 0.5 μmol / L, forward and reverse primers for the internal reference GAPDH each at 0.2 μmol / L to 0.5 μmol / L, the probe for the internal reference GAPDH at 0.1 to 0.5 μmol / L, PCR buffer, MgCl2 at 2 to 8 mmol / L, dNTP at 0.2 to 0.4 mmol / L, and Taq enzyme at 200 U / ml to 1000 U / ml; The described PRAMEF33 PCR reaction solution includes forward and reverse primers for PRAMEF33 each at 0.2 μmol / L to 0.5 μmol / L, methylated and unmethylated probes for PRAMEF33 each at 0.1 to 0.5 μmol / L, forward and reverse primers for the internal reference GAPDH each at 0.2 μmol / L to 0.5 μmol / L, the probe for the internal reference GAPDH at 0.1 to 0.5 μmol / L, PCR buffer, MgCl2 at 2 to 8 mmol / L, dNTP at 0.2 to 0.4 mmol / L, and Taq enzyme at 200 U / ml to 1000 U / ml; The described CPLANE2 PCR reaction solution includes forward and reverse primers for CPLANE2 each at 0.2 μmol / L to 0.5 μmol / L, methylated and unmethylated probes for CPLANE2 each at 0.1 to 0.5 μmol / L, forward and reverse primers for the internal reference GAPDH each at 0.2 μmol / L to 0.5 μmol / L, the probe for the internal reference GAPDH at 0.1 to 0.5 μmol / L, PCR buffer, MgCl2 at 2 to 8 mmol / L, dNTP at 0.2 to 0.4 mmol / L, and Taq enzyme at 200 U / ml to 1000 U / ml.

[0035] Furthermore, for the PAX1 methylated fluorescence probe, the reporter group at the 5' end is FAM and the quencher group at the 3' end is BHQ1; for the PAX1 unmethylated fluorescence probe, the reporter group at the 5' end is VIC and the quencher group at the 3' end is BHQ1; for the fluorescence probe of the internal reference gene, the fluorescence reporter group at the 5' end is ROX and the quencher group at the 3' end is BHQ1.

[0036] For the PAX9 methylated fluorescence probe, the reporter group at the 5' end is FAM and the quencher group at the 3' end is BHQ1; for the PAX9 unmethylated fluorescence probe, the reporter group at the 5' end is VIC and the quencher group at the 3' end is BHQ1; for the fluorescence probe of the internal reference gene, the fluorescence reporter group at the 5' end is ROX and the quencher group at the 3' end is BHQ1.

[0037] The 5'-end luminescent group of the PRAMEF33 methylation fluorescent probe is FAM, and the 3'-end quenching group is BHQ1. The 5'-end luminescent group of the PRAMEF33 unmethylated fluorescent probe is VIC, and the 3'-end quenching group is BHQ1. The 5'-end fluorescent reporter group of the internal reference gene fluorescent probe is ROX, and the 3'-end quenching group is BHQ1.

[0038] The 5'-end reporter group of the CPLANE2 methylation fluorescent probe is FAM, and the 3'-end quenching group is BHQ1. The 5'-end reporter group of the CPLANE2 unmethylated fluorescent probe is VIC, and the 3'-end quenching group is BHQ1. The 5'-end fluorescent reporter group of the internal reference gene fluorescent probe is ROX, and the 3'-end quenching group is BHQ1. The 5'-end fluorescent reporter group of the internal reference gene fluorescent probe is ROX, and the 3'-end quenching group is BHQ1.

[0039] The detection reagents provided by the present invention, namely the PAX1 PCR reaction solution, PAX9 PCR reaction solution, PRAMEF33 PCR reaction solution, and CPLANE2 PCR reaction solution, contain specific primers and fluorescent probes for the target genes, and have high specificity and sensitivity, and can rapidly detect the methylation of four target genes, namely PAX1, PAX9, PRAMEF33, and CPLANE2, in the detection sample.

[0040] Example 2 This example provides a method for applying the oral cancer methylation prognosis marker combination and the oral cancer detection kit to oral cancer prediction, which specifically includes the following steps: S1. Extract the DNA of the detection sample, subject the DNA of the detection sample to bisulfite conversion treatment, deaminate the unmethylated cytosine in the nucleic acid of the target gene in the detection sample to uracil, and obtain the purified Bis-DNA sample; S1.1. Extract the DNA of the detection sample: Extract the DNA from human fecal exfoliated cells. After the extraction is completed, use a Nano-500 type micro-spectrophotometer to measure the concentration and OD260 / OD280 of the DNA, and the OD260 / OD280 is between 1.6 and 2.0; S1.2. Subject the DNA of the detection sample to bisulfite conversion treatment to obtain the purified Bis-DNA sample. The obtained Bis-DNA sample should be detected in time, or can be stored at -20°C for future measurement. The storage period generally does not exceed 4 months. For long-term storage, please place it at -80°C.

[0041] S2. Using the purified Bis-DNA sample as a template, prepare the target gene PCR reaction system with the PAX1 PCR reaction solution, PAX9 PCR reaction solution, PRAMEF33 PCR reaction solution, and CPLANE2 PCR reaction solution in the oral cancer detection kit of the present invention respectively. Amplify the nucleic acid using the specific primers and fluorescent probes in the PAX1 PCR reaction solution, PAX9 PCR reaction solution, PRAMEF33 PCR reaction solution, and CPLANE2 PCR reaction solution, and detect the methylation levels of the PAX1, PAX9, PRAMEF33, and CPLANE2 genes in the DNA of the test sample using a droplet digital PCR instrument.

[0042] S2.1. Reaction system preparation: Composition of the PAX1 gene PCR reaction system: The volume of the PAX1 PCR reaction solution is 15 μL, the volume of the Bis-DNA template is 2 - 5 μL, and the volume of purified water is up to 20 μL; Composition of the PAX9 gene PCR reaction system: The volume of the PAX9 PCR reaction solution is 15 μL, the volume of the Bis-DNA template is 2 - 5 μL, and the volume of purified water is up to 20 μL; Composition of the PRAMEF33 gene PCR reaction system: The volume of the PRAMEF33 PCR reaction solution is 15 μL, the volume of the Bis-DNA template is 2 - 5 μL, and the volume of purified water is up to 20 μL; Composition of the CPLANE2 gene PCR reaction system: The volume of the CPLANE2 PCR reaction solution is 15 μL, the volume of the Bis-DNA template is 2 - 5 μL, and the volume of purified water is up to 20 μL.

[0043] S2.2. Detection of the methylation level of the target gene Add the above target gene PAX1, PAX9, PRAMEF33, and CPLANE2 PCR reaction systems into test tubes for detection respectively: The volume of each tube of the reaction system is 20 μL. Tighten the tube caps, perform instantaneous low-speed centrifugation, and then carry out PCR amplification and detection of the methylation level of the target gene.

[0044] Perform relevant operations for PCR amplification and detection of the methylation level of the target gene using a droplet digital PCR instrument. When using the droplet digital PCR instrument, select the fluorescence detection channel and set the amplification cycle parameters.

[0045] S3. Judgment of the detection results: If the total number of droplets in the test sample is greater than 10,000, the reaction is judged to be valid; if the sum of the methylated copy number and the unmethylated copy number of the test sample is ≥ 100, the test result of the sample is valid; if it is less than 100, the test of the sample is invalid and needs to be retested; The methylation ratio of the test sample is calculated by the following formula: Methylation ratio = Methylated copy number / [(Methylated copy number + Unmethylated copy number)] × 100%. If the methylation ratio of the target gene ≥ 2%, the interpretation result is methylation positive; if the methylation ratio of the target gene < 2%, the interpretation result is methylation negative; When any one of the four target genes PAX1, PAX9, PRAMEF33, and CPLANE2 is methylation positive, the test sample is judged to be cancer positive; when all four target genes PAX1, PAX9, PRAMEF33, and CPLANE2 are methylation negative, the test sample is judged to be cancer negative.

[0046] The oral cancer detection reagent provided by the present invention uses digital PCR technology during application to jointly detect the methylation of PAX1, PAX9, PRAMEF33, and CPLANE2 genes in the plasma-free DNA of patients. After bisulfite treatment of the sample, specific primers and fluorescent probes are used to amplify nucleic acids, so as to achieve rapid detection of the methylation of methylation-related genes PAX1, PAX9, PRAMEF33, and CPLANE2 genes, providing a simpler and more accurate oral cancer screening for clinical use.

[0047] The oral cancer detection reagent provided by the present invention takes DNA methylation abnormality as the detection object. Based on digital PCR technology, it can efficiently and sensitively complete the PCR amplification of target nucleic acid fragments in a microreaction unit, obtain fluorescence signals for statistical analysis, get rid of the dependence on the standard curve and directly give the copy number of the target sequence, improve the stability of experimental results within batches and between batches, improve the sensitivity of nucleic acid detection methods, effectively reduce the occurrence of false negatives, and is suitable for clinical detection.

[0048] Example 3 Example 3 is an application experiment of the oral cancer methylation prognostic marker combination and the oral cancer detection kit of the present invention.

[0049] The postoperative samples and 5-year survival status of 103 oral cancer patients were collected and statistically analyzed. The oral cancer detection reagent provided by the present invention was used to detect and judge the methylation levels of the four target genes PAX1, PAX9, PRAMEF33, and CPLANE2 in the test samples, and the test results were compared with the 5-year survival status of the patients.

[0050] The oral cancer detection reagent of the present invention is used to detect the sample. For the detection object, peripheral blood is drawn from the postoperative samples of 103 oral cancer patients. Using the detection method provided in the application of the oral cancer methylation prognosis marker combination and the oral cancer detection kit of the present invention, the methylation of genes PAX1, PAX9, PRAMEF33, and CPLANE2 in the detection sample is detected.

[0051] When using the oral cancer detection reagent provided by the present invention to detect the samples of 103 detection objects, the positive and negative amplification results of target gene methylation are as Figure 1 shown. It can be seen from Figure 1 that the detection reagent provided by the present invention can specifically detect the methylation levels of six target genes, PAX1, PAX9, PRAMEF33, and CPLANE2, in biological samples.

[0052] Figure 2 This is a comparison table of the detection results of Example 3 application experiment of the present invention and the 5-year survival rate of patients. The comparison of the results of using the detection reagent provided by the present invention to detect the samples with the 5-year survival rate results of the patients is as Figure 2 shown. The detection results show that when using the detection reagent of the present invention to detect the samples, by comparing the ROC curve generated by the detection results and the 5-year survival rate of the patients, it can be obtained that when using the detection reagent of the present invention to detect the samples, the 5-year survival rate of oral cancer patients after surgery can be significantly predicted, and the area under the ROC curve (AUC) is 0.959, 95% CI 0.913-1; P<0.001).

[0053] The application experiment of Example 3 shows that when using the oral cancer detection reagent provided by the present invention to detect four target genes in the detection sample, the sensitivity of the detection reagent is 96.80%, the specificity is 95.1%, and the generated ROC curve can significantly predict the 5-year survival rate of oral cancer patients after surgery.

[0054] Example 4 Example 4 is a verification experiment of the oral cancer methylation prognosis marker combination and the oral cancer detection kit of the present invention.

[0055] To verify the accuracy of the results of detecting oral cancer by the methylation detection of four target genes by the oral cancer detection reagent of the present invention, the specific verification experiment is as follows: The methylation levels of four target genes, PAX1, PAX9, PRAMEF33, and CPLANE2, in the postoperative samples of 73 oral cancer patients are detected. The detection results are as Figure 3 shown, Figure 3 which is a graph of the positive and negative amplification results of target gene methylation in the verification experiment. From Figure 3From the positive amplification results of the methylation of the target genes in the verification experiment, it can be seen that among the 73 test samples, the methylation levels of the target genes PAX1, PAX9, PRAMEF33, and CPLANE2 are all positive. From Figure 3 From the negative amplification results of the methylation of the target genes in the verification experiment, it can be seen that among the 73 test samples, the methylation levels of the target genes PAX1, PAX9, PRAMEF33, and CPLANE2 are all negative.

[0056] Taking the postoperative samples of these 73 oral cancer patients as the test objects, the methylation levels of the four target genes PAX1, PAX9, PRAMEF33, and CPLANE2 in the test samples were detected and judged using the oral cancer detection reagent provided by the present invention, and the test results were compared with the 5-year survival rate of the patients after surgery. Figure 4 For the control of the ROC curve of the methylation detection results and the 5-year survival rate of the patients in the verification experiment of Example 4 of the present invention, from Figure 4 it can be seen that when using the oral cancer detection reagent provided by the present invention to detect the four target genes in the test samples, it can significantly predict the 5-year survival rate of oral cancer patients after surgery, and the area under the ROC curve (AUC) is 0.934 (95% CI 0.823-1; P<0.001).

[0057] The comparison between the detection results of the verification experiment and the 5-year survival status of the patients is as Figure 5 shown.

[0058] The verification experiment of this example shows that when using the oral cancer detection reagent provided by the present invention to detect the four target genes in the test samples, the sensitivity (positive coincidence rate) of the detection reagent is 96.08%, that is, the missed diagnosis rate is 3.92%; the specificity (negative coincidence rate) is 90.00%, that is, the misdiagnosis rate is 10.00%; the total correct rate of all 73 samples in the verification set is 95.89%.

[0059] Using the oral cancer detection reagent provided by the present invention to detect and judge the methylation levels of the four target genes PAX1, PAX9, PRAMEF33, and CPLANE2 in the test samples can effectively predict the postoperative survival rate of oral cancer patients, has high sensitivity and specificity, and can provide a simple and accurate screening method for clinical practice.

Claims

1. Oral cancer methylation prognosis biomarker combination, characterized in that, It consists of four oral cancer DNA methylation prognostic markers, namely PAX1, PAX9, PRAMEF33, and CPLANE2. Among them, the nucleotide sequence of the methylation region to be detected of the marker PAX1 is as shown in SEQ ID No. 1; the nucleotide sequence of the methylation region to be detected of the marker PAX9 is as shown in SEQ ID No. 2; the nucleotide sequence of the methylation region to be detected of the marker PRAMEF33 is as shown in SEQ ID No. 3; the nucleotide sequence of the methylation region to be detected of the marker CPLANE2 is as shown in SEQ ID No.

4.

2. An oral cancer detection kit, characterized in that, The kit contains the following detection reagents: PAX1 PCR reaction solution, which is used to detect the methylation degree in at least one target region in the promoter region of the PAX1 gene. The nucleotide sequence of the methylation region to be detected of PAX1 is as shown in SEQ ID No. 1; PAX9 PCR reaction solution, which is used to detect the methylation degree in at least one target region in the promoter region of the PAX9 gene. The nucleotide sequence of the methylation region to be detected of PAX9 is as shown in SEQ ID No. 2; PRAMEF33 PCR reaction solution, which is used to detect the methylation degree in at least one target region in the promoter region of the PRAMEF33 gene. The nucleotide sequence of the methylation region to be detected of PRAMEF33 is as shown in SEQ ID No. 3; CPLANE2 PCR reaction solution, which is used to detect the methylation degree in at least one target region in the promoter region of the CPLANE2 gene. The nucleotide sequence of the methylation region to be detected of the CPLANE2 gene is as shown in SEQ ID No. 4; Positive control and negative control. The positive control substance contained in the positive control is selected from the DNA of human oral cancer cell NCI-H1703; the negative control is sterile water.

3. The oral cancer detection kit according to claim 2, wherein the PAX1 PCR reaction solution includes PAX1 forward and reverse primers, PAX1 methylation probe, PAX1 unmethylated probe, internal reference GAPDH forward and reverse primers, and internal reference GAPDH probe; among them, the base sequence of the PAX1 forward primer is as shown in SEQ ID No. 5; the base sequence of the PAX1 reverse primer is as shown in SEQ ID No. 6; the base sequence of the PAX1 methylation probe is as shown in SEQ ID No. 7; the base sequence of the PAX1 unmethylated probe is as shown in SEQ ID No. 8; The described PAX9 PCR reaction solution includes forward and reverse primers for PAX9, methylated and unmethylated probes for PAX9, and forward and reverse primers for the internal reference GAPDH; an internal reference GAPDH probe; wherein, the base sequence of the PAX9 forward primer is as shown in SEQ ID No. 9; the base sequence of the PAX9 reverse primer is as shown in SEQ ID No. 10; the base sequence of the PAX9 methylated probe is as shown in SEQ ID No. 11; the base sequence of the PAX9 unmethylated probe is as shown in SEQ ID No. 12; The described PRAMEF33 PCR reaction solution includes forward and reverse primers for PRAMEF33, methylated and unmethylated probes for PRAMEF33, forward and reverse primers for the internal reference GAPDH, and an internal reference GAPDH probe; wherein, the base sequence of the PRAMEF33 forward primer is as shown in SEQ ID No. 13; the base sequence of the PRAMEF33 reverse primer is as shown in SEQ ID No. 14; the base sequence of the PRAMEF33 methylated probe is as shown in SEQ ID No. 15; the base sequence of the PRAMEF33 unmethylated probe is as shown in SEQ ID No. 16; The described CPLANE2 PCR reaction solution includes forward and reverse primers for CPLANE2, methylated and unmethylated probes for CPLANE2, forward and reverse primers for the internal reference GAPDH, and an internal reference GAPDH probe; wherein, the base sequence of the CPLANE2 forward primer is as shown in SEQ ID No. 17; the base sequence of the CPLANE2 reverse primer is as shown in SEQ ID No. 18; the base sequence of the CPLANE2 methylated probe is as shown in SEQ ID No. 19; the base sequence of the CPLANE2 unmethylated probe is as shown in SEQ ID No. 20; The base sequences of the forward and reverse primers and the probe of the internal reference GAPDH in the described PAX1 PCR reaction solution, PAX9 PCR reaction solution, PRAMEF33 PCR reaction solution, and CPLANE2 PCR reaction solution are as follows: the base sequence of the forward primer of the internal reference GAPDH is as shown in SEQ ID No. 21; the base sequence of the reverse primer of the internal reference GAPDH is as shown in SEQ ID No. 22; the base sequence of the probe of the internal reference GAPDH is as shown in SEQ ID No.

23.

4. The oral cancer detection kit according to claim 3, wherein, The described PAX1 PCR reaction solution includes forward and reverse primers of PAX1 at 0.2 μmol / L to 0.5 μmol / L each, methylated and unmethylated probes of PAX1 at 0.1 to 0.5 μmol / L each, forward and reverse primers of internal reference GAPDH at 0.2 μmol / L to 0.5 μmol / L each, internal reference GAPDH probe at 0.1 to 0.5 μmol / L, PCR buffer, MgCl2 at 2 to 8 mmol / L, dNTP at 0.2 to 0.4 mmol / L, and Taq enzyme at 200 U / ml to 1000 U / ml; The described PAX9 PCR reaction solution includes forward and reverse primers of PAX9 at 0.2 μmol / L to 0.5 μmol / L each, methylated and unmethylated probes of PAX9 at 0.1 to 0.5 μmol / L each, forward and reverse primers of internal reference GAPDH at 0.2 μmol / L to 0.5 μmol / L each, internal reference GAPDH probe at 0.1 to 0.5 μmol / L, PCR buffer, MgCl2 at 2 to 8 mmol / L, dNTP at 0.2 to 0.4 mmol / L, and Taq enzyme at 200 U / ml to 1000 U / ml; The described PRAMEF33 PCR reaction solution includes forward and reverse primers of PRAMEF33 at 0.2 μmol / L to 0.5 μmol / L each, methylated and unmethylated probes of PRAMEF33 at 0.1 to 0.5 μmol / L each, forward and reverse primers of internal reference GAPDH at 0.2 μmol / L to 0.5 μmol / L each, internal reference GAPDH probe at 0.1 to 0.5 μmol / L, PCR buffer, MgCl2 at 2 to 8 mmol / L, dNTP at 0.2 to 0.4 mmol / L, and Taq enzyme at 200 U / ml to 1000 U / ml; The described CPLANE2 PCR reaction solution includes forward and reverse primers of CPLANE2 at 0.2 μmol / L to 0.5 μmol / L each, methylated and unmethylated probes of CPLANE2 at 0.1 to 0.5 μmol / L each, forward and reverse primers of internal reference GAPDH at 0.2 μmol / L to 0.5 μmol / L each, internal reference GAPDH probe at 0.1 to 0.5 μmol / L, PCR buffer, MgCl2 at 2 to 8 mmol / L, dNTP at 0.2 to 0.4 mmol / L, and Taq enzyme at 200 U / ml to 1000 U / ml.

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