WO2023274350A1 - 甲状腺结节良恶性相关标志物及其应用 - Google Patents

甲状腺结节良恶性相关标志物及其应用 Download PDF

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WO2023274350A1
WO2023274350A1 PCT/CN2022/102658 CN2022102658W WO2023274350A1 WO 2023274350 A1 WO2023274350 A1 WO 2023274350A1 CN 2022102658 W CN2022102658 W CN 2022102658W WO 2023274350 A1 WO2023274350 A1 WO 2023274350A1
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gene
sequence
genome
genomic
seq
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French (fr)
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苏明扬
杨世方
马建华
郗大勇
何其晔
刘蕊
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上海鹍远生物科技股份有限公司
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes

Definitions

  • the invention belongs to the field of molecular auxiliary diagnosis, and relates to markers related to benign and malignant thyroid nodules and applications thereof.
  • DNA methylation is a mechanism of epigenetic inheritance, a common epigenetic modification of the genome of eukaryotic cells, which can change genetic expression without changing the DNA sequence.
  • the so-called DNA methylation refers to the covalent bonding of a methyl group at the 5th carbon position of cytosine of a genomic CpG dinucleotide under the action of DNA methyltransferase.
  • DNA methylation plays an important role in cell proliferation, differentiation, and development, and is closely related to the occurrence and development of tumors. Its effects include transcriptional inhibition, chromatin structure regulation, X chromosome inactivation, and genomic imprinting. Abnormal DNA methylation can participate in tumorigenesis and progression by affecting chromatin structure and the expression of oncogenes and tumor suppressor genes.
  • CpG dinucleotides are the most important target of DNA methylation in mammals, and they are distributed throughout the genome.
  • CpG sites in CpG islands are usually in an unmethylated state, while CpG sites outside CpG islands are usually hypermethylated, and this methylated form can be stably retained during cell division .
  • the degree of methylation of CpG sites in non-CpG island regions of tumor suppressor genes usually decreases, while CpG in CpG islands is highly methylated.
  • the nucleotide sequence and its composition No change, but it will lead to changes in chromatin structure and a decrease in the expression of tumor suppressor genes.
  • Thyroid nodules are solid or fluid-filled lumps that form in the thyroid after abnormal growth of thyroid cells. They can move up and down with the thyroid, a small gland at the base of the neck, just above the breastbone, as you swallow. . Thyroid nodules are very common, and although most thyroid nodules are not serious and cause no symptoms, some can progress to thyroid cancer. In order to diagnose and treat thyroid cancer earlier and reduce unnecessary surgical treatment, a highly sensitive and specific in vitro diagnostic method (In-Vitro Diagnostics, IVD) is urgently needed to differentiate benign and malignant thyroid nodules.
  • IVD In-Vitro Diagnostics
  • the American Thyroid Association defines a thyroid nodule as a discrete lesion on the thyroid gland. With the help of imaging examination, it can be observed that the nodule is different from the normal thyroid tissue structure and has a relative boundary.
  • the evaluation of thyroid nodules mainly adopts ultrasound (ultrasonography, US), fine needle aspiration biopsy (fine needle aspiration biopsy, FNAB), electronic computed tomography (Computed Tomography), magnetic resonance imaging (Magnetic Resonance Imaging, MRI) and positron emission tomography. Positron Emission Computed Tomography (PET).
  • high-resolution ultrasonography US is the preferred examination for thyroid nodules.
  • thyroid nodules It can screen and evaluate thyroid nodules, and judge their size, type and internal structure of nodules.
  • Nodules detected by ultrasound are usually not associated with abnormal thyroid function, and the misuse of high-resolution thyroid ultrasound has led to the detection of a large number of clinically insignificant nodules, including thyroid microcarcinomas, which may bias the clinical assessment of thyroid dysfunction.
  • the object of the present invention is to provide a composition for identifying benign and malignant individual thyroid nodules, by using the composition to detect a gene containing a gene selected from CDH1 gene, TSHR gene, MCC gene, TBX15 gene, PRR15 gene, DPYS gene, GRIA2 gene , NR5A1 gene, TTC34 gene, RCOR2 gene, F10 gene, ITPKA gene, SLC16A3 gene, RARG gene, FTAP2B gene, SOD3 gene, RP11-867G23.12 gene, EMX2OS gene, TEAD3 gene and DGKG gene one or one of the target loci Multiple methylation conditions can be used to distinguish benign from malignant thyroid nodules in this individual.
  • the first aspect of the present invention provides the application of primer molecules and/or probe molecules in the preparation of detection reagents or diagnostic kits for diagnosing benign and malignant thyroid nodules in individuals, and the device in the preparation of diagnostic kits for diagnosing benign and malignant thyroid nodules in individuals application;
  • the primer molecule is identical to, complementary to, or hybridizes to one or more target markers under stringent conditions and includes at least 9 consecutive nucleotides;
  • the probe molecule is combined with one or more target markers The amplified products of the hybridization under stringent conditions; the device is used to determine the methylation status or level of at least one CpG dinucleotide in one or more target markers.
  • the invention provides at least one reagent or set of reagents for distinguishing between methylated and unmethylated CpG dinucleotides in at least one target region of genomic DNA prepared for detecting and/or classifying thyroid nodules in an individual
  • a kit for a method of benign and malignant wherein said method comprises contacting genomic DNA isolated from said individual biological sample with said at least one reagent or set of reagents, wherein said target region is identical to or complementary to A sequence of at least 16 contiguous nucleotides of one or more markers of interest, wherein the contiguous nucleotides comprise at least one CpG dinucleotide sequence, thereby providing at least in part the detection of benign and malignant thyroid nodules and /or categories.
  • a third aspect of the invention provides one or more reagents, amplification enzymes, and other bases that convert unmethylated cytosine bases at position 5 to uracil or other bases that are detectably different from cytosine in hybridization properties.
  • a fourth aspect of the present invention provides one or more methylation-sensitive restriction enzymes and amplification enzymes and at least one primer comprising at least 9 contiguous nucleotides prepared for detection and/or classification of benign thyroid nodules in individuals
  • the primers are identical to, complementary to, or hybridize under stringent conditions to one or more markers of interest; said method comprising: a) isolating a genome from said individual biological sample DNA; b) digesting the genomic DNA or fragments thereof of a) with the one or more methylation-sensitive restriction enzymes, contacting the resulting digestion product with the amplification enzyme and the at least one primer; and c) determining the methylation status or level of at least one CpG dinucleotide of said one or more markers of interest based on the presence or absence or nature of said amplicon, thereby at least in part detecting and/or classifying Benign and malignant thyroid nodules in individuals.
  • the fifth aspect of the present invention provides the use of the processed nucleic acid derived from one or more target markers in the preparation of a kit for diagnosing benign and malignant thyroid nodules in individuals, wherein the processing is suitable for the one or more At least one unmethylated cytosine base of each marker of interest is converted to uracil or other base that hybridizes detectably different from cytosine.
  • the sixth aspect of the present invention provides a diagnostic reagent or a diagnostic kit for detecting the methylation level or methylation state of at least one CpG dinucleotide of a target marker to diagnose benign and malignant thyroid nodules in an individual, which comprises primers molecule and/or probe molecule; wherein, the primer molecule is identical to, complementary to, or hybridizes to one or more target markers under stringent conditions and comprises at least 9 consecutive nucleotides, and the probe molecule and Amplified products of one or more markers of interest are hybridized under stringent conditions.
  • the seventh aspect of the present invention provides a method for diagnosing benign and malignant thyroid nodules in an individual or patient, the method comprising detecting one or more target markers and at least one formazan of at least one CpG dinucleotide in the biological sample of the individual or patient benign and malignant thyroid nodules based on the methylation level.
  • the eighth aspect of the present invention provides a device for detecting and diagnosing benign and malignant thyroid nodules in individuals, the device includes a memory, a processor, and a computer program stored in the memory and operable on the processor, the processor The following steps are achieved when the procedure is executed: (1) obtaining the methylation level or methylation status of at least one CpG dinucleotide of one or more target markers in the sample, and (2) the methylation status according to (1) Kylation level in judging benign and malignant thyroid nodules.
  • the one or more target markers are selected from: CDH1 gene or genomic CDH1 sequence, TSHR gene or genomic TSHR sequence, MCC gene or genomic MCC sequence, TBX15 gene or TBX15 sequence of genome, PRR15 gene or PRR15 sequence of genome, DPYS gene or DPYS sequence of genome, GRIA2 gene or GRIA2 sequence of genome, NR5A1 gene or NR5A1 sequence of genome, TTC34 gene or TTC34 sequence of genome, RCOR2 gene or genome RCOR2 sequence, F10 gene or genome F10 sequence, ITPKA gene or genome ITPKA sequence, SLC16A3 gene or genome SLC16A3 sequence, RARG gene or genome RARG sequence, FTAP2B gene or genome FTAP2B sequence, SOD3 gene or genome SOD3 sequence , RP11-867G23.12 gene or genome RP11-867G23.12 sequence, EMX2OS gene or genome EMX2
  • the target marker is the SLC16A3 gene or the genome's SLC16A3 sequence and the TSHR gene or the genome's TSHR sequence, the TTC34 gene or the genome's TTC34 sequence, the ROCR2 gene or the genome's ROCR2 sequence, the RARG gene or the genome's RARG sequence
  • MCC gene or genome MCC sequence and ITPKA gene or genome ITPKA sequence is the SLC16A3 gene or the genome's SLC16A3 sequence and the TSHR gene or the genome's TSHR sequence, the TTC34 gene or the genome's TTC34 sequence, the ROCR2 gene or the genome's ROCR2 sequence, the RARG gene or the genome's RARG sequence.
  • the target marker is the SLC16A3 gene or the SLC16A3 sequence of the genome and the TSHR gene or the TSHR sequence of the genome; preferably, the methylation level of the target marker in the thyroid nodule from the individual is detected.
  • the target marker is the SLC16A3 gene or the genome's SLC16A3 sequence and the ROCR2 gene or the genome's ROCR2 sequence, and the TTC34 gene or the genome's TTC34 sequence, the RARG gene or the genome's RARG sequence, the ITPKA gene or the genome's ITPKA sequence and at least one, at least two or at least three of the MCC sequence of the MCC gene or the genome;
  • the target marker is the SLC16A3 gene or the SLC16A3 sequence of the genome, the ROCR2 gene or the ROCR2 sequence of the genome, the TTC34 gene or Genomic TTC34 sequence and RARG gene or genomic RARG sequence
  • the target marker is SLC16A3 gene or genomic SLC16A3 sequence, ROCR2 gene or genomic ROCR2 sequence, ITPKA gene or genomic ITPKA sequence and MCC gene or genomic MCC sequence; preferably, the methylation levels of these target markers are detected in a blood sample from the individual, such as plasma
  • the target marker is the SLC16A3 gene or the genome's SLC16A3 sequence and the RARG gene or the genome's RARG sequence, and is selected from the TTC34 gene or the genome's TTC34 sequence, the ROCR2 gene or the genome's ROCR2 sequence and the ITPKA gene or the genome's ITPKA At least one, at least two, or all three of the sequences; preferably, the target marker is the SLC16A3 gene or the genomic SLC16A3 sequence, the ROCR2 gene or the genomic ROCR2 sequence, the TTC34 gene or the genomic TTC34 sequence and the RARG gene or the genomic sequence RARG sequence, or the target marker is SLC16A3 gene or genome SLC16A3 sequence, TTC34 gene or genome TTC34 sequence, ITPKA gene or genome ITPKA sequence and RARG gene or genome RARG sequence; The methylation levels of these target markers in blood samples such as plasma.
  • the target marker is the SLC16A3 gene or the genome's SLC16A3 sequence and the TTC34 gene or the genome's TTC34 sequence, and the ROCR2 sequence selected from the ROCR2 gene or the genome, the RARG gene or the genome's RARG sequence, and the ITPKA gene or the genome's ITPKA At least one, at least two, or all three of the sequences; preferably, the target marker is the SLC16A3 gene or the genomic SLC16A3 sequence, the ROCR2 gene or the genomic ROCR2 sequence, the TTC34 gene or the genomic TTC34 sequence and the RARG gene or the genomic sequence RARG sequence, or the target marker is SLC16A3 gene or genome SLC16A3 sequence, TTC34 gene or genome TTC34 sequence, ITPKA gene or genome ITPKA sequence and RARG gene or genome RARG sequence; The methylation levels of these target markers in blood samples such as plasma.
  • the target marker is the SLC16A3 gene or the genome's SLC16A3 sequence and the ITPKA gene or the genome's ITPKA sequence, and the ROCR2 sequence selected from the ROCR2 gene or the genome, the MCC gene or the genome's MCC sequence, the TTC34 gene or the genome's TTC34 sequence and at least one, at least two or all three of RARG sequence and RARG gene or genome; preferably, the target marker is SLC16A3 gene or genome SLC16A3 sequence, ROCR2 gene or genome ROCR2 sequence, ITPKA gene or Genomic ITPKA sequence and MCC gene or genomic MCC sequence, or the target marker is SLC16A3 gene or genomic SLC16A3 sequence, TTC34 gene or genomic TTC34 sequence, ITPKA gene or genomic ITPKA sequence and RARG gene or genomic RARG sequence; preferably, the methylation levels of these target markers are detected in a blood sample from the individual, such as plasma.
  • the target marker is the SLC16A3 gene or the genomic SLC16A3 sequence, the TTC34 gene or the genomic TTC34 sequence and the RARG gene or the genomic RARG sequence, and the ROCR2 gene or the genomic ROCR2 sequence or the ITPKA gene or the genomic ITPKA sequence;
  • the methylation levels of these target markers are detected in a blood sample from the individual, such as plasma.
  • the target marker is FTAP2B gene or genomic FTAP2B sequence and EMS2OS gene or genomic EMS2OS sequence, DGKG gene or genomic DGKG sequence, SOD3 gene or genomic SOD3 sequence, RP11-867G23.12 gene or At least one, at least two or at least three of the RP11-867G23.12 sequences of the genome.
  • the target marker is FTAP2B gene or genomic FTAP2B sequence, EMS2OS gene or genomic EMS2OS sequence and optional SOD3 gene or genomic SOD3 sequence; preferably, the target marker is FTAP2B gene or genomic The FTAP2B sequence and the EMS2OS gene or the EMS2OS sequence of the genome, wherein the methylation level of these target markers in the thyroid nodule from the individual is detected; preferably, the target marker is the FTAP2B gene or the FTAP2B sequence of the genome, The EMS2OS gene or the genome's EMS2OS sequence and the SOD3 gene or the genome's SOD3 sequence, wherein the methylation levels of these target markers in blood samples such as plasma from the individual are detected.
  • the target markers are FTAP2B gene or genomic FTAP2B sequence and DGKG gene or genomic DGKG sequence, wherein the methylation levels of these target markers in blood samples such as plasma from the individual are detected.
  • the target marker is the FTAP2B gene or genome FTAP2B sequence and SOD3 gene or genome SOD3 sequence, and the EMS2OS gene or genome EMS2OS sequence or RP11-867G23.12 gene or genome RP11-867G23.12 sequence, wherein, the methylation levels of these target markers in blood samples such as plasma are detected.
  • the target marker is the RARG gene or the RARG sequence of the genome, and is selected from the DPYS gene or the DPYS sequence of the genome, the TSHR gene or the TSHR sequence of the genome, the TTC34 gene or the TTC34 sequence of the genome, the RCOR2 gene or the RCOR2 of the genome sequence, SLC16A3 gene or genomic SLC16A3 sequence and ITPKA gene or genomic ITPKA sequence at least one, at least two, at least three or at least four.
  • the target markers are RARG gene or genomic RARG sequence and DPYS gene or genomic DPYS sequence, wherein the methylation levels of these target markers in thyroid nodules from the individual are detected.
  • the target markers are RARG gene or genomic RARG sequence and TSHR gene or genomic TSHR sequence, wherein the methylation levels of these target markers in blood samples such as plasma from the individual are detected.
  • the target marker is the RARG gene or the genome's RARG sequence and the TTC34 gene or the genome's TTC34 sequence, and the RCOR2 gene or the genome's RCOR2 sequence, the SLC16A3 gene or the genome's SLC16A3 sequence and the ITPKA gene or the genome's ITPKA At least one or at least two of the sequences, wherein the methylation level of these target markers in blood samples such as plasma from the individual is detected; preferably, the target markers are RARG gene or genomic RARG sequence, TTC34 Gene or genome TTC34 sequence, RCOR2 gene or genome RCOR2 sequence, and SLC16A3 gene or genome SLC16A3 sequence wherein the methylation levels of these target markers in blood samples such as plasma from the individual are detected; preferably, the target The markers are the RARG gene or the RARG sequence of the genome, the TTC34 gene or the TTC34 sequence of the genome, the ITPKA gene or the ITPKA sequence of the genome
  • the target marker is RARG gene or genome RARG sequence and SLC16A3 gene or genome SLC16A3 sequence, and selected from TTC34 gene or genome TTC34 sequence, RCOR2 gene or genome RCOR2 sequence and ITPKA gene or genome ITPKA At least one or at least two of the sequences, wherein the methylation level of these target markers in blood samples such as plasma from the individual is detected; preferably, the target markers are RARG gene or genomic RARG sequence, TTC34 Gene or genome TTC34 sequence, RCOR2 gene or genome RCOR2 sequence, and SLC16A3 gene or genome SLC16A3 sequence wherein the methylation levels of these target markers in blood samples such as plasma from the individual are detected; preferably, the target The markers are the RARG gene or the RARG sequence of the genome, the TTC34 gene or the TTC34 sequence of the genome, the ITPKA gene or the ITPKA sequence of the genome, and the SLC16A3 gene or the SLC16A3 sequence of
  • the target marker is RARG gene or genomic RARG sequence, SLC16A3 gene or genomic SLC16A3 sequence and TTC34 gene or genomic TTC34 sequence, and ROCR2 gene or genomic ROCR2 sequence or ITPKA gene or genomic ITPKA sequence;
  • the methylation levels of these target markers are detected in a blood sample from the individual, such as plasma.
  • the target marker is the SOD3 gene or the SOD3 sequence of the genome, and is selected from the group consisting of the EMS2OS gene or the EMS2OS sequence of the genome, the TEAD3 gene or the TEAD3 sequence of the genome, the FTAP2B gene or the FTAP2B sequence of the genome, and the RP11-867G23.12 gene Or at least one or at least two of the RP11-867G23.12 sequences of the genome.
  • the target marker is the SOD3 gene or the SOD3 sequence of the genome and the EMS2OS gene or the EMS2OS sequence of the genome, and the TEAD3 gene or the TEAD3 sequence of the genome or the FTAP2B gene or the FTAP2B sequence of the genome; Methylation levels of these target markers in blood samples such as plasma.
  • the target marker is the SOD3 gene or genomic SOD3 sequence and FTAP2B gene or genomic FTAP2B sequence, and the EMS2OS gene or genomic EMS2OS sequence or RP11-867G23.12 gene or genomic RP11-867G23.12 sequence;
  • the methylation levels of these target markers are detected in a blood sample from the individual, such as plasma.
  • the target marker is TEAD3 gene or genome TEAD3 sequence, and selected from RP11-867G23.12 gene or genome RP11-867G23.12 sequence, SOD3 gene or genome SOD3 sequence and EMS2OS gene or genome EMS2OS At least one or at least two of the sequences; preferably, the target marker is the TEAD3 gene or the genome TEAD3 sequence and the RP11-867G23.12 gene or the genome RP11-867G23.12 sequence; preferably, the detection is from the The methylation levels of these target markers in individual thyroid nodules; preferably, the target markers are TEAD3 gene or genomic TEAD3 sequence, SOD3 gene or genomic SOD3 sequence and EMS2OS gene or genomic EMS2OS sequence; Preferably, the methylation levels of these target markers are detected in a blood sample from the individual, such as plasma.
  • the target marker is TTC34 gene or genomic TTC34 sequence, and selected from RCOR2 gene or genomic RCOR2 sequence, ITPKA gene or genomic ITPKA sequence, CDH1 gene or genomic CDH1 sequence, SLC16A3 gene or genomic SLC16A3 sequence and at least one, at least two or at least three of the RARG gene or genomic RARG sequence; preferably, the methylation level of these target markers in a blood sample such as plasma from the individual is detected.
  • the target marker is TTC34 gene or genomic TTC34 sequence and RCOR2 gene or genomic RCOR2 sequence, and selected from ITPKA gene or genomic ITPKA sequence, CDH1 gene or genomic CDH1 sequence, SLC16A3 gene or genomic SLC16A3 sequence and RARG gene or at least one or at least two of the RARG sequence of the genome; preferably, the methylation level of these target markers in a blood sample such as plasma from the individual is detected.
  • the target marker is the TTC34 gene or the TTC34 sequence of the genome, the RCOR2 gene or the RCOR2 sequence of the genome, the ITPKA gene or the ITPKA sequence of the genome, and the CDH1 gene or the CDH1 sequence of the genome; preferably, the blood from the individual is detected Methylation levels of these target markers in samples such as plasma.
  • the target marker is the TTC34 gene or the TTC34 sequence of the genome, the RCOR2 gene or the RCOR2 sequence of the genome, the SLC16A3 gene or the SLC16A3 sequence of the genome, and the RARG gene or the RARG sequence of the genome; preferably, the blood from the individual is detected Methylation levels of these target markers in samples such as plasma.
  • the target marker is TTC34 gene or genome TTC34 sequence and ITPKA gene or genome ITPKA sequence, and selected from RCOR2 gene or genome RCOR2 sequence, CDH1 gene or genome CDH1 sequence, SLC16A3 gene or genome SLC16A3 sequence and RARG gene or at least one or at least two of the RARG sequence of the genome; preferably, the methylation level of these target markers in a blood sample such as plasma from the individual is detected.
  • the target marker is the TTC34 gene or the TTC34 sequence of the genome, the ITPKA gene or the ITPKA sequence of the genome, the RCOR2 gene or the RCOR2 sequence of the genome, and the CDH1 gene or the CDH1 sequence of the genome; preferably, the blood from the individual is detected Methylation levels of these target markers in samples such as plasma;
  • the target marker is the TTC34 gene or the TTC34 sequence of the genome, the ITPKA gene or the ITPKA sequence of the genome, the SLC16A3 gene or the SLC16A3 sequence of the genome, and the RARG gene or the RARG sequence of the genome; preferably, the blood from the individual is detected Methylation levels of these target markers in samples such as plasma.
  • the target marker is TTC34 gene or genome TTC34 sequence and SLC16A3 gene or genome SLC16A3 sequence, and selected from RCOR2 gene or genome RCOR2 sequence, ITPKA gene or genome ITPKA sequence and RARG gene or genome RARG At least one or at least two of the sequences; preferably, the methylation levels of these target markers are detected in a blood sample, such as plasma, from the individual.
  • the target marker is the TTC34 gene or the TTC34 sequence of the genome, the SLC16A3 gene or the SLC16A3 sequence of the genome, the RCOR2 gene or the RCOR2 sequence of the genome, and the RARG gene or the RARG sequence of the genome; preferably, the blood from the individual is detected
  • the methylation levels of these target markers in samples such as plasma preferably, the target markers are the TTC34 gene or the TTC34 sequence of the genome, the SLC16A3 gene or the SLC16A3 sequence of the genome, the ITPKA gene or the ITPKA sequence of the genome and the RARG gene or The RARG sequence of the genome; preferably, the methylation levels of these target markers are detected in a blood sample, such as plasma, from the individual.
  • the target marker is the TTC34 gene or the genome's TTC34 sequence, the SLC16A3 gene or the genome's SLC16A3 sequence and the RARG gene or the genome's RARG sequence, and the RCOR2 gene or the genome's RCOR2 sequence or the ITPKA gene or the genome's ITPKA sequence; preferably , detecting the methylation levels of these target markers in a blood sample, such as plasma, from the individual.
  • the target marker is the MCC gene or the MCC sequence of the genome, and is selected from the ITPKA gene or the genome's ITPKA sequence, the RCOR2 gene or the genome's RCOR2 sequence, the SLC16A3 gene or the genome's SLC16A3 sequence, and the CDH1 gene or the genome's CDH1 at least one, at least two or at least three of the sequences; preferably, detecting the methylation levels of these target markers in a blood sample from the individual such as plasma; preferably, the target marker is MCC gene or genomic MCC sequence, ITPKA gene or genome ITPKA sequence, RCOR2 gene or genome RCOR2 sequence and SLC16A3 gene or genome SLC16A3 sequence; preferably, detecting the methylation level of these target markers in a blood sample such as plasma from the individual; preferably Preferably, the target markers are the MCC gene or the MCC sequence of the genome and the CDH1 gene or the CDH1 sequence of the genome; preferably, the
  • the target marker is CDH1 gene or genomic CDH1 sequence, and selected from MCC gene or genomic MCC sequence, TTC34 gene or genomic TTC34 sequence, RCOR2 gene or genomic RCOR2 sequence and ITPKA gene or genomic ITPKA At least one, at least two or at least three of the sequences; preferably, detecting the methylation level of these target markers in a blood sample from the individual such as plasma; preferably, the target marker is CDH1 gene or genomic CDH1 sequence and MCC gene or genome MCC sequence; preferably, detect the methylation level of these target markers in a blood sample from the individual such as plasma; preferably, the target marker is CDH1 gene or genome CDH1 sequence, TTC34 gene or genomic TTC34 sequence, RCOR2 gene or genomic RCOR2 sequence, and ITPKA gene or genomic ITPKA sequence; preferably, detecting the methylation levels of these target markers in a blood sample such as plasma from the individual.
  • the Hg19 coordinates of each gene are as follows:
  • the one or more target markers are one or more target regions amplified by each gene using the following primers: the target region of the CDH1 gene: the sequence shown in SEQ ID NO: 1 and 2 or with SEQ ID NO:1 and 2 have at least 90% identical sequence as the fragment of the CDH1 gene that primer amplifies;
  • the target region of TSHR gene the sequence shown in SEQ ID NO:3 and 4 or with SEQ ID NO:3 and 4 have at least 90% identical sequence as a fragment of the TSHR gene obtained by primer amplification;
  • the target region of the MCC gene the sequence shown in SEQ ID NO:5 and 6 or have at least 90% with SEQ ID NO:5 and 6
  • the sequence of % identity is used as a fragment of the MCC gene obtained by primer amplification;
  • the target region of TBX15 gene the sequence shown in SEQ ID NO:7 and 8 or a sequence with at least 90% identity with SEQ ID NO:7 and 8 A fragment of the TBX15 gene amplified as a primer;
  • the Hg19 coordinates of the target region of each gene are as follows: target region of CDH1 gene, chr16: 68770965-68771133; target region of TSHR gene, chr14: 81421913-81422134; target region of MCC gene, chr5: 112538899-112539205 Target region of TBX15 gene, chr1:119535640-119535860; Target region of PRR15 gene, chr7:29606122-29606390; Target region of DPYS gene, chr8:105478808-105479066; Target region of GRIA2 gene, chr4:1581415A-13814
  • the primer molecules of each gene are respectively: CDH1: SEQ ID NO: 1 and 2, or a primer molecule that hybridizes to the fragment obtained by its amplification under stringent conditions; TSHR: SEQ ID NO: 3 and 4, or Primer molecules that hybridize under stringent conditions to its amplified fragments; MCC: SEQ ID NOs: 5 and 6, or primer molecules that hybridize to its amplified fragments under stringent conditions; TBX15: SEQ ID NOs: 7 and 8 , or a primer molecule that hybridizes to its amplified fragment under stringent conditions; PRR15: SEQ ID NO: 9 and 10, or a primer molecule that hybridizes to its amplified fragment under stringent conditions; DPYS: SEQ ID NO: 11 and 12, or primer molecules that hybridize under stringent conditions to its amplified fragments; GRIA2: SEQ ID NO: 13 and 14, or primer molecules that hybridize to its amplified fragments under stringent conditions; NR5A1: SEQ ID NO : 15 and
  • the probe molecules of the amplification products of each gene are respectively: CDH1: SEQ ID NO: 41 or a sequence having at least 90% identity with it; TSHR: SEQ ID NO: 42 or a sequence with at least 90% identity therewith MCC: SEQ ID NO:43 or a sequence having at least 90% identity thereto; TBX15: SEQ ID NO:44 or a sequence having at least 90% identity thereto; PRR15: SEQ ID NO:45 or a sequence having at least 90% identity thereto A sequence with at least 90% identity; DPYS: SEQ ID NO:46 or a sequence with at least 90% identity therewith; GRIA2: SEQ ID NO:47 or a sequence with at least 90% identity therewith; NR5A1: SEQ ID NO: 48 or a sequence having at least 90% identity thereto; TTC34: SEQ ID NO: 49 or a sequence having at least 90% identity thereto; RCOR2: SEQ ID NO: 50 or a sequence having at least 90% identity thereto; F10: SEQ ID NO: 41
  • the detection reagent or diagnostic kit also includes primer molecules and/or probe molecules for detecting the internal reference gene ACTB, and the method further includes using the primer molecules for the internal reference gene ACTB And/or probe molecule;
  • the primer molecule of internal reference gene ACTB is the sequence shown in SEQ ID NO:61 and 62 and by SEQ ID NO:61 and the fragment of the ACTB gene that 62 obtains as primer amplification under stringent conditions
  • the probe of the internal reference gene ACTB comprises the sequence shown in SEQ ID NO: 63 or a sequence having at least 90% identity therewith.
  • the probe used also contains a detectable substance.
  • the detectable substance is a fluorescent reporter group at the 5' end and a labeling quencher group at the 3' end routinely used in the art.
  • the fluorescent reporter gene is selected from Cy5, Texas Red, FAM and VIC.
  • the detection reagent or diagnostic kit further includes one or more substances selected from the following: PCR buffer, polymerase, dNTP, restriction endonuclease, endonuclease Buffer, fluorescent dye, fluorescent quencher, fluorescent reporter, exonuclease, alkaline phosphatase, internal standard, control, KCl, MgCl 2 and (NH 4 ) 2 SO 4 .
  • the detection reagent or diagnostic kit further includes a converted positive standard, wherein unmethylated cytosine is converted into a base that does not bind to guanine.
  • the positive standard is fully methylated.
  • the reagent for detecting methylation is a reagent selected from one or more of the following methods: PCR based on bisulfite conversion (such as methylation-specific PCR ), DNA sequencing (eg, bisulfite sequencing, whole-genome methylation sequencing, simplified methylation sequencing), methylation-sensitive restriction endonuclease assays, fluorometric methods, methylation-sensitive high-resolution Rate melting curve method, chip-based methylation profiling, mass spectrometry (e.g. mass spectrometry of flight).
  • the reagent is selected from one or more of the following: bisulfite and its derivatives, fluorescent dye, fluorescent quencher, fluorescent reporter, internal standard, and control.
  • said individual or patient is a mammal, especially a human.
  • said sample is from a mammalian tissue, cell or body fluid, such as thyroid tissue or blood.
  • the sample is a thyroid nodule biopsy, preferably a fine needle aspiration biopsy.
  • the sample is plasma.
  • the sample is from a subject with benign or malignant thyroid nodules. In one or more embodiments, the sample is from a patient with goiter.
  • the DNA is whole genome DNA and/or cfDNA.
  • the DNA is converted wherein unmethylated cytosines are converted to bases that do not bind guanine.
  • the conversion is carried out using enzymatic methods, preferably deaminase treatment, or the conversion is carried out using non-enzymatic methods, preferably treatment with bisulfite or bisulfate.
  • the detection includes, but is not limited to: PCR based on bisulfite conversion (eg, methylation-specific PCR), fluorometric methods.
  • the detection is a real-time PCR detection, further comprising the use of the probes described herein.
  • the PCR reaction solution comprises Taq DNA polymerase, PCR buffer (buffer), dNTPs, KCl, MgCl 2 and (NH 4 ) 2 SO 4 .
  • the Taq DNA polymerase is a hot-start Taq DNA polymerase.
  • the final concentration of Mg 2+ is 1.0-20.0 mM.
  • the concentration of each primer in PCR is 100-500 nM.
  • the concentration of each probe in the PCR is 100-500 nM.
  • the PCR reaction conditions are: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 15 seconds, annealing and extension at 60°C for 60 seconds, 50 cycles.
  • the interpretation method is as follows: calculating the score according to the methylation level of each gene, if the score is greater than 0, the result is positive, that is, the thyroid nodule is a malignant nodule. In one or more embodiments, a score of less than 0 is considered negative, ie, the thyroid nodule is benign.
  • methylation level number of methylated bases/total number of bases.
  • the main advantage of the present invention is that the sensitivity and specificity of differentiating benign and malignant thyroid nodules are improved through simultaneous detection of multiple target sequences.
  • the operation is simple, the results are easy to interpret, the requirements for the instrument are not high, and the form of a kit is more convenient for clinical promotion and application.
  • Figure 1 CDH1, TSHR, MCC, TBX15, PRR15, DPYS, GRIA2, NR5A1, TTC34, RCOR2, F10, ITPKA, SLC16A3, RARG, FTAP2B, SOD3, RP11 in the tissue DNA of 10 cases of thyroid cancer and 10 cases of benign thyroid nodules - Methylation level map of 867G23.12, EMX2OS, TEAD3 and DGKG genes.
  • Figure 2 CDH1, TSHR, MCC, TBX15, PRR15, DPYS, GRIA2, NR5A1, TTC34, RCOR2, F10, ITPKA, SLC16A3, RARG, FTAP2B, SOD3 in tissue DNA of 10 patients with thyroid cancer and 10 patients with benign thyroid nodules , RP11-867G23.12, EMX2OS, TEAD3 and DGKG gene methylation levels of the ROC curve analysis chart.
  • Figure 3 Alpha expression of CDH1, TSHR, MCC, TBX15, PRR15, DPYS, GRIA2, NR5A1, TTC34, RCOR2, F10, ITPKA, SLC16A3 and RARG genes in plasma cfDNA of 196 patients with thyroid cancer and 148 patients with benign thyroid nodules Baseline level analysis chart.
  • Figure 4 Respective methylation of CDH1, TSHR, MCC, TBX15, PRR15, DPYS, GRIA2, NR5A1, TTC34, RCOR2, F10, ITPKA, SLC16A3 and RARG genes in plasma cfDNA of 196 patients with thyroid cancer and 148 patients with benign thyroid nodules
  • the ROC curve analysis chart of the level The ROC curve analysis chart of the level.
  • Figure 5 The methylation levels of FTAP2B, SOD3, RP11-867G23.12, EMX2OS, TEAD3 and DGKG genes in plasma cfDNA of 79 patients with thyroid cancer and 22 patients with benign thyroid nodules.
  • Figure 6 ROC curve analysis graphs of FTAP2B, SOD3, RP11-867G23.12, EMX2OS, TEAD3 and DGKG genes in plasma cfDNA of 79 patients with thyroid cancer and 22 patients with benign thyroid nodules.
  • Figure 7 ROC curve analysis chart of DPYS and RARG genes in the tissue DNA of 10 patients with thyroid cancer and 10 patients with benign thyroid nodules.
  • Figure 8 ROC curve analysis graph of TSHR and SLC16A3 genes in the tissue DNA of 10 patients with thyroid cancer and 10 patients with benign thyroid nodules.
  • Figure 9 ROC curve analysis graph of FTAP2B and EMX2OS genes in the tissue DNA of 10 patients with thyroid cancer and 10 patients with benign thyroid nodules.
  • Figure 10 ROC curve analysis graph of RP11-867G23.12 gene and TEAD3 gene in tissue DNA of 10 patients with thyroid cancer and 10 patients with benign thyroid nodules.
  • Figure 11 ROC curve analysis of TSHR and RARG genes in plasma cfDNA of 196 patients with thyroid cancer and 148 patients with benign thyroid nodules.
  • Figure 12 ROC curve analysis of MCC and CDH1 genes in plasma cfDNA of 196 patients with thyroid cancer and 148 patients with benign thyroid nodules.
  • Figure 13 ROC curve analysis of SOD3, EMX2OS and TRAD3 genes in plasma cfDNA of 79 patients with thyroid cancer and 22 patients with benign thyroid nodules.
  • Figure 14 ROC curve analysis graph of FTAP2B and DGKG genes in plasma cfDNA of 90 patients with thyroid cancer and 54 patients with benign thyroid nodules.
  • Figure 15 ROC curve analysis graph of TTC34 gene, RCOR2 gene, SLC16A3 gene and RARG gene in plasma cfDNA of 196 patients with thyroid cancer and 148 patients with benign thyroid nodules.
  • Figure 16 ROC curve analysis graph of SOD3 gene, EMX2OS gene and FTAP2B gene in plasma cfDNA of 90 patients with thyroid cancer and 54 patients with benign thyroid nodules.
  • Figure 17 ROC curve analysis chart of TTC34 gene, RCOR2 gene, ITPKA gene and CDH1 gene in plasma cfDNA of 196 patients with thyroid cancer and 148 patients with benign thyroid nodules.
  • Figure 18 ROC curve analysis graph of SOD3 gene, RP11-867G23.12 gene and FTAP2B gene in plasma cfDNA of 90 patients with thyroid cancer and 54 patients with benign thyroid nodules.
  • Figure 19 ROC curve analysis graph of ITPKA gene, RCOR2 gene, SLC16A3 gene and MCC gene in plasma cfDNA of 196 patients with thyroid cancer and 148 patients with benign thyroid nodules.
  • Figure 20 ROC curve analysis graph of TTC34 gene, ITPKA gene, SLC16A3 gene and RARG gene in plasma cfDNA of 196 patients with thyroid cancer and 148 patients with benign thyroid nodules.
  • BTN refers to patients with benign thyroid nodules
  • PTC refers to patients with thyroid cancer. * indicates p ⁇ 0.05, ** indicates p ⁇ 0.01, *** indicates p ⁇ 0.001.
  • target markers related to malignant thyroid nodules include: CDH1 gene or genomic CDH1 sequence, TSHR gene or genomic TSHR sequence, MCC gene or genomic MCC sequence, TBX15 gene or genome TBX15 sequence, PRR15 gene or genome PRR15 sequence, DPYS gene or genome DPYS sequence, GRIA2 gene or genome GRIA2 sequence, NR5A1 gene or genome NR5A1 sequence, TTC34 gene or genome TTC34 sequence, RCOR2 gene or genome RCOR2 sequence, F10 gene or genome F10 sequence, ITPKA gene or genome ITPKA sequence, SLC16A3 gene or genome SLC16A3 sequence, RARG gene or genome RARG sequence, FTAP2B gene or genome FTAP2B sequence, SOD3 gene Or the SOD3 sequence of the genome, the RP11-867G23.12 gene or the RP11-867G23.12 sequence of the genome, the EMX2OS gene
  • target marker refers to a target nucleic acid or gene region whose methylation level indicates benign or malignant nodular thyroid nodules.
  • the term "marker of interest” shall be considered to include all transcript variants thereof and all promoter and regulatory elements thereof.
  • certain genes are known to exhibit allelic variation or single nucleotide polymorphisms ("SNPs") between individuals. SNPs include insertions and deletions of simple repeats (eg, dinucleotide and trinucleotide repeats) of varying lengths. Accordingly, this application should be understood to extend to all forms of the marker/gene resulting from any other mutation, polymorphism or allelic variation.
  • target marker shall include both the sense strand sequence of the marker or gene and the antisense strand sequence of the marker or gene.
  • the term "marker of interest” as used herein is broadly interpreted to include both 1) the original marker (in a specific methylation state) found in a biological sample or genomic DNA, and 2) its processed sequence ( For example the corresponding area after bisulfite conversion or the corresponding area after MSRE treatment).
  • the bisulfite-converted corresponding region differs from the marker of interest in the genomic sequence by one or more unmethylated cytosine residues being converted to a uracil base, a thymine base, or Other bases that behave differently from cytosine.
  • the MSRE-treated corresponding region differs from the target marker in the genomic sequence by being cleaved at one or more MSRE cleavage sites.
  • the target markers involved in the products, uses and methods described herein are CDH1, TSHR, MCC, TBX15, PRR15, DPYS, GRIA2, NR5A1, TTC34, RCOR2, F10, ITPKA, SLC16A3 , RARG, FTAP2B, SOD3, RP11-867G23.12, EMX2OS, TEAD3 and DGKG genes can be described both by reference to their names and by their chromosomal coordinates. The chromosomal coordinates are consistent with the Hg19 version of the Human Genome Database released in February 2009 (referred to herein as "Hg19 coordinates"). It should be understood that the sequence of a gene and its genome described herein also includes fragments of each gene containing at least one CpG dinucleotide sequence. In some embodiments, the fragment is the target region of each gene described herein.
  • the target regions of each gene are:
  • the Hg19 coordinates of the gene and its target region are defined as follows:
  • the target marker of the present invention also includes 5 kb upstream of each start site and 5 kb downstream of each end site of each of the above regions.
  • Specific nucleotide sequences for the above Hg19 coordinates, as well as 5 kb upstream of each start site and 5 kb downstream of each end site for each region, are available in public databases such as UCSC Genome Browser, Ensemble, and the NCBI website.
  • the target marker of the present invention (such as a certain gene and its genome sequence, or a fragment of each gene containing at least one CpG dinucleotide sequence, or the target region, or a sequence comprising an intergenic region) also includes non- Corresponding regions after enzymatic transformations, such as bisulfite conversions, and corresponding regions obtained after enzymatic transformations, such as MSRE conversions.
  • the target markers of the present invention also include various variants of the above-mentioned genes or target regions.
  • Variants include at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to a gene or region described herein from the same region Nucleic acid sequences that are neutral (ie, have one or more deletions, insertions, substitutions, reverse sequences, etc.). Accordingly, the disclosure of this application should be understood to extend to such variants which achieve the same result, notwithstanding the fact that there are minor genetic variations in the actual nucleic acid sequence between individuals.
  • the term “percentage (%) of sequence identity” refers to the same percentage of the amino acid (or nucleic acid) residues of the candidate sequence and the amino acid (or nucleic acid) residues of the reference sequence after sequence alignment, when compared Spacers (if necessary) may be introduced to maximize the number of identical amino acids (or nucleic acids).
  • sequence identity percentage (%) of an amino acid sequence (or nucleic acid sequence) can be calculated by dividing the number of amino acid residues (or bases) identical to the reference sequence by the number of amino acid residues (or bases) in the candidate sequence or the reference sequence ) (whichever is shorter). Conservative substitutions of amino acid residues may or may not be considered to be the same residue.
  • the percent amino acid (or nucleic acid) sequence identity can be determined, for example, using published tools such as BLASTN, BLASTp (available on the website of the National Center for Biotechnology Information (NCBI), see also Altschul S.F. et al., J.Mol.Biol., 215:403–410 (1990); Stephen F. et al., Nucleic Acids Res., 25:3389–3402 (1997)), ClustalW2 (available at European Bioinformatics Research Institute website), see also Higgins D.G. et al., Methods in Enzymology, 266:383-402 (1996); Larkin M.A.
  • the target marker of the present invention also includes the above-mentioned gene or target region, or the corresponding region after non-enzymatic conversion (such as bisulfite conversion) of the 5kb upstream of the start site and 5kb downstream of the end site of the above-mentioned target region or Corresponding regions after enzymatic treatment (such as methylation-sensitive restriction enzyme treatment).
  • the target marker can be from any biological sample of an individual of interest.
  • the term "subject” includes humans and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals. “Subject” may also be a livestock such as cattle, pigs, sheep, poultry, and horses; or a rodent such as a rat, mouse; or a non-human primate such as an ape, monkey, rhesus monkey; or a domesticated Animals, such as dogs or cats.
  • the individual is a human or non-human primate.
  • the individual is a human. In this application, "individual”, “subject” and “subject” are used interchangeably.
  • sequences given in Section I above are human sequences.
  • the corresponding positions and corresponding sequences of the above-mentioned genes in the non-human animal genome can be easily determined by using existing technologies.
  • biological sample refers to a biological composition obtained or derived from an individual comprising cells and/or other molecular entities (such as DNA) to be characterized or identified based on physical, biochemical, chemical and/or physiological characteristics ).
  • a biological sample includes, but is not limited to, cells, tissues, organs and/or biological fluids of an individual obtained by any method known to those skilled in the art.
  • the biological sample is selected from the group consisting of histological sections, tissue biopsies, paraffin-embedded tissues, body fluids, surgical resection samples, isolated blood cells, cells isolated from blood, and any combination thereof.
  • the body fluid is selected from the group consisting of whole blood, serum, plasma, and any combination thereof.
  • the biological sample is whole blood of an individual.
  • the biological sample is plasma from an individual.
  • Various methods of preparing plasma from whole blood are known to those skilled in the art.
  • plasma is obtained by centrifuging whole blood from an individual one, two, three, four, five or more times.
  • the biological sample is a thyroid nodule biopsy, preferably a fine needle aspiration biopsy.
  • the DNA to be detected can be isolated from said biological sample.
  • the DNA to be detected can be isolated and purified from a biological sample by using various methods known in the art. Isolation and purification can be performed using commercially available kits. For example, DNA is isolated from cells and tissues by lysis of raw materials under highly denaturing and reducing conditions, partial use of protein-degrading enzymes, purification of nucleic acid fractions obtained by phenol/chloroform extraction processes, and separation from water by dialysis or ethanol precipitation. Nucleic acids are recovered in phase (see for example Sambrook, J., Fritsch, E.F. in T. Maniatis, CSH, Molecular Cloning, 1989).
  • reagent systems that are particularly suitable for the purification of DNA fragments from agarose gels, the isolation of plasmid DNA from bacterial lysates, and the isolation of longer chains of nucleic acids (genomic DNA, total cellular RNA).
  • Many of these commercially available purification systems are based on the fairly well-known principle of binding nucleic acids to mineral supports in the presence of solutions of various chaotropic salts. In these systems, suspensions of finely ground glass powder, diatomaceous earth or silica gel are used as carrier material.
  • Some other methods of isolating and purifying DNA from biological samples are described in eg US7888006B2 and EP1626085A1. Choosing between methods will be influenced by several factors, including time, expense, and the amount of DNA required.
  • the DNA contained in the biological sample comprises genomic DNA.
  • genomic DNA refers to DNA comprising the complete genome of a cell or organism as well as fragments or parts thereof.
  • Genomic DNA is a large stretch of DNA (e.g., longer than about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, or 300 kb) derived from an individual and may have natural modifications, such as DNA methylation .
  • the DNA contained in the biological sample includes cellular DNA.
  • cellular DNA refers to DNA present in a cell, or DNA obtained from a cell in vivo and isolated in vitro, or otherwise manipulated in vitro, so long as the DNA is not removed from the cell in vivo.
  • the DNA contained in the biological sample includes cell-free extracellular DNA.
  • the term "free extracellular DNA” as used herein refers to DNA fragments present outside cells in vivo. The term may also be used to refer to a segment of DNA obtained from an extracellular source in vivo and isolated, or manipulated in vitro. DNA fragments in extracellular free DNA usually have a length of about 100 to 200 bp, presumably related to the length of DNA fragments wrapped in nucleosomes.
  • Cell-free extracellular DNA includes, for example, cell-free extracellular fetal DNA and circulating tumor DNA.
  • cell-free extracellular fetal DNA circulates in the body (eg, blood) of pregnant women and represents the fetal genome, whereas circulating tumor DNA circulates in the body (eg, blood) of cancer patients.
  • cell-free extracellular DNA can be substantially free of the individual's cellular DNA.
  • the cell-free extracellular DNA can comprise less than about 1,000 ng/mL, less than about 100 ng/mL, less than about 10 ng/mL, less than about 1 ng/mL of cellular DNA.
  • Extracellular episomal DNA can be prepared by using conventional techniques known in the art. For example, blood samples can be centrifuged at about 200-20,000 g, about 200-10,000 g, about 200-5,000 g, about 300-4000 g, etc., for about 3-30 minutes, about 3-15 minutes, about 3-10 minutes , about 3-5 minutes to obtain the extracellular DNA of the blood sample.
  • cell-free extracellular DNA from a blood sample can be obtained by centrifuging the individual's plasma or serum one, two, three, four, five or more times.
  • the biological sample may be obtained by microfiltration in order to separate cells and fragments thereof from cell-free fractions comprising soluble DNA.
  • microfiltration can be performed by using a filter, for example, a 0.1-0.45 micron membrane filter, such as a 0.22 micron membrane filter.
  • cell-free extracellular DNA is extracted from whole blood, serum, or plasma for analysis using commercially available DNA extraction products.
  • This extraction method is reported to provide high recovery (>50%) of circulating DNA, and some products (such as the QIAamp Circulating Nucleic Acid Kit from Qiagen) are reported to extract DNA fragments of small size.
  • Typical sample volumes used are 1-5 mL of serum or plasma.
  • cell-free extracellular DNA includes circulating tumor DNA.
  • Circulating tumor DNA (“ctDNA”) is tumor-derived fragmented DNA in body fluids (eg, blood, urine, saliva, sputum, stool, pleural fluid, cerebrospinal fluid, etc.) that are not associated with cells.
  • body fluids eg, blood, urine, saliva, sputum, stool, pleural fluid, cerebrospinal fluid, etc.
  • ctDNA is highly fragmented, with an average length of approximately 150 base pairs.
  • ctDNA typically comprises a very small fraction of cell-free extracellular DNA in bodily fluids such as plasma, eg ctDNA may constitute less than about 10% of plasma DNA. Typically, this percentage is less than about 1%, such as less than about 0.5% or less than about 0.01%.
  • the total amount of plasma DNA is usually very low, eg, about 10 ng/mL plasma.
  • the amount of ctDNA varies from person to person and depends on the type of tumor, its location and, in the case of cancerous tumors, the stage of the cancer.
  • ctDNA is usually very rare in body fluids and can only be detected by extremely sensitive and specific techniques. Detection of ctDNA may be useful in detecting and diagnosing tumors, guiding tumor-specific therapy, monitoring therapy, and monitoring cancer remission.
  • DNA methylation is the biological process of adding a methyl group to a DNA molecule (eg, to one or more cytosine bases of the DNA molecule) (eg, by the action of a DNA methyltransferase).
  • DNA methylation occurs at the 5' position of cytosine-phosphate-guanine (CpG) dinucleotide (ie, "CpG site"), when it occurs at the promoter or first When present in the 5'-CpG-3' dinucleotide in the exon, it can lead to epigenetic inactivation of the gene. It has been well documented that DNA methylation plays an important role in regulating gene expression, tumorigenesis, and other genetic and epigenetic diseases.
  • methylated cytosine residue refers to a derivative of a cytosine residue in which a methyl group is attached to a carbon atom of the cytosine ring (e.g. C5).
  • unmethylated cytosine residue refers to an underivatized cytosine residue in which, in contrast to "methylated cytosine residue", there is no Methyl linkage.
  • a CpG site in which cytosine residues are methylated is a methylated CpG site, and a CpG site in which cytosine residues are not methylated is an unmethylated CpG site .
  • conversions can occur between bases of DNA or RNA.
  • Conversion refers to the use of non-enzymatic or enzymatic methods to treat DNA to convert unmodified cytosine bases (cytosine, C) into guanine (G ) combined base (such as uracil base (uracil, U)) process.
  • cytosine, C unmodified cytosine bases
  • G guanine
  • uracil base uracil, U
  • Some reagents are able to distinguish between unmethylated and methylated CpG sites in DNA, resulting in processed DNA. This reagent acts selectively on unmethylated cytosine residues but not significantly on methylated cytosine residues.
  • the reagent may act selectively on methylated cytosine residues but not significantly on unmethylated cytosine residues.
  • some reagents can selectively convert unmethylated cytosine residues to uracil, thymine, or another base that hybridizes differently from cytosine, while methylated cytosine residues remain unmethylated. Transformation state; as another example, some reagents can selectively cleave methylated residues, or selectively cleave unmethylated residues.
  • the original DNA is converted into processed DNA in a manner dependent on whether it is methylated or not, so that the processed DNA can be distinguished from the original DNA by its hybridization behavior.
  • processed DNA refers to CpG sites that have been treated to distinguish between unmethylated and methylated DNA, nucleic acid sequences, gene fragments DNA, nucleic acid sequences, and gene fragments treated with the spot reagents.
  • cytosine conversion can be performed using non-enzymatic or enzymatic methods.
  • non-enzymatic methods include bisulfite or bisulfate treatment.
  • reagents used in non-enzymatic methods include bisulfite reagents.
  • bisulfite reagent refers to, for example, those disclosed herein that can be used to distinguish between methylated and unmethylated CpG dinucleotide sequences, including bisulfite, bisulfite, ions or any combination thereof.
  • the treatment of DNA with a bisulfite reagent is also described as a "bisulfite reaction” or “bisulfite treatment” and refers to a reaction that converts unmethylated cytosine residues, especially is the conversion of unmethylated cytosine residues in nucleic acids to uracil bases, thymine bases, or other bases that differ in hybridization behavior from cytosine in the presence of bisulfite ions, while Therein methylated cytosine residues were not significantly converted.
  • bisulfite treatment can be used to distinguish methylated CpG dinucleotides from unmethylated CpG dinucleotides.
  • methylated cytosine residues are not significantly converted does not exclude very small percentages (e.g., less than 0.1%, less than 0.2%, less than 0.3%, less than 0.4%, less than 0.5%, less than 0.6 %, less than 0.7%, less than 0.8%, less than 0.9%, less than 1%, less than 2%, less than 3%, less than 4%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, Less than 10%, less than 11%, less than 12%, less than 13%, less than 14%, less than 15%, less than 16%, less than 17%, less than 18%, less than 19%, less than 20%) of methylated cells Pyrimidine residues are converted to uracil, thymine, or other bases that hybridize differently from cytosine, although it is intended that only unmethylated cytosine residues be converted.
  • very small percentages e.g., less than 0.1%, less than 0.2%, less than 0.3%, less than
  • the bisulfite reagent is selected from the group consisting of ammonium bisulfite, sodium bisulfite, potassium bisulfite, calcium bisulfite, magnesium bisulfite, aluminum bisulfite, sulfurous acid Hydrogen ions, and any combination thereof.
  • the bisulfite reagent is sodium bisulfite.
  • bisulfite reagents are commercially available, eg, MethylCode TM Bisulfite Conversion Kit, EpiMark TM Bisulfite Conversion Kit, EpiJET TM Bisulfite Conversion Kit, EZDNAMethylation-Gold TM Kit, and the like.
  • the bisulfite reaction is performed according to the kit's instructions.
  • Exemplary enzymatic methods include deaminase treatment, and the use of reagents that selectively cleave unmethylated residues but not methylated residues, or selectively cleave methylated residues but not cleave Unmethylated residues.
  • the reagent is a methylation sensitive restriction enzyme (MSRE).
  • methylation-sensitive restriction enzyme refers to an enzyme that selectively digests a nucleic acid based on the methylation status of its recognition site. For restriction enzymes that specifically cleave when the recognition site is unmethylated or hemimethylated, when the recognition site is methylated, cleavage does not occur, or cleaves at a significantly reduced efficiency . For restriction enzymes that specifically cleave when the recognition site is methylated, when the recognition site is not methylated, cleavage does not occur, or cleaves at a significantly reduced efficiency.
  • the recognition sequence for a methylation sensitive restriction enzyme contains a CG dinucleotide (eg, cgcg or cccggg). In some embodiments, when the cytosine in the CG dinucleotide is methylated at the C5 carbon atom, the methylation-sensitive restriction enzyme does not cleavage.
  • Exemplary MSREs are selected from the group consisting of HpaII enzymes, SalI enzymes, ScrFI enzyme, BbeI enzyme, NotI enzyme, SmaI enzyme, XmaI enzyme, MboI enzyme, BstBI enzyme, ClaI enzyme, MluI enzyme, NaeI enzyme, Narl enzyme, PvuI enzyme, SacII enzyme, HhaI enzyme, and any combination thereof.
  • a methylation-sensitive restriction enzyme that distinguishes between methylated and unmethylated CpG dinucleotides in the region of interest or that includes a methylation-sensitive restriction enzyme
  • a range of restriction enzyme reagents can be used to determine methylation, such as, but not limited to, differential methylation hybridization ("DMH").
  • DNA in a biological sample can be cleaved prior to treatment with a methylation-sensitive restriction enzyme.
  • a methylation-sensitive restriction enzyme Such methods are known in the art and may include both physical and enzymatic means. It is particularly preferred to use one or more restriction enzymes which are insensitive to methylation and whose recognition sites are AT-rich and do not contain CG dinucleotides. The use of such enzymes results in the preservation of CpG sites and CpG-rich regions in DNA fragments.
  • such restriction enzymes are selected from the group consisting of MseI enzyme, BfaI enzyme, Csp6I15 enzyme, Trull enzyme, Tru9I enzyme, MaeI enzyme, XspI enzyme, and any combination thereof.
  • the transformed DNA is optionally purified.
  • DNA purification methods suitable for use herein are well known in the art.
  • the methylation status or methylation level is used to distinguish benign from malignant thyroid nodules.
  • the detection reagent and diagnostic kit of the present invention can be used for the detection of the methylation state or methylation level.
  • the "benign” and “malignant” denote properties of thyroid nodules.
  • benign nodules grow slowly, have uniform texture, good mobility, smooth surface, cystic changes, no lymphadenopathy, and no calcification.
  • Malignancy manifests as uncontrolled growth, spread, and tissue infiltration of malignant cells.
  • Ultrasonographic signs that suggest a malignant thyroid nodule include: nodules that are taller than wide, lack of halos, microcalcifications, irregular borders, hypoechoic, solid nodules, and rich blood flow within the nodules.
  • the malignant thyroid nodule comprises thyroid cancer.
  • methylation status refers to the presence or absence of one or more methylated nucleotide bases in a nucleic acid molecule.
  • a nucleic acid molecule that contains methylated cytosines is considered methylated (eg, the methylation status of the nucleic acid molecule is methylated).
  • a nucleic acid molecule that does not contain any methylated nucleotides is considered unmethylated.
  • a nucleic acid may be characterized as "unmethylated” if it is not methylated at a particular locus (e.g., a locus of a particular single CpG dinucleotide) or a particular combination of loci, even if It is methylated at other loci of the same gene or molecule as well.
  • the methylation state describes the state of methylation of a nucleic acid (eg, a genomic sequence or a marker of interest as described herein).
  • methylation status refers to a characteristic of a nucleic acid segment at a particular genomic locus that is associated with methylation. Such characteristics include, but are not limited to, whether any cytosine (C) residues within the DNA sequence are methylated, the location of one or more methylated C residues, methylation throughout any particular region of the nucleic acid Frequency or percentage of C and allelic differences in methylation due to, for example, differences in allelic origin.
  • C cytosine
  • Methods refers to the relative concentration, absolute concentration or pattern of methylated C or unmethylated C throughout any particular region of nucleic acid in a biological sample. For example, if one or more cytosine (C) residues within a nucleic acid sequence are methylated, it may be said to be “hypermethylated” or have “increased methylation”, whereas if within the DNA sequence One or more of the cytosine (C) residues is unmethylated, it can be said to be “demethylated” or have “reduced methylation”.
  • cytosine (C) residues within a nucleic acid sequence are methylated compared to another nucleic acid sequence (e.g., from a different region or from a different individual, etc.), the sequence is considered to be different from the other The nucleic acid sequence is hypermethylated or has increased methylation compared to the nucleic acid sequence.
  • one or more cytosine (C) residues within a DNA sequence are unmethylated compared to another nucleic acid sequence (e.g., from a different region or from a different individual, etc.)
  • the sequence is considered to be different from the other
  • the nucleic acid sequence is demethylated or has reduced methylation compared to.
  • the methylation level represents the proportion (or percentage, fraction, ratio, degree) of one or more sites in the methylation state.
  • the methylation level of a region (or group of sites) is the average of the methylation levels of all sites in the region (or all sites in the group). Therefore, an increase or decrease in the methylation level of a region does not mean that the methylation level of all methylated sites in the region is increased or decreased.
  • the process of converting the results obtained by methods for detecting DNA methylation (such as simplified methylation sequencing) into methylation levels is known in the art. Methylation levels can be determined, for example, by quantitative analysis of the amount of intact DNA present after restriction digestion with a methylation-sensitive restriction enzyme.
  • the methylation level as in the above example can be used as a quantitative indicator of methylation status. This is especially useful when the methylation levels of sequences in a sample need to be compared to a threshold level.
  • the methylation level (eg, Ct value) of a marker of interest is increased or decreased when compared to a reference level.
  • the methylation marker level (eg, Ct value) meets a certain threshold, the thyroid nodule is identified as malignant.
  • a mathematical analysis of the methylation levels of the target markers can be performed to obtain a score. For the detected samples, when the score is greater than or less than the threshold, the result is determined to be positive, that is, the thyroid nodule is malignant.
  • SVM support vector machine
  • a support vector machine is constructed for the training group samples, and the accuracy, sensitivity and specificity of the test results are calculated using the model, as well as the area under the characteristic curve (ROC) (AUC) of the predicted value, Statistical test set sample prediction scores.
  • the methylation level/state of one or more CpG dinucleotide sequences within a DNA sequence can be determined by various analytical methods known in the art, preferably quantitative analytical methods.
  • Exemplary assays include: polymerase chain reaction, including real-time polymerase chain reaction, digital polymerase chain reaction, and bisulfite conversion-based PCR (e.g., methylation-specific PCR , MSP)); nucleic acid sequencing; genome-wide methylation sequencing; simplified methylation sequencing; mass-based separation (e.g., electrophoresis, mass spectrometry); target capture (e.g., hybridization, microarray); methylation-sensitive constraints endonuclease assays; methylation-sensitive high-resolution melting curve assays; chip-based methylation profiling; mass spectrometry; and fluorometric assays.
  • detection includes detection of either strand at a gene or locus.
  • quantitative analysis is performed by real-time PCR.
  • real-time PCR include HeavyMethyl TM PCR described by Cottrell et al., Nucl. Acids Res. 32:e10, 2003; MethyLight TM PCR described by Eads et al., Cancer Res. 59:2302-2306, 1999; Headloop PCR as described by Rand et al., Nucl. Acids Res. 33:e 127, 2005.
  • HeavyMethyl TM PCR refers to an art-recognized real-time PCR technique in which one or more non-extendable nucleic acid (e.g., oligonucleotide) blockers are combined with subgroups in a methylation-specific manner.
  • Bisulfate-treated nucleic acids bind (ie, the blocker binds specifically to unmutated DNA under conditions of moderate to high stringency).
  • the amplification reaction is carried out using one or more primers which may optionally be methylation specific but flanked by one or more blockers.
  • the blocker binds and no PCR product is produced.
  • the level of methylation of nucleic acids in a sample is determined using a TaqMan TM assay essentially as described, eg, by Holland et al., Proc. Natl. Acad. Sci. USA, 88:7276-7280, 1991.
  • Methods of Methods of Methods of Methods of Methods of Methods of Methods of Methods of Methods of Methods refers to an art-recognized fluorescence-based real-time PCR technique in which dual-labeled fluorescent oligonucleotide probes called TaqMan TM probes are employed and designed to Hybridizes to CpG-rich sequences located between forward and reverse amplification primers.
  • the TaqMan (TM) probes comprise a fluorescent "reporter moiety” and "quencher moiety” covalently bound to a linker moiety (eg, phosphoramidite) attached to the nucleotide of the TaqMan (TM) oligonucleotide.
  • linker moiety eg, phosphoramidite
  • TaqMan TM probes that hybridize to CpG-rich sequences are cleaved by the 5' nuclease activity of Taq polymerase, resulting in a signal that is detected in real-time during the PCR reaction.
  • molecular beacons can be used as detectable probes, and the system is independent of the 5'-3' exonuclease activity of the DNA polymerase used (see Mhlanga and Malmberg, Methods 25: 463-471, 2001).
  • Headloop PCR refers to an art-recognized type of real-time PCR that selectively amplifies a target nucleic acid, but suppresses non-enzymatic activity by extending the 3' stem-loop to form a hairpin that does not provide further template for amplification. Amplify the amplification of the variant of interest.
  • the real-time PCR is multiplex real-time PCR.
  • the term “multiplex” may refer to the use of more than one marker, each having at least one distinct detection characteristic, such as a fluorescence characteristic (e.g., excitation wavelength, emission wavelength, emission intensity, FWHM (half maximum Full width at height) or fluorescence lifetime) or unique nucleic acid or protein sequence characteristics, assays or other analytical methods that can simultaneously determine the presence and/or amount of multiple markers (eg, multiple nucleic acid sequences).
  • a fluorescence characteristic e.g., excitation wavelength, emission wavelength, emission intensity, FWHM (half maximum Full width at height) or fluorescence lifetime
  • unique nucleic acid or protein sequence characteristics e.g., assays or other analytical methods that can simultaneously determine the presence and/or amount of multiple markers (eg, multiple nucleic acid sequences).
  • nucleic acid sequencing is performed by nucleic acid sequencing.
  • Exemplary methods of nucleic acid sequencing are known in the art, see, e.g., Frommer et al., Proc. Natl. Acad. Sci. USA 89:1827-1831, 1992; Clark et al., Nucl. Acids Res. 22: 2990-2997,1994.
  • comparison of the sequence obtained from a sample that was not treated with bisulfite or the known nucleotide sequence of the target region with the sequence obtained from a sample that was treated with bisulfite helps to identify methyl groups in the DNA sequence.
  • Cytosine Thymine residues detected at any cytosine site in bisulfite-treated samples compared to untreated samples can be considered mutations caused by bisulfite treatment, i.e., the presence of Methylated cytosine.
  • Methods for sequencing DNA include, for example, the dideoxy chain termination method or the Maxam-Gilbert method (see Sambrook et al., Molecular Cloning, A Laboratory Manual ( 2nd Ed., CSHP, New York 1989) ), pyrosequencing (seeing Uhlmann et al., Electrophoresis, 23:4072-4079, 2002), solid-phase pyrosequencing (seeing Landegren et al., Genome Res., 8(8):769-776, 1998) , solid-phase microsequencing (see, for example, Southern et al., Genomics, 13:1008-1017, 1992), microsequencing using FRET (see, for example, Chen and Kwok, Nucleic Acids Res. 25:347-353, 1997) , sequencing by ligation or ultra-deep sequencing (see Marguiles et al., Nature 437(7057):376-80(2005)).
  • quantitative analysis is performed by mass-based separation (eg, electrophoresis, mass spectrometry).
  • mass-based separation eg, electrophoresis, mass spectrometry
  • COBRA combined bisulfite restriction analysis
  • This method utilizes the presence of restriction enzymes between methylated and unmethylated nucleic acids following treatment with compounds that selectively mutate unmethylated cytosine residues (e.g., bisulfite) Identify differences in loci.
  • the restriction endonuclease Taq1 cuts the sequence TCGA, which after bisulfite treatment of unmethylated nucleic acids will be TTGA and thus will not be cut. Digested and/or undigested nucleic acids are then detected using detection means known in the art, such as electrophoresis and/or mass spectrometry.
  • detection means known in the art such as electrophoresis and/or mass spectrometry.
  • different techniques are used to detect nucleic acid differences in amplified products based on differences in nucleotide sequence and/or secondary structure after treatment with compounds that selectively mutate unmethylated cytosine residues, such as formazan Methylation-specific single-strand conformation analysis (MS-SSCA) (Bianco et al., Hum.
  • MS-SSCA formazan Methylation-specific single-strand conformation analysis
  • MS-DGGE methylation-specific denaturing gradient gel electrophoresis
  • MS-DHPLC methylation-specific denaturing high-performance liquid chromatography
  • target capture eg, hybridization, microarray
  • Suitable detection methods by hybridization are known in the art, such as Southern, dot blot, slot blot or other means of nucleic acid hybridization (Kawai et al., Mol. Cell. Biol. 14:7421-7427, 1994; Gonzalgo et al. al., Cancer Res. 57:594-599, 1997).
  • probes for hybridization analysis are detectably labeled.
  • nucleic acid-based probes used in hybridization assays are unlabeled.
  • Such unlabeled probes can be immobilized on a solid support, such as a microarray, and can hybridize to detectably labeled target nucleic acid molecules.
  • a microarray is a methylation-specific microarray, which can be used to distinguish sequences with converted cytosine residues from sequences with non-converted cytosine residues (see Adorjan et al., Nucl. Acids Res. , 30:e21, 2002).
  • Hybridization-based analysis can also be used on nucleic acids after treatment with methylation-sensitive restriction enzymes.
  • the methylation status of CpG dinucleotide sequences within a DNA sequence can be determined by oligonucleotide probes that hybridize to bisulfite-treated DNA simultaneously with PCR amplification primers (wherein the primers may be methylation-specific primers or standard primers).
  • detection reagent is a reagent used to detect the presence, absence or amount of nucleic acid in a quantitative analysis step.
  • detection reagent is selected from the group consisting of fluorescent probes, intercalating dyes, chromophore-labeled probes, radioisotope-labeled probes, and biotin-labeled probes.
  • exemplary probe sequences are as follows:
  • CDH1 gene probe CGCCCACCCGACCTCGCAT (SEQ ID NO: 41);
  • TSHR gene probe ACAACACCAACTACAACAAATCCGCCGA (SEQ ID NO: 42);
  • TBX15 gene probe ACCCTACTCCTACGCAAACCGAAAT (SEQ ID NO: 44);
  • PRR15 gene probe CCTCCGAAAACAACGTAACGCGC (SEQ ID NO: 45);
  • DPYS gene probe CGAAAACATCGACACACACACGCA (SEQ ID NO: 46);
  • GRIA2 gene probe AAAACGCTTCGCCGCCAACA (SEQ ID NO: 47);
  • NR5A1 gene probe AAACGCTACGCGAAACGCTC (SEQ ID NO: 48);
  • TTC34 gene probe CGAACCGCAACAAACGCTCG (SEQ ID NO: 49);
  • RCOR2 gene probe CCGACTCGCGCCAAACTCGA (SEQ ID NO:50);
  • F10 gene probe CAAACAACGCGACCTCTAAACGC (SEQ ID NO:51);
  • ITPKA gene probe ACGCTAAAATCACCTTCACTACGCC (SEQ ID NO:52);
  • SLC16A3 gene probe ATGTAAGCGGATATAGAGCGGTAGGGTA (SEQ ID NO: 53);
  • RARG gene probe CGCAACCACGCAAAAACACACGC (SEQ ID NO:54);
  • FTAP2B gene probe CGAGTCGTCGTATACGGTTTCGGG (SEQ ID NO: 55);
  • SOD3 gene probe CGAACCCGAACTCTAAAAACGCCAAACG (SEQ ID NO:56);
  • RP11-867G23.12 gene probe CGGTGTTGTCGGTTGTTTTTTACGTACG (SEQ ID NO: 57);
  • EMX2OS gene probe CCAAAACGTACACCGACTCCTAAATTCC (SEQ ID NO:58);
  • TEAD3 gene probe TGGTTGCGGTATCGGATTATTTATACGG (SEQ ID NO: 59);
  • control marker is ACTB
  • nucleotide sequence of its exemplary probe is shown in SEQ ID NO: 63: ACCACCACCACCCAACACACAATAAACAAACACA.
  • Fluorescent probes are usually labeled at the 5' end with a fluorescent dye (such as FAM, HEX/VIC, TAMRA, Texas Red, or Cy5) and at the 3' end with a quencher (such as BHQ1, BHQ2, BHQ3, DABCYL, or TAMRA).
  • a fluorescent dye such as FAM, HEX/VIC, TAMRA, Texas Red, or Cy5
  • a quencher such as BHQ1, BHQ2, BHQ3, DABCYL, or TAMRA
  • Labeling can be done by direct or indirect methods. Direct labeling involves coupling a label directly (covalently or non-covalently) to a reagent. Indirect labeling involves the binding (covalent or non-covalent) of a second reagent to a first reagent. The second reagent should specifically bind to the first reagent. The second reagent may be coupled to a suitable label and/or the second reagent may be a target (receptor) for a third reagent that binds to the second reagent. Using secondary, tertiary or even higher order reagents will usually increase the signal intensity. Suitable secondary and advanced reagents may include antibodies, secondary antibodies and the well known streptavidin-biotin system (Vector Laboratories, Inc.). A reagent or substrate can also be "labeled" with one or more tags known in the art.
  • CDH1 gene probe Texas Red-CGCCCACCCGACCTCGCAT-BHQ2;
  • TSHR gene probe FAM-ACAACACCAACTACAACAAATCCGCCGA-BHQ1;
  • MCC gene probe Cy5-CCTACCGCACGCCTATTCAATAACCT-BHQ1;
  • TBX15 gene probe Texas Red-ACCCTACTCCTACGCAAACCGAAAT-BHQ2;
  • PRR15 gene probe FAM-CCTCCGAAAACAACGTAACGCGC-BHQ1;
  • DPYS gene probe Cy5-CGAAAACATCGACACACACACGCA-BHQ1;
  • GRIA2 gene probe Texas Red-AAAACGCTTCGCCGCCAACA-BHQ2;
  • NR5A1 gene probe FAM-AAACGCTACGCGAAACGCTC-BHQ1;
  • TTC34 gene probe Cy5-CGAACCGCAACAAACGCTCG-BHQ1;
  • RCOR2 gene probe Texas Red-CCGACTCGCGCCAAACTCGA-BHQ2;
  • F10 gene probe FAM-CAAACAACGCGACCTCTAAACGC-BHQ1;
  • ITPKA gene probe Cy5-ACGCTAAAATCACCTTCACTACGCC-BHQ1;
  • SLC16A3 gene probe Texas Red-ATGTAAGCGGATATAGAGCGGTAGGGTA-BHQ2;
  • RARG gene probe FAM-CGCAACCACGCAAAAACACACGC-BHQ1;
  • FTAP2B gene probe Cy5-CGAGTCGTCGTATACGGTTTCGGG-BHQ1;
  • SOD3 gene probe Texas Red-CGAACCCGAACTCTAAAAACGCCAAACG-BHQ2;
  • RP11-867G23.12 gene probe FAM-CGGTGTTGTCGGTTGTTTTTTACGTACG-BHQ1;
  • EMX2OS gene probe Cy5-CCAAAACGTACACCGACTCCTAAATTCC-BHQ1;
  • TEAD3 gene probe Texas Red-TGGTTGCGGTATCGGATTATTTATACGG-BHQ2;
  • ACTB gene probe VIC-ACCACCACCCAACACACAATAACAAACACA-BHQ1.
  • the quantitative analysis comprises amplifying the treated DNA using a quantitative primer pair and a DNA polymerase.
  • the term "quantitative primer pair" refers to one or more primer pairs used in a quantitative analysis step.
  • the quantitative primer pair is capable of hybridizing to at least 9 consecutive nucleotides of the processed DNA under stringent conditions, moderately stringent conditions or highly stringent conditions.
  • the quantitative analysis comprises determining the concentration of one or more markers of interest based on the presence or level of a plurality of CpG dinucleotides, TpG dinucleotides, or CpA dinucleotides in the processed DNA. methylation levels. In some embodiments, the quantitative analysis comprises determining the level of methylation of cytosine residues based on the presence or level of one or more CpG dinucleotides in the processed DNA. In some embodiments, said quantitative analysis comprises determining the level of methylation of cytosine residues based on the presence or level of one or more TpG dinucleotides in said processed DNA. In some embodiments, said quantitative analysis comprises determining the level of methylation of cytosine residues based on the presence of CpA dinucleotides in said processed DNA.
  • the quantifying step is performed by separating the processed DNA product into fractions.
  • a plurality of different quantitative assays are performed on a plurality of fractions, wherein quantification of said processed DNA product (if present in said fraction) is performed in one of the plurality of fractions. different combinations.
  • the control markers in each fraction are quantified.
  • the methylation level of each marker of interest is quantified separately based on the pre-amplified DNA by using MSP (see Herman, supra). For example, by using one or more primers that specifically hybridize to non-transformed sequences under conditions of moderate and/or high stringency, amplification products are generated only when the template contains methylated cytosines at CpG sites.
  • the quantitative primer pair is designed to amplify at least a portion of the processed DNA product, ie the quantitative analysis is designed as a nested PCR.
  • Nested PCR is a modification of PCR designed to increase sensitivity and specificity. Nested PCR involves the use of two primer sets and two consecutive PCR reactions. A first round of amplification is performed to generate the first amplicon, and a second round of amplification is performed using a primer pair where one or both primers anneal to a site within the region bounded by the initial primer pair, i.e. the second A primer pair is said to be "nested" within the first primer pair. In this way, background amplification products from the first PCR reaction that do not contain the correct internal sequence are not further amplified in the second PCR reaction.
  • the PCR reaction solution includes Taq DNA polymerase, PCR buffer, primers, probes, dNTPs, and Mg 2+ .
  • the Taq DNA polymerase is a hot-start Taq DNA polymerase.
  • the final concentration of Mg 2+ is 1.0-20.0 mM; the concentration of each primer is 100-500 nM; the concentration of each probe is 100-500 nM.
  • Exemplary PCR reaction conditions are: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 15s, annealing and extension at 60°C for 60s, 50 cycles.
  • the methods of the invention include a pre-amplification step.
  • One of the purposes of preamplifying a marker of interest is to increase the amount of the marker of interest in the processed DNA.
  • the term “amplification” refers generally to any process capable of resulting in an increase in the copy number of a molecule or group of related molecules.
  • “Amplification” when applied to a polynucleotide molecule refers to the production of multiple copies of a polynucleotide molecule, or multiple copies of a portion of a polynucleotide molecule, usually starting from a small number of polynucleotides, wherein the amplified substance ( Amplicon, PCR amplicon) is usually detectable.
  • Amplification of polynucleotides encompasses multiple chemical and enzymatic processes. Formats of amplification include by polymerase chain reaction (reverse transcription PCR, PCR), strand displacement amplification (SDA) reaction, transcription-mediated amplification (TMA) reaction, nucleic acid sequence-based amplification (NASBA) reaction or ligation Enzyme chain reaction (LCR), which generates multiple copies of DNA from one or a few copies of a template RNA or DNA molecule.
  • polymerase chain reaction reverse transcription PCR, PCR
  • SDA strand displacement amplification
  • TMA transcription-mediated amplification
  • NASBA nucleic acid sequence-based amplification
  • LCR ligation Enzyme chain reaction
  • the target markers in the processed DNA can be preamplified with preamplification primers.
  • primer refers to a single-stranded oligonucleotide capable of reacting in four different nucleoside triphosphates and reagents for polymerization ( For example, in the presence of DNA polymerase), as the starting point for template-directed DNA synthesis.
  • the length of the primer depends, for example, on the intended use of the primer, and typically ranges from 15 to 30 nucleotides. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. Primers do not have to reflect the exact sequence of the template, but must be sufficiently complementary to hybridize to that template.
  • the primer site is the region on the template to which the primer hybridizes.
  • a primer pair is a set of primers that includes a 5' forward primer that hybridizes to the 5' end of the sequence to be amplified and a 3' reverse primer that hybridizes to the complementary strand at the 3' end of the sequence to be amplified.
  • Those skilled in the art can design primers according to the markers to be amplified based on common knowledge in the art (see, for example, PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratories, NY, 1995).
  • several software packages for designing optimal probes and/or primers for use in a wide variety of assays are publicly available, e.g., from the Center for Genome Research, Cambridge, MA, USA.
  • a primer designed for the purposes of the present invention may include at least one CpG site, or an amplification product obtained from the primer may include at least one CpG site.
  • Tools for designing primers for detecting DNA methylation status are also known in the art, such as MethPrimer (Li LC and Dahiya R. MethPrimer: designing primers for methylation PCRs. Bioinformatics. 2002 Nov; 18(11): 1427-31) .
  • any target marker (every at least a portion of the target marker or a subregion of the target marker) in the processed DNA can be pre-amplified by using the pre-amplification primer as a primer pool.
  • complementary refers to hybridization or base pairing between nucleotides or nucleic acids, for example, between the two strands of a double-stranded DNA molecule, or a primer on a single-stranded nucleic acid to be sequenced or amplified Between the binding site and the oligonucleotide primer.
  • Complementary nucleotides are usually A and T (or A and U), or C and G.
  • nucleotides of one strand are optimally aligned and compared, with appropriate nucleotide insertions or deletions, at least about 80% (usually at least about 90% to 95%, more preferably Between about 98% and 100%) of the nucleotide pairs, two single-stranded RNA or DNA molecules are said to be complementary.
  • complementarity exists when a strand of RNA or DNA hybridizes to its complement under selective hybridization conditions.
  • selective hybridization will occur when there is at least about 65% (preferably at least about 75%, more preferably at least about 90%) complementarity over a stretch of at least 14 to 25 nucleotides. See M. Kanehisa, Nucleic Acids Res. 12:203 (1984), incorporated herein by reference.
  • the pool of preamplification primers comprises at least one methylation-specific primer pair. In some embodiments, the pool of preamplification primers comprises a plurality of methylation-specific primer pairs. In some embodiments, the preamplification step is performed by methylation-specific PCR ("MSP"), which is PCR using methylation-specific primers.
  • MSP methylation-specific PCR
  • methylation-specific primer pair refers to a pair of primers that are specifically designed to recognize CpG sites to exploit differences in methylation to amplify a specific marker of interest in processed DNA. Primers work only on molecules with or without a specific methylation state.
  • a primer can be an oligonucleotide that, under stringent conditions, moderately stringent conditions, or highly stringent conditions, can specifically hybridize in a methylation-specific manner to a specific CpG site with methylation, but not to a specific CpG site without methylation. Hybridization of methylated specific CpG sites.
  • the primers will specifically amplify target markers that have methylation at specific CpG sites.
  • the primer can be an oligonucleotide that can specifically hybridize to a specific unmethylated CpG site in a methylation-specific manner under stringent conditions, moderately stringent conditions or highly stringent conditions, but Does not hybridize to methylated specific CpG sites.
  • the primers will specifically amplify target markers that are not methylated at specific CpG sites.
  • methylated and unmethylated CpG sites can be distinguished using methylation-specific primers in the preamplification of at least one target marker within the processed DNA.
  • the methylation-specific primer pairs of the present application comprise at least one primer that hybridizes to a bisulfite-treated CpG dinucleotide.
  • sequence of the primer specific for methylated DNA comprises at least one CpG dinucleotide and the sequence of the primer specific for unmethylated DNA comprises a "T" at the C position of the CpG, and/or contain "A" at the G position in the CpG.
  • a pair of methylation-specific primers typically comprises a forward primer and a reverse primer, each comprising an oligonucleotide sequence that is compatible with one of the target markers (or a subset of the target marker).
  • the acid comprises at least one (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG site.
  • hybridization may refer to a process in which two single-stranded polynucleotides associate non-covalently to form a stable double-stranded polynucleotide.
  • the resulting double-stranded polynucleotide can be a "hybrid” or "double-stranded”.
  • Salt concentrations in “hybridization conditions” are generally less than about 1 M, often less than about 500 mM and can be less than about 200 mM.
  • “Hybridization buffer” includes buffered saline solutions, such as 5% SSPE, or other such buffers known in the art.
  • Hybridization temperatures can be as low as 5°C, but are typically above 22°C, and more typically above about 30°C, and often above 37°C.
  • Hybridization is typically performed under stringent conditions, ie, conditions under which a sequence will hybridize to its target sequence but to no other noncomplementary sequences. Stringent conditions are sequence dependent and will be different in different circumstances. For example, longer fragments may require higher hybridization temperatures than short fragments for specific hybridization. Since other factors may affect the stringency of hybridization, including base composition and length of complementary strands, the presence of organic solvents, and the degree of base mismatching, the combination of parameters is more important than the absolute measurement of any one parameter alone.
  • Tm melting point
  • the Tm can be the temperature at which half of a population of double-stranded nucleic acid molecules are separated into single strands.
  • Tm melting point
  • hybrid stability is a function of ion concentration and temperature.
  • hybridization reactions are performed under less stringent conditions followed by washes in washes of different but higher stringency.
  • Exemplary stringent conditions include a pH of about 7.0 to about 8.3, a temperature of at least 25°C, and a sodium ion (or other salt) concentration of at least 0.01M to no more than 1M.
  • 5x SSPE 750mM NaCl, 50mM sodium phosphate, 5mM EDTA, pH 7.4
  • a temperature of about 30°C are suitable for allele-specific hybridization, although suitable temperatures depend on the length and/or GC content of the hybridization region.
  • the "stringency of hybridization" for determining the percentage of mismatches can be as follows: 1) high stringency: 0.1x SSPE, 0.1% SDS, 65°C; 2) medium stringency (also known as moderate stringency): 0.2 x SSPE, 0.1% SDS, 50°C; 3) Low stringency: 1.0x SSPE, 0.1% SDS, 50°C. It is understood that the same stringency can be achieved using alternative buffers, salts and temperatures.
  • moderately stringent hybridization can refer to conditions that allow a nucleic acid molecule (eg, a probe) to bind a complementary nucleic acid molecule.
  • Hybridizing nucleic acid molecules typically have at least 60% identity, including, for example, at least 70%, 75%, 80%, 85%, 90%, or 95% identity.
  • Moderately stringent conditions can be conditions equivalent to the following conditions: 42°C, 50% formamide, 5x Denhardt solution, 5x SSPE, 0.2% SDS for hybridization, and then wash with 42°C, 0.2x SSPE, 0.2% SDS.
  • Highly stringent conditions can be provided by, for example, 42°C, 50% formamide, 5x Denhardt's solution, 5x SSPE, 0.2% SDS for hybridization, followed by 65°C, 0.1x SSPE and 0.1% SDS for washing.
  • Low stringency hybridization may be equivalent to the following conditions: 22°C, 10% formamide, 5x Denhardt's solution, 6x SSPE, 0.2% SDS, followed by washing in 1x SSPE, 0.2% SDS at 37°C.
  • Denhardt's solution contained 1% polysucrose, 1% polyvinylpyrrolidone and 1% bovine serum albumin (BSA).
  • BSA bovine serum albumin
  • 20x SSPE Sodium Chloride, Sodium Phosphate, EDTA
  • the pool of preamplification primers also includes a control primer pair for amplifying a control marker.
  • a control marker is a nucleic acid with known characteristics (eg, known sequence, known copy number per cell) for comparison to the experimental target (eg, nucleic acid of unknown concentration).
  • a control can be an endogenous, preferably invariant gene, against which the test or target nucleic acid under analysis can be normalized. Such controls for normalization due to inter-sample variability may occur, for example, in sample handling, assay efficiency, etc., and allow accurate inter-sample data comparisons, quantitative analysis of amplification efficiencies and biases.
  • the preamplification primer pair for the target marker described herein can be, for example, the corresponding primer pair described herein as SEQ ID NO: 1-40.
  • the present invention finds that the CDH1 gene or the CDH1 sequence of the genome, the TSHR gene or the TSHR sequence of the genome, the MCC gene or the MCC sequence of the genome, the TBX15 gene or the TBX15 sequence of the genome, the DPYS gene or the DPYS sequence of the genome in the malignant thyroid nodule tissue , GRIA2 gene or genome GRIA2 sequence, NR5A1 gene or genome NR5A1 sequence, TTC34 gene or genome TTC34 sequence, FTAP2B gene or genome FTAP2B sequence, RP11-867G23.12 gene or genome RP11-867G23.12 sequence and DGKG
  • the methylation level (%) of the gene or genomic DGKG sequence is higher than the methylation level of these genes in benign thyroid nodules; PRR15 gene or genomic PRR15 sequence, RCOR2 gene or genomic RCOR2 sequence in malignant thyroid nodules , F10 gene or genome F10 sequence, ITPKA gene or genome I
  • the present invention finds that in the cfDNA of the blood sample of the patient with malignant thyroid nodule, the TSHR gene or the genome's TSHR sequence, the MCC gene or the genome's MCC sequence, the RARG gene or the genome's RARG sequence, the EMX2OS gene or the genome's
  • the methylation level of one or more of the EMX2OS sequence and the TEAD3 gene or the TEAD3 sequence of the genome is higher than the methylation level of the corresponding target region in the blood sample of the control such as a benign thyroid nodule subject; the CDH1 gene or the CDH1 sequence of the genome, TBX15 gene or genome TBX15 sequence, PRR15 gene or genome PRR15 sequence, DPYS gene or genome DPYS sequence, GRIA2 gene or genome GRIA2 sequence, NR5A1 gene or genome NR5A1 sequence, TTC34 gene or genome TTC34 sequence, RCOR2 gene Or the RCOR2 sequence of the genome,
  • the methylation level of one or more of the RP11-867G23.12 sequence of the gene or genome and the DGKG gene or the DGKG sequence of the genome is equal to or lower than the methylation level of the corresponding target region in the blood sample of a control such as a benign thyroid nodule subject level. Therefore, by comparing the detected methylation level with the corresponding reference level, the benign and malignant thyroid nodules of the subject can be identified or diagnosed according to the comparison results.
  • comparison refers to comparing the methylation levels of the target markers contained in the detected biological samples obtained through the quantitative analysis steps with their corresponding reference levels. It should be understood that the terms used herein refer to the comparison of corresponding parameters or values, for example, comparing an absolute quantity with an absolute reference quantity, comparing a concentration with a reference concentration, or comparing an intensity signal obtained from a tested sample with that of a reference sample. Compare with intensity signals of the same type. Comparisons can be made manually or computer-assisted. For computer-assisted comparisons, the value of the determined quantity can be compared with the value of a suitable reference stored in a database by a computer program.
  • the computer program can further evaluate the results of the comparison and automatically provide the desired evaluation in a suitable output format. Based on the comparison of the methylation level of each target marker in the quantitative analysis step with the corresponding reference level, individuals with malignant thyroid nodules or individuals with a higher risk of their thyroid nodules being malignant can be identified.
  • the "reference level” may be derived from one or more reference samples, wherein the reference level is obtained from an experiment performed in parallel to the experiment detecting the sample of interest.
  • reference levels may be obtained in a database comprising a collection of data, standards or levels from one or more reference samples or disease reference samples.
  • sets of data, standards or levels are normalized so that they can be used for comparison purposes with data from one or more samples.
  • Normalization is the process of transforming measured raw data into data that can be directly compared with other standardized data. Normalization is used to overcome analytical method-specific errors caused by different factors in different analytical methods, such as differences in sample loading, differences in binding efficiencies, differences in detection sensitivity, and other types of errors.
  • the reference database includes methylation levels of markers of interest and/or other laboratory and clinical data from one or more reference samples.
  • the reference database includes methylation levels of markers of interest, each normalized to a percentage of the methylation level of a control marker detected under the same conditions as the reference sample. For comparison with such normalized methylation levels of the target markers, the methylation levels of the target markers of the test samples were also measured and calculated as the methylation levels of the control markers detected under the same conditions as the test samples percentage.
  • a reference database is established by aggregating reference level data from reference samples obtained from healthy individuals and/or individuals with non-malignant thyroid nodules (ie, individuals whose thyroid nodules are known to be benign). In some embodiments, a reference database is established by aggregating reference level data obtained from reference samples of individuals being treated for thyroid cancer.
  • reference level can choose the reference level according to the desired sensitivity and specificity.
  • Means for determining suitable reference levels are known to those skilled in the art, for example reference levels may be determined from data collected in clinical studies.
  • “Higher than the corresponding reference level” mentioned herein means that the methylation level of the corresponding target marker or its target region is at least 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10 times or more.
  • “Below the corresponding reference level” as described herein means that the methylation level of the corresponding target marker is 98%, 95%, 93%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or even lower.
  • any one, any two, any three, any four, any five, any six, any seven, any eight of the following , any nine, any ten or all eleven genes or their respective sequences in the genome have methylation levels higher than the corresponding reference levels: CDH1, TSHR, MCC, TBX15, DPYS, GRIA2, NR5A1, TTC34, FTAP2B, RP11-867G23.12, and DGKG, and/or selected from any one, any two, any three, any four, any five, any six, any seven, any eight, or all nine of the following
  • the methylation level of the gene or its respective sequence in the genome is less than or equal to the corresponding reference level: PRR15, RCOR2, F10, ITPKA, SLC16A3, RARG, SOD3, EMX2OS, and TEAD3, indicating that the thyroid nodule of the subject is malignant or have thyroid cancer.
  • the genes include the CDH1 gene or its sequence in the genome, the TBX15 gene or its sequence in the genome, the DPYS gene or its sequence in the genome, the GRIA2 gene or its sequence in the genome , any one or more of the NR5A1 gene or its sequence in the genome, the RARG gene or its sequence in the genome, and the EMX2OS gene or its sequence in the genome.
  • the methylation level of any one, any two, any three, any four or all five genes selected from the following or their respective sequences in the genome is higher than the corresponding Reference levels: TSHR, MCC, RARG, EMX2OS, and TEAD3, and/or selected from any one, any two, any three, any four, any five, any six, any seven, any eight, Methylation levels of any nine, any ten, any eleven genes, any twelve genes, any thirteen genes, any fourteen genes, or all fifteen genes or their respective sequences in the genome Equal to or lower than the corresponding reference levels: CDH1, TBX15, PRR15, DPYS, GRIA2, NR5A1, TTC34, RCOR2, F10, ITPKA, SLC16A3, FTAP2B, SOD3, RP11-867G23.12 and DGKG, the thyroid nodules of the indicated subject are Malignant nodules or thyroid cancer.
  • the gene is the TSHR gene or its sequence in the genome, the RARG gene or its sequence in the genome, the EMX2OS gene or its sequence in the genome, the CDH1 gene or its sequence in the genome , GRIA2 gene or its sequence in the genome, RCOR2 gene or its sequence in the genome, F10 gene or its sequence in the genome, ITPKA gene or its sequence in the genome, FTAP2B gene or its sequence in the genome , any one or more of the SOD3 gene or its sequence in the genome and the DGKG gene or its sequence in the genome.
  • the marker of interest is the SLC16A3 gene or genomic SLC16A3 sequence and is selected from the TSHR gene or genomic TSHR sequence, TTC34 gene or genomic TTC34 sequence, ROCR2 gene or genomic ROCR2 sequence, RARG gene or genomic RARG One or more of sequence, MCC gene or genome MCC sequence and ITPKA gene or genome ITPKA sequence.
  • the target marker is the SLC16A3 gene or genomic SLC16A3 sequence and the TSHR gene or genomic TSHR sequence; preferably, the methylation level of the target marker is detected in a thyroid nodule from the individual.
  • the target marker is the SLC16A3 gene or the genome's SLC16A3 sequence and the ROCR2 gene or the genome's ROCR2 sequence, and is selected from the TTC34 gene or the genome's TTC34 sequence, the RARG gene or the genome's RARG sequence, the ITPKA gene or the genome's sequence At least one, at least two or at least three of ITPKA sequence and MCC gene or genome MCC sequence; preferably, the target marker is SLC16A3 gene or genome SLC16A3 sequence, ROCR2 gene or genome ROCR2 sequence, TTC34 gene or genome
  • the TTC34 sequence and the RARG gene or the RARG sequence of the genome, or the target marker is the SLC16A3 gene or the SLC16A3 sequence of the genome, the ROCR2 gene or the ROCR2 sequence of the genome, the ITPKA gene or the ITPKA sequence of the genome, and the MCC gene or the MCC sequence of the genome; preferably Preferably, the methylation levels of these
  • the target marker is the SLC16A3 sequence of the SLC16A3 gene or genome and the RARG gene or RARG sequence of the genome, and the TTC34 sequence selected from the TTC34 gene or genome, the ROCR2 sequence of the ROCR2 gene or genome, and the ITPKA gene or genome At least one, at least two, or all three of the ITPKA sequences; preferably, the target marker is the SLC16A3 gene or genomic SLC16A3 sequence, the ROCR2 gene or genomic ROCR2 sequence, the TTC34 gene or genomic TTC34 sequence and the RARG gene or genomic
  • the RARG sequence, or marker of interest is the SLC16A3 gene or genomic SLC16A3 sequence, the TTC34 gene or genomic TTC34 sequence, the ITPKA gene or genomic ITPKA sequence, and the RARG gene or genomic RARG sequence; preferably, a blood sample from the individual is detected Such as the methylation levels of these target markers in plasma.
  • the marker of interest is the SLC16A3 sequence of the SLC16A3 gene or genome and the TTC34 sequence of the TTC34 gene or genome, and the ROCR2 sequence selected from the ROCR2 gene or genome, the RARG sequence of the RARG gene or genome, and the ITPKA gene or genome At least one, at least two, or all three of the ITPKA sequences; preferably, the target marker is the SLC16A3 gene or the genomic SLC16A3 sequence, the ROCR2 gene or the genomic ROCR2 sequence, the TTC34 gene or the genomic TTC34 sequence and the RARG gene or the genomic TTC34 sequence
  • the RARG sequence, or marker of interest is the SLC16A3 gene or genomic SLC16A3 sequence, the TTC34 gene or genomic TTC34 sequence, the ITPKA gene or genomic ITPKA sequence, and the RARG gene or genomic RARG sequence; preferably, a blood sample from the individual is detected Such as the methylation levels of these target markers in plasma.
  • the target marker is the SLC16A3 gene or genome's SLC16A3 sequence and the ITPKA gene or genome's ITPKA sequence, and the ROCR2 sequence selected from the ROCR2 gene or genome, the MCC gene or genome's MCC sequence, the TTC34 gene or genome's At least one, at least two, or all three of TTC34 sequence and RARG gene or genomic RARG sequence; preferably, the target marker is SLC16A3 gene or genomic SLC16A3 sequence, ROCR2 gene or genomic ROCR2 sequence, ITPKA gene or genomic The ITPKA sequence and the MCC gene or the MCC sequence of the genome, or the target marker is the SLC16A3 gene or the genome's SLC16A3 sequence, the TTC34 gene or the genome's TTC34 sequence, the ITPKA gene or the genome's ITPKA sequence and the RARG gene or the genome's RARG sequence; preferably Preferably, the methylation levels of these markers of interest are detected in a blood
  • the target marker is the SLC16A3 gene or genomic SLC16A3 sequence, the TTC34 gene or genomic TTC34 sequence and the RARG gene or genomic RARG sequence, and the ROCR2 gene or genomic ROCR2 sequence or ITPKA gene or genomic ITPKA sequence preferably, detecting the methylation levels of these target markers in a blood sample from the individual, such as plasma.
  • the target marker is the FTAP2B gene or the genomic FTAP2B sequence and the EMS2OS gene or the genomic EMS2OS sequence, the DGKG gene or the genomic DGKG sequence, the SOD3 gene or the genomic SOD3 sequence, the RP11-867G23.12 gene Or at least one, at least two or at least three of the RP11-867G23.12 sequences of the genome.
  • the marker of interest is the FTAP2B gene or genomic FTAP2B sequence, the EMS2OS gene or genomic EMS2OS sequence, and optionally the SOD3 gene or genomic SOD3 sequence.
  • the target markers are FTAP2B gene or genomic FTAP2B sequence and EMS2OS gene or genomic EMS2OS sequence, wherein the methylation levels of these target markers in thyroid nodules from the individual are detected.
  • the target marker is the FTAP2B gene or the FTAP2B sequence of the genome, the EMS2OS gene or the EMS2OS sequence of the genome, and the SOD3 gene or the SOD3 sequence of the genome, wherein the methylation of these target markers in blood samples such as plasma from the individual is detected level.
  • the target markers are the FTAP2B gene or genomic FTAP2B sequence and the DGKG gene or genomic DGKG sequence, wherein the methylation levels of these target markers are detected in a blood sample, such as plasma, from the individual.
  • the markers of interest are the FTAP2B gene or genomic FTAP2B sequence and the SOD3 gene or genomic SOD3 sequence, and the EMS2OS gene or genomic EMS2OS sequence or RP11-867G23.12 gene or genomic RP11-867G23.12 sequence , wherein the methylation levels of the markers of interest are detected in a blood sample, such as plasma, from the individual.
  • the target marker is a RARG gene or a genomic RARG sequence, and a DPYS gene or a genomic DPYS sequence, a TSHR gene or a genomic TSHR sequence, a TTC34 gene or a genomic TTC34 sequence, an RCOR2 gene or a genomic sequence At least one, at least two, at least three or at least four of the RCOR2 sequence, the SLC16A3 gene or genomic SLC16A3 sequence, and the ITPKA gene or genomic ITPKA sequence.
  • the target markers are the RARG gene or genomic RARG sequence and the DPYS gene or genomic DPYS sequence, wherein the methylation levels of these target markers are detected in thyroid nodules from the individual.
  • the target markers are the RARG gene or genomic RARG sequence and the TSHR gene or genomic TSHR sequence, wherein the methylation levels of these target markers are detected in a blood sample, such as plasma, from the individual.
  • the target marker is the RARG gene or the RARG sequence of the genome and the TTC34 gene or the TTC34 sequence of the genome, and the RCOR2 sequence selected from the RCOR2 gene or genome, the SLC16A3 gene or genome SLC16A3 sequence and the ITPKA gene or genome At least one or at least two of the ITPKA sequences, wherein the methylation levels of these target markers are detected in a blood sample, such as plasma, from the individual.
  • the target marker is RARG gene or genomic RARG sequence, TTC34 gene or genomic TTC34 sequence, RCOR2 gene or genomic RCOR2 sequence, and SLC16A3 gene or genomic SLC16A3 sequence. Methylation levels of these markers of interest.
  • the target marker is the RARG gene or the RARG sequence of the genome, the TTC34 gene or the TTC34 sequence of the genome, the ITPKA gene or the ITPKA sequence of the genome, and the SLC16A3 gene or the SLC16A3 sequence of the genome. Methylation levels of these markers of interest.
  • the markers of interest are the RARG sequence of the RARG gene or genome and the SLC16A3 sequence of the SLC16A3 gene or genome, and the TTC34 sequence selected from the TTC34 gene or genome, the RCOR2 sequence of the RCOR2 gene or genome, and the ITPKA gene or genome At least one or at least two of the ITPKA sequences, wherein the methylation levels of these target markers are detected in a blood sample, such as plasma, from the individual.
  • the target marker is RARG gene or genomic RARG sequence, TTC34 gene or genomic TTC34 sequence, RCOR2 gene or genomic RCOR2 sequence, and SLC16A3 gene or genomic SLC16A3 sequence.
  • the target marker is the RARG gene or the RARG sequence of the genome, the TTC34 gene or the TTC34 sequence of the genome, the ITPKA gene or the ITPKA sequence of the genome, and the SLC16A3 gene or the SLC16A3 sequence of the genome. Methylation levels of these markers of interest.
  • the markers of interest are the RARG gene or genomic RARG sequence, the SLC16A3 gene or genomic SLC16A3 sequence, and the TTC34 gene or genomic TTC34 sequence, and the ROCR2 gene or genomic ROCR2 sequence or ITPKA gene or genomic ITPKA sequence preferably, detecting the methylation levels of these target markers in a blood sample from the individual, such as plasma.
  • the marker of interest is the SOD3 gene or the SOD3 sequence of the genome, and the EMS2OS sequence selected from the EMS2OS gene or genome, the TEAD3 gene or the TEAD3 sequence of the genome, the FTAP2B gene or the FTAP2B sequence of the genome, and RP11-867G23.12 At least one or at least two of the RP11-867G23.12 sequences of the gene or genome.
  • the markers of interest are the SOD3 gene or genomic SOD3 sequence and the EMS2OS gene or genomic EMS2OS sequence, and the TEAD3 gene or genomic TEAD3 sequence or FTAP2B gene or genomic FTAP2B sequence; The methylation levels of these target markers in blood samples such as plasma.
  • the markers of interest are the SOD3 gene or genome's SOD3 sequence and the FTAP2B gene or genome's FTAP2B sequence, and the EMS2OS gene or genome's EMS2OS sequence or the RP11-867G23.12 gene or genome's RP11-867G23.12 sequence preferably, detecting the methylation levels of these target markers in a blood sample from the individual, such as plasma.
  • the target marker is the TEAD3 gene or the TEAD3 sequence of the genome, and the RP11-867G23.12 sequence selected from the RP11-867G23.12 gene or genome, the SOD3 sequence of the SOD3 gene or genome, and the EMS2OS gene or genome. At least one or at least two of the EMS2OS sequences.
  • the target markers are the TEAD3 gene or the TEAD3 sequence of the genome and the RP11-867G23.12 gene or the RP11-867G23.12 sequence of the genome; basic level.
  • the target marker is the TEAD3 gene or the TEAD3 sequence of the genome, the SOD3 gene or the SOD3 sequence of the genome and the EMS2OS gene or the EMS2OS sequence of the genome; preferably, the formazan of these target markers in blood samples such as plasma from the individual is detected basic level.
  • the target marker is the TTC34 gene or the TTC34 sequence of the genome, and the RCOR2 sequence selected from the RCOR2 gene or the genome, the ITPKA gene or the ITPKA sequence of the genome, the CDH1 gene or the CDH1 sequence of the genome, the SLC16A3 gene or a sequence of the genome At least one, at least two or at least three of the SLC16A3 sequence and the RARG gene or the RARG sequence of the genome; preferably, the methylation levels of these target markers in blood samples such as plasma from the individual are detected.
  • the target marker is the TTC34 gene or the TTC34 sequence of the genome and the RCOR2 gene or the RCOR2 sequence of the genome, and the ITPKA sequence selected from the ITPKA gene or genome, the CDH1 gene or the CDH1 sequence of the genome, the SLC16A3 gene or the genome At least one or at least two of the SLC16A3 sequence and the RARG gene or the RARG sequence of the genome.
  • the methylation levels of these target markers are detected in a blood sample from the individual, such as plasma.
  • the target marker is the TTC34 gene or genomic TTC34 sequence, RCOR2 gene or genomic RCOR2 sequence, ITPKA gene or genomic ITPKA sequence, and CDH1 gene or genomic CDH1 sequence; Methylation levels of these target markers in blood samples such as plasma.
  • the target marker is TTC34 gene or genomic TTC34 sequence, RCOR2 gene or genomic RCOR2 sequence, SLC16A3 gene or genomic SLC16A3 sequence and RARG gene or genomic RARG sequence; Methylation levels of these target markers in blood samples such as plasma.
  • the target marker is TTC34 gene or genome TTC34 sequence and ITPKA gene or genome ITPKA sequence, and RCOR2 sequence selected from RCOR2 gene or genome, CDH1 gene or genome CDH1 sequence, SLC16A3 gene or genome At least one or at least two of the SLC16A3 sequence and the RARG gene or the RARG sequence of the genome.
  • the methylation levels of these target markers are detected in a blood sample from the individual, such as plasma.
  • the target marker is TTC34 gene or genomic TTC34 sequence, ITPKA gene or genomic ITPKA sequence, RCOR2 gene or genomic RCOR2 sequence, and CDH1 gene or genomic CDH1 sequence; Methylation levels of these target markers in blood samples such as plasma.
  • the target marker is the TTC34 gene or genomic TTC34 sequence, ITPKA gene or genomic ITPKA sequence, SLC16A3 gene or genomic SLC16A3 sequence, and RARG gene or genomic RARG sequence; Methylation levels of these target markers in blood samples such as plasma.
  • the target marker is the TTC34 gene or the TTC34 sequence of the genome and the SLC16A3 gene or the SLC16A3 sequence of the genome, and the RCOR2 sequence selected from the RCOR2 gene or genome, the ITPKA gene or genome ITPKA sequence and the RARG gene or genome At least one or at least two of the RARG sequences.
  • the methylation levels of these target markers are detected in a blood sample from the individual, such as plasma.
  • the target marker is TTC34 gene or genomic TTC34 sequence, SLC16A3 gene or genomic SLC16A3 sequence, RCOR2 gene or genomic RCOR2 sequence and RARG gene or genomic RARG sequence; Methylation levels of these target markers in blood samples such as plasma.
  • the target marker is the TTC34 gene or the TTC34 sequence of the genome, the SLC16A3 gene or the SLC16A3 sequence of the genome, the ITPKA gene or the ITPKA sequence of the genome, and the RARG gene or the RARG sequence of the genome; The methylation levels of these target markers in a blood sample, such as plasma, of the individual.
  • the target marker is the TTC34 gene or genomic TTC34 sequence, the SLC16A3 gene or genomic SLC16A3 sequence and the RARG gene or genomic RARG sequence, and the RCOR2 gene or genomic RCOR2 sequence or ITPKA gene or genomic ITPKA sequence preferably, detecting the methylation levels of these target markers in a blood sample from the individual, such as plasma.
  • the target marker is the MCC gene or the MCC sequence of the genome, and the ITPKA sequence of the ITPKA gene or genome, the RCOR2 sequence of the RCOR2 gene or genome, the SLC16A3 sequence of the SLC16A3 gene or genome, and the CDH1 gene or genome At least one, at least two or at least three of the CDH1 sequences; preferably, the methylation levels of these target markers are detected in a blood sample such as plasma from the individual.
  • the target marker is the MCC gene or the MCC sequence of the genome, the ITPKA gene or the ITPKA sequence of the genome, the RCOR2 gene or the RCOR2 sequence of the genome, and the SLC16A3 gene or the SLC16A3 sequence of the genome; Methylation levels of these markers of interest in plasma.
  • the target markers are the MCC gene or the MCC sequence of the genome and the CDH1 gene or the CDH1 sequence of the genome; preferably, the methylation levels of these target markers in blood samples such as plasma from the individual are detected.
  • the marker of interest is the CDH1 gene or the CDH1 sequence of the genome, and the MCC sequence selected from the MCC gene or genome, the TTC34 sequence of the TTC34 gene or genome, the RCOR2 sequence of the RCOR2 gene or genome, and the ITPKA gene or genome At least one, at least two or at least three of the ITPKA sequences; preferably, the methylation levels of these target markers are detected in a blood sample such as plasma from the individual.
  • the target markers are CDH1 gene or genomic CDH1 sequence and MCC gene or genomic MCC sequence; preferably, the methylation levels of these target markers in blood samples such as plasma from the individual are detected.
  • the target marker is the CDH1 gene or the genome's CDH1 sequence, the TTC34 gene or the genome's TTC34 sequence, the RCOR2 gene or the genome's RCOR2 sequence, and the ITPKA gene or the genome's ITPKA sequence; Methylation levels of these markers of interest in plasma.
  • those skilled in the art can also determine whether an individual's thyroid nodule is malignant or the risk of being malignant based on various factors, such as age, gender, medical history, family history, symptoms, etc.
  • Ct value refers to the cycle number at which fluorescence of a PCR product can be detected above background signal. The Ct value is inversely proportional to the amount of the target marker in the sample, that is, the lower the Ct value, the greater the amount of the target marker in the sample.
  • the methylation levels of the DPYS gene or the DPYS sequence of the genome or its target region and the RARG gene or the RARG sequence of the genome or its target region in the thyroid nodule tissue of the subject are detected, wherein the methylation levels of the two genes or their target regions
  • the methylation levels of the FTAP2B gene or the FTAP2B sequence of the genome or its target region and the EMX2OS gene or the genome's EMX2OS sequence or its target region in the thyroid nodule tissue of the subject are detected, wherein the methylation levels of the two genes or their target regions
  • the TTC34 gene or genome's TTC34 sequence or its target region, the RCOR2 gene or genome's RCOR2 sequence or its target region, the SLC16A3 gene or genome's SLC16A3 sequence or its target region, and the RARG gene or genome in the blood of the subject are detected
  • the methylation level of the RARG sequence or its target region, where the binary Logistic regression analysis is performed on the methylation levels of the four genes or their target regions, and the fitting equation is score 2.23+3.57 ⁇ TCC34 methylation level -24.82 ⁇ RCOR2 methylation level-4.66 ⁇ SLC16A3 methylation level-0.75 ⁇ RARG methylation level; among them, if the score is greater than 0, the result is positive and it is a malignant nodule.
  • the SOD3 gene or the genome's SOD3 sequence or its target region, the RP11-867G23.12 gene or the genome's RP11-867G23.12 sequence or its target region, and the FTAP2B gene or the genome's FTAP2B sequence or The methylation level of its target region, among which, binary Logistic regression analysis was performed on the methylation level of the three genes or their target regions, and the fitting equation was score 6.10–1.04 ⁇ SOD3 methylation level+0.60 ⁇ RP11 -867G23.12 methylation level+0.61 ⁇ FTAP2B methylation level; wherein, if the score is greater than 0, the result is determined to be positive and it is a malignant nodule.
  • the ITPKA gene or genome's ITPKA sequence or its target region, the RCOR2 gene or genome's RCOR2 sequence or its target region, the SLC16A3 gene or genome's SLC16A3 sequence or its target region, and the MCC gene or genome in the blood of the subject are detected
  • the TTC34 gene or the genome's TTC34 sequence or its target region, the ITPKA gene or the genome's ITPKA sequence or its target region, the SLC16A3 gene or the genome's SLC16A3 sequence or its target region, and the RARG gene or genome in the blood of the subject are detected
  • the methylation level of the RARG sequence or its target region, where the binary Logistic regression analysis is performed on the methylation levels of the four genes or their target regions, and the fitting equation is score 2.47+3.27 ⁇ TTC34 methylation level –3.71 ⁇ ITPKA methylation level –6.48 ⁇ SLC16A3 methylation level –0.69 ⁇ RARG methylation level; among them, if the score is greater than 0, the result is positive and it is a malignant nodule.
  • kits and compositions for differentiating benign and malignant thyroid nodules, and the kit and composition include primer molecules.
  • the kits and compositions may contain a compound capable of hybridizing to the target marker to be detected or its target region under stringent conditions, moderately stringent conditions or highly stringent conditions. primer pair.
  • the primer pairs in the kits and compositions can be selected from any one or any multiple of the following primer pairs:
  • (3) can amplify the primer pair of the fragment of the MCC gene obtained as primer amplification by SEQ ID NO:5 and 6;
  • the primer pair is selected from at least one or more pairs of primer pairs in the following group: SEQ ID NO: 1 and 2; SEQ ID NO: 3 and 4; SEQ ID NO: 5 and 6; SEQ ID NO: 5 and 6; ID NO: 7 and 8; SEQ ID NO: 9 and 10; SEQ ID NO: 11 and 12; SEQ ID NO: 13 and 14; SEQ ID NO: 15 and 16; SEQ ID NO: 17 and 18; SEQ ID NO SEQ ID NO:21 and 22; SEQ ID NO:23 and 24; SEQ ID NO:25 and 26; SEQ ID NO:27 and 28; SEQ ID NO:29 and 30; SEQ ID NO:31 and 32; SEQ ID NO:33 and 34; SEQ ID NO:35 and 36; SEQ ID NO:37 and 38; and SEQ ID NO:39 and 40.
  • the primer pair contained in the kits and compositions is a primer pair for amplifying a marker of interest or a region of interest thereof selected from the group consisting of: CDH1, TSHR, MCC, TBX15, PRR15, DPYS, GRIA2, NR5A1, TTC34, RCOR2, F10, ITPKA, SLC16A3, RARG, FTAP2B, SOD3, RP11-867G23.12, EMX2OS, TEAD3 or DGKG genes or their respective sequences in the genome or their respective target regions; DPYS or Their respective sequences in the genome or their target regions and the RARG gene or genome's RARG sequence or its target regions; TSHR gene or genome's TSHR sequence or its target region and SLC16A3 gene or genome's SLC16A3 sequence or its target region; FTAP2B gene or genomic FTAP2B sequence or its target region and EMX2OS gene or genomic EMX2OS sequence or its
  • the primer sequences of the primer pair are respectively: SEQ ID NO:1 and 2, SEQ ID NO:3 and 4, SEQ ID NO:5 and 6, SEQ ID NO: 7 and 8, SEQ ID NO:9 and 10, SEQ ID NO:11 and 12, SEQ ID NO:13 and 14, SEQ ID NO:15 and 16, SEQ ID NO:17 and 18, SEQ ID NO:19 and 20, SEQ ID NO:21 and 22, SEQ ID NO:23 and 24, SEQ ID NO:25 and 26, SEQ ID NO:27 and 28, SEQ ID NO:29 and 30, SEQ ID NO:31 and 32, SEQ ID NO:33 and 34, SEQ ID NO:35 and 36, SEQ ID NO:37 and 38, or SEQ ID NO:39 and 40; SEQ ID NO:11 and 12, and SEQ ID NO:27 and 28; SEQ ID NO:3 and 4, and SEQ ID NO:25 and 26; SEQ ID NO:29 and 30, and SEQ ID NO:35 and 36; SEQ ID NO:33 and 34, and SEQ ID NO:31 and 32, SEQ ID NO
  • Primers can also include primers for detecting internal references such as ACTB, for example, primers that can amplify fragments of the ACTB gene amplified by the sequences of SEQ ID NO: 61 and 62 as primers.
  • the primer pair for the internal reference is SEQ ID NO: 61 and 62.
  • the primers are packaged in a single container or packaged in separate containers.
  • the kit further comprises one or more blocking oligonucleotides.
  • kits and compositions further comprise detection reagents.
  • the detection reagent is selected from the group consisting of fluorescent probes, intercalating dyes, chromophore-labeled probes, radioisotope-labeled probes, and biotin-labeled probes.
  • kits and compositions may contain corresponding fluorescent probes.
  • the fluorescent probes in the kits and compositions may be fluorescent probes for detecting the following target markers or their target regions: CDH1, TSHR, MCC, TBX15, PRR15, DPYS, GRIA2, NR5A1, TTC34 , RCOR2, F10, ITPKA, SLC16A3, RARG, FTAP2B, SOD3, RP11-867G23.12, EMX2OS, TEAD3 or DGKG genes and their respective sequences in the genome or their respective target regions; DPYS or their respective sequences in the genome or Its target region and RARG gene or genome's RARG sequence its target region; TSHR gene or genome's TSHR sequence its target region and SLC16A3 gene or genome's SLC16A3 sequence its target region; FTAP2B gene or genome's FTAP2B sequence its target region
  • the nucleotide sequences and specific fluorescent probes of the fluorescent probes for the above-mentioned genes or their target regions can be as described in Section IV above. More specifically, the nucleotide sequence of the fluorescent probe in the kit can be selected from the following group: any one of SEQ ID NO:41-60; SEQ ID NO:46 and SEQ ID NO:54; SEQ ID NO :42 and SEQ ID NO:53; SEQ ID NO:55 and SEQ ID NO:58; SEQ ID NO:57 and SEQ ID NO:59; SEQ ID NO:42 and SEQ ID NO:54; SEQ ID NO:43 and SEQ ID NO:41; SEQ ID NO:56, SEQ ID NO:58 and SEQ ID NO:59; SEQ ID NO:55 and SEQ ID NO:60; SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:50, SEQ ID NO:59 ID NO:53 and SEQ ID NO:54; SEQ ID NO:56, SEQ ID NO:58 and
  • Probes also include probes for detecting internal reference genes such as ACTB.
  • the nucleotide sequence of the probe for detecting the internal reference gene ACTB is shown in SEQ ID NO:63.
  • the kit may further comprise a DNA polymerase and/or a container suitable for storing a biological sample obtained from an individual.
  • the kit further comprises instructions for use and/or an explanation of the test results of the kit.
  • kits and compositions may also include reagents for enzymatic or non-enzymatic transformations.
  • the kits shown also include a bisulfite reagent or a methylation sensitive restriction enzyme (MSRE).
  • the bisulfite reagent is selected from the group consisting of ammonium bisulfite, sodium bisulfite, potassium bisulfite, calcium bisulfite, magnesium bisulfite, aluminum bisulfite, sulfurous acid Hydrogen ions, and any combination thereof.
  • the bisulfite reagent is sodium bisulfite.
  • the MSRE is selected from the group consisting of HpaII enzyme, SalI enzyme, ScrFI enzyme, BbeI enzyme, NotI enzyme, SmaI enzyme, XmaI enzyme, MboI enzyme, BstBI enzyme, ClaI enzyme, MluI enzyme, NaeI enzyme, Narl enzyme, PvuI enzyme, SacII enzyme, HhaI enzyme, and any combination thereof.
  • kits and compositions may also include converted positive standards in which unmethylated cytosines are converted to bases that do not bind guanine.
  • the positive standard can be fully methylated.
  • kits and compositions may also include PCR reaction reagents.
  • the PCR reaction reagents include Taq DNA polymerase, PCR buffer (buffer), dNTPs, Mg 2+ .
  • kits and compositions further comprise standard reagents useful for CpG position-specific methylation analysis, wherein the analysis includes one or more of the following techniques: MS-SNuPE, MSP, MethyLight TM , HeavyMethyl TM , COBRA and nucleic acid sequencing.
  • kits and compositions may comprise additional reagents selected from the group consisting of buffers (e.g. restriction enzymes, PCR, storage or wash buffers), DNA recovery reagents or kits (e.g. precipitation, ultrafiltration, affinity column) and DNA recovery components, etc.
  • buffers e.g. restriction enzymes, PCR, storage or wash buffers
  • DNA recovery reagents or kits e.g. precipitation, ultrafiltration, affinity column
  • DNA recovery components e.g. precipitation, ultrafiltration, affinity column
  • the kit of the present application may further comprise one or more of the following components known in the field of DNA enrichment: a protein component, which selectively binds to methylated DNA; a triple-strand forming nucleic acid component , one or more linkers, optionally in a suitable solution; substances or solutions for performing the ligation, such as ligases, buffers; substances or solutions for performing column chromatography; for performing immunologically based A substance or solution for enrichment (e.g.
  • immunoprecipitation a substance or solution for nucleic acid amplification, such as PCR; a dye or dyes, if suitable for a coupling agent, if suitable for use in solution; for A substance or solution for carrying out hybridization; and/or a substance or solution for carrying out a washing step.
  • a substance or solution for nucleic acid amplification such as PCR
  • a dye or dyes if suitable for a coupling agent, if suitable for use in solution
  • a substance or solution for carrying out hybridization for A substance or solution for carrying out hybridization
  • a substance or solution for carrying out a washing step a substance or solution for carrying out a washing step.
  • the composition of the present invention contains an isolated nucleic acid molecule selected from one or more of the following: (1) a sequence consisting of SEQ ID NO: 1 and 2 or at least 90 therebetween The sequence of % identity is used as the fragment of the CDH1 gene that primer amplifies; (2) by SEQ ID NO:3 and 4 or there is the fragment of the TSHR gene that there is at least 90% identity sequence as primer amplification; (3 ) by SEQ ID NO: 5 and 6 or the sequence that has at least 90% identity with it as the fragment of the MCC gene amplified by primer; (4) by SEQ ID NO: 7 and 8 or it has at least 90% identity with it The sequence is used as a fragment of the TBX15 gene amplified by primers; (5) by SEQ ID NO:9 and 10 or a sequence with at least 90% identity therewith is used as a fragment of the PRR15 gene amplified by primers, (6) by SEQ ID NO:11 and 12 or its sequence of at least 90% identity is used as the fragment of the fragment of the sequence of
  • the composition contains: DPYS gene target region and RARG gene target region; TSHR gene target region and SLC16A3 gene target region; FTAP2B gene target region and EMX2OS gene target region; RP11-867G23.12 gene target region and TEAD3 gene target region; TSHR gene target region and RARG gene target region; MCC gene target region and CDH1 gene target region; SOD3 gene target region, EMX2OS gene target region and TEAD3 gene target region; FTAP2B gene target region and DGKG gene target region ; TTC34 gene target region, RCOR2 gene target region, SLC16A3 gene target region and RARG gene target region; SOD3 gene target region, EMX2OS gene target region and FTAP2B gene target region; TTC34 gene target region, RCOR2 gene target region, ITPKA gene target region and CDH1 gene target region; SOD3 gene target region, RP11-867G23.12 gene target region and FTAP2
  • the present application also includes a medium recording the sequence of the isolated nucleic acid molecule described herein and optionally its methylation information, said medium being used for comparison with gene methylation sequencing data to determine the presence of said nucleic acid molecule, content and/or methylation levels.
  • said medium is a card printed with said sequence and optionally its methylation information, eg paper, plastic, metal, glass card.
  • the medium is a computer-readable medium storing the sequence and optionally its methylation information and a computer program.
  • methylation of the sample The methylation sequencing data is compared with the sequence, so as to obtain the presence, content and/or methylation level of nucleic acid molecules containing the sequence in the sample.
  • the present application also includes a device for distinguishing benign from malignant thyroid nodules, the device includes a memory, a processor, and a computer program stored in the memory and operable on the processor, when the processor executes the program
  • the following steps are achieved: (1) obtaining the methylation level of one or more target markers or target regions selected from the following one or more target markers described herein in the sample, (2) interpreting the thyroid nodule according to the methylation level of (1) benign and malignant.
  • the obtaining step is performed by any one of the methods described in Section IV of the present application; preferably, the interpretation is performed by any one of the methods described in Section V of the present application.
  • the present application also provides the primer pair described herein, the combination of primer pairs and the optional probe or probe combination in the diagnosis of benign and malignant thyroid nodules, as well as the preparation of reagents for diagnosing benign and malignant thyroid nodules or application in the kit.
  • the kit is as described in any scheme in Section VI of the present application.
  • the kit is used to implement the various steps and methods described in any one or more of Sections II, III, IV and V herein.
  • the present application also provides the application of the isolated nucleic acid molecule described in the present application as a detection target in the diagnosis of benign and malignant thyroid nodules.
  • the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.
  • the reagents and materials used in the following examples are commercially available.
  • Example 1 To verify the relationship between benign and malignant thyroid nodules and tissue genome DNA methylation.
  • the applicant performed genomic DNA methylation-specific PCR on the tissues of 10 patients with thyroid cancer and 10 patients with benign thyroid nodules, and found that CDH1 gene, TSHR gene, MCC gene, TBX15 gene, PRR15 gene, DPYS gene, GRIA2 gene, NR5A1 gene, TTC34 gene, RCOR2 gene, F10 gene, ITPKA gene, SLC16A3 gene, RARG gene, FTAP2B gene, SOD3 gene, RP11-867G23.12 gene, EMX2OS gene, TEAD3 gene and DGKG gene in thyroid cancer and benign thyroid nodules
  • the genomic DNA methylation levels of the cases were different, and the results are shown in Figure 1 and Figure 2.
  • the specific experimental steps are as follows:
  • MethylCodeTM Bisulfite Conversion Kit (Thermo, product number: MECOV50) to perform bisulfite conversion on the DNA obtained in step 1, and the unmethylated cytosine (cytosine, C) is converted into uracil (uracil, U); Kylated cytosines are unchanged after conversion.
  • kit including PCR reaction solution, primer mixture (SEQ ID NO:1-40, 61-62), probe mixture (SEQ ID NO:41-60 and 63), carry out the preparation of single sample, As shown in Table 1-7.
  • the primer sequences are as follows:
  • CDH1 gene forward primer F CDH1 gene forward primer F:
  • CDH1 gene reverse primer R CDH1 gene reverse primer R:
  • PRR15 gene forward primer F
  • TTC34 gene forward primer F TTC34 gene forward primer F:
  • ITPKA gene forward primer F ITPKA gene forward primer F:
  • the probe sequences are as follows:
  • CDH1 gene probe CGCCCACCCGACCTCGCAT (SEQ ID NO: 41)
  • TSHR gene probe ACAACACCAACTACAACAAATCCGCCGA (SEQ ID NO: 42)
  • TBX15 gene probe ACCCTACTCCTACGCAAACCGAAAT (SEQ ID NO: 44)
  • PRR15 gene probe CCTCCGAAAACAACGTAACGCGC (SEQ ID NO: 45)
  • DPYS gene probe CGAAAACATCGACACACACACGCA (SEQ ID NO: 46)
  • GRIA2 gene probe AAAACGCTTCGCCGCCAACA (SEQ ID NO: 47)
  • NR5A1 gene probe AAACGCTACGCGAAACGCTC (SEQ ID NO: 48)
  • TTC34 gene probe CGAACCGCAACAAACGCTCG (SEQ ID NO: 49)
  • RCOR2 gene probe CCGACTCGCGCCAAACTCGA (SEQ ID NO:50)
  • F10 gene probe CAAACAACGCGACCTCTAAACGC (SEQ ID NO:51)
  • ITPKA gene probe ACGCTAAAATCACCTTCACTACGCC (SEQ ID NO:52)
  • SLC16A3 gene probe ATGTAAGCGGATATAGAGCGGTAGGGTA (SEQ ID NO:53)
  • RARG gene probe CGCAACCACGCAAAAACACACGC (SEQ ID NO:54)
  • FTAP2B gene probe CGAGTCGTCGTATACGGTTTCGGG (SEQ ID NO: 55)
  • SOD3 gene probe CGAACCCGAACTCTAAAAACGCCAAACG (SEQ ID NO:56)
  • RP11-867G23.12 gene probe CGGTGTTGTCGGTTGTTTTTTACGTACG (SEQ ID NO: 57)
  • EMX2OS gene probe CCAAAACGTACACCGACTCCTAAATTCC (SEQ ID NO:58)
  • TEAD3 gene probe TGGTTGCGGTATCGGATTATTTATACGG (SEQ ID NO: 59)
  • ACTB gene probe ACCACCACCCAACACACAATAACAAACACA (SEQ ID NO: 63)
  • Methylation level 2 - ⁇ Ct sample to be tested / 2 - ⁇ Ct positive standard ⁇ 100%.
  • ⁇ Ct Ct target gene - Ct internal reference gene .
  • CDH1, TSHR, MCC, TBX15, PRR15, DPYS, GRIA2, NR5A1, TTC34, RCOR2, F10, ITPKA, SLC16A3, RARG, FTAP2B, SOD3, RP11-867G23.12, EMX2OS, TEAD3, and DGKG genes are methylated in tissue genomes
  • the methylation level of each gene is shown in Figure 1; the ROC curve analysis of the methylation level of each gene is shown in Figure 2, and the AUC of each gene is greater than 0.6.
  • Example 2 Verifying the relationship between benign and malignant thyroid nodules and cfDNA methylation in plasma
  • the applicant performed methylation-specific PCR on the plasma cfDNA of 196 patients with thyroid cancer and 148 patients with benign thyroid nodules, and found that CDH1, TSHR, MCC, TBX15, PRR15, DPYS, GRIA2, NR5A1, TTC34, RCOR2, F10, ITPKA, SLC16A3 and RARG genes have differences in the methylation levels of plasma cfDNA in thyroid cancer and benign thyroid nodules cases, the results are shown in Figure 3 and Figure 4.
  • the plasma cfDNA of 196 cases of thyroid cancer and 148 patients with benign thyroid nodules were extracted using QIAamp DNA Mini Kit (QIAGEN, catalog number: 51304); the concentration of cfDNA was detected using QubitTM dsDNA HS Assay Kit (Thermo, catalog number: Q32854); Quality checks were performed using 1% agarose gel electrophoresis.
  • MethylCodeTM Bisulfite Conversion Kit (Thermo, product number: MECOV50) to perform bisulfite conversion on the DNA obtained in step 1, and the unmethylated cytosine (cytosine, C) is converted into uracil (uracil, U); Kylated cytosines are unchanged after conversion.
  • kit including PCR reaction solution, primer mixture (SEQ ID NO:1-28, 61-62), probe mixture (SEQ ID NO:41-54 and 63), carry out the preparation of single sample, The details are shown in Table 1-4 and Table 8:
  • Methylation level 2 - ⁇ Ct sample to be tested / 2 - ⁇ Ct positive standard ⁇ 100%.
  • ⁇ Ct Ct target gene - Ct internal reference gene.
  • the methylation levels of CDH1, TSHR, MCC, TBX15, PRR15, DPYS, GRIA2, NR5A1, TTC34, RCOR2, F10, ITPKA, SLC16A3 and RARG genes in the tissue genome are shown in Figure 3; the methylation levels of each gene
  • the ROC curve analysis was performed separately, as shown in Figure 4, and the AUC of each gene was greater than 0.6.
  • Example 3 Verifying the relationship between benign and malignant thyroid nodules and cfDNA methylation in plasma
  • the applicant performed methylation-specific PCR on the plasma cfDNA of 90 patients with thyroid cancer and 54 patients with benign thyroid nodules, and found that FTAP2B, SOD3, RP11-867G23.12, EMX2OS, TEAD3 and DGKG genes were involved in thyroid cancer and thyroid
  • FTAP2B, SOD3, RP11-867G23.12, EMX2OS, TEAD3 and DGKG genes were involved in thyroid cancer and thyroid
  • the methylation levels of plasma cfDNA in the cases of benign nodules were different, the results are shown in Figure 5 and Figure 6.
  • MethylCodeTM Bisulfite Conversion Kit (Thermo, product number: MECOV50) to perform bisulfite conversion on the DNA obtained in step 1, and the unmethylated cytosine (cytosine, C) is converted into uracil (uracil, U); Kylated cytosines are unchanged after conversion.
  • kit including PCR reaction solution, primer mixture (SEQ ID NO:29-40, 61-62), probe mixture (SEQ ID NO:55-60 and 63), carry out the preparation of single sample such as Table 5-7 shows.
  • Methylation level 2 - ⁇ Ct sample to be tested / 2 - ⁇ Ct positive standard ⁇ 100%.
  • ⁇ Ct Ct target gene - Ct internal reference gene.
  • methylation levels of FTAP2B, SOD3, RP11-867G23.12, EMX2OS, TEAD3 and DGKG genes in the tissue genome are shown in Figure 5; the ROC curve analysis of the methylation levels of each gene is shown in Figure 6, and each The AUC of the genes were all greater than 0.6.
  • Example 4 DPYS gene and RARG gene combined for discrimination of benign and malignant thyroid nodules
  • the applicant performed methylation-specific PCR on the tissue DNA of 10 patients with thyroid cancer and 10 patients with benign thyroid nodules, and found that the DNA methylation levels of DPYS and RARG genes were different in the cases of thyroid cancer and benign thyroid nodules , the result is shown in Figure 7.
  • MethylCode TM Bisulfite Conversion Kit (Thermo, product number: MECOV50) to perform bisulfite conversion on the DNA obtained in step 1, and unmethylated cytosine (cytosine, C) is converted into uracil (uracil, U); Methylated cytosines are unchanged after conversion.
  • the PCR mixture includes PCR reaction solution, primer mixture, and probe mixture to prepare a single sample.
  • the primer mix contains a pair of primers for DPYS gene, RARG gene and internal reference gene.
  • Exemplary primers are shown in SEQ ID NO:11,12,27,28,61,62; exemplary probes are shown in SEQ ID NO:46,54,63.
  • the PCR reaction system is as follows:
  • the scores of DPYS and RARG genes are shown in Table 9, and the ROC analysis is shown in Figure 7. According to the interpretation standard, 0 cases of 10 cases of benign thyroid nodules were positive, and 10 cases of 10 cases of thyroid cancer were positive, the specificity reached 100%, and the sensitivity was 100%.
  • Example 5 Combination of TSHR gene and SLC16A3 gene for discrimination of benign and malignant thyroid nodules
  • MethylCode TM Bisulfite Conversion Kit (Thermo, product number: MECOV50) to perform bisulfite conversion on the DNA obtained in step 1, and unmethylated cytosine (cytosine, C) is converted into uracil (uracil, U); Methylated cytosines are unchanged after conversion.
  • the PCR mixture includes PCR reaction solution, primer mixture, and probe mixture to prepare a single sample.
  • the primer mix contains a pair of primers for TSHR gene, SLC16A3 gene and internal reference gene.
  • Exemplary primers are shown in SEQ ID NO:3,4,25,26,61,62; exemplary probes are shown in SEQ ID NO:42,53,63.
  • the PCR reaction system is as follows:
  • the scores of TSHR and SLC16A3 genes are shown in Table 10, and the ROC analysis is shown in Figure 8. According to the interpretation standard, 1 case was positive in 20 cases of benign thyroid nodules, and 9 cases were positive in 20 cases of thyroid cancer, with a specificity of 90% and a sensitivity of 90%.
  • Example 6 Combination of FTAP2B gene and EMX2OS gene for discrimination of benign and malignant thyroid nodules
  • MethylCode TM Bisulfite Conversion Kit (Thermo, product number: MECOV50) to perform bisulfite conversion on the DNA obtained in step 1, and unmethylated cytosine (cytosine, C) is converted into uracil (uracil, U); Methylated cytosines are unchanged after conversion.
  • the PCR mixture includes PCR reaction solution, primer mixture, and probe mixture to prepare a single sample.
  • the primer mix contains a pair of primers for TSHR gene, SLC16A3 gene and internal reference gene.
  • Exemplary primers are shown in SEQ ID NO: 29, 30, 35, 36, 61, 62; exemplary probes are shown in SEQ ID NO: 55, 58, 63.
  • the PCR reaction system is as follows:
  • the scores of FTAP2B and EMX2OS genes are shown in Table 11, and the ROC analysis is shown in Figure 9. According to the interpretation criteria, 2 out of 10 benign thyroid nodules were positive, and 9 out of 10 thyroid cancer were positive, with a specificity of 100% and a sensitivity of 90%.
  • Example 7 Combination of RP11-867G23.12 gene and TEAD3 gene for discrimination of benign and malignant thyroid nodules
  • MethylCode TM Bisulfite Conversion Kit (Thermo, product number: MECOV50) to perform bisulfite conversion on the DNA obtained in step 1, and unmethylated cytosine (cytosine, C) is converted into uracil (uracil, U); Methylated cytosines are unchanged after conversion.
  • the PCR mixture includes PCR reaction solution, primer mixture, and probe mixture to prepare a single sample.
  • the primer mix contains a pair of primers for TSHR gene, SLC16A3 gene and internal reference gene.
  • Exemplary primers are shown in SEQ ID NO: 33, 34, 37, 38, 61, 62; exemplary probes are shown in SEQ ID NO: 57, 59, 63.
  • the PCR reaction system is as follows:
  • the scores of RP11-867G23.12 and TEAD3 genes are shown in Table 12, and the ROC analysis is shown in Figure 10. According to the interpretation criteria, 2 out of 10 benign thyroid nodules were positive, and 7 out of 10 thyroid cancer were positive, with a specificity of 90% and a sensitivity of 70%.
  • Example 8 Combination of TSHR gene and RARG gene for discrimination of benign and malignant thyroid nodules
  • cfDNA was extracted from the plasma of 196 cases of thyroid cancer and 148 cases of benign thyroid nodules using the QIAamp DNA Mini Kit (QIAGEN, product number: 51304); the DNA concentration was detected using the Qubit TM dsDNA HS Assay Kit (Thermo, product number: Q32854); Quality checks were performed using 1% agarose gel electrophoresis.
  • MethylCode TM Bisulfite Conversion Kit (Thermo, product number: MECOV50) to perform bisulfite conversion on the DNA obtained in step 1, and unmethylated cytosine (cytosine, C) is converted into uracil (uracil, U); Methylated cytosines are unchanged after conversion.
  • the PCR mixture includes PCR reaction solution, primer mixture, and probe mixture to prepare a single sample.
  • the primer mix contains a pair of primers for TSHR gene, SLC16A3 gene and internal reference gene.
  • Exemplary primers are shown in SEQ ID NO:3,4,27,28,61,62; exemplary probes are shown in SEQ ID NO:42,54,63.
  • the PCR reaction system is as follows:
  • Example 9 MCC gene and CDH1 gene combined for discrimination of benign and malignant thyroid nodules
  • the applicant performed methylation-specific PCR on the plasma cfDNA of 196 patients with thyroid cancer and 148 patients with benign thyroid nodules, and found that the methylation levels of MCC and CDH1 genes in the tissue DNA of thyroid cancer and benign thyroid nodules have significant The difference is shown in Figure 12.
  • cfDNA was extracted from the plasma of 196 cases of thyroid cancer and 148 cases of benign thyroid nodules using the QIAamp DNA Mini Kit (QIAGEN, product number: 51304); the DNA concentration was detected using the Qubit TM dsDNA HS Assay Kit (Thermo, product number: Q32854); Quality checks were performed using 1% agarose gel electrophoresis.
  • MethylCode TM Bisulfite Conversion Kit (Thermo, product number: MECOV50) to perform bisulfite conversion on the DNA obtained in step 1, and unmethylated cytosine (cytosine, C) is converted into uracil (uracil, U); Methylated cytosines are unchanged after conversion.
  • the PCR mixture includes PCR reaction solution, primer mixture, and probe mixture to prepare a single sample.
  • the primer mix contains a pair of primers for MCC gene, CDH1 gene and internal reference gene.
  • Exemplary primers are shown in SEQ ID NO:1,2,5,6,61,62; exemplary probes are shown in SEQ ID NO:41,42,63.
  • the PCR reaction system is as follows:
  • Example 10 Combination of SOD3 gene, EMX2OS gene and TEAD3 gene for discrimination of benign and malignant thyroid nodules
  • the applicant performed methylation-specific PCR on the plasma cfDNA of 90 patients with thyroid cancer and 54 patients with benign thyroid nodules, and found that the DNA methylation of SOD3, EMX2OS and TRAD3 genes in thyroid cancer and benign thyroid nodules cases There are differences in levels, and the results are shown in Figure 13.
  • cfDNA was extracted from the plasma of 90 cases of thyroid cancer and 54 cases of benign thyroid nodules using the QIAamp DNA Mini Kit (QIAGEN, product number: 51304); the DNA concentration was detected using the Qubit TM dsDNA HS Assay Kit (Thermo, product number: Q32854); Quality checks were performed using 1% agarose gel electrophoresis.
  • MethylCode TM Bisulfite Conversion Kit (Thermo, product number: MECOV50) to perform bisulfite conversion on the DNA obtained in step 1, and unmethylated cytosine (cytosine, C) is converted into uracil (uracil, U); Methylated cytosines are unchanged after conversion.
  • the PCR mixture includes PCR reaction solution, primer mixture, and probe mixture to prepare a single sample.
  • the primer mix contains a pair of primers for each of the SOD3 gene, the EMX2OS gene, the TEAD3 gene and the internal reference gene.
  • Exemplary primers are shown in SEQ ID NO:31,32,35,36,37,38,61,62; exemplary probes are shown in SEQ ID NO:56,58,59,63.
  • the PCR reaction system is as follows:
  • Example 11 Combination of FTAP2B gene and DGKG gene for discrimination of benign and malignant thyroid nodules
  • the applicant performed methylation-specific PCR on the plasma cfDNA of 90 patients with thyroid cancer and 54 patients with benign thyroid nodules, and found that the methylation levels of FTAP2B and DGKG genes in the tissue DNA of thyroid cancer and benign thyroid nodules have significant The difference is shown in Figure 14.
  • cfDNA was extracted from the plasma of 90 cases of thyroid cancer and 54 cases of benign thyroid nodules using the QIAamp DNA Mini Kit (QIAGEN, product number: 51304); the DNA concentration was detected using the Qubit TM dsDNA HS Assay Kit (Thermo, product number: Q32854); Quality checks were performed using 1% agarose gel electrophoresis.
  • MethylCode TM Bisulfite Conversion Kit (Thermo, product number: MECOV50) to perform bisulfite conversion on the DNA obtained in step 1, and unmethylated cytosine (cytosine, C) is converted into uracil (uracil, U); Methylated cytosines are unchanged after conversion.
  • the PCR mixture includes PCR reaction solution, primer mixture, and probe mixture to prepare a single sample.
  • the primer mix contains a pair of primers for the FTAP2B gene, the DGKG gene, and the internal reference gene.
  • Exemplary primers are shown in SEQ ID NO:29,30,39,40,61,62; exemplary probes are shown in SEQ ID NO:55,60,63.
  • the PCR reaction system is as follows
  • Example 12 TTC34 gene, RCOR2 gene, SLC16A3 gene and RARG gene combined for discrimination of benign and malignant thyroid nodules
  • Methylation-specific PCR was performed on the plasma cfDNA of 196 patients with thyroid cancer and 148 patients with benign thyroid nodules. Kylation levels were different, and the results are shown in Figure 15.
  • cfDNA was extracted from the plasma of 196 cases of thyroid cancer and 148 cases of benign thyroid nodules using the QIAamp DNA Mini Kit (QIAGEN, product number: 51304); the DNA concentration was detected using the Qubit TM dsDNA HS Assay Kit (Thermo, product number: Q32854); Quality checks were performed using 1% agarose gel electrophoresis.
  • MethylCode TM Bisulfite Conversion Kit (Thermo, product number: MECOV50) to perform bisulfite conversion on the DNA obtained in step 1, and unmethylated cytosine (cytosine, C) is converted into uracil (uracil, U); Methylated cytosines are unchanged after conversion.
  • the PCR mixture includes PCR reaction solution, primer mixture, and probe mixture to prepare a single sample.
  • the primer mixture comprises a pair of primers for TTC34 gene, RCOR2 gene, SLC16A3 gene, RARG gene and internal reference gene.
  • Exemplary primers are shown in SEQ ID NO: 17, 18, 19, 20, 25, 26, 27, 28, 61, 62;
  • Exemplary probes are shown in SEQ ID NO: 49, 50, 53, 54, 63 shown.
  • the PCR reaction system is as follows:
  • Example 13 The combination of SOD3 gene, EMX2OS gene and FTAP2B gene for distinguishing benign from malignant thyroid nodules
  • Methylation-specific PCR was performed on the plasma cfDNA of 90 patients with thyroid cancer and 54 patients with benign thyroid nodules, and it was found that SOD3, EMX2OS and FTAP2B genes had significant effects on the DNA methylation levels of thyroid cancer and benign thyroid nodules. The difference is shown in Figure 16.
  • cfDNA was extracted from the plasma of 90 cases of thyroid cancer and 54 cases of benign thyroid nodules using the QIAamp DNA Mini Kit (QIAGEN, product number: 51304); the DNA concentration was detected using the Qubit TM dsDNA HS Assay Kit (Thermo, product number: Q32854); Quality checks were performed using 1% agarose gel electrophoresis.
  • MethylCode TM Bisulfite Conversion Kit (Thermo, product number: MECOV50) to perform bisulfite conversion on the DNA obtained in step 1, and unmethylated cytosine (cytosine, C) is converted into uracil (uracil, U); Methylated cytosines are unchanged after conversion.
  • the PCR mixture includes PCR reaction solution, primer mixture, and probe mixture to prepare a single sample.
  • the primer mix contains a pair of primers for SOD3 gene, EMX2OS gene, FTAP2B gene and internal reference gene.
  • Exemplary primers are shown in SEQ ID NO:31,32,35,36,29,30,61,62; exemplary probes are shown in SEQ ID NO:56,58,55,63.
  • the PCR reaction system is as follows:
  • Example 14 Combination of TTC34 gene, RCOR2 gene, ITPKA gene and CDH1 gene for discrimination of benign and malignant thyroid nodules
  • Methylation-specific PCR was performed on the plasma cfDNA of 196 patients with thyroid cancer and 148 patients with benign thyroid nodules. Kylation levels were different, and the results are shown in Figure 17.
  • cfDNA was extracted from the plasma of 196 cases of thyroid cancer and 148 cases of benign thyroid nodules using the QIAamp DNA Mini Kit (QIAGEN, product number: 51304); the DNA concentration was detected using the Qubit TM dsDNA HS Assay Kit (Thermo, product number: Q32854); Quality checks were performed using 1% agarose gel electrophoresis.
  • MethylCode TM Bisulfite Conversion Kit (Thermo, product number: MECOV50) to perform bisulfite conversion on the DNA obtained in step 1, and unmethylated cytosine (cytosine, C) is converted into uracil (uracil, U); Methylated cytosines are unchanged after conversion.
  • the PCR mixture includes PCR reaction solution, primer mixture, and probe mixture to prepare a single sample.
  • the primer mixture comprises a pair of primers for TTC34 gene, RCOR2 gene, ITPKA gene, CDH1 gene and internal reference gene.
  • Exemplary primers are shown in SEQ ID NO: 17, 18, 19, 20, 23, 24, 1, 2, 61, 62; exemplary probes are shown in SEQ ID NO: 49, 50, 52, 41, 63 shown.
  • the PCR reaction system is as follows:
  • Example 15 Combination of SOD3 gene, RP11-867G23.12 gene and FTAP2B gene for discrimination of benign and malignant thyroid nodules
  • Methylation-specific PCR was performed on the plasma cfDNA of 90 patients with thyroid cancer and 54 patients with benign thyroid nodules, and found that the SOD3 gene, RP11-867G23. There are differences in the methylation levels of , and the results are shown in FIG. 18 .
  • cfDNA was extracted from the plasma of 90 cases of thyroid cancer and 54 cases of benign thyroid nodules using the QIAamp DNA Mini Kit (QIAGEN, product number: 51304); the DNA concentration was detected using the Qubit TM dsDNA HS Assay Kit (Thermo, product number: Q32854); Quality checks were performed using 1% agarose gel electrophoresis.
  • MethylCode TM Bisulfite Conversion Kit (Thermo, product number: MECOV50) to perform bisulfite conversion on the DNA obtained in step 1, and unmethylated cytosine (cytosine, C) is converted into uracil (uracil, U); Methylated cytosines are unchanged after conversion.
  • the PCR mixture includes PCR reaction solution, primer mixture, and probe mixture to prepare a single sample.
  • the primer mixture contains a pair of primers for SOD3 gene, RP11-867G23.12 gene, FTAP2B gene and internal reference gene.
  • Exemplary primers are shown in SEQ ID NO:31,32,33,34,29,30,61,62; exemplary probes are shown in SEQ ID NO:56,57,55,63.
  • the PCR reaction system is as follows:
  • Example 16 The combination of ITPKA gene, RCOR2 gene, SLC16A3 gene and MCC gene for distinguishing benign from malignant thyroid nodules
  • Methylation-specific PCR was performed on the plasma cfDNA of 196 patients with thyroid cancer and 148 patients with benign thyroid nodules, and it was found that ITPKA gene, RCOR2 gene, SLC16A3 gene and MCC gene were the most important in the DNA methylation of thyroid cancer and benign thyroid nodules cases. Kylation levels were different, and the results are shown in Figure 19.
  • cfDNA was extracted from the plasma of 196 cases of thyroid cancer and 148 cases of benign thyroid nodules using QIAamp DNA Mini Kit (QIAGEN, catalog number: 51304); the concentration of DNA was detected using Qubit TM dsDNA HS Assay Kit (Thermo, catalog number: Q32854); Quality checks were performed using 1% agarose gel electrophoresis.
  • MethylCode TM Bisulfite Conversion Kit (Thermo, product number: MECOV50) to perform bisulfite conversion on the DNA obtained in step 1, and unmethylated cytosine (cytosine, C) is converted into uracil (uracil, U); Methylated cytosines are unchanged after conversion.
  • the PCR mixture includes PCR reaction solution, primer mixture, and probe mixture to prepare a single sample.
  • the primer mixture comprises a pair of primers for ITPKA gene, RCOR2 gene, SLC16A3 gene, MCC gene and internal reference gene.
  • Exemplary primers are shown in SEQ ID NO:23, 24, 19, 20, 25, 26, 5, 6, 61, 62; exemplary probes are shown in SEQ ID NO: 52, 50, 53, 43, 63 shown.
  • the PCR reaction system is as follows:
  • Example 17 TTC34 gene, ITPKA gene, SLC16A3 gene and RARG gene combined for discrimination of benign and malignant thyroid nodules
  • the applicant performed methylation-specific PCR on the plasma cfDNA of 196 patients with thyroid cancer and 148 patients with benign thyroid nodules, and found that TTC34 gene, ITPKA gene, SLC16A3 gene and RARG gene were significantly different in the tissue DNA of thyroid cancer and benign thyroid nodules cases. There are differences in the methylation levels of , and the results are shown in Figure 20.
  • cfDNA was extracted from the plasma of 196 cases of thyroid cancer and 148 cases of benign thyroid nodules using the QIAamp DNA Mini Kit (QIAGEN, product number: 51304); the DNA concentration was detected using the Qubit TM dsDNA HS Assay Kit (Thermo, product number: Q32854); Quality checks were performed using 1% agarose gel electrophoresis.
  • MethylCode TM Bisulfite Conversion Kit (Thermo, product number: MECOV50) to perform bisulfite conversion on the DNA obtained in step 1, and unmethylated cytosine (cytosine, C) is converted into uracil (uracil, U); Methylated cytosines are unchanged after conversion.
  • the PCR mixture includes PCR reaction solution, primer mixture, and probe mixture to prepare a single sample.
  • the primer mixture comprises a pair of primers for TTC34 gene, ITPKA gene, SLC16A3 gene, RARG gene and internal reference gene.
  • Exemplary primers are shown in SEQ ID NO: 17, 18, 23, 24, 25, 26, 27, 28, 61, 62; exemplary probes are shown in SEQ ID NO: 49, 52, , 53, 54, 63.
  • the PCR reaction system is as follows:

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Abstract

本发明公开了甲状腺结节良恶性相关标志物及其应用，所述应用包括引物分子和/或探针分子在制备诊断甲状腺结节良恶性的检测试剂或诊断试剂盒中的应用，以及装置在制备诊断甲状腺结节良恶性的诊断试剂盒中的应用等；其中，所述应用所涉及的引物分子、探针分子、装置和目标标志物等如文中所述。本发明也包括用于检测并诊断个体甲状腺结节良恶性的装置，其所包含的处理器运行时可获取样品中所述一个或多个目标标志物至少一个CpG二核苷酸的甲基化水平或状态，并根据该甲基化水平或状态判读甲状腺结节良恶性。

Description

甲状腺结节良恶性相关标志物及其应用 技术领域
本发明属于分子辅助诊断领域，涉及甲状腺结节良恶性相关标志物及其应用。
背景技术
DNA甲基化是表观遗传的一种机制，是真核细胞基因组常见的表观遗传学修饰，能够在不改变DNA序列的情况下改变遗传表现。所谓DNA甲基化是指在DNA甲基化转移酶(methyltransferase)的作用下，在基因组CpG二核苷酸的胞嘧啶5号碳位共价结合一个甲基基团。DNA甲基化在细胞增殖、分化、发育等方面起重要作用，与肿瘤的发生、发展关系密切，其效应有转录抑制、染色质结构调节、X染色体失活、基因组印记等。DNA甲基化异常可以通过影响染色质结构以及癌基因和抑癌基因的表达而参与肿瘤的发生和进展。
CpG二核苷酸是哺乳动物发生DNA甲基化的最主要靶点，分布于整个染色体组。健康人基因组中，CpG岛中的CpG位点通常处于非甲基化状态，而在CpG岛外的CpG位点则通常高甲基化，这种甲基化的形式在细胞分裂的过程中能够稳定保留。当肿瘤发生时，通常抑癌基因非CpG岛区的CpG位点甲基化程度降低，而CpG岛中的CpG则呈高度甲基化状态，DNA甲基化后核苷酸顺序及其组成虽未发生改变，但会导致染色质结构改变及抑癌基因表达的降低。
随着过去十几年来遗传学和表观遗传学的不断发展，越来越多的研究者认识到肿瘤的发生并不完全由遗传基因决定，表观遗传学的后天影响同样发挥重要作用。甲状腺癌中的表观遗传学改变主要体现为抑癌基因和甲状腺相关基因的异常甲基化。研究甲状腺癌的DNA甲基化可以为我们提供新的分子标志物，为早期诊断、治疗方案选择和预后评估提供可靠依据。
甲状腺结节(Thyroid nodule)是甲状腺细胞异常增生后在甲状腺内形成的固体或者液体填充的肿块，可随着吞咽动作随甲状腺而上下移动，甲状腺是位于颈部底部、胸骨上方的一个小腺体。甲状腺结节非常常见，尽管大多数甲状腺结节并不严重，不会引起症状，但还是有一部分甲状腺结节进展为甲状腺癌。为了更早的诊断和治疗甲 状腺癌，同时降低非必要的手术治疗，亟需一种高灵敏度和特异性的体外诊断方法(In-Vitro Diagnostics,IVD)对甲状腺结节的良恶性进行鉴别。
美国甲状腺协会(American Thyroid Association,ATA)将甲状腺结节定义为甲状腺上的一种离散型病变，借助影像学检查，可观测到结节与正常甲状腺组织结构不同，存在相对边界。目前对甲状腺结节的评估主要通过超声(ultrasonography,US)、细针穿刺活检(fine needle aspiration biopsy,FNAB)、电子计算机断层扫描(Computed Tomography)、核磁共振成像(Magnetic Resonance Imaging，MRI)和正电子发射计算机断层扫描(Positron Emission Computed Tomography，PET)。在甲状腺结节的诊断程序中，高分辨率的超声(US)是甲状腺结节的首选检查，可对甲状腺结节进行筛查和评估，判断其大小、类型和结节的内部结构。提示甲状腺结节为恶性的US征象包括：结节的高度大于宽度(OR＝10.15)、缺乏声晕(OR＝7.14)、微小钙化(OR＝6.76)、边界不规则(OR＝6.12)、回声减低(OR＝5.07)、实性结节(OR＝4.69)、结节内部血流丰富(OR＝3.76)等。通过超声检测到的结节通常与甲状腺功能异常无关，且高分辨率甲状腺超声的滥用使得大量无临床意义的结节(包括甲状腺微小癌)被检出，可能使临床评估偏离甲状腺功能异常。事实上这些结节(包括甲状腺微小癌)绝大部分对患者的危害甚小，可能根本无需处理，但很多患者却因此背上了沉重的精神负担。目前，细胞学检查结果中还有高达20％的结节为不确定甲状腺结节(indeterminate thyroid nodules)，这部分结节需要结合分子检测，市面上已有
Figure PCTCN2022102658-appb-000001
Gene Expression Classifier和ThyroSeqv2产品，前者阳性预测值(positive predictive value,PPV)很低，只有46％；后者PPV也只有42％-77％。
因此，开发具有低成本、无创、适合临床推广的甲状腺结节良恶性鉴别的高特异性、高灵敏度的分子诊断工具，对有效控制甲状腺癌的发病率和更深入的研究其发病机制具有重要意义。
发明内容
本发明的目的是提供一种用于鉴别个体甲状腺结节良恶性的组合物，通过运用该组合物检测含有选自CDH1基因，TSHR基因，MCC基因，TBX15基因，PRR15基因，DPYS基因，GRIA2基因，NR5A1基因，TTC34基因，RCOR2基因，F10基因，ITPKA基因，SLC16A3基因，RARG基因，FTAP2B基因，SOD3基因，RP11-867G23.12基因，EMX2OS基因，TEAD3基因和DGKG基因中目标位点的一个或多个的甲基化情况，实现该个体甲状腺结节的良恶性鉴别。
本发明第一方面提供引物分子和/或探针分子在制备诊断个体甲状腺结节良恶性的检测试剂或诊断试剂盒中的应用，以及装置在制备诊断个体甲状腺结节良恶性的诊断试剂盒中的应用；所述引物分子相同于、互补于或在严谨条件下杂交于一个或多个目标标志物并包含至少9个连续的核苷酸；所述探针分子与一个或多个目标标志物的扩增产物在严谨条件下杂交；所述装置用于确定一个或多个目标标志物中的至少一个CpG二核苷酸的甲基化状态或水平。
本发明第二方面提供区分基因组DNA至少一个靶区域内甲基化和未甲基化CpG二核苷酸的至少一种试剂或成组试剂在制备用于检测和/或分类个体中甲状腺结节良恶性的方法的试剂盒中的用途，其中所述方法包括使从所述个体生物样品中分离的基因组DNA与所述至少一种试剂或成组试剂接触，其中所述靶区域等同于或互补于一个或多个目标标志物的至少16连续核苷酸的序列，其中所述连续核苷酸包含至少一个CpG二核苷酸序列，由此至少部分地提供对甲状腺结节良恶性的检测和/或分类。
本发明第三方面提供将5位未甲基化的胞嘧啶碱基转化为尿嘧啶或在杂交性能方面可检测地不同于胞嘧啶的其它碱基的一种或多种试剂、扩增酶以及至少一种包含至少9个连续核苷酸的引物在制备用于检测和/或分类个体中甲状腺结节良恶性的方法的试剂盒中的用途，其中所述方法包括：a)从所述个体生物样品分离基因组DNA；b)用所述一种或多种试剂处理a)的所述基因组DNA或其片段；c)使所述经处理的基因组DNA或其经处理的片段与所述扩增酶和所述至少一种引物接触，所述引物相同于、互补于或在严谨条件下杂交于一个或多个目标标志物，其中所述经处理的基因组DNA或其片段被扩增以产生至少一种扩增产物或不被扩增；以及d)基于所述扩增物是否存在或其性质，确定所述一个或多个目标标志物的至少一个CpG二核苷酸的甲基化状态或水平，或者反映所述一个或多个目标标志物的多个CpG二核苷酸平均甲基化状态或水平的均值或值，由此至少部分地检测和/或分类个体中甲状腺结节的良恶性。
本发明第四方面提供一种或多种甲基化敏感限制酶和扩增酶以及至少一种包含至少9个连续核苷酸的引物在制备用于检测和/或分类个体中甲状腺结节良恶性的方法的试剂盒中的用途，其中，所述引物相同于、互补于或在严谨条件下杂交于一个或多个目标标志物；所述方法包括：a)从所述个体生物样品分离基因组DNA；b)以所述一种或多种甲基化敏感限制酶消化a)所述的基因组DNA或其片段，使所得消化产物与所述扩增酶和所述至少一种引物接触；和c)基于所述扩增物是否存在或其性质，确定所述一个或多个目标标志物的至少一个CpG二核苷酸的甲基化状态或水 平，由此至少部分地检测和/或分类个体中甲状腺结节的良恶性。
本发明第五方面提供衍生自一个或多个目标标志物的经处理的核酸在制备用于诊断个体甲状腺结节良恶性的试剂盒中的用途，其中所述处理适合于将所述一个或多个目标标志物的至少一个未甲基化的胞嘧啶碱基转化至尿嘧啶或在杂交上可检测地不同于胞嘧啶的其它碱基。
本发明第六方面提供一种用于检测目标标志物至少一个CpG二核苷酸的甲基化水平或甲基化状态以诊断个体甲状腺结节良恶性的诊断试剂或诊断试剂盒，其包含引物分子和/或探针分子；其中，所述引物分子相同于、互补于或在严谨条件下杂交于一个或多个目标标志物并包含至少9个连续的核苷酸，所述探针分子与一个或多个目标标志物的扩增产物在严谨条件下杂交。
本发明第七方面提供一种诊断个体或患者的甲状腺结节良恶性的方法，所述方法包括检测该个体或患者的生物样品中一个或多个目标标志物至少一个CpG二核苷酸的甲基化水平或甲基化状态，并基于所述甲基化水平来诊断其甲状腺结节良恶性。
本发明第八方面提供一种用于检测并诊断个体甲状腺结节良恶性的装置，所述装置包括存储器、处理器以及存储在存储器上并可在处理器上运行的计算机程序，所述处理器执行所述程序时实现以下步骤：(1)获取样品中一个或多个目标标志物至少一个CpG二核苷酸的甲基化水平或甲基化状态，和(2)根据(1)的甲基化水平判读甲状腺结节良恶性。
本发明上述各方面所述的方案中，所述一个或多个目标标志物选自：CDH1基因或基因组的CDH1序列、TSHR基因或基因组的TSHR序列、MCC基因或基因组的MCC序列、TBX15基因或基因组的TBX15序列、PRR15基因或基因组的PRR15序列、DPYS基因或基因组的DPYS序列、GRIA2基因或基因组的GRIA2序列、NR5A1基因或基因组的NR5A1序列、TTC34基因或基因组的TTC34序列、RCOR2基因或基因组的RCOR2序列、F10基因或基因组的F10序列、ITPKA基因或基因组的ITPKA序列、SLC16A3基因或基因组的SLC16A3序列、RARG基因或基因组的RARG序列、FTAP2B基因或基因组的FTAP2B序列、SOD3基因或基因组的SOD3序列、RP11-867G23.12基因或基因组的RP11-867G23.12序列、EMX2OS基因或基因组的EMX2OS序列、TEAD3基因或基因组的TEAD3序列和DGKG基因或基因组的DGKG序列。
优选地，所述目标标志物为SLC16A3基因或基因组的SLC16A3序列以及选自TSHR基因或基因组的TSHR序列、TTC34基因或基因组的TTC34序列、ROCR2基 因或基因组的ROCR2序列、RARG基因或基因组的RARG序列、MCC基因或基因组的MCC序列和ITPKA基因或基因组的ITPKA序列中的一种或多种。
优选地，所述目标标志物为SLC16A3基因或基因组的SLC16A3序列和TSHR基因或基因组的TSHR序列；优选地，检测来自该个体的甲状腺结节中的该目标标志物的甲基化水平。
优选地，所述目标标志物为SLC16A3基因或基因组的SLC16A3序列和ROCR2基因或基因组的ROCR2序列，以及选自TTC34基因或基因组的TTC34序列、RARG基因或基因组的RARG序列、ITPKA基因或基因组的ITPKA序列和MCC基因或基因组的MCC序列中的至少一个、至少两个或至少三个；优选地，所述目标标志物为SLC16A3基因或基因组的SLC16A3序列、ROCR2基因或基因组的ROCR2序列、TTC34基因或基因组的TTC34序列和RARG基因或基因组的RARG序列，或所述目标标志物为SLC16A3基因或基因组的SLC16A3序列、ROCR2基因或基因组的ROCR2序列、ITPKA基因或基因组的ITPKA序列和MCC基因或基因组的MCC序列；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。
优选地，所述目标标志物为SLC16A3基因或基因组的SLC16A3序列和RARG基因或基因组的RARG序列，以及选自TTC34基因或基因组的TTC34序列、ROCR2基因或基因组的ROCR2序列和ITPKA基因或基因组的ITPKA序列的至少一个、至少两个或全部三个；优选地，所述目标标志物为SLC16A3基因或基因组的SLC16A3序列、ROCR2基因或基因组的ROCR2序列、TTC34基因或基因组的TTC34序列和RARG基因或基因组的RARG序列，或所述目标标志物为SLC16A3基因或基因组的SLC16A3序列、TTC34基因或基因组的TTC34序列、ITPKA基因或基因组的ITPKA序列和RARG基因或基因组的RARG序列；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。
优选地，所述目标标志物为SLC16A3基因或基因组的SLC16A3序列和TTC34基因或基因组的TTC34序列，以及选自ROCR2基因或基因组的ROCR2序列、RARG基因或基因组的RARG序列和ITPKA基因或基因组的ITPKA序列的至少一个、至少两个或全部三个；优选地，所述目标标志物为SLC16A3基因或基因组的SLC16A3序列、ROCR2基因或基因组的ROCR2序列、TTC34基因或基因组的TTC34序列和RARG基因或基因组的RARG序列，或所述目标标志物为SLC16A3基因或基因组的SLC16A3序列、TTC34基因或基因组的TTC34序列、ITPKA基因或基因组的ITPKA序列和RARG基因或基因组的RARG序列；优选地，检测来自该个体的血液样品如 血浆中这些目标标志物的甲基化水平。
优选地，所述目标标志物为SLC16A3基因或基因组的SLC16A3序列和ITPKA基因或基因组的ITPKA序列，以及选自ROCR2基因或基因组的ROCR2序列、MCC基因或基因组的MCC序列、TTC34基因或基因组的TTC34序列和RARG基因或基因组的RARG序列和的至少一个、至少两个或全部三个；优选地，所述目标标志物为SLC16A3基因或基因组的SLC16A3序列、ROCR2基因或基因组的ROCR2序列、ITPKA基因或基因组的ITPKA序列和MCC基因或基因组的MCC序列，或所述目标标志物为SLC16A3基因或基因组的SLC16A3序列、TTC34基因或基因组的TTC34序列、ITPKA基因或基因组的ITPKA序列和RARG基因或基因组的RARG序列；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。
优选地，所述目标标志物为SLC16A3基因或基因组的SLC16A3序列、TTC34基因或基因组的TTC34序列和RARG基因或基因组的RARG序列，以及ROCR2基因或基因组的ROCR2序列或ITPKA基因或基因组的ITPKA序列；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。
优选地，所述目标标志物为FTAP2B基因或基因组的FTAP2B序列以及选自EMS2OS基因或基因组的EMS2OS序列、DGKG基因或基因组的DGKG序列、SOD3基因或基因组的SOD3序列、RP11-867G23.12基因或基因组的RP11-867G23.12序列中的至少一个、至少两个或至少三个。
优选地，所述目标标志物为FTAP2B基因或基因组的FTAP2B序列、EMS2OS基因或基因组的EMS2OS序列以及任选的SOD3基因或基因组的SOD3序列；优选地，所述目标标志物为FTAP2B基因或基因组的FTAP2B序列和EMS2OS基因或基因组的EMS2OS序列，其中，检测来自该个体的甲状腺结节中的这些目标标志物的甲基化水平；优选地，所述目标标志物为FTAP2B基因或基因组的FTAP2B序列、EMS2OS基因或基因组的EMS2OS序列以及SOD3基因或基因组的SOD3序列，其中，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。
优选地，所述目标标志物为为FTAP2B基因或基因组的FTAP2B序列和DGKG基因或基因组的DGKG序列，其中，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。
优选地，所述目标标志物为FTAP2B基因或基因组的FTAP2B序列和SOD3基因或基因组的SOD3序列，以及EMS2OS基因或基因组的EMS2OS序列或RP11-867G23.12基因或基因组的RP11-867G23.12序列，其中，检测血液样品如血浆 中这些目标标志物的甲基化水平。
优选地，所述目标标志物为RARG基因或基因组的RARG序列，以及选自DPYS基因或基因组的DPYS序列、TSHR基因或基因组的TSHR序列、TTC34基因或基因组的TTC34序列、RCOR2基因或基因组的RCOR2序列、SLC16A3基因或基因组的SLC16A3序列和ITPKA基因或基因组的ITPKA序列中的至少一个、至少两个、至少三个或至少四个。
优选地，所述目标标志物为RARG基因或基因组的RARG序列和DPYS基因或基因组的DPYS序列，其中，检测来自该个体的甲状腺结节中的这些目标标志物的甲基化水平。
优选地，所述目标标志物为RARG基因或基因组的RARG序列和TSHR基因或基因组的TSHR序列，其中，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。
优选地，所述目标标志物为RARG基因或基因组的RARG序列和TTC34基因或基因组的TTC34序列，以及选自RCOR2基因或基因组的RCOR2序列、SLC16A3基因或基因组的SLC16A3序列和ITPKA基因或基因组的ITPKA序列中的至少一个或至少两个，其中，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平；优选地，所述目标标志物为RARG基因或基因组的RARG序列、TTC34基因或基因组的TTC34序列、RCOR2基因或基因组的RCOR2序列和SLC16A3基因或基因组的SLC16A3序列其中，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平；优选地，所述目标标志物为RARG基因或基因组的RARG序列、TTC34基因或基因组的TTC34序列、ITPKA基因或基因组的ITPKA序列和SLC16A3基因或基因组的SLC16A3序列其中，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。
优选地，所述目标标志物为RARG基因或基因组的RARG序列和SLC16A3基因或基因组的SLC16A3序列，以及选自TTC34基因或基因组的TTC34序列、RCOR2基因或基因组的RCOR2序列和ITPKA基因或基因组的ITPKA序列中的至少一个或至少两个，其中，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平；优选地，所述目标标志物为RARG基因或基因组的RARG序列、TTC34基因或基因组的TTC34序列、RCOR2基因或基因组的RCOR2序列和SLC16A3基因或基因组的SLC16A3序列其中，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平；优选地，所述目标标志物为RARG基因或基因组的RARG序列、TTC34基 因或基因组的TTC34序列、ITPKA基因或基因组的ITPKA序列和SLC16A3基因或基因组的SLC16A3序列其中，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。
优选地，所述目标标志物为RARG基因或基因组的RARG序列、SLC16A3基因或基因组的SLC16A3序列和TTC34基因或基因组的TTC34序列，以及ROCR2基因或基因组的ROCR2序列或ITPKA基因或基因组的ITPKA序列；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。
优选地，所述目标标志物为SOD3基因或基因组的SOD3序列，以及选自EMS2OS基因或基因组的EMS2OS序列、TEAD3基因或基因组的TEAD3序列、FTAP2B基因或基因组的FTAP2B序列和RP11-867G23.12基因或基因组的RP11-867G23.12序列中的至少一个或至少两个。
优选地，所述目标标志物为SOD3基因或基因组的SOD3序列和EMS2OS基因或基因组的EMS2OS序列，以及TEAD3基因或基因组的TEAD3序列或FTAP2B基因或基因组的FTAP2B序列；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。
优选地，所述目标标志物为SOD3基因或基因组的SOD3序列和FTAP2B基因或基因组的FTAP2B序列，以及EMS2OS基因或基因组的EMS2OS序列或RP11-867G23.12基因或基因组的RP11-867G23.12序列；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。
优选地，所述目标标志物为TEAD3基因或基因组的TEAD3序列，以及选自RP11-867G23.12基因或基因组的RP11-867G23.12序列、SOD3基因或基因组的SOD3序列和EMS2OS基因或基因组的EMS2OS序列中的至少一种或至少两种；优选地，所述目标标志物为TEAD3基因或基因组的TEAD3序列和RP11-867G23.12基因或基因组的RP11-867G23.12序列；优选地，检测来自该个体的甲状腺结节中的这些目标标志物的甲基化水平；优选地，所述目标标志物为TEAD3基因或基因组的TEAD3序列，SOD3基因或基因组的SOD3序列和EMS2OS基因或基因组的EMS2OS序列；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。
优选地，所述目标标志物为TTC34基因或基因组的TTC34序列，以及选自RCOR2基因或基因组的RCOR2序列、ITPKA基因或基因组的ITPKA序列、CDH1基因或基因组的CDH1序列、SLC16A3基因或基因组的SLC16A3序列和RARG基因或基因组的RARG序列中的至少一个、至少两个或至少三个；优选地，检测来自 该个体的血液样品如血浆中这些目标标志物的甲基化水平。
优选地，所述目标标志物为TTC34基因或基因组的TTC34序列和RCOR2基因或基因组的RCOR2序列，以及选自ITPKA基因或基因组的ITPKA序列、CDH1基因或基因组的CDH1序列、SLC16A3基因或基因组的SLC16A3序列和RARG基因或基因组的RARG序列中的至少一个或至少两个；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。
优选地，所述目标标志物为TTC34基因或基因组的TTC34序列、RCOR2基因或基因组的RCOR2序列、ITPKA基因或基因组的ITPKA序列和CDH1基因或基因组的CDH1序列；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。
优选地，所述目标标志物为TTC34基因或基因组的TTC34序列、RCOR2基因或基因组的RCOR2序列、SLC16A3基因或基因组的SLC16A3序列和RARG基因或基因组的RARG序列；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。
优选地，所述目标标志物为TTC34基因或基因组的TTC34序列和ITPKA基因或基因组的ITPKA序列，以及选自RCOR2基因或基因组的RCOR2序列、CDH1基因或基因组的CDH1序列、SLC16A3基因或基因组的SLC16A3序列和RARG基因或基因组的RARG序列中的至少一个或至少两个；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。
优选地，所述目标标志物为TTC34基因或基因组的TTC34序列、ITPKA基因或基因组的ITPKA序列、RCOR2基因或基因组的RCOR2序列和CDH1基因或基因组的CDH1序列；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平；
优选地，所述目标标志物为TTC34基因或基因组的TTC34序列、ITPKA基因或基因组的ITPKA序列、SLC16A3基因或基因组的SLC16A3序列和RARG基因或基因组的RARG序列；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。
优选地，所述目标标志物为TTC34基因或基因组的TTC34序列和SLC16A3基因或基因组的SLC16A3序列，以及选自RCOR2基因或基因组的RCOR2序列、ITPKA基因或基因组的ITPKA序列和RARG基因或基因组的RARG序列中的至少一个或至少两个；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。
优选地，所述目标标志物为TTC34基因或基因组的TTC34序列、SLC16A3基因或基因组的SLC16A3序列、RCOR2基因或基因组的RCOR2序列和RARG基因或基因组的RARG序列；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平；优选地，所述目标标志物为TTC34基因或基因组的TTC34序列、SLC16A3基因或基因组的SLC16A3序列、ITPKA基因或基因组的ITPKA序列和RARG基因或基因组的RARG序列；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。
优选地，目标标志物为TTC34基因或基因组的TTC34序列、SLC16A3基因或基因组的SLC16A3序列和RARG基因或基因组的RARG序列，以及RCOR2基因或基因组的RCOR2序列或ITPKA基因或基因组的ITPKA序列；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。
优选地，所述目标标志物为MCC基因或基因组的MCC序列，以及选自ITPKA基因或基因组的ITPKA序列、RCOR2基因或基因组的RCOR2序列、SLC16A3基因或基因组的SLC16A3序列和CDH1基因或基因组的CDH1序列中的至少一个、至少两个或至少三个；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平；优选地，目标标志物为MCC基因或基因组的MCC序列、ITPKA基因或基因组的ITPKA序列、RCOR2基因或基因组的RCOR2序列和SLC16A3基因或基因组的SLC16A3序列；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平；优选地，目标标志物为MCC基因或基因组的MCC序列和CDH1基因或基因组的CDH1序列；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。
优选地，所述目标标志物为CDH1基因或基因组的CDH1序列，以及选自MCC基因或基因组的MCC序列、TTC34基因或基因组的TTC34序列、RCOR2基因或基因组的RCOR2序列和ITPKA基因或基因组的ITPKA序列中的至少一个、至少两个或至少三个；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平；优选地，目标标志物为CDH1基因或基因组的CDH1序列和MCC基因或基因组的MCC序列；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平；优选地，所述目标标志物为CDH1基因或基因组的CDH1序列、TTC34基因或基因组的TTC34序列、RCOR2基因或基因组的RCOR2序列和ITPKA基因或基因组的ITPKA序列；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。
优选地，所述各基因的Hg19坐标如下：
CDH1基因，chr16:68771195-68869439；TSHR基因，chr14:81422025-81610697；MCC基因，chr5:112357803-112630635；TBX15基因，chr1:119427355-119530418；PRR15基因，chr7:29605149-29606911；DPYS基因，chr8:105391659-105479283；GRIA2基因，chr4:158141272-158287226；NR5A1基因，chr9:127245034-127265678；TTC34基因，chr1:2572708-2717433；RCOR2基因，chr11:63678702-63684636；F10基因，chr13:113777113-113803843；ITPKA基因，chr15:41786456-41795364；SLC16A3基因，chr17:80193650-80197375；RARG基因，chr12:53604350-53626040；FTAP2B基因，chr6:50786439-50815326；SOD3基因，chr4:24797195-24802464；RP11-867G23.12基因，chr11:66101965-66107346；EMX2OS基因，chr10:119243804-119302100；TEAD3基因，chr6:35441374-35464856；DGKG基因，chr3:185864990-186080023。
优选地，所述一个或多个目标标志物为所述各基因采用以下引物扩增得到的一个或多个目标区域：CDH1基因的目标区域：SEQ ID NO:1和2所示的序列或与SEQ ID NO:1和2具有至少90％相同性的序列作为引物扩增得到的CDH1基因的片段；TSHR基因的目标区域：SEQ ID NO:3和4所示的序列或与SEQ ID NO:3和4具有至少90％相同性的序列作为引物扩增得到的TSHR基因的片段；MCC基因的目标区域：SEQ ID NO:5和6所示的序列或与SEQ ID NO:5和6具有至少90％相同性的序列作为引物扩增得到的MCC基因的片段；TBX15基因的目标区域：SEQ ID NO:7和8所示的序列或与SEQ ID NO:7和8具有至少90％相同性的序列作为引物扩增得到的TBX15基因的片段；PRR15基因的目标区域：SEQ ID NO:9和10所示的序列或与SEQ ID NO:9和10具有至少90％相同性的序列作为引物扩增得到的PRR15基因的片段；DPYS基因的目标区域：SEQ ID NO:11和12所示的序列或与SEQ ID NO:11和12具有至少90％相同性的序列作为引物扩增得到的DPYS基因的片段；GRIA2基因的目标区域：SEQ ID NO:13和14所示的序列或与SEQ ID NO:13和14具有至少90％相同性的序列作为引物扩增得到的GRIA2基因的片段；NR5A1基因的目标区域：SEQ ID NO:15和16所示的序列或与SEQ ID NO:15和16具有至少90％相同性的序列作为引物扩增得到的NR5A1基因的片段；TTC34基因的目标区域：SEQ ID NO:17和18所示的序列或与SEQ ID NO:17和18具有至少90％相同性的序列作为引物扩增得到的TTC34基因的片段；RCOR2基因的目标区域：SEQ ID NO:19和20所示的序列或与SEQ ID NO:19和20具有至少90％相同性的序列作为引物扩增得到的RCOR2基因的片段；F10基因的目标区域：SEQ ID NO:21和22所示的序列或与SEQ ID NO:21和22具有 至少90％相同性的序列作为引物扩增得到的F10基因的片段；ITPKA基因的目标区域：SEQ ID NO:23和24所示的序列或与SEQ ID NO:23和24具有至少90％相同性的序列作为引物扩增得到的ITPKA基因的片段；SLC16A3基因的目标区域：SEQ ID NO:25和26所示的序列或与SEQ ID NO:25和26具有至少90％相同性的序列作为引物扩增得到的SLC16A3基因的片段；RARG基因的目标区域：SEQ ID NO:27和28所示的序列或与SEQ ID NO:27和28具有至少90％相同性的序列作为引物扩增得到的RARG基因的片段；FTAP2B基因的目标区域：SEQ ID NO:29和30所示的序列或与SEQ ID NO:29和30具有至少90％相同性的序列作为引物扩增得到的FTAP2B基因的片段；SOD3基因的目标区域：SEQ ID NO:31和32所示的序列或与SEQ ID NO:31和32具有至少90％相同性的序列作为引物扩增得到的SOD3基因的片段；RP11-867G23.12基因的目标区域：SEQ ID NO:33和34所示的序列或与SEQ ID NO:33和34具有至少90％相同性的序列作为引物扩增得到的RP11-867G23.12基因的片段；EMX2OS基因的目标区域：SEQ ID NO:35和36所示的序列或与SEQ ID NO:35和36具有至少90％相同性的序列作为引物扩增得到的EMX2OS基因的片段；TEAD3基因的目标区域：SEQ ID NO:37和38所示的序列或与SEQ ID NO:37和38具有至少90％相同性的序列作为引物扩增得到的TEAD3基因的片段；DGKG基因的目标区域：SEQ ID NO:39和40所示的序列或与SEQ ID NO:39和40具有至少90％相同性的序列作为引物扩增得到的DGKG基因的片段。
优选地，所述各基因的目标区域的Hg19坐标如下：CDH1基因的目标区域，chr16:68770965-68771133；TSHR基因的目标区域，chr14:81421913-81422134；MCC基因的目标区域，chr5:112538899-112539205；TBX15基因的目标区域，chr1:119535640-119535860；PRR15基因的目标区域，chr7:29606122-29606390；DPYS基因的目标区域，chr8:105478808-105479066；GRIA2基因的目标区域，chr4:158141305-158141513；NR5A1基因的目标区域，chr9:127265730-127265942；TTC34基因的目标区域，chr1:2706494-2706702；RCOR2基因的目标区域，chr11:63687060-63687339；F10基因的目标区域，chr13:113807232-113807480；ITPKA基因的目标区域，chr15:41793298-41793573；SLC16A3基因的目标区域，chr17:80189548-80189879；RARG基因的目标区域，chr12:53613026-53613303；FTAP2B基因的目标区域，chr6:50818141-50818368；SOD3基因的目标区域，chr4:24801668-24801925；RP11-867G23.12基因的目标区域，chr11:66104360-66104678；EMX2OS基因的目标区域，chr10:119295848-119296135；TEAD3基因的目标区域， chr6:35454128-35454333；DGKG基因的目标区域，chr3:185973693-185973973。
优选地，所述各基因的引物分子分别为：CDH1：SEQ ID NO:1和2，或与其扩增得到的片段在严谨条件下杂交的引物分子；TSHR：SEQ ID NO:3和4，或与其扩增得到的片段在严谨条件下杂交的引物分子；MCC：SEQ ID NO:5和6，或与其扩增得到的片段在严谨条件下杂交的引物分子；TBX15：SEQ ID NO:7和8，或与其扩增得到的片段在严谨条件下杂交的引物分子；PRR15：SEQ ID NO:9和10，或与其扩增得到的片段在严谨条件下杂交的引物分子；DPYS：SEQ ID NO:11和12，或与其扩增得到的片段在严谨条件下杂交的引物分子；GRIA2：SEQ ID NO:13和14，或与其扩增得到的片段在严谨条件下杂交的引物分子；NR5A1：SEQ ID NO:15和16，或与其扩增得到的片段在严谨条件下杂交的引物分子；TTC34：SEQ ID NO:17和18，或与其扩增得到的片段在严谨条件下杂交的引物分子；RCOR2：SEQ ID NO:19和20，或与其扩增得到的片段在严谨条件下杂交的引物分子；F10：SEQ ID NO:21和22，或与其扩增得到的片段在严谨条件下杂交的引物分子；ITPKA：SEQ ID NO:23和24，或与其扩增得到的片段在严谨条件下杂交的引物分子；SLC16A3：SEQ ID NO:25和26，或与其扩增得到的片段在严谨条件下杂交的引物分子；RARG：SEQ ID NO:27和28，或与其扩增得到的片段在严谨条件下杂交的引物分子；FTAP2B：SEQ ID NO:29和30，或与其扩增得到的片段在严谨条件下杂交的引物分子；SOD3：SEQ ID NO:31和32，或与其扩增得到的片段在严谨条件下杂交的引物分子；RP11-867G23.12：SEQ ID NO:33和34，或与其扩增得到的片段在严谨条件下杂交的引物分子；EMX2OS：SEQ ID NO:35和36，或与其扩增得到的片段在严谨条件下杂交的引物分子；TEAD3：SEQ ID NO:37和38，或与其扩增得到的片段在严谨条件下杂交的引物分子；DGKG：SEQ ID NO:39和40，或与其扩增得到的片段在严谨条件下杂交的引物分子。
优选地，所述各基因的扩增产物的探针分子分别为：CDH1：SEQ ID NO:41或与其具有至少90％相同性的序列；TSHR：SEQ ID NO:42或与其具有至少90％相同性的序列；MCC：SEQ ID NO:43或与其具有至少90％相同性的序列；TBX15：SEQ ID NO:44或与其具有至少90％相同性的序列；PRR15：SEQ ID NO:45或与其具有至少90％相同性的序列；DPYS：SEQ ID NO:46或与其具有至少90％相同性的序列；GRIA2：SEQ ID NO:47或与其具有至少90％相同性的序列；NR5A1：SEQ ID NO:48或与其具有至少90％相同性的序列；TTC34：SEQ ID NO:49或与其具有至少90％相同性的序列；RCOR2：SEQ ID NO:50或与其具有至少90％相同性的序列；F10：SEQ ID NO:51或与其具有至少90％相同性的序列；ITPKA：SEQ ID NO:52或与其具有至少90％相 同性的序列；SLC16A3：SEQ ID NO:53或与其具有至少90％相同性的序列；RARG：SEQ ID NO:54或与其具有至少90％相同性的序列；FTAP2B：SEQ ID NO:55或与其具有至少90％相同性的序列；SOD3：SEQ ID NO:56或与其具有至少90％相同性的序列；RP11-867G23.12：SEQ ID NO:57或与其具有至少90％相同性的序列；EMX2OS：SEQ ID NO:58或与其具有至少90％相同性的序列；TEAD3：SEQ ID NO:59或与其具有至少90％相同性的序列；DGKG：SEQ ID NO:60或与其具有至少90％相同性的序列。
优选地，所述各方面的各实施方案中，所述检测试剂或诊断试剂盒还包括检测内参基因ACTB的引物分子和/或探针分子，以及所述方法还包括使用内参基因ACTB的引物分子和/或探针分子；优选地，内参基因ACTB的引物分子为SEQ ID NO:61和62所示的序列与由SEQ ID NO:61和62作为引物扩增得到的ACTB基因的片段在严谨条件下杂交的序列，内参基因ACTB的探针包含SEQ ID NO:63所示序列或与其有至少90％相同性的序列。
优选地，所述各方面的各实施方案中，所用的探针还含有可检测物。在一个或多个实施方案中，所述可检测物是本领域常规使用的5’端荧光报告基团和3’端标记淬灭基团。在一个或多个实施方案中，所述荧光报告基因选自Cy5、Texas Red、FAM和VIC。
优选地，所述各方面的各实施方案中，所述检测试剂或诊断试剂盒还包括选自以下一种或多种物质：PCR缓冲液、聚合酶、dNTP、限制性内切酶、酶切缓冲液、荧光染料、荧光淬灭剂、荧光报告剂、外切核酸酶、碱性磷酸酶、内标、对照物、KCl、MgCl 2和(NH 4) 2SO 4。
优选地，所述各方面的各实施方案中，所述检测试剂或诊断试剂盒还包括经转化的阳性标准品，其中未甲基化的胞嘧啶转化为不与鸟嘌呤结合的碱基。在一个或多个实施方案中，所述阳性标准品是完全甲基化的。
优选地，所述各方面的各实施方案中，检测甲基化的试剂是选自以下方法的一个或多个中所用的试剂：基于重亚硫酸盐转化的PCR(例如甲基化特异性PCR)、DNA测序(如亚硫酸氢盐测序、全基因组甲基化测序、简化甲基化测序)、甲基化敏感的限制性内切酶分析法、荧光定量法、甲基化敏感性高分辨率熔解曲线法、基于芯片的甲基化图谱分析、质谱(例如飞行质谱)。优选地，所述试剂选自以下一种或多种：重亚硫酸盐及其衍生物、荧光染料、荧光淬灭剂、荧光报告剂、内标、对照物。
优选地，所述各方面的各实施方案中，所述个体或患者为哺乳动物，尤其是人。
优选地，所述各方面的各实施方案中，所述样品来自哺乳动物的组织、细胞或者体液，例如甲状腺组织或血液。在一个或多个实施方案中，所述样品是甲状腺结节活检物，优选是细针穿刺活检物。在一个或多个实施方案中，所述样品是血浆。
在一个或多个实施方案中，所述样品来自具有甲状腺良性或恶性结节的对象。在一个或多个实施方案中，所述样品来自甲状腺肿大的患者。
在一个或多个实施方案中，所述DNA是全基因组DNA和/或cfDNA。
在一个或多个实施方案中，所述DNA经转化，其中未甲基化的胞嘧啶转化为不与鸟嘌呤结合的碱基。所述转化使用酶促方法进行，优选脱氨酶处理，或所述转化使用非酶促方法进行，优选用亚硫酸氢盐或重硫酸盐处理。
在一个或多个实施方案中，所述检测包括但不限于：基于重亚硫酸盐转化的PCR(例如甲基化特异性PCR)、荧光定量法。
在一个或多个实施方案中，所述检测是荧光定量PCR检测，还包括使用本文所述探针。
在一个或多个实施方案中，PCR的反应液包含Taq DNA聚合酶、PCR缓冲液(buffer)、dNTPs、KCl、MgCl 2和(NH 4) 2SO 4。优选地，Taq DNA聚合酶为热启动Taq DNA聚合酶。优选地，Mg 2+终浓度为1.0-20.0mM。
在一个或多个实施方案中，PCR中各引物浓度为100-500nM。
在一个或多个实施方案中，PCR中各探针浓度为100-500nM。
在一个或多个实施方案中，PCR反应条件为，95℃预变性5min；95℃变性15s，60℃退火延伸60s，50个循环。
在一个或多个实施方案中，判读方法为：根据各基因的甲基化水平计算得分，得分大于0则结果为阳性，即甲状腺结节为恶性结节。在一个或多个实施方案中，得分小于0则结果为阴性，即甲状腺结节为良性结节。
在PCR实施方案中，优选地，甲基化水平＝2 –ΔCt待检样品/2 –ΔCt阳性标准品×100％，其中，ΔCt＝Ct 目的基因–Ct 内参基因。
在测序实施方案中，甲基化水平＝甲基化碱基数/总碱基数。
本发明的主要优点在于：通过同时进行多个靶序列的检测，提高了甲状腺结节良恶性鉴别的灵敏度和特异性。采用PCR法，操作简单，结果易于判读，对仪器要求不高，以试剂盒的形式更方便临床上的推广应用。
附图说明
图1：10例甲状腺癌和10例甲状腺良性结节组织DNA中CDH1、TSHR、MCC、TBX15、PRR15、DPYS、GRIA2、NR5A1、TTC34、RCOR2、F10、ITPKA、SLC16A3、RARG、FTAP2B、SOD3、RP11-867G23.12、EMX2OS、TEAD3和DGKG基因的甲基化水平图。
图2：10例甲状腺癌患者和10例甲状腺良性结节患者组织DNA中CDH1、TSHR、MCC、TBX15、PRR15、DPYS、GRIA2、NR5A1、TTC34、RCOR2、F10、ITPKA、SLC16A3、RARG、FTAP2B、SOD3、RP11-867G23.12、EMX2OS、TEAD3和DGKG基因各自甲基化水平的ROC曲线分析图。
图3：196例甲状腺癌患者血浆和148例甲状腺良性结节患者血浆cfDNA中CDH1、TSHR、MCC、TBX15、PRR15、DPYS、GRIA2、NR5A1、TTC34、RCOR2、F10、ITPKA、SLC16A3和RARG基因的甲基化水平分析图。
图4：196例甲状腺癌患者和148例甲状腺良性结节患者血浆cfDNA中CDH1、TSHR、MCC、TBX15、PRR15、DPYS、GRIA2、NR5A1、TTC34、RCOR2、F10、ITPKA、SLC16A3和RARG基因各自甲基化水平的ROC曲线分析图。
图5：79例甲状腺癌患者和22例甲状腺良性结节患者的血浆cfDNA中FTAP2B、SOD3、RP11-867G23.12、EMX2OS、TEAD3和DGKG基因的甲基化水平图。
图6：79例甲状腺癌患者和22例甲状腺良性结节患者的血浆cfDNA中FTAP2B、SOD3、RP11-867G23.12、EMX2OS、TEAD3和DGKG基因各自的ROC曲线分析图。
图7：10例甲状腺癌患者和10例甲状腺良性结节患者的组织DNA中DPYS和RARG基因的ROC曲线分析图。
图8：10例甲状腺癌患者和10例甲状腺良性结节患者的组织DNA中TSHR和SLC16A3基因的ROC曲线分析图。
图9：10例甲状腺癌患者和10例甲状腺良性结节患者的组织DNA中FTAP2B和EMX2OS基因的ROC曲线分析图。
图10：10例甲状腺癌患者和10例甲状腺良性结节患者的组织DNA中RP11-867G23.12基因和TEAD3基因的ROC曲线分析图。
图11：196例甲状腺癌患者和148例甲状腺良性结节患者血浆cfDNA中TSHR和RARG基因的ROC曲线分析图。
图12：196例甲状腺癌患者和148例甲状腺良性结节患者血浆cfDNA中MCC和CDH1基因的ROC曲线分析图。
图13：79例甲状腺癌患者和22例甲状腺良性结节患者的血浆cfDNA中SOD3、 EMX2OS和TRAD3基因的ROC曲线分析图。
图14：90例甲状腺癌患者和54例甲状腺良性结节患者的血浆cfDNA中FTAP2B和DGKG基因的ROC曲线分析图。
图15：196例甲状腺癌患者和148例甲状腺良性结节患者的血浆cfDNA中TTC34基因、RCOR2基因、SLC16A3基因和RARG基因的ROC曲线分析图。
图16：90例甲状腺癌患者和54例甲状腺良性结节患者的血浆cfDNA中SOD3基因、EMX2OS基因和FTAP2B基因的ROC曲线分析图。
图17：196例甲状腺癌患者和148例甲状腺良性结节患者的血浆cfDNA中TTC34基因、RCOR2基因、ITPKA基因和CDH1基因的ROC曲线分析图。
图18：90例甲状腺癌患者和54例甲状腺良性结节患者的血浆cfDNA中SOD3基因、RP11-867G23.12基因和FTAP2B基因的ROC曲线分析图。
图19：196例甲状腺癌患者和148例甲状腺良性结节患者的血浆cfDNA中ITPKA基因、RCOR2基因、SLC16A3基因和MCC基因的ROC曲线分析图。
图20：196例甲状腺癌患者和148例甲状腺良性结节患者的血浆cfDNA中TTC34基因、ITPKA基因、SLC16A3基因和RARG基因的ROC曲线分析图。
图中，BTN指甲状腺良性结节患者，PTC指甲状腺癌患者。*表示p<0.05，**表示p<0.01，***表示p<0.001。
具体实施方式
虽然本申请公开了本申请的各个方面和各种实施方式，但是本领域技术人员可以在不脱离本申请的精神和范围的前提下做出各种等同改变或修改。本申请公开的各个方面和各种实施方式均是示例性的，并不旨在限制本申请的范围，本申请的实际保护范围以权利要求书为准。除非另有说明，否则本申请中使用的所有技术和科学术语均是本领域技术人员通常理解的含义。本申请引用的所有参考文献、专利和专利申请均通过引用并入本申请。
需注意的是，在本申请的说明书和权利要求书中，单数形式的“一个”、“一种”和“所述”均包括其复数形式，除非上下文另有说明。因此，例如，“一种试剂”包括多种试剂。
在本申请的说明书和权利要求书，除非另有说明，否则术语“包含”、“包括”或“含有”是指含有所列出的数值、步骤或成分，但也不排除还含有其他数值、步骤或成分。
经过深入的研究，本发明人发现了一些与恶性甲状腺结节相关的目标标志物，这些目标标志物包括：CDH1基因或基因组的CDH1序列、TSHR基因或基因组的TSHR序列、MCC基因或基因组的MCC序列、TBX15基因或基因组的TBX15序列、PRR15基因或基因组的PRR15序列、DPYS基因或基因组的DPYS序列、GRIA2基因或基因组的GRIA2序列、NR5A1基因或基因组的NR5A1序列、TTC34基因或基因组的TTC34序列、RCOR2基因或基因组的RCOR2序列、F10基因或基因组的F10序列、ITPKA基因或基因组的ITPKA序列、SLC16A3基因或基因组的SLC16A3序列、RARG基因或基因组的RARG序列、FTAP2B基因或基因组的FTAP2B序列、SOD3基因或基因组的SOD3序列、RP11-867G23.12基因或基因组的RP11-867G23.12序列、EMX2OS基因或基因组的EMX2OS序列、TEAD3基因或基因组的TEAD3序列和DGKG基因或基因组的DGKG序列。通过对来自个体的包含DNA的生物样品中的目标标志物中的一个或多个的甲基化水平进行检测可鉴别甲状腺结节良恶性。
I.目标标志物及其目标区域
如本文所用，术语“目标标志物”是指这样的目的核酸或基因区域：其甲基化水平指示着结甲状腺结节良恶性。术语“目标标志物”应被认为包括其所有转录变体及其所有启动子和调控元件。如本领域技术人员所理解的，已知某些基因在个体之间表现出等位基因变异或单核苷酸多态性(“SNP”)。SNP包括不同长度的简单的重复序列(例如二核苷酸和三核苷酸重复)的插入和缺失。因此，本申请应被理解为扩展到由任何其他突变、多态性或等位基因变异产生的标志物/基因的所有形式。另外，应当理解，术语“目标标志物”应既包括标志物或基因的正义链序列，也包括标志物或基因的反义链序列。
本文所用的术语“目标标志物”被宽泛地解释为既包括1)在生物样品或基因组DNA中发现的原始标志物(处于特定的甲基化状态)，也包括2)其经过处理的序列(例如亚硫酸氢盐转化后的对应区域或MSRE处理后的对应区域)。亚硫酸氢盐转化后的对应区域与基因组序列中的目标标志物不同之处在于，一个或多个未甲基化的胞嘧啶残基被转化为尿嘧啶碱基、胸腺嘧啶碱基或在杂交行为上与胞嘧啶不同的其他碱基。经MSRE处理的对应区域与基因组序列中的目标标志物不同之处在于，该序列在一个或多个MSRE切割位点处被切割。
在本文中，应该理解的是，本文所述各产品、用途和方法中所涉及的目标标志物CDH1、TSHR、MCC、TBX15、PRR15、DPYS、GRIA2、NR5A1、TTC34、RCOR2、 F10、ITPKA、SLC16A3、RARG、FTAP2B、SOD3、RP11-867G23.12、EMX2OS、TEAD3和DGKG基因既可通过引用其名称又可通过其染色体坐标来进行描述。所述染色体坐标与2009年2月发布的人类基因组数据库Hg19版本一致(在本文中称为“Hg19坐标”)。应理解的是，本所述的某个基因及其基因组的序列也包括各基因的含有至少一个CpG二核苷酸序列的片段。在一些实施方案中，该片段为本文所述的各基因的目标区域。
在一些实施方案中，所述各基因的目标区域分别为：
(1)由SEQ ID NO:1和2或与其有至少90％相同性的序列作为引物扩增得到的CDH1基因的片段；
(2)由SEQ ID NO:3和4或与其有至少90％相同性的序列作为引物扩增得到的TSHR基因的片段；
(3)由SEQ ID NO:5和6或与其有至少90％相同性的序列作为引物扩增得到的MCC基因的片段；
(4)由SEQ ID NO:7和8或与其有至少90％相同性的序列作为引物扩增得到的TBX15基因的片段；
(5)由SEQ ID NO:9和10或与其有至少90％相同性的序列作为引物扩增得到的PRR15基因的片段；
(6)由SEQ ID NO:11和12或与其有至少90％相同性的序列作为引物扩增得到的DPYS基因的片段；
(7)由SEQ ID NO:13和14或与其有至少90％相同性的序列作为引物扩增得到的GRIA2基因的片段；
(8)由SEQ ID NO:15和16或与其有至少90％相同性的序列作为引物扩增得到的NR5A1基因的片段；
(9)由SEQ ID NO:17和18或与其有至少90％相同性的序列作为引物扩增得到的TTC34基因的片段；
(10)由SEQ ID NO:19和20或与其有至少90％相同性的序列作为引物扩增得到的RCOR2基因的片段；
(11)由SEQ ID NO:21和22或与其有至少90％相同性的序列作为引物扩增得到的F10基因的片段；
(12)由SEQ ID NO:23和24或与其有至少90％相同性的序列作为引物扩增得到的ITPKA基因的片段；
(13)由SEQ ID NO:25和26或与其有至少90％相同性的序列作为引物扩增得到的SLC16A3基因的片段；
(14)由SEQ ID NO:27和28或与其有至少90％相同性的序列作为引物扩增得到的RARG基因的片段；
(15)由SEQ ID NO:29和30或与其有至少90％相同性的序列作为引物扩增得到的FTAP2B基因的片段；
(16)由SEQ ID NO:31和32或与其有至少90％相同性的序列作为引物扩增得到的SOD3基因的片段；
(17)由SEQ ID NO:33和34或与其有至少90％相同性的序列作为引物扩增得到的RP11-867G23.12基因的片段；
(18)由SEQ ID NO:35和36或与其有至少90％相同性的序列作为引物扩增得到的EMX2OS基因的片段；
(19)由SEQ ID NO:37和38或与其有至少90％相同性的序列作为引物扩增得到的TEAD3基因的片段；和
(20)由SEQ ID NO:39和40或与其有至少90％相同性的序列作为引物扩增得到的DGKG基因的片段。
在优选的实施方案中，所述基因及其目标区域的Hg19坐标定义如下：
目标标志物 目标区域 基因位置
CDH1 chr16:68770965-68771133 chr16:68771195-68869439
TSHR chr14:81421913-81422134 chr14:81422025-81610697
MCC chr5:112538899-112539205 chr5:112357803-112630635
TBX15 chr1:119535640-119535860 chr1:119427355-119530418
PRR15 chr7:29606122-29606390 chr7:29605149-29606911
DPYS chr8:105478808-105479066 chr8:105391659-105479283
GRIA2 chr4:158141305-158141513 chr4:158141272-158287226
NR5A1 chr9:127265730-127265942 chr9:127245034-127265678
TTC34 chr1:2706494-2706702 chr1:2572708-2717433
RCOR2 chr11:63687060-63687339 chr11:63678702-63684636
F10 chr13:113807232-113807480 chr13:113777113-113803843
ITPKA chr15:41793298-41793573 chr15:41786456-41795364
SLC16A3 chr17:80189548-80189879 chr17:80193650-80197375
RARG chr12:53613026-53613303 chr12:53604350-53626040
FTAP2B chr6:50818141-50818368 chr6:50786439-50815326
SOD3 chr4:24801668-24801925 chr4:24797195-24802464
RP11-867G23.12 chr11:66104360-66104678 chr11:66101965-66107346
EMX2OS chr10:119295848-119296135 chr10:119243804-119302100
TEAD3 chr6:35454128-35454333 chr6:35441374-35464856
DGKG chr3:185973693-185973973 chr3:185864990-186080023
本发明的目标标志物也包括上述每个区域的各个起始位点的上游5kb和各个末端位点的下游5kb。可在公共数据库(例如UCSC Genome Browser、Ensemble和NCBI网站)中获得上述Hg19坐标的特定核苷酸序列，以及每个区域的各个起始位点的上游5kb和各个末端位点的下游5kb。
本发明的目标标志物(如某个基因及其基因组的序列、或各基因的含有至少一个CpG二核苷酸序列的片段、或所述目标区域、或包含基因间隔区的序列)还包括非酶促法转化(如亚硫酸氢盐转化后的对应区域，以及酶促法转化(如MSRE转化)后获得的对应区域。
在一些实施方式中，本发明的目标标志物也包括上述各基因或各目标区域的各类变体。变体包括来自相同区域的、与本文所述的基因或区域具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的序列同一性(即，具有一个或多个缺失、插入、取代、反向序列等)的核酸序列。因此，本申请内容应理解为延伸至实现相同结果的此类变体，尽管事实上个体间的实际核酸序列具有微小的遗传变异。
如本文所用，术语“序列同一性的百分比(％)”是指候选序列的氨基酸(或核酸)残基和参考序列的氨基酸(或核酸)残基进行序列比对后的相同百分比，比对时可以引入间隔(如有必要)以使得相同的氨基酸(或核酸)数目达到最多。换言之，氨基酸序列(或核酸序列)的序列同一性百分比(％)可以通过用与参考序列相同的氨基酸残基(或碱基)的数目除以候选序列或参考序列中氨基酸残基(或碱基)的总数(以较短者为准)来计算。氨基酸残基的保守取代可以被认为或可以不被认为是相同的残基。可以通过以下方式来确定氨基酸(或核酸)序列同一性的百分比，例如， 可以使用公开的工具如BLASTN、BLASTp(可在美国国家生物技术信息中心(NCBI)的网站上获得，也可参见Altschul S.F.et al.,J.Mol.Biol.,215:403–410(1990)；Stephen F.et al.,Nucleic Acids Res.,25:3389–3402(1997))、ClustalW2(可在欧洲生物信息研究所的网站上找到)，也可参见Higgins D.G.et al.,Methods in Enzymology,266:383-402(1996)；Larkin M.A.et al.,Bioinformatics(Oxford,England),23(21):2947-8(2007))和ALIGN或Megalign(DNASTAR)软件。本领域技术人员可以使用所述工具提供的默认参数，或者可以(例如，通过选择合适的算法)定制适合比对的参数。
本发明的目标标志物也包括上述基因或目标区域、或上述目标区域的起始位点上游5kb和末端位点下游5kb经非酶促法转化(如亚硫酸氢盐转化)后的对应区域或经酶促方法处理(如甲基化敏感限制酶处理)后的对应区域。
II.目标标志物的来源及制备
本文中，所述目标标志物可以来自任何感兴趣的个体的生物样品。本文所用的术语“个体”包括人类和非人类的动物。非人类动物包括所有脊椎动物，例如哺乳动物和非哺乳动物。“个体”也可以是家畜，例如牛、猪、绵羊、家禽和马；或啮齿动物，例如大鼠、小鼠；或非人类灵长类动物，例如猿、猴、恒河猴；或家养的动物，例如狗或猫。在一些实施方式中，个体是人类或非人类灵长类动物。在一些实施方式中，个体是人类。在本申请中，“个体”、“对象”和“受试者”可互换使用。
应理解，上述第I部分给出的序列为人的序列。当涉及非人动物的序列时，可采用现有技术容易地确定上述基因在非人动物基因组中的对应位置和对应序列。
本文所用的术语“生物样品”是指获自或衍生自个体的生物组合物，其包含基于物理、生化、化学和/或生理特征待表征或待识别的细胞和/或其他分子实体(例如DNA)。生物样品包括但不限于通过本领域技术人员已知的任何方法获得的个体的细胞、组织、器官和/或生物体液。在一些实施方式中，所述生物样品选自下组：组织学切片、组织活检、石蜡包埋的组织、体液、手术切除样本、分离的血细胞、分离自血液的细胞，及其任意组合。在一些实施方式中，所述体液选自下组：全血、血清、血浆，及其任意组合。选择最适合的样品将取决于情境的性质。在一些实施方式中，所述生物样品为个体的全血。在一些实施方式中，所述生物样品为个体的血浆。本领域技术人员知道从全血制备血浆的各种方法。例如，在一些实施方式中，血浆通过将来自个体的全血离心一次、两次、三次、四次、五次或更多次来获得。在一些实施方 式中，所述生物样品是甲状腺结节活检物，优选是细针穿刺活检物。
待检测的DNA可分离自所述生物样品。可以通过使用本领域已知的各种方法从生物样品中分离和纯化出待检测的DNA。可使用市售试剂盒来进行分离和纯化。例如，通过以下方式从细胞和组织中分离DNA：在高度变性和还原条件下裂解原材料、部分使用蛋白质降解酶、纯化通过苯酚/氯仿提取工艺获得的核酸组分，并通过渗析或乙醇沉淀从水相中回收核酸(参见例如Sambrook,J.,Fritsch,E.F.in T.Maniatis,C S H,Molecular Cloning,1989)。又例如，现在有许多试剂体系特别适用于从琼脂糖凝胶中纯化DNA片段、从细菌裂解物中分离质粒DNA，以及从血液、组织或细胞培养物中分离较长链的核酸(基因组DNA、总细胞RNA)。许多这些可商购的纯化体系中是基于相当众所周知的原理，即，在不同离液盐的溶液的存在下将核酸与矿物载体相结合。在这些体系中，细磨的玻璃粉、硅藻土或硅胶的悬浮液被用作载体材料。在例如US7888006B2和EP1626085A1中描述了从生物样品中分离和纯化DNA的一些其他方法。在方法之间进行选择将受到几个因素的影响，包括时间、费用和所需的DNA数量。
在一些实施方式中，生物样品中包含的DNA包括基因组DNA。本文所用的术语“基因组DNA”是指包含细胞或生物体的完整基因组及其片段或部分的DNA。基因组DNA是来源于个体的大段DNA(例如长于大约10、20、30、40、50、60、70、80、90、100、200或300kb)，并且可以具有天然修饰，例如DNA甲基化。
在一些实施方式中，生物样品中包含的DNA包括细胞DNA。本文所用的术语“细胞DNA”是指存在于细胞内的DNA，或从体内细胞中获取DNA并在体外分离、或以其他方式在体外操作，只要该DNA未从体内细胞中移除。
在一些实施方式中，生物样品中包含的DNA包括细胞外游离DNA。本文所用的术语“细胞外游离DNA”是指在体内的细胞外存在的DNA片段。该术语也可以被用于指代获取自体内的细胞外来源并在体外分离、或操作的DNA片段。细胞外游离DNA中的DNA片段通常具有约100到200bp的长度，推测与被包裹于核小体的DNA片段的长度有关。细胞外游离DNA(cfDNA)包括例如细胞外游离胎儿DNA和循环肿瘤DNA。细胞外游离胎儿DNA在孕妇的体内(例如血液)中循环，代表胎儿基因组，而循环肿瘤DNA在癌症患者的体内(例如血液)中循环。在一些实施方式中，细胞外游离DNA可基本上不含个体的细胞DNA。例如，所述细胞外游离DNA可包含小于约1,000ng/mL、小于约100ng/mL、小于约10ng/mL、小于约1ng/mL的细胞DNA。
可以通过使用本领域已知的常规技术来制备细胞外游离DNA。例如，可以通过以约200-20,000g、约200-10,000g、约200-5,000g、约300-4000g等的速度离心血液样品约3-30分钟、约3-15分钟、约3-10分钟、约3-5分钟来获得血液样品的细胞外游离DNA。例如，在一些实施方式中，可以通过将个体的血浆或血清离心一、二、三、四、五次或更多次来获得血液样本的细胞外游离DNA。在一些实施方式中，为了从包含可溶性DNA的无细胞组分中分离细胞及其片段，可以通过微滤来获得所述生物样品。通常来说，微滤可以通过使用过滤器来进行，例如，0.1微米～0.45微米的膜过滤器，诸如0.22微米的膜过滤器。
在一些实施方式中，使用商购的DNA提取产品从全血、血清或血浆中提取细胞外游离DNA用于分析。这种提取方法据称对循环DNA的回收率高(>50％)，某些产品(例如Qiagen生产的QIAamp Circulating Nucleic Acid Kit)据称可提取小尺寸的DNA片段。所使用的典型样品量为1-5mL血清或血浆。
在一些实施方式中，细胞外游离DNA包括循环肿瘤DNA。循环肿瘤DNA(“ctDNA”)是与细胞无关的体液(例如血液、尿液、唾液、痰、粪便、胸膜液、脑脊液等)中肿瘤来源的片段化DNA。通常，ctDNA高度片段化，平均长度约为150个碱基对。ctDNA通常包括体液(例如血浆)中细胞外游离DNA的极小部分，例如ctDNA可能构成血浆DNA的不到约10％。通常，该百分比小于约1％，例如小于约0.5％或小于约0.01％。另外，血浆DNA的总量通常非常低，例如约10ng/mL血浆。ctDNA的数量因人而异，并且取决于肿瘤的类型、位置，对于癌性肿瘤，则取决于癌症的阶段。但是，ctDNA通常在体液中非常罕见，只能通过极其敏感和特异性的技术进行检测。检测ctDNA可能有助于检测和诊断肿瘤、指导肿瘤特异性治疗、监测治疗以及监测癌症的缓解。
III.碱基转化
本文中，DNA甲基化是(例如，通过DNA甲基转移酶的作用)将甲基添加到DNA分子上(例如，添加至DNA分子的一个或多个胞嘧啶碱基)的生物学过程。在哺乳动物中，DNA甲基化出现于胞嘧啶-磷酸-鸟嘌呤(CpG)二核苷酸(即“CpG位点”)的5’位置，当其出现在基因的启动子或第一个外显子中的5’-CpG-3’二核苷酸中时，会导致基因的表观遗传失活。已充分证明了DNA甲基化在调节基因表达、肿瘤发生、以及其他遗传和表观遗传疾病中起重要作用。
如本文所用，术语“甲基化的胞嘧啶残基”是指胞嘧啶残基的衍生物，其中一个 甲基连接至胞嘧啶环的碳原子上(例如C5)。术语“未甲基化的胞嘧啶残基”是指未衍生化的胞嘧啶残基，其中与“甲基化的胞嘧啶残基”相反，在胞嘧啶环的碳原子(例如C5)上没有甲基连接。其内的胞嘧啶残基被甲基化的CpG位点就是甲基化的CpG位点，而其内的胞嘧啶残基未被甲基化的CpG位点是未甲基化的CpG位点。
如本文所述，DNA或RNA的碱基之间可发生转化。本文所述“转化”、“胞嘧啶转化”或“CT转化”是利用非酶促或酶促方法处理DNA，将未修饰的胞嘧啶碱基(cytosine，C)转化为不与鸟嘌呤(G)结合的碱基(例如尿嘧啶碱基(uracil，U))的过程。一些试剂能够区分DNA中的未甲基化和甲基化的CpG位点，从而获得经处理的DNA。该试剂可以选择性地作用于未甲基化的胞嘧啶残基，但不能显著地作用于甲基化的胞嘧啶残基。或者该试剂可以选择性地作用于甲基化的胞嘧啶残基，而不显著地作用于未甲基化的胞嘧啶残基。例如，一些试剂可以选择性地将未甲基化的胞嘧啶残基转化为尿嘧啶、胸腺嘧啶或杂交上与胞嘧啶不同的另一碱基，而甲基化的胞嘧啶残基依然处于未转化状态；又例如，一些试剂可以选择性地切割甲基化的残基，或者选择性地切割未甲基化的残基。由此，原始DNA以取决于是否被甲基化的方式转化为经处理的DNA，从而可以通过其杂交行为将经处理的DNA与原始DNA区分开。
如本文所用，“经处理的DNA”、“经处理的序列”、“经处理的片段”是指已经用能够区分DNA、核酸序列、基因片段中的未甲基化和甲基化的CpG位点的试剂处理后的DNA、核酸序列、基因片段。
更具体而言，可采用非酶促或酶促方法进行胞嘧啶转化。示例性地，非酶促方法包括亚硫酸氢盐或重硫酸盐处理。在一些实施方式中，非酶促方法所用的试剂包括亚硫酸氢盐试剂。如本文所用，术语“亚硫酸氢盐试剂”是指，例如本申请所公开的可用于区分甲基化和未甲基化的CpG二核苷酸序列的包括亚硫酸氢盐、亚硫酸氢根离子或其任意组合的试剂。在本申请中，用亚硫酸氢盐试剂处理DNA也被描述为“亚硫酸氢盐反应”或“亚硫酸氢盐处理”，指的是转化未甲基化的胞嘧啶残基的反应，特别是在亚硫酸氢根离子存在的情况下，核酸中未甲基化的胞嘧啶残基被转化为尿嘧啶碱基、胸腺嘧啶碱基或在杂交行为上与胞嘧啶不同的其他碱基，而其中甲基化的胞嘧啶残基未被显著地转化。换言之，亚硫酸氢盐处理可用于区分甲基化的CpG二核苷酸和未甲基化的CpG二核苷酸。Frommer,M.,et al.,Proc Natl Acad Sci USA 89(1992)1827-31和Grigg,G.,Clark,S.,Bioessays 16(1994)431-6中详细描述了用于检测甲基化的胞嘧啶残基的亚硫酸氢盐反应。亚硫酸氢盐反应包括脱氨基步骤和脱磺酸基步 骤(参见Grigg and Clark,同上)。“甲基化的胞嘧啶残基未被显著地转化”这一陈述，不排除非常小的百分比(例如，小于0.1％、小于0.2％、小于0.3％、小于0.4％、小于0.5％、小于0.6％、小于0.7％、小于0.8％、小于0.9％、小于1％、小于2％、小于3％、小于4％、小于5％、小于6％、小于7％、小于8％、小于9％、小于10％、小于11％、小于12％、小于13％、小于14％、小于15％、小于16％、小于17％、小于18％、小于19％、小于20％)的甲基化的胞嘧啶残基被转化为尿嘧啶、胸腺嘧啶或在杂交行为上与胞嘧啶不同的其他碱基，尽管其意在仅仅转化未甲基化的胞嘧啶残基。
在例如参考Frommer M.,et al.(同上)或Grigg and Clark(同上)的情况下(它们公开了亚硫酸氢盐处理的基本参数)，本领域技术人员知道如何进行亚硫酸氢盐处理，特别是脱氨基步骤和脱磺酸基步骤。孵育时间和温度对脱氨基效率的影响、以及影响DNA降解的参数都已公开。
在一些实施方式中，所述亚硫酸氢盐试剂选自下组：亚硫酸氢铵、亚硫酸氢钠、亚硫酸氢钾、亚硫酸氢钙、亚硫酸氢镁、亚硫酸氢铝、亚硫酸氢根离子，及其任意组合。在一些实施方式中，所述亚硫酸氢盐试剂是亚硫酸氢钠。在一些实施方式中，亚硫酸氢盐试剂是可商购的，例如，MethylCode TM Bisulfite Conversion Kit、EpiMark TM Bisulfite Conversion Kit、EpiJET TM Bisulfite Conversion Kit、EZDNAMethylation-Gold TM Kit等。在一些实施方式中，根据试剂盒的使用说明书进行亚硫酸氢盐反应。
示例性的酶促方法包括脱氨酶处理，以及使用试剂选择性地切割未甲基化的残基但不切割甲基化的残基，或者选择性地切割甲基化的残基但不切割未甲基化的残基。优选地，所述试剂是甲基化敏感限制酶(MSRE)。
术语“甲基化敏感限制酶”是指根据其识别位点的甲基化状态而选择性地消化核酸的酶。对于当识别位点未被甲基化或半甲基化时才特异剪切的限制酶来说，当识别位点被甲基化时，不会发生剪切，或以显著降低的效率剪切。对于当识别位点被甲基化时才特异剪切的限制酶来说，当识别位点未被甲基化时，不会发生剪切，或以显著降低的效率剪切。在一些实施方式中，甲基化敏感限制酶的识别序列含有CG二核苷酸(例如cgcg或cccggg)。在一些实施方式中，当该CG二核苷酸中的胞嘧啶在C5碳原子处被甲基化时，甲基化敏感限制酶不进行剪切。
示例性的MSRE选自下组：HpaII酶、SalI酶、
Figure PCTCN2022102658-appb-000002
ScrFI酶、BbeI酶、NotI酶、SmaI酶、XmaI酶、MboI酶、BstBI酶、ClaI酶、MluI酶、NaeI酶、NarI酶、PvuI酶、SacII酶、HhaI酶及其任意组合。
使用本领域已知的方法，使用能区分目标区域内的甲基化的CpG二核苷酸和未甲 基化的CpG二核苷酸的甲基化敏感限制酶或包含甲基化敏感限制酶的一系列限制酶试剂来确定甲基化，例如但不限于，差异性甲基化杂交(“DMH”)。
在一些实施方式中，生物样品中的DNA可以在用甲基化敏感限制酶处理之前被切割。这样的方法是本领域已知的，并且可以既包括物理方式也包括酶促方式。特别优选的是使用一种或多种对甲基化不敏感的并且其识别位点富含AT并且不包含CG二核苷酸的限制酶。使用此类酶使得DNA片段中的CpG位点和CpG富集区域得以保存。在一些实施方式中，此类限制酶选自MseI酶、BfaI酶、Csp6I15酶、Tru1I酶、Tru9I酶、MaeI酶、XspI酶及其任意组合。
经转化的DNA任选经纯化。适用于本文的DNA纯化方法本领域周知。
IV.定量分析
可检测本文所述任意1种、任意2种、任意3种、任意4种、任意5种、任意6种、任意7种、任意8种、任意9种、任意10种、任意11种、任意12种、任意13种、任意14种、任意15种、任意16种、任意17种、任意18种、任意19种和全部20种目标标志物或其目标区域中的至少一个CpG二核苷酸的甲基化状态或甲基化水平，用以鉴别甲状腺结节良恶性。本发明所述的检测试剂和诊断试剂盒可用于所述甲基化状态或甲基化水平的检测。
本文中，所述“良性”和“恶性”表示甲状腺结节的性质。通常，良性表现为结节生长缓慢、质地均匀、活动度好、表面光滑、呈囊性改变、无淋巴结肿大、无钙化等。恶性表现为不可控的恶性细胞生长、扩散和组织浸润。提示甲状腺结节为恶性的超声征象包括：结节的高度大于宽度、缺乏声晕、微小钙化、边界不规则、回声减低、实性结节、结节内部血流丰富等。在一些实施方式中，恶性甲状腺结节包括甲状腺癌。
本文中，“甲基化状态”是指一种或多种甲基化核苷酸碱基在核酸分子中的存在或不存在。例如，含有甲基化胞嘧啶的核酸分子被认为是甲基化的(例如核酸分子的甲基化状态是甲基化的)。不含有任何甲基化核苷酸的核酸分子被认为是未甲基化的。在一些实施方案中，如果核酸在特定基因座(例如特定单一CpG二核苷酸的基因座)或基因座特定组合处不是甲基化的，则核酸可表征为“未甲基化”，即使它在相同基因或分子的其他基因座处为甲基化的，也如此。
因此，甲基化状态描述了核酸(例如基因组序列或本文所述的目标标志物)的甲基化的状态。另外，甲基化状态是指在特定基因组基因座处的核酸区段与甲基化相关的特征。此类特征包括但不限于此DNA序列内的任何胞嘧啶(C)残基是否为甲基化 的、一个或多个甲基化C残基的位置、贯穿核酸的任何特定区域的甲基化C的频率或百分比以及由于例如等位基因起点的差异而导致的甲基化等位基因差异。“甲基化状态”是指在生物样品中贯穿核酸的任何特定区域的甲基化C或未甲基化C的相对浓度、绝对浓度或模式。例如，如果核酸序列内的一个或多个胞嘧啶(C)残基是甲基化的，则其可称为“超甲基化”或具有“增加的甲基化”，而如果DNA序列内的一个或多个胞嘧啶(C)残基是未甲基化的，则其可称为“去甲基化”或具有“减少的甲基化”。同样地，如果核酸序列内的一个或多个胞嘧啶(C)残基与另一个核酸序列(例如来自不同区域或来自不同个体等)相比是甲基化的，则该序列被认为与其他核酸序列相比是超甲基化的或具有增加的甲基化。或者，如果DNA序列内的一个或多个胞嘧啶(C)残基与另一个核酸序列(例如来自不同区域或来自不同个体等)相比是未甲基化的，则该序列被认为与其他核酸序列相比是去甲基化的或具有减少的甲基化。
本文中，甲基化水平代表一个或多个位点处于甲基化状态的比例(或百分比、份数、比率、程度)。一个区域(或一组位点)的甲基化水平是该区域中所有位点(或组中所有位点)的甲基水平的均值。因此，区域的甲基化水平上升或下降并不表示区域中所有甲基化位点的甲基化水平都上升或下降。本领域知晓将检测DNA甲基化的方法(例如简化甲基化测序)所得结果转化为甲基化水平的过程。甲基化水平可以通过例如定量分析在用甲基化敏感性限制性酶进行限制性消化后存在的完整DNA的量来确定。在该例中，如果使用定量PCR对DNA中的特定序列进行定量分析，模板DNA的量大约等于模拟处理的对照则表明该序列未高度甲基化，而模板量明显少于模拟处理的样品中的模板量则表明该序列中存在甲基化DNA。因此，如上述例子中的甲基化水平可以用作甲基化状态的定量指标。当需要将样品中序列的甲基化水平与阈值水平进行比较时，这尤其有用。
在一个或多个实施方案中，与参考水平比较时，目标标志物的甲基化水平(例如Ct值)升高或降低。当甲基化标志物水平(例如Ct值)满足某一阈值时，则鉴定甲状腺结节为恶性。或者，可对目标标志物的甲基化水平进行数学分析，获得得分。对于检测的样品而言，当得分大于或小于阈值，则判定结果为阳性，即甲状腺结节为恶性。本领域知晓常规数学分析的方法以及确定阈值的过程，示例性的方法是支持向量机(SVM)数学模型。例如，对于差异甲基化标志物，对训练组样本构建支持向量机(SVM)，利用模型统计检测结果的准确率，敏感性和特异性以及预测值特征曲线(ROC)下面积(AUC)，统计测试集样本预测得分。
DNA序列(例如目标标志物)内的一个或多个CpG二核苷酸序列的甲基化水平/状态可以通过本领域中已知的各种分析方法来确定，优选为定量分析方法。示例性的分析方法包括：聚合酶链式反应，包括实时聚合酶链式反应，数字聚合酶链式反应，和基于重亚硫酸盐转化的PCR(例如甲基化特异性PCR(Methylation-specific PCR，MSP))；核酸测序；全基因组甲基化测序；简化甲基化测序；基于质量的分离(例如电泳法、质谱法)；靶标捕获(例如杂交、微阵列)；甲基化敏感的限制性内切酶分析法；甲基化敏感性高分辨率熔解曲线法；基于芯片的甲基化图谱分析；质谱；和荧光定量法。本文中，检测包括检测基因或位点处的任一条链。
在一些实施方式中，通过实时PCR进行定量分析。实时PCR的非限制性实例包括Cottrell et al.，Nucl.Acids Res.32:e10，2003描述的HeavyMethyl TM PCR；Eads et al.，Cancer Res.59:2302-2306，1999描述的MethyLight TM PCR；Rand et al.，Nucl.Acids Res.33:e 127，2005描述的Headloop PCR。
如本文所用，术语“HeavyMethyl TM PCR”是指本领域公认的一种实时PCR技术，其中一个或多个不可延伸性核酸(例如，寡核苷酸)封闭物以甲基化特异性方式与亚硫酸氢盐处理的核酸结合(即，封闭物在中等至高等严谨条件下与未突变的DNA特异性结合)。使用一种或多种引物进行扩增反应，所述引物可以任选地是甲基化特异性的，但旁侧分布一个或多个封闭物。在未甲基化的核酸(即突变的DNA)存在的情况下，封闭物结合并且无PCR产物产生。使用基本上像例如Holland et al.,Proc.Natl.Acad.Sci.USA,88:7276-7280,1991所述的TaqMan TM分析方法，样品中核酸的甲基化水平得以确定。
如本文所用，术语“MethyLight TM PCR”是指基于本领域公认的一种基于荧光的实时PCR技术，其中采用了称为TaqMan TM探针的双标记荧光寡核苷酸探针，并且被设计为可同位于正向和反向扩增引物之间的富含CpG的序列杂交。所述的TaqMan TM探针包含一个荧光“报告因子部分”和“淬灭剂部分”共价结合到与TaqMan TM寡核苷酸的核苷酸相连的接头部分(例如，亚磷酰胺)。在PCR扩增过程中，与富含CpG的序列杂交的TaqMan TM探针被Taq聚合酶的5’核酸酶活性切割，从而在PCR反应过程中产生以实时方式检测的信号。在该方法中，可以将分子信标用作可检测的探针，并且该系统不依赖于所使用的DNA聚合酶的5’-3’核酸外切酶活性(参见Mhlanga and Malmberg，Methods 25:463-471，2001)。
如本文所用，术语“Headloop PCR”是指本领域公认的一种实时PCR，其选择性地扩增目标核酸，但是通过将3’茎环延伸形成不能进一步提供扩增模板的发卡结构来 抑制非扩增目标变体的扩增。
在一些实施方式中，所述实时PCR是多重实时PCR。如本文所用，术语“多重”可指，通过使用一个以上的标志物，每个标志物具有至少一个不同的检测特征，例如荧光特征(例如，激发波长、发射波长、发射强度、FWHM(半峰高处的全宽度)或荧光寿命)或独特的核酸或蛋白序列特征，可以同时对多个标志物(例如多个核酸序列)的存在和/或量进行测定的分析或其他分析方法。
在一些实施方式中，通过核酸测序进行定量分析。核酸测序的示例性方法是本领域已知的，参见，例如Frommer et al.,Proc.Natl.Acad.Sci.USA 89:1827-1831,1992；Clark et al.,Nucl.Acids Res.22:2990-2997,1994。例如，通过将未使用亚硫酸氢盐处理的样品获得的序列或目标区域的已知核苷酸序列与使用亚硫酸氢盐处理的样品获得的序列进行比较，有助于鉴定DNA序列中甲基化胞嘧啶。与未处理的样品相比，在亚硫酸氢盐处理的样品中的任意胞嘧啶位点检测到的胸腺嘧啶残基都可以认为是由亚硫酸氢盐处理而引起的突变，即该位点存在甲基化的胞嘧啶。
用于测序DNA的方法是本领域已知的，包括例如双脱氧链终止法或Maxam-Gilbert法(参见Sambrook et al.,Molecular Cloning,A Laboratory Manual(2 nd Ed.,CSHP,New York 1989))、焦磷酸测序(参见Uhlmann et al.,Electrophoresis,23:4072-4079,2002)、固相焦磷酸测序(参见Landegren et al.,Genome Res.,8(8):769-776,1998)、固相微测序(参见例如，Southern et al.,Genomics,13:1008-1017,1992)、采用FRET的微测序(参见例如，Chen and Kwok,Nucleic Acids Res.25:347-353,1997)、连接法测序或超深度测序(参见Marguiles et al.,Nature 437(7057):376-80(2005))。
在一些实施方式中，通过基于质量的分离(例如电泳、质谱法)进行定量分析。例如，甲基化胞嘧啶残基的存在可以通过联合亚硫酸氢盐限制分析法(COBRA)进行检测，基本如Xiong and Laird,Nucl.Acids Res.,25:2532-2534,2001所述。这种方法利用了在使用可以选择性地突变未甲基化的胞嘧啶残基的化合物(例如，亚硫酸氢盐)处理之后，在甲基化和未甲基化的核酸之间的限制酶识别位点的差异。例如，限制性核酸内切酶Taq1切割序列TCGA，在对未甲基化核酸进行亚硫酸氢盐处理后该序列将是TTGA，因此将不被切割。然后使用本领域已知的检测手段例如电泳和/或质谱法，检测消化的和/或未消化的核酸。又例如，在用选择性突变未甲基化胞嘧啶残基的化合物处理后，基于核苷酸序列和/或二级结构的差异，使用不同的技术来检测扩增产物中核酸差异，例如甲基化特异性单链构象分析(MS-SSCA)(Bianco et al.,Hum. Mutat.,14:289-293,1999)、甲基化特异性变性梯度凝胶电泳(MS-DGGE)(Abrams and Stanton,Methods Enzymol.,212:71-74,1992)和甲基化特异性变性高效液相色谱(MS-DHPLC)(Deng et al.,Chin.J.Cancer Res.,12:171-191,2000)。
在一些实施方式中，通过靶标捕获(例如杂交、微阵列)进行定量分析。通过杂交的合适的检测方法是本领域已知的，例如Southern、斑点印迹、狭缝印迹或其他核酸杂交方式(Kawai et al.,Mol.Cell.Biol.14:7421-7427,1994；Gonzalgo et al.,Cancer Res.57:594-599,1997)。在一些实施方式中，用于杂交分析的探针被可检测地标记。在一些实施方式中，用于杂交分析的基于核酸的探针是未标记的。这种未标记的探针可以固定在固体载体如微阵列上，并且可以与被可检测地标记的目标核酸分子杂交。微阵列的一个实例是甲基化特异性微阵列，其可用于区分具有转化的胞嘧啶残基的序列和具有未转化的胞嘧啶残基的序列(参见Adorjan et al.,Nucl.Acids Res.,30:e21,2002)。基于杂交的分析还可被用于用甲基化敏感的限制酶处理后的核酸。又例如，可通过寡核苷酸探针确定DNA序列内CpG二核苷酸序列的甲基化状态，所述寡核苷酸探针与PCR扩增引物同时与亚硫酸氢盐处理的DNA杂交(其中所述引物可以是甲基化特异性引物或标准引物)。
在一些实施方式中，定量分析在检测试剂的存在下进行。如本文所用，术语“检测试剂”是在定量分析步骤中用于检测核酸的存在、不存在或量的试剂。本领域已知的各种检测试剂在本申请中都可使用。在一些实施方式中，检测试剂选自下组：荧光探针、嵌入染料、生色团标记的探针、放射性同位素标记的探针和生物素标记的探针。
优选地，本文中，示例性探针序列如下：
CDH1基因探针：CGCCCACCCGACCTCGCAT(SEQ ID NO:41)；
TSHR基因探针：ACAACACCAACTACAACAAATCCGCCGA(SEQ ID NO:42)；
MCC基因探针：CCTACCGCACGCCTATTCAATAACCT(SEQ ID NO:43)；
TBX15基因探针：ACCCTACTCCTACGCAAACCGAAAT(SEQ ID NO:44)；
PRR15基因探针：CCTCCGAAAACAACGTAACGCGC(SEQ ID NO:45)；
DPYS基因探针：CGAAAACATCGACACACACACGCA(SEQ ID NO:46)；
GRIA2基因探针：AAAACGCTTCGCCGCCAACA(SEQ ID NO:47)；
NR5A1基因探针：AAACGCTACGCGAAACGCTC(SEQ ID NO:48)；
TTC34基因探针：CGAACCGCAACAAACGCTCG(SEQ ID NO:49)；
RCOR2基因探针：CCGACTCGCGCCAAACTCGA(SEQ ID NO:50)；
F10基因探针：CAAACAACGCGACCTCTAAACGC(SEQ ID NO:51)；
ITPKA基因探针：ACGCTAAAATCACCTTCACTACGCC(SEQ ID NO:52)；
SLC16A3基因探针：ATGTAAGCGGATATAGAGCGGTAGGGTA(SEQ ID NO:53)；
RARG基因探针：CGCAACCACGCAAAAACACACGC(SEQ ID NO:54)；
FTAP2B基因探针：CGAGTCGTCGTATACGGTTTCGGG(SEQ ID NO:55)；
SOD3基因探针：CGAACCCGAACTCTAAAAACGCCAAACG(SEQ ID NO:56)；
RP11-867G23.12基因探针：CGGTGTTGTCGGTTGTTTTTTACGTACG(SEQ ID NO:57)；
EMX2OS基因探针：CCAAAACGTACACCGACTCCTAAATTCC(SEQ ID NO:58)；
TEAD3基因探针：TGGTTGCGGTATCGGATTATTTATACGG(SEQ ID NO:59)；
DGKG基因探针：AGGTTCGGGTAGGTTTTCGGCG(SEQ ID NO:60)。
在一些实施方式中，对照标志物为ACTB，其示例性的探针的核苷酸序列如SEQ ID NO:63所示：ACCACCACCCAACACACAATAACAAACACA。
荧光探针的5’端通常标记有荧光染料(例如FAM、HEX/VIC、TAMRA、Texas Red或Cy5)，3’端标记有猝灭剂(例如BHQ1、BHQ2、BHQ3、DABCYL或TAMRA)。
标记可以通过直接或间接方法来完成。直接标记涉及将标记直接(共价或非共价)偶联至试剂上。间接标记涉及第二试剂与第一试剂的结合(共价或非共价)。第二试剂应与第一试剂特异性结合。所述第二试剂可以与合适的标记偶联和/或第二试剂是可与第二试剂结合的第三试剂的目标(受体)。使用二级、三级甚至更高阶的试剂通常会增加信号强度。合适的二级和高级试剂可以包括抗体、二级抗体和众所周知的链霉亲和素-生物素系统(Vector Laboratories，Inc.)。试剂或底物也可以被本领域中已知的一个或多个标签“标记”。
示例性的经标记的探针如下：
CDH1基因探针：Texas Red-CGCCCACCCGACCTCGCAT-BHQ2；
TSHR基因探针：FAM-ACAACACCAACTACAACAAATCCGCCGA-BHQ1；
MCC基因探针：Cy5-CCTACCGCACGCCTATTCAATAACCT-BHQ1；
TBX15基因探针：Texas Red-ACCCTACTCCTACGCAAACCGAAAT-BHQ2；
PRR15基因探针：FAM-CCTCCGAAAACAACGTAACGCGC-BHQ1；
DPYS基因探针：Cy5-CGAAAACATCGACACACACACGCA-BHQ1；
GRIA2基因探针：Texas Red-AAAACGCTTCGCCGCCAACA-BHQ2；
NR5A1基因探针：FAM-AAACGCTACGCGAAACGCTC-BHQ1；
TTC34基因探针：Cy5-CGAACCGCAACAAACGCTCG-BHQ1；
RCOR2基因探针：Texas Red-CCGACTCGCGCCAAACTCGA-BHQ2；
F10基因探针：FAM-CAAACAACGCGACCTCTAAACGC-BHQ1；
ITPKA基因探针：Cy5-ACGCTAAAATCACCTTCACTACGCC-BHQ1；
SLC16A3基因探针：Texas Red-ATGTAAGCGGATATAGAGCGGTAGGGTA-BHQ2；
RARG基因探针：FAM-CGCAACCACGCAAAAACACACGC-BHQ1；
FTAP2B基因探针：Cy5-CGAGTCGTCGTATACGGTTTCGGG-BHQ1；
SOD3基因探针：Texas Red-CGAACCCGAACTCTAAAAACGCCAAACG-BHQ2；
RP11-867G23.12基因探针：FAM-CGGTGTTGTCGGTTGTTTTTTACGTACG-BHQ1；
EMX2OS基因探针：Cy5-CCAAAACGTACACCGACTCCTAAATTCC-BHQ1；
TEAD3基因探针：Texas Red-TGGTTGCGGTATCGGATTATTTATACGG-BHQ2；
DGKG基因探针：FAM-AGGTTCGGGTAGGTTTTCGGCG-BHQ1；
ACTB基因探针：VIC-ACCACCACCCAACACACAATAACAAACACA-BHQ1。
在一些实施方式中，定量分析包含使用定量引物对和DNA聚合酶对经处理的DNA进行扩增。如本文所用，术语“定量引物对”是指在定量分析步骤中使用的一个或多个引物对。优选地，所述定量引物对能够与所述经处理的DNA的至少9个连续核苷酸在严谨条件下、中等严谨条件下或高度严谨条件下杂交。
在一些实施方式中，所述定量分析包括基于经处理的DNA中多个CpG二核苷酸、TpG二核苷酸或CpA二核苷酸的存在或水平，确定一个或多个目标标志物的甲基化水平。在一些实施方式中，所述定量分析包括基于经处理的DNA中一个或多个CpG二核苷酸的存在或水平来确定胞嘧啶残基的甲基化水平。在一些实施方式中，所述定量分析包括基于所述经处理的DNA中一个或多个TpG二核苷酸的存在或水平来确定胞嘧啶残基的甲基化水平。在一些实施方式中，所述定量分析包括基于所述经处理的DNA中CpA二核苷酸的存在来确定胞嘧啶残基的甲基化水平。
在一些实施方式中，定量分析步骤是通过将经处理的DNA产物分为多个组分来进行的。在一些实施方式中，对多个组分进行多个不同的定量分析测试，其中在多个组分之一中定量分析所述经处理的DNA产物(如果存在于所述组分中的话)的不同 组合。在一些实施方式中，定量分析每个组分中的对照标志物。
在一些实施方式中，基于预扩增的DNA通过使用MSP(参见Herman，同上)分别定量分析每个目标标志物的甲基化水平。例如，通过使用在中等和/或高度严谨条件下与未转化序列特异性杂交的一种或多种引物，仅当模板在CpG位点包含甲基化胞嘧啶时才产生扩增产物。
在一些实施方式中，所述定量引物对被设计为扩增所述经处理的DNA产物中的至少一部分，即定量分析被设计为巢式PCR。巢式PCR是PCR的一种改进，旨在提高灵敏度和特异性。巢式PCR涉及使用两个引物组和两个连续的PCR反应。进行第一轮扩增以产生第一扩增子，并使用一个引物对进行第二轮扩增，其中一个或两个引物与由初始引物对界定的区域内的位点退火，即第二个引物对被认为是“嵌套”在第一对引物中。以这种方式，不包含正确内部序列的来自第一次PCR反应的背景扩增产物在第二次PCR反应中不再被进一步扩增。
通常，PCR的反应液包含Taq DNA聚合酶、PCR缓冲液、引物、探针、dNTPs、Mg 2+。优选地，Taq DNA聚合酶为热启动Taq DNA聚合酶。示例性地，Mg 2+终浓度为1.0-20.0mM；各引物浓度为100-500nM；各探针浓度为100-500nM。示例性的PCR反应条件为，95℃预变性5min；95℃变性15s，60℃退火延伸60s，50个循环。
在一些实施方案中，本发明的方法包括预扩增步骤。对目标标志物进行预扩增的目的之一是增加经处理的DNA中的目标标志物的数量。如本文所用，术语“扩增”大体上指任何能够导致分子或一组相关分子的拷贝数增加的过程。当“扩增”被用于多核苷酸分子时，是指通常从少量多核苷酸开始产生多拷贝的多核苷酸分子或多核苷酸分子的一部分的多份拷贝，其中被扩增的物质(扩增子，PCR扩增子)通常是可被检测到的。多核苷酸的扩增涵盖多个化学和酶促过程。扩增的形式包括通过聚合酶链式反应(逆转录PCR、PCR)、链置换扩增(SDA)反应、转录介导扩增(TMA)反应、基于核酸序列的扩增(NASBA)反应或连接酶链反应(LCR)，从一个或几个拷贝的模板RNA或DNA分子生成多个DNA拷贝。
可用预扩增引物预扩增经处理的DNA中的所述目标标志物。如本文所用，术语“引物”是指这样的单链寡核苷酸，其能够在合适的条件(例如缓冲液和温度)下，在四种不同的三磷酸核苷和用于聚合的试剂(例如DNA聚合酶)的存在下，作为模板指导的DNA合成的起始点。在任何给定的情况下，引物的长度取决于例如引物的预期用途，并且通常在15至30个核苷酸的范围内。短的引物分子通常需要较低的温度才能与模板形成足够稳定的杂交复合物。引物不必反映模板的确切序列，但必须足够 互补以能与该模板杂交。引物位点是模板上与引物杂交的区域。引物对是一组引物，其包括与待扩增的序列的5’末端杂交的5’正向引物和与待扩增的序列的3’末端的互补链杂交的3’反向引物。本领域技术人员可以基于本领域的公知常识根据待扩增的标志物设计引物(参见，例如PCR Primer:A Laboratory Manual,Cold Spring Harbor Laboratories,NY,1995)。此外，一些用于设计在各种各样分析中使用的最佳探针和/或引物的软件包是公开的，例如可从美国马萨诸塞州剑桥市的基因组研究中心(the Center for Genome Research,Cambridge,Mass.,USA)获得的Primer 3。显然，在设计探针或引物时其潜在用途也应考虑在内。例如，设计用于本发明目的的引物可以包括至少一个CpG位点，或者从该引物获得的扩增产物可以包括至少一个CpG位点。用于设计检测DNA甲基化状态的引物的工具也是本领域已知的，例如MethPrimer(Li LC and Dahiya R.MethPrimer:designing primers for methylation PCRs.Bioinformatics.2002Nov；18(11):1427-31)。在本申请中，通过将预扩增引物作为引物池，经处理的DNA中的任何目标标志物(目标标志物的每至少一部分或目标标志物的一个亚区域)均可以被预扩增。
如本文所用，术语“互补”是指核苷酸或核酸之间的杂交或碱基配对，例如，双链DNA分子的两条链之间，或待测序或扩增的单链核酸上的引物结合位点和寡核苷酸引物之间。互补核苷酸通常是A和T(或A和U)，或C和G。当一条链的核苷酸以最佳的方式对齐、并比较、并有适当的核苷酸插入或缺失后，与另一链的至少约80％(通常至少约90％至95％，更优选地为约98％至100％)的核苷酸配对，两条单链RNA或DNA分子就被称为是互补的。或者，当RNA链或DNA链在选择性杂交条件下与其互补序列杂交时，互补存在。通常，当在至少14至25个核苷酸的一段上具有至少约65％(优选至少约75％、更优选至少约90％)的互补性时，将发生选择性杂交。参见M.Kanehisa,Nucleic Acids Res.12:203(1984)，作为参考并入本文。
在一些实施方式中，预扩增引物池包含至少一个甲基化特异性引物对。在一些实施方式中，预扩增引物池包含多个甲基化特异性引物对。在一些实施方式中，预扩增步骤通过甲基化特异性PCR(“MSP”)进行，甲基化特异性PCR是使用甲基化特异性引物的PCR。Herman et al.,Methylation-specific PCR:a novelPCRassay for methylation status ofCpGislands.Proc Natl Acad Sci USA.1996 September 3；93(18):9821-6和United States Patent No.6,265,171中已描述了该技术(即MSP)。
如本文所用，术语“甲基化特异性引物对”是指经特异性设计以识别CpG位点以 利用甲基化的差异来扩增经处理的DNA中的特定目标标志物的引物对。引物仅作用于具有特定甲基化状态或没有特定甲基化状态的分子。例如，引物可以是寡核苷酸，在严谨条件、中等严谨条件或高度严谨条件下，其可以以甲基化特异性方式与具有甲基化的特定CpG位点特异性杂交，但不能与没有甲基化的特定CpG位点杂交。因此，引物将特异性扩增在特定CpG位点具有甲基化的目标标志物。又例如，引物可以是寡核苷酸，在严谨条件、中等严谨条件或高度严谨条件下，其可以以甲基化特异性的方式与未甲基化的特定的CpG位点特异性杂交，但是不能与甲基化的特定的CpG位点杂交。因此，引物将特异性扩增在特定CpG位点没有甲基化的目标标志物。因此，在本申请中，对在经处理的DNA内的至少一个目标标志物的预扩增中使用甲基化特异性引物，可以区分甲基化的和未甲基化的CpG位点。本申请的甲基化特异性引物对包含至少一个与亚硫酸氢盐处理的CpG二核苷酸杂交的引物。因此，所述特异性针对甲基化DNA的引物的序列包含至少一个CpG二核苷酸，并且所述特异性针对未甲基化DNA的引物的序列在CpG的C位置上包含“T”，和/或在CpG中G位置上包含“A”。
甲基化特异性引物对通常包含正向引物和反向引物，所述引物均包含寡核苷酸序列，所述寡核苷酸序列与所述目标标志物之一(或目标标志物的亚区域)的至少9个连续核苷酸在严谨条件下、中等严谨条件下或高度严谨条件下杂交，其中所述目标标志物之一(或目标标志物的亚区域)的至少9个连续核苷酸包含至少一个(例如1、2、3、4、5、6、7、8、9、10或更多个)CpG位点。
如本文所用，术语“杂交”可以指其中两条单链多核苷酸非共价形式结合以形成稳定的双链多核苷酸的过程。在一个方面，所得的双链多核苷酸可以是“杂交物”或“双链”。“杂交条件”中的盐浓度通常约小于1M，经常小于约500mM并且可以小于约200mM。“杂交缓冲液”包括缓冲盐溶液，例如5％SSPE，或本领域已知的其他此类缓冲液。杂交温度可以低至5℃，但是通常高于22℃，并且更为通常地高于约30℃，并且通常超过37℃。杂交通常在严谨条件下进行，即在该条件下序列将与其目标序列杂交但不与其他非互补序列杂交。严谨条件取决于序列，且在不同情况下有所不同。例如，更长的片段可能需要比短片段更高的杂交温度才能进行特异性杂交。由于其他因素可能会影响杂交的严谨性，包括碱基组成和互补链的长度，有机溶剂的存在以及碱基错配的程度，因此参数组合比单独使用任何一个参数的绝对测量更为重要。通常严谨条件被选定为比特定序列在特定的离子强度和pH下的解链温度(Tm)低约5℃。Tm可以是双链核酸分子群体中的一半被分离成单链的温度。用于计算核酸 的Tm的几个方程式是本领域众所周知的。如标准参考文献所示，当核酸在1M NaCl水溶液中时，可以通过公式Tm＝81.5+0.41(％G+C)计算出简单估算的Tm值(参见例如Anderson and Young,Quantitative Filter Hybridization,in Nucleic Acid Hybridization(1985))。其他参考文献(例如Allawi and SantaLucia,Jr.,Biochemistry,36:10581-94(1997))包括替代的计算方法，其计算Tm时将结构和环境以及序列特征等考虑在内。
通常，杂交物的稳定性是关于离子浓度和温度的函数。通常，杂交反应在较低严谨条件下进行，然后在具有不同但较高严谨性的洗涤液中洗涤。示例性的严谨条件包括pH约7.0至约8.3、温度至少25℃、钠离子(或其他盐)浓度为至少0.01M至不超过1M。例如，5x SSPE(750mM NaCl，50mM磷酸钠，5mM EDTA，pH 7.4)和约30℃的温度适合于等位基因特异性杂交，尽管合适的温度取决于杂交区域的长度和/或GC含量。在一个方面，确定错配百分比的“杂交严谨性”可以如下：1)高度严谨性：0.1x SSPE，0.1％SDS，65℃；2)中等严谨性(也称为中度严谨性)：0.2x SSPE，0.1％SDS，50℃；3)低严谨性：1.0x SSPE，0.1％SDS，50℃。应当理解，使用替代的缓冲剂、盐和温度可以达到相同的严谨性。例如，中等严谨杂交可以是指允许核酸分子(例如探针)结合互补核酸分子的条件。杂交的核酸分子通常具有至少60％的同一性，包括例如至少70％、75％、80％、85％、90％或95％的同一性。中等严谨条件可以是与下述条件达到同等效果的条件：42℃，50％甲酰胺，5x Denhardt溶液，5x SSPE，0.2％SDS杂交，然后用42℃，0.2x SSPE，0.2％SDS进行洗涤。高度严谨条件可以通过如下条件提供，例如，42℃，50％甲酰胺，5x Denhardt溶液，5x SSPE，0.2％SDS杂交，然后65℃，0.1x SSPE和0.1％SDS中洗涤。低严谨性杂交可以是与下述条件达到同等效果的条件：22℃，10％甲酰胺，5x Denhardt溶液，6x SSPE，0.2％SDS杂交，然后在1x SSPE，0.2％SDS中于37℃洗涤。Denhardt的溶液包含1％聚蔗糖，1％聚乙烯吡咯烷酮和1％牛血清白蛋白(BSA)。20x SSPE(氯化钠，磷酸钠，EDTA)包含3M氯化钠、0.2M磷酸钠和0.025M EDTA。其他合适的中等严谨性和高度严谨性杂交缓冲液和条件是本领域技术人员众所周知的，并且描述于例如Sambrook et al.,Molecular Cloning:A Laboratory Manual,2nd ed.,Cold Spring Harbor Press,Plainview,N.Y.(1989)和Ausubel et al.,Short Protocols in Molecular Biology,4th ed.,John Wiley & Sons(1999)。
在一些实施方式中，预扩增引物池还包含用于扩增对照标志物的对照引物对。通常，对照标志物是具有已知特征(例如，序列已知，每个细胞的拷贝数已知)的核酸 ，用于与实验目标(例如，浓度未知的核酸)进行比较。对照可以是内源的，优选为不变的基因，可以将分析中的实验核酸或目标核酸相对其进行标准化。此类因为样品间差异而标准化的对照可能发生在例如样品处理，分析效率等，并且允许精确的样品间数据比较，定量分析扩增效率和偏差。
在一些实施方案中，针对本文所述目标标志物的预扩增引物对可以是例如本文SEQ ID NO:1-40所述的相应引物对。
V.甲状腺结节良恶性鉴定
本发明发现，恶性甲状腺结节组织中的CDH1基因或基因组的CDH1序列、TSHR基因或基因组的TSHR序列、MCC基因或基因组的MCC序列、TBX15基因或基因组的TBX15序列、DPYS基因或基因组的DPYS序列、GRIA2基因或基因组的GRIA2序列、NR5A1基因或基因组的NR5A1序列、TTC34基因或基因组的TTC34序列、FTAP2B基因或基因组的FTAP2B序列、RP11-867G23.12基因或基因组的RP11-867G23.12序列和DGKG基因或基因组的DGKG序列的甲基化水平(％)高于这些基因在良性甲状腺结节的甲基化水平；恶性甲状腺结节中的PRR15基因或基因组的PRR15序列、RCOR2基因或基因组的RCOR2序列、F10基因或基因组的F10序列、ITPKA基因或基因组的ITPKA序列、SLC16A3基因或基因组的SLC16A3序列、RARG基因或基因组的RARG序列、SOD3基因或基因组的SOD3序列、EMX2OS基因或基因组的EMX2OS序列和TEAD3基因或基因组的TEAD3序列的甲基化水平小于或等于这些基因在良性甲状腺结节中的甲基化水平。在一些实施方案中，本发明发现，恶性甲状腺结节患者血液样品的cfDNA中，TSHR基因或基因组的TSHR序列、MCC基因或基因组的MCC序列、RARG基因或基因组的RARG序列、EMX2OS基因或基因组的EMX2OS序列和TEAD3基因或基因组的TEAD3序列中的一个或多个的甲基化水平高于对照如良性甲状腺结节对象血液样品中相应目标区域的甲基化水平；CDH1基因或基因组的CDH1序列、TBX15基因或基因组的TBX15序列、PRR15基因或基因组的PRR15序列、DPYS基因或基因组的DPYS序列、GRIA2基因或基因组的GRIA2序列、NR5A1基因或基因组的NR5A1序列、TTC34基因或基因组的TTC34序列、RCOR2基因或基因组的RCOR2序列、F10基因或基因组的F10序列、ITPKA基因或基因组的ITPKA序列、SLC16A3基因或基因组的SLC16A3序列、FTAP2B基因或基因组的FTAP2B序列、SOD3基因或基因组的SOD3序列、RP11-867G23.12基因或基因组的RP11-867G23.12序列和DGKG基因或基因组的 DGKG序列中的一个或多个的甲基化水平等于或低于对照如良性甲状腺结节对象血液样品中相应目标区域的甲基化水平。因此，通过将检测到的甲基化水平与相应的参考水平进行比较，根据比较结果可对对象甲状腺结节的良恶性进行鉴定或诊断。
本文所用的术语“比较”是指分别对检测的生物样本所含有的通过定量分析步骤获取的目标标志物的甲基化水平与其相应的参考水平进行对比。应当理解，本文所用的术语是指相应参数或值的比较，例如，将绝对量与绝对参考量进行比较，将浓度与参考浓度进行比较，或从检测的样本中获得的强度信号同参考样本的同类型的强度信号进行比较。可以通过手动或计算机辅助进行比较。对于计算机辅助进行的比较，可以将所确定的量的值与通过计算机程序存储在数据库中的合适参考的值进行比较。该计算机程序可以进一步评估比较的结果，并以合适的输出格式自动提供期望的评估。基于定量分析步骤中每个目标标志物的甲基化水平与相应参考水平的比较，可以鉴定出具有恶性甲状腺结节的个体或其甲状腺结节为恶性的风险较高的个体。
所述“参考水平”可以源自一个或多个参考样品，其中参考水平获自与检测目的样品的实验平行进行的实验。或者，可以在数据库中获得参考水平，该数据库包括来自一个或多个参考样品或疾病参考样品的数据、标准或水平的集合。在一些实施方式中，此类数据、标准或水平的集合被标准化，以便可用于与来自一个或多个样品的数据进行比较的目的。“标准化”是将测量原始数据转换为可以直接与其他标准化数据进行比较的数据的过程。标准化被用于克服因不同的分析方法里因素不同而导致的、分析方法特异性的误差，例如上样量的不同、结合效率的不同、检测灵敏度的不同和其他各类的误差。
在一些实施方式中，参考数据库包括来自一个或多个参考样品的目标标志物和/或其他实验室和临床数据的甲基化水平。在一些实施方式中，参考数据库包括目标标志物的甲基化水平，其各自被标准化为在与参考样品相同的条件下检测的对照标志物的甲基化水平的百分比。为了与目标标志物的如此标准化甲基化水平进行比较，测试样品的目标标志物的甲基化水平也被测量并计算为在与测试样品相同的条件下检测的对照标志物的甲基化水平的百分比。
在一些实施方式中，通过汇总获自健康个体和/或非恶性甲状腺结节个体(即已知其甲状腺结节为良性的个体)的参考样品的参考水平数据来建立参考数据库。在一些实施方式中，通过汇总获自正在接受甲状腺癌治疗的个体的参考样品的参考水平数据来建立参考数据库。
本领域技术人员可以根据期望的灵敏度和特异性来选择参考水平。确定合适的参 考水平的手段是本领域技术人员已知的，例如参考水平可以从临床研究中收集的数据来确定。
本文所述的“高于相应的参考水平”指相应的目标标志物或其目标区域的甲基化水平至少是其相应参考水平的1.05、1.1、1.2、1.3、1.4、1.5、2、3、4、5、6、7、8、9、10倍或更多倍。本文所述的“低于相应的参考水”指相应的目标标志物的甲基化水平是其相应参考水平的98％、95％、93％、90％、85％、80％、75％、70％、65％、60％、55％、50％、45％、40％、35％、30％、25％、20％、15％、10％甚至更低。
具体而言，若检测发现来自个体甲状腺结节组织的样品中，选自以下任意一个、任意两个、任意三个、任意四个、任意五个、任意六个、任意七个、任意八个、任意九个、任意十个或全部十一个基因或其各自在基因组中的序列的甲基化水平高于相应的参考水平：CDH1、TSHR、MCC、TBX15、DPYS、GRIA2、NR5A1、TTC34、FTAP2B、RP11-867G23.12和DGKG，和/或选自以下任意一个、任意两个、任意三个、任意四个、任意五个、任意六个、任意七个、任意八个或全部九个基因或其各自在基因组中的序列的甲基化水平小于或等于相应的参考水平：PRR15、RCOR2、F10、ITPKA、SLC16A3、RARG、SOD3、EMX2OS和TEAD3，则指示对象甲状腺结节为恶性结节或患有甲状腺癌。在优选的实施方案中，所述基因包括CDH1基因或其在基因组中的序列、TBX15基因或其在基因组中的序列、DPYS基因或其在基因组中的序列、GRIA2基因或其在基因组中的序列、NR5A1基因或其在基因组中的序列、RARG基因或其在基因组中的序列和EMX2OS基因或其在基因组中的序列中的任意一种或多种。
或者，若检测到血液样品的cfDNA中，选自以下任意一个、任意两个、任意三个、任意四个或全部五个基因或其各自在基因组中的序列的甲基化水平高于相应的参考水平：TSHR、MCC、RARG、EMX2OS和TEAD3，和/或选自以下任意一个、任意两个、任意三个、任意四个、任意五个、任意六个、任意七个、任意八个、任意九个、任意十个、任意十一个基因、任意十二个基因、任意十三个基因、任意十四个基因或全部十五个基因或其各自在基因组中的序列的甲基化水平等于或低于相应的参考水平：CDH1、TBX15、PRR15、DPYS、GRIA2、NR5A1、TTC34、RCOR2、F10、ITPKA、SLC16A3、FTAP2B、SOD3、RP11-867G23.12和DGKG，则指示对象甲状腺结节为恶性结节或患有甲状腺癌。在优选的实施方案中，所述基因为TSHR基因或其在基因组中的序列、RARG基因或其在基因组中的序列、EMX2OS基因或其在基因组中的序列、CDH1基因或其在基因组中的序列、GRIA2基因或其在基因组中的序列、RCOR2基因或其在基因组中的序列、F10基因或其在基因组中的序列、ITPKA 基因或其在基因组中的序列、FTAP2B基因或其在基因组中的序列、SOD3基因或其在基因组中的序列和DGKG基因或其在基因组中的序列中的任意一个或多个。
在一些实施方案中，目标标志物为SLC16A3基因或基因组的SLC16A3序列以及选自TSHR基因或基因组的TSHR序列、TTC34基因或基因组的TTC34序列、ROCR2基因或基因组的ROCR2序列、RARG基因或基因组的RARG序列、MCC基因或基因组的MCC序列和ITPKA基因或基因组的ITPKA序列中的一种或多种。
在一些实施方案中，目标标志物为SLC16A3基因或基因组的SLC16A3序列和TSHR基因或基因组的TSHR序列；优选地，检测来自该个体的甲状腺结节中的该目标标志物的甲基化水平。
在一些实施方案中，目标标志物为SLC16A3基因或基因组的SLC16A3序列和ROCR2基因或基因组的ROCR2序列，以及选自TTC34基因或基因组的TTC34序列、RARG基因或基因组的RARG序列、ITPKA基因或基因组的ITPKA序列和MCC基因或基因组的MCC序列中的至少一个、至少两个或至少三个；优选地，目标标志物为SLC16A3基因或基因组的SLC16A3序列、ROCR2基因或基因组的ROCR2序列、TTC34基因或基因组的TTC34序列和RARG基因或基因组的RARG序列，或目标标志物为SLC16A3基因或基因组的SLC16A3序列、ROCR2基因或基因组的ROCR2序列、ITPKA基因或基因组的ITPKA序列和MCC基因或基因组的MCC序列；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。
在一些实施方案中，目标标志物为SLC16A3基因或基因组的SLC16A3序列和RARG基因或基因组的RARG序列，以及选自TTC34基因或基因组的TTC34序列、ROCR2基因或基因组的ROCR2序列和ITPKA基因或基因组的ITPKA序列的至少一个、至少两个或全部三个；优选地，目标标志物为SLC16A3基因或基因组的SLC16A3序列、ROCR2基因或基因组的ROCR2序列、TTC34基因或基因组的TTC34序列和RARG基因或基因组的RARG序列，或目标标志物为SLC16A3基因或基因组的SLC16A3序列、TTC34基因或基因组的TTC34序列、ITPKA基因或基因组的ITPKA序列和RARG基因或基因组的RARG序列；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。
在一些实施方案中，目标标志物为SLC16A3基因或基因组的SLC16A3序列和TTC34基因或基因组的TTC34序列，以及选自ROCR2基因或基因组的ROCR2序列、RARG基因或基因组的RARG序列和ITPKA基因或基因组的ITPKA序列的至少一个、至少两个或全部三个；优选地，目标标志物为SLC16A3基因或基因组的SLC16A3 序列、ROCR2基因或基因组的ROCR2序列、TTC34基因或基因组的TTC34序列和RARG基因或基因组的RARG序列，或目标标志物为SLC16A3基因或基因组的SLC16A3序列、TTC34基因或基因组的TTC34序列、ITPKA基因或基因组的ITPKA序列和RARG基因或基因组的RARG序列；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。
在一些实施方案中，目标标志物为SLC16A3基因或基因组的SLC16A3序列和ITPKA基因或基因组的ITPKA序列，以及选自ROCR2基因或基因组的ROCR2序列、MCC基因或基因组的MCC序列、TTC34基因或基因组的TTC34序列和RARG基因或基因组的RARG序列和的至少一个、至少两个或全部三个；优选地，目标标志物为SLC16A3基因或基因组的SLC16A3序列、ROCR2基因或基因组的ROCR2序列、ITPKA基因或基因组的ITPKA序列和MCC基因或基因组的MCC序列，或目标标志物为SLC16A3基因或基因组的SLC16A3序列、TTC34基因或基因组的TTC34序列、ITPKA基因或基因组的ITPKA序列和RARG基因或基因组的RARG序列；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。
在一些实施方案中，目标标志物为SLC16A3基因或基因组的SLC16A3序列、TTC34基因或基因组的TTC34序列和RARG基因或基因组的RARG序列，以及ROCR2基因或基因组的ROCR2序列或ITPKA基因或基因组的ITPKA序列；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。
在一些实施方案中，目标标志物为FTAP2B基因或基因组的FTAP2B序列以及选自EMS2OS基因或基因组的EMS2OS序列、DGKG基因或基因组的DGKG序列、SOD3基因或基因组的SOD3序列、RP11-867G23.12基因或基因组的RP11-867G23.12序列中的至少一个、至少两个或至少三个。
在一些实施方案中，目标标志物为FTAP2B基因或基因组的FTAP2B序列、EMS2OS基因或基因组的EMS2OS序列以及任选的SOD3基因或基因组的SOD3序列。优选地，目标标志物为FTAP2B基因或基因组的FTAP2B序列和EMS2OS基因或基因组的EMS2OS序列，其中，检测来自该个体的甲状腺结节中的这些目标标志物的甲基化水平。优选地，目标标志物为FTAP2B基因或基因组的FTAP2B序列、EMS2OS基因或基因组的EMS2OS序列以及SOD3基因或基因组的SOD3序列，其中，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。
在一些实施方案中，目标标志物为为FTAP2B基因或基因组的FTAP2B序列和DGKG基因或基因组的DGKG序列，其中，检测来自该个体的血液样品如血浆中这 些目标标志物的甲基化水平。
在一些实施方案中，目标标志物为FTAP2B基因或基因组的FTAP2B序列和SOD3基因或基因组的SOD3序列，以及EMS2OS基因或基因组的EMS2OS序列或RP11-867G23.12基因或基因组的RP11-867G23.12序列，其中，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。
在一些实施方案中，目标标志物为RARG基因或基因组的RARG序列，以及选自DPYS基因或基因组的DPYS序列、TSHR基因或基因组的TSHR序列、TTC34基因或基因组的TTC34序列、RCOR2基因或基因组的RCOR2序列、SLC16A3基因或基因组的SLC16A3序列和ITPKA基因或基因组的ITPKA序列中的至少一个、至少两个、至少三个或至少四个。
在一些实施方案中，目标标志物为RARG基因或基因组的RARG序列和DPYS基因或基因组的DPYS序列，其中，检测来自该个体的甲状腺结节中的这些目标标志物的甲基化水平。
在一些实施方案中，目标标志物为RARG基因或基因组的RARG序列和TSHR基因或基因组的TSHR序列，其中，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。
在一些实施方案中，目标标志物为RARG基因或基因组的RARG序列和TTC34基因或基因组的TTC34序列，以及选自RCOR2基因或基因组的RCOR2序列、SLC16A3基因或基因组的SLC16A3序列和ITPKA基因或基因组的ITPKA序列中的至少一个或至少两个，其中，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。优选地，目标标志物为RARG基因或基因组的RARG序列、TTC34基因或基因组的TTC34序列、RCOR2基因或基因组的RCOR2序列和SLC16A3基因或基因组的SLC16A3序列其中，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。优选地，目标标志物为RARG基因或基因组的RARG序列、TTC34基因或基因组的TTC34序列、ITPKA基因或基因组的ITPKA序列和SLC16A3基因或基因组的SLC16A3序列其中，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。
在一些实施方案中，目标标志物为RARG基因或基因组的RARG序列和SLC16A3基因或基因组的SLC16A3序列，以及选自TTC34基因或基因组的TTC34序列、RCOR2基因或基因组的RCOR2序列和ITPKA基因或基因组的ITPKA序列中的至少一个或至少两个，其中，检测来自该个体的血液样品如血浆中这些目标标志物 的甲基化水平。优选地，目标标志物为RARG基因或基因组的RARG序列、TTC34基因或基因组的TTC34序列、RCOR2基因或基因组的RCOR2序列和SLC16A3基因或基因组的SLC16A3序列其中，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。优选地，目标标志物为RARG基因或基因组的RARG序列、TTC34基因或基因组的TTC34序列、ITPKA基因或基因组的ITPKA序列和SLC16A3基因或基因组的SLC16A3序列其中，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。
在一些实施方案中，目标标志物为RARG基因或基因组的RARG序列、SLC16A3基因或基因组的SLC16A3序列和TTC34基因或基因组的TTC34序列，以及ROCR2基因或基因组的ROCR2序列或ITPKA基因或基因组的ITPKA序列；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。
在一些实施方案中，目标标志物为SOD3基因或基因组的SOD3序列，以及选自EMS2OS基因或基因组的EMS2OS序列、TEAD3基因或基因组的TEAD3序列、FTAP2B基因或基因组的FTAP2B序列和RP11-867G23.12基因或基因组的RP11-867G23.12序列中的至少一个或至少两个。
在一些实施方案中，目标标志物为SOD3基因或基因组的SOD3序列和EMS2OS基因或基因组的EMS2OS序列，以及TEAD3基因或基因组的TEAD3序列或FTAP2B基因或基因组的FTAP2B序列；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。
在一些实施方案中，目标标志物为SOD3基因或基因组的SOD3序列和FTAP2B基因或基因组的FTAP2B序列，以及EMS2OS基因或基因组的EMS2OS序列或RP11-867G23.12基因或基因组的RP11-867G23.12序列；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。
在一些实施方案中，目标标志物为TEAD3基因或基因组的TEAD3序列，以及选自RP11-867G23.12基因或基因组的RP11-867G23.12序列、SOD3基因或基因组的SOD3序列和EMS2OS基因或基因组的EMS2OS序列中的至少一种或至少两种。优选地，目标标志物为TEAD3基因或基因组的TEAD3序列和RP11-867G23.12基因或基因组的RP11-867G23.12序列；优选地，检测来自该个体的甲状腺结节中的这些目标标志物的甲基化水平。优选地，目标标志物为TEAD3基因或基因组的TEAD3序列，SOD3基因或基因组的SOD3序列和EMS2OS基因或基因组的EMS2OS序列；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。
在一些实施方案中，目标标志物为TTC34基因或基因组的TTC34序列，以及选自RCOR2基因或基因组的RCOR2序列、ITPKA基因或基因组的ITPKA序列、CDH1基因或基因组的CDH1序列、SLC16A3基因或基因组的SLC16A3序列和RARG基因或基因组的RARG序列中的至少一个、至少两个或至少三个；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。
在一些实施方案中，目标标志物为TTC34基因或基因组的TTC34序列和RCOR2基因或基因组的RCOR2序列，以及选自ITPKA基因或基因组的ITPKA序列、CDH1基因或基因组的CDH1序列、SLC16A3基因或基因组的SLC16A3序列和RARG基因或基因组的RARG序列中的至少一个或至少两个。优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。
在一些实施方案中，目标标志物为TTC34基因或基因组的TTC34序列、RCOR2基因或基因组的RCOR2序列、ITPKA基因或基因组的ITPKA序列和CDH1基因或基因组的CDH1序列；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。
在一些实施方案中，目标标志物为TTC34基因或基因组的TTC34序列、RCOR2基因或基因组的RCOR2序列、SLC16A3基因或基因组的SLC16A3序列和RARG基因或基因组的RARG序列；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。
在一些实施方案中，目标标志物为TTC34基因或基因组的TTC34序列和ITPKA基因或基因组的ITPKA序列，以及选自RCOR2基因或基因组的RCOR2序列、CDH1基因或基因组的CDH1序列、SLC16A3基因或基因组的SLC16A3序列和RARG基因或基因组的RARG序列中的至少一个或至少两个。优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。
在一些实施方案中，目标标志物为TTC34基因或基因组的TTC34序列、ITPKA基因或基因组的ITPKA序列、RCOR2基因或基因组的RCOR2序列和CDH1基因或基因组的CDH1序列；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。
在一些实施方案中，目标标志物为TTC34基因或基因组的TTC34序列、ITPKA基因或基因组的ITPKA序列、SLC16A3基因或基因组的SLC16A3序列和RARG基因或基因组的RARG序列；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。
在一些实施方案中，目标标志物为TTC34基因或基因组的TTC34序列和SLC16A3基因或基因组的SLC16A3序列，以及选自RCOR2基因或基因组的RCOR2序列、ITPKA基因或基因组的ITPKA序列和RARG基因或基因组的RARG序列中的至少一个或至少两个。优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。
在一些实施方案中，目标标志物为TTC34基因或基因组的TTC34序列、SLC16A3基因或基因组的SLC16A3序列、RCOR2基因或基因组的RCOR2序列和RARG基因或基因组的RARG序列；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。在一个或多个实施方案中，目标标志物为TTC34基因或基因组的TTC34序列、SLC16A3基因或基因组的SLC16A3序列、ITPKA基因或基因组的ITPKA序列和RARG基因或基因组的RARG序列；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。
在一些实施方案中，目标标志物为TTC34基因或基因组的TTC34序列、SLC16A3基因或基因组的SLC16A3序列和RARG基因或基因组的RARG序列，以及RCOR2基因或基因组的RCOR2序列或ITPKA基因或基因组的ITPKA序列；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。
在一些实施方案中，目标标志物为MCC基因或基因组的MCC序列，以及选自ITPKA基因或基因组的ITPKA序列、RCOR2基因或基因组的RCOR2序列、SLC16A3基因或基因组的SLC16A3序列和CDH1基因或基因组的CDH1序列中的至少一个、至少两个或至少三个；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。优选地，目标标志物为MCC基因或基因组的MCC序列、ITPKA基因或基因组的ITPKA序列、RCOR2基因或基因组的RCOR2序列和SLC16A3基因或基因组的SLC16A3序列；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。优选地，目标标志物为MCC基因或基因组的MCC序列和CDH1基因或基因组的CDH1序列；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。
在一些实施方案中，目标标志物为CDH1基因或基因组的CDH1序列，以及选自MCC基因或基因组的MCC序列、TTC34基因或基因组的TTC34序列、RCOR2基因或基因组的RCOR2序列和ITPKA基因或基因组的ITPKA序列中的至少一个、至少两个或至少三个；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。优选地，目标标志物为CDH1基因或基因组的CDH1序列和MCC基 因或基因组的MCC序列；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。优选地，目标标志物为CDH1基因或基因组的CDH1序列、TTC34基因或基因组的TTC34序列、RCOR2基因或基因组的RCOR2序列和ITPKA基因或基因组的ITPKA序列；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。
除上述比较之外，本领域技术人员还可以基于各种因素，例如年龄、性别、病史、家族史、症状等，来确定个体的甲状腺结节是否为恶性或为恶性的风险。
本文中，各基因或其目标区域的甲基化水平的计算方式为：在PCR实施方案中，甲基化水平＝2 –ΔCt待检样品/2 –ΔCt阳性标准品×100％，其中，ΔCt＝Ct 目的基因–Ct 内参基因；在测序实施方案中，甲基化水平＝甲基化碱基数/总碱基数。如本文所用，术语“Ct值”是指在背景信号以上可以检测到PCR产物的荧光的循环数。Ct值与样品中目标标志物的数量成反比，即Ct值越低，样品中目标标志物的数量就越大。
本文中，当检测两个以上基因或其目标区域时，对两个以上基因或其目标区域的甲基化水平进行二元Logistic回归分析，得到拟合方程。其中，若得分大于0，则判定结果为阳性，即为恶性结节
在一些实施方案中，检测对象甲状腺结节组织中DPYS基因或基因组的DPYS序列或其目标区域和RARG基因或基因组的RARG序列或其目标区域的甲基化水平，其中，对两个基因或其目标区域的甲基化水平进行二元Logistic回归分析，拟合方程为得分＝0.36+0.22×DPYS甲基化水平–0.03×RARG甲基化水平；其中，若得分大于0，则判定结果为阳性，即为恶性结节。
在一些实施方案中，检测对象甲状腺结节组织中TSHR基因或基因组的TSHR序列或其目标区域和SLC16A3基因或基因组的SLC16A3序列或其目标区域的甲基化水平，其中，对两个基因或其目标区域的甲基化水平进行二元Logistic回归分析，拟合方程为得分＝0.5528+0.21×TSHR甲基化水平–0.11×SLC16A3甲基化水平；其中，若得分大于0，则判定结果为阳性，即为恶性结节。
在一些实施方案中，检测对象甲状腺结节组织中FTAP2B基因或基因组的FTAP2B序列或其目标区域和EMX2OS基因或基因组的EMX2OS序列或其目标区域的甲基化水平，其中，对两个基因或其目标区域的甲基化水平进行二元Logistic回归分析，拟合方程为得分＝1.85+0.11×FTAP2G甲基化水平–0.17×EMX2OS甲基化水平；其中，若得分大于0，则判定结果为阳性，即为恶性结节。
在一些实施方案中，检测对象甲状腺结节组织中RP11-867G23.12基因或基因组 的RP11-867G23.12序列或其目标区域和TEAD3基因或基因组的TEAD3序列或其目标区域的甲基化水平，其中，对两个基因或其目标区域的甲基化水平进行二元Logistic回归分析，拟合方程为得分＝0.61+0.15×RP11-867G23.12甲基化水平–0.05×TEAD3甲基化水平；其中，若得分大于0，则判定结果为阳性，即为恶性结节。
在一些实施方案中，检测对象血液中TSHR基因或基因组的TSHR序列或其目标区域和RARG基因或基因组的RARG序列或其目标区域的甲基化水平，其中，对两个基因或其目标区域的甲基化水平进行二元Logistic回归分析，拟合方程为得分＝-8.34+8.11×TSHR甲基化水平+14.13×RARG甲基化水平；其中，若得分大于0，则判定结果为阳性，即为恶性结节。
在一些实施方案中，检测对象血液中MCC基因或基因组的MCC序列或其目标区域和CDH1基因或基因组的CDH1序列或其目标区域的甲基化水平，其中，对两个基因或其目标区域的甲基化水平进行二元Logistic回归分析，拟合方程为得分＝-2.84+14.53×MCC甲基化水平–98.54×CDH1甲基化水平；其中，若得分大于0，则判定结果为阳性，即为恶性结节。
在一些实施方案中，检测对象血液中SOD3基因或基因组的SOD3序列或其目标区域、EMX2OS基因或基因组的EMX2OS序列或其目标区域和TEAD3基因或基因组的TEAD3序列或其目标区域的甲基化水平，其中，对三个基因或其目标区域的甲基化水平进行二元Logistic回归分析，拟合方程为得分＝13.46–0.67×SOD3甲基化水平–1.45×EMX2OS甲基化水平+TEAD3甲基化水平；其中，若得分大于0，则判定结果为阳性，即为恶性结节。
在一些实施方案中，检测对象血液中FTAP2B基因或基因组的FTAP2B序列或其目标区域和DGKG基因或基因组的DGKG序列或其目标区域的甲基化水平，其中，对两个基因或其目标区域的甲基化水平进行二元Logistic回归分析，拟合方程为得分＝2.50–0.37×FTAP2B甲基化水平–0.42×DGKG甲基化水平；其中，若得分大于0，则判定结果为阳性，即为恶性结节。
在一些实施方案中，检测对象血液中TTC34基因或基因组的TTC34序列或其目标区域、RCOR2基因或基因组的RCOR2序列或其目标区域、SLC16A3基因或基因组的SLC16A3序列或其目标区域和RARG基因或基因组的RARG序列或其目标区域的甲基化水平，其中，对四个基因或其目标区域的甲基化水平进行二元Logistic回归分析，拟合方程为得分＝2.23+3.57×TCC34甲基化水平–24.82×RCOR2甲基化水平–4.66×SLC16A3甲基化水平–0.75×RARG甲基化水平；其中，若得 分大于0，则判定结果为阳性，即为恶性结节。
在一些实施方案中，检测对象血液中SOD3基因或基因组的SOD3序列或其目标区域、EMX2OS基因或基因组的EMX2OS序列或其目标区域和FTAP2B基因或基因组的FTAP2B序列或其目标区域的甲基化水平，其中，对三个基因或其目标区域的甲基化水平进行二元Logistic回归分析，拟合方程为得分＝6.53–1.02×SOD3甲基化水平–1.21×EMX2OS甲基化水平+1.40×FTAP2B甲基化水平；其中，若得分大于0，则判定结果为阳性，即为恶性结节。
在一些实施方案中，检测对象血液中TTC34基因或基因组的TTC34序列或其目标区域、RCOR2基因或基因组的RCOR2序列或其目标区域、ITPKA基因或基因组的ITPKA序列或其目标区域和CDH1基因或基因组的CDH1序列或其目标区域的甲基化水平，其中，对四个基因或其目标区域的甲基化水平进行二元Logistic回归分析，拟合方程为得分＝1.16+1.75×TTC34甲基化水平–52.62×RCOR2甲基化水平–7.62×ITPKA甲基化水平+75.98×CDH1甲基化水平；其中，若得分大于0，则判定结果为阳性，即为恶性结节。
在一些实施方案中，检测对象血液中SOD3基因或基因组的SOD3序列或其目标区域、RP11-867G23.12基因或基因组的RP11-867G23.12序列或其目标区域和FTAP2B基因或基因组的FTAP2B序列或其目标区域的甲基化水平，其中，对三个基因或其目标区域的甲基化水平进行二元Logistic回归分析，拟合方程为得分＝6.10–1.04×SOD3甲基化水平+0.60×RP11-867G23.12甲基化水平+0.61×FTAP2B甲基化水平；其中，若得分大于0，则判定结果为阳性，即为恶性结节。
在一些实施方案中，检测对象血液中ITPKA基因或基因组的ITPKA序列或其目标区域、RCOR2基因或基因组的RCOR2序列或其目标区域、SLC16A3基因或基因组的SLC16A3序列或其目标区域和MCC基因或基因组的MCC序列或其目标区域的甲基化水平，其中，对四个基因或其目标区域的甲基化水平进行二元Logistic回归分析，拟合方程为得分＝-1.61–2.37×SLC16A3甲基化水平–9.34×RCOR2甲基化水平–17.82×ITPKA甲基化水平+1.62×MCC甲基化水平；其中，若得分大于0，则判定结果为阳性，即为恶性结节。
在一些实施方案中，检测对象血液中TTC34基因或基因组的TTC34序列或其目标区域、ITPKA基因或基因组的ITPKA序列或其目标区域、SLC16A3基因或基因组的SLC16A3序列或其目标区域和RARG基因或基因组的RARG序列或其目标区域的甲基化水平，其中，对四个基因或其目标区域的甲基化水平进行二元Logistic回归分 析，拟合方程为得分＝2.47+3.27×TTC34甲基化水平–3.71×ITPKA甲基化水平–6.48×SLC16A3甲基化水平–0.69×RARG甲基化水平；其中，若得分大于0，则判定结果为阳性，即为恶性结节。
VI.组合物和试剂盒
本发明提供一种用于甲状腺结节良恶性鉴别的甲基化检测试剂盒和组合物，所述试剂盒和组合物包括引物分子。根据待检测的目标标志物或其目标区域，试剂盒和组合物中可含有能够与所述待检测的目标标志物或其目标区域在严谨条件下、中等严谨条件下或高度严谨条件下杂交的引物对。
在一些实施方案中，试剂盒和组合物中的引物对可选自以下引物对中的任意一对或任意多对：
(1)能扩增出由SEQ ID NO:1和2作为引物扩增得到的CDH1基因的片段的引物对；
(2)能扩增出由SEQ ID NO:3和4作为引物扩增得到的TSHR基因的片段的引物对；
(3)能扩增出由SEQ ID NO:5和6作为引物扩增得到的MCC基因的片段的引物对；
(4)能扩增出由SEQ ID NO:7和8作为引物扩增得到的TBX15基因的片段的引物对；
(5)能扩增出由SEQ ID NO:9和10作为引物扩增得到的PRR15基因的片段的引物对；
(6)能扩增出由SEQ ID NO:11和12作为引物扩增得到的DPYS基因的片段的引物对；
(7)能扩增出由SEQ ID NO:13和14作为引物扩增得到的GRIA2基因的片段的引物对；
(8)能扩增出由SEQ ID NO:15和16作为引物扩增得到的NR5A1基因的片段的引物对；
(9)能扩增出由SEQ ID NO:17和18作为引物扩增得到的TTC34基因的片段的引物对；
(10)能扩增出由SEQ ID NO:19和20作为引物扩增得到的RCOR2基因的片段的引物对；
(11)能扩增出由SEQ ID NO:21和22作为引物扩增得到的F10基因的片段的引物对；
(12)能扩增出由SEQ ID NO:23和24作为引物扩增得到的ITPKA基因的片段的引物对；
(13)能扩增出由SEQ ID NO:25和26作为引物扩增得到的SLC16A3基因的片段的引物对；
(14)能扩增出由SEQ ID NO:27和28作为引物扩增得到的RARG基因的片段的引物对；
(15)能扩增出由SEQ ID NO:29和30作为引物扩增得到的FTAP2B基因的片段的引物对；
(16)能扩增出由SEQ ID NO:31和32作为引物扩增得到的SOD3基因的片段的引物对；
(17)能扩增出由SEQ ID NO:33和34作为引物扩增得到的RP11-867G23.12基因的片段的引物对；
(18)能扩增出由SEQ ID NO:35和36作为引物扩增得到的EMX2OS基因的片段的引物对；
(19)能扩增出由SEQ ID NO:37和38作为引物扩增得到的TEAD3基因的片段的引物对；
(20)能扩增出由SEQ ID NO:39和40作为引物扩增得到的DGKG基因的片段的引物对。
在一些实施方案中，所述引物对选自下组引物对中的至少一对或多对：SEQ ID NO:1和2；SEQ ID NO:3和4；SEQ ID NO:5和6；SEQ ID NO:7和8；SEQ ID NO:9和10；SEQ ID NO:11和12；SEQ ID NO:13和14；SEQ ID NO:15和16；SEQ ID NO:17和18；SEQ ID NO:19和20；SEQ ID NO:21和22；SEQ ID NO:23和24；SEQ ID NO:25和26；SEQ ID NO:27和28；SEQ ID NO:29和30；SEQ ID NO:31和32；SEQ ID NO:33和34；SEQ ID NO:35和36；SEQ ID NO:37和38；和SEQ ID NO:39和40。
在一些实施方案中，所述试剂盒和组合物中所含的引物对为用于扩增选自下组的目标标志物或其目标区域的引物对：CDH1、TSHR、MCC、TBX15、PRR15、DPYS、GRIA2、NR5A1、TTC34、RCOR2、F10、ITPKA、SLC16A3、RARG、FTAP2B、SOD3、RP11-867G23.12、EMX2OS、TEAD3或DGKG基因或其各自在基因组中的序列或其各自目标区域；DPYS或其各自在基因组中的序列或其目标区域和RARG基 因或基因组的RARG序列或其目标区域；TSHR基因或基因组的TSHR序列或其目标区域和SLC16A3基因或基因组的SLC16A3序列或其目标区域；FTAP2B基因或基因组的FTAP2B序列或其目标区域和EMX2OS基因或基因组的EMX2OS序列或其目标区域；RP11-867G23.12基因或基因组的RP11-867G23.12序列或其目标区域和TEAD3基因或基因组的TEAD3序列或其目标区域；TSHR基因或基因组的TSHR序列或其目标区域和RARG基因或基因组的RARG序列或其目标区域；MCC基因或基因组的MCC序列或其目标区域和CDH1基因或基因组的CDH1序列或其目标区域；SOD3基因或基因组的SOD3序列或其目标区域、EMX2OS基因或基因组的EMX2OS序列或其目标区域和TEAD3基因或基因组的TEAD3序列或其目标区域；FTAP2B基因或基因组的FTAP2B序列或其目标区域和DGKG基因或基因组的DGKG序列或其目标区域；TTC34基因或基因组的TTC34序列或其目标区域、RCOR2基因或基因组的RCOR2序列或其目标区域、SLC16A3基因或基因组的SLC16A3序列或其目标区域和RARG基因或基因组的RARG序列或其目标区域；SOD3基因或基因组的SOD3序列或其目标区域、EMX2OS基因或基因组的EMX2OS序列或其目标区域和FTAP2B基因或基因组的FTAP2B序列或其目标区域；TTC34基因或基因组的TTC34序列或其目标区域、RCOR2基因或基因组的RCOR2序列或其目标区域、ITPKA基因或基因组的ITPKA序列或其目标区域和CDH1基因或基因组的CDH1序列或其目标区域；SOD3基因或基因组的SOD3序列或其目标区域、RP11-867G23.12基因或基因组的RP11-867G23.12序列或其目标区域和FTAP2B基因或基因组的FTAP2B序列或其目标区域；ITPKA基因或基因组的ITPKA序列或其目标区域、RCOR2基因或基因组的RCOR2序列或其目标区域、SLC16A3基因或基因组的SLC16A3序列或其目标区域和MCC基因或基因组的MCC序列或其目标区域；TTC34基因或基因组的TTC34序列或其目标区域、ITPKA基因或基因组的ITPKA序列或其目标区域、SLC16A3基因或基因组的SLC16A3序列或其目标区域和RARG基因或基因组的RARG序列或其目标区域。在这些实施方案中的一些优选实施方案中，所述引物对的引物序列分别为：SEQ ID NO:1和2，SEQ ID NO:3和4，SEQ ID NO:5和6，SEQ ID NO:7和8，SEQ ID NO:9和10，SEQ ID NO:11和12，SEQ ID NO:13和14，SEQ ID NO:15和16，SEQ ID NO:17和18，SEQ ID NO:19和20，SEQ ID NO:21和22，SEQ ID NO:23和24，SEQ ID NO:25和26，SEQ ID NO:27和28，SEQ ID NO:29和30，SEQ ID NO:31和32，SEQ ID NO:33和34，SEQ ID NO:35和36，SEQ ID NO:37和38，或SEQ ID NO:39和40；SEQ ID NO:11和12，和SEQ ID NO:27和28；SEQ ID  NO:3和4，和SEQ ID NO:25和26；SEQ ID NO:29和30，和SEQ ID NO:35和36；SEQ ID NO:33和34，和SEQ ID NO:37和38；SEQ ID NO:3和4，和SEQ ID NO:27和28；SEQ ID NO:5和6，SEQ ID NO:1和2；SEQ ID NO:31和32、SEQ ID NO:35和36、和SEQ ID NO:37和38；SEQ ID NO:29和30，和SEQ ID NO:39和40；SEQ ID NO:17和18、SEQ ID NO:19和20、SEQ ID NO:25和26、和SEQ ID NO:27和28；SEQ ID NO:31和32、SEQ ID NO:35和36、和SEQ ID NO:29和30；SEQ ID NO:17和18、SEQ ID NO:19和20、SEQ ID NO:23和24、和SEQ ID NO:1和2；SEQ ID NO:31和32、SEQ ID NO:33和34、和SEQ ID NO:29和30；SEQ ID NO:23和24、SEQ ID NO:19和20、SEQ ID NO:25和26、和SEQ ID NO:5和6；SEQ ID NO:17和18、SEQ ID NO:23和24、SEQ ID NO:25和26、和SEQ ID NO:27和28。
引物还可包括检测内参如ACTB的引物，例如能扩增出由SEQ ID NO:61和62序列作为引物扩增得到的ACTB基因的片段的引物。在一些实施方案中，内参的引物对为SEQ ID NO:61和62。
在一些实施方式中，所述引物被包装在单一容器内或被包装在独立容器内。在一些实施方式中，所述试剂盒进一步包含一个或多个封闭寡核苷酸。
在一些实施方式中，所述试剂盒和组合物进一步包含检测试剂。在一些实施方式中，所述检测试剂选自下组：荧光探针，嵌入染料、生色团标记的探针，放射性同位素标记的探针和生物素标记的探针。
在优选的实施方案中，根据待检测的目标标志物或其目标区域，试剂盒和组合物中可含有向对应的荧光探针。具体而言，试剂盒和组合物中的荧光探针可以是用于检测下组目标标志物或其目标区域的荧光探针：CDH1、TSHR、MCC、TBX15、PRR15、DPYS、GRIA2、NR5A1、TTC34、RCOR2、F10、ITPKA、SLC16A3、RARG、FTAP2B、SOD3、RP11-867G23.12、EMX2OS、TEAD3或DGKG基因其各自在基因组中的序列或其各自目标区域；DPYS或其各自在基因组中的序列或其目标区域和RARG基因或基因组的RARG序列其目标区域；TSHR基因或基因组的TSHR序列其目标区域和SLC16A3基因或基因组的SLC16A3序列其目标区域；FTAP2B基因或基因组的FTAP2B序列其目标区域和EMX2OS基因或基因组的EMX2OS序列其目标区域；RP11-867G23.12基因或基因组的RP11-867G23.12序列其目标区域和TEAD3基因或基因组的TEAD3序列其目标区域；TSHR基因或基因组的TSHR序列其目标区域和RARG基因或基因组的RARG序列其目标区域；MCC基因或基因组的MCC序列其目标区域和CDH1基因或基因组的CDH1序列其目标区域；SOD3基因或基因组的 SOD3序列其目标区域、EMX2OS基因或基因组的EMX2OS序列其目标区域和TEAD3基因或基因组的TEAD3序列其目标区域；FTAP2B基因或基因组的FTAP2B序列其目标区域和DGKG基因或基因组的DGKG序列其目标区域；TTC34基因或基因组的TTC34序列其目标区域、RCOR2基因或基因组的RCOR2序列其目标区域、SLC16A3基因或基因组的SLC16A3序列其目标区域和RARG基因或基因组的RARG序列其目标区域；SOD3基因或基因组的SOD3序列其目标区域、EMX2OS基因或基因组的EMX2OS序列其目标区域和FTAP2B基因或基因组的FTAP2B序列其目标区域；TTC34基因或基因组的TTC34序列其目标区域、RCOR2基因或基因组的RCOR2序列其目标区域、ITPKA基因或基因组的ITPKA序列其目标区域和CDH1基因或基因组的CDH1序列其目标区域；SOD3基因或基因组的SOD3序列其目标区域、RP11-867G23.12基因或基因组的RP11-867G23.12序列其目标区域和FTAP2B基因或基因组的FTAP2B序列其目标区域；ITPKA基因或基因组的ITPKA序列其目标区域、RCOR2基因或基因组的RCOR2序列其目标区域、SLC16A3基因或基因组的SLC16A3序列其目标区域和MCC基因或基因组的MCC序列其目标区域；TTC34基因或基因组的TTC34序列其目标区域、ITPKA基因或基因组的ITPKA序列其目标区域、SLC16A3基因或基因组的SLC16A3序列其目标区域和RARG基因或基因组的RARG序列其目标区域。优选的实施方案中，针对上述各基因或其目标区域的荧光探针的核苷酸序列以及具体的荧光探针可如前文第IV部分所述。更具体而言，试剂盒中的荧光探针的核苷酸序列可以是选自下组：SEQ ID NO:41-60中任意一条；SEQ ID NO:46和SEQ ID NO:54；SEQ ID NO:42和SEQ ID NO:53；SEQ ID NO:55和SEQ ID NO:58；SEQ ID NO:57和SEQ ID NO:59；SEQ ID NO:42和SEQ ID NO:54；SEQ ID NO:43和SEQ ID NO:41；SEQ ID NO:56、SEQ ID NO:58和SEQ ID NO:59；SEQ ID NO:55和SEQ ID NO:60；SEQ ID NO:49、SEQ ID NO:50、SEQ ID NO:53和SEQ ID NO:54；SEQ ID NO:56、SEQ ID NO:58和SEQ ID NO:55；SEQ ID NO:49、SEQ ID NO:50、SEQ ID NO:52和SEQ ID NO:41；SEQ ID NO:56、SEQ ID NO:57和SEQ ID NO:55；SEQ ID NO:52、SEQ ID NO:50、SEQ ID NO:53和SEQ ID NO:43；SEQ ID NO:49、SEQ ID NO:52、SEQ ID NO:53和SEQ ID NO:54。
探针还包括检测内参基因如ACTB的探针。在一些实施方案中，检测内参基因ACTB的探针的核苷酸序列如SEQ ID NO:63所示。
在一些实施方式中，所述试剂盒还可包含DNA聚合酶和/或适合存放从个体获取的生物样品的容器。在一些实施方式中，所述试剂盒进一步含使用说明书和/或对试 剂盒检测结果的解释。
在一些实施方式中，所述试剂盒和组合物还可包括用于酶促法或非酶促法进行转化的试剂。在优选的实施方案中，所示试剂盒还包括亚硫酸氢盐试剂或甲基化敏感限制酶(MSRE)。在一些实施方式中，所述亚硫酸氢盐试剂选自下组：亚硫酸氢铵、亚硫酸氢钠、亚硫酸氢钾、亚硫酸氢钙、亚硫酸氢镁、亚硫酸氢铝、亚硫酸氢根离子，及其任意组合。在一些实施方式中，亚硫酸氢盐试剂是亚硫酸氢钠。在一些实施方式中，所述MSRE选自下组：HpaII酶、SalI酶、
Figure PCTCN2022102658-appb-000003
ScrFI酶、BbeI酶、NotI酶、SmaI酶、XmaI酶、MboI酶、BstBI酶、ClaI酶、MluI酶、NaeI酶、NarI酶、PvuI酶、SacII酶、HhaI酶及其任意组合。
所述试剂盒和组合物还可包括经转化的阳性标准品，其中未甲基化的胞嘧啶转化为不与鸟嘌呤结合的碱基。所述阳性标准品可以是完全甲基化的。
所述试剂盒和组合物还可包括PCR反应试剂。优选地，所述PCR反应试剂包括Taq DNA聚合酶、PCR缓冲液(buffer)、dNTPs、Mg 2+。
在一些实施方式中，所述试剂盒和组合物还包含可用于进行CpG位置特异性甲基化分析的标准试剂，其中所述分析包括以下一种或多种技术：MS-SNuPE、MSP、MethyLight TM、HeavyMethyl TM、COBRA和核酸测序。
在一些实施方式中，所述试剂盒和组合物可包含选自下组的额外的试剂：缓冲液(例如限制酶、PCR、保存或洗涤缓冲液)、DNA回收试剂或试剂盒(例如沉淀、超滤、亲和柱)和DNA回收组件等。
本申请的试剂盒可进一步包含在DNA富集领域中已知的以下组分的一种或几种：蛋白组分，所述蛋白选择性地结合甲基化的DNA；三链形成核酸组分，一个或多个接头，任选地在合适的溶液中；用于进行连接的物质或溶液，例如连接酶、缓冲液；用于进行柱层析的物质或溶液；用于进行免疫学为基础的富集(例如免疫沉淀)的物质或溶液；用于进行核酸扩增的物质或溶液，例如PCR；一种染料或几种染料，若适用于偶联剂，若适用于溶液中；用于进行杂交的物质或溶液；和/或用于进行洗涤步骤的物质或溶液。
在其他一些实施方案中，本发明的组合物含有分离的核酸分子，所述分离的核酸分子选自以下的一种或多种：(1)由SEQ ID NO:1和2或与其有至少90％相同性的序列作为引物扩增得到的CDH1基因的片段；(2)由SEQ ID NO:3和4或与其有至少90％相同性的序列作为引物扩增得到的TSHR基因的片段；(3)由SEQ ID NO:5 和6或与其有至少90％相同性的序列作为引物扩增得到的MCC基因的片段；(4)由SEQ ID NO:7和8或与其有至少90％相同性的序列作为引物扩增得到的TBX15基因的片段；(5)由SEQ ID NO:9和10或与其有至少90％相同性的序列作为引物扩增得到的PRR15基因的片段，(6)由SEQ ID NO:11和12或与其有至少90％相同性的序列作为引物扩增得到的DPYS基因的片段；(7)由SEQ ID NO:13和14或与其有至少90％相同性的序列作为引物扩增得到的GRIA2基因的片段；(8)由SEQ ID NO:15和16或与其有至少90％相同性的序列作为引物扩增得到的NR5A1基因的片段；(9)由SEQ ID NO:17和18或与其有至少90％相同性的序列作为引物扩增得到的TTC34基因的片段；(10)由SEQ ID NO:19和20或与其有至少90％相同性的序列作为引物扩增得到的RCOR2基因的片段；(11)由SEQ ID NO:21和22或与其有至少90％相同性的序列作为引物扩增得到的F10基因的片段；(12)由SEQ ID NO:23和24或与其有至少90％相同性的序列作为引物扩增得到的ITPKA基因的片段；(13)由SEQ ID NO:25和26或与其有至少90％相同性的序列作为引物扩增得到的SLC16A3基因的片段；(14)由SEQ ID NO:27和28或与其有至少90％相同性的序列作为引物扩增得到的RARG基因的片段；(15)由SEQ ID NO:29和30或与其有至少90％相同性的序列作为引物扩增得到的FTAP2B基因的片段；(16)由SEQ ID NO:31和32或与其有至少90％相同性的序列作为引物扩增得到的SOD3基因的片段；(17)由SEQ ID NO:33和34或与其有至少90％相同性的序列作为引物扩增得到的RP11-867G23.12基因的片段；(18)由SEQ ID NO:35和36或与其有至少90％相同性的序列作为引物扩增得到的EMX2OS基因的片段；(19)由SEQ ID NO:37和38或与其有至少90％相同性的序列作为引物扩增得到的TEAD3基因的片段；(20)由SEQ ID NO:39和40或与其有至少90％相同性的序列作为引物扩增得到的DGKG基因的片段。
在优选的实施方案中，所述组合物含有：DPYS目标区域和RARG基因目标区域；TSHR基因目标区域和SLC16A3基因目标区域；FTAP2B基因目标区域和EMX2OS基因目标区域；RP11-867G23.12基因目标区域和TEAD3基因目标区域；TSHR基因目标区域和RARG基因目标区域；MCC基因目标区域和CDH1基因目标区域；SOD3基因目标区域、EMX2OS基因目标区域和TEAD3基因目标区域；FTAP2B基因目标区域和DGKG基因目标区域；TTC34基因目标区域、RCOR2基因目标区域、SLC16A3 基因目标区域和RARG基因目标区域；SOD3基因目标区域、EMX2OS基因目标区域和FTAP2B基因目标区域；TTC34基因目标区域、RCOR2基因目标区域、ITPKA基因目标区域和CDH1基因目标区域；SOD3基因目标区域、RP11-867G23.12基因目标区域和FTAP2B基因目标区域；ITPKA基因目标区域、RCOR2基因目标区域、SLC16A3基因目标区域和MCC基因目标区域；TTC34基因目标区域、ITPKA基因目标区域、SLC16A3基因目标区域和RARG基因目标区域。
本申请还包括记载有本文所述分离的核酸分子的序列和任选的其甲基化信息的介质，所述介质用于与基因甲基化测序数据比对以确定所述核酸分子的存在、含量和/或甲基化水平。优选地，所述介质是印有所述序列和任选的其甲基化信息的卡片，例如纸质、塑料、金属、玻璃卡片。优选地，所述介质是存储有所述序列和任选的其甲基化信息和计算机程序的计算机可读介质，当所述计算机程序被处理器执行时，实现下述步骤：将样品的甲基化测序数据与所述序列比较，从而获得所述样品中含所述序列的核酸分子的存在在、含量和/或甲基化水平。
本申请还包括一种用于鉴别甲状腺结节良恶性的装置，所述装置包括存储器、处理器以及存储在存储器上并可在处理器上运行的计算机程序，所述处理器执行所述程序时实现以下步骤：(1)获取样品中选自以下一种或多种本文所述的目标标志物或其目标区域的甲基化水平，(2)根据(1)的甲基化水平判读甲状腺结节良恶性。优选地，所述获取步骤采用本申请第IV部分所述的任意一种方法进行；优选地，所述判读采取本申请第V部分所述的任意一种方法进行。
VII.用途
本申请还提供本文所述的引物对、引物对的组合以及任选的探针或探针组合在诊断甲状腺结节良恶性中的应用，以及在制备用于诊断甲状腺结节良恶性的试剂或试剂盒中的应用。
优选地，所述试剂盒如本申请第VI部分中任一方案所述。优选地，所述试剂盒用于实施本文第II部分、第III部分、第IV部分和第V部分中任意一个或多个部分所述的各个步骤和方法。
本申请还提供本申请所述的分离的核酸分子做为检测靶标在甲状腺结节良恶性诊断中的应用。
以下结合附图和具体实施例来进一步说明本发明，但实施例并不对本发明做任何形式的限定。除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。除非特别说明，以下实施例所用试剂和材料均为市购。
实施例
实施例1：验证甲状腺结节良恶性与组织基因组DNA甲基化的关系。
申请人对10例甲状腺癌患者和10例甲状腺良性结节患者的组织进行基因组DNA甲基化特异的PCR，发现CDH1基因，TSHR基因，MCC基因，TBX15基因，PRR15基因，DPYS基因，GRIA2基因，NR5A1基因，TTC34基因，RCOR2基因，F10基因，ITPKA基因，SLC16A3基因，RARG基因，FTAP2B基因，SOD3基因，RP11-867G23.12基因，EMX2OS基因，TEAD3基因和DGKG基因在甲状腺癌和甲状腺良性结节病例的基因组DNA甲基化水平具有差异，结果如图1和图2所示。具体实验步骤如下：
1.1样本准备
使用QIAamp DNA Mini Kit(QIAGEN，货号：51304)对10例甲状腺癌和10例甲状腺良性结节患者的组织DNA进行抽提；使用Qubit TM dsDNA HS Assay Kit(Thermo，货号：Q32854)检测DNA的浓度；使用1％琼脂糖凝胶电泳进行质检。
1.2 DNA转化
使用MethylCodeTM Bisulfite Conversion Kit(Thermo，货号：MECOV50)对步骤1得到的DNA进行重亚硫酸盐转化，未甲基化的胞嘧啶(cytosine,C)经过转化变为尿嘧啶(uracil,U)；甲基化的胞嘧啶转化后不发生改变。
1.3 PCR混合物准备
使用本发明提供的试剂盒，包括PCR反应液、引物混合物(SEQ ID NO:1-40、61-62)、探针混合物(SEQ ID NO:41-60和63)，进行单个样本的配制，如表1-7所示。
表1：PCR反应体系组成
Figure PCTCN2022102658-appb-000004
Figure PCTCN2022102658-appb-000005
表2：PCR反应体系组成
组分 体积(μl)
2x PCR反应液 10
水 2.88
内参基因F,100μM 0.12
内参基因R,100μM 0.12
TBX15基因F,100μM 0.12
TBX15基因R,100μM 0.12
PRR15基因F,100μM 0.12
PRR15基因R,100μM 0.12
DPYS基因F,100μM 0.12
DPYS基因R,100μM 0.12
内参基因探针,100μM(VIC/BHQ1) 0.04
TBX15基因探针,100μM(FAM/BHQ1) 0.04
PRR15基因探针,100μM(Texas/BHQ2) 0.04
DPYS基因,100μM(Cy5/BHQ2) 0.04
样本DNA(10ng)/阳性对照/阴性对照 6
总体积 20
表3：PCR反应体系组成
组分 体积(μl)
2x PCR反应液 10
水 2.88
内参基因F,100μM 0.12
内参基因R,100μM 0.12
GRIA2基因F,100μM 0.12
GRIA2基因R,100μM 0.12
NR5A1基因F,100μM 0.12
NR5A1基因R,100μM 0.12
TTC34基因F,100μM 0.12
TTC34基因R,100μM 0.12
内参基因探针,100μM(VIC/BHQ1) 0.04
GRIA2基因探针,100μM(FAM/BHQ1) 0.04
NR5A1基因探针,100μM(Texas/BHQ2) 0.04
TTC34基因,100μM(Cy5/BHQ2) 0.04
样本DNA(10ng)/阳性对照/阴性对照 6
总体积 20
表4：PCR反应体系组成
Figure PCTCN2022102658-appb-000006
Figure PCTCN2022102658-appb-000007
表5：PCR反应体系组成
组分 体积(μl)
2x PCR反应液 10
水 2.88
内参基因F,100μM 0.12
内参基因R,100μM 0.12
SLC16A3基因F,100μM 0.12
SLC16A3基因R,100μM 0.12
RARG基因F,100μM 0.12
RARG基因R,100μM 0.12
FTAP2B基因F,100μM 0.12
FTAP2B基因R,100μM 0.12
内参基因探针,100μM(VIC/BHQ1) 0.04
SLC16A3基因探针,100μM(FAM/BHQ1) 0.04
RARG基因探针,100μM(Texas/BHQ2) 0.04
FTAP2B基因,100μM(Cy5/BHQ2) 0.04
样本DNA(10ng)/阳性对照/阴性对照 6
总体积 20
表6：PCR反应体系组成
Figure PCTCN2022102658-appb-000008
Figure PCTCN2022102658-appb-000009
表7：PCR反应体系组成
组分 体积(μl)
2x PCR反应液 10
水 3.16
内参基因F,100μM 0.12
内参基因R,100μM 0.12
TEAD3基因F,100μM 0.12
TEAD3基因R,100μM 0.12
DGKG基因F,100μM 0.12
DGKG基因R,100μM 0.12
内参基因探针,100μM(VIC/BHQ1) 0.04
TEAD3基因探针,100μM(FAM/BHQ1) 0.04
DGKG基因探针,100μM(Texas/BHQ2) 0.04
样本DNA(10ng)/阳性对照/阴性对照 6
总体积 20
引物序列如下：
CDH1基因正向引物F:
GTAATTTTAGGTTAGAGGGTTATYG(SEQ ID NO:1)，
CDH1基因反向引物R:
CCCTCCCCAAAACRAAAC(SEQ ID NO:2)，
TSHR基因正向引物F:
TGTAGAGTTGAGAATGAGGTGATTTC(SEQ ID NO:3)，
TSHR基因反向引物R:
GCCCAAATCCCTAAACAAATCG(SEQ ID NO:4)，
MCC基因正向引物F:
AAAAACACAATCCGAAATTAATCTCG(SEQ ID NO:5)，
MCC基因反向引物R:
GGGAGGGATAGCGTTGAGT(SEQ ID NO:6)，
TBX15基因正向引物F:
ACCTAACCACACAAAACTCCC(SEQ ID NO:7)，
TBX15基因反向引物R:
TAAAATTGCGCGATTGTTCG(SEQ ID NO:8)，
PRR15基因正向引物F:
ACCTCCTCCGAAAACCTACC(SEQ ID NO:9)，
PRR15基因反向引物R:
GAAGATTTCGCGTTTCGGTC(SEQ ID NO:10)，
DPYS基因正向引物F:
GCCGACAAACTCGTCCTAC(SEQ ID NO:11)，
DPYS基因反向引物R:
TCGATGGATCGCGAGTTTATG(SEQ ID NO:12)，
GRIA2基因正向引物F:
TGTTGTGTAGAAGTYGAATTGT(SEQ ID NO:13)，
GRIA2基因反向引物R:
TATAACRTAAAACAACAAAACCTAAT(SEQ ID NO:14)，
NR5A1基因正向引物F:
TTTGTGTTTAGGYGTTGTY(SEQ ID NO:15)，
NR5A1基因反向引物R:
AAACTACACCRAACCCAC(SEQ ID NO:16)，
TTC34基因正向引物F:
CCCRCAAAATCCTCAAAAC(SEQ ID NO:17)，
TTC34基因反向引物R:
GAGTGTTTGTTGTTGGTAGG(SEQ ID NO:18)，
RCOR2基因正向引物F:
CGAAAAAAACATTCCCGAAAAC(SEQ ID NO:19)，
RCOR2基因反向引物R:
TCGAGAATTCGGGGTTTTTA(SEQ ID NO:20)，
F10基因正向引物F:
CCCGAAACGACTCAACAC(SEQ ID NO:21)，
F10基因反向引物R:
AGGAGAGACGAGTTCGTAG(SEQ ID NO:22)，
ITPKA基因正向引物F:
ACGCAAACCGAAAACTTC(SEQ ID NO:23)，
ITPKA基因反向引物R:
GTGTGGYGGGTGTTGATA(SEQ ID NO:24)，
SLC16A3基因正向引物F:
GTAAATGAGGTTTGTGTGTTTGTTT(SEQ ID NO:25)，
SLC16A3基因反向引物R:
CACCTCTAACCCCCRCAAA(SEQ ID NO:26)，
RARG基因正向引物F:
CRAACACAACACTTTCCAAAACC(SEQ ID NO:27)，
RARG基因反向引物R:
GTTTGTGAGGGGATGTTTGTG(SEQ ID NO:28)，
FTAP2B基因正向引物F:
TTGGGAGATTGGGTAATAATATAC(SEQ ID NO:29)，
FTAP2B基因反向引物R:
CCTAAAAAARGTACCTAAACGC(SEQ ID NO:30)，
SOD3基因正向引物F:
ACTAACCTACTACGTAATAAACGT(SEQ ID NO:31)，
SOD3基因反向引物R:
TTGCGTTTTGAGTGTTTTCG(SEQ ID NO:32)，
RP11-867G23.12基因正向引物F:
GTTGTGAGGTYGTTAGTATATG(SEQ ID NO:33)，
RP11-867G23.12基因反向引物R:
ACCTCRACGAAAAAAATAAATACG(SEQ ID NO:34)，
EMX2OS基因正向引物F:
GCGTTTCGTTTCGTTTTTTATG(SEQ ID NO:35)，
EMX2OS基因反向引物R:
GTAACAAATCCTTCCCATCG(SEQ ID NO:36)，
TEAD3基因正向引物F:
AGTTTTTYGGGAGTTAAGGAAG(SEQ ID NO:37)，
TEAD3基因反向引物R:
CGAATAAACTACATAAAACTCTCCG(SEQ ID NO:38)，
DGKG基因正向引物F:
GGTAATTGAAAGGACGAAGTTG(SEQ ID NO:39)，
DGKG基因反向引物R:
CCTACTACGTTTACTTCACTACC(SEQ ID NO:40)，
ACTB基因正向引物F:
TGGAGGAGGTTTAGTAAGTTTTTTG(SEQ ID NO:61)，
ACTB基因反向引物R:
CCTCCCTTAAAAATTACAAAAACCA(SEQ ID NO:62)。
探针序列如下：
CDH1基因探针：CGCCCACCCGACCTCGCAT(SEQ ID NO:41)
TSHR基因探针：ACAACACCAACTACAACAAATCCGCCGA(SEQ ID NO:42)
MCC基因探针：CCTACCGCACGCCTATTCAATAACCT(SEQ ID NO:43)
TBX15基因探针：ACCCTACTCCTACGCAAACCGAAAT(SEQ ID NO:44)
PRR15基因探针：CCTCCGAAAACAACGTAACGCGC(SEQ ID NO:45)
DPYS基因探针：CGAAAACATCGACACACACACGCA(SEQ ID NO:46)
GRIA2基因探针：AAAACGCTTCGCCGCCAACA(SEQ ID NO:47)
NR5A1基因探针：AAACGCTACGCGAAACGCTC(SEQ ID NO:48)
TTC34基因探针：CGAACCGCAACAAACGCTCG(SEQ ID NO:49)
RCOR2基因探针：CCGACTCGCGCCAAACTCGA(SEQ ID NO:50)
F10基因探针：CAAACAACGCGACCTCTAAACGC(SEQ ID NO:51)
ITPKA基因探针：ACGCTAAAATCACCTTCACTACGCC(SEQ ID NO:52)
SLC16A3基因探针：ATGTAAGCGGATATAGAGCGGTAGGGTA(SEQ ID NO:53)
RARG基因探针：CGCAACCACGCAAAAACACACGC(SEQ ID NO:54)
FTAP2B基因探针：CGAGTCGTCGTATACGGTTTCGGG(SEQ ID NO:55)
SOD3基因探针：CGAACCCGAACTCTAAAAACGCCAAACG(SEQ ID NO:56)
RP11-867G23.12基因探针：CGGTGTTGTCGGTTGTTTTTTACGTACG(SEQ ID NO:57)
EMX2OS基因探针：CCAAAACGTACACCGACTCCTAAATTCC(SEQ ID NO:58)
TEAD3基因探针：TGGTTGCGGTATCGGATTATTTATACGG(SEQ ID NO:59)
DGKG基因探针：AGGTTCGGGTAGGTTTTCGGCG(SEQ ID NO:60)
ACTB基因探针：ACCACCACCCAACACACAATAACAAACACA(SEQ ID NO:63)
1.4荧光定量PCR
设置PCR程序为95℃预变性5min；95℃变性20s，60℃退火延伸45s，50个循环。60℃退火延伸阶段收集荧光信号。
1.5检测结果分析
利用下式计算各样品中基因的甲基化水平：
甲基化水平＝2 –ΔCt待检样品/2 –ΔCt阳性标准品×100％。
ΔCt＝Ct 目的基因–Ct 内参基因。
CDH1、TSHR、MCC、TBX15、PRR15、DPYS、GRIA2、NR5A1、TTC34、RCOR2、F10、ITPKA、SLC16A3、RARG、FTAP2B、SOD3、RP11-867G23.12、EMX2OS、TEAD3和DGKG基因在组织基因组中甲基化水平如图1所示；对各个基因的甲基化水平分别进行ROC曲线分析如图2所示，各个基因的AUC均大于0.6。
实施例2：验证甲状腺结节良恶性与血浆中cfDNA甲基化的关系
申请人对196例甲状腺癌患者和148例甲状腺良性结节患者的血浆cfDNA进行甲基化特异的PCR，发现CDH1、TSHR、MCC、TBX15、PRR15、DPYS、GRIA2、 NR5A1、TTC34、RCOR2、F10、ITPKA、SLC16A3和RARG基因，在甲状腺癌和甲状腺良性结节病例血浆cfDNA的甲基化水平具有差异，结果如图3和图4所示。
2.1样本准备
使用QIAamp DNA Mini Kit(QIAGEN，货号：51304)对196例甲状腺癌和148例甲状腺良性结节患者的血浆cfDNA进行抽提；使用QubitTM dsDNA HS Assay Kit(Thermo，货号：Q32854)检测cfDNA的浓度；使用1％琼脂糖凝胶电泳进行质检。
2.2 DNA转化
使用MethylCodeTM Bisulfite Conversion Kit(Thermo，货号：MECOV50)对步骤1得到的DNA进行重亚硫酸盐转化，未甲基化的胞嘧啶(cytosine,C)经过转化变为尿嘧啶(uracil,U)；甲基化的胞嘧啶转化后不发生改变。
2.3 PCR混合物准备
使用本发明提供的试剂盒，包括PCR反应液、引物混合物(SEQ ID NO:1-28、61-62)、探针混合物(SEQ ID NO:41-54和63)，进行单个样本的配制，具体如表1-4和表8所示：
表8：PCR反应体系组成
组分 体积(μl)
2x PCR反应液 10
水 3.16
内参基因F,100μM 0.12
内参基因R,100μM 0.12
SLC16A3基因F,100μM 0.12
SLC16A3基因R,100μM 0.12
RARG基因F,100μM 0.12
RARG基因R,100μM 0.12
内参基因探针,100μM(VIC/BHQ1) 0.04
SLC16A3基因探针,100μM(FAM/BHQ1) 0.04
RARG基因探针,100μM(Texas/BHQ2) 0.04
样本DNA(10ng)/阳性对照/阴性对照 6
总体积 20
2.4荧光定量PCR
设置PCR程序为95℃预变性5min；95℃变性20s，60℃退火延伸45s，50个循环。60℃退火延伸阶段收集荧光信号。
2.5检测结果分析
利用下式计算各样品中基因的甲基化水平：
甲基化水平＝2 –ΔCt待检样品/2 –ΔCt阳性标准品×100％。
ΔCt＝Ct目的基因–Ct内参基因。
CDH1、TSHR、MCC、TBX15、PRR15、DPYS、GRIA2、NR5A1、TTC34、RCOR2、F10、ITPKA、SLC16A3和RARG基因在组织基因组中甲基化水平如图3所示；对各个基因的甲基化水平分别进行ROC曲线分析如图4所示，各个基因的AUC均大于0.6。
实施例3：验证甲状腺结节良恶性与血浆中cfDNA甲基化的关系
申请人对90例甲状腺癌患者和54例甲状腺良性结节患者的血浆cfDNA进行甲基化特异的PCR，发现FTAP2B、SOD3、RP11-867G23.12、EMX2OS、TEAD3和DGKG基因，在甲状腺癌和甲状腺良性结节病例血浆cfDNA的甲基化水平具有差异，结果如图5和图6所示。
3.1样本准备
使用QIAamp DNA Mini Kit(QIAGEN，货号：51304)对79例甲状腺癌和22例甲状腺良性结节患者的血浆cfDNA进行抽提；使用QubitTM dsDNA HS Assay Kit(Thermo，货号：Q32854)检测cfDNA的浓度；使用1％琼脂糖凝胶电泳进行质检。
3.2 DNA转化
使用MethylCodeTM Bisulfite Conversion Kit(Thermo，货号：MECOV50)对步骤1得到的DNA进行重亚硫酸盐转化，未甲基化的胞嘧啶(cytosine,C)经过转化变为尿嘧啶(uracil,U)；甲基化的胞嘧啶转化后不发生改变。
3.3 PCR混合物准备
使用本发明提供的试剂盒，包括PCR反应液、引物混合物(SEQ ID NO:29-40、61-62)、探针混合物(SEQ ID NO:55-60和63)，进行单个样本的配制如表5-7所示。
3.4荧光定量PCR
设置PCR程序为95℃预变性5min；95℃变性20s，60℃退火延伸45s，50个循环。60℃退火延伸阶段收集荧光信号。
3.5检测结果分析
利用下式计算各样品中基因的甲基化水平：
甲基化水平＝2 –ΔCt待检样品/2 –ΔCt阳性标准品×100％。
ΔCt＝Ct目的基因–Ct内参基因。
FTAP2B、SOD3、RP11-867G23.12、EMX2OS、TEAD3和DGKG基因在组织基因组中甲基化水平如图5所示；对各个基因的甲基化水平分别进行ROC曲线分析如图6所示，各个基因的AUC均大于0.6。
实施例4：DPYS基因和RARG基因联合用于甲状腺结节良恶性判别
申请人对10例甲状腺癌患者和10例甲状腺良性结节患者的组织DNA进行甲基化特异的PCR，发现DPYS和RARG基因在甲状腺癌和甲状腺良性结节病例组织DNA的甲基化水平具有差异，结果如图7所示。
4.1样本准备
使用QIAamp DNA Mini Kit(QIAGEN，货号：51304)对10例甲状腺癌和10例甲状腺良性结节的组织进行DNA抽提；使用Qubit TM dsDNA HS Assay Kit(Thermo，货号：Q32854)检测DNA的浓度；使用1％琼脂糖凝胶电泳进行质检。
4.2 DNA转化
使用MethylCode TM Bisulfite Conversion Kit(Thermo，货号：MECOV50)对步骤1得到的DNA进行重亚硫酸盐转化，未甲基化的胞嘧啶(cytosine,C)经过转化变为尿嘧啶(uracil,U)；甲基化的胞嘧啶转化后不发生改变。
4.3 PCR混合物准备
采用多重甲基化特异的PCR法(Multiplex MSP)，PCR混合物包括PCR反应液、引物混合物、探针混合物，进行单个样本的配制。引物混合物包含DPYS基因、RARG基因和内参基因的各一对引物。示例性的引物如SEQ ID NO:11、12、27、28、61、62所示；示例性的探针如SEQ ID NO:46、54、63所示。
PCR反应体系如下：
Figure PCTCN2022102658-appb-000010
Figure PCTCN2022102658-appb-000011
4.4 PCR反应
设置PCR程序为94℃预变性2min；94℃变性30s，60℃退火延伸1min，45个循环。60℃退火延伸阶段收集荧光信号。
4.5检测结果分析
甲基化水平(methylation level)＝2 –ΔCt待检样品/2 –ΔCt阳性标准品×100，其中，ΔCt＝Ct 目的基因–Ct 内参基因。
对DPYS基因以及RARG基因的甲基化水平进行二元Logistic回归分析，拟合方程为得分(Score)＝0.36+0.22×DPYS甲基化水平–0.03×RARG甲基化水平，判读方法为所检测DPYS和RARG基因的得分大于0，则判定结果为阳性，即为恶性结节。
DPYS和RARG基因的得分如表9，ROC分析如图7。根据判读标准，10例甲状腺良性结节有0例阳性，10例甲状腺癌有10例阳性，特异性达到100％，灵敏度为100％。
表9
组别 得分 组别 得分
良性结节 -1.08 恶性结节 1.22
良性结节 -1.18 恶性结节 0.22
良性结节 -0.25 恶性结节 1.91
良性结节 -0.77 恶性结节 0.23
良性结节 -0.22 恶性结节 0.49
良性结节 -1.63 恶性结节 0.25
良性结节 -1.22 恶性结节 0.77
良性结节 -0.48 恶性结节 1.87
良性结节 -1.26 恶性结节 1.42
良性结节 -1.55 恶性结节 2.39
实施例5：TSHR基因和SLC16A3基因联合用于甲状腺结节良恶性判别
申请人对10例甲状腺癌患者和10例甲状腺良性结节患者的组织DNA进行甲基化特异的PCR，发现TSHR和SLC16A3基因，在甲状腺癌和甲状腺良性结节病例组 织DNA的甲基化水平具有差异，结果如图8所示。
5.1样本准备
使用QIAamp DNA Mini Kit(QIAGEN，货号：51304)对10例甲状腺癌和10例甲状腺良性结节的组织进行DNA抽提；使用Qubit TM dsDNA HS Assay Kit(Thermo，货号：Q32854)检测DNA的浓度；使用1％琼脂糖凝胶电泳进行质检。
5.2 DNA转化
使用MethylCode TM Bisulfite Conversion Kit(Thermo，货号：MECOV50)对步骤1得到的DNA进行重亚硫酸盐转化，未甲基化的胞嘧啶(cytosine,C)经过转化变为尿嘧啶(uracil,U)；甲基化的胞嘧啶转化后不发生改变。
5.3 PCR混合物准备
采用多重甲基化特异的PCR法(Multiplex MSP)，PCR混合物包括PCR反应液、引物混合物、探针混合物，进行单个样本的配制。引物混合物包含TSHR基因、SLC16A3基因和内参基因的各一对引物。示例性的引物如SEQ ID NO:3、4、25、26、61、62所示；示例性的探针如SEQ ID NO:42、53、63所示。
PCR反应体系如下：
组分 体积(μl)
2x PCR反应液 12.50
水 4.10
多重引物混合物,100μM 3.40
样本cfDNA/阳性对照/阴性对照 5.00
总共 25.00
5.4 PCR反应
设置PCR程序为94℃预变性2min；94℃变性30s，60℃退火延伸1min，45个循环。60℃退火延伸阶段收集荧光信号。
5.5检测结果分析
甲基化水平(methylation level)＝2 –ΔCt待检样品/2 –ΔCt阳性标准品×100，其中，ΔCt＝Ct 目的基因–Ct 内参基因。
对TSHR基因以及SLC16A3基因的甲基化水平进行二元Logistic回归分析，拟合方程为得分(Score)＝0.5528+0.21×TSHR甲基化水平–0.11×SLC16A3甲基化水平，判读方法为所检测TSHR和SLC16A3基因的得分大于0，则判定结果为阳性，即为恶性结节。
TSHR和SLC16A3基因的得分如表10，ROC分析如图8。根据判读标准，20例甲状腺良性结节有1例阳性，20例甲状腺癌有9例阳性，特异性达到90％，灵敏度为90％。
表10
组别 得分 组别 得分
良性结节 -0.04 恶性结节 0.80
良性结节 -6.32 恶性结节 -0.42
良性结节 -0.28 恶性结节 3.72
良性结节 -2.52 恶性结节 0.07
良性结节 2.28 恶性结节 0.93
良性结节 -2.01 恶性结节 0.23
良性结节 -10.02 恶性结节 2.26
良性结节 -2.30 恶性结节 3.58
良性结节 -1.61 恶性结节 3.34
良性结节 -3.82 恶性结节 5.80
实施例6：FTAP2B基因和EMX2OS基因联合用于甲状腺结节良恶性判别
申请人对10例甲状腺癌患者和10例甲状腺良性结节患者的组织DNA进行甲基化特异的PCR，发现FTAP2B和EMX2OS基因，在甲状腺癌和甲状腺良性结节病例组织DNA的甲基化水平具有差异，结果如图9所示。
6.1样本准备
使用QIAamp DNA Mini Kit(QIAGEN，货号：51304)对10例甲状腺癌和10例甲状腺良性结节的组织进行DNA抽提；使用Qubit TM dsDNA HS Assay Kit(Thermo，货号：Q32854)检测DNA的浓度；使用1％琼脂糖凝胶电泳进行质检。
6.2 DNA转化
使用MethylCode TM Bisulfite Conversion Kit(Thermo，货号：MECOV50)对步骤1得到的DNA进行重亚硫酸盐转化，未甲基化的胞嘧啶(cytosine,C)经过转化变为尿嘧啶(uracil,U)；甲基化的胞嘧啶转化后不发生改变。
6.3 PCR混合物准备
采用多重甲基化特异的PCR法(Multiplex MSP)，PCR混合物包括PCR反应液、 引物混合物、探针混合物，进行单个样本的配制。引物混合物包含TSHR基因、SLC16A3基因和内参基因的各一对引物。示例性的引物如SEQ ID NO：29、30、35、36、61、62所示；示例性的探针如SEQ ID NO:55、58、63所示。
PCR反应体系如下：
组分 体积(μl)
2x PCR反应液 12.50
水 4.10
多重引物混合物,100μM 3.40
样本cfDNA/阳性对照/阴性对照 5.00
总共 25.00
6.4 PCR反应
设置PCR程序为94℃预变性2min；94℃变性30s，60℃退火延伸1min，45个循环。60℃退火延伸阶段收集荧光信号。
6.5检测结果分析
甲基化水平(methylation level)＝2 –ΔCt待检样品/2 –ΔCt阳性标准品×100，其中，ΔCt＝Ct 目的基因–Ct 内参基因。
对FTAP2B基因以及EMX2OS基因的甲基化水平进行二元Logistic回归分析，拟合方程为得分(Score)＝1.85+0.11×FTAP2B甲基化水平–0.17×EMX2OS甲基化水平，判读方法为所检测FTAP2B和EMX2OS基因的得分大于0，则判定结果为阳性，即为恶性结节。
FTAP2B和EMX2OS基因的得分如表11，ROC分析如图9。根据判读标准，10例甲状腺良性结节有2例阳性，10例甲状腺癌有9例阳性，特异性达到100％，灵敏度为90％。
表11
Figure PCTCN2022102658-appb-000012
Figure PCTCN2022102658-appb-000013
实施例7：RP11-867G23.12基因和TEAD3基因联合用于甲状腺结节良恶性判别
申请人对10例甲状腺癌患者和10例甲状腺良性结节患者的组织DNA进行甲基化特异的PCR，发现RP11-867G23.12和TEAD3基因，在甲状腺癌和甲状腺良性结节病例组织DNA的甲基化水平具有差异，结果如图10所示。
7.1样本准备
使用QIAamp DNA Mini Kit(QIAGEN，货号：51304)对10例甲状腺癌和10例甲状腺良性结节的组织进行DNA抽提；使用Qubit TM dsDNA HS Assay Kit(Thermo，货号：Q32854)检测DNA的浓度；使用1％琼脂糖凝胶电泳进行质检。
7.2 DNA转化
使用MethylCode TM Bisulfite Conversion Kit(Thermo，货号：MECOV50)对步骤1得到的DNA进行重亚硫酸盐转化，未甲基化的胞嘧啶(cytosine,C)经过转化变为尿嘧啶(uracil,U)；甲基化的胞嘧啶转化后不发生改变。
7.3 PCR混合物准备
采用多重甲基化特异的PCR法(Multiplex MSP)，PCR混合物包括PCR反应液、引物混合物、探针混合物，进行单个样本的配制。引物混合物包含TSHR基因、SLC16A3基因和内参基因的各一对引物。示例性的引物如SEQ ID NO：33、34、37、38、61、62所示；示例性的探针如SEQ ID NO:57、59、63所示。
PCR反应体系如下：
组分 体积(μl)
2x PCR反应液 12.50
水 4.10
多重引物混合物,100μM 3.40
样本cfDNA/阳性对照/阴性对照 5.00
总共 25.00
7.4 PCR反应
设置PCR程序为94℃预变性2min；94℃变性30s，60℃退火延伸1min，45个循环。60℃退火延伸阶段收集荧光信号。
7.5检测结果分析
甲基化水平(methylation level)＝2 –ΔCt待检样品/2 –ΔCt阳性标准品×100，其中，ΔCt＝Ct 目的基因–Ct 内参基因。
对FTAP2B基因以及EMX2OS基因的甲基化水平进行二元Logistic回归分析，拟合方程为得分(Score)＝0.61+0.15×RP11-867G23.12甲基化水平–0.05×TEAD3甲基化水平，判读方法为所检测RP11-867G23.12和TEAD3基因的得分大于0，则判定结果为阳性，即为恶性结节。
RP11-867G23.12和TEAD3基因的得分如表12，ROC分析如图10。根据判读标准，10例甲状腺良性结节有2例阳性，10例甲状腺癌有7例阳性，特异性达到90％，灵敏度为70％。
表12
组别 得分 组别 得分
良性结节 -1.09 恶性结节 1.63
良性结节 -2.69 恶性结节 1.30
良性结节 0.80 恶性结节 1.30
良性结节 1.05 恶性结节 1.12
良性结节 -0.17 恶性结节 0.83
良性结节 -1.22 恶性结节 -0.01
良性结节 -0.74 恶性结节 0.88
良性结节 -2.91 恶性结节 -1.14
良性结节 -0.47 恶性结节 1.67
良性结节 -0.13 恶性结节 -1.38
实施例8：TSHR基因和RARG基因联合用于甲状腺结节良恶性判别
申请人对196例甲状腺癌患者和148例甲状腺良性结节患者的血浆cfDNA进行甲基化特异的PCR，发现TSHR和RARG基因，在甲状腺癌和甲状腺良性结节病例组织DNA的甲基化水平具有差异，结果如图11所示。
8.1样本准备
使用QIAamp DNA Mini Kit(QIAGEN，货号：51304)对196例甲状腺癌和148例甲状腺良性结节的血浆进行cfDNA抽提；使用Qubit TM dsDNA HS Assay Kit(Thermo，货号：Q32854)检测DNA的浓度；使用1％琼脂糖凝胶电泳进行质检。
8.2 DNA转化
使用MethylCode TM Bisulfite Conversion Kit(Thermo，货号：MECOV50)对步骤1得到的DNA进行重亚硫酸盐转化，未甲基化的胞嘧啶(cytosine,C)经过转化变为尿嘧啶(uracil,U)；甲基化的胞嘧啶转化后不发生改变。
8.3 PCR混合物准备
采用多重甲基化特异的PCR法(Multiplex MSP)，PCR混合物包括PCR反应液、引物混合物、探针混合物，进行单个样本的配制。引物混合物包含TSHR基因、SLC16A3基因和内参基因的各一对引物。示例性的引物如SEQ ID NO:3、4、27、28、61、62所示；示例性的探针如SEQ ID NO:42、54、63所示。
PCR反应体系如下：
组分 体积(μl)
2x PCR反应液 12.50
水 4.10
多重引物混合物,100μM 3.40
样本cfDNA/阳性对照/阴性对照 5.00
总共 25.00
8.4 PCR反应
设置PCR程序为94℃预变性2min；94℃变性30s，60℃退火延伸1min，45个循环。60℃退火延伸阶段收集荧光信号。
8.5检测结果分析
甲基化水平(methylation level)＝2 –ΔCt待检样品/2 –ΔCt阳性标准品×100，其中，ΔCt＝Ct 目的基因–Ct 内参基因。
对TSHR基因以及RARG基因的甲基化水平进行二元Logistic回归分析，拟合方程为得分(Score)＝-8.34+8.11×TSHR甲基化水平+14.13×RARG甲基化水平，判读方法为所检测TSHR和RARG基因的得分大于0，则判定结果为阳性，即为恶性结节。
TSHR和RARG基因的ROC分析如图11，特异性达到90％，灵敏度为80％。
实施例9：MCC基因和CDH1基因联合用于甲状腺结节良恶性判别
申请人对196例甲状腺癌患者和148例甲状腺良性结节患者的血浆cfDNA进行甲基化特异的PCR，发现MCC和CDH1基因，在甲状腺癌和甲状腺良性结节病例组织DNA的甲基化水平具有差异，结果如图12所示。
9.1样本准备
使用QIAamp DNA Mini Kit(QIAGEN，货号：51304)对196例甲状腺癌和148例甲状腺良性结节的血浆进行cfDNA抽提；使用Qubit TM dsDNA HS Assay Kit(Thermo，货号：Q32854)检测DNA的浓度；使用1％琼脂糖凝胶电泳进行质检。
9.2 DNA转化
使用MethylCode TM Bisulfite Conversion Kit(Thermo，货号：MECOV50)对步骤1得到的DNA进行重亚硫酸盐转化，未甲基化的胞嘧啶(cytosine,C)经过转化变为尿嘧啶(uracil,U)；甲基化的胞嘧啶转化后不发生改变。
9.3 PCR混合物准备
采用多重甲基化特异的PCR法(Multiplex MSP)，PCR混合物包括PCR反应液、引物混合物、探针混合物，进行单个样本的配制。引物混合物包含MCC基因、CDH1基因和内参基因的各一对引物。示例性的引物如SEQ ID NO:1、2、5、6、61、62所示；示例性的探针如SEQ ID NO:41、42、63所示。
PCR反应体系如下：
组分 体积(μl)
2x PCR反应液 12.50
水 4.10
多重引物混合物,100μM 3.40
样本cfDNA/阳性对照/阴性对照 5.00
总共 25.00
9.4 PCR反应
设置PCR程序为94℃预变性2min；94℃变性30s，60℃退火延伸1min，45个循环。60℃退火延伸阶段收集荧光信号。
9.5检测结果分析
甲基化水平(methylation level)＝2 –ΔCt待检样品/2 –ΔCt阳性标准品×100，其中，ΔCt＝Ct 目的基因–Ct 内参基因。
对MCC基因以及CDH1基因的甲基化水平进行二元Logistic回归分析，拟合方程为得分(Score)＝-2.84+14.53×MCC甲基化水平–98.54×CDH1甲基化水平，判读方法为所检测MCC和CDH1基因的得分大于0，则判定结果为阳性，即为恶性结节。
MCC和CDH1基因的ROC分析如图12，特异性达到82％，灵敏度为80％。
实施例10：SOD3基因、EMX2OS基因和TEAD3基因联合用于甲状腺结节良恶性判别
申请人对90例甲状腺癌患者和54例甲状腺良性结节患者的血浆cfDNA进行甲基化特异的PCR，发现SOD3、EMX2OS和TRAD3基因，在甲状腺癌和甲状腺良性结节病例组织DNA的甲基化水平具有差异，结果如图13所示。
10.1样本准备
使用QIAamp DNA Mini Kit(QIAGEN，货号：51304)对90例甲状腺癌和54例甲状腺良性结节的血浆进行cfDNA抽提；使用Qubit TM dsDNA HS Assay Kit(Thermo，货号：Q32854)检测DNA的浓度；使用1％琼脂糖凝胶电泳进行质检。
10.2 DNA转化
使用MethylCode TM Bisulfite Conversion Kit(Thermo，货号：MECOV50)对步骤1得到的DNA进行重亚硫酸盐转化，未甲基化的胞嘧啶(cytosine,C)经过转化变为尿嘧啶(uracil,U)；甲基化的胞嘧啶转化后不发生改变。
10.3 PCR混合物准备
采用多重甲基化特异的PCR法(Multiplex MSP)，PCR混合物包括PCR反应液、引物混合物、探针混合物，进行单个样本的配制。引物混合物包含SOD3基因、EMX2OS基因、TEAD3基因和内参基因的各一对引物。示例性的引物如SEQ ID NO:31、32、35、36、37、38、61、62所示；示例性的探针如SEQ ID NO:56、58、59、63所示。
PCR反应体系如下：
Figure PCTCN2022102658-appb-000014
Figure PCTCN2022102658-appb-000015
10.4 PCR反应
设置PCR程序为94℃预变性2min；94℃变性30s，60℃退火延伸1min，45个循环。60℃退火延伸阶段收集荧光信号。
10.5检测结果分析
甲基化水平(methylation level)＝2 –ΔCt待检样品/2 –ΔCt阳性标准品×100，其中，ΔCt＝Ct 目的基因–Ct 内参基因。
对SOD3基因以及EMX2OS基因的甲基化水平进行二元Logistic回归分析，拟合方程为得分(Score)＝12.66–1.22×SOD3甲基化水平–0.63×EMX2OS甲基化水平+0.64×TEAD3甲基化水平，判读方法为所检测SOD3、EMX2OS和TEAD3基因的得分大于0，则判定结果为阳性，即为恶性结节。
SOD3、EMX2OS和TEAD3基因的ROC分析如图13，特异性达到82％，灵敏度为72％。
实施例11：FTAP2B基因和DGKG基因联合用于甲状腺结节良恶性判别
申请人对90例甲状腺癌患者和54例甲状腺良性结节患者的血浆cfDNA进行甲基化特异的PCR，发现FTAP2B和DGKG基因，在甲状腺癌和甲状腺良性结节病例组织DNA的甲基化水平具有差异，结果如图14所示。
11.1样本准备
使用QIAamp DNA Mini Kit(QIAGEN，货号：51304)对90例甲状腺癌和54例甲状腺良性结节的血浆进行cfDNA抽提；使用Qubit TM dsDNA HS Assay Kit(Thermo，货号：Q32854)检测DNA的浓度；使用1％琼脂糖凝胶电泳进行质检。
11.2 DNA转化
使用MethylCode TM Bisulfite Conversion Kit(Thermo，货号：MECOV50)对步骤1得到的DNA进行重亚硫酸盐转化，未甲基化的胞嘧啶(cytosine,C)经过转化变为尿嘧啶(uracil,U)；甲基化的胞嘧啶转化后不发生改变。
11.3 PCR混合物准备
采用多重甲基化特异的PCR法(Multiplex MSP)，PCR混合物包括PCR反应液、引物混合物、探针混合物，进行单个样本的配制。引物混合物包含FTAP2B基因、DGKG基因和内参基因的各一对引物。示例性的引物如SEQ ID NO:29、30、39、40、61、62所示；示例性的探针如SEQ ID NO:55、60、63所示。
PCR反应体系如下
组分 体积(μl)
2x PCR反应液 12.50
水 4.10
多重引物混合物,100μM 3.40
样本cfDNA/阳性对照/阴性对照 5.00
总共 25.00
11.4 PCR反应
设置PCR程序为94℃预变性2min；94℃变性30s，60℃退火延伸1min，45个循环。60℃退火延伸阶段收集荧光信号。
11.5检测结果分析
甲基化水平(methylation level)＝2 –ΔCt待检样品/2 –ΔCt阳性标准品×100，其中，ΔCt＝Ct 目的基因–Ct 内参基因。
对FTAP2B基因以及DGKG基因的甲基化水平进行二元Logistic回归分析，拟合方程为得分(Score)＝-7.33+1.86×FTAP2B甲基化水平–1.25×DGKG甲基化水平，判读方法为所检测FTAP2B和DGKG基因的得分大于0，则判定结果为阳性，即为恶性结节。
FTAP2B和DGKG基因的ROC分析如图14，特异性达到84％，灵敏度为72％。
实施例12：TTC34基因、RCOR2基因、SLC16A3基因和RARG基因联合用于甲状腺结节良恶性判别
对196例甲状腺癌患者和148例甲状腺良性结节患者的血浆cfDNA进行甲基化特异的PCR，发现TTC34基因、RCOR2基因、SLC16A3基因和RARG基因在甲状腺癌和甲状腺良性结节病例组织DNA的甲基化水平具有差异，结果如图15所示。
12.1样本准备
使用QIAamp DNA Mini Kit(QIAGEN，货号：51304)对196例甲状腺癌和148例甲状腺良性结节的血浆进行cfDNA抽提；使用Qubit TM dsDNA HS Assay Kit(Thermo，货号：Q32854)检测DNA的浓度；使用1％琼脂糖凝胶电泳进行质检。
12.2 DNA转化
使用MethylCode TM Bisulfite Conversion Kit(Thermo，货号：MECOV50)对步骤1得到的DNA进行重亚硫酸盐转化，未甲基化的胞嘧啶(cytosine,C)经过转化变为 尿嘧啶(uracil,U)；甲基化的胞嘧啶转化后不发生改变。
12.3 PCR混合物准备
采用多重甲基化特异的PCR法(Multiplex MSP)，PCR混合物包括PCR反应液、引物混合物、探针混合物，进行单个样本的配制。引物混合物包含TTC34基因、RCOR2基因、SLC16A3基因、RARG基因和内参基因的各一对引物。示例性的引物如SEQ ID NO:17、18、19、20、25、26、27、28、61、62所示；示例性的探针如SEQ ID NO:49、50、53、54、63所示。
PCR反应体系如下：
组分 体积(μl)
2x PCR反应液 12.50
水 4.10
多重引物混合物,100μM 3.40
样本cfDNA/阳性对照/阴性对照 5.00
总共 25.00
12.4 PCR反应
设置PCR程序为94℃预变性2min；94℃变性30s，60℃退火延伸1min，45个循环。60℃退火延伸阶段收集荧光信号。
12.5检测结果分析
甲基化水平(methylation level)＝2 –ΔCt待检样品/2 –ΔCt阳性标准品×100，其中，ΔCt＝Ct 目的基因–Ct 内参基因。
对TTC34基因、RCOR2基因、SLC16A3基因和RARG基因的甲基化水平进行二元Logistic回归分析，拟合方程为得分(Score)＝2.23+3.57×TTC34甲基化水平–24.82×RCOR2甲基化水平–4.66×SLC16A3甲基化水平–0.75×RARG甲基化水平，判读方法为所检测TTC34、RCOR2、SLC16A3和RARG基因的得分大于0，则判定结果为阳性，即为恶性结节。
TTC34、RCOR2、SLC16A3和RARG基因的ROC分析如图15，特异性达到68％，灵敏度为68％。
实施例13：SOD3基因、EMX2OS基因和FTAP2B基因联合用于甲状腺结节良恶性判别
对90例甲状腺癌患者和54例甲状腺良性结节患者的血浆cfDNA进行甲基化特 异的PCR，发现SOD3、EMX2OS和FTAP2B基因，在甲状腺癌和甲状腺良性结节病例组织DNA的甲基化水平具有差异，结果如图16所示。
13.1样本准备
使用QIAamp DNA Mini Kit(QIAGEN，货号：51304)对90例甲状腺癌和54例甲状腺良性结节的血浆进行cfDNA抽提；使用Qubit TM dsDNA HS Assay Kit(Thermo，货号：Q32854)检测DNA的浓度；使用1％琼脂糖凝胶电泳进行质检。
13.2 DNA转化
使用MethylCode TM Bisulfite Conversion Kit(Thermo，货号：MECOV50)对步骤1得到的DNA进行重亚硫酸盐转化，未甲基化的胞嘧啶(cytosine,C)经过转化变为尿嘧啶(uracil,U)；甲基化的胞嘧啶转化后不发生改变。
13.3 PCR混合物准备
采用多重甲基化特异的PCR法(Multiplex MSP)，PCR混合物包括PCR反应液、引物混合物、探针混合物，进行单个样本的配制。引物混合物包含SOD3基因、EMX2OS基因、FTAP2B基因和内参基因的各一对引物。示例性的引物如SEQ ID NO:31、32、35、36、29、30、61、62所示；示例性的探针如SEQ ID NO:56、58、55、63所示。
PCR反应体系如下：
组分 体积(μl)
2x PCR反应液 12.50
水 4.10
多重引物混合物,100μM 3.40
样本cfDNA/阳性对照/阴性对照 5.00
总共 25.00
13.4 PCR反应
设置PCR程序为94℃预变性2min；94℃变性30s，60℃退火延伸1min，45个循环。60℃退火延伸阶段收集荧光信号。
13.5检测结果分析
甲基化水平(methylation level)＝2 –ΔCt待检样品/2 –ΔCt阳性标准品×100，其中，ΔCt＝Ct 目的基因–Ct 内参基因。
对SOD3基因以及EMX2OS基因的甲基化水平进行二元Logistic回归分析，拟合方程为得分(Score)＝6.53–1.02×SOD3甲基化水平–1.21×EMX2OS甲基 化水平+1.40×FTAP2B甲基化水平，判读方法为所检测SOD3、EMX2OS和FTAP2B基因的得分大于0，则判定结果为阳性，即为恶性结节。
SOD3、EMX2OS和FTAP2B基因的ROC分析如图16，特异性达到76％，灵敏度为78％。
实施例14：TTC34基因、RCOR2基因、ITPKA基因和CDH1基因联合用于甲状腺结节良恶性判别
对196例甲状腺癌患者和148例甲状腺良性结节患者的血浆cfDNA进行甲基化特异的PCR，发现TTC34基因、RCOR2基因、ITPKA基因和CDH1基因在甲状腺癌和甲状腺良性结节病例组织DNA的甲基化水平具有差异，结果如图17所示。
14.1样本准备
使用QIAamp DNA Mini Kit(QIAGEN，货号：51304)对196例甲状腺癌和148例甲状腺良性结节的血浆进行cfDNA抽提；使用Qubit TM dsDNA HS Assay Kit(Thermo，货号：Q32854)检测DNA的浓度；使用1％琼脂糖凝胶电泳进行质检。
14.2 DNA转化
使用MethylCode TM Bisulfite Conversion Kit(Thermo，货号：MECOV50)对步骤1得到的DNA进行重亚硫酸盐转化，未甲基化的胞嘧啶(cytosine,C)经过转化变为尿嘧啶(uracil,U)；甲基化的胞嘧啶转化后不发生改变。
14.3 PCR混合物准备
采用多重甲基化特异的PCR法(Multiplex MSP)，PCR混合物包括PCR反应液、引物混合物、探针混合物，进行单个样本的配制。引物混合物包含TTC34基因、RCOR2基因、ITPKA基因、CDH1基因和内参基因的各一对引物。示例性的引物如SEQ ID NO:17、18、19、20、23、24、1、2、61、62所示；示例性的探针如SEQ ID NO:49、50、52、41、63所示。
PCR反应体系如下：
组分 体积(μl)
2x PCR反应液 12.50
水 4.10
多重引物混合物,100μM 3.40
样本cfDNA/阳性对照/阴性对照 5.00
总共 25.00
14.4 PCR反应
设置PCR程序为94℃预变性2min；94℃变性30s，60℃退火延伸1min，45个循环。60℃退火延伸阶段收集荧光信号。
14.5检测结果分析
甲基化水平(methylation level)＝2 –ΔCt待检样品/2 –ΔCt阳性标准品×100，其中，ΔCt＝Ct 目的基因–Ct 内参基因。
对TTC34基因、RCOR2基因、ITPKA基因和CDH1基因的甲基化水平进行二元Logistic回归分析，拟合方程为得分(Score)＝1.16+1.75×TTC34甲基化水平–52.62×RCOR2甲基化水平–7.62×ITPKA甲基化水平+75.98×CDH1甲基化水平，判读方法为所检测TTC34、RCOR2、ITPKA和CDH1基因的得分大于0，则判定结果为阳性，即为恶性结节。
TTC34、RCOR2、ITPKA和CDH1基因的ROC分析如图17，特异性达到70％，灵敏度为65％。
实施例15：SOD3基因、RP11-867G23.12基因和FTAP2B基因联合用于甲状腺结节良恶性判别
对90例甲状腺癌患者和54例甲状腺良性结节患者的血浆cfDNA进行甲基化特异的PCR，发现SOD3基因、RP11-867G23.12基因和FTAP2B基因，在甲状腺癌和甲状腺良性结节病例组织DNA的甲基化水平具有差异，结果如图18所示。
15.1样本准备
使用QIAamp DNA Mini Kit(QIAGEN，货号：51304)对90例甲状腺癌和54例甲状腺良性结节的血浆进行cfDNA抽提；使用Qubit TM dsDNA HS Assay Kit(Thermo，货号：Q32854)检测DNA的浓度；使用1％琼脂糖凝胶电泳进行质检。
15.2 DNA转化
使用MethylCode TM Bisulfite Conversion Kit(Thermo，货号：MECOV50)对步骤1得到的DNA进行重亚硫酸盐转化，未甲基化的胞嘧啶(cytosine,C)经过转化变为尿嘧啶(uracil,U)；甲基化的胞嘧啶转化后不发生改变。
15.3 PCR混合物准备
采用多重甲基化特异的PCR法(Multiplex MSP)，PCR混合物包括PCR反应液、引物混合物、探针混合物，进行单个样本的配制。引物混合物包含SOD3基因、RP11-867G23.12基因、FTAP2B基因和内参基因的各一对引物。示例性的引物如SEQ  ID NO:31、32、33、34、29、30、61、62所示；示例性的探针如SEQ ID NO:56、57、55、63所示。
PCR反应体系如下：
组分 体积(μl)
2x PCR反应液 12.50
水 4.10
多重引物混合物,100μM 3.40
样本cfDNA/阳性对照/阴性对照 5.00
总共 25.00
15.4 PCR反应
设置PCR程序为94℃预变性2min；94℃变性30s，60℃退火延伸1min，45个循环。60℃退火延伸阶段收集荧光信号。
15.5检测结果分析
甲基化水平(methylation level)＝2 –ΔCt待检样品/2 –ΔCt阳性标准品×100，其中，ΔCt＝Ct 目的基因–Ct 内参基因。
对SOD3基因以及EMX2OS基因的甲基化水平进行二元Logistic回归分析，拟合方程为得分(Score)＝6.10–1.04×SOD3甲基化水平+0.60×RP11-867G23.12甲基化水平+0.61×FTAP2B甲基化水平，判读方法为所检测SOD3、RP11-867G23.12和FTAP2B基因的得分大于0，则判定结果为阳性，即为恶性结节。
SOD3、RP11-867G23.12和FTAP2B基因的ROC分析如图18，特异性达到79％，灵敏度为77％。
实施例16：ITPKA基因、RCOR2基因、SLC16A3基因和MCC基因联合用于甲状腺结节良恶性判别
对196例甲状腺癌患者和148例甲状腺良性结节患者的血浆cfDNA进行甲基化特异的PCR，发现ITPKA基因、RCOR2基因、SLC16A3基因和MCC基因在甲状腺癌和甲状腺良性结节病例组织DNA的甲基化水平具有差异，结果如图19所示。
16.1样本准备
使用QIAamp DNA Mini Kit(QIAGEN，货号：51304)对196例甲状腺癌和148例甲状腺良性结节的血浆进行cfDNA抽提；使用Qubit TM dsDNA HS Assay Kit (Thermo，货号：Q32854)检测DNA的浓度；使用1％琼脂糖凝胶电泳进行质检。
16.2 DNA转化
使用MethylCode TM Bisulfite Conversion Kit(Thermo，货号：MECOV50)对步骤1得到的DNA进行重亚硫酸盐转化，未甲基化的胞嘧啶(cytosine,C)经过转化变为尿嘧啶(uracil,U)；甲基化的胞嘧啶转化后不发生改变。
16.3 PCR混合物准备
采用多重甲基化特异的PCR法(Multiplex MSP)，PCR混合物包括PCR反应液、引物混合物、探针混合物，进行单个样本的配制。引物混合物包含ITPKA基因、RCOR2基因、SLC16A3基因、MCC基因和内参基因的各一对引物。示例性的引物如SEQ ID NO:23、24、19、20、25、26、5、6、61、62所示；示例性的探针如SEQ ID NO:52、50、53、43、63所示。
PCR反应体系如下：
组分 体积(μl)
2x PCR反应液 12.50
水 4.10
多重引物混合物,100μM 3.40
样本cfDNA/阳性对照/阴性对照 5.00
总共 25.00
16.4 PCR反应
设置PCR程序为94℃预变性2min；94℃变性30s，60℃退火延伸1min，45个循环。60℃退火延伸阶段收集荧光信号。
16.5检测结果分析
甲基化水平(methylation level)＝2 –ΔCt待检样品/2 –ΔCt阳性标准品×100，其中，ΔCt＝Ct 目的基因–Ct 内参基因。
对ITPKA基因、RCOR2基因、SLC16A3基因、MCC基因的甲基化水平进行二元Logistic回归分析，拟合方程为得分(Score)＝-1.61-2.37×SLC16A3甲基化水平–9.34×RCOR2甲基化水平–17.82×ITPKA甲基化水平+1.62×MCC甲基化水平，判读方法为所检测ITPKA、RCOR2、SLC16A3和MCC基因的得分大于0，则判定结果为阳性，即为恶性结节。
ITPKA、RCOR2、SLC16A3和MCC基因的ROC分析如图19，特异性达到80％，灵敏度为79％。
实施例17：TTC34基因、ITPKA基因、SLC16A3基因和RARG基因联合用于甲状腺结节良恶性判别
申请人对196例甲状腺癌患者和148例甲状腺良性结节患者的血浆cfDNA进行甲基化特异的PCR，发现TTC34基因、ITPKA基因、SLC16A3基因和RARG基因在甲状腺癌和甲状腺良性结节病例组织DNA的甲基化水平具有差异，结果如图20所示。
17.1样本准备
使用QIAamp DNA Mini Kit(QIAGEN，货号：51304)对196例甲状腺癌和148例甲状腺良性结节的血浆进行cfDNA抽提；使用Qubit TM dsDNA HS Assay Kit(Thermo，货号：Q32854)检测DNA的浓度；使用1％琼脂糖凝胶电泳进行质检。
17.2 DNA转化
使用MethylCode TM Bisulfite Conversion Kit(Thermo，货号：MECOV50)对步骤1得到的DNA进行重亚硫酸盐转化，未甲基化的胞嘧啶(cytosine,C)经过转化变为尿嘧啶(uracil,U)；甲基化的胞嘧啶转化后不发生改变。
17.3 PCR混合物准备
采用多重甲基化特异的PCR法(Multiplex MSP)，PCR混合物包括PCR反应液、引物混合物、探针混合物，进行单个样本的配制。引物混合物包含TTC34基因、ITPKA基因、SLC16A3基因、RARG基因和内参基因的各一对引物。示例性的引物如SEQ ID NO:17、18、23、24、25、26、27、28、61、62所示；示例性的探针如SEQ ID NO:49、52、、53、54、63所示。
PCR反应体系如下：
组分 体积(μl)
2x PCR反应液 12.50
水 4.10
多重引物混合物,100μM 3.40
样本cfDNA/阳性对照/阴性对照 5.00
总共 25.00
17.4 PCR反应
设置PCR程序为94℃预变性2min；94℃变性30s，60℃退火延伸1min，45个循环。60℃退火延伸阶段收集荧光信号。
17.5检测结果分析
甲基化水平(methylation level)＝2 –ΔCt待检样品/2 –ΔCt阳性标准品×100，其中，ΔCt＝Ct 目的基因–Ct 内参基因。
对TTC34基因、ITPKA基因、SLC16A3基因和RARG基因的甲基化水平进行二元Logistic回归分析，拟合方程为得分(Score)＝2.47+3.27×TTC34甲基化水平–3.71×ITPKA甲基化水平–6.48×SLC16A3甲基化水平–0.69×RARG甲基化水平，判读方法为所检测TTC34、ITPKA、SLC16A3和RARG基因的得分大于0，则判定结果为阳性，即为恶性结节。
TTC34、ITPKA、SLC16A3和RARG基因的ROC分析如图20，特异性达到64％，灵敏度为68％。
以上所述实施例仅表达了本发明的实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (32)

  1. 引物分子和/或探针分子在制备诊断个体甲状腺结节良恶性的检测试剂或诊断试剂盒中的应用，以及装置在制备诊断个体甲状腺结节良恶性的诊断试剂盒中的应用；其特征在于：
    所述引物分子相同于、互补于或在严谨条件下杂交于一个或多个目标标志物并包含至少9个连续的核苷酸；
    所述探针分子与一个或多个目标标志物的扩增产物在严谨条件下杂交；
    所述装置用于确定一个或多个目标标志物的至少一个CpG二核苷酸的甲基化状态或水平；
    其中，所述一个或多个目标标志物选自：CDH1基因或基因组的CDH1序列、TSHR基因或基因组的TSHR序列、MCC基因或基因组的MCC序列、TBX15基因或基因组的TBX15序列、PRR15基因或基因组的PRR15序列、DPYS基因或基因组的DPYS序列、GRIA2基因或基因组的GRIA2序列、NR5A1基因或基因组的NR5A1序列、TTC34基因或基因组的TTC34序列、RCOR2基因或基因组的RCOR2序列、F10基因或基因组的F10序列、ITPKA基因或基因组的ITPKA序列、SLC16A3基因或基因组的SLC16A3序列、RARG基因或基因组的RARG序列、FTAP2B基因或基因组的FTAP2B序列、SOD3基因或基因组的SOD3序列、RP11-867G23.12基因或基因组的RP11-867G23.12序列、EMX2OS基因或基因组的EMX2OS序列、TEAD3基因或基因组的TEAD3序列和DGKG基因或基因组的DGKG序列中的任意一个或任意多个。
  2. 如权利要求1所述的应用，其特征在于，
    所述目标标志物为SLC16A3基因或基因组的SLC16A3序列以及选自TSHR基因或基因组的TSHR序列、TTC34基因或基因组的TTC34序列、ROCR2基因或基因组的ROCR2序列、RARG基因或基因组的RARG序列、MCC基因或基因组的MCC序列和ITPKA基因或基因组的ITPKA序列中的一种或多种；
    优选地，目标标志物为SLC16A3基因或基因组的SLC16A3序列和TSHR基因或基因组的TSHR序列；优选地，检测来自该个体的甲状腺结节中的该目 标标志物的甲基化水平；
    优选地，所述目标标志物为SLC16A3基因或基因组的SLC16A3序列和ROCR2基因或基因组的ROCR2序列，以及选自TTC34基因或基因组的TTC34序列、RARG基因或基因组的RARG序列、ITPKA基因或基因组的ITPKA序列和MCC基因或基因组的MCC序列中的至少一个、至少两个或至少三个；优选地，所述目标标志物为SLC16A3基因或基因组的SLC16A3序列、ROCR2基因或基因组的ROCR2序列、TTC34基因或基因组的TTC34序列和RARG基因或基因组的RARG序列，或所述目标标志物为SLC16A3基因或基因组的SLC16A3序列、ROCR2基因或基因组的ROCR2序列、ITPKA基因或基因组的ITPKA序列和MCC基因或基因组的MCC序列；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平；
    优选地，所述目标标志物为SLC16A3基因或基因组的SLC16A3序列和RARG基因或基因组的RARG序列，以及选自TTC34基因或基因组的TTC34序列、ROCR2基因或基因组的ROCR2序列和ITPKA基因或基因组的ITPKA序列的至少一个、至少两个或全部三个；优选地，所述目标标志物为SLC16A3基因或基因组的SLC16A3序列、ROCR2基因或基因组的ROCR2序列、TTC34基因或基因组的TTC34序列和RARG基因或基因组的RARG序列，或所述目标标志物为SLC16A3基因或基因组的SLC16A3序列、TTC34基因或基因组的TTC34序列、ITPKA基因或基因组的ITPKA序列和RARG基因或基因组的RARG序列；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平；
    优选地，所述目标标志物为SLC16A3基因或基因组的SLC16A3序列和TTC34基因或基因组的TTC34序列，以及选自ROCR2基因或基因组的ROCR2序列、RARG基因或基因组的RARG序列和ITPKA基因或基因组的ITPKA序列的至少一个、至少两个或全部三个；优选地，所述目标标志物为SLC16A3基因或基因组的SLC16A3序列、ROCR2基因或基因组的ROCR2序列、TTC34基因或基因组的TTC34序列和RARG基因或基因组的RARG序列，或所述目标标志物为SLC16A3基因或基因组的SLC16A3序列、TTC34基因或基因组的TTC34序列、ITPKA基因或基因组的ITPKA序列和RARG基因或基因组的 RARG序列；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平；
    优选地，所述目标标志物为SLC16A3基因或基因组的SLC16A3序列和ITPKA基因或基因组的ITPKA序列，以及选自ROCR2基因或基因组的ROCR2序列、MCC基因或基因组的MCC序列、TTC34基因或基因组的TTC34序列和RARG基因或基因组的RARG序列和的至少一个、至少两个或全部三个；优选地，所述目标标志物为SLC16A3基因或基因组的SLC16A3序列、ROCR2基因或基因组的ROCR2序列、ITPKA基因或基因组的ITPKA序列和MCC基因或基因组的MCC序列，或所述目标标志物为SLC16A3基因或基因组的SLC16A3序列、TTC34基因或基因组的TTC34序列、ITPKA基因或基因组的ITPKA序列和RARG基因或基因组的RARG序列；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平；
    优选地，所述目标标志物为SLC16A3基因或基因组的SLC16A3序列、TTC34基因或基因组的TTC34序列和RARG基因或基因组的RARG序列，以及ROCR2基因或基因组的ROCR2序列或ITPKA基因或基因组的ITPKA序列；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。
  3. 如权利要求1所述的应用，其特征在于，
    所述目标标志物为FTAP2B基因或基因组的FTAP2B序列以及选自EMS2OS基因或基因组的EMS2OS序列、DGKG基因或基因组的DGKG序列、SOD3基因或基因组的SOD3序列、RP11-867G23.12基因或基因组的RP11-867G23.12序列中的至少一个、至少两个或至少三个；
    优选地，所述目标标志物为FTAP2B基因或基因组的FTAP2B序列、EMS2OS基因或基因组的EMS2OS序列以及任选的SOD3基因或基因组的SOD3序列；优选地，所述目标标志物为FTAP2B基因或基因组的FTAP2B序列和EMS2OS基因或基因组的EMS2OS序列，其中，检测来自该个体的甲状腺结节中的这些目标标志物的甲基化水平；优选地，所述目标标志物为FTAP2B基因或基因组的FTAP2B序列、EMS2OS基因或基因组的EMS2OS序列以及SOD3基因或基因组的SOD3序列，其中，检测血液样品如血浆中这些目标标 志物的甲基化水平；
    优选地，所述目标标志物为为FTAP2B基因或基因组的FTAP2B序列和DGKG基因或基因组的DGKG序列，其中，检测血液样品如血浆中这些目标标志物的甲基化水平；
    优选地，所述目标标志物为FTAP2B基因或基因组的FTAP2B序列和SOD3基因或基因组的SOD3序列，以及EMS2OS基因或基因组的EMS2OS序列或RP11-867G23.12基因或基因组的RP11-867G23.12序列，其中，检测血液样品如血浆中这些目标标志物的甲基化水平。
  4. 如权利要求1所述的应用，其特征在于，
    所述目标标志物为RARG基因或基因组的RARG序列，以及选自DPYS基因或基因组的DPYS序列、TSHR基因或基因组的TSHR序列、TTC34基因或基因组的TTC34序列、RCOR2基因或基因组的RCOR2序列、SLC16A3基因或基因组的SLC16A3序列和ITPKA基因或基因组的ITPKA序列中的至少一个、至少两个、至少三个或至少四个；
    优选地，所述目标标志物为RARG基因或基因组的RARG序列和DPYS基因或基因组的DPYS序列，其中，检测来自该个体的甲状腺结节中的这些目标标志物的甲基化水平；
    优选地，所述目标标志物为RARG基因或基因组的RARG序列和TSHR基因或基因组的TSHR序列，其中，检测血液样品如血浆中这些目标标志物的甲基化水平；
    优选地，所述目标标志物为RARG基因或基因组的RARG序列和TTC34基因或基因组的TTC34序列，以及选自RCOR2基因或基因组的RCOR2序列、SLC16A3基因或基因组的SLC16A3序列和ITPKA基因或基因组的ITPKA序列中的至少一个或至少两个，其中，检测血液样品如血浆中这些目标标志物的甲基化水平；优选地，所述目标标志物为RARG基因或基因组的RARG序列、TTC34基因或基因组的TTC34序列、RCOR2基因或基因组的RCOR2序列和SLC16A3基因或基因组的SLC16A3序列其中，检测血液样品如血浆中这些目标标志物的甲基化水平；优选地，所述目标标志物为RARG基因或基因组的RARG序列、TTC34基因或基因组的TTC34序列、ITPKA基因或基因组的 ITPKA序列和SLC16A3基因或基因组的SLC16A3序列其中，检测血液样品如血浆中这些目标标志物的甲基化水平；
    优选地，所述目标标志物为RARG基因或基因组的RARG序列和SLC16A3基因或基因组的SLC16A3序列，以及选自TTC34基因或基因组的TTC34序列、RCOR2基因或基因组的RCOR2序列和ITPKA基因或基因组的ITPKA序列中的至少一个或至少两个，其中，检测血液样品如血浆中这些目标标志物的甲基化水平；优选地，所述目标标志物为RARG基因或基因组的RARG序列、TTC34基因或基因组的TTC34序列、RCOR2基因或基因组的RCOR2序列和SLC16A3基因或基因组的SLC16A3序列其中，检测血液样品如血浆中这些目标标志物的甲基化水平；优选地，所述目标标志物为RARG基因或基因组的RARG序列、TTC34基因或基因组的TTC34序列、ITPKA基因或基因组的ITPKA序列和SLC16A3基因或基因组的SLC16A3序列其中，检测血液样品如血浆中这些目标标志物的甲基化水平；
    优选地，所述目标标志物为RARG基因或基因组的RARG序列、SLC16A3基因或基因组的SLC16A3序列和TTC34基因或基因组的TTC34序列，以及ROCR2基因或基因组的ROCR2序列或ITPKA基因或基因组的ITPKA序列；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。
  5. 如权利要求1所述的应用，其特征在于，
    所述目标标志物为SOD3基因或基因组的SOD3序列，以及选自EMS2OS基因或基因组的EMS2OS序列、TEAD3基因或基因组的TEAD3序列、FTAP2B基因或基因组的FTAP2B序列和RP11-867G23.12基因或基因组的RP11-867G23.12序列中的至少一个或至少两个；
    优选地，所述目标标志物为SOD3基因或基因组的SOD3序列和EMS2OS基因或基因组的EMS2OS序列，以及TEAD3基因或基因组的TEAD3序列或FTAP2B基因或基因组的FTAP2B序列；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平；
    优选地，所述目标标志物为SOD3基因或基因组的SOD3序列和FTAP2B基因或基因组的FTAP2B序列，以及EMS2OS基因或基因组的EMS2OS序列或RP11-867G23.12基因或基因组的RP11-867G23.12序列；优选地，检测来自 该个体的血液样品如血浆中这些目标标志物的甲基化水平；
    优选地，所述目标标志物为TEAD3基因或基因组的TEAD3序列，以及选自RP11-867G23.12基因或基因组的RP11-867G23.12序列、SOD3基因或基因组的SOD3序列和EMS2OS基因或基因组的EMS2OS序列中的至少一种或至少两种；优选地，所述目标标志物为TEAD3基因或基因组的TEAD3序列和RP11-867G23.12基因或基因组的RP11-867G23.12序列；优选地，检测来自该个体的甲状腺结节中的这些目标标志物的甲基化水平；优选地，所述目标标志物为TEAD3基因或基因组的TEAD3序列，SOD3基因或基因组的SOD3序列和EMS2OS基因或基因组的EMS2OS序列；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。
  6. 如权利要求1所述的应用，其特征在于，
    所述目标标志物为TTC34基因或基因组的TTC34序列，以及选自RCOR2基因或基因组的RCOR2序列、ITPKA基因或基因组的ITPKA序列、CDH1基因或基因组的CDH1序列、SLC16A3基因或基因组的SLC16A3序列和RARG基因或基因组的RARG序列中的至少一个、至少两个或至少三个；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平；
    优选地，所述目标标志物为TTC34基因或基因组的TTC34序列和RCOR2基因或基因组的RCOR2序列，以及选自ITPKA基因或基因组的ITPKA序列、CDH1基因或基因组的CDH1序列、SLC16A3基因或基因组的SLC16A3序列和RARG基因或基因组的RARG序列中的至少一个或至少两个；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平；
    优选地，所述目标标志物为TTC34基因或基因组的TTC34序列、RCOR2基因或基因组的RCOR2序列、ITPKA基因或基因组的ITPKA序列和CDH1基因或基因组的CDH1序列；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平；
    优选地，所述目标标志物为TTC34基因或基因组的TTC34序列、RCOR2基因或基因组的RCOR2序列、SLC16A3基因或基因组的SLC16A3序列和RARG基因或基因组的RARG序列；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平；
    优选地，所述目标标志物为TTC34基因或基因组的TTC34序列和ITPKA基因或基因组的ITPKA序列，以及选自RCOR2基因或基因组的RCOR2序列、CDH1基因或基因组的CDH1序列、SLC16A3基因或基因组的SLC16A3序列和RARG基因或基因组的RARG序列中的至少一个或至少两个；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平；
    优选地，所述目标标志物为TTC34基因或基因组的TTC34序列、ITPKA基因或基因组的ITPKA序列、RCOR2基因或基因组的RCOR2序列和CDH1基因或基因组的CDH1序列；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平；
    优选地，所述目标标志物为TTC34基因或基因组的TTC34序列、ITPKA基因或基因组的ITPKA序列、SLC16A3基因或基因组的SLC16A3序列和RARG基因或基因组的RARG序列；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平；
    优选地，所述目标标志物为TTC34基因或基因组的TTC34序列和SLC16A3基因或基因组的SLC16A3序列，以及选自RCOR2基因或基因组的RCOR2序列、ITPKA基因或基因组的ITPKA序列和RARG基因或基因组的RARG序列中的至少一个或至少两个；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平；
    优选地，所述目标标志物为TTC34基因或基因组的TTC34序列、SLC16A3基因或基因组的SLC16A3序列、RCOR2基因或基因组的RCOR2序列和RARG基因或基因组的RARG序列；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平；优选地，所述目标标志物为TTC34基因或基因组的TTC34序列、SLC16A3基因或基因组的SLC16A3序列、ITPKA基因或基因组的ITPKA序列和RARG基因或基因组的RARG序列；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平；
    优选地，目标标志物为TTC34基因或基因组的TTC34序列、SLC16A3基因或基因组的SLC16A3序列和RARG基因或基因组的RARG序列，以及RCOR2基因或基因组的RCOR2序列或ITPKA基因或基因组的ITPKA序列；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。
  7. 如权利要求1所述的应用，其特征在于，
    所述目标标志物为MCC基因或基因组的MCC序列，以及选自ITPKA基因或基因组的ITPKA序列、RCOR2基因或基因组的RCOR2序列、SLC16A3基因或基因组的SLC16A3序列和CDH1基因或基因组的CDH1序列中的至少一个、至少两个或至少三个；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平；优选地，目标标志物为MCC基因或基因组的MCC序列、ITPKA基因或基因组的ITPKA序列、RCOR2基因或基因组的RCOR2序列和SLC16A3基因或基因组的SLC16A3序列；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平；优选地，目标标志物为MCC基因或基因组的MCC序列和CDH1基因或基因组的CDH1序列；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平；或
    所述目标标志物为CDH1基因或基因组的CDH1序列，以及选自MCC基因或基因组的MCC序列、TTC34基因或基因组的TTC34序列、RCOR2基因或基因组的RCOR2序列和ITPKA基因或基因组的ITPKA序列中的至少一个、至少两个或至少三个；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平；优选地，目标标志物为CDH1基因或基因组的CDH1序列和MCC基因或基因组的MCC序列；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平；优选地，所述目标标志物为CDH1基因或基因组的CDH1序列、TTC34基因或基因组的TTC34序列、RCOR2基因或基因组的RCOR2序列和ITPKA基因或基因组的ITPKA序列；优选地，检测来自该个体的血液样品如血浆中这些目标标志物的甲基化水平。
  8. 如权利要求1-7中任一项所述的应用，其特征在于，所述各基因的Hg19坐标如下：
    CDH1基因，chr16:68771195-68869439；TSHR基因，chr14:81422025-81610697；MCC基因，chr5:112357803-112630635；TBX15基因，chr1:119427355-119530418；PRR15基因，chr7:29605149-29606911；DPYS基因，chr8:105391659-105479283；GRIA2基因，chr4:158141272-158287226；NR5A1基因，chr9:127245034-127265678；TTC34基因，chr1:2572708-2717433； RCOR2基因，chr11:63678702-63684636；F10基因，chr13:113777113-113803843；ITPKA基因，chr15:41786456-41795364；SLC16A3基因，chr17:80193650-80197375；RARG基因，chr12:53604350-53626040；FTAP2B基因，chr6:50786439-50815326；SOD3基因，chr4:24797195-24802464；RP11-867G23.12基因，chr11:66101965-66107346；EMX2OS基因，chr10:119243804-119302100；TEAD3基因，chr6:35441374-35464856；DGKG基因，chr3:185864990-186080023。
  9. 如权利要求1-7中任一项所述的应用，其特征在于，所述一个或多个目标标志物为所述各基因采用以下引物扩增得到的一个或多个目标区域：
    CDH1基因的目标区域：SEQ ID NO:1和2所示的序列或与SEQ ID NO:1和2具有至少90％相同性的序列作为引物扩增得到的CDH1基因的片段；
    TSHR基因的目标区域：SEQ ID NO:3和4所示的序列或与SEQ ID NO:3和4具有至少90％相同性的序列作为引物扩增得到的TSHR基因的片段；
    MCC基因的目标区域：SEQ ID NO:5和6所示的序列或与SEQ ID NO:5和6具有至少90％相同性的序列作为引物扩增得到的MCC基因的片段；
    TBX15基因的目标区域：SEQ ID NO:7和8所示的序列或与SEQ ID NO:7和8具有至少90％相同性的序列作为引物扩增得到的TBX15基因的片段；
    PRR15基因的目标区域：SEQ ID NO:9和10所示的序列或与SEQ ID NO:9和10具有至少90％相同性的序列作为引物扩增得到的PRR15基因的片段；
    DPYS基因的目标区域：SEQ ID NO:11和12所示的序列或与SEQ ID NO:11和12具有至少90％相同性的序列作为引物扩增得到的DPYS基因的片段；
    GRIA2基因的目标区域：SEQ ID NO:13和14所示的序列或与SEQ ID NO:13和14具有至少90％相同性的序列作为引物扩增得到的GRIA2基因的片段；
    NR5A1基因的目标区域：SEQ ID NO:15和16所示的序列或与SEQ ID NO:15和16具有至少90％相同性的序列作为引物扩增得到的NR5A1基因的片段；
    TTC34基因的目标区域：SEQ ID NO:17和18所示的序列或与SEQ ID  NO:17和18具有至少90％相同性的序列作为引物扩增得到的TTC34基因的片段；
    RCOR2基因的目标区域：SEQ ID NO:19和20所示的序列或与SEQ ID NO:19和20具有至少90％相同性的序列作为引物扩增得到的RCOR2基因的片段；
    F10基因的目标区域：SEQ ID NO:21和22所示的序列或与SEQ ID NO:21和22具有至少90％相同性的序列作为引物扩增得到的F10基因的片段；
    ITPKA基因的目标区域：SEQ ID NO:23和24所示的序列或与SEQ ID NO:23和24具有至少90％相同性的序列作为引物扩增得到的ITPKA基因的片段；
    SLC16A3基因的目标区域：SEQ ID NO:25和26所示的序列或与SEQ ID NO:25和26具有至少90％相同性的序列作为引物扩增得到的SLC16A3基因的片段；
    RARG基因的目标区域：SEQ ID NO:27和28所示的序列或与SEQ ID NO:27和28具有至少90％相同性的序列作为引物扩增得到的RARG基因的片段；
    FTAP2B基因的目标区域：SEQ ID NO:29和30所示的序列或与SEQ ID NO:29和30具有至少90％相同性的序列作为引物扩增得到的FTAP2B基因的片段；
    SOD3基因的目标区域：SEQ ID NO:31和32所示的序列或与SEQ ID NO:31和32具有至少90％相同性的序列作为引物扩增得到的SOD3基因的片段；
    RP11-867G23.12基因的目标区域：SEQ ID NO:33和34所示的序列或与SEQ ID NO:33和34具有至少90％相同性的序列作为引物扩增得到的RP11-867G23.12基因的片段；
    EMX2OS基因的目标区域：SEQ ID NO:35和36所示的序列或与SEQ ID NO:35和36具有至少90％相同性的序列作为引物扩增得到的EMX2OS基因的片段；
    TEAD3基因的目标区域：SEQ ID NO:37和38所示的序列或与SEQ ID  NO:37和38具有至少90％相同性的序列作为引物扩增得到的TEAD3基因的片段；
    DGKG基因的目标区域：SEQ ID NO:39和40所示的序列或与SEQ ID NO:39和40具有至少90％相同性的序列作为引物扩增得到的DGKG基因的片段。
  10. 如权利要求9所述的应用，其特征在于，所述各基因的目标区域的Hg19坐标如下：
    CDH1基因的目标区域，chr16:68770965-68771133；TSHR基因的目标区域，chr14:81421913-81422134；MCC基因的目标区域，chr5:112538899-112539205；TBX15基因的目标区域，chr1:119535640-119535860；PRR15基因的目标区域，chr7:29606122-29606390；DPYS基因的目标区域，chr8:105478808-105479066；GRIA2基因的目标区域，chr4:158141305-158141513；NR5A1基因的目标区域，chr9:127265730-127265942；TTC34基因的目标区域，chr1:2706494-2706702；RCOR2基因的目标区域，chr11:63687060-63687339；F10基因的目标区域，chr13:113807232-113807480；ITPKA基因的目标区域，chr15:41793298-41793573；SLC16A3基因的目标区域，chr17:80189548-80189879；RARG基因的目标区域，chr12:53613026-53613303；FTAP2B基因的目标区域，chr6:50818141-50818368；SOD3基因的目标区域，chr4:24801668-24801925；RP11-867G23.12基因的目标区域，chr11:66104360-66104678；EMX2OS基因的目标区域，chr10:119295848-119296135；TEAD3基因的目标区域，chr6:35454128-35454333；DGKG基因的目标区域，chr3:185973693-185973973。
  11. 如权利要求1-10中任一项所述的应用，其特征在于，
    所述各基因的引物分子分别为：
    CDH1：SEQ ID NO:1和2，或与其扩增得到的片段在严谨条件下杂交的引物分子；TSHR：SEQ ID NO:3和4，或与其扩增得到的片段在严谨条件下杂交的引物分子；MCC：SEQ ID NO:5和6，或与其扩增得到的片段在严谨条件下杂交的引物分子；TBX15：SEQ ID NO:7和8，或与其扩增得到的片段在严谨条件下杂交的引物分子；PRR15：SEQ ID NO:9和10，或与其扩增得到的片 段在严谨条件下杂交的引物分子；DPYS：SEQ ID NO:11和12，或与其扩增得到的片段在严谨条件下杂交的引物分子；GRIA2：SEQ ID NO:13和14，或与其扩增得到的片段在严谨条件下杂交的引物分子；NR5A1：SEQ ID NO:15和16，或与其扩增得到的片段在严谨条件下杂交的引物分子；TTC34：SEQ ID NO:17和18，或与其扩增得到的片段在严谨条件下杂交的引物分子；RCOR2：SEQ ID NO:19和20，或与其扩增得到的片段在严谨条件下杂交的引物分子；F10：SEQ ID NO:21和22，或与其扩增得到的片段在严谨条件下杂交的引物分子；ITPKA：SEQ ID NO:23和24，或与其扩增得到的片段在严谨条件下杂交的引物分子；SLC16A3：SEQ ID NO:25和26，或与其扩增得到的片段在严谨条件下杂交的引物分子；RARG：SEQ ID NO:27和28，或与其扩增得到的片段在严谨条件下杂交的引物分子；FTAP2B：SEQ ID NO:29和30，或与其扩增得到的片段在严谨条件下杂交的引物分子；SOD3：SEQ ID NO:31和32，或与其扩增得到的片段在严谨条件下杂交的引物分子；RP11-867G23.12：SEQ ID NO:33和34，或与其扩增得到的片段在严谨条件下杂交的引物分子；EMX2OS：SEQ ID NO:35和36，或与其扩增得到的片段在严谨条件下杂交的引物分子；TEAD3：SEQ ID NO:37和38，或与其扩增得到的片段在严谨条件下杂交的引物分子；DGKG：SEQ ID NO:39和40，或与其扩增得到的片段在严谨条件下杂交的引物分子；
    所述各基因的扩增产物的探针分子分别为：
    CDH1：SEQ ID NO:41或与其具有至少90％相同性的序列；TSHR：SEQ ID NO:42或与其具有至少90％相同性的序列；MCC：SEQ ID NO:43或与其具有至少90％相同性的序列；TBX15：SEQ ID NO:44或与其具有至少90％相同性的序列；PRR15：SEQ ID NO:45或与其具有至少90％相同性的序列；DPYS：SEQ ID NO:46或与其具有至少90％相同性的序列；GRIA2：SEQ ID NO:47或与其具有至少90％相同性的序列；NR5A1：SEQ ID NO:48或与其具有至少90％相同性的序列；TTC34：SEQ ID NO:49或与其具有至少90％相同性的序列；RCOR2：SEQ ID NO:50或与其具有至少90％相同性的序列；F10：SEQ ID NO:51或与其具有至少90％相同性的序列；ITPKA：SEQ ID NO:52或与其具有至少90％相同性的序列；SLC16A3：SEQ ID NO:53或与其具有至少90％相 同性的序列；RARG：SEQ ID NO:54或与其具有至少90％相同性的序列；FTAP2B：SEQ ID NO:55或与其具有至少90％相同性的序列；SOD3：SEQ ID NO:56或与其具有至少90％相同性的序列；RP11-867G23.12：SEQ ID NO:57或与其具有至少90％相同性的序列；EMX2OS：SEQ ID NO:58或与其具有至少90％相同性的序列；TEAD3：SEQ ID NO:59或与其具有至少90％相同性的序列；DGKG：SEQ ID NO:60或与其具有至少90％相同性的序列。
  12. 如权利要求1所述的应用，其特征在于，所述检测试剂或诊断试剂盒还包括检测内参基因ACTB的引物分子和/或探针分子；优选地，内参基因ACTB的引物分子为SEQ ID NO:61和62所示的序列与由SEQ ID NO:61和62作为引物扩增得到的ACTB基因的片段在严谨条件下杂交的序列，内参基因ACTB的探针包含SEQ ID NO:63所示序列或与其有至少90％相同性的序列。
  13. 一种用于检测目标标志物至少一个CpG二核苷酸的甲基化状态或甲基化水平以诊断甲状腺结节良恶性的诊断试剂或诊断试剂盒，其包含引物分子和/或探针分子；其中，所述引物分子相同于、互补于或在严谨条件下杂交于一个或多个目标标志物并包含至少9个连续的核苷酸，所述探针分子与一个或多个目标标志物的扩增产物在严谨条件下杂交，所述一个或多个目标标志物选自：CDH1基因或基因组的CDH1序列、TSHR基因或基因组的TSHR序列、MCC基因或基因组的MCC序列、TBX15基因或基因组的TBX15序列、PRR15基因或基因组的PRR15序列、DPYS基因或基因组的DPYS序列、GRIA2基因或基因组的GRIA2序列、NR5A1基因或基因组的NR5A1序列、TTC34基因或基因组的TTC34序列、RCOR2基因或基因组的RCOR2序列、F10基因或基因组的F10序列、ITPKA基因或基因组的ITPKA序列、SLC16A3基因或基因组的SLC16A3序列、RARG基因或基因组的RARG序列、FTAP2B基因或基因组的FTAP2B序列、SOD3基因或基因组的SOD3序列、RP11-867G23.12基因或基因组的RP11-867G23.12序列、EMX2OS基因或基因组的EMX2OS序列、TEAD3基因或基因组的TEAD3序列和DGKG基因或基因组的DGKG序列。
  14. 如权利要求13所述的诊断试剂或诊断试剂盒，其特征在于，所述一个或多个目标标志物如权利要求2-10中任一项所述；优选地，所述引物分子 和探针分子如权利要求11所述。
  15. 如权利要求13或14所述的诊断试剂或诊断试剂盒，其特征在于，所述所述诊断试剂或诊断试剂盒还包括检测内参基因ACTB的引物分子和/或探针分子；优选地，内参基因ACTB的引物分子为SEQ ID NO:61和62所示的序列与由SEQ ID NO:61和62作为引物扩增得到的ACTB基因的片段在严谨条件下杂交的序列，内参基因ACTB的探针包含SEQ ID NO:63所示序列或与其有至少90％相同性的序列。
  16. 如权利要求13所述的诊断试剂或诊断试剂盒，其特征在于，所述诊断试剂盒还包括选自以下一种或多种物质：PCR缓冲液、聚合酶、dNTP、限制性内切酶、酶切缓冲液、荧光染料、荧光淬灭剂、荧光报告剂、外切核酸酶、碱性磷酸酶、内标、对照物、KCl、MgCl 2和(NH 4) 2SO 4。
  17. 如权利要求13所述的诊断试剂或诊断试剂盒，其特征在于，检测DNA甲基化的试剂还包括以下一个或多个方法中所用的试剂：基于重亚硫酸盐转化的PCR、DNA测序、甲基化敏感的限制性内切酶分析法、荧光定量法、甲基化敏感性高分辨率熔解曲线法、基于芯片的甲基化图谱分析和质谱。
  18. 如权利要求17所述的诊断试剂或诊断试剂盒，其特征在于，所述试剂选自以下一种或多种：重亚硫酸盐及其衍生物、荧光染料、荧光淬灭剂、荧光报告剂、内标和对照物。
  19. 区分基因组DNA至少一个靶区域内甲基化和未甲基化CpG二核苷酸的至少一种试剂或成组试剂在制备用于检测和/或分类个体中甲状腺结节良恶性的方法的试剂盒中的用途，其中所述方法包括使从所述个体生物样品中分离的基因组DNA与所述至少一种试剂或成组试剂接触，其中所述靶区域等同于或互补于一个或多个目标标志物的至少16连续核苷酸的序列，其中所述连续核苷酸包含至少一个CpG二核苷酸序列，由此至少部分地提供对甲状腺结节良恶性的检测和/或分类，其中，所述一个或多个目标标志物选自：CDH1基因或基因组的CDH1序列、TSHR基因或基因组的TSHR序列、MCC基因或基因组的MCC序列、TBX15基因或基因组的TBX15序列、PRR15基因或基因组的PRR15序列、DPYS基因或基因组的DPYS序列、GRIA2基因或基因组的GRIA2序列、NR5A1基因或基因组的NR5A1序列、TTC34基因或基因组 的TTC34序列、RCOR2基因或基因组的RCOR2序列、F10基因或基因组的F10序列、ITPKA基因或基因组的ITPKA序列、SLC16A3基因或基因组的SLC16A3序列、RARG基因或基因组的RARG序列、FTAP2B基因或基因组的FTAP2B序列、SOD3基因或基因组的SOD3序列、RP11-867G23.12基因或基因组的RP11-867G23.12序列、EMX2OS基因或基因组的EMX2OS序列、TEAD3基因或基因组的TEAD3序列和DGKG基因或基因组的DGKG序列。
  20. 如权利要求19所述的用途，其特征在于，所述一个或多个目标标志物如权利要求2-10中任一项所述。
  21. 将5位未甲基化的胞嘧啶碱基转化为尿嘧啶或在杂交性能方面可检测地不同于胞嘧啶的其它碱基的一种或多种试剂、扩增酶以及至少一种包含至少9个连续核苷酸的引物在制备用于检测和/或分类个体中甲状腺结节良恶性的方法的试剂盒中的用途，其中所述方法包括：
    a)从所述个体生物样品分离基因组DNA；
    b)用所述一种或多种试剂处理a)的所述基因组DNA或其片段；
    c)使所述经处理的基因组DNA或其经处理的片段与所述扩增酶和所述至少一种引物接触，所述引物相同于、互补于或在严谨条件下杂交于一个或多个目标标志物，其中所述经处理的基因组DNA或其片段被扩增以产生至少一种扩增产物或不被扩增；以及
    d)基于所述扩增物是否存在或其性质，确定所述一个或多个目标标志物的至少一个CpG二核苷酸的甲基化状态或水平，或者反映所述一个或多个目标标志物的多个CpG二核苷酸平均甲基化状态或水平的均值或值，由此至少部分地检测和/或分类个体中甲状腺结节的良恶性；
    其中，所述一个或多个目标标志物选自：CDH1基因或基因组的CDH1序列、TSHR基因或基因组的TSHR序列、MCC基因或基因组的MCC序列、TBX15基因或基因组的TBX15序列、PRR15基因或基因组的PRR15序列、DPYS基因或基因组的DPYS序列、GRIA2基因或基因组的GRIA2序列、NR5A1基因或基因组的NR5A1序列、TTC34基因或基因组的TTC34序列、RCOR2基因或基因组的RCOR2序列、F10基因或基因组的F10序列、ITPKA基因或基因组的ITPKA序列、SLC16A3基因或基因组的SLC16A3序列、RARG 基因或基因组的RARG序列、FTAP2B基因或基因组的FTAP2B序列、SOD3基因或基因组的SOD3序列、RP11-867G23.12基因或基因组的RP11-867G23.12序列、EMX2OS基因或基因组的EMX2OS序列、TEAD3基因或基因组的TEAD3序列和DGKG基因或基因组的DGKG序列。
  22. 如权利要求21所述的用途，其特征在于，所述一个或多个目标标志物如权利要求2-10中任一项所述；优选地，所述引物分子如权利要求11所述。
  23. 如权利要求21所述的用途，其中步骤b)中，使用选自亚硫酸氢盐、酸式亚硫酸盐、焦亚硫酸盐及其组合的试剂处理所述基因组DNA或其片段。
  24. 如权利要求21所述的用途，其中c)中，通过使用耐热DNA聚合酶作为所述扩增酶、使用缺乏5’-3’外切酶活性的聚合酶、使用聚合酶链式反应和/或产生带有可检测标记的扩增产物进行核酸分子的接触或扩增。
  25. 如权利要求21所述的用途，其中c)中的接触或扩增包括使用甲基化特异的引物。
  26. 一种或多种甲基化敏感限制酶和扩增酶以及至少一种包含至少9个连续核苷酸的引物在制备用于检测和/或分类个体中甲状腺结节良恶性的方法的试剂盒中的用途，其中，所述引物相同于、互补于或在严谨条件下杂交于一个或多个目标标志物；所述方法包括：
    a)从所述个体生物样品分离基因组DNA；
    b)以所述一种或多种甲基化敏感限制酶消化a)所述的基因组DNA或其片段，使所得消化产物与所述扩增酶和所述至少一种引物接触；和
    c)基于所述扩增物是否存在或其性质，确定所述一个或多个目标标志物的至少一个CpG二核苷酸的甲基化状态或水平，由此至少部分地检测和/或分类个体中甲状腺结节的良恶性；
    其中，所述一个或多个目标标志物选自：CDH1基因或基因组的CDH1序列、TSHR基因或基因组的TSHR序列、MCC基因或基因组的MCC序列、TBX15基因或基因组的TBX15序列、PRR15基因或基因组的PRR15序列、DPYS基因或基因组的DPYS序列、GRIA2基因或基因组的GRIA2序列、NR5A1基因或基因组的NR5A1序列、TTC34基因或基因组的TTC34序列、RCOR2基因或基因组的RCOR2序列、F10基因或基因组的F10序列、ITPKA 基因或基因组的ITPKA序列、SLC16A3基因或基因组的SLC16A3序列、RARG基因或基因组的RARG序列、FTAP2B基因或基因组的FTAP2B序列、SOD3基因或基因组的SOD3序列、RP11-867G23.12基因或基因组的RP11-867G23.12序列、EMX2OS基因或基因组的EMX2OS序列、TEAD3基因或基因组的TEAD3序列和DGKG基因或基因组的DGKG序列。
  27. 如权利要求26所述的用途，其特征在于，所述一个或多个目标标志物如权利要求2-10中任一项所述；优选地，所述引物分子如权利要求11所述。
  28. 如权利要求26所述的用途，其特征在于，通过杂交至少一种核酸或肽核酸来确定扩增产物的存在与否，所述至少一种核酸或肽核酸等同于或互补于选自所述一个或多个目标标志物的序列的至少16碱基长片段。
  29. 衍生自一个或多个目标标志物的经处理的核酸在制备用于诊断甲状腺结节良恶性的试剂盒中的用途，其中所述处理适合于将所述一个或多个目标标志物的至少一个未甲基化的胞嘧啶碱基转化至尿嘧啶或在杂交上可检测地不同于胞嘧啶的其它碱基，所述一个或多个目标标志物选自：CDH1基因或基因组的CDH1序列、TSHR基因或基因组的TSHR序列、MCC基因或基因组的MCC序列、TBX15基因或基因组的TBX15序列、PRR15基因或基因组的PRR15序列、DPYS基因或基因组的DPYS序列、GRIA2基因或基因组的GRIA2序列、NR5A1基因或基因组的NR5A1序列、TTC34基因或基因组的TTC34序列、RCOR2基因或基因组的RCOR2序列、F10基因或基因组的F10序列、ITPKA基因或基因组的ITPKA序列、SLC16A3基因或基因组的SLC16A3序列、RARG基因或基因组的RARG序列、FTAP2B基因或基因组的FTAP2B序列、SOD3基因或基因组的SOD3序列、RP11-867G23.12基因或基因组的RP11-867G23.12序列、EMX2OS基因或基因组的EMX2OS序列、TEAD3基因或基因组的TEAD3序列和DGKG基因或基因组的DGKG序列。
  30. 如权利要求29所述的用途，其特征在于，所述一个或多个目标标志物如权利要求2-10中任一项所述。
  31. 用于检测并诊断个体甲状腺结节良恶性的装置，所述装置包括存储器、处理器以及存储在存储器上并可在处理器上运行的计算机程序，所述处理器执行所述程序时实现以下步骤：(1)获取样品中一个或多个目标标志物至少一 个CpG二核苷酸的甲基化水平或甲基化状态，和(2)根据(1)的甲基化水平或甲基化状态判读甲状腺结节良恶性；
    其中，所述一个或多个目标标志物选自：CDH1基因或基因组的CDH1序列、TSHR基因或基因组的TSHR序列、MCC基因或基因组的MCC序列、TBX15基因或基因组的TBX15序列、PRR15基因或基因组的PRR15序列、DPYS基因或基因组的DPYS序列、GRIA2基因或基因组的GRIA2序列、NR5A1基因或基因组的NR5A1序列、TTC34基因或基因组的TTC34序列、RCOR2基因或基因组的RCOR2序列、F10基因或基因组的F10序列、ITPKA基因或基因组的ITPKA序列、SLC16A3基因或基因组的SLC16A3序列、RARG基因或基因组的RARG序列、FTAP2B基因或基因组的FTAP2B序列、SOD3基因或基因组的SOD3序列、RP11-867G23.12基因或基因组的RP11-867G23.12序列、EMX2OS基因或基因组的EMX2OS序列、TEAD3基因或基因组的TEAD3序列和DGKG基因或基因组的DGKG序列。
  32. 如权利要求31所述的装置，其特征在于，所述一个或多个目标标志物如权利要求2-10中任一项所述。
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