Reagents for detecting DNA methylation and uses thereof

Abstract

Disclosed herein is a method of identifying the nature of a thyroid nodule, comprising detecting the level of DNA methylation in a sample in a region selected from (1) and (2) below: (1) a fragment of one or more genes selected from the group consisting of PRDM1, ACRBP, TPO, MCC, TBX15, UNCX, RPS7P5, FOXI2, (2) a nucleic acid region within 10 Kb upstream or downstream of the genes of (1).

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of molecular auxiliary diagnosis, and particularly relates to application in benign and malignant screening of thyroid nodules. BACKGROUND

[0002] DNA methylation is a mechanism of epigenetics, is a common epigenetic modification of eukaryotic genome, and is also an important natural chemical modification of DNA of vertebrates without changing the DNA sequence, which plays an important role in cell proliferation, differentiation, development, and is closely related to the occurrence and development of tumors. DNA methylation plays an important role in vivo, and its effects include transcription inhibition, chromatin structure regulation, X chromosome inactivation, genomic imprinting, etc. Abnormal DNA methylation can participate in the occurrence and progression of tumors by affecting chromatin structure and expression of oncogenes and tumor suppressor genes.

[0003] CpG dinucleotide is the main target of DNA methylation in mammals, which is distributed throughout the genome. In healthy human genome, CpG sites in CpG islands are usually in a non-methylated state, while CpG sites outside CpG islands are usually highly methylated, and this form of methylation can be stably retained during cell division. When tumor occurs, the methylation degree of CpG sites in non-CpG island region of tumor suppressor genes decreases, while CpG in CpG island is in a highly methylated state, which leads to changes in chromatin structure and reduction of tumor suppressor gene expression.

[0004] With the continuous development of genetics and epigenetics in the past decade, more and more researchers have realized that the occurrence of tumors is not completely determined by genetic genes, and epigenetic effects also play an important role. The main epigenetic changes in thyroid cancer are abnormal methylation of tumor suppressor genes and thyroid-related genes. Studying DNA methylation in thyroid cancer can provide new molecular markers for early diagnosis, treatment plan selection and prognosis evaluation.

[0005] Thyroid nodules are lumps formed in thyroid tissue after abnormal proliferation of thyroid cells. Thyroid nodules are very common, although most thyroid nodules are benign, but a small part of thyroid nodules progress to thyroid cancer. In order to diagnose and treat thyroid cancer earlier, and reduce unnecessary surgery, it is necessary to differentiate benign and malignant thyroid nodules.

[0006] Currently, the evaluation of thyroid nodules is mainly through ultrasonography (US) and fine needle aspiration biopsy (FNAB). In the diagnostic procedure of thyroid nodules, US is the most sensitive examination method, which can measure the size of the nodule, determine the internal structure of the nodule, etc. The US signs suggesting malignancy of thyroid nodules include: the height of the nodule is greater than the width (OR = 10.15), lack of halo (OR = 7.14), microcalcification (OR = 6.76), irregular border (OR = 6.12), hypoechogenicity (OR = 5.07), solid nodule (OR = 4.69), rich blood flow in the nodule (OR = 3.76), etc. FNAB is used to determine the nature of the nodule for the nodule with a diameter > 1 cm and US signs of malignancy. There are still up to 20% of the nodules with indeterminate cytology results, which need to be combined with molecular testing. There are already Gene Expression Classifier and ThyroSeq v2 products on the market, the former has a very low positive predictive value (PPV) of only 46%; the latter also has a PPV of only 42%-77%. Therefore, more accurate molecular diagnostic tools are needed. Gene ExpressionClassifierandThyroSeqv2products, the former has a very low positive predictive value (PPV) of only 46%; the latter also has a PPV of only 42%-77%. Therefore, more accurate molecular diagnostic tools are needed.

[0007] There is still a need in the art for a high specificity and high sensitivity method for the diagnosis of thyroid nodules. SUMMARY

[0008] The purpose of the present application is to provide a reagent for detecting DNA methylation and its use in the screening of benign and malignant thyroid nodules.

[0009] The present application provides in a first aspect an isolated nucleic acid molecule from a mammal, the nucleic acid molecule having a sequence of a nucleic acid selected from (1) and (2) below or a variant thereof having at least 70% identity: (1) a fragment of a nucleic acid of one or more genes selected from the group consisting of PRDM1, ACRBP, TPO, MCC, TBX15, UNCX, RPS7P5, FOXI2, the fragment being 50-1000 bp in length, wherein the fragment of the PRDM1 gene comprises one or more or all of positions 106429711, 106429722, 106429731, 106429747, 106429750, 106429761, 106429769, 106429771 of the PRDM1 gene, the fragment of the ACRBP gene comprises one or more or all of positions 6756182, 6756187, 6756191, 6756195, 6756211, 6756225, 6756230, 6756270 of the ACRBP gene, the fragment of the TPO gene comprises one or more or all of positions 1481013, 1481015, 1481022, 1481039 of the TPO gene, the fragment of the MCC gene comprises one or more or all of positions 112538999, 112539011, 112539018, 112539022, 112539061, 112539084, 112539104, 112539128 of the MCC gene, the fragment of the TBX15 gene comprises one or more or all of positions 119535725, 119535730, 119535740, 119535742, 119535750, 119535759, 119535766, 119535812, 119535817, 119535821, 119535823, 119535876, 119535879, 119535884, 119535891 of the TBX15 gene, the fragment of the UNCX gene comprises one or more or all of positions 1263643, 1263655, 1263659, 1263664, 1263676, 1263694, 1263716, 1263723 of the UNCX gene, the fragment of the RPS7P5 gene comprises one or more or all of positions 240161502, 240161507, 240161511, 240161516, 240161523, 240161527, 240161530, 240161535, 240161546, 240161558, 240161560 of the RPS7P5 gene, the fragment of the FOXI2 gene comprises one or more or all of positions 119535725, 119535730, 119535740, 119535742, 119535750, 119535759, 119535766, 119535812, 119535817, 119535821, 119535823, 119535876, 119535879, 119535884, 119535891 of the FOXI2 gene, and (2) a fragment of a nucleic acid of one or more genes selected from the group consisting of PRDM1, ACRBP, TPO, MCC, TBX15, UNCX, RPS7P5, FOXI2, the fragment being 50-1000 bp in length, wherein the fragment of the PRDM1 gene comprises one or more or all of positions 106429711, 106429722, 106429731, 106429747, 106429750, 106429761, 106429769, 106429771 of the PRDM1 gene, the fragment of the ACRBP gene comprises one or more or all of positions 6756182, 6756187, 6756191, 6756195, 6756211, 6756225, 6756230, 6756270 of the ACRBP gene, the fragment of the TPO gene comprises one or more or all of positions 1481013, 1481015, 1481022, 1481039 of the TPO gene, the fragment of the MCC gene comprises one or more or all of positions 112538999, 112539011, 112539018, 112539022, 112539061, 112539084, 112539104, 112539128 of the MCC gene, the fragment of the TBX15 gene comprises one or more or all of positions 119535725, 119535730, 119535740, 119535742, 119535750, 119535759, 119535766, 119535812, 119535817, 119535821, 119535823, 119535876, 119535879, 119535884, 119535891 of the TBX15 gene, the fragment of the UNCX gene comprises one or more or all of positions 1263643, 1263655, 1263659, 1263664, 1263676, 1263694, 1263716, 1263723 of the UNCX gene, the fragment of the RPS7P5 gene comprises one or more or all of positions 240161502, 240161507, 240161511, 240161516, 240161523, 240161527, 240161530, 240161535, 240161546, 240161558, 240161560 of the RPS7P5 gene, the fragment of the FOXI2 gene comprises one or more or all of positions 119535725, 119535730, 119535740, 119535742, 119535750, 119535759, 119535766, 119535812, 119535817, 119535821, 119535823, 119535876, 119535879, 119535884, 119535891 of the FOXI2 gene.The fragment of the FOXI2 gene comprises one or more or all of the positions: 129534843, 129534853, 129534866, 129534879, 129534891, 129534910, 129534912, 129534924 of the FOXI2 gene, (2) a nucleic acid region within 10 Kb upstream and downstream of the gene of (1) in which the above-mentioned positions in the variant are not mutated.

[0010] In one or more embodiments, the fragment of the PRDM1 gene comprises one or more or all of the positions: 106429722, 106429731, 106429747, 106429750, 106429761, 106429769, 106429771 of the PRDM1 gene, the fragment of the ACRBP gene comprises one or more or all of the positions: 6756182, 6756187, 6756191, 6756195, 6756211, 6756225, 6756230 of the ACRBP gene, the fragment of the TPO gene comprises one or more or all of the positions: 1481013, 1481015, 1481022, 1481039 of the TPO gene, the fragment of the MCC gene comprises one or more or all of the positions: 112538999, 112539011, 112539018, 112539022, 112539061 of the MCC gene, the fragment of the TBX15 gene comprises one or more or all of the positions: 119535740, 119535742, 119535750, 119535759, 119535766 of the TBX15 gene, the fragment of the UNCX gene comprises one or more or all of the positions: 1263643, 1263655, 1263659, 1263664, 1263676 of the UNCX gene, the fragment of the RPS7P5 gene comprises one or more or all of the positions: 240161502, 240161507, 240161511, 240161516, 240161523, 240161527, 240161530, 240161535, 240161546 of the RPS7P5 gene, the fragment of the FOXI2 gene comprises one or more or all of the positions: 129534843, 129534853, 129534866, 129534879, 129534891, 129534910, 129534912, 129534924 of the FOXI2 gene.

[0011] In one or more embodiments, the fragment of the PRDM1 gene comprises one or more or all of the positions: 106429722, 106429731, 106429747, 106429750, 106429761, 106429769, 106429771 of the PRDM1 gene, the fragment of the ACRBP gene comprises one or more or all of the positions: 6756182, 6756187, 6756191, 6756195, 6756211, 6756225, 6756230 of the ACRBP gene, the fragment of the TPO gene comprises one or more or all of the positions: 1481013, 1481015, 1481022, 1481039 of the TPO gene, the fragment of the MCC gene comprises one or more or all of the positions: 112538999, 112539011, 112539018, 112539022, 112539061 of the MCC gene, the fragment of the TBX15 gene comprises one or more or all of the positions: 119535740, 119535742, 119535750, 119535759, 119535766 of the TBX15 gene, the fragment of the UNCX gene comprises one or more or all of the positions: 1263659, 1263664, 1263676 of the UNCX gene, the fragment of the RPS7P5 gene comprises one or more or all of the positions: 240161502, 240161507, 240161511, 240161516, 240161523, 240161527, 240161530, 240161535, 240161546 of the RPS7P5 gene, the fragment of the FOXI2 gene comprises one or more or all of the positions: 129534843, 129534853, 129534866, 129534879, 129534891, 129534910, 129534912, 129534924 of the FOXI2 gene.

[0012] In one or more embodiments, the nucleic acid molecule comprises one or more fragments selected from the group consisting of: a fragment of the PRDM1 gene amplified with SEQ ID NO: 4 and 5 as primers, a fragment of the ACRBP gene amplified with SEQ ID NO: 6 and 7 as primers, a fragment of the TPO gene amplified with SEQ ID NO: 8 and 9 as primers, a fragment of the MCC gene amplified with SEQ ID NO: 10 and 11 as primers, a fragment of the TBX15 gene amplified with SEQ ID NO: 12 and 13 as primers, a fragment of the UNCX gene amplified with SEQ ID NO: 14 and 15 as primers, a fragment of the RPS7P5 gene amplified with SEQ ID NO: 16 and 17 as primers, a fragment of the FOXI2 gene amplified with SEQ ID NO: 18 and 19 as primers.

[0013] The second aspect of the present application provides a reagent for detecting DNA methylation, the reagent comprising a reagent for detecting the level of DNA methylation in a region selected from (1) and (2) described below: (1) a fragment of one or more genes selected from the group consisting of ZMIZ1, C15orf52, SLC16A3, ZNF512B, SLC17A5, LIMK1, PLEC, TOR4A, TMEM131L, DNM2, IL17C, PRDM16, MT1JP, TBX3, BIN1, TIMP2, CFAP65, TSHR, KIF1A, DAPK, CDH1, TPO, RARG, PRR15, DPYS, MCC, TBX15, COL23A1, ILDR2, DHRS3, GDNF, TBX18, SIM2, HOXA9, EHBP1L1, GJC2, RCOR2, PRDM1, UNCX, RPS7P5, FOXI2, ACRBP, GAS6, MCRIP2, LINC01977, EGR3, SOX17, PAX5, NEURL1, IRX4, RUSC1, the fragment being 50-1000 bp in length, (2) a nucleic acid region within 5Kb or 10Kb upstream and downstream of the gene of (1).

[0014] In one or more embodiments, the reagent for detecting the level of DNA methylation detects the level of methylation of a fragment of one or more genes selected from the group consisting of: (a) one or more genes selected from the group consisting of: PRDMl, ACRBP, TPO, MCC, TBX15, UNCX, RPS7P5, FOXI2, (b) PRDMl, ACRBP, (c) PRDMl, ACRBP and one or two genes selected from the group consisting of TPO, MCC and TBX15, (d) PRDMl, ACRBP and one or two genes selected from the group consisting of UNCX, RPS7P5 and FOXI2, (e) a nucleic acid region within 5Kb or 10Kb upstream and downstream of any of the genes in (a)-(d).

[0015] In one or more embodiments, the fragment of each gene comprises one or more sites corresponding to a site selected from the group consisting of:

[0016] ZMIZ1: 81001968, 81001996, 81002041, 81002052, 81002054, 81002056, 81002062, 81002083, 81002110, 81002116, 81002123, 81002129, 81002133, 81002137, 81002139, 81002164, 81002168, 81002223, 81002241, 81002253, of chromosome 10,

[0017] C15orf52: 40626309, 40626312, 40626386, of chromosome 15,

[0018] SLC16A3: 80189165, 80189174, 80189177, 80189197, 80189225, 80189230, 80189239, 80189645, 80189671, 80189674, 80189684, 80189687, 80189698, 80189709, 80189719, 80189726, 80189728, 80189739, 80189757, 80189787, 80189792, 80189811, 80189817, 80189832, 80189841, of chromosome 17,

[0019] ZNF512B: 62588634, 62588638, 62588672, of chromosome 20,

[0020] SLC17A5: 74290205, 74290207, 74290220, 74290225, 74290228 of chromosome 6,

[0021] LIMK1 : 73508994, 73509017, 73509055, 73509062, 73509073, 73509075, 73509112, 73509133, 73509138, 73509148, 73509160 of chromosome 7,

[0022] PLEC: 145013661, 145013673 of chromosome 8,

[0023] TOR4A: 140172787, 140172790, 140172812 of chromosome 9,

[0024] TMEM131L: 154409945, 154409963, 154409972, 154409978, 154409997, 154410003, 154410006 of chromosome 4,

[0025] DNM2: 10870373, 10870377, 10870427, 10870429, 10870441, 10870448 of chromosome 19,

[0026] IL17C: 88700818, 88700826, 88700844, 88700849, 88700857, 88700869, 88700875, 88700891, 88700897, 88700916, 88700920, 88700937, 887 00943、88700948、88700967、88700970、88700993、88701004、88701021、88701029、88701036、88701043、88701051、88701060、88701074、88701081、88 701090、88701099、88701111、88701115、88701133、88701140、88701148、88701159、88701161、88701176、88701178、88701180、88701183、88701190、8 8701201、88701204、88701210、88701212、88701236、88701240、88701266、88701278、88701281、88701285、88701305、88701421、88701442、88701451,

[0027] PRDM16: 3229914, 3229921, 3229950, 3229968, 3229973, 3310213, 3310229, 3310235, 3310238, 3310240, 3310268, 3310287, 3310312, 3310314, 3310317, 3310329 on chromosome 1,

[0028] TSHR: 81421983, 81421989, 81422010, 81422017, 81422032, 81422035, 81422063, 81422084 on chromosome 14,

[0029] KIF1A: 241759696, 241759701, 241759714, 241759716 on chromosome 2,

[0030] DAPK: 90112842, 90112853, 90112861, 90112866 on chromosome 9,

[0031] CDH1 : 68771035, 68771037, 68771045, 68771051, 68771059, 68771064, 68771073 of chromosome 16,

[0032] TPO: 1481013, 1481015, 1481022, 1481039 of chromosome 2,

[0033] RARG: 53613176, 53613182, 53613190, 53613202, 53613210, 53613218 of chromosome 12,

[0034] MT1JP: 56669271, 56669292, 56669295, 56669300, 56669318, 56669322, 56669324, 56669327, 56669344, 56669351, 56669353, 56669402, 56669414, 56669423, 56669430, 56669433, 56669437, 56669451, 56669453, 56669455, 56669463, 56669474, 56669480, 56669482, 56669485, 56669487, 56669490, 56669519, 56669533, 56669553, 56669564, 56669573, 56669578, 56669588, 56669590, 56669606, 56669610 of chromosome 16,

[0035] TBX3: 115174750, 115174773, 115174780 of chromosome 12,

[0036] BIN1: 127822478, 127822492, 127822495, 127822514, 127822551, 127822568, 127822582, 127822593, 127822616, 127822644 of chromosome 2,

[0037] TIMP2: 76921845, 76921853, 76921860 of chromosome 17,

[0038] CFAP65: 219866132, 219866139, 219866148, 219866158, 219866165, 219866168, 219866199, 219866218 of chromosome 2,

[0039] PRR15: 29605992, 29606026, 29606040, 29606047, 29606056, 29606062, 29606073, 29606179, 29606191, 29606201, 29606204, 29606220, 29606222, 29606227, 29606231, 29606255, 29606257, 29606262, 29606271, 29606277, 29606289, 29606320 on chromosome 7,

[0040] DPYS: 105478870, 105478873, 105478878, 105478905, 105478908, 105478916, 105478918, 105478945, 105478956, 105478965, 105478974, 105478983, 105478986, 105478989 of chromosome 8,

[0041] MCC: 112538999, 112539011, 112539018, 112539022, 112539061, 112539084, 112539104, 112539128 on chromosome 5,

[0042] TBX15: 119535725, 119535730, 119535740, 119535742, 119535750, 119535759, 119535766, 119535812, 119535817, 119535821, 119535823, 119535876, 119535879, 119535884, 119535891 on chromosome 1,

[0043] COL23A1: 178003785, 178003798, 178003803, 178003814, 178003823, 178003825, 178003834, 178003841, 178003844 on chromosome 5,

[0044] ILDR2: 166890429, 166890436, 166890440, 166890442, 166890448, 166890452, 166890456, 166890461, 166890468, 166890473, 166890475, 166890480, 166890492, 166890500, 166890503, 166890509, 166890516, 166890528, 166890535, 166890543, 166890555, 166890559, 166890568, 166890573, 166890584, 166890586 of chromosome 1,

[0045] DHRS3: 12656091, 12656114, 12656132, 12656152, 12656170, 12656175, 12656182, 12656187, 12656197, 12656200, 12656211, 12656315, 12656323, 12656340, 12656355, 12656367 of chromosome 1,

[0046] GDNF: 37834763, 37834770, 37834772, 37834774, 37834777, 37834780, 37834784, 37834792, 37834799, 37834802, 37834806, 37834811 of chromosome 5,

[0047] TBX18: 85477032, 85477035, 85477070, 85477083, 85477106, 85477124, 85477151, 85477153, 85477166 of chromosome 6,

[0048] SIM2: 38069563, 38069579, 38069619, 38069625, 38069638, 38069650, 38069662, 38069664, 38069676, 38069681 of chromosome 21,

[0049] HOXA9: 27204848, 27204854, 27204858, 27204861, 27204863, 27204879, 27204884, 27204894, 27204897, 27204918, 27204929, 27204938, 27204945, 27204948, 27204951, 27204958, 27204981, 27204984 of chromosome 7,

[0050] EHBP1L1: 65352612, 65352621, 65352635, 65352639, 65352642, 65352651, 65352654, 65352665, 65352670 of chromosome 11,

[0051] GJC2: 228345954, 228345957, 228345965, 228345978, 228345980, 228345989 of chromosome 1,

[0052] RCOR2: 63687223, 63687238, 63687247, 63687250, 63687259, 63687282, 63687288, 63687299, 63687318, 63687325 of chromosome 11,

[0053] PRDM1: 106429711, 106429722, 106429731, 106429747, 106429750, 106429761, 106429769, 106429771 of chromosome 6,

[0054] UNCX: 1263643, 1263655, 1263659, 1263664, 1263676, 1263694, 1263716, 1263723 of chromosome 7,

[0055] RPS7P5: 240161502, 240161507, 240161511, 240161516, 240161523, 240161527, 240161530, 240161535, 240161546, 240161558, 240161560 of chromosome 1,

[0056] FOXI2: 129534843, 129534853, 129534866, 129534879, 129534891, 129534910, 129534912, 129534924 of chromosome 10,

[0057] ACRBP: 6756182, 6756187, 6756191, 6756195, 6756211, 6756225, 6756230, 6756270 of chromosome 12,

[0058] GAS6: 114524043, 114524062, 114524068, 114524084, 114524095, 114524131, 114524138, 114524142, 114524150, 114524158 of chromosome 13,

[0059] MCRIP2: 698072, 698142, 698153, 698168, 698208, 698218, 698222, 698230 of chromosome 16,

[0060] LINC01977: 77789596, 77789601, 77789612, 77789620, 77789628, 77789632, 77789635, 77789640 of chromosome 17,

[0061] EGR3: 22548250, 22548260, 22548269, 22548279, 22548283, 22548287, 22548296, 22548299 of chromosome 8,

[0062] SOX17: 55379566, 55379568, 55379573, 55379579, 55379583, 55379591, 55379599, 55379602, 55379608, 55379617, 55379620 of chromosome 8,

[0063] PAX5: 36986087, 36986093, 36986098, 36986101, 36986103, 36986117, 36986131, 36986138, 36986141, 36986143, 36986147, 36986149, 36986156 of chromosome 9,

[0064] NEURL1 : one or more of positions 105344464, 105344482, 105344493, 105344495, 105344497, 105344503, 105344506, 105344513, 105344516, 105344519, 105344526 of chromosome 10,

[0065] IRX4 : one or more of positions 1876386, 1876395, 1876397, 1876403, 1876420, 1876424, 1876432, 1876436, 1876449, 1876456, 1876459, 1876463 of chromosome 5,

[0066] RUSC1 : one or more of positions 155295135, 155295171, 155295181, 155295192, 155295196, 155295212, 155295229, 155295236 of chromosome 1.

[0067] Preferably, the fragment of the ZMIZ1 gene comprises one or more of positions 81002041, 81002052, 81002054, 81002056, 81002062, 81002083 of the ZMIZ1 gene,

[0068] the fragment of the C15orf52 gene comprises one or more of positions 40626309, 40626312 of the C15orf52 gene,

[0069] the fragment of the SLC16A3 gene comprises one or more of positions 80189671, 80189674, 80189684, 80189687, 80189698, 80189709, 80189719, 80189726, 80189728, 80189739, 80189757 of the SLC16A3 gene,

[0070] the fragment of the ZNF512B gene comprises one or more of positions 62588634, 62588638, 62588672 of the ZNF512B gene,

[0071] the fragment of the SLC17A5 gene comprises one or more of positions 74290205, 74290207, 74290220, 74290225, 74290228 of the SLC17A5 gene,

[0072] a fragment of the LIMK1 gene comprises one or more of positions 73509112, 73509133, 73509138, 73509148, 73509160 of the LIMK1 gene,

[0073] a fragment of the PLEC gene comprises one or more of positions 145013661, 145013673 of the PLEC gene,

[0074] a fragment of the TOR4A gene comprises one or more of positions 140172787, 140172790, 140172812 of the TOR4A gene,

[0075] a fragment of the TMEM131L gene comprises one or more of positions 154409945, 154409963, 154409972, 154409978, 154409997 of the TMEM131L gene,

[0076] a fragment of the DNM2 gene comprises one or more of positions 10870427, 10870429, 10870441, 10870448 of the DNM2 gene,

[0077] a fragment of the IL17C gene comprises one or more of positions 88701004, 88701021, 88701029, 88701036, 88701043, 88701051, 88701060 of the IL17C gene,

[0078] a fragment of the PRDM16 gene comprises one or more of positions 3229950, 3229968, 3229973 of the PRDM16 gene,

[0079] a fragment of the MT1JP gene comprises one or more of positions 56669271, 56669292, 56669295, 56669300, 56669318, 56669322, 56669324, 56669327, 56669344 of the MT1JP gene,

[0080] a fragment of the TBX3 gene comprises one or more of positions 115174750, 115174773, 115174780 of the TBX3 gene,

[0081] a fragment of the BIN1 gene comprises one or more of positions 127822478, 127822492, 127822495, 127822514, 127822551, 127822568, 127822582, 127822593, 127822616 of the BIN1 gene,

[0082] the fragment of the TIMP2 gene comprises one or more of positions 76921845, 76921853, 76921860 of the TIMP2 gene,

[0083] the fragment of the CFAP65 gene comprises one or more of positions 219866199, 219866218 of the CFAP65 gene,

[0084] the fragment of the TSHR gene comprises one or more of positions 81421983, 81421989, 81422010, 81422017, 81422032, 81422035, 81422063, 81422084 of the TSHR gene,

[0085] the fragment of the KIF1A gene comprises one or more of positions 241759696, 241759701, 241759714, 241759716 of the KIF1A gene,

[0086] the fragment of the DAPK gene comprises one or more of positions 90112842, 90112853, 90112861, 90112866 of the DAPK gene,

[0087] the fragment of the CDH1 gene comprises one or more of positions 68771035, 68771037, 68771045, 68771051, 68771059, 68771064, 68771073 of the CDH1 gene,

[0088] the fragment of the TPO gene comprises one or more of positions 1481013, 1481015, 1481022, 1481039 of the TPO gene,

[0089] the fragment of the RARG gene comprises one or more of positions 53613176, 53613182, 53613190, 53613202, 53613210, 53613218 of the RARG gene,

[0090] the fragment of the PRR15 gene comprises one or more of positions 29606026, 29606040, 29606047, 29606056, 29606062, 29606073, 29606220, 29606222, 29606227, 29606231, 29606255, 29606257, 29606262, 29606271, 29606277, 29606289 of the PRR15 gene,

[0091] a fragment of the DPYS gene comprises one or more of positions 105478905, 105478908, 105478916, 105478918, 105478945, 105478956, 105478965, 105478974, 105478983 of the DPYS gene,

[0092] a fragment of the MCC gene comprises one or more of positions 112538999, 112539011, 112539018, 112539022, 112539061 of the MCC gene,

[0093] a fragment of the TBX15 gene comprises one or more of positions 119535740, 119535742, 119535750, 119535759, 119535766 of the TBX15 gene,

[0094] a fragment of the COL23A1 gene comprises one or more of positions 178003798, 178003803, 178003814, 178003823, 178003825, 178003834, 178003841, 178003844 of the COL23A1 gene,

[0095] a fragment of the ILDR2 gene comprises one or more of positions 166890516, 166890528, 166890535, 166890543, 166890555, 166890559, 166890568, 166890573, 166890584, 166890586 of the ILDR2 gene,

[0096] a fragment of the DHRS3 gene comprises one or more of positions 12656340, 12656355, 12656367 of the DHRS3 gene,

[0097] a fragment of the GDNF gene comprises one or more of positions 37834770, 37834772, 37834774, 37834777, 37834780, 37834784, 37834792, 37834799, 37834802, 37834806, 37834811 of the GDNF gene,

[0098] a fragment of the TBX18 gene comprises one or more of positions 85477035, 85477070, 85477083, 85477106 of the TBX18 gene,

[0099] The fragment of the SIM2 gene comprises one or more of the positions: 38069638, 38069650, 38069662, 38069664, 38069676, 38069681 of the SIM2 gene,

[0100] The fragment of the HOXA9 gene comprises one or more of the positions: 27204854, 27204858, 27204861, 27204863, 27204879 of the HOXA9 gene,

[0101] The fragment of the EHBP1L1 gene comprises one or more of the positions: 65352621, 65352635, 65352639, 65352642, 65352651, 65352654, 65352665, 65352670 of the EHBP1L1 gene,

[0102] The fragment of the GJC2 gene comprises one or more of the positions: 228345965, 228345978, 228345980, 228345989 of the GJC2 gene,

[0103] The fragment of the RCOR2 gene comprises one or more of the positions: 63687223, 63687238, 63687247, 63687250, 63687259 of the RCOR2 gene,

[0104] The fragment of the PRDM1 gene comprises one or more of the positions: 106429722, 106429731, 106429747, 106429750, 106429761, 106429769, 106429771 of the PRDM1 gene,

[0105] The fragment of the UNCX gene comprises one or more of the positions: 1263643, 1263655, 1263659, 1263664, 1263676 of the UNCX gene,

[0106] The fragment of the RPS7P5 gene comprises one or more of the positions: 240161511, 240161516, 240161523, 240161527, 240161530 of the RPS7P5 gene,

[0107] The fragment of the FOXI2 gene comprises one or more of the positions: 129534910, 129534912, 129534924 of the FOXI2 gene,

[0108] A fragment of the ACRBP gene comprises one or more of the following positions of the ACRBP gene: 6756182, 6756187, 6756191, 6756195, 6756211,

[0109] A fragment of the GAS6 gene comprises one or more of the following positions of the GAS6 gene: 114524062, 114524068, 114524084, 114524095, 114524131, 114524138,

[0110] A fragment of the MCRIP2 gene comprises one or more of the following positions of the MCRIP2 gene: 698072, 698142, 698153, 698168, 698208,

[0111] A fragment of the LINC01977 gene comprises one or more of the following positions of the LINC01977 gene: 77789596, 77789601, 77789612, 77789620,

[0112] A fragment of the EGR3 gene comprises one or more of the following positions of the EGR3 gene: 22548269, 22548279, 22548283, 22548287, 22548296, 22548299,

[0113] A fragment of the SOX17 gene comprises one or more of the following positions of the SOX17 gene: 55379602, 55379608, 55379617, 55379620,

[0114] A fragment of the PAX5 gene comprises one or more of the following positions of the PAX5 gene: 36986087, 36986093, 36986098, 36986101, 36986103,

[0115] A fragment of the NEURL1 gene comprises one or more of the following positions of the NEURL1 gene: 105344493, 105344495, 105344497,

[0116] A fragment of the IRX4 gene comprises one or more of the following positions of the IRX4 gene: 1876386, 1876395, 1876397, 1876403,

[0117] A fragment of the RUSC1 gene comprises one or more of the following positions of the RUSC1 gene: 155295192, 155295196, 155295212.

[0118] In any embodiment of the application, the numbering of the positions with respect to each gene corresponds to the base numbering of the chromosome on which the gene is located.

[0119] In a preferred embodiment of the second aspect, the reagent for detecting the level of DNA methylation detects the level of DNA methylation of a fragment of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 genes selected from the group consisting of SLC16A3, CDH1, TSHR, RARG, PRR15, MCC, TBX15, DPYS, COL23A1, ILDR2, NEURL1, BIN1, DNM2, IL17C. In one or more embodiments, the reagent for detecting the level of DNA methylation detects the level of DNA methylation of a fragment of 2 genes selected from the group consisting of SLC16A3 and CDH1, SLC16A3 and TSHR, SLC16A3 and RARG, SLC16A3 and PRR15, SLC16A3 and MCC, SLC16A3 and TBX15, SLC16A3 and DPYS, SLC16A3 and COL23A1, SLC16A3 and ILDR2, SLC16A3 and NEURL1, SLC16A3 and BIN1, SLC16A3 and DNM2, SLC16A3 and IL17C, CDH1 and TSHR, CDH1 and RARG, CDH1 and PRR15, CDH1 and MCC, CDH1 and TBX15, CDH1 and DPYS, CDH1 and COL23A1, CDH1 and ILDR2, CDH1 and NEURL1, CDH1 and BIN1, CDH1 and DNM2, CDH1 and IL17C, TSHR and RARG, TSHR and PRR15, TSHR and MCC, TSHR and TBX15, TSHR and DPYS, TSHR and COL23A1, TSHR and ILDR2, TSHR and NEURL1, TSHR and BIN1, TSHR and DNM2, TSHR and IL17C, RARG and PRR15, RARG and MCC, RARG and TBX15, RARG and DPYS, RARG and COL23A1, RARG and ILDR2, RARG and NEURL1, RARG and BIN1, RARG and DNM2, RARG and IL17C, PRR15 and MCC, PRR15 and TBX15, PRR15 and DPYS, PRR15 and COL23A1, PRR15 and ILDR2, PRR15 and NEURL1, PRR15 and BIN1, PRR15 and DNM2, PRR15 and IL17C, MCC and TBX15, MCC and DPYS, MCC and COL23A1, MCC and ILDR2, MCC and NEURL1, MCC and BIN1, MCC and DNM2, MCC and IL17C.TBX15 and DPYS, TBX15 and COL23A1, TBX15 and ILDR2, TBX15 and NEURL1, TBX15 and BIN1, TBX15 and DNM2, TBX15 and IL17C, DPYS and COL23A1, DPYS and ILDR2, DPYS and NEURL1, DPYS and BIN1, DPYS and DNM2, DPYS and IL17C, COL23A1 and ILDR2, COL23A1 and NEURL1, COL23A1 and BIN1, COL23A1 and DNM2, COL23A1 and IL17C, ILDR2 and NEURL1, ILDR2 and BIN1, ILDR2 and DNM2, ILDR2 and IL17C, NEURL1 and BIN1, NEURL1 and DNM2, NEURL1 and IL17C, BIN1 and DNM2, BIN1 and IL17C, or DNM2 and IL17C. In one or more embodiments, the reagent that detects the level of DNA methylation detects the level of DNA methylation of a fragment of three genes selected from the group consisting of SLC16A3 and CDH1 and TSHR, CDH1 and TSHR and RARG, TSHR and RARG and PRR15, RARG and PRR15 and MCC, PRR15 and MCC and TBX15, MCC and TBX15 and DPYS, TBX15 and DPYS and COL23A1, DPYS and COL23A1 and ILDR2, COL23A1 and ILDR2 and NEURL1, ILDR2 and NEURL1 and BIN1, NEURL1 and BIN1 and DNM2, or BIN1 and DNM2 and IL17C. In one or more embodiments, the reagent that detects the level of DNA methylation detects the level of DNA methylation of a fragment of four genes selected from the group consisting of SLC16A3 and CDH1 and TSHR and RARG, SLC16A3 and CDH1 and TSHR and PRR15, SLC16A3 and CDH1 and TSHR and MCC, SLC16A3 and CDH1 and TSHR and TBX15, SLC16A3 and CDH1 and TSHR and DPYS, SLC16A3 and CDH1 and TSHR and COL23A1, SLC16A3 and CDH1 and TSHR and ILDR2, SLC16A3 and CDH1 and TSHR and NEURL1, SLC16A3 and CDH1 and TSHR and BIN1, SLC16A3 and CDH1 and TSHR and DNM2, or SLC16A3 and CDH1 and TSHR and IL17C. In one or more embodiments,The reagent for detecting the level of DNA methylation detects the level of DNA methylation of a fragment of five genes selected from SLC16A3 and CDH1 and TSHR and RARG and PRR15, SLC16A3 and CDH1 and TSHR and PRR15 and MCC, SLC16A3 and CDH1 and TSHR and MCC and TBX15, SLC16A3 and CDH1 and TSHR and TBX15 and DPYS, SLC16A3 and CDH1 and TSHR and DPYS and COL23A1, SLC16A3 and CDH1 and TSHR and COL23A1 and ILDR2, SLC16A3 and CDH1 and TSHR and ILDR2 and NEURL1, SLC16A3 and CDH1 and TSHR and NEURL1 and BIN1, SLC16A3 and CDH1 and TSHR and BIN1 and DNM2, or SLC16A3 and CDH1 and TSHR and DNM2 and IL17C. In one or more embodiments, the reagent for detecting the level of DNA methylation detects the level of DNA methylation of a fragment of six genes selected from SLC16A3 and CDH1 and TSHR and RARG and PRR15 and MCC, SLC16A3 and CDH1 and TSHR and PRR15 and MCC and TBX15, SLC16A3 and CDH1 and TSHR and MCC and TBX15 and DPYS, SLC16A3 and CDH1 and TSHR and TBX15 and DPYS and COL23A1, SLC16A3 and CDH1 and TSHR and DPYS and COL23A1 and ILDR2, SLC16A3 and CDH1 and TSHR and COL23A1 and ILDR2 and NEURL1, SLC16A3 and CDH1 and TSHR and ILDR2 and NEURL1 and BIN1, SLC16A3 and CDH1 and TSHR and NEURL1 and BIN1 and DNM2, or SLC16A3 and CDH1 and TSHR and BIN1 and DNM2 and IL17C. In one or more embodiments,The reagents for detecting DNA methylation levels detected the DNA methylation levels of the following seven gene fragments: SLC16A3 and CDH1 and TSHR and RARG and PRR15 and MCC and TBX15, CDH1 and TSHR and RARG and PRR15 and MCC and TBX15 and DPYS, TSHR and RARG and PRR15 and MCC and TBX15 and DPYS and COL23A1, RARG and PRR15 and MCC and TBX15 and DPYS and COL 23A1 and ILDR2, PRR15 and MCC and TBX15 and DPYS and COL23A1 and ILDR2 and NEURL1, MCC and TBX15 and DPYS and COL23A1 and ILDR2 and NEURL1 and BIN1, TBX15 and DPYS and COL23A1 and ILDR2 and NEURL1 and BIN1 and DNM2, DPYS and COL23A1 and ILDR2 and NEURL1 and BIN1 and DNM2 and IL17C. In one or more embodiments, the reagent for detecting DNA methylation levels detects DNA methylation levels of fragments of 8, 9, 10, 11, 12, 13, 14, or all 15 genes selected from the group consisting of SLC16A3, CDH1, TSHR, RARG, PRR15, MCC, TBX15, DPYS, COL23A1, ILDR2, NEURL1, BIN1, DNM2, and IL17C.

[0120] In one or more embodiments, the reagent detects the level of DNA methylation at a site of one or more of the following (al)-(a8): (al) a site of the PRDMl gene: one or more or all of 106429711, 106429722, 106429731, 106429747, 106429750, 106429761, 106429769, 106429771, (a2) a site of the ACRBP gene: one or more or all of 6756182, 6756187, 6756191, 6756195, 6756211, 6756225, 6756230, 6756270, (a3) a site of the TPO gene: one or more or all of 1481013, 1481015, 1481022, 1481039, (a4) a site of the MCC gene: one or more or all of 112538999, 112539011, 112539018, 112539022, 112539061, 112539084, 112539104, 112539128, (a5) a site of the TBX15 gene: one or more or all of 119535725, 119535730, 119535740, 119535742, 119535750, 119535759, 119535766, 119535812, 119535817, 119535821, 119535823, 119535876, 119535879, 119535884, 119535891, (a6) a site of the UNCX gene: one or more or all of 1263643, 1263655, 1263659, 1263664, 1263676, 1263694, 1263716, 1263723, (a7) a site of the RPS7P5 gene: one or more or all of 240161502, 240161507, 240161511, 240161516, 240161523, 240161527, 240161530, 240161535, 240161546, 240161558, 240161560, (a8) a site of the FOXI2 gene: one or more or all of 129534843, 129534853, 129534866, 129534879, 129534891, 129534910, 129534912, 129534924, or (2) detects the level of DNA methylation of a nucleic acid fragment of one or more genes selected from the group consisting of: PRDMl, ACRBP, TPO, MCC, TBX15, UNCX, RPS7P5, FOXI2, the fragment being 50-1000 bp in length,wherein the fragment of PRDM1 gene comprises one or more or all of the positions: 106429711, 106429722, 106429731, 106429747, 106429750, 106429761, 106429769, 106429771 of PRDM1 gene, the fragment of ACRBP gene comprises one or more or all of the positions: 6756182, 6756187, 6756191, 6756195, 6756211, 6756225, 6756230, 6756270 of ACRBP gene, the fragment of TPO gene comprises one or more or all of the positions: 1481013, 1481015, 1481022, 1481039 of TPO gene, the fragment of MCC gene comprises one or more or all of the positions: 112538999, 112539011, 112539018, 112539022, 112539061, 112539084, 112539104, 112539128 of MCC gene, the fragment of TBX15 gene comprises one or more or all of the positions: 119535725, 119535730, 119535740, 119535742, 119535750, 119535759, 119535766, 119535812, 119535817, 119535821, 119535823, 119535876, 119535879, 119535884, 119535891 of TBX15 gene, the fragment of UNCX gene comprises one or more or all of the positions: 1263643, 1263655, 1263659, 1263664, 1263676, 1263694, 1263716, 1263723 of UNCX gene, the fragment of RPS7P5 gene comprises one or more or all of the positions: 240161502, 240161507, 240161511, 240161516, 240161523, 240161527, 240161530, 240161535, 240161546, 240161558, 240161560 of RPS7P5 gene, the fragment of FOXI2 gene comprises one or more or all of the positions: 129534843, 129534853, 129534866, 129534879, 129534891, 129534910, 129534912, 129534924 of FOXI2 gene, or (3) detecting the DNA methylation level of a nucleic acid region within 10 Kb up- or downstream of the gene mentioned in (2). The positions are referred to human reference genome version hg19.

[0121] In one or more embodiments, (a1)-(a8) are: one or more or all of positions: 106429722, 106429731, 106429747, 106429750, 106429761, 106429769, 106429771 of the PRDM1 gene, one or more or all of positions: 6756182, 6756187, 6756191, 6756195, 6756211, 6756225, 6756230 of the ACRBP gene, one or more or all of positions: 1481013, 1481015, 1481022, 1481039 of the TPO gene, one or more or all of positions: 112538999, 112539011, 112539018, 112539022, 112539061 of the MCC gene, one or more or all of positions: 119535740, 119535742, 119535750, 119535759, 119535766 of the TBX15 gene, one or more or all of positions: 1263643, 1263655, 1263659, 1263664, 1263676 of the UNCX gene, one or more or all of positions: 240161502, 240161507, 240161511, 240161516, 240161523, 240161527, 240161530, 240161535, 240161546 of the RPS7P5 gene, one or more or all of positions: 129534843, 129534853, 129534866, 129534879, 129534891, 129534910, 129534912, 129534924 of the FOXI2 gene.

[0122] In one or more embodiments, (a1)-(a8) are: one or more or all of the sites: 106429722, 106429731, 106429747, 106429750, 106429761, 106429769, 106429771 of the PRDM1 gene, one or more or all of the sites: 6756182, 6756187, 6756191, 6756195, 6756211, 6756225, 6756230 of the ACRBP gene, one or more or all of the sites: 1481013, 1481015, 1481022, 1481039 of the TPO gene, one or more or all of the sites: 112538999, 112539011, 112539018, 112539022, 112539061 of the MCC gene, one or more or all of the sites: 119535740, 119535742, 119535750, 119535759, 119535766 of the TBX15 gene, one or more or all of the sites: 1263659, 1263664, 1263676 of the UNCX gene, one or more or all of the sites: 240161502, 240161507, 240161511, 240161516, 240161523, 240161527, 240161530, 240161535, 240161546 of the RPS7P5 gene, one or more or all of the sites: 129534843, 129534853, 129534866, 129534879, 129534891, 129534910, 129534912, 129534924 of the FOXI2 gene.

[0123] In one or more embodiments, the reagent that detects the level of DNA methylation detects the level of DNA methylation at one or more of the following sets of sites: (a1)-(a2), and optionally one or more of (a3)-(a8); preferably, the sites comprise one or more of (a1)-(a2) and optionally (a3)-(a5), or, one or more of (a1)-(a2) and optionally (a6)-(a8); more preferably, the sites comprise one or more of (a1)-(a2) and optionally (a3)-(a5) or optionally (a6)-(a8). In one or more embodiments, the reagent that detects the level of DNA methylation detects the level of DNA methylation at (a1)-(a5), or, (a1)-(a2) and (a6)-(a8).

[0124] In one or more embodiments, the agent is a primer capable of amplifying one or more fragments selected from the group consisting of: (bl) a fragment of the PRDMl gene amplified with SEQ ID NOs: 4 and 5 as primers, (b2) a fragment of the ACRBP gene amplified with SEQ ID NOs: 6 and 7 as primers, (b3) a fragment of the TPO gene amplified with SEQ ID NOs: 8 and 9 as primers, (b4) a fragment of the MCC gene amplified with SEQ ID NOs: 10 and 11 as primers, (b5) a fragment of the TBX15 gene amplified with SEQ ID NOs: 12 and 13 as primers, (b6) a fragment of the UNCX gene amplified with SEQ ID NOs: 14 and 15 as primers, (b7) a fragment of the RPS7P5 gene amplified with SEQ ID NOs: 16 and 17 as primers, and (b8) a fragment of the FOXI2 gene amplified with SEQ ID NOs: 18 and 19 as primers. Preferably, the primer is capable of amplifying: one or more of (bl)-(b2), and optionally one or more of (b3)-(b8); more preferably, the primer is capable of amplifying: (bl)-(b2) and optionally one or more of (b3)-(b5), or, (bl)-(b2) and optionally one or more of (b6)-(b8); more preferably, the primer is capable of amplifying: (bl)-(b2) and optionally one or more of (b3)-(b5) or (b6)-(b8). In one or more embodiments, the primer is any one of SEQ ID NOs: 4-19 or a sequence having 90% identity thereto. Preferably, the primer is selected from the group consisting of (1) one or more or all of SEQ ID NOs: 4-7, (2) one or more or all of SEQ ID NOs: 4-13, (3) one or more or all of SEQ ID NOs: 4-7, 14-19, or (4) a sequence having 90% identity to any of (1)-(3).

[0125] In one or more embodiments, the agent is a probe that is capable of hybridizing to one or more fragments selected from the group consisting of: (bl) a fragment of the PRDMl gene amplified with SEQ ID NOs: 4 and 5 as primers, (b2) a fragment of the ACRBP gene amplified with SEQ ID NOs: 6 and 7 as primers, (b3) a fragment of the TPO gene amplified with SEQ ID NOs: 8 and 9 as primers, (b4) a fragment of the MCC gene amplified with SEQ ID NOs: 10 and 11 as primers, (b5) a fragment of the TBX15 gene amplified with SEQ ID NOs: 12 and 13 as primers, (b6) a fragment of the UNCX gene amplified with SEQ ID NOs: 14 and 15 as primers, (b7) a fragment of the RPS7P5 gene amplified with SEQ ID NOs: 16 and 17 as primers, and (b8) a fragment of the FOXI2 gene amplified with SEQ ID NOs: 18 and 19 as primers. Preferably, the probe is capable of hybridizing to one or more fragments selected from the group consisting of: (bl)-(b2), and optionally one or more of (b3)-(b8); more preferably, the probe is capable of hybridizing to one or more fragments selected from the group consisting of: (bl)-(b2) and optionally one or more of (b3)-(b5), or (bl)-(b2) and optionally one or more of (b6)-(b8). More preferably, the probe is capable of hybridizing to one or more fragments selected from the group consisting of: (bl)-(b2) and optionally one or more of (b3)-(b5) or (b6)-(b8). More preferably, the probe is any one of SEQ ID NOs: 20-27 or a sequence having 90% identity thereto. Preferably, the probe is selected from the group consisting of (1) one or more or all of SEQ ID NOs: 20-21, 25-27, (2) one or more or all of SEQ ID NOs: 20-24, (3) one or more or all of SEQ ID NOs: 20-21, 25-27, or (4) a sequence having 90% identity to any of (1)-(3).

[0126] In one or more embodiments, the fragment is 30-2000 bp, 30-1500 bp, 50-1000 bp, 50-800 bp, 50-500 bp, 50-400 bp, 50-350 bp, 50-300 bp, 50-250 bp, 50-200 bp, 60-180 bp, 60-170 bp, 60-160 bp, 60-150 bp, 60-140 bp, 60-130 bp, 60-120 bp, 70-110 bp, or 80-100 bp in length, preferably 50-350 bp or 60-180 bp.

[0127] In one or more embodiments of each of the above aspects, the mammal is a human.

[0128] In one or more embodiments of each of the above aspects, the gene or locus comprises a DNA sense or antisense strand.

[0129] In one or more embodiments of each of the above aspects, the locus is referenced to the human reference genome hg19 version.

[0130] In one or more embodiments of each of the above aspects, the reagent for detecting DNA methylation is selected from one or more of the following methods: bisulfite conversion based PCR (e.g. methylation specific PCR), DNA sequencing (e.g. bisulfite sequencing, whole genome methylation sequencing, reduced representation methylation sequencing), methylation sensitive restriction enzyme analysis, fluorescence quantification, methylation sensitive high resolution melting curve, chip based methylation profiling, mass spectrometry (e.g. flight mass spectrometry). Preferably, the reagent is selected from one or more of the following: bisulfite and derivatives thereof, PCR buffer, polymerase, dNTP, primer, probe, methylation sensitive or insensitive restriction enzyme, enzyme digestion buffer, fluorescent dye, fluorescence quencher, fluorescence reporter, exonuclease, alkaline phosphatase, internal standard, control.

[0131] Preferably, the reagent comprises a primer. The sequence of the primer is methylation specific or non-specific. Preferably, the sequence of the primer comprises a non-methylation specific blocker. Preferably, the primer is any one of SEQ ID NO: 4-19 or a sequence having 90% identity thereto.

[0132] Preferably, the reagent comprises a probe. The sequence of the probe is labeled with a fluorescence reporter group at the 5’ end and a quencher group at the 3’ end. Preferably, the sequence of the probe comprises a MGB (Minor groove binder) or LNA (Locked nucleic acid). Preferably, the probe is any one of SEQ ID NO: 20-27 or a sequence having 90% identity thereto.

[0133] In one or more embodiments, the kit further comprises reagents for detecting a mutation in the V600E locus of the BRAF gene and / or a mutation in the C228T / C250T locus of the TERT gene.

[0134] In one or more embodiments, the kit further comprises reagents for detecting a mutation in the V600E locus of the BRAF gene and / or a mutation in the C228T / C250T locus of the TERT gene.

[0135] In one or more embodiments, the kit further comprises reagents for detecting a mutation in the V600E locus of the BRAF gene and / or a mutation in the C228T / C250T locus of the TERT gene.

[0136] In another aspect, the present application provides a use of a reagent for detecting DNA methylation and optionally a nucleic acid molecule described herein in the manufacture of a kit for identifying the nature of a thyroid nodule, the reagent detecting the level of DNA methylation in a sample in a region selected from (1) and (2) below: (1) a fragment of one or more genes selected from the group consisting of ZMIZ1, C15orf52, SLC16A3, ZNF512B, SLC17A5, LIMK1, PLEC, TOR4A, TMEM131L, DNM2, IL17C, PRDM16, MT1JP, TBX3, BIN1, TIMP2, CFAP65, TSHR, KIF1A, DAPK, CDH1, TPO, RARG, PRR15, DPYS, MCC, TBX15, COL23A1, ILDR2, DHRS3, GDNF, TBX18, SIM2, HOXA9, EHBP1L1, GJC2, RCOR2, PRDM1, UNCX, RPS7P5, FOXI2, ACRBP, GAS6, MCRIP2, LINC01977, EGR3, SOX17, PAX5, NEURL1, IRX4, RUSC1, (2) a nucleic acid region within 5Kb or 10Kb upstream or downstream of the genes of (1). Preferably, the reagent detects the methylation level of one or more genes selected from the group consisting of PRDM1, ACRBP, TPO, MCC, TBX15, UNCX, RPS7P5, FOXI2.

[0137] In one or more embodiments, the reagent detects the methylation level of a fragment of one or more groups of genes selected from (1)-(4) below in a sample: (1) PRDM1, ACRBP, (2) PRDM1, ACRBP and one or two selected from TPO, MCC and TBX15, (3) PRDM1, ACRBP and one or two selected from UNCX, RPS7P5 and FOXI2, (4) a nucleic acid region within 5Kb or 10Kb upstream or downstream of the genes of any one of (1)-(3).

[0138] In one or more embodiments, the detection site of each gene is selected from one or more of the following sites or a nucleic acid region within 500bp upstream or downstream thereof:

[0139] PRDM1: 106429711, 106429722, 106429731, 106429747, 106429750, 106429761, 106429769, 106429771 of chromosome 6,

[0140] TPO: 1481013, 1481015, 1481022, 1481039 of chromosome 2,

[0141] MCC: 112538999, 112539011, 112539018, 112539022, 112539061, 112539084, 112539104, 112539128 of chromosome 5,

[0142] TBX15: 119535725, 119535730, 119535740, 119535742, 119535750, 119535759, 119535766, 119535812, 119535817, 119535821, 119535823, 119535876, 119535879, 119535884, 119535891 of chromosome 1,

[0143] UNCX: 1263643, 1263655, 1263659, 1263664, 1263676, 1263694, 1263716, 1263723 of chromosome 7,

[0144] RPS7P5: 240161502, 240161507, 240161511, 240161516, 240161523, 240161527, 240161530, 240161535, 240161546, 240161558, 240161560 of chromosome 1,

[0145] FOXI2: 129534843, 129534853, 129534866, 129534879, 129534891, 129534910, 129534912, 129534924 of chromosome 10,

[0146] ACRBP: 6756182, 6756187, 6756191, 6756195, 6756211, 6756225, 6756230, 6756270 of chromosome 12.

[0147] In one or more embodiments, the agent that detects DNA methylation detects the methylation level of one or more or all of the following (a1)-(a8): (a1) one or more or all of the following sites of the PRDM1 gene: 106429711, 106429722, 106429731, 106429747, 106429750, 106429761, 106429769, 106429771, (a2) one or more or all of the following sites of the ACRBP gene: 6756182, 6756187, 6756191, 6756195, 6756211, 6756225, 6756230, 6756270, (a3) one or more or all of the following sites of the TPO gene: 1481013, 1481015, 1481022, 1481039, (a4) one or more or all of the following sites of the MCC gene: 112538999, 112539011, 112539018, 112539022, 112539061, 112539084, 112539104, 112539128, (a5) one or more or all of the following sites of the TBX15 gene: 119535725, 119535730, 119535740, 119535742, 119535750, 119535759, 119535766, 119535812, 119535817, 119535821, 119535823, 119535876, 119535879, 119535884, 119535891, (a6) one or more or all of the following sites of the UNCX gene: 1263643, 1263655, 1263659, 1263664, 1263676, 1263694, 1263716, 1263723, (a7) one or more or all of the following sites of the RPS7P5 gene: 240161502, 240161507, 240161511, 240161516, 240161523, 240161527, 240161530, 240161535, 240161546, 240161558, 240161560, (a8) one or more or all of the following sites of the FOXI2 gene: 129534843, 129534853, 129534866, 129534879, 129534891, 129534910, 129534912, 129534924. The agent that detects DNA methylation is as described in other embodiments herein.

[0148] In one or more embodiments, (a1)-(a8) are: one or more or all of positions: 106429722, 106429731, 106429747, 106429750, 106429761, 106429769, 106429771 of the PRDM1 gene, one or more or all of positions: 6756182, 6756187, 6756191, 6756195, 6756211, 6756225, 6756230 of the ACRBP gene, one or more or all of positions: 1481013, 1481015, 1481022, 1481039 of the TPO gene, one or more or all of positions: 112538999, 112539011, 112539018, 112539022, 112539061 of the MCC gene, one or more or all of positions: 119535740, 119535742, 119535750, 119535759, 119535766 of the TBX15 gene, one or more or all of positions: 1263643, 1263655, 1263659, 1263664, 1263676 of the UNCX gene, one or more or all of positions: 240161502, 240161507, 240161511, 240161516, 240161523, 240161527, 240161530, 240161535, 240161546 of the RPS7P5 gene, one or more or all of positions: 129534843, 129534853, 129534866, 129534879, 129534891, 129534910, 129534912, 129534924 of the FOXI2 gene.

[0149] In one or more embodiments, (a1)-(a8) are: one or more or all of the sites: 106429722, 106429731, 106429747, 106429750, 106429761, 106429769, 106429771 of the PRDM1 gene, one or more or all of the sites: 6756182, 6756187, 6756191, 6756195, 6756211, 6756225, 6756230 of the ACRBP gene, one or more or all of the sites: 1481013, 1481015, 1481022, 1481039 of the TPO gene, one or more or all of the sites: 112538999, 112539011, 112539018, 112539022, 112539061 of the MCC gene, one or more or all of the sites: 119535740, 119535742, 119535750, 119535759, 119535766 of the TBX15 gene, one or more or all of the sites: 1263659, 1263664, 1263676 of the UNCX gene, one or more or all of the sites: 240161502, 240161507, 240161511, 240161516, 240161523, 240161527, 240161530, 240161535, 240161546 of the RPS7P5 gene, one or more or all of the sites: 129534843, 129534853, 129534866, 129534879, 129534891, 129534910, 129534912, 129534924 of the FOXI2 gene.

[0150] In one or more embodiments, the reagent for detecting the level of DNA methylation detects the level of DNA methylation at one or more groups of (a1)-(a2), and optionally one or more groups of (a3)-(a8); preferably, the sites comprise one or more groups of (a1)-(a2) and optionally (a3)-(a5), or one or more groups of (a1)-(a2) and optionally (a6)-(a8); more preferably, the sites comprise (a1)-(a2) and optionally (a3)-(a5) or optionally (a6)-(a8).

[0151] In one or more embodiments, the kit further comprises a reagent for detecting the level of mutation at the V600E site of the BRAF gene.

[0152] In one or more embodiments of the use, the kit further comprises reagents for detecting the level of mutation of the C228T / C250T site of the TERT gene.

[0153] In one or more embodiments of the use, the gene or site comprises the DNA sense or antisense strand.

[0154] In one or more embodiments of the use, the site is referenced to the human reference genome version hg19.

[0155] In one or more embodiments of the use, the kit further comprises reagents for detecting the level of mutation of the V600E site of the BRAF gene and / or the level of mutation of the C228T / C250T site of the TERT gene.

[0156] In one or more embodiments of the use, identifying the nature of the thyroid nodule comprises obtaining a score from the level of methylation and / or the level of mutation, comparing to a control sample, or identifying the nature of the thyroid nodule from the comparison result or the score.

[0157] In one or more embodiments of the use, the sample is from a human, preferably from a tissue, a cell or a body fluid, such as thyroid tissue or blood. In one or more embodiments of the use, the sample contains genomic DNA or cfDNA.

[0158] In one or more embodiments of the use, the reagents for detecting DNA methylation are as described in the second aspect of the application.

[0159] In one or more embodiments of the use, the reagents for detecting DNA methylation are selected from the reagents used in one or more of the following methods: bisulfite conversion based PCR (e.g. methylation specific PCR), DNA sequencing (e.g. bisulfite sequencing, whole genome methylation sequencing, reduced representation methylation sequencing), methylation sensitive restriction enzyme analysis, fluorescence quantification, methylation sensitive high resolution melting curve, chip based methylation profiling, mass spectrometry (e.g. flight mass spectrometry). Preferably, the reagents are selected from one or more of the following: bisulfite and its derivatives, PCR buffer, polymerase, dNTP, primer, probe, methylation sensitive or insensitive restriction enzyme, enzyme digestion buffer, fluorescent dye, fluorescence quencher, fluorescence reporter, exonuclease, alkaline phosphatase, internal standard, control.

[0160] Preferably, the primer sequence is methylation specific or non-specific. Preferably, the sequence of the primer comprises a non-methylation specific blocker. Preferably, the primer is any of SEQ ID NO: 4-19 or a sequence having 90% identity thereto.

[0161] Preferably, the sequence of the probe is labeled with a fluorescent reporter group at the 5' end and a quencher group at the 3' end. Preferably, the sequence of the probe comprises MGB (Minor groove binder) or LNA (Locked nucleic acid). Preferably, the probe is any one of SEQ ID NO: 20-27 or a sequence having 90% identity thereto.

[0162] The present application also provides a primer which detects the methylation level of DNA in a region selected from (1) and (2) below: (1) a fragment of one or more genes selected from ZMIZ1, C15orf52, SLC16A3, ZNF512B, SLC17A5, LIMK1, PLEC, TOR4A, TMEM131L, DNM2, IL17C, PRDM16, MT1JP, TBX3, BIN1, TIMP2, CFAP65, TSHR, KIF1A, DAPK, CDH1, TPO, RARG, PRR15, DPYS, MCC, TBX15, COL23A1, ILDR2, DHRS3, GDNF, TBX18, SIM2, HOXA9, EHBP1L1, GJC2, RCOR2, PRDM1, UNCX, RPS7P5, FOXI2, ACRBP, GAS6, MCRIP2, LINC01977, EGR3, SOX17, PAX5, NEURL1, IRX4, RUSC1, (2) a nucleic acid region within 5Kb or 10Kb upstream or downstream of the genes of (1). Preferably, the primer detects the methylation level of the site described herein.

[0163] The present application also provides a probe for detecting the methylation level of DNA in a region selected from (1) and (2) below: (1) a fragment of one or more genes selected from the group consisting of ZMIZ1, C15orf52, SLC16A3, ZNF512B, SLC17A5, LIMK1, PLEC, TOR4A, TMEM131L, DNM2, IL17C, PRDM16, MT1JP, TBX3, BIN1, TIMP2, CFAP65, TSHR, KIF1A, DAPK, CDH1, TPO, RARG, PRR15, DPYS, MCC, TBX15, COL23A1, ILDR2, DHRS3, GDNF, TBX18, SIM2, HOXA9, EHBP1L1, GJC2, RCOR2, PRDM1, UNCX, RPS7P5, FOXI2, ACRBP, GAS6, MCRIP2, LINC01977, EGR3, SOX17, PAX5, NEURL1, IRX4, RUSC1, (2) a nucleic acid region within 5Kb or 10Kb upstream or downstream of the genes in (1). Preferably, the probe detects the methylation level of a site described herein.

[0164] The present application also provides a method for screening the benign or malignant nature of a thyroid nodule, comprising: (1) detecting the methylation level of a gene, site or nucleic acid region described herein in a sample from a subject; optionally (2) detecting the mutation level of the V600E site of the BRAF gene and / or the mutation level of the C228T / C250T site of the TERT gene; (3) comparing with a control sample, or obtaining a score based on the methylation level and / or mutation level, for example by calculation; (4) identifying the nature of the thyroid nodule based on the comparison result or score of step (3).

[0165] The present application also provides a method for screening the benign or malignant nature of a thyroid nodule, comprising: (1) detecting the mutation level of the V600E site of the BRAF gene and / or the mutation level of the C228T / C250T site of the TERT gene; optionally (2) detecting the methylation level of a gene, site or nucleic acid region described herein in a sample from a subject; (3) comparing with a control sample, or obtaining a score based on the mutation level and / or methylation level, for example by calculation; (4) identifying the nature of the thyroid nodule based on the comparison result or score of step (3).

[0166] In one or more embodiments, the method further comprises, before step (1): extraction, quality control, and / or conversion of unmethylated cytosine on the DNA into a base that does not bind to guanine, of the DNA of the sample.

[0167] In one or more embodiments, the conversion is performed using an enzymatic method, preferably a deaminase treatment, or the conversion is performed using a non-enzymatic method, preferably a bisulfite or a pyrosulfate treatment, more preferably a calcium bisulfite, sodium bisulfite, potassium bisulfite, ammonium bisulfite, sodium pyrosulfate, potassium pyrosulfate and ammonium pyrosulfate treatment.

[0168] In one or more embodiments, the detection includes, but is not limited to, PCR based on bisulfite conversion (e.g. methylation specific PCR), DNA sequencing (e.g. bisulfite sequencing, whole genome methylation sequencing, reduced representation methylation sequencing), methylation sensitive restriction enzyme assay, fluorescence quantification, methylation sensitive high resolution melting curve, chip based methylation profiling, mass spectrometry (e.g. flight mass spectrometry).

[0169] In one or more embodiments, step (4) comprises comparing the methylation level and / or mutation level of the subject sample with a control sample, and identifying the thyroid nodule as a benign or malignant nodule when the methylation level and / or mutation level meets a threshold.

[0170] In one or more embodiments, step (4) comprises identifying the thyroid nodule as a benign or malignant nodule when the score meets a threshold.

[0171] In one or more embodiments, the sample is from a human, preferably from a tissue, a cell or a body fluid, e.g. thyroid tissue or blood. In one or more embodiments, the sample is a thyroid nodule biopsy, preferably a fine needle aspiration biopsy. In one or more embodiments, the sample is plasma.

[0172] In one or more embodiments, the sample is from a subject having a benign or malignant thyroid nodule. In one or more embodiments, the sample is from a patient with thyroid goiter.

[0173] In one or more embodiments, the sample comprises genomic DNA or cfDNA.

[0174] The present application also provides a kit for identifying the nature of a thyroid nodule, comprising primers and / or probes for detecting the methylation level of the genes, sites, nucleic acid regions described herein. BRIEF DESCRIPTION OF DRAWINGS

[0175] Figure 1 LabChip is used to detect the distribution of single library fragments in the present application.

[0176] Figure 2A -C is the ROC curve analysis of the present application for detecting 10 cases of thyroid cancer and 10 cases of benign thyroid nodules. A: tissue samples; B, C: plasma samples.

[0177] Figure 3 1 is an ROC curve analysis of 20 plasma samples of thyroid cancer and 20 plasma samples of benign thyroid nodules according to one embodiment of the present invention.

[0178] Figure 4 1 is an ROC curve analysis of 20 plasma samples of thyroid cancer and 20 plasma samples of benign thyroid nodules according to one embodiment of the present invention. DETAILED DESCRIPTION

[0179] The inventors discovered that specific chromosomes, genes or methylation sites are associated with malignant thyroid nodules.

[0180] When referring to thyroid nodules, the terms "benign" and "malignant" as used herein refer to the nature of the thyroid nodule. Generally, benign nodules are characterized by slow growth, uniform texture, good mobility, a smooth surface, cystic changes, absence of lymphadenopathy, and absence of calcification. Malignant nodules are characterized by uncontrolled growth, spread, and tissue infiltration of malignant cells. Ultrasound signs suggesting malignancy include: nodules that are taller than they are wide, lack of an acoustic halo, microcalcifications, irregular borders, hypoechoic properties, solid nodules, and abundant internal blood flow. In some embodiments, malignant thyroid nodules include thyroid cancer.

[0181] The inventors found that the nature of thyroid nodules is associated with the methylation level of fragments of one or more genes selected from the group consisting of ZMIZ1, C15orf52, SLC16A3, ZNF512B, SLC17A5, LIMK1, PLEC, TOR4A, TMEM131L, DNM2, IL17C, PRDM16, MT1JP, TBX3, BIN1, TIMP2, CFAP65, TSHR, KIF1A, DAPK, CDH1, TPO, RARG, PRR15, DPYS, MCC, TBX15, COL23A1, ILDR2, DHRS3, GDNF, TBX18, SIM2, HOXA9, EHBP1L1, GJC2, RCOR2, PRDM1, UNCX, RPS7P5, FOXI2, ACRBP, GAS6, MCRIP2, LINC01977, EGR3, SOX17, PAX5, NEURL1, IRX4, RUSC1. Preferably, the genes are selected from the group consisting of (1) LIMK1 and SLC17A5, (2) BIN1 and DNM2, (3) BIN1 and SLC16A3, (4) SLC16A3, DNM2 and IL17C, (5) SLC16A3, DNM2, IL17C, CDH1 and TSHR, (6) PRDM1 and ACRBP, (3) PRDM1, ACRBP, UNCX, RPS7P5 and FOXI2, (7) PRDM1, ACRBP, TPO, MCC and TBX15, (8) PRDM1, ACRBP, UNCX, RPS7P5 and FOXI2.

[0182] The inventors also found that the nature of thyroid nodules is associated with the methylation level of one or more loci selected from the group consisting of:

[0183] ZMIZ1: 81001968, 81001996, 81002041, 81002052, 81002054, 81002056, 81002062, 81002083, 81002110, 81002116, 81002123, 81002129, 81002133, 81002137, 81002139, 81002164, 81002168, 81002223, 81002241, 81002253, of chromosome 10,

[0184] C15orf52: 40626309, 40626312, 40626386 of chromosome 15, SLC16A3: 80189165, 80189174, 80189177, 80189197, 80189225, 80189230, 80189239, 80189645, 80189671, 80189674, 80189684, 80189687, 80189698, 80189709, 80189719, 80189726, 80189728, 80189739, 80189757, 80189787, 80189792, 80189811, 80189817, 80189832, 80189841, of chromosome 17,

[0185] ZNF512B: 62588634, 62588638, 62588672 of chromosome 20,

[0186] SLC17A5: 74290205, 74290207, 74290220, 74290225, 74290228 of chromosome 6,

[0187] LIMK1: 73508994, 73509017, 73509055, 73509062, 73509073, 73509075, 73509112, 73509133, 73509138, 73509148, 73509160 of chromosome 7,

[0188] PLEC: 145013661, 145013673 of chromosome 8,

[0189] TOR4A: 140172787, 140172790, 140172812 of chromosome 9,

[0190] TMEM131L: 154409945, 154409963, 154409972, 154409978, 154409997, 154410003, 154410006 of chromosome 4,

[0191] DNM2: 10870373, 10870377, 10870427, 10870429, 10870441, 10870448 of chromosome 19,

[0192] IL17C: 88700818, 88700826, 88700844, 88700849, 88700857, 88700869, 88700875, 88700891, 88700897, 88700916, 88700920, 88700937, 887 00943、88700948、88700967、88700970、88700993、88701004、88701021、88701029、88701036、88701043、88701051、88701060、88701074、88701081、88 701090、88701099、88701111、88701115、88701133、88701140、88701148、88701159、88701161、88701176、88701178、88701180、88701183、88701190、8 8701201、88701204、88701210、88701212、88701236、88701240、88701266、88701278、88701281、88701285、88701305、88701421、88701442、88701451,

[0193] PRDM16: 3229914, 3229921, 3229950, 3229968, 3229973, 3310213, 3310229, 3310235, 3310238, 3310240, 3310268, 3310287, 3310312, 3310314, 3310317, 3310329 on chromosome 1,

[0194] TSHR: 81421983, 81421989, 81422010, 81422017, 81422032, 81422035, 81422063, 81422084 on chromosome 14,

[0195] KIF1A: 241759696, 241759701, 241759714, 241759716 on chromosome 2,

[0196] DAPK: 90112842, 90112853, 90112861, 90112866 on chromosome 9,

[0197] CDH1 : 68771035, 68771037, 68771045, 68771051, 68771059, 68771064, 68771073 of chromosome 16,

[0198] TPO: 1481013, 1481015, 1481022, 1481039 of chromosome 2,

[0199] RARG: 53613176, 53613182, 53613190, 53613202, 53613210, 53613218 of chromosome 12,

[0200] MT1JP: 56669271, 56669292, 56669295, 56669300, 56669318, 56669322, 56669324, 56669327, 56669344, 56669351, 56669353, 56669402, 56669414, 56669423, 56669430, 56669433, 56669437, 56669451, 56669453, 56669455, 56669463, 56669474, 56669480, 56669482, 56669485, 56669487, 56669490, 56669519, 56669533, 56669553, 56669564, 56669573, 56669578, 56669588, 56669590, 56669606, 56669610 of chromosome 16,

[0201] TBX3: 115174750, 115174773, 115174780 of chromosome 12,

[0202] BIN1: 127822478, 127822492, 127822495, 127822514, 127822551, 127822568, 127822582, 127822593, 127822616, 127822644 of chromosome 2,

[0203] TIMP2: 76921845, 76921853, 76921860 of chromosome 17,

[0204] CFAP65: 219866132, 219866139, 219866148, 219866158, 219866165, 219866168, 219866199, 219866218 of chromosome 2,

[0205] PRR15: 29605992, 29606026, 29606040, 29606047, 29606056, 29606062, 29606073, 29606179, 29606191, 29606201, 29606204, 29606220, 29606222, 29606227, 29606231, 29606255, 29606257, 29606262, 29606271, 29606277, 29606289, 29606320 of chromosome 7,

[0206] DPYS: 105478870, 105478873, 105478878, 105478905, 105478908, 105478916, 105478918, 105478945, 105478956, 105478965, 105478974, 105478983, 105478986, 105478989 of chromosome 8,

[0207] MCC: 112538999, 112539011, 112539018, 112539022, 112539061, 112539084, 112539104, 112539128 of chromosome 5,

[0208] TBX15: 119535725, 119535730, 119535740, 119535742, 119535750, 119535759, 119535766, 119535812, 119535817, 119535821, 119535823, 119535876, 119535879, 119535884, 119535891 of chromosome 1,

[0209] COL23A1: 178003785, 178003798, 178003803, 178003814, 178003823, 178003825, 178003834, 178003841, 178003844 of chromosome 5,

[0210] ILDR2: 166890429, 166890436, 166890440, 166890442, 166890448, 166890452, 166890456, 166890461, 166890468, 166890473, 166890475, 166890480, 166890492, 166890500, 166890503, 166890509, 166890516, 166890528, 166890535, 166890543, 166890555, 166890559, 166890568, 166890573, 166890584, 166890586 of chromosome 1,

[0211] DHRS3: 12656091, 12656114, 12656132, 12656152, 12656170, 12656175, 12656182, 12656187, 12656197, 12656200, 12656211, 12656315, 12656323, 12656340, 12656355, 12656367 of chromosome 1,

[0212] GDNF: 37834763, 37834770, 37834772, 37834774, 37834777, 37834780, 37834784, 37834792, 37834799, 37834802, 37834806, 37834811 of chromosome 5,

[0213] TBX18: 85477032, 85477035, 85477070, 85477083, 85477106, 85477124, 85477151, 85477153, 85477166 of chromosome 6,

[0214] SIM2: 38069563, 38069579, 38069619, 38069625, 38069638, 38069650, 38069662, 38069664, 38069676, 38069681 of chromosome 21,

[0215] HOXA9: 27204848, 27204854, 27204858, 27204861, 27204863, 27204879, 27204884, 27204894, 27204897, 27204918, 27204929, 27204938, 27204945, 27204948, 27204951, 27204958, 27204981, 27204984 of chromosome 7,

[0216] EHBP1L1: 65352612, 65352621, 65352635, 65352639, 65352642, 65352651, 65352654, 65352665, 65352670 of chromosome 11,

[0217] GJC2: 228345954, 228345957, 228345965, 228345978, 228345980, 228345989 of chromosome 1,

[0218] RCOR2: 63687223, 63687238, 63687247, 63687250, 63687259, 63687282, 63687288, 63687299, 63687318, 63687325 of chromosome 11,

[0219] PRDM1: 106429711, 106429722, 106429731, 106429747, 106429750, 106429761, 106429769, 106429771 of chromosome 6,

[0220] UNCX: 1263643, 1263655, 1263659, 1263664, 1263676, 1263694, 1263716, 1263723 of chromosome 7,

[0221] RPS7P5: 240161502, 240161507, 240161511, 240161516, 240161523, 240161527, 240161530, 240161535, 240161546, 240161558, 240161560 of chromosome 1,

[0222] FOXI2: 129534843, 129534853, 129534866, 129534879, 129534891, 129534910, 129534912, 129534924 of chromosome 10,

[0223] ACRBP: 6756182, 6756187, 6756191, 6756195, 6756211, 6756225, 6756230, 6756270 of chromosome 12,

[0224] GAS6: 114524043, 114524062, 114524068, 114524084, 114524095, 114524131, 114524138, 114524142, 114524150, 114524158 of chromosome 13,

[0225] MCRIP2: 698072, 698142, 698153, 698168, 698208, 698218, 698222, 698230 of chromosome 16,

[0226] LINC01977: 77789596, 77789601, 77789612, 77789620, 77789628, 77789632, 77789635, 77789640 of chromosome 17,

[0227] EGR3: 22548250, 22548260, 22548269, 22548279, 22548283, 22548287, 22548296, 22548299 of chromosome 8,

[0228] SOX17: 55379566, 55379568, 55379573, 55379579, 55379583, 55379591, 55379599, 55379602, 55379608, 55379617, 55379620 of chromosome 8,

[0229] PAX5: 36986087, 36986093, 36986098, 36986101, 36986103, 36986117, 36986131, 36986138, 36986141, 36986143, 36986147, 36986149, 36986156 of chromosome 9,

[0230] NEURL1: 105344464, 105344482, 105344493, 105344495, 105344497, 105344503, 105344506, 105344513, 105344516, 105344519, 105344526 of chromosome 10,

[0231] IRX4: 1876386, 1876395, 1876397, 1876403, 1876420, 1876424, 1876432, 1876436, 1876449, 1876456, 1876459, 1876463 of chromosome 5,

[0232] RUSC1: 155295135, 155295171, 155295181, 155295192, 155295196, 155295212, 155295229, 155295236 of chromosome 1.

[0233] The inventors have also found that the nature of thyroid nodules is also associated with the mutation level of the V600E site of the BRAF gene and / or the mutation level of the C228T / C250T site of the TERT gene.

[0234] In the present document, methods for detecting DNA methylation are well known in the art, such as PCR based on the conversion of bisulfite (e.g. Methylation-specific PCR (MSP)), DNA sequencing (e.g. Bisulfite sequencing (BS), Whole-genome bisulfite sequencing (WGBS), Reduced Representation Bisulfite Sequencing (RRBS)), Methylation-Sensitive Dependent Restriction Enzymes, Fluorescence quantification, Methylation-sensitivity High-resolution Melting (MS-HRM), Chip-based methylation profiling, Mass spectrometry (e.g. Flight Mass Spectrometry). In one or more embodiments, the detection comprises detecting either strand at the gene or site. Detecting the methylation level of the above-mentioned sites comprises detecting the methylation level of a nucleic acid region within 500bp upstream and downstream of the site.

[0235] Accordingly, the present application relates to reagents for detecting DNA methylation. Reagents used in the above-mentioned methods for detecting DNA methylation are well known in the art. Exemplarily, the reagents for detecting DNA methylation can be selected from one or more of the reagents used in the following methods: bisulfite conversion based PCR (e.g. methylation specific PCR), DNA sequencing (e.g. bisulfite sequencing, whole genome methylation sequencing, reduced representation methylation sequencing), methylation sensitive restriction enzyme analysis, fluorescence quantification, methylation sensitive high resolution melting curve analysis, chip based methylation profiling, mass spectrometry (e.g. flight mass spectrometry).

[0236] The reagents for detecting DNA methylation can comprise one or more of the following: bisulfite and its derivatives, PCR buffer, polymerase, dNTP, primer, probe, restriction enzyme which is sensitive or insensitive to methylation, enzyme digestion buffer, fluorescent dye, fluorescence quencher, fluorescence reporter, exonuclease, alkaline phosphatase, internal standard, control. In the detection methods involving DNA amplification, the reagents for detecting DNA methylation comprise primers. The sequence of the primers is methylation specific or non-specific. Preferably, the sequence of the primers comprises a non-methylation specific blocker. The blocker can improve the specificity of methylation detection. The reagents for detecting DNA methylation can also comprise a probe. Typically, the sequence of the probe is labeled with a fluorescence reporter group at the 5' end and a quencher group at the 3' end. Preferably, the sequence of the probe comprises MGB or LNA.

[0237] Herein, methods for detecting genetic mutations and reagents therefor are well known in the art. Exemplarily, the methods for detecting genetic mutations comprise PCR-single strand conformation polymorphism, heteroduplex analysis, mutation enrichment PCR, mutation gradient gel electrophoresis, chemical cleavage mismatch, allele specific oligonucleotide analysis, ligase chain reaction, allele specific amplification, RNase A cleavage, in situ hybridization to chromosomes, fluorescence in situ hybridization, DNA sequence analysis, enzymatic cleavage of mismatches, cleaved fragment length polymorphism, dideoxy fingerprinting, mismatch binding protein truncation test, primer extension, oligonucleotide ligation assay, capillary electrophoresis, chip based methods, etc. In one or more embodiments, the detection comprises detection of either strand at the gene or locus.

[0238] Accordingly, the present application relates to reagents for detecting genetic mutations. Reagents used in the above-mentioned methods for detecting genetic mutations are well known in the art. Exemplarily, the reagents for detecting genetic mutations comprise: primer, probe, buffer, polymerase, dNTP, restriction enzyme, enzyme digestion buffer, fluorescent dye, fluorescence quencher, fluorescence reporter, exonuclease, alkaline phosphatase, internal standard, control.

[0239] The present application also relates to a kit for identifying the nature of a thyroid nodule, comprising the reagents described herein, in particular the reagents described in the second and / or third aspects described herein. The kit can also comprise a nucleic acid molecule described herein, in particular the nucleic acid molecule described in the first aspect, as an internal standard or positive control. The kit can comprise reagents for detecting a genetic mutation. A "primer" as described herein refers to a nucleic acid molecule with a specific nucleotide sequence that directs synthesis at the initiation of nucleotide polymerization. Primers are usually two oligonucleotide sequences synthesized artificially, one primer is complementary to one DNA template strand at one end of the target region, and the other primer is complementary to the other DNA template strand at the other end of the target region, and its function is to serve as the starting point of nucleotide polymerization. Artificially designed primers in vitro are widely used in polymerase chain reaction (PCR), qPCR, sequencing and probe synthesis, etc. Usually, the primers are designed to have a product length of 50-150 bp, 60-140, 70-130, 80-120 bp for amplification. Preferably, the product length is 80-100 bp.

[0240] In one or more embodiments, the reagent for detecting DNA methylation comprises a probe. The sequence of the probe is labeled with a fluorescent reporter group at the 5' end and a quencher group at the 3' end. Preferably, the sequence of the probe comprises MGB (Minor groove binder) or LNA (Locked nucleic acid). MGB and LNA are used to increase the Tm (melting temperature) value, increase the specificity of the analysis, and increase the flexibility of probe design.

[0241] The term "variant" or "mutant" as used herein refers to a polynucleotide that has been altered from a reference sequence by the insertion, deletion or substitution of one or more nucleotides while retaining its ability to hybridize to other nucleic acids. Mutants as described in any of the embodiments herein include nucleotide sequences that have at least 70%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97% sequence identity to a reference sequence and retain the biological activity of the reference sequence. Sequence identity between two aligned sequences can be calculated using, for example, BLASTn of NCBI. Mutants also include nucleotide sequences that have one or more mutations (insertions, deletions or substitutions) in the reference sequence and still retain the biological activity of the reference sequence. The number of mutations typically refers to 1-10, for example 1-8, 1-5 or 1-3. The substitution can be between a purine nucleotide and a pyrimidine nucleotide or between purine nucleotides or between pyrimidine nucleotides. The substitution is preferably a conservative substitution. For example, in the art, conservative substitutions are often made with nucleotides that have similar or identical properties and do not substantially alter the stability and function of the polynucleotide. Conservative substitutions are, for example, the interchange between purine nucleotides (A and G) or between pyrimidine nucleotides (T or U and C). Thus, in the polynucleotides of the present application, substitution of one or several positions with nucleotides from the same residue will not substantially affect its activity. Specifically, the variants in the present application contain the positions described herein that are not mutated. That is, the method of the present application detects the methylation status of the positions in the corresponding sequence, and mutations can occur in the bases outside of these positions.

[0242] The present application also provides a method for screening the benign or malignant thyroid nodules, comprising: (1) detecting the methylation level of the genes, positions or nucleic acid regions described herein in the sample of the subject; optionally (2) detecting the mutation level of the V600E position of the BRAF gene and / or the mutation level of the C228T / C250T position of the TERT gene; (3) comparing with the control sample, or calculating the score, to measure the methylation level; (4) identifying the subject as benign or malignant nodules when the interpretation criteria are met. Typically, the method further comprises, before step (1): extraction, quality control of the sample DNA, and / or conversion of the unmethylated cytosine on the DNA into a base that does not bind to guanine.

[0243] DNA" or "DNA molecule" as used herein refers to deoxyribonucleic acid. DNA is a long chain molecule made up of deoxyribonucleotides condensed by phosphodiester bonds. Each deoxyribonucleotide is made up of a phosphate, a deoxyribose, and a base. The bases of DNA are adenine (A), guanine (G), cytosine (C), and thymine (T). In the double helix structure of double stranded DNA, A pairs with T via hydrogen bonding and G pairs with C via hydrogen bonding. DNA forms include cDNA, genomic DNA, fragmented DNA, or artificially synthesized DNA. DNA can be single stranded or double stranded. DNA can be of any length, for example, 50-500 bp, 100-400 bp, 150-300 bp, or 200-250 bp.

[0244] "Uracil" or "U" as used herein is a component of RNA. "RNA" or "RNA molecule" refers to ribonucleic acid. RNA is a long chain molecule made up of ribonucleotides condensed by phosphodiester bonds. Each ribonucleotide molecule is made up of a phosphate, a ribose, and a base. The bases of RNA are adenine (A), guanine (G), cytosine (C), and uracil (U). In the base pairing of RNA, U replaces the position of T in DNA, i.e., A pairs with U via hydrogen bonding and G pairs with C via hydrogen bonding.

[0245] A transformation between the bases of DNA or RNA can occur. "Transformation," "cytosine transformation," or "CT transformation" as used herein refers to a process of converting unmodified cytosine bases (cytosine, C) to bases that do not bind to guanine (e.g., uracil bases (uracil, U)) using non-enzymatic or enzymatic methods on DNA. Non-enzymatic or enzymatic methods for performing cytosine transformation are well known in the art. Exemplarily, non-enzymatic methods include bisulfite or bisulfate treatment, such as calcium bisulfite, sodium bisulfite, potassium bisulfite, ammonium bisulfite, sodium bisulfate, potassium bisulfate, and ammonium bisulfate, etc. Exemplarily, enzymatic methods include deaminase treatment. The transformed DNA is optionally purified. Methods for DNA purification suitable for use herein are well known in the art.

[0246] "Modification" when referring to cytosine means the introduction or removal of a chemical group on the cytosine base. In the process of cytosine transformation, the modified cytosine base is more stable than the unmodified cytosine base and is less likely or not likely to be converted to U by the transformation process. In one or more embodiments, the modification refers to methylation. "Methylation" or "DNA methylation" as used herein refers to the covalent binding of a methyl group to the 5' carbon position of cytosine in the CpG dinucleotide of genomic DNA, becoming 5-methylcytosine (5mC).

[0247] Optionally, the modified cytosine can be protected from downstream conversion or deamination by non-enzymatic or enzymatic methods prior to the conversion of cytosine described herein. Non-enzymatic or enzymatic methods suitable for protecting the modified cytosine are known in the art. For example, TET2 (ten-eleven translocation 2) and / or an oxidation enhancer can protect the modified cytosine. TET2 can oxidize 5mC and 5hmC to 5caC through a cascade reaction. An oxidation enhancer can convert 5hmC to 5ghmC through glycosylation. Oxidation enhancers suitable for performing the glycosylation are known in the art.

[0248] In one or more embodiments, the interpretation criteria is that the methylation level and / or mutation level of the subject sample is increased or decreased when compared to the control sample. When the methylation level and / or mutation level meets a certain threshold, the nodule is identified as malignant. The methylation level of the detected gene is mathematically analyzed to obtain a fitting equation of the score. For the detected sample, when the score is greater than the threshold, the result is determined as positive, i.e. the nodule is malignant, otherwise, the result is determined as negative, i.e. the nodule is benign. Methods of conventional mathematical analysis and the process of determining the threshold are known in the art, and an exemplary method is binary Logistic regression analysis. Generally, the threshold is 0.

[0249] For example, when the nodule property is identified by the methylation levels of BIN1 and SLC16A3 genes, the methylation levels of BIN1 and SLC16A3 genes are subjected to binary Logistic regression analysis, and the fitting equation is:

[0250] Score = 3.45 - 0.08 x methylation level of BIN1 + 0.01 x methylation level of SLC16A3.

[0251] Therefore, if the score of BIN1 and SLC16A3 genes of the sample is greater than 0, the result is determined as positive, i.e. the nodule is malignant.

[0252] In this disclosure, the sample is from a mammal, preferably a human. The sample can be from any organ (e.g. thyroid), tissue (e.g. epithelial tissue, connective tissue, muscle tissue and neural tissue), cell (e.g. thyroid nodule biopsy) or body fluid (e.g. blood, plasma, serum, tissue fluid, urine). Generally, as long as the sample contains genomic DNA or cfDNA (Circulating free DNA or Cell free DNA). cfDNA is referred to as circulating free DNA or cell free DNA, which is a degraded DNA fragment released into the plasma. Exemplarily, the sample is a thyroid nodule biopsy, preferably a fine needle biopsy. Alternatively, the sample is plasma.

[0253] Exemplary embodiments:

[0254] 1. An isolated nucleic acid molecule from a mammal selected from one or more of the following groups or variants thereof having at least 70% identity: (a) a fragment of chromosome 7 and a fragment of chromosome 6, (b) a fragment of chromosome 2 and a fragment of chromosome 19, (c) a fragment of chromosome 2 and a fragment of chromosome 17, (d) a fragment of chromosome 17, a fragment of chromosome 19 and a fragment of chromosome 16, said fragments being 50-5000 bp, preferably 50-1000 bp in length, wherein

[0255] the fragment of chromosome 7 comprises one or more of the positions 73508994, 73509017, 73509055, 73509062, 73509073, 73509075, 73509112, 73509133, 73509138, 73509148, 73509160 on chromosome 7,

[0256] the fragment of chromosome 6 comprises one or more of the positions 74290205, 74290207, 74290220, 74290225, 74290228 on chromosome 6;

[0257] the fragment of chromosome 2 comprises one or more of the positions 127822478, 127822492, 127822495, 127822514, 127822551, 127822568, 127822582, 127822593, 127822616, 127822644 on chromosome 2,

[0258] the fragment of chromosome 19 comprises one or more of the positions 10870373, 10870377, 10870427, 10870429, 10870441, 10870448 on chromosome 19;

[0259] the fragment of chromosome 17 comprises one or more of the positions 80189165, 80189174, 80189177, 80189197, 80189225, 80189230, 80189239, 80189645, 80189671, 80189674, 80189684, 80189687, 80189698, 80189709, 80189719, 80189726, 80189728, 80189739, 80189757, 80189787, 80189792, 80189811, 80189817, 80189832, 80189841 on chromosome 17,

[0260] The fragment of chromosome 16 comprises one or more of the sites 88700818, 88700826, 88700844, 88700849, 88700857, 88700869, 88700875, 88700891, 88700897, 88700916, 88700920, 88700937, 88700943, 88700948, 88700967, 88700970, 88700993, 88701004, 88701021, 88701029, 88701036, 88701043, 88701051, 88701060, 88701074, 88701081, 88701090, 88701099, 88701111, 88701115, 88701133, 88701140, 88701148, 88701159, 88701161, 88701176, 88701178, 88701180, 88701183, 88701190, 88701201, 88701204, 88701210, 88701212, 88701236, 88701240, 88701266, 88701278, 88701281, 88701285, 88701305, 88701421, 88701442, 88701451 on chromosome 16,

[0261] The above-mentioned sites in the variant are not mutated.

[0262] 2. The nucleic acid molecule according to embodiment 1, wherein the nucleic acid molecule further comprises a fragment of chromosome 14 comprising one or more of the sites 81421983, 81421989, 81422010, 81422017, 81422032, 81422035, 81422063, 81422084 on chromosome 14, which fragment is 50-5000 bp, preferably 50-1000 bp in length, and a fragment of chromosome 16 further comprising one or more of the sites 68771035, 68771037, 68771045, 68771051, 68771059, 68771064, 68771073 on chromosome 16,

[0263] The above-mentioned sites in the variant are not mutated.

[0264] 3. A reagent for detecting DNA, the reagent for detecting DNA comprising a reagent for detecting the methylation level of DNA in a region selected from the group consisting of (1) and (2) below:

[0265] (1) a fragment of one or more genes selected from the group consisting of ZMIZ1, C15orf52, SLC16A3, ZNF512B, SLC17A5, LIMK1, PLEC, TOR4A, TMEM131L, DNM2, IL17C, PRDM16, MT1JP, TBX3, BIN1, TIMP2, CFAP65, TSHR, KIF1A, DAPK, CDH1, TPO, RARG, PRR15, DPYS, MCC, TBX15, COL23A1, ILDR2, DHRS3, GDNF, TBX18, SIM2, HOXA9, EHBP1L1, GJC2, RCOR2, PRDM1, UNCX, RPS7P5, FOXI2, ACRBP, GAS6, MCRIP2, LINC01977, EGR3, SOX17, PAX5, NEURL1, IRX4, RUSC1, the fragment being 50-1000 bp in length,

[0266] (2) a nucleic acid region within 10Kb upstream and downstream of the gene in (1),

[0267] wherein the fragment of ZMIZ1 gene comprises one or more of the following sites of ZMIZ1 gene: 81001968, 81001996, 81002041, 81002052, 81002054, 81002056, 81002062, 81002083, 81002110, 81002116, 81002123, 81002129, 81002133, 81002137, 81002139, 81002164, 81002168, 81002223, 81002241, 81002253,

[0268] the fragment of C15orf52 gene comprises one or more of the following sites of C15orf52 gene: 40626309, 40626312, 40626386,

[0269] A fragment of the SLC16A3 gene comprises one or more of the positions: 80189165, 80189174, 80189177, 80189197, 80189225, 80189230, 80189239, 80189645, 80189671, 80189674, 80189684, 80189687, 80189698, 80189709, 80189719, 80189726, 80189728, 80189739, 80189757, 80189787, 80189792, 80189811, 80189817, 80189832, 80189841, of the SLC16A3 gene,

[0270] A fragment of the ZNF512B gene comprises one or more of the positions: 62588634, 62588638, 62588672, of the ZNF512B gene,

[0271] A fragment of the SLC17A5 gene comprises one or more of the positions: 74290205, 74290207, 74290220, 74290225, 74290228, of the SLC17A5 gene,

[0272] A fragment of the LIMK1 gene comprises one or more of the positions: 73508994, 73509017, 73509055, 73509062, 73509073, 73509075, 73509112, 73509133, 73509138, 73509148, 73509160, of the LIMK1 gene,

[0273] A fragment of the PLEC gene comprises one or more of the positions: 145013661, 145013673, of the PLEC gene,

[0274] A fragment of the TOR4A gene comprises one or more of the positions: 140172787, 140172790, 140172812, of the TOR4A gene,

[0275] A fragment of the TMEM131L gene comprises one or more of the positions: 154409945, 154409963, 154409972, 154409978, 154409997, 154410003, 154410006, of the TMEM131L gene,

[0276] a fragment of the DNM2 gene comprises one or more of positions: 10870373, 10870377, 10870427, 10870429, 10870441, 10870448, of the DNM2 gene,

[0277] a fragment of the IL17C gene comprises one or more of positions: 88700818, 88700826, 88700844, 88700849, 88700857, 88700869, 88700875, 88700891, 88700897, 88700916, 88700920, 88700937, 88700943, 88700948, 88700967, 88700970, 88700993, 88701004, 88701021, 88701029, 88701036, 88701043, 88701051, 88701060, 88701074, 88701081, 88701090, 88701099, 88701111, 88701115, 88701133, 88701140, 88701148, 88701159, 88701161, 88701176, 88701178, 88701180, 88701183, 88701190, 88701201, 88701204, 88701210, 88701212, 88701236, 88701240, 88701266, 88701278, 88701281, 88701285, 88701305, 88701421, 88701442, 88701451, of the IL17C gene,

[0278] a fragment of the PRDM16 gene comprises one or more of positions: 3229914, 3229921, 3229950, 3229968, 3229973, 3310213, 3310229, 3310235, 3310238, 3310240, 3310268, 3310287, 3310312, 3310314, 3310317, 3310329, of the PRDM16 gene,

[0279] a fragment of the TSHR gene comprises one or more of positions: 81421983, 81421989, 81422010, 81422017, 81422032, 81422035, 81422063, 81422084, of the TSHR gene,

[0280] The fragment of the KIF1A gene comprises one or more of the positions: 241759696, 241759701, 241759714, 241759716 of the KIF1A gene,

[0281] The fragment of the DAPK gene comprises one or more of the positions: 90112842, 90112853, 90112861, 90112866 of the DAPK gene,

[0282] The fragment of the CDH1 gene comprises one or more of the positions: 68771035, 68771037, 68771045, 68771051, 68771059, 68771064, 68771073 of the CDH1 gene,

[0283] The fragment of the TPO gene comprises one or more of the positions: 1481013, 1481015, 1481022, 1481039 of the TPO gene,

[0284] The fragment of the RARG gene comprises one or more of the positions: 53613176, 53613182, 53613190, 53613202, 53613210, 53613218 of the RARG gene,

[0285] The fragment of the MT1JP gene comprises one or more of the positions: 56669271, 56669292, 56669295, 56669300, 56669318, 56669322, 56669324, 56669327, 56669344, 56669351, 56669353, 56669402, 56669414, 56669423, 56669430, 56669433, 56669437, 56669451, 56669453, 56669455, 56669463, 56669474, 56669480, 56669482, 56669485, 56669487, 56669490, 56669519, 56669533, 56669553, 56669564, 56669573, 56669578, 56669588, 56669590, 56669606, 56669610 of the MT1JP gene,

[0286] The fragment of the TBX3 gene comprises one or more of the positions: 115174750, 115174773, 115174780 of the TBX3 gene,

[0287] The fragment of the BIN1 gene comprises one or more of the positions 127822478, 127822492, 127822495, 127822514, 127822551, 127822568, 127822582, 127822593, 127822616, 127822644 of the BIN1 gene,

[0288] The fragment of the TIMP2 gene comprises one or more of the positions 76921845, 76921853, 76921860 of the TIMP2 gene,

[0289] The fragment of the CFAP65 gene comprises one or more of the positions 219866132, 219866139, 219866148, 219866158, 219866165, 219866168, 219866199, 219866218 of the CFAP65 gene,

[0290] The fragment of the PRR15 gene comprises one or more of the positions 29605992, 29606026, 29606040, 29606047, 29606056, 29606062, 29606073, 29606179, 29606191, 29606201, 29606204, 29606220, 29606222, 29606227, 29606231, 29606255, 29606257, 29606262, 29606271, 29606277, 29606289, 29606320 of the PRR15 gene,

[0291] The fragment of the DPYS gene comprises one or more of the positions 105478870, 105478873, 105478878, 105478905, 105478908, 105478916, 105478918, 105478945, 105478956, 105478965, 105478974, 105478983, 105478986, 105478989 of the DPYS gene,

[0292] The fragment of the MCC gene comprises one or more of the positions 112538999, 112539011, 112539018, 112539022, 112539061, 112539084, 112539104, 112539128 of the MCC gene,

[0293] a fragment of the TBX15 gene comprises one or more of the positions: 119535725, 119535730, 119535740, 119535742, 119535750, 119535759, 119535766, 119535812, 119535817, 119535821, 119535823, 119535876, 119535879, 119535884, 119535891 of the TBX15 gene,

[0294] a fragment of the COL23A1 gene comprises one or more of the positions: 178003785, 178003798, 178003803, 178003814, 178003823, 178003825, 178003834, 178003841, 178003844 of the COL23A1 gene,

[0295] a fragment of the ILDR2 gene comprises one or more of the positions: 166890429, 166890436, 166890440, 166890442, 166890448, 166890452, 166890456, 166890461, 166890468, 166890473, 166890475, 166890480, 166890492, 166890500, 166890503, 166890509, 166890516, 166890528, 166890535, 166890543, 166890555, 166890559, 166890568, 166890573, 166890584, 166890586 of the ILDR2 gene,

[0296] a fragment of the DHRS3 gene comprises one or more of the positions: 12656091, 12656114, 12656132, 12656152, 12656170, 12656175, 12656182, 12656187, 12656197, 12656200, 12656211, 12656315, 12656323, 12656340, 12656355, 12656367 of the DHRS3 gene,

[0297] a fragment of the GDNF gene comprises one or more of the following positions of the GDNF gene: 37834763, 37834770, 37834772, 37834774, 37834777, 37834780, 37834784, 37834792, 37834799, 37834802, 37834806, 37834811,

[0298] a fragment of the TBX18 gene comprises one or more of the following positions of the TBX18 gene: 85477032, 85477035, 85477070, 85477083, 85477106, 85477124, 85477151, 85477153, 85477166,

[0299] a fragment of the SIM2 gene comprises one or more of the following positions of the SIM2 gene: 38069563, 38069579, 38069619, 38069625, 38069638, 38069650, 38069662, 38069664, 38069676, 38069681,

[0300] a fragment of the HOXA9 gene comprises one or more of the following positions of the HOXA9 gene: 27204848, 27204854, 27204858, 27204861, 27204863, 27204879, 27204884, 27204894, 27204897, 27204918, 27204929, 27204938, 27204945, 27204948, 27204951, 27204958, 27204981, 27204984,

[0301] a fragment of the EHBP1L1 gene comprises one or more of the following positions of the EHBP1L1 gene: 65352612, 65352621, 65352635, 65352639, 65352642, 65352651, 65352654, 65352665, 65352670,

[0302] a fragment of the GJC2 gene comprises one or more of the following positions of the GJC2 gene: 228345954, 228345957, 228345965, 228345978, 228345980, 228345989,

[0303] a fragment of the RCOR2 gene comprises one or more of the following positions of the RCOR2 gene: 63687223, 63687238, 63687247, 63687250, 63687259, 63687282, 63687288, 63687299, 63687318, 63687325,

[0304] a fragment of the PRDM1 gene comprises one or more of the following positions of the PRDM1 gene: 106429711, 106429722, 106429731, 106429747, 106429750, 106429761, 106429769, 106429771,

[0305] a fragment of the UNCX gene comprises one or more of the following positions of the UNCX gene: 1263643, 1263655, 1263659, 1263664, 1263676, 1263694, 1263716, 1263723,

[0306] a fragment of the RPS7P5 gene comprises one or more of the following positions of the RPS7P5 gene: 240161502, 240161507, 240161511, 240161516, 240161523, 240161527, 240161530, 240161535, 240161546, 240161558, 240161560,

[0307] a fragment of the FOXI2 gene comprises one or more of the following positions of the FOXI2 gene: 129534843, 129534853, 129534866, 129534879, 129534891, 129534910, 129534912, 129534924,

[0308] a fragment of the ACRBP gene comprises one or more of the following positions of the ACRBP gene: 6756182, 6756187, 6756191, 6756195, 6756211, 6756225, 6756230, 6756270,

[0309] a fragment of the GAS6 gene comprises one or more of the following positions of the GAS6 gene: 114524043, 114524062, 114524068, 114524084, 114524095, 114524131, 114524138, 114524142, 114524150, 114524158,

[0310] A fragment of the MCRIP2 gene comprises one or more of the positions: 698072, 698142, 698153, 698168, 698208, 698218, 698222, 698230 of the MCRIP2 gene,

[0311] A fragment of the LINC01977 gene comprises one or more of the positions: 77789596, 77789601, 77789612, 77789620, 77789628, 77789632, 77789635, 77789640 of the LINC01977 gene,

[0312] A fragment of the EGR3 gene comprises one or more of the positions: 22548250, 22548260, 22548269, 22548279, 22548283, 22548287, 22548296, 22548299 of the EGR3 gene,

[0313] A fragment of the SOX17 gene comprises one or more of the positions: 55379566, 55379568, 55379573, 55379579, 55379583, 55379591, 55379599, 55379602, 55379608, 55379617, 55379620 of the SOX17 gene,

[0314] A fragment of the PAX5 gene comprises one or more of the positions: 36986087, 36986093, 36986098, 36986101, 36986103, 36986117, 36986131, 36986138, 36986141, 36986143, 36986147, 36986149, 36986156 of the PAX5 gene,

[0315] A fragment of the NEURL1 gene comprises one or more of the positions: 105344464, 105344482, 105344493, 105344495, 105344497, 105344503, 105344506, 105344513, 105344516, 105344519, 105344526 of the NEURL1 gene,

[0316] A fragment of the IRX4 gene comprises one or more of the positions: 1876386, 1876395, 1876397, 1876403, 1876420, 1876424, 1876432, 1876436, 1876449, 1876456, 1876459, 1876463 of the IRX4 gene,

[0317] The fragment of the RUSC1 gene comprises one or more of the positions 155295135, 155295171, 155295181, 155295192, 155295196, 155295212, 155295229, 155295236 of the RUSC1 gene.

[0318] 4. The reagent for detecting DNA according to Embodiment 3, characterized by,

[0319] The fragment of the ZMIZ1 gene comprises one or more of the positions 81002041, 81002052, 81002054, 81002056, 81002062, 81002083 of the ZMIZ1 gene,

[0320] The fragment of the C15orf52 gene comprises one or more of the positions 40626309, 40626312 of the C15orf52 gene,

[0321] The fragment of the SLC16A3 gene comprises one or more of the positions 80189671, 80189674, 80189684, 80189687, 80189698, 80189709, 80189719, 80189726, 80189728, 80189739, 80189757 of the SLC16A3 gene,

[0322] The fragment of the ZNF512B gene comprises one or more of the positions 62588634, 62588638, 62588672 of the ZNF512B gene,

[0323] The fragment of the SLC17A5 gene comprises one or more of the positions 74290205, 74290207, 74290220, 74290225, 74290228 of the SLC17A5 gene,

[0324] The fragment of the LIMK1 gene comprises one or more of the positions 73509112, 73509133, 73509138, 73509148, 73509160 of the LIMK1 gene,

[0325] The fragment of the PLEC gene comprises one or more of the positions 145013661, 145013673 of the PLEC gene,

[0326] The fragment of the TOR4A gene comprises one or more of the positions 140172787, 140172790, 140172812 of the TOR4A gene,

[0327] a fragment of the TMEM131L gene comprises one or more of positions 154409945, 154409963, 154409972, 154409978, 154409997 of the TMEM131L gene,

[0328] a fragment of the DNM2 gene comprises one or more of positions 10870427, 10870429, 10870441, 10870448 of the DNM2 gene,

[0329] a fragment of the IL17C gene comprises one or more of positions 88701004, 88701021, 88701029, 88701036, 88701043, 88701051, 88701060 of the IL17C gene,

[0330] a fragment of the PRDM16 gene comprises one or more of positions 3229950, 3229968, 3229973 of the PRDM16 gene,

[0331] a fragment of the MT1JP gene comprises one or more of positions 56669271, 56669292, 56669295, 56669300, 56669318, 56669322, 56669324, 56669327, 56669344 of the MT1JP gene,

[0332] a fragment of the TBX3 gene comprises one or more of positions 115174750, 115174773, 115174780 of the TBX3 gene,

[0333] a fragment of the BIN1 gene comprises one or more of positions 127822478, 127822492, 127822495, 127822514, 127822551, 127822568, 127822582, 127822593, 127822616 of the BIN1 gene,

[0334] a fragment of the TIMP2 gene comprises one or more of positions 76921845, 76921853, 76921860 of the TIMP2 gene,

[0335] a fragment of the CFAP65 gene comprises one or more of positions 219866199, 219866218 of the CFAP65 gene,

[0336] a fragment of the TSHR gene comprises one or more of positions 81421983, 81421989, 81422010, 81422017, 81422032, 81422035, 81422063, 81422084 of the TSHR gene,

[0337] a fragment of the KIF1A gene comprises one or more of positions 241759696, 241759701, 241759714, 241759716 of the KIF1A gene,

[0338] a fragment of the DAPK gene comprises one or more of positions 90112842, 90112853, 90112861, 90112866 of the DAPK gene,

[0339] a fragment of the CDH1 gene comprises one or more of positions 68771035, 68771037, 68771045, 68771051, 68771059, 68771064, 68771073 of the CDH1 gene,

[0340] a fragment of the TPO gene comprises one or more of positions 1481013, 1481015, 1481022, 1481039 of the TPO gene,

[0341] a fragment of the RARG gene comprises one or more of positions 53613176, 53613182, 53613190, 53613202, 53613210, 53613218 of the RARG gene,

[0342] a fragment of the PRR15 gene comprises one or more of positions 29606026, 29606040, 29606047, 29606056, 29606062, 29606073, 29606220, 29606222, 29606227, 29606231, 29606255, 29606257, 29606262, 29606271, 29606277, 29606289 of the PRR15 gene,

[0343] a fragment of the DPYS gene comprises one or more of positions 105478905, 105478908, 105478916, 105478918, 105478945, 105478956, 105478965, 105478974, 105478983 of the DPYS gene,

[0344] a fragment of the MCC gene comprises one or more of positions 112538999, 112539011, 112539018, 112539022, 112539061 of the MCC gene,

[0345] a fragment of the TBX15 gene comprises one or more of positions 119535740, 119535742, 119535750, 119535759, 119535766 of the TBX15 gene,

[0346] a fragment of the COL23A1 gene comprises one or more of positions 178003798, 178003803, 178003814, 178003823, 178003825, 178003834, 178003841, 178003844 of the COL23A1 gene,

[0347] a fragment of the ILDR2 gene comprises one or more of positions 166890516, 166890528, 166890535, 166890543, 166890555, 166890559, 166890568, 166890573, 166890584, 166890586 of the ILDR2 gene,

[0348] a fragment of the DHRS3 gene comprises one or more of positions 12656340, 12656355, 12656367 of the DHRS3 gene,

[0349] a fragment of the GDNF gene comprises one or more of positions 37834770, 37834772, 37834774, 37834777, 37834780, 37834784, 37834792, 37834799, 37834802, 37834806, 37834811 of the GDNF gene,

[0350] a fragment of the TBX18 gene comprises one or more of positions 85477035, 85477070, 85477083, 85477106 of the TBX18 gene,

[0351] a fragment of the SIM2 gene comprises one or more of positions 38069638, 38069650, 38069662, 38069664, 38069676, 38069681 of the SIM2 gene,

[0352] The fragment of the HOXA9 gene includes one or more of the following sites: 27204854, 27204858, 27204861, 27204863, and 27204879 of the HOXA9 gene.

[0353] The fragment of the EHBP1L1 gene includes one or more of the following sites: 65352621, 65352635, 65352639, 65352642, 65352651, 65352654, 65352665, and 65352670 of the EHBP1L1 gene.

[0354] The fragment of the GJC2 gene contains one or more of the following sites: 228345965, 228345978, 228345980, and 228345989 of the GJC2 gene.

[0355] The fragment of the RCOR2 gene includes one or more of the RCOR2 gene sites: 63687223, 63687238, 63687247, 63687250, and 63687259.

[0356] The fragment of the PRDM1 gene includes one or more of the sites of the PRDM1 gene: 106429722, 106429731, 106429747, 106429750, 106429761, 106429769, 106429771,

[0357] The fragment of the UNCX gene contains one or more of the following sites: 1263643, 1263655, 1263659, 1263664, 1263676.

[0358] The fragment of the RPS7P5 gene includes one or more of the sites of the RPS7P5 gene: 240161511, 240161516, 240161523, 240161527, 240161530,

[0359] The fragment of the FOXI2 gene includes one or more of the FOXI2 gene sites: 129534910, 129534912, and 129534924.

[0360] The fragment of the ACRBP gene contains one or more of the following sites: 6756182, 6756187, 6756191, 6756195, and 6756211.

[0361] The fragment of the GAS6 gene comprises one or more of the following sites of the GAS6 gene: 114524062, 114524068, 114524084, 114524095, 114524131, 114524138,

[0362] The fragment of the MCRIP2 gene comprises one or more of the following sites of the MCRIP2 gene: 698072, 698142, 698153, 698168, 698208,

[0363] The fragment of the LINC01977 gene comprises one or more of the following sites of the LINC01977 gene: 77789596, 77789601, 77789612, 77789620,

[0364] The fragment of the EGR3 gene comprises one or more of the following sites of the EGR3 gene: 22548269, 22548279, 22548283, 22548287, 22548296, 22548299,

[0365] The fragment of the SOX17 gene comprises one or more of the following sites of the SOX17 gene: 55379602, 55379608, 55379617, 55379620,

[0366] The fragment of the PAX5 gene comprises one or more of the following sites of the PAX5 gene: 36986087, 36986093, 36986098, 36986101, 36986103,

[0367] The fragment of the NEURL1 gene comprises one or more of the following sites of the NEURL1 gene: 105344493, 105344495, 105344497,

[0368] The fragment of the IRX4 gene comprises one or more of the following sites of the IRX4 gene: 1876386, 1876395, 1876397, 1876403,

[0369] The fragment of the RUSC1 gene comprises one or more of the following sites of the RUSC1 gene: 155295192, 155295196, 155295212.

[0370] 5. The reagent for detecting DNA according to embodiment 3, wherein the reagent for detecting DNA further comprises a reagent for detecting the mutation level of the V600E site of the BRAF gene.

[0371] 6. The reagent for detecting DNA according to claim 3, wherein the reagent for detecting DNA further comprises a reagent for detecting the mutation level of the C228T / C250T site of the TERT gene.

[0372] 7. A reagent for detecting DNA methylation, which detects the methylation level of one or more groups of sites in (a) to (d) below:

[0373] a. (1) one or more of 73508994, 73509017, 73509055, 73509062, 73509073, 73509075, 73509112, 73509133, 73509138, 73509148, 73509160 on chromosome 7, and

[0374] (2) one or more of 74290205, 74290207, 74290220, 74290225, 74290228 on chromosome 6;

[0375] b. (1) one or more of 127822478, 127822492, 127822495, 127822514, 127822551, 127822568, 127822582, 127822593, 127822616, 127822644 on chromosome 2, and

[0376] (2) one or more of 10870373, 10870377, 10870427, 10870429, 10870441, 10870448 on chromosome 19;

[0377] c. (1) one or more of 127822478, 127822492, 127822495, 127822514, 127822551, 127822568, 127822582, 127822593, 127822616, 127822644 on chromosome 2, and

[0378] (2) one or more of 80189165, 80189174, 80189177, 80189197, 80189225, 80189230, 80189239, 80189645, 80189671, 80189674, 80189684, 80189687, 80189698, 80189709, 80189719, 80189726, 80189728, 80189739, 80189757, 80189787, 80189792, 80189811, 80189817, 80189832, 80189841 on chromosome 17;

[0379] d. (1) one or more of 80189165, 80189174, 80189177, 80189197, 80189225, 80189230, 80189239, 80189645, 80189671, 80189674, 80189684, 80189687, 80189698, 80189709, 80189719, 80189726, 80189728, 80189739, 80189757, 80189787, 80189792, 80189811, 80189817, 80189832, 80189841 on chromosome 17,

[0380] (2) one or more of 10870373, 10870377, 10870427, 10870429, 10870441, 10870448 on chromosome 19, and

[0381] (3) one or more of 88700818, 88700826, 88700844, 88700849, 88700857, 88700869, 88700875, 88700891, 88700897, 88700916, 88700920, 88700937, 88700943, 88700948, 88700967, 88700970, 88700993, 88701004, 88701021, 88701029, 88701036, 88701043, 88701051, 88701060, 88701074, 88701081, 88701090, 88701099, 88701111, 88701115, 88701133, 88701140, 88701148, 88701159, 88701161, 88701176, 88701178, 88701180, 88701183, 88701190, 88701201, 88701204, 88701210, 88701212, 88701236, 88701240, 88701266, 88701278, 88701281, 88701285, 88701305, 88701421, 88701442, 88701451 on chromosome 16.

[0382] 8. The agent of embodiment 7, further detecting the methylation level at:

[0383] e. (1) one or more of 68771035, 68771037, 68771045, 68771051, 68771059, 68771064, 68771073 on chromosome 16, and (2) one or more of 81421983, 81421989, 81422010, 81422017, 81422032, 81422035, 81422063, 81422084 on chromosome 14.

[0384] 9. The agent of any one of embodiments 2-8, further having one or more features selected from:

[0385] the fragment comprises a DNA sense strand or an antisense strand,

[0386] The reagents for detecting DNA methylation are selected from one or more of the following methods: bisulfite conversion based PCR, DNA sequencing, methylation sensitive restriction enzyme analysis, fluorescence quantification, methylation sensitive high resolution melting curve analysis, chip based methylation profiling, mass spectrometry,

[0387] Preferably, the reagents for detecting DNA methylation are selected from one or more of the following: bisulfite and derivatives thereof, PCR buffer, polymerase, dNTPs, primers, probes, methylation sensitive or insensitive restriction enzymes, enzyme digestion buffer, fluorescent dyes, fluorescence quenchers, fluorescence reporters, exonucleases, alkaline phosphatase, internal standards, controls,

[0388] Preferably, the primers are methylation specific or non-specific, preferably the sequence of the primers comprises a non-methylation specific blocker sequence, preferably the primers are SEQ ID NO: 1, 2, 4, 5, 7, 8 or a sequence having 90% identity thereto,

[0389] Preferably, the probes have a reporter sequence, preferably the probes are SEQ ID NO: 3, 6, 9 or a sequence having 90% identity thereto,

[0390] The reagents for detecting gene mutations are selected from one or more of the following methods: PCR-single strand conformation polymorphism, heteroduplex analysis, mutation enrichment PCR, mutation gradient gel electrophoresis, chemical cleavage mismatch, allele specific oligonucleotide analysis, ligase chain reaction, allele specific amplification, RNase A cleavage, in situ hybridization to chromosomes, fluorescence in situ hybridization, DNA sequence analysis, enzymatic cleavage of mismatches, cleaved fragment length polymorphism, dideoxy fingerprinting, mismatch binding protein truncation test, primer extension, oligonucleotide ligation assay, capillary electrophoresis, chip based methods,

[0391] Preferably, the reagents for detecting gene mutations comprise: primers, probes, buffers, polymerase, dNTPs, restriction enzymes, enzyme digestion buffer, fluorescent dyes, fluorescence quenchers, fluorescence reporters, exonucleases, alkaline phosphatase, internal standards, controls.

[0392] 10. A kit for identifying the nature of a thyroid nodule comprising the reagents of any one of embodiments 2-9 and optionally the nucleic acid molecule of embodiment 1.

[0393] 11. Use of reagents for detecting DNA and optionally the nucleic acid molecule of embodiment 1 for detecting the methylation level of one or more sets of loci (a)-(d):

[0394] a. (1) one or more of 73508994, 73509017, 73509055, 73509062, 73509073, 73509075, 73509112, 73509133, 73509138, 73509148, 73509160 on chromosome 7, and

[0395] (2) one or more of 74290205, 74290207, 74290220, 74290225, 74290228 on chromosome 6;

[0396] b. (1) one or more of 127822478, 127822492, 127822495, 127822514, 127822551, 127822568, 127822582, 127822593, 127822616, 127822644 on chromosome 2, and

[0397] (2) one or more of 10870373, 10870377, 10870427, 10870429, 10870441, 10870448 on chromosome 19;

[0398] c. (1) one or more of 127822478, 127822492, 127822495, 127822514, 127822551, 127822568, 127822582, 127822593, 127822616, 127822644 on chromosome 2, and

[0399] (2) one or more of 80189165, 80189174, 80189177, 80189197, 80189225, 80189230, 80189239, 80189645, 80189671, 80189674, 80189684, 80189687, 80189698, 80189709, 80189719, 80189726, 80189728, 80189739, 80189757, 80189787, 80189792, 80189811, 80189817, 80189832, 80189841 on chromosome 17;

[0400] d.(1) one or more of 80189165, 80189174, 80189177, 80189197, 80189225, 80189230, 80189239, 80189645, 80189671, 80189674, 80189684, 80189687, 80189698, 80189709, 80189719, 80189726, 80189728, 80189739, 80189757, 80189787, 80189792, 80189811, 80189817, 80189832, 80189841 on chromosome 17,

[0401] (2) one or more of 10870373, 10870377, 10870427, 10870429, 10870441, 10870448 on chromosome 19, and

[0402] (3) one or more of 88700818, 88700826, 88700844, 88700849, 88700857, 88700869, 88700875, 88700891, 88700897, 88700916, 88700920, 88700937, 88700943, 88700948, 88700967, 88700970, 88700993, 88701004, 88701021, 88701029, 88701036, 88701043, 88701051, 88701060, 88701074, 88701081, 88701090, 88701099, 88701111, 88701115, 88701133, 88701140, 88701148, 88701159, 88701161, 88701176, 88701178, 88701180, 88701183, 88701190, 88701201, 88701204, 88701210, 88701212, 88701236, 88701240, 88701266, 88701278, 88701281, 88701285, 88701305, 88701421, 88701442, 88701451 on chromosome 16,

[0403] Optionally, e. (1) one or more of 68771035, 68771037, 68771045, 68771051, 68771059, 68771064, 68771073 on chromosome 16, and (2) one or more of 81421983, 81421989, 81422010, 81422017, 81422032, 81422035, 81422063, 81422084 on chromosome 14,

[0404] Preferably, the agent is as described in any one of embodiments 8-9.

[0405] 12. The use according to embodiment 11, characterized in that the use has one or more features selected from the group consisting of:

[0406] The kit further comprises reagents for detecting the mutation level of the V600E site of the BRAF gene and / or the mutation level of the C228T / C250T site of the TERT gene,

[0407] The identifying the nature of the thyroid nodule comprises comparing with a control sample, or obtaining a score based on the methylation level and / or mutation level, and identifying the nature of the thyroid nodule based on the comparison result or the score,

[0408] The sample is from a human, preferably from a tissue, a cell or a body fluid, such as thyroid tissue or blood,

[0409] The sample contains genomic DNA or cfDNA.

[0410] 13. A method for identifying the nature of a thyroid nodule, comprising:

[0411] (1) detecting the methylation level of a gene, site or nucleic acid region in a sample of the subject, the gene, site or nucleic acid region being as described in embodiments 2-9;

[0412] Optionally, (2) detecting the mutation level of the V600E site of the BRAF gene and / or the mutation level of the C228T / C250T site of the TERT gene;

[0413] (3) comparing with a control sample, or obtaining a score based on the methylation level and / or mutation level;

[0414] (4) identifying the nature of the thyroid nodule based on the comparison result or the score of step (3),

[0415] Preferably, step (4) comprises:

[0416] comparing the methylation level and / or mutation level of the subject sample with the control sample, and identifying the thyroid nodule as a benign or malignant nodule when the methylation level and / or mutation level meets a threshold, or

[0417] identifying the thyroid nodule as a benign or malignant nodule when the score meets a threshold.

[0418] Embodiments

[0419] The application will be further described in conjunction with the accompanying drawings and specific embodiments. In the following examples, the experimental methods not specified in the specific conditions are generally carried out according to the methods described in the conventional conditions.

[0420] Example 1: Reduced Representation Bisulfite Sequencing (RRBS) screening of methylation sites differentially expressed in benign and malignant thyroid nodules

[0421] 1) Sample preparation

[0422] DNA extraction was performed on 37 thyroid cancer and 37 benign thyroid nodules using QIAamp DNA Mini Kit (QIAGEN, Cat No: 51304); the concentration of DNA was detected using QubitTM dsDNA HS Assay Kit (Thermo, Cat No: Q32854); and the quality was detected using 1% agarose gel electrophoresis.

[0423] 2) MspI digestion

[0424] The reaction system was prepared as follows:

[0425] Components Volume (μl) 10×Buffer Tango 2.0 MspI (10 U / μl) 1.0 Nuclease-free water + DNA 17.0 total 20.0

[0426] The reaction program was as follows: 37°C for 2 hours, 4°C for storage.

[0427] 3) End repair and A addition

[0428] The reaction system was prepared as follows:

[0429] Components Volume (μl) Enzyme-digested DNA products 20.0 End Repair & A-Tailing Buffer 4.0 End Repair&A-Tailing Enzyme Mix 2.0 Nuclease-free water 14.0 total 40.0

[0430] The reaction program was as follows: 20°C for 30 minutes, 65°C for 30 minutes, 4°C for storage.

[0431] 4) Adapter ligation

[0432] The reaction system was prepared as follows:

[0433] Components Volume (μl) End repair and A-addition products 40.0 Indexed methylated adapter 2.0 T4 DNA Ligase Buffer (10×) 5.0 T4 DNA Ligase 1.0 Nuclease-free water 2.0 total 50.0

[0434] The reaction procedure was: 16°C overnight, 65°C for 10 minutes, and storage at 4°C.

[0435] 5) Post-ligation purification

[0436] i. Transfer the ligated solution to 50 μl of AMPure beads, vortex to mix, incubate at room temperature for 5 minutes, and briefly centrifuge at low speed. Place the tube on a magnetic rack until the solution is clear.

[0437] ii. Wash twice with 80% ethanol solution;

[0438] iii. Dry the magnetic beads at room temperature;

[0439] iv. Add 32 μl of ddH2O and incubate at room temperature for 2 minutes. Place the centrifuge tube on a magnetic rack until the solution is clear. Pipette 30 μl of the supernatant solution and transfer it to a new centrifuge tube.

[0440] 6) Bisulfite conversion

[0441] Using MethylCode TM The DNA obtained in step 5 was subjected to bisulfite conversion using the Bisulfite Conversion Kit (Thermo, Catalog No.: MECOV50). Unmethylated cytosine (C) was converted to uracil (U); methylated cytosine remained unchanged after conversion.

[0442] The conversion reagent was prepared as follows:

[0443] Components Volume (μl) Nuclease-free water 800.0 Dilution Buffer 300.0 Resuspension Buffer 50.0

[0444] Add 120 μl of the prepared conversion reagent to 30 μl of the ligation and purification product obtained in step 5 and mix thoroughly. The reaction procedure is: 98°C for 10 minutes, 64°C for 2.5 hours, and store at 4°C.

[0445] Recover the treated DNA according to the instructions, finally elute the DNA with 43 μl of elution buffer, and transfer 41.6 μl for the next reaction.

[0446] 7) Library amplification

[0447] The reaction system was prepared as follows:

[0448] Components Volume (μl) 10×PfuTurbo Cx reaction buffer 5.0 dNTPs (25mM each dNTP) 0.4 Primer mix 2.0 PfuTurbo Cx hotstart DNA polymerase(2.5U / μl) 1.0 Bisulfite-converted DNA 41.6 total 50.0

[0449] The reaction program was as follows: 95°C for 2 minutes; 95°C for 30 seconds, 65°C for 30 seconds, 72°C for 1 minute, 15 cycles; 72°C for 5 minutes, and storage at 4°C.

[0450] 8) Library purification

[0451] i. Add 50 μΐ AMPure beads to the library amplification product, vortex to mix, incubate at room temperature for 5 minutes, and briefly centrifuge at low speed. Place the tube on a magnetic stand until the solution is clear;

[0452] ii. Wash twice with 80% ethanol solution;

[0453] iii. Air dry the beads at room temperature;

[0454] iv. Add 40 μΐ ddH20, incubate at room temperature for 2 minutes, place the tube on a magnetic stand until the solution is clear, and transfer 38 μΐ of the supernatant to a new tube.

[0455] 9) Library quality control

[0456] Determine the library concentration using Qubit, and determine the library fragment distribution using LabChip (PerkinElmer), as shown in Figure 1

[0457] 10) Sequencing

[0458] Sequencing was performed using the Illumina platform HiSeq X Ten with PE150.

[0459] 11) Data analysis

[0460] After bioinformatics analysis, the CpG sites with methylation differences between benign and malignant thyroid nodules were obtained, as shown in Table 1, including the chromosome where the CpG is located, the starting site of the CpG, the corresponding gene, the statistical comparison P value, and the ratio between the methylation CpG sites of the thyroid malignant nodules and the benign nodules.

[0461] Table 1, 566 CpG sites with methylation differences between benign and malignant thyroid nodules and the corresponding 51 genes

[0462]

[0463]

[0464]

[0465]

[0466]

[0467]

[0468]

[0469] ​

[0470]

[0471]

[0472] Example 2, Methylation-specific PCR (MSP) and quantitative methylation-specific PCR (Q-MSP) validation of differential methylation sites

[0473] 1) Sample preparation

[0474] DNA extraction was performed on 10 cases of thyroid cancer and 10 cases of thyroid benign nodule tissues or plasma using QIAamp DNA Mini Kit (QIAGEN, Cat No: 51304); DNA concentration was detected using Qubit dsDNA HS Assay Kit (Thermo, Cat No: Q32854); and quality inspection was performed using 1% agarose gel electrophoresis. TM

[0475] 2) DNA transformation

[0476] DNA obtained in step 1 was subjected to bisulfite conversion using MethylCode Bisulfite Conversion Kit (Thermo, Cat No: MECOV50), unmethylated cytosine (C) was converted to uracil (U) after conversion; methylated cytosine did not change after conversion. TM

[0477] 3) PCR mixture preparation

[0478] The preparation of individual samples included PCR reaction solution, primer mixture, and probe mixture as follows:

[0479] MSP reaction system composition

[0480] Components Volume (μl) Platinum TM II Hot-Start PCR Master Mix (2x) 10.00 water 7.44 Target gene forward primer F, 100 μM 0.12 Target gene reverse primer R, 100 μM 0.12 Internal reference gene forward primer F, 100 μM 0.12 Internal reference gene reverse primer R, 100 μM 0.12 Target gene probe P, 100 μM (FAM / BHQ1) 0.04 Internal reference gene probe P, 100 μM (HEX / BHQ1) 0.04 Sample DNA (10.0ng) / positive control / negative control 2.00 Total 20.00

[0481] Q-MSP reaction system composition

[0482]

[0483]

[0484] Note: The gradient-diluted standard was 30 ng of 4-fold gradient-diluted 6 gradients of fully methylated positive standard subjected to bisulfite conversion.

[0485] 4) PCR reaction ​​

[0486] PCR program was set as 94°C pre-denaturation for 2 min; 94°C denaturation for 30 s, 60°C annealing and extension for 1 min, 45 cycles. Fluorescence signal was collected at the stage of 60°C annealing and extension.

[0487] 5) Analysis of detection results

[0488] ROC curve analysis was performed for the methylation level of each gene, as shown in FIG. 5C, the AUC of each gene was greater than 0.6. Figure 2A

[0489] Example 3: Multiple pre-amplification methylation-specific PCR (preAMP-MSP) for distinguishing between benign and malignant thyroid nodules

[0490] 1) Sample preparation

[0491] QIAamp Circulating Nucleic Acid Kit (QIAGEN, item number: 55114) was used to extract cfDNA from the plasma of 20 cases of thyroid cancer and 20 cases of benign thyroid nodules; Qubit TM dsDNA HS Assay Kit (Thermo, item number: Q32854) was used to detect the concentration of cfDNA; LabChip 3K Assay was used for quality control.

[0492] 2) DNA transformation

[0493] MethylCode TM Bisulfite Conversion Kit (Thermo, item number: MECOV50) was used to perform bisulfite conversion on the cfDNA obtained in step 1, unmethylated cytosine (C) was converted to uracil (U) after conversion; methylated cytosine did not change after conversion.

[0494] 3) Pre-amplification PCR reaction

[0495] The pre-amplification PCR mixture includes PCR reaction solution and primer mixture. The primer mixture contains one pair of primers for each of PRDM1 (SEQ ID NO: 4 and 5), ACRBP (SEQ ID NO: 6 and 7), UNCX (SEQ ID NO: 14 and 15), RPS7P5 (SEQ ID NO: 16 and 17), FOXI2 (SEQ ID NO: 18 and 19), and the internal reference gene (SEQ ID NO: 1 and 2).

[0496] Pre-amplification PCR reaction system composition

[0497] ​ Components Volume (μl) Platinum TM II Hot-Start PCR Master Mix (2x) 12.50 water 4.10 Multiplex primer mix, 100 μM 3.40 Sample cfDNA / positive control / negative control 5.00 Total 25.00

[0498] PCR program was set as 94℃ pre-denaturation 2 min; 94℃ denaturation 15 s, 60℃ annealing 45 s, 68℃ extension 1 min, 15 cycles; 72℃ extension 1 min, 4℃ preservation.

[0499] 4) MSP reaction

[0500] The reaction system was prepared according to the reagent kit instructions, including Platinum TM II Hot-Start PCR MasterMix (2x), water, primer mixture (as above), probe mixture (probe sequences as shown in SEQ ID NO: 20 (PRDM1), SEQ ID NO: 21 (ACRBP), SEQ ID NO: 25 (UNCX), SEQ ID NO: 26 (RPS7P5), SEQ ID NO: 27 (FOXI2) and SEQ ID NO: 3 (internal reference)), 1:100 diluted pre-amplification PCR product. Among them, the PRDM1 gene includes sites 106429722, 106429731, 106429747, 106429750, 106429761, 106429769, 106429771, the ACRBP gene includes sites 6756182, 6756187, 6756191, 6756195, 6756211, 6756225, 6756230, the UNCX gene includes sites: 1263659, 1263664, 1263676, the RPS7P5 gene includes sites: 240161502, 240161507, 240161511, 240161516, 240161523, 240161527, 240161530, 240161535, 240161546, the FOXI2 gene includes sites: 129534843, 129534853, 129534866, 129534879, 129534891, 129534910, 129534912, 129534924. The PCR program was set as 94℃ pre-denaturation 2 min; 94℃ denaturation 30 s, 60℃ annealing and extension 1 min, 45 cycles. The fluorescence signal was collected during the 60℃ annealing and extension stage.

[0501] 5) Analysis of detection results

[0502] Methylation level = 2 –ΔCt待检样品 / 2 –ΔCt阳性标准品 x 100. Among them, ΔCt = Ct 目的基因 -Ct 内参基因 .

[0503] The methylation levels of PRDMl gene, UNCX gene, RPS7P5 gene, FOXI2 gene and ACRBP gene were subjected to binary Logistic regression analysis, and the fitting equation was Score = -0.46 - 3.08 x PRDMl methylation level - 1.02 x UNCX methylation level + 12.06 x RPS7P5 methylation level - 11.71 x FOXI2 methylation level - 5.00 x ACRBP methylation level. The interpretation method was that if the score of the detected PRDMl, UNCX, RPS7P5, FOXI2 and ACRBP genes was greater than 0, the result was positive, i.e. malignant nodule.

[0504] The score is shown in Table 2, and the ROC analysis is shown in Figure 3 According to the interpretation criteria, 7 of 20 cases of benign thyroid nodules were positive, and 17 of 20 cases of thyroid cancer were positive, with a specificity of 80.0% and a sensitivity of 66.0%.

[0505] Table 2

[0506] Group Score Group Score Benign nodules -1.85 Malignant nodules -0.82 Benign nodules -6.26 Malignant nodules 2.47 Benign nodules -0.24 Malignant nodules 0.31 Benign nodules 1.19 Malignant nodules 7.76 Benign nodules 0.78 Malignant nodules 0.97 Benign nodules 0.98 Malignant nodules 0.17 Benign nodules -2.37 Malignant nodules 1.43 Benign nodules 0.84 Malignant nodules 7.90 Benign nodules 0.51 Malignant nodules 1.35 Benign nodules -0.33 Malignant nodules -0.47 Benign nodules -0.77 Malignant nodules 1.04 Benign nodules 0.32 Malignant nodules 1.66 Benign nodules -13.56 Malignant nodules 0.52 Benign nodules -2.61 Malignant nodules 0.56 Benign nodules -4.78 Malignant nodules 0.57 Benign nodules -3.27 Malignant nodules 1.33 Benign nodules 0.01 Malignant nodules 0.19 Benign nodules -3.82 Malignant nodules 0.11 Benign nodules -8.38 Malignant nodules 25.41 Benign nodules -8.60 Malignant nodules -0.09

[0507] Example 4, Multiple pre-amplification methylation-specific PCR (preAMP-MSP) for distinguishing between benign and malignant thyroid nodules

[0508] Steps 1) - 4) were the same as in Example 3, except that in step 3), the primer mixture contained each pair of primers for PRDM1 (SEQ ID NO: 4 and 5), ACRBP (SEQ ID NO: 6 and 7), TPO (SEQ ID NO: 8 and 9), MCC (SEQ ID NO: 10 and 11), TBX15 (SEQ ID NO: 12 and 13) and the internal reference gene (SEQ ID NO: 1 and 2). The probes for each gene were as shown in SEQ ID NO: 20 (PRDM1), SEQ ID NO: 21 (ACRBP), SEQ ID NO: 22 (TPO), SEQ ID NO: 23 (MCC), SEQ ID NO: 24 (TBX15) and SEQ ID NO: 3 (internal reference). In step 4), the PRDM1 gene sites included 106429722, 106429731, 106429747, 106429750, 106429761, 106429769, 106429771, the TPO gene sites included 1481013, 1481015, 1481022, 1481039, the ACRBP gene sites included 6756182, 6756187, 6756191, 6756195, 6756211, 6756225, 6756230, the MCC gene sites included 112538999, 112539011, 112539018, 112539022, 112539061, and the TBX15 gene sites included 119535740, 119535742, 119535750, 119535759, 119535766.

[0509] 5) Analysis of the detection results

[0510] Methylation level = 2 –ΔCt待检样品 / 2 –ΔCt阳性标准品 x 100. Wherein, ΔCt = Ct 目的基因 -Ct 内参基因 .

[0511] Binary Logistic regression analysis was performed on the methylation levels of the PRDM1 gene, the TPO gene, the ACRBP gene, the MCC gene and the TBX15 gene, and the fitting equation was Score = 1.83 - 4.26 x PRDM1 methylation level - 0.85 x TPO methylation level - 3.94 x ACRBP methylation level - 0.88 x MCC methylation level + 7.61 x TBX15 methylation level, and the interpretation method was that if the score of the detected PRDM1, TPO, ACRBP, MCC and TBX15 genes was greater than 0, the result was determined to be positive, i.e. malignant nodule.

[0512] The scores are shown in Table 3, and the ROC analysis is shown in Figure 4 According to the interpretation criteria, 6 of 20 cases of benign thyroid nodules were positive, 16 of 20 cases of thyroid cancer were positive, the specificity reached 80.0%, and the sensitivity was 80.0%.

[0513] Table 3

[0514] Group Score Group Score Benign nodules -0.58 Malignant nodules -2.03 Benign nodules -3.68 Malignant nodules 0.43 Benign nodules 0.82 Malignant nodules -0.08 Benign nodules 0.71 Malignant nodules 0.45 Benign nodules -2.77 Malignant nodules -0.16 Benign nodules 0.28 Malignant nodules 0.11 Benign nodules -2.63 Malignant nodules 0.50 Benign nodules 0.63 Malignant nodules 0.96 Benign nodules -0.10 Malignant nodules 0.12 Benign nodules -0.48 Malignant nodules 0.54 Benign nodules -2.44 Malignant nodules 0.78 Benign nodules 2.05 Malignant nodules 0.76 Benign nodules -10.72 Malignant nodules 0.93 Benign nodules -0.63 Malignant nodules 0.25 Benign nodules -2.71 Malignant nodules 0.66 Benign nodules -2.41 Malignant nodules 0.53 Benign nodules 0.08 Malignant nodules 0.82 Benign nodules -4.35 Malignant nodules 1.43 Benign nodules -6.52 Malignant nodules -1.29 Benign nodules -3.85 Malignant nodules 1.01

[0515] The above-described embodiments only express the implementation of the present application, and the description is more specific and detailed, but it cannot be understood as limiting the scope of the patent of the present application. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are within the scope of protection of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims. SEQUENCE LISTING <110> Jiangsu Yuyuan Biotechnology Co., Ltd. <120> Reagent for detecting DNA methylation and use <130> 20A602 <150> 202010038550.8 <151> 2020-01-14 <160> 27 <170> SIPOSequenceListing 1.0 <210> 1 <211> 27 <212> DNA <213> Artificial Sequence <400> 1 cccttaaaaa ttacaaaaac cacaacc 27 <210> 2 <211> 27 <212> DNA <213> Artificial Sequence <400> 2 aggaggttta gtaagttttt tggattg 27 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <400> 3 accaccaccc aacacacaat aacaaacaca 30 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <400> 4 tcctttcatc ccttccgc 18 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <400> 5 ttaggttagc gttggtttcg g 21 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <400> 6 ggtgttagag gtaggagtag ta 22 <210> 7 <211> 25 <212> DNA <213> Artificial Sequence <400> 7 cgaaacgaat tttaaaattc gatcc 25 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <400> 8 cgttttttgg gaatttgttt acgg 24 <210> 9 <211> 22 <212> DNA <213> Artificial Sequence <400> 9 ttcgacacta atccaattcc gc 22 <210> 10 <211> 26 <212> DNA <213> Artificial Sequence <400> 10 aaaaacacaa tccgaaatta atctcg 26 <210> 11 <211> 19 <212> DNA <213> Artificial Sequence <400> 11 gggagggata gcgttgagt 19 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <400> 12 acctaaccac acaaaactcc c 21 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <400> 13 taaaattgcg cgattgttcg 20 <210> 14 <211> 27 <212> DNA <213> Artificial Sequence <400> 14 acctaaaata aaacctcttc raattcc 27 <210> 15 <211> 22 <212> DNA <213> Artificial Sequence <400> 15 tgggtatgag ygttggtatt tg 22 <210> 16 <211> 15 <212> DNA <213> Artificial Sequence <400> 16 tttatgtgcg gggcg 15 <210> 17 <211> 18 <212> DNA <213> Artificial Sequence <400> 17 ccaaccgaaa caactacg 18 <210> 18 <211> 19 <212> DNA <213> Artificial Sequence <400> 18 agttcgttgt ttttcgggt 19 <210> 19 <211> 22 <212> DNA <213> Artificial Sequence <400> 19 acgaaataaa aaacgcgaaa ct 22 <210> 20 <211> 21 <212> DNA <213> Artificial Sequence <400> 20 ccacccgtat aaccctcgcc g 21 <210> 21 <211> 16 <212> DNA <213> Artificial Sequence <400> 21 cggagcgggc gaacgg 16 <210> 22 <211> 27 <212> DNA <213> Artificial Sequence <400> 22 aactaccata cacgataaac gcgtcca 27 <210> 23 <211> 26 <212> DNA <213> Artificial Sequence <400> 23 cctaccgcac gcctattcaa taacct 26 <210> 24 <211> 25 <212> DNA <213> Artificial Sequence <400> 24 accctactcc tacgcaaacc gaaat 25 <210> 25 <211> 24 <212> DNA <213> Artificial Sequence <400> 25 cctcccgaaa aaaaccacgc aacg 24 <210> 26 <211> 24 <212> DNA <213> Artificial Sequence <400> 26 tcgtttcgtt attcgtacgg cgtt 24 <210> 27 <211> 28 <212> DNA <213> Artificial Sequence <400> 27 cgaggatttt agacgatttt agggtcgt 28

Claims

1. Use of a reagent for detecting DNA methylation levels in preparing a kit for identifying the nature of thyroid nodules, wherein the reagent is a primer, and the reagent detects the DNA methylation level of a gene site, wherein: The loci of the gene include (a1) loci of the PRDM1 gene: 106429722, 106429731, 106429747, 106429750, 106429761, 106429769 and 106429771 of chromosome 6, and the loci refer to the human reference genome hg19 version.

2. Use of a reagent for detecting DNA methylation levels in the preparation of a kit for identifying the nature of thyroid nodules, wherein the reagent is a primer, and the reagent detects the DNA methylation level of a gene fragment, wherein: The gene includes PRDM1, the fragment of the PRDM1 gene is 50-1000 bp long, and the fragment of the PRDM1 gene includes (a1) the sites of the PRDM1 gene: 106429722, 106429731, 106429747, 106429750, 106429761, 106429769 and 106429771 of chromosome 6, and the sites are referenced to the human reference genome hg19 version.

3. The use according to claim 1 or 2, characterized in that The reagent also detects the DNA methylation level of (a2) sites of the ACRBP gene: 6756182, 6756187, 6756191, 6756195, 6756211, 6756225 and 6756230 on chromosome 12, with reference to the human reference genome hg19 version.

4. The use according to claim 3, characterized in that The reagent also detects the DNA methylation level of the loci of one or more groups of genes in (a3)-(a8): (a3) TPO gene loci: 1481013, 1481015, 1481022 and 1481039 on chromosome 2, (a4) MCC gene loci: 112538999, 112539011, 112539018, 112539022, and 112539061 on chromosome 5, (a5) The loci of the TBX15 gene: 119535740, 119535742, 119535750, 119535759 and 119535766 on chromosome 1, (a6) UNCX gene loci: 1263659, 1263664, and 1263676 on chromosome 7, (a7) The loci of the RPS7P5 gene are 240161502, 240161507, 240161511, 240161516, 240161523, 240161527, 240161530, 240161535, and 240161546 on chromosome 1. (a8) FOXI2 gene loci: 129534843, 129534853, 129534866, 129534879, 129534891, 129534910, 129534912, and 129534924 on chromosome 10; The loci are referenced to the human reference genome hg19 version.

5. The use according to claim 1 or 2, characterized in that The reagent also detects the DNA methylation level of a fragment of the ACRBP gene, wherein the fragment is 50-1000 bp in length, and the fragment of the ACRBP gene includes the sites of the (a2) ACRBP gene: 6756182, 6756187, 6756191, 6756195, 6756211, 6756225 and 6756230 of chromosome 12, and the sites are referenced to the human reference genome hg19 version.

6. The use according to claim 5, characterized in that The reagent also detects the DNA methylation level of a nucleic acid fragment of one or more genes selected from the group consisting of: TPO, MCC, TBX15, UNCX, RPS7P5, FOXI2, wherein the fragment is 50-1000 bp in length, The fragment of the TPO gene contains (a3) ​​TPO gene sites: 1481013, 1481015, 1481022 and 1481039 on chromosome 2, The fragment of the MCC gene contains the sites of (a4) MCC gene: 112538999, 112539011, 112539018, 112539022 and 112539061 on chromosome 5, The fragment of TBX15 gene includes the loci of (a5) TBX15 gene: 119535740, 119535742, 119535750, 119535759 and 119535766 on chromosome 1, The fragment of the UNCX gene contains the sites of the (a6) UNCX gene: 1263659, 1263664 and 1263676 on chromosome 7, The fragment of RPS7P5 gene includes the loci of (a7)RPS7P5 gene: 240161502, 240161507, 240161511, 240161516, 240161523, 240161527, 240161530, 240161535 and 240161546 on chromosome 1, The fragment of the FOXI2 gene includes (a8) FOXI2 gene loci: 129534843, 129534853, 129534866, 129534879, 129534891, 129534910, 129534912, and 129534924 of chromosome 10; The loci are referenced to the human reference genome hg19 version.

7. The use according to claim 4 or 6, characterized in that The sites include: (a1)-(a2) and one or more groups selected from (a3)-(a5), or (a1)-(a2) and one or more groups selected from (a6)-(a8).

8. The use according to claim 1 or 2, characterized in that The primers can amplify (b1) a fragment of the PRDM1 gene amplified by using SEQ ID NOs: 4 and 5 as primers.

9. The use according to claim 8, characterized in that The primers can also amplify (b2) a fragment of the ACRBP gene amplified using SEQ ID NOs: 6 and 7 as primers.

10. The use according to claim 9, characterized in that The primers are also capable of amplifying one or more fragments selected from the group consisting of: (b3) a fragment of the TPO gene amplified using SEQ ID NOs: 8 and 9 as primers, (b4) a fragment of the MCC gene amplified using SEQ ID NOs: 10 and 11 as primers, (b5) a fragment of the TBX15 gene amplified using SEQ ID NOs: 12 and 13 as primers, (b6) a fragment of the UNCX gene amplified using SEQ ID NOs: 14 and 15 as primers, (b7) a fragment of the RPS7P5 gene amplified using SEQ ID NOs: 16 and 17 as primers, and (b8) A fragment of the FOXI2 gene amplified using SEQ ID NOs: 18 and 19 as primers.

11. The use according to claim 10, characterized in that The primers are capable of amplifying: (b1)-(b2) and one or more groups selected from (b3)-(b5), or (b1)-(b2) and one or more groups selected from (b6)-(b8).

12. The use according to claim 1 or 2, characterized in that The reagents further include a probe capable of hybridizing with (b1) a fragment of the PRDM1 gene amplified using SEQ ID NOs: 4 and 5 as primers.

13. The use according to claim 12, characterized in that The probe can also hybridize with (b2) a fragment of the ACRBP gene amplified using SEQ ID NOs: 6 and 7 as primers.

14. The use according to claim 13, characterized in that The probe is also capable of hybridizing to one or more fragments selected from the group consisting of: (b3) a fragment of the TPO gene amplified using SEQ ID NOs: 8 and 9 as primers, (b4) a fragment of the MCC gene amplified using SEQ ID NOs: 10 and 11 as primers, (b5) a fragment of the TBX15 gene amplified using SEQ ID NOs: 12 and 13 as primers, (b6) a fragment of the UNCX gene amplified using SEQ ID NOs: 14 and 15 as primers, (b7) a fragment of the RPS7P5 gene amplified using SEQ ID NOs: 16 and 17 as primers, and (b8) A fragment of the FOXI2 gene amplified using SEQ ID NOs: 18 and 19 as primers.

15. The use according to claim 14, characterized in that The probe is capable of hybridizing to the following fragments: (b1)-(b2) and one or more groups selected from (b3)-(b5), or (b1)-(b2) and one or more groups selected from (b6)-(b8).

16. The use according to any one of claims 2 to 15, characterized in that The fragments include the sense or antisense strand of DNA.

17. The use according to any one of claims 1 to 15, characterized in that Reagents for detecting DNA methylation also include reagents used in one or more of the following methods: PCR based on bisulfite conversion, DNA sequencing, methylation-sensitive restriction endonuclease analysis, fluorescence quantification, methylation-sensitive high-resolution melting curve method, chip-based methylation mapping analysis, and mass spectrometry.

18. The use according to any one of claims 1 to 15, characterized in that Reagents for detecting DNA methylation also include one or more reagents selected from the following: bisulfite, PCR buffer, polymerase, dNTP, methylation-sensitive or -insensitive restriction endonuclease, enzyme digestion buffer, fluorescent dye, fluorescence quencher, fluorescence reporter, exonuclease, alkaline phosphatase.

19. The use according to any one of claims 1 to 15, characterized in that The reagent for detecting DNA methylation also includes an internal standard.

20. The use according to any one of claims 1 to 15, characterized in that The reagents for detecting DNA methylation also include controls.

21. Use of a reagent for detecting DNA methylation in the preparation of a kit for identifying the nature of thyroid nodules in a sample, wherein the reagent detects the DNA methylation level of a gene site, wherein the gene site includes (a1) sites of the PRDM1 gene: 106429722, 106429731, 106429747, 106429750, 106429761, 106429769 and 106429771 of chromosome 6, and the sites are referenced to the human reference genome hg19 version.

22. The use according to claim 21, characterized in that The reagent also includes a nucleic acid molecule having a nucleic acid sequence of a fragment of the PRDM1 gene, wherein the fragment of the PRDM1 gene is 50-1000 bp in length, and the fragment of the PRDM1 gene includes (a1) sites of the PRDM1 gene: 106429722, 106429731, 106429747, 106429750, 106429761, 106429769 and 106429771 of chromosome 6, and the sites are referenced to the human reference genome hg19 version.

23. The use according to claim 22, characterized in that The nucleic acid molecule comprises a fragment of the PRDM1 gene amplified using SEQ ID NOs: 4 and 5 as primers.

24. The use according to claim 22, characterized in that The reagent also detects the DNA methylation level of (a2) sites of the ACRBP gene: 6756182, 6756187, 6756191, 6756195, 6756211, 6756225 and 6756230 on chromosome 12, with reference to the human reference genome hg19 version.

25. The use according to claim 24, characterized in that The nucleic acid molecule also has a nucleic acid sequence of a fragment of the ACRBP gene, wherein the fragment is 50-1000 bp long, and the fragment of the ACRBP gene includes (a2) sites of the ACRBP gene: 6756182, 6756187, 6756191, 6756195, 6756211, 6756225 and 6756230 of chromosome 12, and the sites are referenced to the human reference genome hg19 version.

26. The use according to claim 24, characterized in that The reagent also detects the DNA methylation level of the loci of one or more groups of genes in (a3)-(a8): (a3) The loci of the TPO gene include: 1481013, 1481015, 1481022 and 1481039 on chromosome 2, (a4) The loci of the MCC gene include: 112538999, 112539011, 112539018, 112539022 and 112539061 on chromosome 5, (a5) The loci of the TBX15 gene include: 119535740, 119535742, 119535750, 119535759 and 119535766 on chromosome 1, (a6) The loci of the UNCX gene include: 1263659, 1263664 and 1263676 on chromosome 7, (a7) The loci of the RPS7P5 gene include: 240161502, 240161507, 240161511, 240161516, 240161523, 240161527, 240161530, 240161535 and 240161546 on chromosome 1, (a8) The loci of the FOXI2 gene include: 129534843, 129534853, 129534866, 129534879, 129534891, 129534910, 129534912, and 129534924 on chromosome 10; The loci are referenced to the human reference genome hg19 version.

27. The use according to claim 26, characterized in that The nucleic acid molecule further comprises a nucleic acid sequence of a fragment of a gene selected from the group consisting of TPO, MCC, TBX15, UNCX, RPS7P5, and FOXI2, wherein the fragment is 50-1000 bp in length, The fragment of the TPO gene contains (a3) ​​TPO gene sites: 1481013, 1481015, 1481022 and 1481039 on chromosome 2, The fragment of the MCC gene contains the sites of (a4) MCC gene: 112538999, 112539011, 112539018, 112539022 and 112539061 on chromosome 5, The fragment of TBX15 gene includes the loci of (a5) TBX15 gene: 119535740, 119535742, 119535750, 119535759 and 119535766 on chromosome 1, The fragment of the UNCX gene contains the sites of the (a6) UNCX gene: 1263659, 1263664 and 1263676 on chromosome 7, The fragment of RPS7P5 gene includes the loci of (a7)RPS7P5 gene: 240161502, 240161507, 240161511, 240161516, 240161523, 240161527, 240161530, 240161535 and 240161546 on chromosome 1, The fragment of the FOXI2 gene includes (a8) FOXI2 gene loci: 129534843, 129534853, 129534866, 129534879, 129534891, 129534910, 129534912, and 129534924 of chromosome 10; The loci are referenced to the human reference genome hg19 version.

28. The use according to any one of claims 21 to 27, characterized in that The use has one or more characteristics selected from the following: The kit further comprises a reagent for detecting the mutation level of the V600E site of the BRAF gene and / or the mutation level of the C228T / C250T site of the TERT gene. The identifying the nature of the thyroid nodule comprises: comparing with a control sample, or obtaining a score according to the methylation level and / or mutation level, and identifying the nature of the thyroid nodule according to the comparison result or the score, The sample is from a human.

29. The use according to claim 28, characterized in that The sample contains genomic DNA.

30. The use according to claim 28, wherein The sample contains cfDNA.

31. The use according to claim 28, wherein The sample was from thyroid tissue.

32. The use according to claim 28, wherein The sample is from blood.

33. The use according to claim 28, wherein The samples were from plasma.

34. The use according to claim 28, wherein The samples were from thyroid nodule biopsies.

Images