Reagents and applications for detecting DNA methylation

Abstract

This article discloses a method for identifying the nature of thyroid nodules, including detecting the DNA methylation level in a sample selected from the following regions (1) and (2): (1) fragments of one or more of the following genes: COL23A1, ILDR2, DHRS3, KIF1A, GDNF, TBX18, and (2) nucleic acid regions within 10 kb upstream and downstream of the genes (1).

Description

Technical Field

[0001] This invention belongs to the field of molecular-assisted diagnosis, specifically relating to its application in screening for benign and malignant thyroid nodules. Background Technology

[0002] DNA methylation is an epigenetic mechanism and a common epigenetic modification of the eukaryotic genome. It is also an important natural chemical modification of vertebrate DNA without altering the DNA sequence, playing a crucial role in cell proliferation, differentiation, and development, and is closely related to tumorigenesis and progression. DNA methylation has significant effects in vivo, including transcriptional repression, chromatin structure regulation, X chromosome inactivation, and genomic imprinting. Abnormal DNA methylation can participate in tumorigenesis and progression by affecting chromatin structure and the expression of oncogenes and tumor suppressor genes.

[0003] CpG dinucleotides are the primary targets of DNA methylation in mammals, distributed throughout the chromosome set. In the genome of healthy individuals, CpG sites within CpG islands are typically unmethylated, while those outside the islands are usually hypermethylated. This form of methylation is stably maintained during cell division. When tumors develop, the methylation level of CpG sites outside CpG islands in tumor suppressor genes is usually reduced, while CpG sites within CpG islands are highly methylated, leading to altered chromatin structure and decreased expression of tumor suppressor genes.

[0004] With the continuous development of genetics and epigenetics over the past decade, more and more researchers have realized that tumorigenesis is not entirely determined by genetic factors; acquired epigenetic influences also play a significant role. Epigenetic alterations in thyroid cancer are mainly manifested as abnormal methylation of tumor suppressor genes and thyroid-related genes. Studying DNA methylation in thyroid cancer can provide us with new molecular markers, offering reliable evidence for early diagnosis, treatment selection, and prognostic assessment.

[0005] Thyroid nodules are clumps that form in the thyroid tissue due to abnormal proliferation of thyroid cells. Thyroid nodules are very common, and although most are benign, a small percentage can progress to thyroid cancer. To facilitate earlier diagnosis and treatment of thyroid cancer, and to reduce unnecessary surgery, it is essential to differentiate between benign and malignant thyroid nodules.

[0006] Currently, the evaluation of thyroid nodules mainly relies on ultrasonography (US) and fine needle aspiration biopsy (FNAB). In the diagnostic process for thyroid nodules, US is currently the most sensitive examination method, capable of measuring nodule size and determining its internal structure. US signs suggesting malignancy include: nodule height greater than width (OR = 10.15), lack of halo (OR = 7.14), microcalcifications (OR = 6.76), irregular borders (OR = 6.12), decreased echogenicity (OR = 5.07), solid nodule (OR = 4.69), and abundant internal blood flow (OR = 3.76). Nodules larger than 1 cm in diameter with malignant features on ultrasound are then subjected to FNAB to determine their nature. Up to 20% of nodules on cytological examination are indeterminate thyroid nodules; these require further molecular testing. Commercially available... The Gene Expression Classifier and ThyroSeqv2 products have very low positive predictive value (PPV), with the former having a very low PPV of only 46% and the latter only 42%-77%. Therefore, more accurate molecular diagnostic tools are needed.

[0007] There is still a need in this field for highly specific and sensitive methods for the diagnosis of thyroid nodules. Summary of the Invention

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

[0009] The first aspect of the present invention provides an isolated nucleic acid molecule from a mammal, said nucleic acid molecule having a sequence of nucleic acids selected from the following (1) and (2) or a variant having at least 70% identity with them: (1) a fragment of nucleic acid selected from one or more of the following genes: COL23A1, ILDR2, DHRS3, KIF1A, GDNF, TBX18, said fragment being 50-1000 bp in length, wherein the fragment of the COL23A1 gene contains the following COL23A1 gene sites: 178003785, 178003798, 178003803, 178003814, 178003823, 178003825, 17800 One or more of the following ILDR2 gene fragments: 3834, 178003841, and 178003844; ILDR2 gene fragments containing the following ILDR2 gene loci: 166890429, 166890436, 166890440, 166890442, 166890448, 166890452, 166890456, 166890461, 166890468, 166890473, 166890475, 166890480, 166890492, 166890500, 166890503, 166890509, 166890516, 166890528, 1 One or more of the following sequences are included: 66890535, 166890543, 166890555, 166890559, 166890568, 166890573, 166890584, and 166890586. The fragments containing the DHRS3 gene at the following loci are: 12656091, 12656114, 12656132, 12656152, 12656170, 12656175, 12656182, 12656187, 12656197, 12656200, 12656211, 12656315, 12656323, and 12656340. One or more of 2656355 and 12656367; KIF1A gene fragments containing KIF1A gene loci: one or more of 241759696, 241759701, 241759714, and 241759716; GDNF gene fragments containing GDNF gene loci: one or more of 37834763, 37834770, 37834772, 37834774, 37834777, 37834780, 37834784, 37834792, 37834799, 37834802, 37834806, and 37834811.The fragment of the TBX18 gene contains one or all of the following sites of the TBX18 gene: 85477032, 85477035, 85477070, 85477083, 85477106, 85477124, 85477151, 85477153, and 85477166, and (2) the nucleic acid region within 10 kb upstream and downstream of the gene in (1), wherein the above-mentioned sites in the variant are not mutated.

[0010] In one or more embodiments, the fragment of the COL23A1 gene contains one or more of the following COL23A1 gene sites: 178003798, 178003803, 178003814, 178003823, 178003825, 178003834, 178003841, and 178003844, and the fragment of the ILDR2 gene contains the following ILDR2 gene sites: 166890516, 166890528, and 166890. One or more of the following sequences are included: 535, 166890543, 166890555, 166890559, 166890568, 166890573, 166890584, and 166890586. The fragments containing the DHRS3 gene at the following loci are: 12656091, 12656114, 12656132, 12656152, 12656170, 12656175, 12656182, and 12656187. The fragments containing one or more of the following KIF1A gene loci: 12656197, 12656200, 12656211, 12656315, 12656323, 12656340, 12656355, and 12656367; the fragments containing the following KIF1A gene loci: 241759696, 241759701, 241759714, and 241759716; and the fragments containing the following GDNF gene loci: One or more of the following TBX18 gene fragments: 37834770, 37834772, 37834774, 37834777, 37834780, 37834784, 37834792, 37834799, 37834802, 37834806, and 37834811; and one or more of the following TBX18 gene fragments: 85477035, 85477070, 85477083, and 85477106.

[0011] In one or more embodiments, the fragment of the COL23A1 gene contains one or more of the following COL23A1 gene loci: 178003798, 178003803, 178003814, 178003823, 178003825, 178003834, 178003841, and 178003844, and the fragment of the ILDR2 gene contains the following ILDR2 gene locus: 1668905. One or more of the following sequences are included: 16, 166890528, 166890535, 166890543, 166890555, 166890559, 166890568, 166890573, 166890584, and 166890586. The following loci contain the DHRS3 gene: 12656170, 12656175, 12656182, and 1265619. 7. One or all of the following KIF1A gene fragments: 12656200, 12656211, 12656315, 12656323; KIF1A gene fragments containing KIF1A gene loci: one or all of the following 241759696, 241759701, 241759714, 241759716; GDNF gene fragments containing GDNF gene loci: 37834770, 37834772. One or more of the following 37834774, 37834777, 37834780, 37834784, 37834792, 37834799, 37834802, 37834806, and 37834811; and the following TBX18 gene fragments containing the following TBX18 gene loci: one or more of the following 85477035, 85477070, 85477083, and 85477106.

[0012] In one or more embodiments, the nucleic acid molecule comprises one or more fragments selected from the following: a fragment of the COL23A1 gene amplified using SEQ ID NO:4 and 5 as primers, a fragment of the ILDR2 gene amplified using SEQ ID NO:6 and 7 as primers, a fragment of the DHRS3 gene amplified using SEQ ID NO:8 and 9 as primers, a fragment of the KIF1A gene amplified using SEQ ID NO:10 and 11 as primers, a fragment of the GDNF gene amplified using SEQ ID NO:12 and 13 as primers, and a fragment of the TBX18 gene amplified using SEQ ID NO:14 and 15 as primers.

[0013] A second aspect of the present invention provides a reagent for detecting DNA methylation, the reagent comprising a reagent for detecting the level of DNA methylation selected from the regions described in (1) and (2) below: (1) fragments selected from one or more genes: ZMIZ1, C15orf52, SLC16A3, ZNF512B, SLC17A5, LIMK1, PLEC, TOR4A, TMEM131L, DNM2, IL17C, PRDM16, MT1JP, TBX3, BIN1, TIMP2, CFAP65, TSHR, KIF1A, DAPK, CDH1, TPO, RAR G, 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 length is 50-1000bp, (2) (1) the nucleic acid region within 5Kb or 10Kb upstream and downstream of the gene.

[0014] In one or more embodiments, the reagent for detecting DNA methylation levels detects the methylation levels of fragments of one or more of the following groups of genes: (a) selected from one or more of the following genes: COL23A1, ILDR2, DHRS3, KIF1A, GDNF, TBX18; (b) COL23A1, ILDR2, DHRS3; (c) COL23A1, ILDR2, DHRS3, and KIF1A; (d) COL23A1, ILDR2, DHRS3, and one or two of GDNF and TBX18; and (e) nucleic acid regions within 5 kb or 10 kb upstream or downstream of any one of the groups of genes in (a)-(d).

[0015] In one or more embodiments, the fragments of each gene contain a corresponding nucleic acid region within 500 bp upstream or downstream of one or more sites selected from the following sites:

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

[0017] C15orf52: Chromosome 15, numbers 40626309, 40626312, and 40626386.

[0018] SLC16A3: Chromosome 17, numbers 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.

[0019] ZNF512B: Chromosome 20, numbers 62588634, 62588638, and 62588672.

[0020] SLC17A5: Chromosome 6, numbers 74290205, 74290207, 74290220, 74290225, and 74290228.

[0021] LIMK1: Chromosome 7, numbers 73508994, 73509017, 73509055, 73509062, 73509073, 73509075, 73509112, 73509133, 73509138, 73509148, 73509160.

[0022] PLEC: Chromosome 8, 145013661, 145013673,

[0023] TOR4A: Chromosome 9, digits 140172787, 140172790, and 140172812.

[0024] TMEM131L: Chromosome 4, numbers 154409945, 154409963, 154409972, 154409978, 154409997, 154410003, 154410006.

[0025] DNM2: Chromosome 19, numbers 10870373, 10870377, 10870427, 10870429, 10870441, and 10870448.

[0026] IL17C: Chromosome 16, numbers 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: Chromosome 1, numbers 3229914, 3229921, 3229950, 3229968, 3229973, 3310213, 3310229, 3310235, 3310238, 3310240, 3310268, 3310287, 3310312, 3310314, 3310317, 3310329.

[0028] TSHR: Chromosome 14, numbers 81421983, 81421989, 81422010, 81422017, 81422032, 81422035, 81422063, 81422084.

[0029] KIF1A: Chromosome 2, numbers 241759696, 241759701, 241759714, and 241759716.

[0030] DAPK: Chromosome 90112842, 90112853, 90112861, 90112866,

[0031] CDH1: Chromosome 16, numbers 68771035, 68771037, 68771045, 68771051, 68771059, 68771064, and 68771073.

[0032] TPO: chromosome 2, numbers 1481013, 1481015, 1481022, and 1481039.

[0033] RARG: Chromosome 12, numbers 53613176, 53613182, 53613190, 53613202, 53613210, 53613218.

[0034] MT1JP: Chromosome 16, numbers 56669271, 56669292, 56669295, 56669300, 56669318, 56669322, 56669324, 56669327, 56669344, 56669351, 56669353, 56669402, 56669414, 56669423, 56669430, 56669433, 56669437, 5666945 1. 56669453, 56669455, 56669463, 56669474, 56669480, 56669482, 56669485, 56669487, 56669490, 56669519, 56669533, 56669553, 56669564, 56669573, 56669578, 56669588, 56669590, 56669606, 56669610

[0035] TBX3: Chromosome 12, numbers 115174750, 115174773, and 115174780.

[0036] BIN1: Chromosome 2, numbers 127822478, 127822492, 127822495, 127822514, 127822551, 127822568, 127822582, 127822593, 127822616, 127822644.

[0037] TIMP2: Chromosome 17, numbers 76921845, 76921853, and 76921860.

[0038] CFAP65: Chromosome 2, numbers 219866132, 219866139, 219866148, 219866158, 219866165, 219866168, 219866199, 219866218.

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

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

[0041] MCC: Chromosome 5, numbers 112538999, 112539011, 112539018, 112539022, 112539061, 112539084, 112539104, 112539128.

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

[0043] COL23A1: Chromosome 5, numbers 178003785, 178003798, 178003803, 178003814, 178003823, 178003825, 178003834, 178003841, 178003844.

[0044] ILDR2: Chromosome 1, numbers 166890429, 166890436, 166890440, 166890442, 166890448, 166890452, 166890456, 166890461, 166890468, 166890473, 166890475, 166890480, 1668 90492, 166890500, 166890503, 166890509, 166890516, 166890528, 166890535, 166890543, 166890555, 166890559, 166890568, 166890573, 166890584, 166890586,

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

[0046] GDNF: Chromosome 5, numbers 37834763, 37834770, 37834772, 37834774, 37834777, 37834780, 37834784, 37834792, 37834799, 37834802, 37834806, 37834811.

[0047] TBX18: Chromosome 6, numbers 85477032, 85477035, 85477070, 85477083, 85477106, 85477124, 85477151, 85477153, 85477166.

[0048] SIM2: Chromosome 21, numbers 38069563, 38069579, 38069619, 38069625, 38069638, 38069650, 38069662, 38069664, 38069676, 38069681.

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

[0050] EHBP1L1: Chromosome 11, numbers 65352612, 65352621, 65352635, 65352639, 65352642, 65352651, 65352654, 65352665, 65352670.

[0051] GJC2: Chromosome 1, numbers 228345954, 228345957, 228345965, 228345978, 228345980, 228345989.

[0052] RCOR2: Chromosome 11, numbers 63687223, 63687238, 63687247, 63687250, 63687259, 63687282, 63687288, 63687299, 63687318, 63687325.

[0053] PRDM1: Chromosome 6, numbers 106429711, 106429722, 106429731, 106429747, 106429750, 106429761, 106429769, 106429771.

[0054] UNCX: Chromosome 7, numbers 1263643, 1263655, 1263659, 1263664, 1263676, 1263694, 1263716, 1263723.

[0055] RPS7P5: Chromosome 1, numbers 240161502, 240161507, 240161511, 240161516, 240161523, 240161527, 240161530, 240161535, 240161546, 240161558, 240161560.

[0056] FOXI2: Chromosome 10, numbers 129534843, 129534853, 129534866, 129534879, 129534891, 129534910, 129534912, and 129534924.

[0057] ACRBP: Chromosome 12, numbers 6756182, 6756187, 6756191, 6756195, 6756211, 6756225, 6756230, 6756270.

[0058] GAS6: Chromosome 13, numbers 114524043, 114524062, 114524068, 114524084, 114524095, 114524131, 114524138, 114524142, 114524150, 114524158.

[0059] MCRIP2: Chromosome 16, numbers 698072, 698142, 698153, 698168, 698208, 698218, 698222, 698230.

[0060] LINC01977: Chromosome 17 numbers 77789596, 77789601, 77789612, 77789620, 77789628, 77789632, 77789635, 77789640.

[0061] EGR3: Chromosome 8, numbers 22548250, 22548260, 22548269, 22548279, 22548283, 22548287, 22548296, 22548299.

[0062] SOX17: Chromosome 8, numbers 55379566, 55379568, 55379573, 55379579, 55379583, 55379591, 55379599, 55379602, 55379608, 55379617, 55379620.

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

[0064] NEURL1: Chromosome 10, numbers 105344464, 105344482, 105344493, 105344495, 105344497, 105344503, 105344506, 105344513, 105344516, 105344519, 105344526.

[0065] IRX4: Chromosome 5, numbers 1876386, 1876395, 1876397, 1876403, 1876420, 1876424, 1876432, 1876436, 1876449, 1876456, 1876459, 1876463.

[0066] RUSC1: Chromosome 1, numbers 155295135, 155295171, 155295181, 155295192, 155295196, 155295212, 155295229, and 155295236.

[0067] Preferably, the fragment of the ZMIZ1 gene includes one or more of the following ZMIZ1 gene loci: 81002041, 81002052, 81002054, 81002056, 81002062, and 81002083.

[0068] The fragment of the C15orf52 gene contains one or more of the C15orf52 gene sites 40626309 and 40626312.

[0069] The fragment of the SLC16A3 gene contains one or more of the following SLC16A3 gene loci: 80189671, 80189674, 80189684, 80189687, 80189698, 80189709, 80189719, 80189726, 80189728, 80189739, and 80189757.

[0070] The fragment of the ZNF512B gene contains one or more of the following ZNF512B gene loci: 62588634, 62588638, and 62588672.

[0071] The fragment of the SLC17A5 gene contains one or more of the following SLC17A5 gene loci: 74290205, 74290207, 74290220, 74290225, and 74290228.

[0072] The fragment of the LIMK1 gene contains one or more of the following LIMK1 gene loci: 73509112, 73509133, 73509138, 73509148, and 73509160.

[0073] The PLEC gene fragment contains one or more of the PLEC gene loci 145013661 and 145013673.

[0074] The fragment of the TOR4A gene contains one or more of the TOR4A gene loci 140172787, 140172790, and 140172812.

[0075] The fragment of the TMEM131L gene contains one or more of the following TMEM131L gene loci: 154409945, 154409963, 154409972, 154409978, and 154409997.

[0076] The fragment of the DNM2 gene contains one or more of the following DNM2 gene loci: 10870427, 10870429, 10870441, and 10870448.

[0077] The fragment of the IL17C gene contains one or more of the following IL17C gene loci: 88701004, 88701021, 88701029, 88701036, 88701043, 88701051, and 88701060.

[0078] The fragment of the PRDM16 gene contains one or more of the PRDM16 gene loci 3229950, 3229968, and 3229973.

[0079] The fragment of the MT1JP gene contains one or more of the following MT1JP gene loci: 56669271, 56669292, 56669295, 56669300, 56669318, 56669322, 56669324, 56669327, and 56669344.

[0080] The fragment of the TBX3 gene contains one or more of the TBX3 gene loci 115174750, 115174773, and 115174780.

[0081] The fragment of the BIN1 gene contains one or more of the following BIN1 gene loci: 127822478, 127822492, 127822495, 127822514, 127822551, 127822568, 127822582, 127822593, and 127822616.

[0082] The fragment of the TIMP2 gene contains one or more of the TIMP2 gene loci 76921845, 76921853, and 76921860.

[0083] The fragment of the CFAP65 gene contains one or more of the CFAP65 gene loci 219866199 and 219866218.

[0084] The TSHR gene fragment contains one or more of the following TSHR gene loci: 81421983, 81421989, 81422010, 81422017, 81422032, 81422035, 81422063, and 81422084.

[0085] The fragment of the KIF1A gene contains one or more of the following KIF1A gene loci: 241759696, 241759701, 241759714, and 241759716.

[0086] The fragment of the DAPK gene contains one or more of the following DAPK gene sites: 90112842, 90112853, 90112861, and 90112866.

[0087] The fragment of the CDH1 gene contains one or more of the following CDH1 gene sites: 68771035, 68771037, 68771045, 68771051, 68771059, 68771064, and 68771073.

[0088] The fragment of the TPO gene contains one or more of the following TPO gene loci: 1481013, 1481015, 1481022, and 1481039.

[0089] The fragment of the RARG gene contains one or more of the following RARG gene loci: 53613176, 53613182, 53613190, 53613202, 53613210, and 53613218.

[0090] The fragment of the PRR15 gene contains one or more of the following PRR15 gene loci: 29606026, 29606040, 29606047, 29606056, 29606062, 29606073, 29606220, 29606222, 29606227, 29606231, 29606255, 29606257, 29606262, 29606271, 29606277, and 29606289.

[0091] The DPYS gene fragment contains one or more of the following DPYS gene loci: 105478905, 105478908, 105478916, 105478918, 105478945, 105478956, 105478965, 105478974, and 105478983.

[0092] The fragment of the MCC gene contains one or more of the following MCC gene sites: 112538999, 112539011, 112539018, 112539022, and 112539061.

[0093] The fragment of the TBX15 gene contains one or more of the following TBX15 gene loci: 119535740, 119535742, 119535750, 119535759, and 119535766.

[0094] The fragment of the COL23A1 gene contains one or more of the following COL23A1 gene loci: 178003798, 178003803, 178003814, 178003823, 178003825, 178003834, 178003841, and 178003844.

[0095] The fragment of the ILDR2 gene contains one or more of the following ILDR2 gene loci: 166890516, 166890528, 166890535, 166890543, 166890555, 166890559, 166890568, 166890573, 166890584, and 166890586.

[0096] The fragment of the DHRS3 gene contains one or more of the following DHRS3 gene loci: 12656340, 12656355, and 12656367.

[0097] The fragment containing the GDNF gene contains one or more of the following GDNF gene loci: 37834770, 37834772, 37834774, 37834777, 37834780, 37834784, 37834792, 37834799, 37834802, 37834806, and 37834811.

[0098] The fragments of the TBX18 gene contain one or more of the following TBX18 gene loci: 85477035, 85477070, 85477083, and 85477106.

[0099] The fragment containing the SIM2 gene contains one or more of the following SIM2 gene loci: 38069638, 38069650, 38069662, 38069664, 38069676, and 38069681.

[0100] The fragment of the HOXA9 gene contains one or more of the following HOXA9 gene loci: 27204854, 27204858, 27204861, 27204863, and 27204879.

[0101] The fragment of the EHBP1L1 gene contains one or more of the following EHBP1L1 gene loci: 65352621, 65352635, 65352639, 65352642, 65352651, 65352654, 65352665, and 65352670.

[0102] The fragment containing the GJC2 gene contains one or more of the following GJC2 gene loci: 228345965, 228345978, 228345980, and 228345989.

[0103] The fragment containing the RCOR2 gene contains one or more of the following RCOR2 gene loci: 63687223, 63687238, 63687247, 63687250, and 63687259.

[0104] The fragment containing the PRDM1 gene contains one or more of the following PRDM1 gene sites: 106429722, 106429731, 106429747, 106429750, 106429761, 106429769, and 106429771.

[0105] The fragment containing the UNCX gene contains one or more of the following UNCX gene sites: 1263643, 1263655, 1263659, 1263664, and 1263676.

[0106] The fragment of the RPS7P5 gene contains one or more of the following RPS7P5 gene sites: 240161511, 240161516, 240161523, 240161527, and 240161530.

[0107] The FOXI2 gene fragment contains one or more of the following FOXI2 gene loci: 129534910, 129534912, and 129534924.

[0108] The fragment of the ACRBP gene contains one or more of the following ACRBP gene sites: 6756182, 6756187, 6756191, 6756195, and 6756211.

[0109] The fragment containing the GAS6 gene contains one or more of the following GAS6 gene sites: 114524062, 114524068, 114524084, 114524095, 114524131, and 114524138.

[0110] The fragment containing the MCRIP2 gene contains one or more of the following MCRIP2 gene sites: 698072, 698142, 698153, 698168, and 698208.

[0111] The fragment of the LINC01977 gene contains one or more of the following LINC01977 gene loci: 77789596, 77789601, 77789612, and 77789620.

[0112] The fragment containing the EGR3 gene contains one or more of the following EGR3 gene loci: 22548269, 22548279, 22548283, 22548287, 22548296, and 22548299.

[0113] The SOX17 gene fragment contains one or more of the following SOX17 gene loci: 55379602, 55379608, 55379617, and 55379620.

[0114] The PAX5 gene fragment contains one or more of the following PAX5 gene loci: 36986087, 36986093, 36986098, 36986101, and 36986103.

[0115] The fragment of the NEURL1 gene contains one or more of the following NEURL1 gene sites: 105344493, 105344495, and 105344497.

[0116] The fragment of the IRX4 gene contains one or more of the following IRX4 gene loci: 1876386, 1876395, 1876397, and 1876403.

[0117] The fragment of the RUSC1 gene contains one or more of the following RUSC1 gene sites: 155295192, 155295196, and 155295212.

[0118] In any embodiment of the present invention, the locus number for each gene corresponds to the base number of the chromosome in which the gene is located.

[0119] In a preferred embodiment of the second aspect, the reagent for detecting DNA methylation levels detects the DNA methylation levels of fragments selected from the following genes: SLC16A3, CDH1, TSHR, RARG, PRR15, MCC, TBX15, DPYS, COL23A1, ILDR2, NEURL1, BIN1, DNM2, and IL17C. In one or more embodiments, the reagent for detecting DNA methylation levels detects the DNA methylation levels of fragments selected from two of the following genes: 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, SLC16A... 3 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, COL 23A1 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 for detecting DNA methylation levels detects DNA methylation levels in fragments selected from the following three genes: 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 for detecting DNA methylation levels detects the DNA methylation levels of fragments selected from the following four genes: 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 DNA methylation levels measures the DNA methylation levels of fragments selected from the following five genes: 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 TSH R 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 DNA methylation levels detects DNA methylation levels in fragments selected from the following six genes: 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 COL23A. 1. 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 reagent for detecting DNA methylation levels measures the DNA methylation levels of fragments from the following seven genes: 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 COL23A1. 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 the DNA methylation levels of fragments selected from 8, 9, 10, 11, 12, 13, 14, or all 15 genes: SLC16A3, CDH1, TSHR, RARG, PRR15, MCC, TBX15, DPYS, COL23A1, ILDR2, NEURL1, BIN1, DNM2, and IL17C. In one or more embodiments, the reagent: (1) detects the DNA methylation level at one or more of the following (a1)-(a6): (a1) COL23A1 gene sites: one or more or all of 178003785, 178003798, 178003803, 178003814, 178003823, 178003825, 178003834, 178003841, 178003844; (a2) ILDR2 gene sites: 166890429, 166890436, 166890440, 166890442, 166 One or more of the following: 890448, 166890452, 166890456, 166890461, 166890468, 166890473, 166890475, 166890480, 166890492, 166890500, 166890503, 166890509, 166890516, 166890528, 166890535, 166890543, 166890555, 166890559, 166890568, 166890573, 166890584, 166890586.(a3) Loci of the DHRS3 gene: one or all of the following: 12656091, 12656114, 12656132, 12656152, 12656170, 12656175, 12656182, 12656187, 12656197, 12656200, 12656211, 12656315, 12656323, 12656340, 12656355, 12656367; (a4) Loci of the KIF1A gene: one or all of the following: 241759696, 241759701, 241759714, 241759716; (a5) GDNF gene. The loci are: one or all of the following: 37834763, 37834770, 37834772, 37834774, 37834777, 37834780, 37834784, 37834792, 37834799, 37834802, 37834806, 37834811; (a6) The loci of the TBX18 gene are: one or all of the following: 85477032, 85477035, 85477070, 85477083, 85477106, 85477124, 85477151, 85477153, 85477166; or (2) Detection of one or more of the following gene loci. The DNA methylation level of the nucleic acid fragments, the fragments being 50-1000bp in length: COL23A1, ILDR2, DHRS3, KIF1A, GDNF, TBX18, wherein the COL23A1 gene fragment contains one or more of the following COL23A1 gene sites: 178003785, 178003798, 178003803, 178003814, 178003823, 178003825, 178003834, 178003841, 178003844, and the ILDR2 gene fragment contains the following ILDR2 gene sites: 166890429, 166890436, 16689... One or more of the following: 0440, 166890442, 166890448, 166890452, 166890456, 166890461, 166890468, 166890473, 166890475, 166890480, 166890492, 166890500, 166890503, 166890509, 166890516, 166890528, 166890535, 166890543, 166890555, 166890559, 166890568, 166890573, 166890584, 166890586The DHRS3 gene fragment contains one or more of the following DHRS3 gene loci: 12656091, 12656114, 12656132, 12656152, 12656170, 12656175, 12656182, 12656187, 12656197, 12656200, 12656211, 12656315, 12656323, 12656340, 12656355, and 12656367. The KIF1A gene fragment contains one or more of the following KIF1A gene loci: 241759696, 241759701, 241759714, and 241759716. The GDNF gene fragment contains the following GDNF gene loci: One or all of the following 37834763, 37834770, 37834772, 37834774, 37834777, 37834780, 37834784, 37834792, 37834799, 37834802, 37834806, and 37834811, and the fragment of the TBX18 gene containing the following TBX18 gene sites: 85477032, 85477035, 85477070, 85477083, 85477106, 85477124, 85477151, 85477153, and 85477166, or (3) detecting the DNA methylation level of the nucleic acid region within 10Kb upstream and downstream of the gene described in (2). The site is referenced from the human reference genome, version hg19.

[0120] In one or more embodiments, (a1)-(a6) are: one or all of the following COL23A1 gene loci: 178003798, 178003803, 178003814, 178003823, 178003825, 178003834, 178003841, 178003844; and the following ILDR2 gene loci: 166890516, 166890528, 16689 One or more of the following loci: 0535, 166890543, 166890555, 166890559, 166890568, 166890573, 166890584, 166890586; and the following loci of the DHRS3 gene: 12656091, 12656114, 12656132, 12656152, 12656170, 12656175, 12656182, 126... One or all of the following loci: 56187, 12656197, 12656200, 12656211, 12656315, 12656323, 12656340, 12656355, 12656367; KIF1A gene loci: one or all of the following loci: 241759696, 241759701, 241759714, 241759716; GDNF gene locus: 378. One or more of the following: 34770, 37834772, 37834774, 37834777, 37834780, 37834784, 37834792, 37834799, 37834802, 37834806, 37834811; and one or more of the following: TBX18 gene loci: 85477035, 85477070, 85477083, 85477106.

[0121] In one or more embodiments, (a1)-(a6) are: one or all of the following COL23A1 gene loci: 178003798, 178003803, 178003814, 178003823, 178003825, 178003834, 178003841, 178003844; and the ILDR2 gene locus: 1668905. One or more of the following loci: 16, 166890528, 166890535, 166890543, 166890555, 166890559, 166890568, 166890573, 166890584, and 166890586; DHRS3 gene loci: 12656170, 12656175, 12656182, and 126... One or all of the following loci: 56197, 12656200, 12656211, 12656315, 12656323; KIF1A gene loci: one or all of the following loci: 241759696, 241759701, 241759714, 241759716; GDNF gene loci: 37834770, 37834772, 378 One or more of the following: 34774, 37834777, 37834780, 37834784, 37834792, 37834799, 37834802, 37834806, 37834811; and one or more of the following: TBX18 gene loci: 85477035, 85477070, 85477083, 85477106.

[0122] In one or more embodiments, the reagent for detecting DNA methylation levels detects DNA methylation levels at the following sites: one or more groups of (a1)-(a3), and optionally one or more groups of (a4)-(a6). Preferably, the sites comprise: (a1)-(a3) and optionally (a4), or, (a1)-(a3) and optionally (a5)-(a6); more preferably, the sites comprise: (a1)-(a3) and optionally (a4) or optionally (a5)-(a6). In one or more embodiments, the reagent for detecting DNA methylation levels detects DNA methylation levels at the following sites: (a1)-(a4), or, (a1)-(a3) and (a5)-(a6).

[0123] In one or more embodiments, the reagent is a primer capable of amplifying one or more fragments selected from: (b1) a fragment of the COL23A1 gene amplified using SEQ ID NO:4 and 5 as primers, (b2) a fragment of the ILDR2 gene amplified using SEQ ID NO:6 and 7 as primers, (b3) a fragment of the DHRS3 gene amplified using SEQ ID NO:8 and 9 as primers, (b4) a fragment of the KIF1A gene amplified using SEQ ID NO:10 and 11 as primers, (b5) a fragment of the GDNF gene amplified using SEQ ID NO:12 and 13 as primers, and (b6) a fragment of the TBX18 gene amplified using SEQ ID NO:14 and 15 as primers. Preferably, the primers amplify one or more sets of (b1)-(b3) and optionally one or more sets of (b4)-(b6); more preferably, the primers amplify one or more sets of (b1)-(b3) and optionally (b4), or (b1)-(b3) and optionally (b5)-(b6); even more preferably, the primers amplify (b1)-(b3) and optionally (b4) or optionally (b5)-(b6). In one or more embodiments, the primers are any of SEQ ID NO:4-15 or sequences having 90% identity with them. Preferably, the primers are selected from one or more or all of (1) SEQ ID NO:4-9, (2) one or more or all of SEQ ID NO:4-11, (3) one or more or all of SEQ ID NO:4-9, 12-15, or (4) any sequence that has 90% identity with (1)-(3).

[0124] In one or more embodiments, the reagent is a probe capable of hybridizing with one or more fragments selected from: (b1) a fragment of the COL23A1 gene amplified using SEQ ID NO:4 and 5 as primers, (b2) a fragment of the ILDR2 gene amplified using SEQ ID NO:6 and 7 as primers, (b3) a fragment of the DHRS3 gene amplified using SEQ ID NO:8 and 9 as primers, (b4) a fragment of the KIF1A gene amplified using SEQ ID NO:10 and 11 as primers, (b5) a fragment of the GDNF gene amplified using SEQ ID NO:12 and 13 as primers, and (b6) a fragment of the TBX18 gene amplified using SEQ ID NO:14 and 15 as primers. Preferably, the primers hybridize with one or more of the following fragments: (b1)-(b3), and optionally one or more of (b4)-(b6); or, the primers hybridize with one or more of the following fragments: (b1)-(b3) and optionally (b4), or, (b1)-(b3) and optionally (b5)-(b6); or, the primers hybridize with the following fragments: (b1)-(b3) and optionally (b4) or optionally (b5)-(b6). In one or more embodiments, the probe is any one of SEQ ID NO:16-21 or a sequence having 90% identity with it. Preferably, the probe is selected from one or all of (1) SEQ ID NO:16-18, (2) one or all of SEQ ID NO:16-19, (3) one or all of SEQ ID NO:16-18, 20-21, or (4) any sequence that has 90% identity with (1)-(3).

[0125] In one or more embodiments, the length of the fragment is 30-2000bp, 30-1500bp, 50-1000bp, 50-800bp, 50-500bp, 50-400bp, 50-350bp, 50-300bp, 50-250bp, 50-200bp, 60-180bp, 60-170bp, 60-160bp, 60-150bp, 60-140bp, 60-130bp, 60-120bp, 70-110bp, or 80-100bp, preferably 50-350bp or 60-180bp.

[0126] In one or more embodiments of the above aspects, the mammal is a human.

[0127] In one or more embodiments of the above aspects, the gene or site includes the sense or antisense strand of DNA.

[0128] In one or more embodiments of the above aspects, the site refers to the human reference genome hg19 version.

[0129] In one or more embodiments of the above aspects, the reagent for detecting DNA methylation is selected from one or more of the following methods: bisulfite-based PCR (e.g., methylation-specific PCR), DNA sequencing (e.g., bisulfite sequencing, whole-genome methylation sequencing, simplified methylation sequencing), methylation-sensitive restriction endonuclease analysis, quantitative fluorescence assay, methylation-sensitive high-resolution melting curve assay, chip-based methylation mapping analysis, and mass spectrometry (e.g., mass spectrometry of flight). Preferably, the reagent is selected from one or more of the following: bisulfite and its derivatives, PCR buffer, polymerase, dNTP, primers, probes, methylation-sensitive or insensitive restriction endonucleases, enzyme digestion buffers, fluorescent dyes, fluorescence quenchers, fluorescent reporter agents, exonucleases, alkaline phosphatase, internal standards, and controls.

[0130] Preferably, the reagent comprises primers. The primer sequences are methylation-specific or non-specific. Preferably, the primer sequences include non-methylation-specific blocking sequences. Preferably, the primers are any of SEQ ID NO:4-15 or sequences having 90% identity with them.

[0131] Preferably, the reagent comprises a probe. The probe sequence is labeled with a fluorescent reporter group at the 5' end and a quencher group at the 3' end. Preferably, the probe sequence contains MGB (Minor Groove Binder) or LNA (Locked Nucleic Acid). Preferably, the probe is any one of SEQ ID NO:16-21 or has 90% identity with it.

[0132] Another aspect of the present invention provides a kit for identifying the nature of thyroid nodules, comprising the reagents described in the second aspect of the present invention and optionally the nucleic acid molecules described in the first aspect of the present invention. In one or more embodiments, the reagent for detecting DNA methylation is selected from one or more of the following methods: bisulfite-based PCR (e.g., methylation-specific PCR), DNA sequencing (e.g., bisulfite sequencing, whole-genome methylation sequencing, simplified methylation sequencing), methylation-sensitive restriction endonuclease analysis, quantitative fluorescence assay, methylation-sensitive high-resolution melting curve assay, chip-based methylation mapping analysis, and mass spectrometry (e.g., mass spectrometry of flight). Preferably, the reagent is selected from one or more of the following: bisulfite and its derivatives, PCR buffer, polymerase, dNTP, primers, probes, methylation-sensitive or insensitive restriction endonucleases, enzyme digestion buffers, fluorescent dyes, fluorescence quenchers, fluorescent reporter agents, exonucleases, alkaline phosphatases, internal standards, and controls. In one or more embodiments, the kit further comprises a reagent for detecting gene mutations. In one or more embodiments, 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 mismatch cleavage, allele-specific oligonucleotide analysis, ligase chain reaction, allele-specific amplification, RNase A cleavage, chromosome in situ hybridization, fluorescence in situ hybridization, DNA sequence analysis, enzymatic mismatch cleavage, fragment length polymorphism, dideoxy fingerprinting, mismatch-binding protein truncation assay, primer extension, oligonucleotide linking detection, capillary electrophoresis, and chip-based methods. Preferably, the reagents for detecting gene mutations include: primers, probes, buffers, polymerases, dNTPs, restriction endonucleases, enzyme digestion buffers, fluorescent dyes, fluorescence quenchers, fluorescent reporters, exonucleases, alkaline phosphatases, internal standards, and controls.

[0133] In one or more embodiments, the kit further includes reagents for detecting the mutation level at the V600E site of the BRAF gene and / or the mutation level at the C228T / C250T site of the TERT gene.

[0134] Another aspect of the present invention provides the use of the nucleic acid molecules and / or reagents described herein in the preparation of kits for identifying the nature of thyroid nodules in samples. The reagents include the reagents for detecting DNA methylation described in any embodiment herein and optionally reagents for detecting gene mutations. The gene mutations are selected from mutations at the V600E site of the BRAF gene and mutations at the C228T / C250T site of the TERT gene. The reagents for detecting DNA methylation are as described in aspects two through four herein.

[0135] Another aspect of the present invention provides the use of reagents for detecting DNA methylation and, optionally, the nucleic acid molecules described herein, in the preparation of a kit for identifying the nature of thyroid nodules, said reagents detecting the level of DNA methylation in a sample selected from the following regions (1) and (2): (1) fragments selected from one or more of the following genes: ZMIZ1, C15orf52, SLC16A3, ZNF512B, SLC17A5, LIMK1, PLEC, TOR4A, TMEM131L, DNM2, IL17C, PRDM16, MT1JP, TBX3, BIN1, TIMP2, CFAP65, TSH R, 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) and (1) the nucleic acid regions within 5Kb or 10Kb upstream and downstream of the genes. Preferably, the reagent detects the methylation level of one or more genes selected from the following: COL23A1, ILDR2, DHRS3, KIF1A, GDNF, TBX18.

[0136] In one or more embodiments, the reagent detects the methylation level of fragments of one or more of the following groups of genes in the sample: (1) COL23A1, ILDR2, DHRS3, (2) COL23A1, ILDR2, DHRS3 and KIF1A, (3) COL23A1, ILDR2, DHRS3 and one or two selected from GDNF and TBX18, (4) nucleic acid regions within 5Kb or 10Kb upstream or downstream of any one of the groups of genes in (1)-(3).

[0137] In one or more embodiments, the detection sites for each gene are selected from one or more of the following sites or nucleic acid regions within 500 bp upstream and downstream:

[0138] KIF1A: Chromosome 2, numbers 241759696, 241759701, 241759714, and 241759716.

[0139] COL23A1: Chromosome 5, numbers 178003785, 178003798, 178003803, 178003814, 178003823, 178003825, 178003834, 178003841, 178003844.

[0140] ILDR2: Chromosome 1, numbers 166890429, 166890436, 166890440, 166890442, 166890448, 166890452, 166890456, 166890461, 166890468, 166890473, 166890475, 166890480, 1668 90492, 166890500, 166890503, 166890509, 166890516, 166890528, 166890535, 166890543, 166890555, 166890559, 166890568, 166890573, 166890584, 166890586,

[0141] DHRS3: Chromosome 1, numbers 12656091, 12656114, 12656132, 12656152, 12656170, 12656175, 12656182, 12656187, 12656197, 12656200, 12656211, 12656315, 12656323, 12656340, 12656355, 12656367.

[0142] GDNF: Chromosome 5, numbers 37834763, 37834770, 37834772, 37834774, 37834777, 37834780, 37834784, 37834792, 37834799, 37834802, 37834806, 37834811.

[0143] TBX18: Chromosome 6, numbers 85477032, 85477035, 85477070, 85477083, 85477106, 85477124, 85477151, 85477153, 85477166.

[0144] Preferably, the detection sites for each gene are selected from one or more of the following sites or nucleic acid regions within 500 bp upstream and downstream of them:

[0145] KIF1A: Chromosome 2, numbers 241759696, 241759701, 241759714, and 241759716.

[0146] COL23A1: Chromosome 5, numbers 178003798, 178003803, 178003814, 178003823, 178003825, 178003834, 178003841, 178003844.

[0147] ILDR2: Chromosome 1, numbers 166890516, 166890528, 166890535, 166890543, 166890555, 166890559, 166890568, 166890573, 166890584, 166890586.

[0148] DHRS3: chromosome 1, numbers 12656340, 12656355, and 12656367.

[0149] GDNF: Chromosome 5, numbers 37834770, 37834772, 37834774, 37834777, 37834780, 37834784, 37834792, 37834799, 37834802, 37834806, 37834811.

[0150] TBX18: chromosome 6, numbers 85477035, 85477070, 85477083, and 85477106.

[0151] In one or more embodiments, the reagent for detecting DNA methylation detects the methylation level of one or more of the following (a1)-(a6): (a1) COL23A1 gene sites: one or more or all of 178003785, 178003798, 178003803, 178003814, 178003823, 178003825, 178003834, 178003841, 178003844; (a2) ILDR2 gene sites: 166890429, 166890436, 166890440, 166890442, 16689044. 8. One or all of the following: 166890452, 166890456, 166890461, 166890468, 166890473, 166890475, 166890480, 166890492, 166890500, 166890503, 166890509, 166890516, 166890528, 166890535, 166890543, 166890555, 166890559, 166890568, 166890573, 166890584, 166890586, (a3)DHRS3 The loci of the gene are: one or all of the following: 12656091, 12656114, 12656132, 12656152, 12656170, 12656175, 12656182, 12656187, 12656197, 12656200, 12656211, 12656315, 12656323, 12656340, 12656355, 12656367; (a4) The loci of the KIF1A gene are: one or all of the following: 241759696, 241759701, 241759714, 241759716; (a5) GD The NF gene loci are one or more of the following: 37834763, 37834770, 37834772, 37834774, 37834777, 37834780, 37834784, 37834792, 37834799, 37834802, 37834806, and 37834811; and the TBX18 gene loci are one or more of the following: 85477032, 85477035, 85477070, 85477083, 85477106, 85477124, 85477151, 85477153, and 85477166. The reagents for detecting DNA methylation are as described in other embodiments herein.

[0152] In one or more embodiments, (a1)-(a6) are: one or all of the following COL23A1 gene loci: 178003798, 178003803, 178003814, 178003823, 178003825, 178003834, 178003841, 178003844; and the following ILDR2 gene loci: 166890516, 166890528, 16689 One or more of the following loci: 0535, 166890543, 166890555, 166890559, 166890568, 166890573, 166890584, 166890586; and the following loci of the DHRS3 gene: 12656091, 12656114, 12656132, 12656152, 12656170, 12656175, 12656182, 126... One or all of the following loci: 56187, 12656197, 12656200, 12656211, 12656315, 12656323, 12656340, 12656355, 12656367; KIF1A gene loci: one or all of the following loci: 241759696, 241759701, 241759714, 241759716; GDNF gene locus: 378. One or more of the following: 34770, 37834772, 37834774, 37834777, 37834780, 37834784, 37834792, 37834799, 37834802, 37834806, 37834811; and one or more of the following: TBX18 gene loci: 85477035, 85477070, 85477083, 85477106.

[0153] In one or more embodiments, (a1)-(a6) are: one or all of the following COL23A1 gene loci: 178003798, 178003803, 178003814, 178003823, 178003825, 178003834, 178003841, 178003844; and the ILDR2 gene locus: 1668905. One or more of the following loci: 16, 166890528, 166890535, 166890543, 166890555, 166890559, 166890568, 166890573, 166890584, and 166890586; DHRS3 gene loci: 12656170, 12656175, 12656182, and 126... One or all of the following loci: 56197, 12656200, 12656211, 12656315, 12656323; KIF1A gene loci: one or all of the following loci: 241759696, 241759701, 241759714, 241759716; GDNF gene loci: 37834770, 37834772, 378 One or more of the following: 34774, 37834777, 37834780, 37834784, 37834792, 37834799, 37834802, 37834806, 37834811; and one or more of the following: TBX18 gene loci: 85477035, 85477070, 85477083, 85477106.

[0154] In one or more embodiments, the reagent for detecting DNA methylation levels detects DNA methylation levels at the following sites: one or more groups of (a1)-(a3), and optionally one or more groups of (a4)-(a6); preferably, the sites comprise: (a1)-(a3) and optionally (a4), or, (a1)-(a3) and optionally (a5)-(a6); more preferably, the sites comprise: (a1)-(a3) and optionally (a4) or optionally (a5)-(a6). In one or more embodiments, the reagent for detecting DNA methylation levels detects DNA methylation levels at the following sites: (a1)-(a4), or, (a1)-(a3) and (a5)-(a6).

[0155] In one or more embodiments, the kit further comprises reagents for detecting the mutation level at the V600E site of the BRAF gene.

[0156] In one or more embodiments, the kit further comprises reagents for detecting mutation levels at the C228T / C250T sites of the TERT gene.

[0157] In one or more embodiments of the use, the gene or site comprises the sense or antisense strand of DNA.

[0158] In one or more embodiments of the use, the site is referenced to the human reference genome version hg19.

[0159] In one or more embodiments of the use, the kit further includes reagents for detecting the mutation level at the V600E site of the BRAF gene and / or the mutation level at the C228T / C250T sites of the TERT gene.

[0160] In one or more embodiments of the use, identifying the nature of a thyroid nodule includes: comparing it with a control sample, or obtaining a score based on methylation level and / or mutation level, and identifying the nature of the thyroid nodule based on the comparison result or score.

[0161] In one or more embodiments of the use, the sample is derived from a human being, preferably from tissues, cells, or bodily fluids, such as thyroid tissue or blood. In one or more embodiments of the use, the sample contains genomic DNA or cfDNA.

[0162] In one or more embodiments of the application, the reagent for detecting DNA methylation is as described in the second aspect of the invention.

[0163] In one or more embodiments of the application, the reagent for detecting DNA methylation is selected from one or more of the following methods: bisulfite-based PCR (e.g., methylation-specific PCR), DNA sequencing (e.g., bisulfite sequencing, whole-genome methylation sequencing, simplified methylation sequencing), methylation-sensitive restriction endonuclease assays, quantitative fluorescence assays, methylation-sensitive high-resolution melting curve assays, chip-based methylation mapping, and mass spectrometry (e.g., mass spectrometry of flight). Preferably, the reagent is selected from one or more of the following: bisulfite and its derivatives, PCR buffer, polymerase, dNTPs, primers, probes, methylation-sensitive or insensitive restriction endonucleases, enzyme digestion buffers, fluorescent dyes, fluorescence quenchers, fluorescent reporter agents, exonucleases, alkaline phosphatases, internal standards, and controls.

[0164] Preferably, the primer sequence is methylation-specific or non-specific. Preferably, the primer sequence includes a non-methylation-specific blocking sequence. Preferably, the primer is any one of SEQ ID NO:4-15 or a sequence having 90% identity with it.

[0165] Preferably, the probe sequence is labeled with a fluorescent reporter group at the 5' end and a quencher group at the 3' end. Preferably, the probe sequence contains MGB (Minor Groove Binder) or LNA (Locked Nucleic Acid). Preferably, the probe is any one of SEQ ID NO:16-21 or has 90% identity with it.

[0166] The present invention also provides a primer for detecting DNA methylation levels selected from the regions described in (1) and (2) below: (1) a fragment selected from one or more of the following genes: ZMIZ1, C15orf52, SLC16A3, ZNF512B, SLC17A5, LIMK1, PLEC, TOR4A, TMEM131L, DNM2, IL17C, PRDM16, MT1JP, TBX3, BIN1, TIMP2, CFAP65, TSHR, KIF1A, DAPK, CDH1, TPO, RARG The primers are located within 5 kb or 10 kb upstream and downstream of the genes described herein. (1) 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) and (1) respectively. Preferably, the primers detect the methylation level of the sites described herein.

[0167] The present invention also provides a probe for detecting DNA methylation levels selected from the regions described in (1) and (2) below: (1) fragments selected from one or more of the following genes: ZMIZ1, C15orf52, SLC16A3, ZNF512B, SLC17A5, LIMK1, PLEC, TOR4A, TMEM131L, DNM2, IL17C, PRDM16, MT1JP, TBX3, BIN1, TIMP2, CFAP65, TSHR, KIF1A, DAPK, CDH1, TPO, RARG The nucleic acid regions within 5Kb or 10Kb upstream and downstream of the genes described in (1) are: 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) and (1) respectively. Preferably, the probe detects the methylation level of the sites described herein.

[0168] The present invention also provides a method for screening benign and malignant thyroid nodules, comprising: (1) detecting the methylation level of the gene, site or nucleic acid region described herein in the sample of the 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; and (4) identifying the nature of the thyroid nodule based on the comparison result or score of step (3).

[0169] The present invention also provides a method for screening benign and malignant thyroid nodules, 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 the gene, site or nucleic acid region described herein in the sample of the subject; (3) comparing with a control sample, or obtaining a score based on the mutation level and / or methylation level, for example by calculation; and (4) identifying the nature of the thyroid nodule based on the comparison result or score of step (3).

[0170] In one or more embodiments, the method further includes, prior to step (1): extraction of sample DNA, quality control, and / or conversion of unmethylated cytosine on the DNA into bases that do not bind to guanine.

[0171] In one or more embodiments, the conversion is carried out using an enzymatic method, preferably deaminase treatment, or the conversion is carried out using a non-enzymatic method, preferably with bisulfite or disulfite treatment, more preferably with calcium bisulfite, sodium bisulfite, potassium bisulfite, ammonium bisulfite, sodium disulfite, potassium disulfite, and ammonium disulfite.

[0172] In one or more embodiments, the detection includes, but is not limited to: bisulfite-based PCR (e.g., methylation-specific PCR), DNA sequencing (e.g., bisulfite sequencing, whole-genome methylation sequencing, simplified methylation sequencing), methylation-sensitive restriction endonuclease assays, quantitative fluorescence assays, methylation-sensitive high-resolution melting curve assays, chip-based methylation mapping analysis, and mass spectrometry (e.g., mass spectrometry of flight).

[0173] In one or more embodiments, step (4) includes: comparing the methylation level and / or mutation level of the target sample with a control sample, and identifying the thyroid nodule as benign or malignant when the methylation level and / or mutation level meets a threshold.

[0174] In one or more embodiments, step (4) includes: when the score meets a threshold, the thyroid nodule is identified as benign or malignant.

[0175] In one or more embodiments, the sample is derived from a human body, preferably from tissues, cells, or bodily fluids, such as 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.

[0176] In one or more embodiments, the sample is derived from a subject with benign or malignant thyroid nodules. In one or more embodiments, the sample is derived from a patient with goiter.

[0177] In one or more embodiments, the sample comprises genomic DNA or cfDNA.

[0178] The present invention also provides a kit for identifying the nature of thyroid nodules, comprising primers and / or probes for detecting the methylation levels of the genes, sites, and nucleic acid regions described herein. Attached Figure Description

[0179] Figure 1 This is a distribution map of a single library fragment detected by the LabChip method of this invention.

[0180] Figure 2A -C represents the ROC curve analysis of 10 cases of thyroid cancer and 10 cases of benign thyroid nodules detected in this invention. A: Tissue sample; B, C: Plasma sample.

[0181] Figure 3 This is an ROC curve analysis of plasma samples from 20 cases of thyroid cancer and 20 cases of benign thyroid nodules in one embodiment of the present invention.

[0182] Figure 4 This is an ROC curve analysis of plasma samples from 20 cases of thyroid cancer and 20 cases of benign thyroid nodules in one embodiment of the present invention. Detailed Implementation

[0183] The inventors discovered that specific chromosomes, genes, or methylation sites are associated with malignant thyroid nodules.

[0184] When referring to thyroid nodules, the terms "benign" and "malignant" in this article indicate the nature of the nodule. Generally, benign nodules are characterized by slow growth, homogeneous texture, good mobility, smooth surface, cystic changes, absence of lymph node enlargement, and no calcification. Malignant nodules are characterized by uncontrolled growth, spread, and tissue infiltration of malignant cells. Ultrasound signs suggesting malignancy in a thyroid nodule include: nodule height greater than width, lack of halo, microcalcifications, irregular borders, decreased echogenicity, solid nodule, and abundant blood flow within the nodule. In some implementations, malignant thyroid nodules include thyroid cancer.

[0185] The inventors discovered that the nature of thyroid nodules is associated with the methylation level of fragments selected from one or more of the following genes: ZMIZ1, C15orf52, SLC16A3, ZNF512B, SLC17A5, LIMK1, PLEC, TOR4A, TMEM131L, DNM2, IL17C, PRDM16, MT1JP, TBX3, BIN1, TIMP2, CFAP65, TSHR, KIF1A, DAPK, CDH1, TP. O, RARG, PRR15, DPYS, MCC, TBX15, COL23A1, ILDR2, DHRS3, GDNF, TBX18, SIM2, HOXA9, EHBP1L1, GJC2, RCOR 2. PRDM1, UNCX, RPS7P5, FOXI2, ACRBP, GAS6, MCRIP2, LINC01977, EGR3, SOX17, PAX5, NEURL1, IRX4, RUSC1. Preferably, the genes are selected from the following group: (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) COL23A1, ILDR2 and DHRS3, (7) COL23A1, ILDR2, DHRS3 and KIF1A, (8) COL23A1, ILDR2, DHRS3, GDNF and TBX18.

[0186] The inventors also discovered that the nature of thyroid nodules is associated with the methylation levels of one or more sites selected from the following sites, numbered with reference to the human reference genome hg19:

[0187] ZMIZ1: Chromosome 10, numbers 81001968, 81001996, 81002041, 81002052, 81002054, 81002056, 81002062, 81002083, 81002110, 81002116, 81002123, 81002129, 81002133, 81002137, 81002139, 81002164, 81002168, 81002223, 81002241, 81002253.

[0188] C15orf52: chromosome 15, numbers 40626309, 40626312, 40626386; SLC16A3: chromosome 17, numbers 80189165, 80189174, 80189177, 80189197, 80189225, 80189230, 80189239, 80189645, 80189671, 80 189674, 80189684, 80189687, 80189698, 80189709, 80189719, 80189726, 80189728, 80189739, 80189757, 80189787, 80189792, 80189811, 80189817, 80189832, 80189841,

[0189] ZNF512B: Chromosome 20, numbers 62588634, 62588638, and 62588672.

[0190] SLC17A5: Chromosome 6, numbers 74290205, 74290207, 74290220, 74290225, and 74290228.

[0191] LIMK1: Chromosome 7, numbers 73508994, 73509017, 73509055, 73509062, 73509073, 73509075, 73509112, 73509133, 73509138, 73509148, 73509160.

[0192] PLEC: Chromosome 8, 145013661, 145013673,

[0193] TOR4A: Chromosome 9, digits 140172787, 140172790, and 140172812.

[0194] TMEM131L: Chromosome 4, numbers 154409945, 154409963, 154409972, 154409978, 154409997, 154410003, 154410006.

[0195] DNM2: Chromosome 19, numbers 10870373, 10870377, 10870427, 10870429, 10870441, and 10870448.

[0196] IL17C: Chromosome 16, numbers 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,

[0197] PRDM16: Chromosome 1, numbers 3229914, 3229921, 3229950, 3229968, 3229973, 3310213, 3310229, 3310235, 3310238, 3310240, 3310268, 3310287, 3310312, 3310314, 3310317, 3310329.

[0198] TSHR: Chromosome 14, numbers 81421983, 81421989, 81422010, 81422017, 81422032, 81422035, 81422063, 81422084.

[0199] KIF1A: Chromosome 2, numbers 241759696, 241759701, 241759714, and 241759716.

[0200] DAPK: Chromosome 90112842, 90112853, 90112861, 90112866,

[0201] CDH1: Chromosome 16, numbers 68771035, 68771037, 68771045, 68771051, 68771059, 68771064, and 68771073.

[0202] TPO: chromosome 2, numbers 1481013, 1481015, 1481022, and 1481039.

[0203] RARG: Chromosome 12, numbers 53613176, 53613182, 53613190, 53613202, 53613210, 53613218.

[0204] MT1JP: Chromosome 16, numbers 56669271, 56669292, 56669295, 56669300, 56669318, 56669322, 56669324, 56669327, 56669344, 56669351, 56669353, 56669402, 56669414, 56669423, 56669430, 56669433, 56669437, 5666945 1. 56669453, 56669455, 56669463, 56669474, 56669480, 56669482, 56669485, 56669487, 56669490, 56669519, 56669533, 56669553, 56669564, 56669573, 56669578, 56669588, 56669590, 56669606, 56669610

[0205] TBX3: Chromosome 12, numbers 115174750, 115174773, and 115174780.

[0206] BIN1: Chromosome 2, numbers 127822478, 127822492, 127822495, 127822514, 127822551, 127822568, 127822582, 127822593, 127822616, 127822644.

[0207] TIMP2: Chromosome 17, numbers 76921845, 76921853, and 76921860.

[0208] CFAP65: Chromosome 2, numbers 219866132, 219866139, 219866148, 219866158, 219866165, 219866168, 219866199, 219866218.

[0209] PRR15: Chromosome 7, numbers 29605992, 29606026, 29606040, 29606047, 29606056, 29606062, 29606073, 29606179, 29606191, 29606201, 29606204, 29606220, 29606222, 29606227, 29606231, 29606255, 29606257, 29606262, 29606271, 29606277, 29606289, 29606320.

[0210] DPYS: Chromosome 8, numbers 105478870, 105478873, 105478878, 105478905, 105478908, 105478916, 105478918, 105478945, 105478956, 105478965, 105478974, 105478983, 105478986, 105478989.

[0211] MCC: Chromosome 5, numbers 112538999, 112539011, 112539018, 112539022, 112539061, 112539084, 112539104, 112539128.

[0212] TBX15: Chromosome 1, numbers 119535725, 119535730, 119535740, 119535742, 119535750, 119535759, 119535766, 119535812, 119535817, 119535821, 119535823, 119535876, 119535879, 119535884, 119535891.

[0213] COL23A1: Chromosome 5, numbers 178003785, 178003798, 178003803, 178003814, 178003823, 178003825, 178003834, 178003841, 178003844.

[0214] ILDR2: Chromosome 1, numbers 166890429, 166890436, 166890440, 166890442, 166890448, 166890452, 166890456, 166890461, 166890468, 166890473, 166890475, 166890480, 1668 90492, 166890500, 166890503, 166890509, 166890516, 166890528, 166890535, 166890543, 166890555, 166890559, 166890568, 166890573, 166890584, 166890586,

[0215] DHRS3: Chromosome 1, numbers 12656091, 12656114, 12656132, 12656152, 12656170, 12656175, 12656182, 12656187, 12656197, 12656200, 12656211, 12656315, 12656323, 12656340, 12656355, 12656367.

[0216] GDNF: Chromosome 5, numbers 37834763, 37834770, 37834772, 37834774, 37834777, 37834780, 37834784, 37834792, 37834799, 37834802, 37834806, 37834811.

[0217] TBX18: Chromosome 6, numbers 85477032, 85477035, 85477070, 85477083, 85477106, 85477124, 85477151, 85477153, 85477166.

[0218] SIM2: Chromosome 21, numbers 38069563, 38069579, 38069619, 38069625, 38069638, 38069650, 38069662, 38069664, 38069676, 38069681.

[0219] HOXA9: Chromosome 7, numbers 27204848, 27204854, 27204858, 27204861, 27204863, 27204879, 27204884, 27204894, 27204897, 27204918, 27204929, 27204938, 27204945, 27204948, 27204951, 27204958, 27204981, 27204984.

[0220] EHBP1L1: Chromosome 11, numbers 65352612, 65352621, 65352635, 65352639, 65352642, 65352651, 65352654, 65352665, 65352670.

[0221] GJC2: Chromosome 1, numbers 228345954, 228345957, 228345965, 228345978, 228345980, 228345989.

[0222] RCOR2: Chromosome 11, numbers 63687223, 63687238, 63687247, 63687250, 63687259, 63687282, 63687288, 63687299, 63687318, 63687325.

[0223] PRDM1: Chromosome 6, numbers 106429711, 106429722, 106429731, 106429747, 106429750, 106429761, 106429769, 106429771.

[0224] UNCX: Chromosome 7, numbers 1263643, 1263655, 1263659, 1263664, 1263676, 1263694, 1263716, 1263723.

[0225] RPS7P5: Chromosome 1, numbers 240161502, 240161507, 240161511, 240161516, 240161523, 240161527, 240161530, 240161535, 240161546, 240161558, 240161560.

[0226] FOXI2: Chromosome 10, numbers 129534843, 129534853, 129534866, 129534879, 129534891, 129534910, 129534912, and 129534924.

[0227] ACRBP: Chromosome 12, numbers 6756182, 6756187, 6756191, 6756195, 6756211, 6756225, 6756230, 6756270.

[0228] GAS6: Chromosome 13, numbers 114524043, 114524062, 114524068, 114524084, 114524095, 114524131, 114524138, 114524142, 114524150, 114524158.

[0229] MCRIP2: Chromosome 16, numbers 698072, 698142, 698153, 698168, 698208, 698218, 698222, 698230.

[0230] LINC01977: Chromosome 17 numbers 77789596, 77789601, 77789612, 77789620, 77789628, 77789632, 77789635, 77789640.

[0231] EGR3: Chromosome 8, numbers 22548250, 22548260, 22548269, 22548279, 22548283, 22548287, 22548296, 22548299.

[0232] SOX17: Chromosome 8, numbers 55379566, 55379568, 55379573, 55379579, 55379583, 55379591, 55379599, 55379602, 55379608, 55379617, 55379620.

[0233] PAX5: Chromosome 9, numbers 36986087, 36986093, 36986098, 36986101, 36986103, 36986117, 36986131, 36986138, 36986141, 36986143, 36986147, 36986149, 36986156.

[0234] NEURL1: Chromosome 10, numbers 105344464, 105344482, 105344493, 105344495, 105344497, 105344503, 105344506, 105344513, 105344516, 105344519, 105344526.

[0235] IRX4: Chromosome 5, numbers 1876386, 1876395, 1876397, 1876403, 1876420, 1876424, 1876432, 1876436, 1876449, 1876456, 1876459, 1876463.

[0236] RUSC1: Chromosome 1, numbers 155295135, 155295171, 155295181, 155295192, 155295196, 155295212, 155295229, and 155295236.

[0237] The inventors also discovered that the nature of thyroid nodules is also related to the mutation level at the V600E site of the BRAF gene and / or the mutation level at the C228T / C250T site of the TERT gene.

[0238] In this document, methods for detecting DNA methylation are well known in the art, such as bisulfite-based PCR (e.g., methylation-specific PCR, MSP), DNA sequencing (e.g., bisulfite sequencing, whole-genome bisulfite sequencing, WGBS, reduced representation bisulfite sequencing, RRBS), methylation-sensitive restriction enzyme assays, quantitative fluorescence assays, methylation-sensitive high-resolution melting (MS-HRM), microarray-based methylation mapping, and mass spectrometry (e.g., mass spectrometry of flight). In one or more embodiments, detection includes detecting any strand at a gene or site. Detecting the methylation level at the aforementioned site includes detecting the methylation level of nucleic acid regions within 500 bp upstream and downstream of the site.

[0239] Therefore, the present invention relates to reagents for detecting DNA methylation. Reagents used in the above-described methods for detecting DNA methylation are well known in the art. Exemplarily, reagents for detecting DNA methylation may be those selected from one or more of the following methods: bisulfite-based PCR (e.g., methylation-specific PCR), DNA sequencing (e.g., bisulfite sequencing, whole-genome methylation sequencing, simplified methylation sequencing), methylation-sensitive restriction endonuclease analysis, quantitative fluorescence assays, methylation-sensitive high-resolution melting curve analysis, chip-based methylation mapping analysis, and mass spectrometry (e.g., mass spectrometry of flight).

[0240] Reagents for detecting DNA methylation may contain one or more of the following: bisulfite and its derivatives, PCR buffer, polymerase, dNTPs, primers, probes, methylation-sensitive or insensitive restriction endonucleases, enzyme digestion buffers, fluorescent dyes, fluorescent quenchers, fluorescent reporter agents, exonucleases, alkaline phosphatases, internal standards, and controls. In detection methods involving DNA amplification, reagents for detecting DNA methylation include primers. The primer sequences may be methylation-specific or non-specific. Preferably, the primer sequences include non-methylation-specific blocking sequences. Blocking sequences can improve the specificity of methylation detection. Reagents for detecting DNA methylation may also include probes. Typically, the 5' end of the probe sequence is labeled with a fluorescent reporter group, and the 3' end is labeled with a quencher group. Preferably, the probe sequence contains MGB or LNA.

[0241] In this document, methods and reagents for detecting gene mutations are well known in the art. Exemplary methods for detecting gene mutations include PCR-single-strand conformation polymorphism, heteroduplex analysis, mutation enrichment PCR, mutation gradient gel electrophoresis, chemical mismatch cleavage, allele-specific oligonucleotide analysis, ligase chain reaction, allele-specific amplification, RNase A cleavage, chromosome in situ hybridization, fluorescence in situ hybridization, DNA sequence analysis, enzymatic mismatch cleavage, fragment length polymorphism, dideoxy fingerprinting, mismatch-binding protein truncation assay, primer extension, oligonucleotide linking detection, capillary electrophoresis, and microarray-based methods. In one or more embodiments, detection includes detecting any one strand at a gene or site.

[0242] Therefore, this invention relates to reagents for detecting gene mutations. Reagents used in the above-described methods for detecting gene mutations are well known in the art. Exemplary reagents for detecting gene mutations include: primers, probes, buffers, polymerases, dNTPs, restriction endonucleases, enzyme digestion buffers, fluorescent dyes, fluorescence quenchers, fluorescent reporter agents, exonucleases, alkaline phosphatases, internal standards, and controls.

[0243] This invention also relates to a kit for identifying the nature of thyroid nodules, comprising the reagents described herein, particularly those described in the second and / or third aspects herein. The kit may also contain nucleic acid molecules described herein, particularly those described in the first aspect, as internal standards or positive controls. The kit may contain reagents for detecting gene mutations. The term "primer" as used herein refers to a nucleic acid molecule with a specific nucleotide sequence that guides the synthesis of a nucleotide polymerase chain at the initiation of nucleotide polymerization. Primers are typically two artificially synthesized oligonucleotide sequences, one complementary to one DNA template strand at one end of the target region, and the other complementary to another DNA template strand at the other end of the target region, serving as the initiation point for nucleotide polymerization. Artificially designed primers are widely used in polymerase chain reaction (PCR), qPCR, sequencing, and probe synthesis. Typically, primers are designed to amplify products with lengths of 50–150 bp, 60–140, 70–130, or 80–120 bp. Preferably, the product length is 80–100 bp.

[0244] In one or more embodiments, the reagent for detecting DNA methylation includes a probe. The probe sequence has a fluorescent reporter group labeled at the 5' end and a quencher group labeled at the 3' end. Preferably, the probe sequence contains either MGB (Minor Groovebinder) or LNA (Locked Nucleic Acid). MGB and LNA are used to increase the melting temperature (Tm) value, enhance the specificity of the analysis, and improve the flexibility of probe design.

[0245] The term "variant" or "mutant" in this document refers to a polynucleotide whose nucleic acid sequence is altered compared to a reference sequence by the insertion, deletion, or substitution of one or more nucleotides while retaining its ability to hybridize with other nucleic acids. A mutant described in any embodiment of this document comprises a nucleotide sequence having 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 with a reference sequence and retaining the biological activity of the reference sequence. Sequence identity between two aligned sequences can be calculated using, for example, NCBI's BLASTn. A mutant also includes a nucleotide sequence having one or more mutations (insertions, deletions, or substitutions) in the reference sequence and its nucleotide sequence while still retaining the biological activity of the reference sequence. The multiple mutations typically refer to 1-10, for example, 1-8, 1-5, or 1-3. Substitution can be between purine nucleotides and pyrimidine nucleotides, or between purine nucleotides or pyrimidine nucleotides. Substitution is preferably conserved. For example, in the art, conserved substitution with nucleotides of similar or comparable properties generally does not alter the stability and function of the polynucleotide. Conservative substitutions include, for example, the interchange of (A and G) between purine nucleotides and the interchange of (T or U and C) between pyrimidine nucleotides. Therefore, replacing one or more sites with residues from the same source in the polynucleotides of this invention will not substantially affect their activity. Specifically, the sites described herein contained in the variants of this invention are not mutated. That is, the method of this invention detects the methylation status of the sites described in the corresponding sequence; mutations can occur at bases outside these sites.

[0246] This invention also provides a method for screening benign and malignant thyroid nodules, comprising: (1) detecting the methylation level of the genes, loci, or nucleic acid regions described herein in the sample of the subject; optionally (2) detecting the mutation level of the V600E locus of the BRAF gene and / or the mutation level of the C228T / C250T locus of the TERT gene; (3) measuring the methylation level by comparing with a control sample or by calculating a score; and (4) identifying the subject as a benign or malignant nodule when the interpretation criteria are met. Typically, the method further includes, prior to step (1): extraction of sample DNA, quality control, and / or conversion of unmethylated cytosine on the DNA into bases that do not bind to guanine.

[0247] The "DNA" or "DNA molecule" mentioned in this article refers to deoxyribonucleic acid. The basic building block of DNA is the deoxyribonucleotide, which is formed into a long chain molecule through phosphodiester condensation. Each deoxyribonucleotide consists of a phosphate group, a deoxyribose sugar, and a base. The main bases (bp) of DNA are adenine (A), guanine (G), cytosine (C), and thymine (T). In the double helix structure of double-stranded DNA, A and T are paired by hydrogen bonds, and G and C are paired by hydrogen bonds. 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.

[0248] The "uracil" or "U" mentioned in this article refers to a component of RNA. "RNA" or "RNA molecule" is ribonucleic acid. RNA is a long, chain-like molecule formed by the condensation of ribonucleotides via phosphodiester bonds. Each ribonucleotide molecule consists of a phosphate group, a sugar sugar, and a base. RNA has four main bases: adenine (A), guanine (G), cytosine (C), and uracil (U). In RNA base pairing, U replaces the T position found in DNA; that is, A pairs with U via hydrogen bonds, and G pairs with C via hydrogen bonds.

[0249] Transformation can occur between bases in DNA or RNA. The terms "transformation," "cytosine transformation," or "CT transformation" used herein refer to the process of treating DNA using non-enzymatic or enzymatic methods to convert unmodified cytosine bases (C) into bases that do not bind to guanine (e.g., uracil bases (U)). Non-enzymatic or enzymatic methods for performing cytosine transformation are well known in the art. Exemplarily, non-enzymatic methods include bisulfite or disulfite treatments, such as calcium bisulfite, sodium bisulfite, potassium bisulfite, ammonium bisulfite, sodium disulfite, potassium disulfite, and ammonium disulfite. Exemplarily, enzymatic methods include deaminase treatment. The transformed DNA may optionally be purified. DNA purification methods suitable for use herein are well known in the art.

[0250] When referring to cytosine, "modification" means the introduction or removal of a chemical group on a cytosine base. During cytosine transformation, modified cytosine bases are more stable than unmodified cytosine bases and are less susceptible to or unaffected by the transformation process to U. In one or more embodiments, modification refers to methylation. As used herein, "methylation" or "DNA methylation" means the covalent bonding of a methyl group to the 5' carbon position of the cytosine in a CpG dinucleotide of genomic DNA, resulting in 5-methylcytosine (5mC).

[0251] Optionally, the modified cytosine described herein can be protected from downstream transformation or deamination by non-enzymatic or enzymatic methods prior to transformation. Non-enzymatic or enzymatic methods suitable for protecting modified cytosine are well known in the art. For example, TET2 (ten-eleven translocation 2) and / or oxidative enhancers can protect modified cytosine. TET2 can oxidize 5mC and 5hmC to 5caC via a cascade reaction. Oxidative enhancers can convert 5hmC to 5ghmC via glycosylation. Oxidative enhancers suitable for performing said glycosylation are well known in the art.

[0252] In one or more embodiments, the interpretation criteria are: an increase or decrease in the methylation level and / or mutation level of the target sample compared to a control sample. When the methylation level and / or mutation level meets a certain threshold, it is identified as a malignant nodule. Mathematical analysis is performed on the methylation level of the tested genes to obtain a fitted equation for the score. For the tested sample, if the score is greater than the threshold, the result is considered positive, i.e., a malignant nodule; otherwise, it is considered negative, i.e., a benign nodule. Conventional mathematical analysis methods and procedures for determining the threshold are known in the art; an exemplary method is binary logistic regression analysis. Typically, the threshold is 0.

[0253] For example, when identifying nodule characteristics based on the methylation levels of the BIN1 and SLC16A3 genes, a binary logistic regression analysis was performed on the methylation levels of the BIN1 and SLC16A3 genes, and the fitted equation was:

[0254] Score = 3.45 – 0.08 × methylation level of BIN1 + 0.01 × methylation level of SLC16A3.

[0255] Therefore, if the scores of the BIN1 and SLC16A3 genes in the sample are greater than 0, the result is positive, which means it is a malignant nodule.

[0256] In this document, the samples are derived from mammals, preferably humans. Samples can be derived from any organ (e.g., thyroid gland), tissue (e.g., epithelial tissue, connective tissue, muscle tissue, and nerve tissue), cell (e.g., thyroid nodule biopsy), or bodily fluid (e.g., blood, plasma, serum, tissue fluid, urine). Generally, the sample is acceptable as long as it contains genomic DNA or cfDNA (circulating free DNA or cell free DNA). cfDNA, also known as circulating cell-free DNA or cell-free DNA, is a fragment of degraded DNA released into the plasma. Exemplarily, the sample is a thyroid nodule biopsy, preferably a fine-needle aspiration biopsy. Alternatively, the sample is plasma.

[0257] Exemplary implementation scheme:

[0258] 1. An isolated nucleic acid molecule derived from a mammal, selected from one or more of the following groups or variants having at least 70% identity with: (a) fragments of chromosome 7 and chromosome 6, (b) fragments of chromosome 2 and chromosome 19, (c) fragments of chromosome 2 and chromosome 17, (d) fragments of chromosome 17, chromosome 19, and chromosome 16, wherein the fragments are 50-5000 bp in length, preferably 50-1000 bp, wherein...

[0259] The segment of chromosome 7 contains one or more of the following loci on chromosome 7: 73508994, 73509017, 73509055, 73509062, 73509073, 73509075, 73509112, 73509133, 73509138, 73509148, and 73509160.

[0260] The segment of chromosome 6 contains one or more of the following sites on chromosome 6: 74290205, 74290207, 74290220, 74290225, and 74290228.

[0261] The segment of chromosome 2 contains one or more of the following loci on chromosome 2: 127822478, 127822492, 127822495, 127822514, 127822551, 127822568, 127822582, 127822593, 127822616, and 127822644.

[0262] The segment of chromosome 19 contains one or more of the following loci on chromosome 19: 10870373, 10870377, 10870427, 10870429, 10870441, and 10870448.

[0263] The segment of chromosome 17 contains one or more of the following loci on chromosome 17: 80189165, 80189174, 80189177, 80189197, 80189225, 80189230, 80189239, 80189645, 80189671, 80189674, 80189684, 80189687, 80189698, 80189709, 80189719, 80189726, 80189728, 80189739, 80189757, 80189787, 80189792, 80189811, 80189817, 80189832, and 80189841.

[0264] The segment of chromosome 16 includes loci 88700818, 88700826, 88700844, 88700849, 88700857, 88700869, 88700875, 88700891, 88700897, 88700916, 88700920, and 88700937 on chromosome 16. 88700943, 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, 8870 One or more of the following: 1201, 88701204, 88701210, 88701212, 88701236, 88701240, 88701266, 88701278, 88701281, 88701285, 88701305, 88701421, 88701442, and 88701451

[0265] The aforementioned sites in the variant were not mutated.

[0266] 2. The nucleic acid molecule as described in Embodiment 1, wherein the nucleic acid molecule further comprises a segment of chromosome 14 or a variant having at least 70% identity with it, the segment of chromosome 14 comprising one or more of the sites 81421983, 81421989, 81422010, 81422017, 81422032, 81422035, 81422063, and 81422084 on chromosome 14, the segment being 50-5000 bp in length, preferably 50-1000 bp, and the segment of chromosome 16 further comprising one or more of the sites 68771035, 68771037, 68771045, 68771051, 68771059, 68771064, and 68771073 on chromosome 16.

[0267] The aforementioned sites in the variant were not mutated.

[0268] 3. A reagent for detecting DNA, said reagent comprising a reagent for detecting DNA methylation levels selected from the regions described in (1) and (2) below:

[0269] (1) Fragments selected from one or more of the following genes: 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, The fragments are named 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, and RUSC1, with a length of 50-1000 bp.

[0270] (2)(1) The nucleic acid regions within 10Kb upstream and downstream of the gene,

[0271] The ZMIZ1 gene fragment contains one or more of the following ZMIZ1 gene loci: 81001968, 81001996, 81002041, 81002052, 81002054, 81002056, 81002062, 81002083, 81002110, 81002116, 81002123, 81002129, 81002133, 81002137, 81002139, 81002164, 81002168, 81002223, 81002241, and 81002253.

[0272] The fragment of the C15orf52 gene contains one or more of the following C15orf52 gene loci: 40626309, 40626312, and 40626386.

[0273] The following SLC16A3 gene fragments contain the following SLC16A3 gene loci: 80189165, 80189174, 80189177, 80189197, 80189225, 80189230, 80189239, 80189645, 80189671, 80189674, 80189684, 801 One or more of the following: 89687, 80189698, 80189709, 80189719, 80189726, 80189728, 80189739, 80189757, 80189787, 80189792, 80189811, 80189817, 80189832, and 80189841

[0274] The fragment of the ZNF512B gene contains one or more of the following ZNF512B gene loci: 62588634, 62588638, and 62588672.

[0275] The fragment of the SLC17A5 gene contains one or more of the following SLC17A5 gene loci: 74290205, 74290207, 74290220, 74290225, and 74290228.

[0276] The fragment of the LIMK1 gene contains one or more of the following LIMK1 gene loci: 73508994, 73509017, 73509055, 73509062, 73509073, 73509075, 73509112, 73509133, 73509138, 73509148, and 73509160.

[0277] The PLEC gene fragment contains one or more of the following PLEC gene loci: 145013661 and 145013673.

[0278] The fragment containing the TOR4A gene contains one or more of the following TOR4A gene sites: 140172787, 140172790, and 140172812.

[0279] The fragment of the TMEM131L gene contains one or more of the following TMEM131L gene loci: 154409945, 154409963, 154409972, 154409978, 154409997, 154410003, and 154410006.

[0280] The fragment containing the DNM2 gene contains one or more of the following DNM2 gene loci: 10870373, 10870377, 10870427, 10870429, 10870441, and 10870448.

[0281] The IL17C gene fragment contains the following IL17C gene loci: 88700818, 88700826, 88700844, 88700849, 88700857, 88700869, 88700875, 88700891, 88700897, 88700916, 88700920, and 8870093. 7, 88700943, 88700948, 88700967, 88700970, 88700993, 88701004, 88701021, 88701029, 88701036, 88701043, 88701051, 88701060, 88701074, 88701081, 8 8701090, 88701099, 88701111, 88701115, 88701133, 88701140, 88701148, 88701159, 88701161, 88701176, 88701178, 88701180, 88701183, 88701190, 8870 One or more of the following: 1201, 88701204, 88701210, 88701212, 88701236, 88701240, 88701266, 88701278, 88701281, 88701285, 88701305, 88701421, 88701442, and 88701451

[0282] The fragment containing the PRDM16 gene contains one or more of the following PRDM16 gene loci: 3229914, 3229921, 3229950, 3229968, 3229973, 3310213, 3310229, 3310235, 3310238, 3310240, 3310268, 3310287, 3310312, 3310314, 3310317, and 3310329.

[0283] The TSHR gene fragment contains one or more of the following TSHR gene loci: 81421983, 81421989, 81422010, 81422017, 81422032, 81422035, 81422063, and 81422084.

[0284] The fragment containing the KIF1A gene contains one or more of the following KIF1A gene loci: 241759696, 241759701, 241759714, and 241759716.

[0285] The fragment containing the DAPK gene contains one or more of the following DAPK gene sites: 90112842, 90112853, 90112861, and 90112866.

[0286] The fragment containing the CDH1 gene contains one or more of the following CDH1 gene sites: 68771035, 68771037, 68771045, 68771051, 68771059, 68771064, and 68771073.

[0287] The fragment containing the TPO gene loci are one or more of the following: 1481013, 1481015, 1481022, and 1481039.

[0288] The fragment containing the RARG gene contains one or more of the following RARG gene loci: 53613176, 53613182, 53613190, 53613202, 53613210, and 53613218.

[0289] The MT1JP gene fragment contains the following loci: 56669271, 56669292, 56669295, 56669300, 56669318, 56669322, 56669324, 56669327, 56669344, 56669351, 56669353, 56669402, 56669414, 56669423, 56669430, 56669433, 56669437, and 56669. One or more of the following: 451, 56669453, 56669455, 56669463, 56669474, 56669480, 56669482, 56669485, 56669487, 56669490, 56669519, 56669533, 56669553, 56669564, 56669573, 56669578, 56669588, 56669590, 56669606, 56669610

[0290] The fragment of the TBX3 gene contains one or more of the following TBX3 gene loci: 115174750, 115174773, and 115174780.

[0291] The fragment containing the BIN1 gene contains one or more of the following BIN1 gene loci: 127822478, 127822492, 127822495, 127822514, 127822551, 127822568, 127822582, 127822593, 127822616, and 127822644.

[0292] The fragment of the TIMP2 gene contains one or more of the following TIMP2 gene sites: 76921845, 76921853, and 76921860.

[0293] The fragment containing the CFAP65 gene contains one or more of the following CFAP65 gene loci: 219866132, 219866139, 219866148, 219866158, 219866165, 219866168, 219866199, and 219866218.

[0294] The fragment containing the PRR15 gene contains one or more of the following sites: 29605992, 29606026, 29606040, 29606047, 29606056, 29606062, 29606073, 29606179, 29606191, 29606201, 29606204, 29606220, 29606222, 29606227, 29606231, 29606255, 29606257, 29606262, 29606271, 29606277, 29606289, and 29606320.

[0295] The DPYS gene fragment contains one or more of the following DPYS gene loci: 105478870, 105478873, 105478878, 105478905, 105478908, 105478916, 105478918, 105478945, 105478956, 105478965, 105478974, 105478983, 105478986, and 105478989.

[0296] The fragment containing the MCC gene contains one or more of the following MCC gene loci: 112538999, 112539011, 112539018, 112539022, 112539061, 112539084, 112539104, and 112539128.

[0297] The fragment containing the TBX15 gene contains one or more of the following TBX15 gene loci: 119535725, 119535730, 119535740, 119535742, 119535750, 119535759, 119535766, 119535812, 119535817, 119535821, 119535823, 119535876, 119535879, 119535884, and 119535891.

[0298] The fragment containing the COL23A1 gene contains one or more of the following COL23A1 gene loci: 178003785, 178003798, 178003803, 178003814, 178003823, 178003825, 178003834, 178003841, and 178003844.

[0299] The ILDR2 gene fragment contains the following ILDR2 gene loci: 166890429, 166890436, 166890440, 166890442, 166890448, 166890452, 166890456, 166890461, 166890468, 166890473, 166890475, 166890480, 16 One or more of the following: 6890492, 166890500, 166890503, 166890509, 166890516, 166890528, 166890535, 166890543, 166890555, 166890559, 166890568, 166890573, 166890584, and 166890586.

[0300] The fragment containing the DHRS3 gene contains one or more of the following DHRS3 gene loci: 12656091, 12656114, 12656132, 12656152, 12656170, 12656175, 12656182, 12656187, 12656197, 12656200, 12656211, 12656315, 12656323, 12656340, 12656355, and 12656367.

[0301] The fragment containing the GDNF gene contains one or more of the following GDNF gene loci: 37834763, 37834770, 37834772, 37834774, 37834777, 37834780, 37834784, 37834792, 37834799, 37834802, 37834806, and 37834811.

[0302] The fragment containing the TBX18 gene contains one or more of the following TBX18 gene loci: 85477032, 85477035, 85477070, 85477083, 85477106, 85477124, 85477151, 85477153, and 85477166.

[0303] The fragment containing the SIM2 gene contains one or more of the following SIM2 gene loci: 38069563, 38069579, 38069619, 38069625, 38069638, 38069650, 38069662, 38069664, 38069676, and 38069681.

[0304] The fragment of the HOXA9 gene contains one or more of the following HOXA9 gene loci: 27204848, 27204854, 27204858, 27204861, 27204863, 27204879, 27204884, 27204894, 27204897, 27204918, 27204929, 27204938, 27204945, 27204948, 27204951, 27204958, 27204981, and 27204984.

[0305] The fragment of the EHBP1L1 gene contains one or more of the following EHBP1L1 gene loci: 65352612, 65352621, 65352635, 65352639, 65352642, 65352651, 65352654, 65352665, and 65352670.

[0306] The fragment containing the GJC2 gene contains one or more of the following GJC2 gene loci: 228345954, 228345957, 228345965, 228345978, 228345980, and 228345989.

[0307] The fragment containing the RCOR2 gene contains one or more of the following sites: 63687223, 63687238, 63687247, 63687250, 63687259, 63687282, 63687288, 63687299, 63687318, and 63687325.

[0308] The fragment containing the PRDM1 gene contains one or more of the following PRDM1 gene sites: 106429711, 106429722, 106429731, 106429747, 106429750, 106429761, 106429769, and 106429771.

[0309] The fragment containing the UNCX gene contains one or more of the following UNCX gene sites: 1263643, 1263655, 1263659, 1263664, 1263676, 1263694, 1263716, and 1263723.

[0310] The fragment containing the RPS7P5 gene contains one or more of the following RPS7P5 gene sites: 240161502, 240161507, 240161511, 240161516, 240161523, 240161527, 240161530, 240161535, 240161546, 240161558, and 240161560.

[0311] The FOXI2 gene fragment contains one or more of the following FOXI2 gene loci: 129534843, 129534853, 129534866, 129534879, 129534891, 129534910, 129534912, and 129534924.

[0312] The fragment of the ACRBP gene contains one or more of the following ACRBP gene loci: 6756182, 6756187, 6756191, 6756195, 6756211, 6756225, 6756230, and 6756270.

[0313] The fragment containing the GAS6 gene contains one or more of the following GAS6 gene loci: 114524043, 114524062, 114524068, 114524084, 114524095, 114524131, 114524138, 114524142, 114524150, and 114524158.

[0314] The fragment containing the MCRIP2 gene loci are one or more of the following: 698072, 698142, 698153, 698168, 698208, 698218, 698222, and 698230.

[0315] The fragment of the LINC01977 gene contains one or more of the following LINC01977 gene loci: 77789596, 77789601, 77789612, 77789620, 77789628, 77789632, 77789635, and 77789640.

[0316] The fragment containing the EGR3 gene contains one or more of the following EGR3 gene loci: 22548250, 22548260, 22548269, 22548279, 22548283, 22548287, 22548296, and 22548299.

[0317] The SOX17 gene fragment contains one or more of the following SOX17 gene loci: 55379566, 55379568, 55379573, 55379579, 55379583, 55379591, 55379599, 55379602, 55379608, 55379617, and 55379620.

[0318] The PAX5 gene fragment contains one or more of the following PAX5 gene loci: 36986087, 36986093, 36986098, 36986101, 36986103, 36986117, 36986131, 36986138, 36986141, 36986143, 36986147, 36986149, and 36986156.

[0319] The fragment of the NEURL1 gene contains one or more of the following NEURL1 gene sites: 105344464, 105344482, 105344493, 105344495, 105344497, 105344503, 105344506, 105344513, 105344516, 105344519, and 105344526.

[0320] The fragment containing the IRX4 gene contains one or more of the following sites: 1876386, 1876395, 1876397, 1876403, 1876420, 1876424, 1876432, 1876436, 1876449, 1876456, 1876459, and 1876463.

[0321] The fragment of the RUSC1 gene contains one or more of the following RUSC1 gene loci: 155295135, 155295171, 155295181, 155295192, 155295196, 155295212, 155295229, and 155295236.

[0322] 4. The DNA detection reagent as described in embodiment 3, characterized in that,

[0323] The fragment of the ZMIZ1 gene contains one or more of the following ZMIZ1 gene loci: 81002041, 81002052, 81002054, 81002056, 81002062, and 81002083.

[0324] The fragment of the C15orf52 gene contains one or more of the C15orf52 gene sites 40626309 and 40626312.

[0325] The fragment of the SLC16A3 gene contains one or more of the following SLC16A3 gene loci: 80189671, 80189674, 80189684, 80189687, 80189698, 80189709, 80189719, 80189726, 80189728, 80189739, and 80189757.

[0326] The fragment of the ZNF512B gene contains one or more of the following ZNF512B gene loci: 62588634, 62588638, and 62588672.

[0327] The fragment of the SLC17A5 gene contains one or more of the following SLC17A5 gene loci: 74290205, 74290207, 74290220, 74290225, and 74290228.

[0328] The fragment of the LIMK1 gene contains one or more of the following LIMK1 gene loci: 73509112, 73509133, 73509138, 73509148, and 73509160.

[0329] The PLEC gene fragment contains one or more of the PLEC gene loci 145013661 and 145013673.

[0330] The fragment of the TOR4A gene contains one or more of the TOR4A gene loci 140172787, 140172790, and 140172812.

[0331] The fragment of the TMEM131L gene contains one or more of the following TMEM131L gene loci: 154409945, 154409963, 154409972, 154409978, and 154409997.

[0332] The fragment of the DNM2 gene contains one or more of the following DNM2 gene loci: 10870427, 10870429, 10870441, and 10870448.

[0333] The fragment of the IL17C gene contains one or more of the following IL17C gene loci: 88701004, 88701021, 88701029, 88701036, 88701043, 88701051, and 88701060.

[0334] The fragment of the PRDM16 gene contains one or more of the PRDM16 gene loci 3229950, 3229968, and 3229973.

[0335] The fragment of the MT1JP gene contains one or more of the following MT1JP gene loci: 56669271, 56669292, 56669295, 56669300, 56669318, 56669322, 56669324, 56669327, and 56669344.

[0336] The fragment of the TBX3 gene contains one or more of the TBX3 gene loci 115174750, 115174773, and 115174780.

[0337] The fragment of the BIN1 gene contains one or more of the following BIN1 gene loci: 127822478, 127822492, 127822495, 127822514, 127822551, 127822568, 127822582, 127822593, and 127822616.

[0338] The fragment of the TIMP2 gene contains one or more of the TIMP2 gene loci 76921845, 76921853, and 76921860.

[0339] The fragment of the CFAP65 gene contains one or more of the CFAP65 gene loci 219866199 and 219866218.

[0340] The TSHR gene fragment contains one or more of the following TSHR gene loci: 81421983, 81421989, 81422010, 81422017, 81422032, 81422035, 81422063, and 81422084.

[0341] The fragment of the KIF1A gene contains one or more of the following KIF1A gene loci: 241759696, 241759701, 241759714, and 241759716.

[0342] The fragment of the DAPK gene contains one or more of the following DAPK gene sites: 90112842, 90112853, 90112861, and 90112866.

[0343] The fragment of the CDH1 gene contains one or more of the following CDH1 gene sites: 68771035, 68771037, 68771045, 68771051, 68771059, 68771064, and 68771073.

[0344] The fragment of the TPO gene contains one or more of the following TPO gene loci: 1481013, 1481015, 1481022, and 1481039.

[0345] The fragment of the RARG gene contains one or more of the following RARG gene loci: 53613176, 53613182, 53613190, 53613202, 53613210, and 53613218.

[0346] The fragment of the PRR15 gene contains one or more of the following PRR15 gene loci: 29606026, 29606040, 29606047, 29606056, 29606062, 29606073, 29606220, 29606222, 29606227, 29606231, 29606255, 29606257, 29606262, 29606271, 29606277, and 29606289.

[0347] The DPYS gene fragment contains one or more of the following DPYS gene loci: 105478905, 105478908, 105478916, 105478918, 105478945, 105478956, 105478965, 105478974, and 105478983.

[0348] The fragment of the MCC gene contains one or more of the following MCC gene sites: 112538999, 112539011, 112539018, 112539022, and 112539061.

[0349] The fragment of the TBX15 gene contains one or more of the following TBX15 gene loci: 119535740, 119535742, 119535750, 119535759, and 119535766.

[0350] The fragment of the COL23A1 gene contains one or more of the following COL23A1 gene loci: 178003798, 178003803, 178003814, 178003823, 178003825, 178003834, 178003841, and 178003844.

[0351] The fragment of the ILDR2 gene contains one or more of the following ILDR2 gene loci: 166890516, 166890528, 166890535, 166890543, 166890555, 166890559, 166890568, 166890573, 166890584, and 166890586.

[0352] The fragment of the DHRS3 gene contains one or more of the following DHRS3 gene loci: 12656340, 12656355, and 12656367.

[0353] The fragment containing the GDNF gene contains one or more of the following GDNF gene loci: 37834770, 37834772, 37834774, 37834777, 37834780, 37834784, 37834792, 37834799, 37834802, 37834806, and 37834811.

[0354] The fragments of the TBX18 gene contain one or more of the following TBX18 gene loci: 85477035, 85477070, 85477083, and 85477106.

[0355] The fragment containing the SIM2 gene contains one or more of the following SIM2 gene loci: 38069638, 38069650, 38069662, 38069664, 38069676, and 38069681.

[0356] The fragment of the HOXA9 gene contains one or more of the following HOXA9 gene loci: 27204854, 27204858, 27204861, 27204863, and 27204879.

[0357] The fragment of the EHBP1L1 gene contains one or more of the following EHBP1L1 gene loci: 65352621, 65352635, 65352639, 65352642, 65352651, 65352654, 65352665, and 65352670.

[0358] The fragment containing the GJC2 gene contains one or more of the following GJC2 gene loci: 228345965, 228345978, 228345980, and 228345989.

[0359] The fragment containing the RCOR2 gene contains one or more of the following RCOR2 gene loci: 63687223, 63687238, 63687247, 63687250, and 63687259.

[0360] The fragment containing the PRDM1 gene contains one or more of the following PRDM1 gene sites: 106429722, 106429731, 106429747, 106429750, 106429761, 106429769, and 106429771.

[0361] The fragment containing the UNCX gene contains one or more of the following UNCX gene sites: 1263643, 1263655, 1263659, 1263664, and 1263676.

[0362] The fragment of the RPS7P5 gene contains one or more of the following RPS7P5 gene sites: 240161511, 240161516, 240161523, 240161527, and 240161530.

[0363] The FOXI2 gene fragment contains one or more of the following FOXI2 gene loci: 129534910, 129534912, and 129534924.

[0364] The fragment of the ACRBP gene contains one or more of the following ACRBP gene sites: 6756182, 6756187, 6756191, 6756195, and 6756211.

[0365] The fragment containing the GAS6 gene contains one or more of the following GAS6 gene sites: 114524062, 114524068, 114524084, 114524095, 114524131, and 114524138.

[0366] The fragment containing the MCRIP2 gene contains one or more of the following MCRIP2 gene sites: 698072, 698142, 698153, 698168, and 698208.

[0367] The fragment of the LINC01977 gene contains one or more of the following LINC01977 gene loci: 77789596, 77789601, 77789612, and 77789620.

[0368] The fragment containing the EGR3 gene contains one or more of the following EGR3 gene loci: 22548269, 22548279, 22548283, 22548287, 22548296, and 22548299.

[0369] The SOX17 gene fragment contains one or more of the following SOX17 gene loci: 55379602, 55379608, 55379617, and 55379620.

[0370] The PAX5 gene fragment contains one or more of the following PAX5 gene loci: 36986087, 36986093, 36986098, 36986101, and 36986103.

[0371] The fragment of the NEURL1 gene contains one or more of the following NEURL1 gene sites: 105344493, 105344495, and 105344497.

[0372] The fragment of the IRX4 gene contains one or more of the following IRX4 gene loci: 1876386, 1876395, 1876397, and 1876403.

[0373] The fragment of the RUSC1 gene contains one or more of the following RUSC1 gene sites: 155295192, 155295196, and 155295212.

[0374] 5. The DNA detection reagent as described in embodiment 3, characterized in that the DNA detection reagent further includes a reagent for detecting the mutation level at the V600E site of the BRAF gene.

[0375] 6. The DNA detection reagent as described in embodiment 3, characterized in that the DNA detection reagent further includes a reagent for detecting the mutation level at the C228T / C250T site of the TERT gene.

[0376] 7. A reagent for detecting DNA methylation, said reagent detecting the methylation level of one or more of the following (a)-(d):

[0377] a.(1) One or more of the following on chromosome 7: 73508994, 73509017, 73509055, 73509062, 73509073, 73509075, 73509112, 73509133, 73509138, 73509148, and 73509160.

[0378] (2) One or more of the following on chromosome 6: 74290205, 74290207, 74290220, 74290225, and 74290228;

[0379] b.(1) One or more of the following on chromosome 2: 127822478, 127822492, 127822495, 127822514, 127822551, 127822568, 127822582, 127822593, 127822616, 127822644, and

[0380] (2) One or more of the following on chromosome 19: 10870373, 10870377, 10870427, 10870429, 10870441, and 10870448;

[0381] c.(1) One or more of the following on chromosome 2: 127822478, 127822492, 127822495, 127822514, 127822551, 127822568, 127822582, 127822593, 127822616, 127822644, and

[0382] (2) One or more of the following on chromosome 17: 80189165, 80189174, 80189177, 80189197, 80189225, 80189230, 80189239, 80189645, 80189671, 80189674, 80189684, 80189687, 80189698, 80189709, 80189719, 80189726, 80189728, 80189739, 80189757, 80189787, 80189792, 80189811, 80189817, 80189832, and 80189841;

[0383] d.(1) One or more of the following on chromosome 17: 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.

[0384] (2) One or more of the following on chromosome 19: 10870373, 10870377, 10870427, 10870429, 10870441, and 10870448.

[0385] (3) 88700818, 88700826, 88700844, 88700849, 88700857, 88700869, 88700875, 88700891, 88700897, 88700916, 88700920, 88700937, 8870094 on chromosome 16 3. 88700948, 88700967, 88700970, 88700993, 88701004, 88701021, 88701029, 88701036, 88701043, 88701051, 88701060, 88701074, 88701081, 8870109 0, 88701099, 88701111, 88701115, 88701133, 88701140, 88701148, 88701159, 88701161, 88701176, 88701178, 88701180, 88701183, 88701190, 8870120 1. One or more of the following: 88701204, 88701210, 88701212, 88701236, 88701240, 88701266, 88701278, 88701281, 88701285, 88701305, 88701421, 88701442, and 88701451.

[0386] 8. The reagent as described in embodiment 7, characterized in that the reagent further detects the methylation level at the following sites:

[0387] e. (1) One or more of the following on chromosome 16: 68771035, 68771037, 68771045, 68771051, 68771059, 68771064, 68771073, and (2) One or more of the following on chromosome 14: 81421983, 81421989, 81422010, 81422017, 81422032, 81422035, 81422063, 81422084.

[0388] 9. The reagent according to any one of embodiments 2-8, characterized in that it further comprises one or more features selected from the following:

[0389] The fragment includes either the sense or antisense strand of DNA.

[0390] The reagents for detecting DNA methylation are selected from one or more of the following methods: PCR based on bisulfite conversion, DNA sequencing, methylation-sensitive restriction endonuclease analysis, quantitative fluorescence method, methylation-sensitive high-resolution melting curve method, chip-based methylation mapping analysis, and mass spectrometry.

[0391] Preferably, the reagent for detecting DNA methylation is selected from one or more of the following: bisulfite and its derivatives, PCR buffer, polymerase, dNTPs, primers, probes, methylation-sensitive or non-sensitive restriction endonucleases, enzyme digestion buffers, fluorescent dyes, fluorescence quenchers, fluorescent reporter reagents, exonucleases, alkaline phosphatase, internal standards, and controls.

[0392] Preferably, the primers are methylation-specific or non-specific primers; preferably, the primer sequence includes a non-methylation-specific blocking sequence; preferably, the primers are SEQ ID NO: 1, 2, 4, 5, 7, 8 or sequences having 90% identity with them.

[0393] Preferably, the probe has a reporter sequence; more preferably, the probe is SEQ ID NO:3, 6, 9 or a sequence having 90% identity with it.

[0394] The reagents used to detect 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 mismatch cleavage, allele-specific oligonucleotide analysis, ligase chain reaction, allele-specific amplification, RNase A cleavage, chromosome in situ hybridization, fluorescence in situ hybridization, DNA sequence analysis, enzymatic mismatch cleavage, fragment length polymorphism, dideoxy fingerprinting, mismatch-binding protein truncation assay, primer extension, oligonucleotide linking detection, capillary electrophoresis, and microarray-based methods.

[0395] Preferably, the reagents for detecting gene mutations include: primers, probes, buffer solutions, polymerases, dNTPs, restriction endonucleases, enzyme digestion buffers, fluorescent dyes, fluorescent quenchers, fluorescent reporter reagents, exonucleases, alkaline phosphatases, internal standards, and controls.

[0396] 10. A kit for identifying the nature of thyroid nodules, comprising the reagent of any one of embodiments 2-9 and optionally the nucleic acid molecule of embodiment 1.

[0397] 11. Reagents for DNA detection and optional embodiment 1: Use of the nucleic acid molecules described in embodiment 1 in the preparation of a kit for identifying the nature of thyroid nodules in a sample, wherein the reagent detects the methylation level of one or more of the following (a)-(d):

[0398] a.(1) One or more of the following on chromosome 7: 73508994, 73509017, 73509055, 73509062, 73509073, 73509075, 73509112, 73509133, 73509138, 73509148, and 73509160.

[0399] (2) One or more of the following on chromosome 6: 74290205, 74290207, 74290220, 74290225, and 74290228;

[0400] b.(1) One or more of the following on chromosome 2: 127822478, 127822492, 127822495, 127822514, 127822551, 127822568, 127822582, 127822593, 127822616, 127822644, and

[0401] (2) One or more of the following on chromosome 19: 10870373, 10870377, 10870427, 10870429, 10870441, and 10870448;

[0402] c.(1) One or more of the following on chromosome 2: 127822478, 127822492, 127822495, 127822514, 127822551, 127822568, 127822582, 127822593, 127822616, 127822644, and

[0403] (2) One or more of the following on chromosome 17: 80189165, 80189174, 80189177, 80189197, 80189225, 80189230, 80189239, 80189645, 80189671, 80189674, 80189684, 80189687, 80189698, 80189709, 80189719, 80189726, 80189728, 80189739, 80189757, 80189787, 80189792, 80189811, 80189817, 80189832, and 80189841;

[0404] d.(1) One or more of the following on chromosome 17: 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.

[0405] (2) One or more of the following on chromosome 19: 10870373, 10870377, 10870427, 10870429, 10870441, and 10870448.

[0406] (3) 88700818, 88700826, 88700844, 88700849, 88700857, 88700869, 88700875, 88700891, 88700897, 88700916, 88700920, 88700937, 8870094 on chromosome 16 3. 88700948, 88700967, 88700970, 88700993, 88701004, 88701021, 88701029, 88701036, 88701043, 88701051, 88701060, 88701074, 88701081, 8870109 0, 88701099, 88701111, 88701115, 88701133, 88701140, 88701148, 88701159, 88701161, 88701176, 88701178, 88701180, 88701183, 88701190, 8870120 1. One or more of the following: 88701204, 88701210, 88701212, 88701236, 88701240, 88701266, 88701278, 88701281, 88701285, 88701305, 88701421, 88701442, and 88701451.

[0407] The optional e.(1) one or more of the following on chromosome 16: 68771035, 68771037, 68771045, 68771051, 68771059, 68771064, 68771073, and (2) one or more of the following on chromosome 14: 81421983, 81421989, 81422010, 81422017, 81422032, 81422035, 81422063, 81422084.

[0408] Preferably, the reagent is as described in any one of embodiments 8-9.

[0409] 12. The use as described in embodiment 11, characterized in that the use has one or more features selected from the following:

[0410] The kit also includes reagents for detecting the mutation level at the V600E site of the BRAF gene and / or the mutation level at the C228T / C250T sites of the TERT gene.

[0411] The identification of the nature of thyroid nodules includes: 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 nodules based on the comparison results or the score.

[0412] The sample is derived from a human body, preferably from tissues, cells, or bodily fluids, such as thyroid tissue or blood.

[0413] The sample contains genomic DNA or cfDNA.

[0414] 13. A method for identifying the nature of thyroid nodules, comprising:

[0415] (1) The methylation level of genes, loci or nucleic acid regions in the sample of the test subject, wherein the genes, loci or nucleic acid regions are as described in Implementation Scheme 2-9;

[0416] Optional (2) detect the mutation level at the V600E site of the BRAF gene and / or the mutation level at the C228T / C250T site of the TERT gene;

[0417] (3) Compare with control samples, or obtain a score based on the stated methylation level and / or mutation level;

[0418] (4) Identify the nature of the thyroid nodules based on the comparison results or scores from step (3).

[0419] Preferably, step (4) includes:

[0420] Compared with the control sample, the changes in methylation level and / or mutation level of the subject sample are analyzed. When the methylation level and / or mutation level meet the threshold, the thyroid nodule is identified as benign or malignant.

[0421] When the score meets the threshold, the thyroid nodule is identified as either benign or malignant.

[0422] Example

[0423] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments. In the following embodiments, experimental methods without specific conditions are generally performed according to the methods described under conventional conditions.

[0424] Example 1: Reduced Representation Bisulfite Sequencing (RRBS) Screening for methylation sites differentiating benign and malignant thyroid nodules

[0425] 1) Sample preparation

[0426] DNA was extracted from tissues of 37 thyroid cancer patients and 37 benign thyroid nodules using the QIAamp DNA Mini Kit (QIAGEN, catalog number: 51304); DNA concentration was detected using the Qubit™ dsDNA HS Assay Kit (Thermo, catalog number: Q32854); and quality control was performed using 1% agarose gel electrophoresis.

[0427] 2) MspI digestion

[0428] The reaction system is prepared as follows:

[0429] Components Volume (μl) 10×Buffer Tango 2.0 MspI (10 U / μl) 1.0 Nuclease-free water + DNA 17.0 total 20.0

[0430] The reaction procedure is as follows: 37℃ for 2 hours, then store at 4℃.

[0431] 3) End repair and A addition

[0432] The reaction system is prepared as follows:

[0433] Components Volume (μl) DNA digestion 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

[0434] The reaction procedure was as follows: 20℃ for 30 minutes, 65℃ for 30 minutes, and stored at 4℃.

[0435] 4) Connector connection

[0436] The reaction system is prepared as follows:

[0437] Components Volume (μl) End-stage repair and A-added 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

[0438] The reaction procedure was as follows: overnight at 16°C, 10 minutes at 65°C, and stored at 4°C.

[0439] 5) Purification after ligation

[0440] i. After ligation, transfer the 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 centrifuge tube on a magnetic rack until the solution becomes clear;

[0441] ii. Rinse twice with 80% ethanol solution;

[0442] iii. Dry the magnetic beads at room temperature;

[0443] iv. Add 32 μl ddH2O, incubate at room temperature for 2 minutes, place the centrifuge tube on a magnetic rack until the solution is clear, and transfer 30 μl of the supernatant solution to a new centrifuge tube.

[0444] 6) Bisulfite conversion

[0445] Use MethylCode TM The Bisulfite Conversion Kit (Thermo, catalog number: MECOV50) performs bisulfite conversion on the DNA obtained in step 5. Unmethylated cytosine (C) is converted into uracil (U); methylated cytosine remains unchanged after conversion.

[0446] The conversion reagent is prepared as follows:

[0447] Components Volume (μl) Nuclease-free water 800.0 Dilution Buffer 300.0 Resuspension Buffer 50.0

[0448] Add 120 μl of the prepared conversion reagent to 30 μl of the ligation and purification product obtained in step 5, and mix well. The reaction program is as follows: 98 °C for 10 minutes, 64 °C for 2.5 hours, and store at 4 °C.

[0449] The processed DNA was recovered according to the instructions, and finally eluted with 43 μl of elution buffer. 41.6 μl of the elution buffer was then transferred for the next reaction.

[0450] 7) Library expansion

[0451] The reaction system is prepared as follows:

[0452] Components Volume (μ1) 10×PfuTurbo Cx reaction buffer 5.0 dNTPs (25mM each) 0.4 Primer mix 2.0 PfuTurbo Cx hotstart DNA polymerase(2.5U / μl) 1.0 Bisulfite conversion of DNA 41.6 total 50.0

[0453] The reaction procedure was as follows: 95℃ for 2 minutes; 95℃ for 30 seconds, 65℃ for 30 seconds, 72℃ for 1 minute, 15 cycles; 72℃ for 5 minutes, and stored at 4℃.

[0454] 8) Library purification

[0455] i. Add the library amplification product to 50 μl of AMPure beads, vortex to mix, incubate at room temperature for 5 minutes, and briefly centrifuge at low speed. Place the centrifuge tube on a magnetic rack until the solution becomes clear;

[0456] ii. Rinse twice with 80% ethanol solution;

[0457] iii. Dry the magnetic beads at room temperature;

[0458] iv. Add 40 μl ddH2O, incubate at room temperature for 2 minutes, place the centrifuge tube on a magnetic rack until the solution is clear, and transfer 38 μl of the supernatant solution to a new centrifuge tube.

[0459] 9) Document Quality Control

[0460] Qubit determines library concentration, and LabChip (PerkinElmer) detects library fragment distribution, such as... Figure 1 As shown.

[0461] 10) Sequencing

[0462] Sequencing was performed using the Illumina platform HiSeq X Ten with PE150.

[0463] 11) Data Analysis

[0464] Bioinformatics analysis yielded the CpG sites with methylation differences between benign and malignant thyroid nodules, as shown in Table 1. These sites include the chromosome where the CpG is located, the CpG initiation site, the corresponding gene, the statistical comparison P-value, and the ratio of methylated CpG sites between malignant and benign thyroid nodules.

[0465] Table 1. CpG sites and corresponding 51 genes showing differential methylation between benign and malignant thyroid nodules.

[0466] chromosome CpG initiation site Gene name p-value Malignant / benign chr10 81001968 ZMIZ1 1.2E-125 0.47 chr10 81001996 ZMIZ1 1.80E-13 0.35

[0467] chromosome CpG initiation site Gene name p-value Malignant / benign chr10 81002041 ZMIZ1 1.3E-28 0.44 chr10 81002052 ZMIZ1 5.7E-19 0.40 chr10 81002054 ZMIZ1 9.5E-16 0.45 chr10 81002056 ZMIZ1 1.6E-87 0.24 chr10 81002062 ZMIZ1 4.52E-40 0.21 chr10 81002083 ZMIZ1 2.08E-24 0.24 chr10 81002110 ZMIZ1 5.7E-19 0.40 chr10 81002116 ZMIZ1 1.78E-94 0.14 chr10 81002123 ZMIZ1 1.6E-87 0.24 chr10 81002129 ZMIZ1 2.6E-37 0.49 chr10 81002133 ZMIZ1 1.4E-50 0.33 chr10 81002137 ZMIZ1 1.4E-50 0.33 chr10 81002139 ZMIZ1 1.2E-125 0.47 chr10 81002164 ZMIZ1 5.7E-19 0.40 chr10 81002168 ZMIZ1 9.32E-79 0.24 chr10 81002223 ZMIZ1 0.000069 0.47 chr10 81002241 ZMIZ1 9.5E-16 0.45 chr10 81002253 ZMIZ1 3.11E-06 0.48 chr15 40626309 C15orf52 1.4E-50 68.49 chr15 40626312 C15orf52 1.2E-125 28.89 chr15 40626386 C15orf52 4.34E-19 4.11 chr17 80189165 SLC16A3 7.75E-14 0.35 chr17 80189174 SLC16A3 0.00022 0.51 chr17 80189177 SLC16A3 1.2E-125 0.47 chr17 80189197 SLC16A3 1.82E-59 0.13 chr17 80189225 SLC16A3 1.72E-62 0.28 chr17 80189230 SLC16A3 9.8E-86 0.28 chr17 80189239 SLC16A3 1.96E-51 0.26 chr17 80189645 SLC16A3 1.97E-02 0.30 chr17 80189671 SLC16A3 1.57E-02 0.58 chr17 80189674 SLC16A3 4.69E-02 0.54 chr17 80189684 SLC16A3 1.20E-02 0.44 chr17 80189687 SLC16A3 7.44E-03 0.31 chr17 80189698 SLC16A3 1.62E-03 0.58 chr17 80189709 SLC16A3 1.54E-04 0.48 chr17 80189719 SLC16A3 3.22E-02 0.49 chr17 80189726 SLC16A3 1.61E-04 0.34 chr17 80189728 SLC16A3 2.62E-02 0.52 chr17 80189739 SLC16A3 1.82E-02 0.58 chr17 80189757 SLC16A3 6.02E-03 0.51 chr17 80189787 SLC16A3 2.67E-02 0.51 chr17 80189792 SLC16A3 0.005194188 0.32 chr17 80189811 SLC16A3 0.011602734 0.40 chr17 80189817 SLC16A3 0.007298955 0.40 chr17 80189832 SLC16A3 0.000530622 0.37 chr17 80189841 SLC16A3 1.86E-04 0.37 chr20 62588634 ZNF512B 1.80E-13 0.35 chr20 62588638 ZNF512B 9.8E-86 0.28 chr20 62588672 ZNF512B 2.6E-37 0.49 chr6 74290205 SLC17A5 2.64E-34 0.47 chr6 74290207 SLC17A5 1.2E-125 0.47 chr6 74290220 SLC17A5 1.4E-50 0.33 chr6 74290225 SLC17A5 9.8E-86 0.28 chr6 74290228 SLC17A5 4.8E-14 0.45 chr7 73508994 LIMK1 8.18E-77 0.28 chr7 73509017 LIMK1 1.4E-50 0.33 chr7 73509055 LIMK1 1.95E-20 0.14 chr7 73509062 LIMK1 1.70E-24 0.13 chr7 73509073 LIMK1 4.8E-14 0.45 chr7 73509075 LIMK1 1.2E-125 0.47 chr7 73509112 LIMK1 1.2E-125 0.47 chr7 73509133 LIMK1 3.88E-18 0.10

[0468] chromosome CpG initiation site Gene name p-value Malignant / benign chr7 73509138 LIMK1 1.2E-125 0.47 chr7 73509148 LIMK1 4.8E-14 0.45 chr7 73509160 LIMK1 5.7E-19 0.40 chr8 145013661 PLEC 1.2E-125 0.47 chr8 145013673 PLEC 9.8E-86 0.28 chr9 140172787 TOR4A 1.25E-12 0.19 chr9 140172790 TOR4A 1.4E-50 0.33 chr9 140172812 TOR4A 1.4E-50 0.33 chr4 154409945 TMEM131L 9.8E-86 0.28 chr4 154409963 TMEM131L 9.32E-12 0.28 chr4 154409972 TMEM131L 0.00860178 0.16 chr4 154409978 TMEM131L 9.8E-86 0.28 chr4 154409997 TMEM131L 1.2E-125 0.47 chr4 154410003 TMEM131L 1.80E-13 0.35 chr4 154410006 TMEM131L 1.4E-50 0.33 chr19 10870373 DNM2 0.000182451 0.49 chr19 10870377 DNM2 6.91506E-08 0.29 chr19 10870427 DNM2 1.48631E-06 0.40 chr19 10870429 DNM2 1.13381E-06 0.36 chr19 10870441 DNM2 4.15659E-06 0.39 chr19 10870448 DNM2 0.000144712 0.47 chr16 88700818 IL17C 1.04E-27 0.23 chr16 88700826 IL17C 1.92E-14 0.28 chr16 88700844 IL17C 1.53E-25 0.33 chr16 88700849 IL17C 5.7E-19 0.40 chr16 88700857 IL17C 9.8E-86 0.28 chr16 88700869 IL17C 8.85E-86 0.06 chr16 88700875 IL17C 1.3E-28 0.44 chr16 88700891 IL17C 5.39E-11 0.54 chr16 88700897 IL17C 2.6E-37 0.49 chr16 88700916 IL17C 2.05E-39 0.28 chr16 88700920 IL17C 1.2E-125 0.47 chr16 88700937 IL17C 3.42E-37 0.30 chr16 88700943 IL17C 1.2E-125 0.47 chr16 88700948 IL17C 1.6E-87 0.24 chr16 88700967 IL17C 9.8E-86 0.28 chr16 88700970 IL17C 4.8E-14 0.45 chr16 88700993 IL17C 1.2E-125 0.47 chr16 88701004 IL17C 0.00022 0.51 chr16 88701021 IL17C 1.25E-80 0.09 chr16 88701029 IL17C 1.36E-87 0.15 chr16 88701036 IL17C 5.7E-19 0.40 chr16 88701043 IL17C 3.06E-33 0.15 chr16 88701051 IL17C 2.6E-37 0.49 chr16 88701060 IL17C 6.7E-37 0.40 chr16 88701074 IL17C 1.2E-125 0.47 chr16 88701081 IL17C 1.2E-125 0.47 chr16 88701090 IL17C 9.5E-16 0.45 chr16 88701099 IL17C 5.7E-19 0.40 chr16 88701111 IL17C 4.10E-28 0.28 chr16 88701115 IL17C 0.000069 0.47 chr16 88701133 IL17C 1.4E-50 0.33 chr16 88701140 IL17C 1.64E-50 0.22 chr16 88701148 IL17C 9.8E-86 0.28 chr16 88701159 IL17C 6.7E-37 0.40 chr16 88701161 IL17C 9.8E-86 0.28 chr16 88701176 IL17C 1.2E-125 0.47 chr16 88701178 IL17C 8.05E-12 0.58 chr16 88701180 IL17C 1.2E-125 0.47 chr16 88701183 IL17C 1.2E-125 0.47 chr16 88701190 IL17C 1.45E-18 0.15 chr16 88701201 IL17C 1.19E-18 0.33

[0469] chromosome CpG initiation site Gene name p-value Malignant / benign chr16 88701204 IL17C 1.4E-50 0.33 chr16 88701210 IL17C 1.6E-87 0.24 chr16 88701212 IL17C 1.80E-13 0.35 chr16 88701236 IL17C 1.2E-125 0.47 chr16 88701240 IL17C 1.2E-125 0.47 chr16 88701266 IL17C 1.2E-125 0.47 chr16 88701278 IL17C 9.64E-36 0.11 chr16 88701281 IL17C 0.036965614 0.48 chr16 88701285 IL17C 1.6E-87 0.24 chr16 88701305 IL17C 1.80E-13 0.35 chr16 88701421 IL17C 1.14E-28 0.17 chr16 88701442 IL17C 1.6E-87 0.24 chr16 88701451 IL17C 2.6E-37 0.49 chr1 3229914 PRDM16 1.4E-50 0.33 chr1 3229921 PRDM16 1.2E-125 0.47 chr1 3229950 PRDM16 1.4E-50 0.33 chr1 3229968 PRDM16 0.00022 0.51 chr1 3229973 PRDM16 9.8E-86 0.28 chr1 3310213 PRDM16 9.8E-86 0.28 chr1 3310229 PRDM16 1.4E-50 0.33 chr1 3310235 PRDM16 1.77E-76 0.27 chr1 3310238 PRDM16 2.42E-31 0.15 chr1 3310240 PRDM16 4.31E-14 0.23 chr1 3310268 PRDM16 4.8E-14 0.45 chr1 3310287 PRDM16 9.8E-86 0.28 chr1 3310312 PRDM16 1.4E-50 0.33 chr1 3310314 PRDM16 1.10E-21 0.10 chr1 3310317 PRDM16 9.8E-86 0.28 chr1 3310329 PRDM16 1.27E-20 0.23 chr14 81421983 TSHR 1.37E-04 7.11 chr14 81421989 TSHR 7.31E-04 4.75 chr14 81422010 TSHR 4.71E-04 4.85 chr14 81422017 TSHR 4.65E-03 4.97 chr14 81422032 TSHR 4.51E-03 1.46 chr14 81422035 TSHR 1.22E-03 1.88 chr14 81422063 TSHR 8.18E-03 1.93 chr14 81422084 TSHR 1.95E-03 1.71 chr2 241759696 KIF1A 7.08E-03 2.47 chr2 241759701 KIF1A 5.29E-02 2.20 chr2 241759714 KIF1A 3.77E-02 1.17 chr2 241759716 KIF1A 1.85E-02 2.35 chr9 90112842 DAPK 1.66E-02 13.18 chr9 90112853 DAPK 1.59E-02 2.99 chr9 90112861 DAPK 9.22E-03 11.52 chr9 90112866 DAPK 4.43E-02 2.26 chr16 68771035 CDH1 7.57E-03 3.16 chr16 68771037 CDH1 9.10E-03 3.82 chr16 68771045 CDH1 9.29E-03 1.96 chr16 68771051 CDH1 7.76E-03 1.36 chr16 68771059 CDH1 1.29E-02 4.22 chr16 68771064 CDH1 3.14E-02 1.90 chr16 68771073 CDH1 1.52E-02 1.88 chr2 1481013 TPO 6.48E-03 0.49 chr2 1481015 TPO 7.07E-03 0.49 chr2 1481022 TPO 8.63E-03 0.49 chr2 1481039 TPO 3.60E-02 0.49 chr12 53613176 RARG 1.03E-04 0.14 chr12 53613182 RARG 1.03E-04 0.14 chr12 53613190 RARG 1.03E-04 0.14 chr12 53613202 RARG 1.03E-04 0.14 chr12 53613210 RARG 1.03E-04 0.14 chr12 53613218 RARG 1.03E-04 0.14

[0470] Chromosome CpG start site Gene name P value Malignant / Benign chr16 56669271 MT1JP 2.6E-37 0.49 chr16 56669292 MT1JP 1.4E-50 0.33 chr16 56669295 MT1JP 1.4E-50 0.33 chr16 56669300 MT1JP 1.3E-28 0.44 chr16 56669318 MT1JP 5.7E-19 0.40 chr16 56669322 MT1JP 1.12E-77 0.29 ​ 56669324 ​ 8.78E-26 0.07 ​ 56669327 ​ 5.7E-19 0.40 ​ 56669344 ​ 5.42E-08 0.48 ​ 56669351 ​ 1.3E-28 0.44 ​ 56669353 ​ 1.4E-50 0.33 ​ 56669402 ​ 1.2E-125 0.47 ​ 56669414 ​ 9.8E-86 0.28 ​ 56669423 ​ 1.4E-50 0.33 ​ 56669430 ​ 1.2E-125 0.47 ​ 56669433 ​ 4.8E-14 0.45 ​ 56669437 ​ 9.8E-86 0.28 ​ 56669451 ​ 9.8E-86 0.28 ​ 56669453 ​ 2.6E-37 0.49 ​ 56669455 ​ 1.4E-50 0.33 ​ 56669463 ​ 1.3E-28 0.44 ​ 56669474 ​ 9.8E-86 0.28 ​ 56669480 ​ 1.2E-125 0.47 ​ 56669482 ​ 1.2E-125 0.47 ​ 56669485 ​ 1.2E-125 0.47 ​ 56669487 ​ 5.68E-47 0.30 ​ 56669490 ​ 0.000069 0.47 ​ 56669519 ​ 1.77E-36 0.23 ​ 56669533 ​ 1.4E-50 0.33 ​ 56669553 ​ 1.2E-125 0.47 ​ 56669564 ​ 1.6E-87 0.24 ​ 56669573 ​ 9.8E-86 0.28 ​ 56669578 ​ 1.4E-50 0.33 ​ 56669588 ​ 4.8E-14 0.45 ​ 56669590 ​ 3.05E-06 0.50 ​ 56669606 ​ 6.7E-37 0.40 ​ 56669610 ​ 5.54E-67 0.25 ​ 115174750 ​ 1.80E-13 0.35 ​ 115174773 ​ 1.32E-27 0.05 ​ 115174780 ​ 5.7E-19 0.40 ​ 127822447 ​ 1.2E-10 0.47 ​ 127822478 ​ 1.1E-08 0.18 ​ 127822492 ​ 1.3E-06 0.43 ​ 127822495 ​ 1.9E-09 0.27 ​ 127822514 ​ 5.9E-09 0.25 ​ 127822551 ​ 1.4E-09 0.26 ​ 127822568 ​ 4.3E-08 0.33 ​ 127822582 ​ 5.5E-10 0.24 ​ 127822593 ​ 2.5E-08 0.42 ​ 127822616 ​ 9.8E-10 0.30 ​ 127822644 ​ 8.6E-07 0.37 ​ 76921845 ​ 9.8E-86 0.28 ​ 76921853 ​ 9.8E-86 0.28 ​ 76921860 ​ 1.2E-125 0.47 ​ 219866132 ​ 9.8E-86 0.28 ​ 219866139 ​ 1.4E-50 0.33 ​ 219866148 ​ 1.4E-50 0.33 ​ 219866158 ​ 1.4E-50 0.33 ​ 219866165 ​ 1.2E-125 0.47 ​ 219866168 ​ 0.029973178 0.39 ​ 219866199 ​ 1.2E-125 0.47 ​ 219866218 ​ 7.40E-75 0.20

[0471] ​ CpG initiation site Gene name p-value Malignant / benign chr7 29605992 PRR15 0.013790853 0.54 chr7 29606026 PRR15 0.013790853 0.54 chr7 29606040 PRR15 0.013790853 0.54 chr7 29606047 PRR15 0.013790853 0.54 chr7 29606056 PRR15 0.013790853 0.54 chr7 29606062 PRR15 0.013790853 0.54 chr7 29606073 PRR15 0.013790853 0.54 chr7 29606179 PRR15 0.033193547 3.56 chr7 29606191 PRR15 0.033193547 3.56 chr7 29606201 PRR15 0.033193547 3.56 chr7 29606204 PRR15 0.033193547 3.56 chr7 29606220 PRR15 0.033193547 3.56 chr7 29606222 PRR15 0.033193547 3.56 chr7 29606227 PRR15 0.033193547 3.56 chr7 29606231 PRR15 0.033193547 3.56 chr7 29606255 PRR15 0.033193547 3.56 chr7 29606257 PRR15 0.033193547 3.56 chr7 29606262 PRR15 0.033193547 3.56 chr7 29606271 PRR15 0.033193547 3.56 chr7 29606277 PRR15 0.033193547 3.56 chr7 29606289 PRR15 0.033193547 3.56 chr7 29606320 PRR15 0.033193547 3.56 chr8 105478870 DPYS 0.041031982 0.22 chr8 105478873 DPYS 0.041031982 0.22 chr8 105478878 DPYS 0.041031982 0.22 chr8 105478905 DPYS 0.025407639 0.37 chr8 105478908 DPYS 0.025407639 0.37 chr8 105478916 DPYS 0.048472944 0.08 chr8 105478918 DPYS 0.048472944 0.08 chr8 105478945 DPYS 0.048472944 0.08 chr8 105478956 DPYS 0.048472944 0.08 chr8 105478965 DPYS 0.048472944 0.08 chr8 105478974 DPYS 0.048472944 0.08 chr8 105478983 DPYS 0.048472944 0.08 chr8 105478986 DPYS 0.048472944 0.08 chr8 105478989 DPYS 0.048472944 0.08 chr5 112538999 MCC 0.047944772 #DIV / 0! chr5 112539011 MCC 0.047944772 #DIV / 0! chr5 112539018 MCC 0.047944772 #DIV / 0! chr5 112539022 MCC 0.047944772 #DIV / 0! chr5 112539061 MCC 0.047944772 #DIV / 0! chr5 112539084 MCC 0.047944772 #DIV / 0! chr5 112539104 MCC 0.047944772 #DIV / 0! chr5 112539128 MCC 0.047944772 #DIV / 0! chr1 119535725 TBX15 0.023314728 5.92 chr1 119535730 TBX15 0.023314728 5.92 chr1 119535740 TBX15 0.023314728 5.92 chr1 119535742 TBX15 0.023314728 5.92 chr1 119535750 TBX15 0.023314728 5.92 chr1 119535759 TBX15 0.047700167 5.15 chr1 119535766 TBX15 0.047700167 5.15 chr1 119535812 TBX15 0.047700167 5.15 chr1 119535817 TBX15 0.047700167 5.15 chr1 119535821 TBX15 0.047700167 5.15 chr1 119535823 TBX15 0.047700167 5.15 chr1 119535876 TBX15 0.047700167 5.15 chr1 119535879 TBX15 0.047700167 5.15 chr1 119535884 TBX15 0.047700167 5.15 chr1 119535891 TBX15 0.047700167 5.15 chr5 178003785 COL23A1 0.033010452 14.95 chr5 178003798 COL23A1 0.033010452 14.95 chr5 178003803 COL23A1 0.033010452 14.95

[0472]

[0473] chromosome CpG initiation site Gene name p-value Malignant / benign chr6 85477070 TBX18 0.039952571 0.06 chr6 85477083 TBX18 0.039952571 0.06 chr6 85477106 TBX18 0.039952571 0.06 chr6 85477124 TBX18 0.039952571 0.06 chr6 85477151 TBX18 0.039952571 0.06 chr6 85477153 TBX18 0.039952571 0.06 chr6 85477166 TBX18 0.039952571 0.06 chr21 38069563 SIM2 0.048899033 7.48 chr21 38069579 SIM2 0.048899033 7.48 chr21 38069619 SIM2 0.048899033 7.48 chr21 38069625 SIM2 0.048899033 7.48 chr21 38069638 SIM2 0.048899033 7.48 chr21 38069650 SIM2 0.048899033 7.48 chr21 38069662 SIM2 0.048899033 7.48 chr21 38069664 SIM2 0.048899033 7.48 chr21 38069676 SIM2 0.048899033 7.48 chr21 38069681 SIM2 0.048899033 7.48 chr7 27204848 HOXA9 0.030970577 0.08 chr7 27204854 HOXA9 0.030970577 0.08 chr7 27204858 HOXA9 0.030970577 0.08 chr7 27204861 HOXA9 0.030970577 0.08 chr7 27204863 HOXA9 0.030970577 0.08 chr7 27204879 HOXA9 0.030970577 0.08 chr7 27204884 HOXA9 0.030970577 0.08 chr7 27204894 HOXA9 0.030970577 0.08 chr7 27204897 HOXA9 0.030970577 0.08 chr7 27204918 HOXA9 0.030970577 0.08 chr7 27204929 HOXA9 0.030970577 0.08 chr7 27204938 HOXA9 0.030970577 0.08 chr7 27204945 HOXA9 0.030970577 0.08 chr7 27204948 HOXA9 0.030970577 0.08 chr7 27204951 HOXA9 0.030970577 0.08 chr7 27204958 HOXA9 0.030970577 0.08 chr7 27204981 HOXA9 0.030970577 0.08 chr7 27204984 HOXA9 0.030970577 0.08 chr11 65352612 EHBP1L1 0.013349063 0.03 chr11 65352621 EHBP1L1 0.013349063 0.03 chr11 65352635 EHBP1L1 0.013349063 0.03 chr11 65352639 EHBP1L1 0.013349063 0.03 chr11 65352642 EHBP1L1 0.013349063 0.03 chr11 65352651 EHBP1L1 0.013349063 0.03 chr11 65352654 EHBP1L1 0.013349063 0.03 chr11 65352665 EHBP1L1 0.013349063 0.03 chr11 65352670 EHBP1L1 0.013349063 0.03 chr1 228345954 GJC2 0.001966241 0.58 chr1 228345957 GJC2 0.001966241 0.58 chr1 228345965 GJC2 0.001966241 0.58 chr1 228345978 GJC2 0.001966241 0.58 chr1 228345980 GJC2 0.001966241 0.58 chr1 228345989 GJC2 0.001966241 0.58 chr11 63687223 RCOR2 0.003031617 1.77 chr11 63687238 RCOR2 0.003031617 1.77 chr11 63687247 RCOR2 0.003031617 1.77 chr11 63687250 RCOR2 0.003031617 1.77 chr11 63687259 RCOR2 0.003031617 1.77 chr11 63687282 RCOR2 0.003031617 1.77 chr11 63687288 RCOR2 0.003031617 1.77 chr11 63687299 RCOR2 0.003031617 1.77 chr11 63687318 RCOR2 0.003031617 1.77 chr11 63687325 RCOR2 0.003031617 1.77 chr6 106429711 PRDM1 0.026046614 0.57 chr6 106429722 PRDM1 0.026046614 0.57

[0474] chromosome CpG initiation site Gene name p-value Malignant / benign chr6 106429731 PRDM1 0.026046614 0.57 chr6 106429747 PRDM1 0.026046614 0.57 chr6 106429750 PRDM1 0.026046614 0.57 chr6 106429761 PRDM1 0.026046614 0.57 chr6 106429769 PRDM1 0.026046614 0.57 chr6 106429771 PRDM1 0.026046614 0.57 chr7 1263643 UNCX 0.015900327 0.70 chr7 1263655 UNCX 0.015900327 0.70 chr7 1263659 UNCX 0.03233225 0.53 chr7 1263664 UNCX 0.03233225 0.53 chr7 1263676 UNCX 0.03233225 0.53 chr7 1263694 UNCX 0.03233225 0.53 chr7 1263716 UNCX 0.03233225 0.53 chr7 1263723 UNCX 0.03233225 0.53 chr1 240161502 RPS7P5 0.020591357 6.23 chr1 240161507 RPS7P5 0.020591357 6.23 chr1 240161511 RPS7P5 0.047939677 8.62 chr1 240161516 RPS7P5 0.047939677 8.62 chr1 240161523 RPS7P5 0.047939677 8.62 chr1 240161527 RPS7P5 0.047939677 8.62 chr1 240161530 RPS7P5 0.047939677 8.62 chr1 240161535 RPS7P5 0.047939677 8.62 chr1 240161546 RPS7P5 0.047939677 8.62 chr1 240161558 RPS7P5 0.047939677 8.62 chr1 240161560 RPS7P5 0.047939677 8.62 chr10 129534843 FOXI2 0.046306111 0.02 chr10 129534853 FOXI2 0.046306111 0.02 chr10 129534866 FOXI2 0.046306111 0.02 chr10 129534879 FOXI2 0.046306111 0.02 chr10 129534891 FOXI2 0.046306111 0.02 chr10 129534910 FOXI2 0.046306111 0.02 chr10 129534912 FOXI2 0.046306111 0.02 chr10 129534924 FOXI2 0.046306111 0.02 chr12 6756182 ACRBP 0.014411356 2.78 chr12 6756187 ACRBP 0.014411356 2.78 chr12 6756191 ACRBP 0.014411356 2.78 chr12 6756195 ACRBP 0.014411356 2.78 chr12 6756211 ACRBP 0.014411356 2.78 chr12 6756225 ACRBP 0.014411356 2.78 chr12 6756230 ACRBP 0.014411356 2.78 chr12 6756270 ACRBP 0.014411356 2.78 chr13 114524043 GAS6 0.010185433 1.56 chr13 114524062 GAS6 0.010185433 1.56 chr13 114524068 GAS6 0.010185433 1.56 chr13 114524084 GAS6 0.010185433 1.56 chr13 114524095 GAS6 0.010185433 1.56 chr13 114524131 GAS6 0.010185433 1.56 chr13 114524138 GAS6 0.010185433 1.56 chr13 114524142 GAS6 0.010185433 1.56 chr13 114524150 GAS6 0.010185433 1.56 chr13 114524158 GAS6 0.010185433 1.56 chr16 698072 MCRIP2 0.016573854 2.31 chr16 698142 MCRIP2 0.016573854 2.31 chr16 698153 MCRIP2 0.016573854 2.31 chr16 698168 MCRIP2 0.016573854 2.31 chr16 698208 MCRIP2 0.016573854 2.31 chr16 698218 MCRIP2 0.016573854 2.31 chr16 698222 MCRIP2 0.016573854 2.31 chr16 698230 MCRIP2 0.016573854 2.31 chr17 77789596 LINC01977 0.017448956 0.54 chr17 77789601 LINC01977 0.017448956 0.54 chr17 77789612 LINC01977 0.017448956 0.54

[0475] chromosome CpG initiation site Gene name p-value Malignant / benign chr17 77789620 LINC01977 0.017448956 0.54 chr17 77789628 LINC01977 0.017448956 0.54 chr17 77789632 LINC01977 0.017448956 0.54 chr17 77789635 LINC01977 0.017448956 0.54 chr17 77789640 LINC01977 0.017448956 0.54 chr8 22548250 EGR3 0.018100911 0.54 chr8 22548260 EGR3 0.018100911 0.54 chr8 22548269 EGR3 0.018100911 0.54 chr8 22548279 EGR3 0.018100911 0.54 chr8 22548283 EGR3 0.018100911 0.54 chr8 22548287 EGR3 0.018100911 0.54 chr8 22548296 EGR3 0.018100911 0.54 chr8 22548299 EGR3 0.018100911 0.54 chr8 55379566 SOX17 0.0474443 2.35 chr8 55379568 SOX17 0.0474443 2.35 chr8 55379573 SOX17 0.0474443 2.35 chr8 55379579 SOX17 0.0474443 2.35 chr8 55379583 SOX17 0.0474443 2.35 chr8 55379591 SOX17 0.0474443 2.35 chr8 55379599 SOX17 0.0474443 2.35 chr8 55379602 SOX17 0.0474443 2.35 chr8 55379608 SOX17 0.0474443 2.35 chr8 55379617 SOX17 0.0474443 2.35 chr8 55379620 SOX17 0.0474443 2.35 chr9 36986087 PAX5 0.028288349 2.80 chr9 36986093 PAX5 0.028288349 2.80 chr9 36986098 PAX5 0.028288349 2.80 chr9 36986101 PAX5 0.028288349 2.80 chr9 36986103 PAX5 0.028288349 2.80 chr9 36986117 PAX5 0.028288349 2.80 chr9 36986131 PAX5 0.028288349 2.80 chr9 36986138 PAX5 0.028288349 2.80 chr9 36986141 PAX5 0.028288349 2.80 chr9 36986143 PAX5 0.028288349 2.80 chr9 36986147 PAX5 0.028288349 2.80 chr9 36986149 PAX5 0.028288349 2.80 chr9 36986156 PAX5 0.028288349 2.80 chr10 105344464 NEURL1 0.033337757 4.95 chr10 105344482 NEURL1 0.033337757 4.95 chr10 105344493 NEURL1 0.033337757 4.95 chr10 105344495 NEURL1 0.033337757 4.95 chr10 105344497 NEURL1 0.033337757 4.95 chr10 105344503 NEURL1 0.033337757 4.95 chr10 105344506 NEURL1 0.033337757 4.95 chr10 105344513 NEURL1 0.033337757 4.95 chr10 105344516 NEURL1 0.033337757 4.95 chr10 105344519 NEURL1 0.033337757 4.95 chr10 105344526 NEURL1 0.033337757 4.95 chr5 1876386 IRX4 0.049364479 3.19 chr5 1876395 IRX4 0.049364479 3.19 chr5 1876397 IRX4 0.049364479 3.19 chr5 1876403 IRX4 0.049364479 3.19 chr5 1876420 IRX4 0.049364479 3.19 chr5 1876424 IRX4 0.049364479 3.19 chr5 1876432 IRX4 0.049364479 3.19 chr5 1876436 IRX4 0.049364479 3.19 chr5 1876449 IRX4 0.049364479 3.19 chr5 1876456 IRX4 0.049364479 3.19 chr5 1876459 IRX4 0.049364479 3.19 chr5 1876463 IRX4 0.049364479 3.19 chr1 155295135 RUSC1 0.02636741 2.20 chr1 155295171 RUSC1 0.02636741 2.20

[0476] chromosome CpG initiation site Gene name p-value Malignant / benign chr1 155295181 RUSC1 0.02636741 2.20 chr1 155295192 RUSC1 0.02636741 2.20 chr1 155295196 RUSC1 0.02636741 2.20 chr1 155295212 RUSC1 0.02636741 2.20 chr1 155295229 RUSC1 0.02636741 2.20 chr1 155295236 RUSC1 0.02636741 2.20

[0477] Example 2: Verification of differentially methylated sites using methylation-specific PCR (MSP) and quantitative methylation-specific PCR (Q-MSP).

[0478] 1) Sample preparation

[0479] DNA was extracted from tissues or plasma from 10 patients with thyroid cancer and 10 patients with benign thyroid nodules using the QIAamp DNA Mini Kit (QIAGEN, catalog number: 51304); Qubit was used for DNA extraction. TM DNA concentration was detected using the dsDNA HS Assay Kit (Thermo, catalog number: Q32854); quality control was performed using 1% agarose gel electrophoresis.

[0480] 2) DNA transformation

[0481] Use MethylCode TM The Bisulfite Conversion Kit (Thermo, catalog number: MECOV50) performs bisulfite conversion on the DNA obtained in step 1. Unmethylated cytosine (C) is converted to uracil (U); methylated cytosine remains unchanged after conversion.

[0482] 3) Preparation of PCR mixture

[0483] The following steps are taken to prepare a single sample, including PCR reaction solution, primer mixture, and probe mixture:

[0484] MSP reaction system composition

[0485] Components Volume (μl) <![CDATA[Platinum TM II Hot-Start PCR Master Mix(2×)]]> 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 (10.0 ng) / Positive control / Negative control 2.00 Total 20.00

[0486] Q-MSP reaction system composition

[0487]

[0488]

[0489] Note: The serially diluted standards are 6 fully methylated positive standards, each a 4-fold serially diluted 30 ng of bisulfite-converted standard.

[0490] 4) PCR reaction

[0491] The PCR program was set as follows: 94℃ pre-denaturation for 2 min; 94℃ denaturation for 30 s; 60℃ annealing and extension for 1 min; 45 cycles. Fluorescence signals were collected during the 60℃ annealing and extension phase.

[0492] 5) Analysis of test results

[0493] ROC curve analysis was performed on the methylation levels of each gene, as follows: Figure 2A As shown in -C, the AUC of each gene is greater than 0.6.

[0494] Example 3: Determining the benign or malignant nature of thyroid nodules using multiplex pre-amplified methylation-specific PCR (preAMP-MSP).

[0495] 1) Sample preparation

[0496] cfDNA was extracted from plasma from 20 patients with thyroid cancer and 20 patients with benign thyroid nodules using the QIAamp Circulating Nucleic Acid Kit (QIAGEN, catalog number: 55114); Qubit was used for cfDNA extraction. TM The concentration of cfDNA was detected using the dsDNA HS Assay Kit (Thermo, catalog number: Q32854); quality control was performed using the LabChip 3K Assay.

[0497] 2) DNA transformation

[0498] Use MethylCode TM The Bisulfite Conversion Kit (Thermo, catalog number: MECOV50) performs bisulfite conversion on the cfDNA obtained in step 1. Unmethylated cytosine (C) is converted to uracil (U); methylated cytosine remains unchanged after conversion.

[0499] 3) Pre-amplification PCR reaction

[0500] The pre-amplification PCR mixture includes PCR reaction solution and primer mixture. The primer mixture contains one pair of primers each for COL23A1 (SEQ ID NO:4 and 5), ILDR2 (SEQ ID NO:6 and 7), DHRS3 (SEQ ID NO:8 and 9), GDNF (SEQ ID NO:12 and 13), TBX18 (SEQ ID NO:14 and 15), and internal reference gene (SEQ ID NO:1 and 2).

[0501] Composition of pre-amplification PCR reaction system

[0502]

[0503]

[0504] The PCR program was set as follows: 94℃ pre-denaturation for 2 min; 94℃ denaturation for 15 s, 60℃ annealing for 45 s, 68℃ extension for 1 min, 15 cycles; 72℃ extension for 1 min, and storage at 4℃.

[0505] 4) MSP reaction

[0506] Prepare the reaction system according to the manufacturer's kit instructions, including Platinum. TMII. Hot-Start PCR MasterMix (2×), water, primer mixture (same as above), probe mixture (probe sequences as shown in SEQ ID NO:16 (COL23A1), SEQ ID NO:17 (ILDR2), SEQ ID NO:18 (DHRS3), SEQ ID NO:20 (GDNF), SEQ ID NO:21 (TBX18) and SEQ ID NO:3 (internal reference)), and 1:100 diluted pre-amplified PCR products. The COL23A1 gene includes loci 178003798, 178003803, 178003814, 178003823, 178003825, 178003834, 178003841, and 178003844; the ILDR2 gene includes loci 166890516, 166890528, 166890535, 166890543, 166890555, 166890559, 166890568, 166890573, 166890584, and 166890586; and the DHRS3 gene includes loci 166890516 and 166890528. The GDNF gene includes loci 37834770, 37834772, 37834774, 37834777, 37834780, 37834784, 37834792, 37834799, 37834802, 37834806, and 37834811. The TBX18 gene includes loci 85477035, 85477070, 85477083, and 85477106. The PCR program was set as follows: 94℃ pre-denaturation for 2 min; 94℃ denaturation for 30 s; 60℃ annealing and extension for 1 min; 45 cycles. Fluorescence signals were collected during the 60℃ annealing and extension phase.

[0507] 5) Analysis of test results

[0508] Methylation level = 2 –ΔCt待检样品 / 2 –ΔCt阳性标准品 ×100. Where, ΔCt=Ct 目的基因 –Ct 内参基因 .

[0509] Binary logistic regression analysis was performed on the methylation levels of the COL23A1, ILDR2, DHRS3, GDNF, and TBX18 genes. The fitted equation was: Score = -2.53 + 1.78 × COL23A1 methylation level + 1.64 × ILDR2 methylation level + 7.28 × DHRS3 methylation level – 2.47 × GDNF methylation level – 1.35 × TBX18 methylation level. The interpretation method was that if the scores of the tested COL23A1, ILDR2, DHRS3, GDNF, and TBX18 genes were greater than 0, the result was considered positive, i.e., malignant nodules.

[0510] The scores are shown in Table 2, and the ROC analysis is as follows: Figure 3 According to the interpretation criteria, 5 out of 20 benign thyroid nodules were positive, and 13 out of 20 thyroid cancers were positive, with a specificity of 80.0% and a sensitivity of 65.0%.

[0511] Table 2

[0512] Group Score Group Score benign nodules -1.17 Malignant nodules -0.29 benign nodules -1.84 Malignant nodules -1.08 benign nodules -0.75 Malignant nodules 1.24 benign nodules -0.62 Malignant nodules 0.41 benign nodules -0.37 Malignant nodules -0.49 benign nodules -1.09 Malignant nodules 1.15 benign nodules -0.91 Malignant nodules 0.73 benign nodules 0.43 Malignant nodules 1.73 benign nodules 0.30 Malignant nodules -1.98 benign nodules 0.50 Malignant nodules 0.79 benign nodules -0.96 Malignant nodules -0.34 benign nodules 0.79 Malignant nodules 2.11 benign nodules -1.55 Malignant nodules 2.39 benign nodules -0.20 Malignant nodules 2.89 benign nodules -0.93 Malignant nodules 0.77 benign nodules -1.84 Malignant nodules 2.98 benign nodules -0.52 Malignant nodules 2.05 benign nodules 1.32 Malignant nodules 2.10 benign nodules -2.86 Malignant nodules -0.59 benign nodules -1.74 Malignant nodules -0.59

[0513] Example 4: Determining the benign or malignant nature of thyroid nodules using multiplex pre-amplified methylation-specific PCR (preAMP-MSP).

[0514] Steps 1)-4) are the same as in Example 3, except that in step 3), the primer mixture contains one pair of primers for each of the following genes: COL23A1 (SEQ ID NO:4 and 5), ILDR2 (SEQ ID NO:6 and 7), DHRS3 (SEQ ID NO:8 and 9), KIF1A (SEQ ID NO:10 and 11), and internal reference genes (SEQ ID NO:1 and 2). The probes for each gene are shown in SEQ ID NO:16 (COL23A1), SEQ ID NO:17 (ILDR2), SEQ ID NO:18 (DHRS3), SEQ ID NO:19 (KIF1A), and SEQ ID NO:3 (internal reference). In step 4), the COL23A1 gene loci include 178003798, 178003803, 178003814, 178003823, 178003825, 178003834, 178003841, and 178003844; the KIF1A gene loci include 241759696, 241759701, 241759714, and 241759716; and the ILDR2 gene loci include 166890516, 1... The DHRS3 gene loci include 66890528, 166890535, 166890543, 166890555, 166890559, 166890568, 166890573, 166890584, and 166890586, and 12656170, 12656175, 12656182, 12656197, 12656200, 12656211, 12656315, and 12656323.

[0515] 5) Analysis of test results

[0516] Methylation level = 2 –ΔCt待检样品 / 2 –ΔCt阳性标准品 ×100. Where, ΔCt=Ct 目的基因 –Ct 内参基因 .

[0517] Binary logistic regression analysis was performed on the methylation levels of the COL23A1, KIF1A, ILDR2, and DHRS3 genes. The fitted equation was: Score = -3.37 + 2.03 × COL23A1 methylation level + 15.86 × KIF1A methylation level + 1.32 × ILDR2 methylation level + 5.74 × DHRS3 methylation level. The interpretation method was that if the scores of the tested COL23A1, KIF1A, ILDR2, and DHRS3 genes were greater than 0, the result was considered positive, i.e., malignant nodules.

[0518] The scores are shown in Table 3, and the ROC analysis is as follows: Figure 4 According to the interpretation criteria, 7 out of 20 benign thyroid nodules were positive, and 12 out of 20 thyroid cancers were positive, with a specificity of 90.0% and a sensitivity of 60.0%.

[0519] Table 3

[0520] Group Score Group Score benign nodules -1.64 Malignant nodules -0.01 benign nodules -0.73 Malignant nodules -1.13 benign nodules -1.27 Malignant nodules 1.42 benign nodules 0.12 Malignant nodules 1.00 benign nodules -0.24 Malignant nodules -0.90 benign nodules -1.12 Malignant nodules 0.56 benign nodules -1.13 Malignant nodules 0.58 benign nodules 0.89 Malignant nodules 2.17 benign nodules -0.55 Malignant nodules -0.29 benign nodules 0.30 Malignant nodules 1.34 benign nodules 0.13 Malignant nodules -0.66 benign nodules 0.41 Malignant nodules 1.13 benign nodules 0.17 Malignant nodules 2.10 benign nodules -0.82 Malignant nodules 2.00 benign nodules -1.26 Malignant nodules -0.01 benign nodules -2.12 Malignant nodules 1.46 benign nodules -0.21 Malignant nodules 1.18 benign nodules 0.66 Malignant nodules 1.77 benign nodules -1.37 Malignant nodules -1.69 benign nodules -0.51 Malignant nodules -0.99

[0521] The embodiments described above are merely illustrative of implementation methods of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims. sequence list <110> Shanghai Kunyuan Biotechnology Co., Ltd. <120> Reagents and applications for detecting DNA methylation <130> 20A600 <150> 202010038550.8 <151> 2020-01-14 <160> twenty one <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> 19 <212> DNA <213> Artificial Sequence <400> 4 atcctatcct ccgaaacgc 19 <210> 5 <211> 18 <212> DNA <213> Artificial Sequence <400> 5 yggcgttgtt cgagaggt 18 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <400> 6 rcgaaaaaac ttcgccacg 19 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <400> 7 ggtcgtagga gttagcgaag 20 <210> 8 <211> 19 <212> DNA <213> Artificial Sequence <400> 8 ctaaccgcta cgtaaaccg 19 <210> 9 <211> 15 <212> DNA <213> Artificial Sequence <400> 9 gggagcgagg tcgtt 15 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <400> 10 taaattagtt ggygattgga g 21 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <400> 11 actactctac rctataacra c 21 <210> 12 <211> 27 <212> DNA <213> Artificial Sequence <400> 12 aaacrattct tacaatcact actcaac 27 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <400> 13 tttcgaggcg ttcgtcgaag 20 <210> 14 <211> 24 <212> DNA <213> Artificial Sequence <400> 14 actatcactc rtcactctca aaac 24 <210> 15 <211> 24 <212> DNA <213> Artificial Sequence <400> 15 tcgaattttt tttgggtatg ggag 24 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <400> 16 aacctccgcc tccaacgcga 20 <210> 17 <211> 28 <212> DNA <213> Artificial Sequence <400> 17 ccgaccgttt ccataaacga actaacga 28 <210> 18 <211> 19 <212> DNA <213> Artificial Sequence <400> 18 cgccgcccct ctaacgaac 19 <210> 19 <211> 27 <212> DNA <213> Artificial Sequence <400> 19 cctcccgaaa cgctaattaa ctacgcg 27 <210> 20 <211> 18 <212> DNA <213> Artificial Sequence <400> 20 acgcgcgacg acgaccga 18 <210> 21 <211> 21 <212> DNA <213> Artificial Sequence <400> 21 cactccgccc aaccaactcg a 21

Claims

1. Use of a reagent for detecting DNA methylation levels in the preparation of a kit for identifying the nature of thyroid nodules, said reagent being primers that detect DNA methylation levels at a gene locus, wherein, The gene loci include: (a1) Loci of the DHRS3 gene: 12656091, 12656114, 12656132, 12656152, 12656170, 12656175, 12656182, 12656187, 12656197, 12656200, 12656211, 12656315, 12656323, 12656340, 12656355 and 12656367 on chromosome 1. (a2) Loci of the COL23A1 gene: 178003785, 178003798, 178003803, 178003814, 178003823, 178003825, 178003834, 178003841 and 178003844 on chromosome 5. (a3) Loci of the ILDR2 gene: 166890429, 166890436, 166890440, 166890442, 166890448, 166890452, 166890456, 166890461, 166890468, 166890473, 166890475, 166890480 on chromosome 1 166890492, 166890500, 166890503, 166890509, 166890516, 166890528, 166890535, 166890543, 166890555, 166890559, 166890568, 166890573, 166890584 and 166890586, (a5) GDNF gene loci: 37834763, 37834770, 37834772, 37834774, 37834777, 37834780, 37834784, 37834792, 37834799, 37834802, 37834806, and 37834811 on chromosome 5. (a6) Loci of the TBX18 gene: 85477032, 85477035, 85477070, 85477083, 85477106, 85477124, 85477151, 85477153 and 85477166 on chromosome 6. The site is referenced from the human reference genome version hg19.

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 that detects the DNA methylation level of a nucleic acid fragment of a gene, wherein, The genes include DHRS3, COL23A1, ILDR2, GDNF, and TBX18, and the fragment length of the genes is 50-1000 bp. The DHRS3 gene fragment contains (a1) DHRS3 gene loci: 12656091, 12656114, 12656132, 12656152, 12656170, 12656175, 12656182, 12656187, 12656197, 12656200, 12656211, 12656315, 12656323, 12656340, 12656355, and 12656367 on chromosome 1. The COL23A1 gene fragment includes (a2) COL23A1 gene loci: 178003785, 178003798, 178003803, 178003814, 178003823, 178003825, 178003834, 178003841 and 178003844 on chromosome 5. The ILDR2 gene fragment contains (a3) ​​ILDR2 gene loci: 166890429, 166890436, 166890440, 166890442, 166890448, 166890452, 166890456, 166890461, 166890468, 166890473, 166890475, 16 6890480, 166890492, 166890500, 166890503, 166890509, 166890516, 166890528, 166890535, 166890543, 166890555, 166890559, 166890568, 166890573, 166890584 and 166890586, The fragment of the GDNF gene contains the (a5) GDNF gene loci: 37834763, 37834770, 37834772, 37834774, 37834777, 37834780, 37834784, 37834792, 37834799, 37834802, 37834806, and 37834811 on chromosome 5. The TBX18 gene fragment contains (a6) TBX18 gene loci: 85477032, 85477035, 85477070, 85477083, 85477106, 85477124, 85477151, 85477153, and 85477166 on chromosome 6. The site is referenced from the human reference genome version hg19.

3. 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 that detects the DNA methylation level at a gene locus, wherein... The gene loci include: (a1) Loci of the DHRS3 gene: 12656091, 12656114, 12656132, 12656152, 12656170, 12656175, 12656182, 12656187, 12656197, 12656200, 12656211, 12656315, 12656323, 12656340, 12656355 and 12656367 on chromosome 1. (a2) Loci of the COL23A1 gene: 178003785, 178003798, 178003803, 178003814, 178003823, 178003825, 178003834, 178003841 and 178003844 on chromosome 5. (a3) Loci of the ILDR2 gene: 166890429, 166890436, 166890440, 166890442, 166890448, 166890452, 166890456, 166890461, 166890468, 166890473, 166890475, 166890480 on chromosome 1. 166890492, 166890500, 166890503, 166890509, 166890516, 166890528, 166890535, 166890543, 166890555, 166890559, 166890568, 166890573, 166890584, and 166890586, and (a4) KIF1A gene loci: 241759696, 241759701, 241759714 and 241759716 on chromosome 2. The site is referenced from the human reference genome version hg19.

4. 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 that detects the DNA methylation level of a nucleic acid fragment of a gene, wherein... The genes include DHRS3, COL23A1, ILDR2, and KIF1A, and the fragment length of the genes is 50-1000 bp. The DHRS3 gene fragment contains (a1) DHRS3 gene loci: 12656091, 12656114, 12656132, 12656152, 12656170, 12656175, 12656182, 12656187, 12656197, 12656200, 12656211, 12656315, 12656323, 12656340, 12656355, and 12656367 on chromosome 1. The COL23A1 gene fragment includes (a2) COL23A1 gene loci: 178003785, 178003798, 178003803, 178003814, 178003823, 178003825, 178003834, 178003841 and 178003844 on chromosome 5. The ILDR2 gene fragment contains (a3) ​​ILDR2 gene loci: 166890429, 166890436, 166890440, 166890442, 166890448, 166890452, 166890456, 166890461, 166890468, 166890473, 166890475, 16 6890480, 166890492, 166890500, 166890503, 166890509, 166890516, 166890528, 166890535, 166890543, 166890555, 166890559, 166890568, 166890573, 166890584 and 166890586, The fragment of the KIF1A gene contains (a4) KIF1A gene loci: 241759696, 241759701, 241759714 and 241759716 on chromosome 2. The site is referenced from the human reference genome version hg19.

5. The use as described in claim 1 or 2, characterized in that, The primers can amplify (b1) a fragment of the DHRS3 gene amplified using SEQ ID NO:8 and 9 as primers, (b2) a fragment of the COL23A1 gene amplified using SEQ ID NO:4 and 5 as primers, (b3) a fragment of the ILDR2 gene amplified using SEQ ID NO:6 and 7 as primers, (b5) a fragment of the GDNF gene amplified using SEQ ID NO:12 and 13 as primers, and (b6) a fragment of the TBX18 gene amplified using SEQ ID NO:14 and 15 as primers.

6. The use as described in claim 3 or 4, characterized in that, The primers can amplify (b1) a fragment of the DHRS3 gene amplified using SEQ ID NO:8 and 9 as primers, (b2) a fragment of the COL23A1 gene amplified using SEQ ID NO:4 and 5 as primers, (b3) a fragment of the ILDR2 gene amplified using SEQ ID NO:6 and 7 as primers, and (b4) a fragment of the KIF1A gene amplified using SEQ ID NO:10 and 11 as primers.

7. The use as described in claim 1 or 2, characterized in that, The reagent also includes probes that can hybridize with the following fragments: (b1) a fragment of the DHRS3 gene amplified using SEQ ID NO:8 and 9 as primers, (b2) a fragment of the COL23A1 gene amplified using SEQ ID NO:4 and 5 as primers, (b3) a fragment of the ILDR2 gene amplified using SEQ ID NO:6 and 7 as primers, (b5) a fragment of the GDNF gene amplified using SEQ ID NO:12 and 13 as primers, and (b6) a fragment of the TBX18 gene amplified using SEQ ID NO:14 and 15 as primers.

8. The use as described in claim 3 or 4, characterized in that, The reagent also includes probes that can hybridize with the following fragments: (b1) a fragment of the DHRS3 gene amplified using SEQ ID NO:8 and 9 as primers, (b2) a fragment of the COL23A1 gene amplified using SEQ ID NO:4 and 5 as primers, (b3) a fragment of the ILDR2 gene amplified using SEQ ID NO:6 and 7 as primers, and (b4) a fragment of the KIF1A gene amplified using SEQ ID NO:10 and 11 as primers.

9. The use as described in claim 2 or 4, characterized in that, The fragments include either the sense or antisense strand of DNA.

10. The use as described in any one of claims 1-4, characterized in that, Reagents for detecting DNA methylation also include those selected from one or more of the following methods: PCR based on bisulfite conversion, DNA sequencing, methylation-sensitive restriction endonuclease analysis, quantitative fluorescence method, methylation-sensitive high-resolution melting curve method, chip-based methylation mapping analysis, and mass spectrometry.

11. The use as described in any one of claims 1-4, characterized in that, Reagents for detecting DNA methylation also include one or more of the following: bisulfite, PCR buffer, polymerase, dNTP, methylation-sensitive or non-methylation-sensitive restriction endonuclease, enzyme digestion buffer, fluorescent dye, fluorescence quencher, fluorescent reporter, exonuclease, and alkaline phosphatase.

12. The use as described in any one of claims 1-4, characterized in that, The reagent for detecting DNA methylation also includes an internal standard.

13. The use as described in any one of claims 1-4, characterized in that, The reagent for detecting DNA methylation also includes a control.

14. Use of a reagent for detecting DNA methylation in the preparation of a kit for identifying the nature of thyroid nodules in a sample, said reagent detecting the methylation level of genes including DHRS3, COL23A1, ILDR2, GDNF, and TBX18, the gene sites including: (a1) Loci of the DHRS3 gene: 12656091, 12656114, 12656132, 12656152, 12656170, 12656175, 12656182, 12656187, 12656197, 12656200, 12656211, 12656315, 12656323, 12656340, 12656355 and 12656367 on chromosome 1. (a2) Loci of the COL23A1 gene: 178003785, 178003798, 178003803, 178003814, 178003823, 178003825, 178003834, 178003841 and 178003844 on chromosome 5. (a3) Loci of the ILDR2 gene: 166890429, 166890436, 166890440, 166890442, 166890448, 166890452, 166890456, 166890461, 166890468, 166890473, 166890475, 166890480 on chromosome 1 166890492, 166890500, 166890503, 166890509, 166890516, 166890528, 166890535, 166890543, 166890555, 166890559, 166890568, 166890573, 166890584 and 166890586, (a5) GDNF gene loci: 37834763, 37834770, 37834772, 37834774, 37834777, 37834780, 37834784, 37834792, 37834799, 37834802, 37834806, and 37834811 on chromosome 5. (a6) Loci of the TBX18 gene: 85477032, 85477035, 85477070, 85477083, 85477106, 85477124, 85477151, 85477153 and 85477166 on chromosome 6. The site is referenced from the human reference genome version hg19.

15. The use as described in claim 14, characterized in that, The reagent also includes nucleic acid molecules having nucleic acid sequences of fragments from the DHRS3, COL23A1, ILDR2, GDNF, and TBX18 genes, wherein the gene fragments are 50-1000 bp in length. The DHRS3 gene fragment contains (a1) DHRS3 gene loci: 12656091, 12656114, 12656132, 12656152, 12656170, 12656175, 12656182, 12656187, 12656197, 12656200, 12656211, 12656315, 12656323, 12656340, 12656355, and 12656367 on chromosome 1. The COL23A1 gene fragment includes (a2) COL23A1 gene loci: 178003785, 178003798, 178003803, 178003814, 178003823, 178003825, 178003834, 178003841 and 178003844 on chromosome 5. The ILDR2 gene fragment contains (a3) ​​ILDR2 gene loci: 166890429, 166890436, 166890440, 166890442, 166890448, 166890452, 166890456, 166890461, 166890468, 166890473, 166890475, 16 6890480, 166890492, 166890500, 166890503, 166890509, 166890516, 166890528, 166890535, 166890543, 166890555, 166890559, 166890568, 166890573, 166890584 and 166890586, The fragment of the GDNF gene contains the (a5) GDNF gene loci: 37834763, 37834770, 37834772, 37834774, 37834777, 37834780, 37834784, 37834792, 37834799, 37834802, 37834806, and 37834811 on chromosome 5. The TBX18 gene fragment contains (a6) TBX18 gene loci: 85477032, 85477035, 85477070, 85477083, 85477106, 85477124, 85477151, 85477153, and 85477166 on chromosome 6. The site is referenced from the human reference genome version hg19.

16. Use of a reagent for detecting DNA methylation in the preparation of a kit for identifying the nature of thyroid nodules in a sample, said reagent detecting the methylation level of genes including DHRS3, COL23A1, ILDR2, and KIF1A, the gene sites including: (a1) Loci of the DHRS3 gene: 12656091, 12656114, 12656132, 12656152, 12656170, 12656175, 12656182, 12656187, 12656197, 12656200, 12656211, 12656315, 12656323, 12656340, 12656355 and 12656367 on chromosome 1. (a2) Loci of the COL23A1 gene: 178003785, 178003798, 178003803, 178003814, 178003823, 178003825, 178003834, 178003841 and 178003844 on chromosome 5. (a3) Loci of the ILDR2 gene: 166890429, 166890436, 166890440, 166890442, 166890448, 166890452, 166890456, 166890461, 166890468, 166890473, 166890475, 166890480 on chromosome 1. 166890492, 166890500, 166890503, 166890509, 166890516, 166890528, 166890535, 166890543, 166890555, 166890559, 166890568, 166890573, 166890584, and 166890586, and (a4) KIF1A gene loci: 241759696, 241759701, 241759714 and 241759716 on chromosome 2. The site is referenced from the human reference genome version hg19.

17. The use as described in claim 16, characterized in that, The reagent also includes nucleic acid molecules having nucleic acid sequences of fragments from the DHRS3, COL23A1, ILDR2, and KIF1A genes, wherein the gene fragments are 50-1000 bp in length. The DHRS3 gene fragment contains (a1) DHRS3 gene loci: 12656091, 12656114, 12656132, 12656152, 12656170, 12656175, 12656182, 12656187, 12656197, 12656200, 12656211, 12656315, 12656323, 12656340, 12656355, and 12656367 on chromosome 1. The COL23A1 gene fragment includes (a2) COL23A1 gene loci: 178003785, 178003798, 178003803, 178003814, 178003823, 178003825, 178003834, 178003841 and 178003844 on chromosome 5. The ILDR2 gene fragment contains (a3) ​​ILDR2 gene loci: 166890429, 166890436, 166890440, 166890442, 166890448, 166890452, 166890456, 166890461, 166890468, 166890473, 166890475, 16 6890480, 166890492, 166890500, 166890503, 166890509, 166890516, 166890528, 166890535, 166890543, 166890555, 166890559, 166890568, 166890573, 166890584 and 166890586, The fragment of the KIF1A gene contains (a4) KIF1A gene loci: 241759696, 241759701, 241759714 and 241759716 on chromosome 2. The site is referenced from the human reference genome version hg19.

18. The use as described in any one of claims 14-17, characterized in that, The use has one or more features selected from the following: The kit also includes reagents for detecting the mutation level at the V600E site of the BRAF gene and / or the mutation level at the C228T / C250T sites of the TERT gene. The identification of the nature of thyroid nodules includes: 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 nodules based on the comparison results or the score. The sample was from a person.

19. The use as described in claim 18, characterized in that, The sample contained genomic DNA.

20. The use as described in claim 18, characterized in that, The sample contained cfDNA.

21. The use as described in claim 18, characterized in that, The sample was derived from tissue.

22. The use as described in claim 21, characterized in that, The sample was derived from thyroid tissue.

23. The use as described in claim 18, characterized in that, The sample was derived from blood.

24. The use as described in claim 23, characterized in that, The sample was derived from blood plasma.

25. The use as described in claim 18, characterized in that, The sample was derived from cells.

26. The use as described in claim 18, characterized in that, The sample was derived from bodily fluids.

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