CN113122631A

CN113122631A - Reagent for detecting DNA methylation and application

Info

Publication number: CN113122631A
Application number: CN202110039797.6A
Authority: CN
Inventors: 刘蕊; 苏明扬; 何其晔
Original assignee: Singlera Genomics Inc
Current assignee: Singlera Genomics Inc
Priority date: 2020-01-14
Filing date: 2021-01-13
Publication date: 2021-07-16
Also published as: CN113122635A; CN113122634A; CN113122632A; CN113122636A; CN113122637A; CN113122633A; WO2021143709A1

Abstract

Disclosed herein is a method for identifying a thyroid nodule property comprising detecting a DNA methylation level in a sample from a region selected from the group consisting of the following (1) and (2): (1) a fragment of one or more genes selected from the group consisting of: COL23A1, ILDR2, DHRS3, KIF1A, GDNF, TBX18, (2) (1) a nucleic acid region within 10Kb of upstream and downstream of the gene.

Description

Reagent for detecting DNA methylation and application

Technical Field

The invention belongs to the field of molecular auxiliary diagnosis, and particularly relates to application of the molecular auxiliary diagnosis in screening benign and malignant thyroid nodules.

Background

DNA methylation is a mechanism of epigenetics, is a common epigenetics modification of eukaryotic cell genomes, is also an important natural chemical modification mode of vertebrate DNA under the condition of not changing DNA sequences, plays an important role in aspects of cell proliferation, differentiation, development and the like, and is closely related to the occurrence and development of tumors. DNA methylation plays an important role in vivo, and its effects are transcription repression, chromatin structure regulation, X chromosome inactivation, genomic imprinting, etc., and DNA methylation abnormalities can be involved in tumor development and progression by affecting chromatin structure and the expression of oncogenes and tumor suppressor genes.

CpG dinucleotides are the most major target for DNA methylation in mammals and are distributed throughout the entire chromosome set. In a healthy human genome, CpG sites in CpG islands are usually unmethylated, whereas CpG sites outside CpG islands are usually hypermethylated, which is stably retained during cell division. When tumors occur, the methylation degree of CpG sites in non-CpG island regions of cancer suppressor genes is generally reduced, and CpG in the CpG island is in a high methylation state, so that the chromatin structure is changed and the expression of the cancer suppressor genes is reduced.

With the continued development of genetics and epigenetics over the last decade, more and more researchers are recognizing that tumor development is not completely genetically determined, and that the acquired effects of epigenetics play an important role as well. Epigenetic alterations in thyroid cancer are mainly manifested by abnormal methylation of tumor suppressor genes and thyroid-associated genes. The research on the DNA methylation of the thyroid cancer can provide a new molecular marker for people, and provide a reliable basis for early diagnosis, treatment scheme selection and prognosis evaluation.

Thyroid nodules (Thyroid nodules) are masses that form in Thyroid tissue following abnormal proliferation of Thyroid cells. Thyroid nodules are very common, and although most thyroid nodules are benign, a small percentage of them progress to thyroid cancer. For earlier diagnosis and treatment of thyroid cancer, while reducing unnecessary surgery, benign and malignant identification of thyroid nodules is required.

Presently, thyroid nodules are mainly evaluated by Ultrasonography (US) and Fine Needle Aspiration Biopsy (FNAB). Among diagnostic procedures for thyroid nodules, US is the most sensitive examination at presentMethods, which can measure the size of a nodule, determine the internal structure of a nodule, etc. US signs suggesting that thyroid nodules are malignant include: nodule height greater than width (OR ═ 10.15), lack of acoustic halo (OR ═ 7.14), microcalcifications (OR ═ 6.76), border irregularity (OR ═ 6.12), echo reduction (OR ═ 5.07), solid nodules (OR ═ 4.69), nodule internal blood flow abundance (OR ═ 3.76), and the like. The diameter is more than 1cm, and the nodules with malignant signs are examined by ultrasonic wave and FNAB is used for judging the properties of the nodules. Up to 20% of the nodules in cytological examination are indeterminate thyroid nodules (indexaminate thyroid nodules), which require detection of binding molecules. Has been on the market

Gene Expression Classifier and Thyroseq 2 products, the Positive Predictive Value (PPV) of the former is very low, only 46%; the latter PPV is also only 42% -77%. More accurate molecular diagnostic tools are therefore needed.

There remains a need in the art for highly specific, highly sensitive methods for thyroid nodule diagnosis.

Disclosure of Invention

The invention aims to provide a reagent for detecting DNA methylation and application thereof in screening benign and malignant thyroid nodules.

In a first aspect, the present invention provides an isolated nucleic acid molecule from a mammal having a sequence of a nucleic acid selected from the group consisting of (1) and (2) below, or a variant thereof having at least 70% identity thereto: (1) a fragment of a nucleic acid of one or more genes selected from the group consisting of: COL23A1, ILDR2, DHRS3, KIF1A, GDNF, TBX18, the length of the fragment is 50-1000bp, wherein, the fragment of the COL23A1 gene comprises the site of the COL23A1 gene: 178003785, 178003798, 178003803, 178003814, 178003823, 178003825, 178003834, 178003841, 178003844, the fragment of ILDR2 gene comprises the site of ILDR2 gene: 166890429, 166890436, 166890440, 166890442, 166890448, 166890452, 166890456, 166890461, 166890468, 166890473, 166890475, 166890480, 166890492, 166890500, 166890503, 166890509, 166890516, 166890528, 166890535, 166890543, 166890555, 166890559, 166890568, 166890573, 166890584, 166890586, or a fragment of the DHRS3 gene comprising the site of the DHRS3 gene: 12656091, 12656114, 12656132, 12656152, 12656170, 12656175, 12656182, 12656187, 12656197, 12656200, 12656211, 12656315, 12656323, 12656340, 12656355, 12656367, a fragment of KIF1A gene comprising the site of KIF1A gene: 241759696, 241759701, 241759714, 241759716, the segment of the GDNF gene comprising the site of the GDNF gene: 37834763, 37834770, 37834772, 37834774, 37834777, 37834780, 37834784, 37834792, 37834799, 37834802, 37834806, 37834811, a fragment of the TBX18 gene comprising the position of the TBX18 gene: 85477032, 85477035, 85477070, 85477083, 85477106, 85477124, 85477151, 85477153, 85477166, (2) (1) a nucleic acid region within 10Kb of the upstream and downstream of the gene, wherein the above-mentioned sites in the variant are not mutated.

In one or more embodiments, the fragment of the COL23a1 gene comprises the site of the COL23a1 gene: 178003798, 178003803, 178003814, 178003823, 178003825, 178003834, 178003841, 178003844, the fragment of the ILDR2 gene comprises one or more or all of positions 166890516, 166890528, 166890535, 166890543, 166890555, 166890559, 166890568, 166890573, 166890584, 166890586 of the ILDR2 gene, the fragment of the DHRS3 gene comprises the positions of the DHRS3 gene: 12656091, 12656114, 12656132, 12656152, 12656170, 12656175, 12656182, 12656187, 12656197, 12656200, 12656211, 12656315, 12656323, 12656340, 12656355, 12656367, a fragment of KIF1A gene comprising the site of KIF1A gene: 241759696, 241759701, 241759714, 241759716, the segment of the GDNF gene comprising the site of the GDNF gene: 37834770, 37834772, 37834774, 37834777, 37834780, 37834784, 37834792, 37834799, 37834802, 37834806, 37834811, a fragment of the TBX18 gene comprising the position of the TBX18 gene: 85477035, 85477070, 85477083, 85477106.

In one or more embodiments, the fragment of the COL23a1 gene comprises the site of the COL23a1 gene: 178003798, 178003803, 178003814, 178003823, 178003825, 178003834, 178003841, 178003844, the fragment of the ILDR2 gene comprising the site of the ILDR2 gene: 166890516, 166890528, 166890535, 166890543, 166890555, 166890559, 166890568, 166890573, 166890584, 166890586, a fragment of the DHRS3 gene comprising the site of the DHRS3 gene: 12656170, 12656175, 12656182, 12656197, 12656200, 12656211, 12656315, 12656323, a fragment of the KIF1A gene comprising the site of the KIF1A gene: 241759696, 241759701, 241759714, 241759716, the segment of the GDNF gene comprising the site of the GDNF gene: 37834770, 37834772, 37834774, 37834777, 37834780, 37834784, 37834792, 37834799, 37834802, 37834806, 37834811, a fragment of the TBX18 gene comprising the position of the TBX18 gene: 85477035, 85477070, 85477083, 85477106.

In one or more embodiments, the nucleic acid molecule comprises one or more fragments selected from the group consisting of: a fragment of COL23A1 gene amplified by SEQ ID NOS.4 and 5 as primers, a fragment of ILDR2 gene amplified by SEQ ID NOS.6 and 7 as primers, a fragment of DHRS3 gene amplified by SEQ ID NOS.8 and 9 as primers, a fragment of KIF1A gene amplified by SEQ ID NOS.10 and 11 as primers, a fragment of GDNF gene amplified by SEQ ID NOS.12 and 13 as primers, and a fragment of TBX18 gene amplified by SEQ ID NOS.14 and 15 as primers.

The second aspect of the present invention provides a reagent for detecting DNA methylation, which comprises a reagent for detecting the level of DNA methylation in a region selected from the following regions (1) and (2): (1) a fragment of one or more genes selected from the group consisting of: ZMIZ1, C15orf52, SLC16A3, ZNF512B, SLC17A5, LIMK1, PLEC, TOR4A, TMEM131L, DNM2, IL17C, PRDM16, MT1JP, TBX3, BIN1, TIMP2, CFAP65, TSHR, KIF1A, DAPK, CDH1, TPO, RARG, PRR15, DPYS, MCC, TBX15, COL23A 15, ILDR 15, DHRS 15, GDNF, TBX15, SIM 15, HOXA 15, EHBP1L 15, GJC 15, RCOR 15, PRDM 15, UNCX, RPS7P 15, FOXI 15, ACRBP, GAS 15, MCRIP 15, SORIC 019C 3677, SOJC 15, SOIRX 15, SODM 15, and the region with length of the up-down-stream region of the gene (up-and down-stream region of the said nucleic acid) is up-KKBX 365, or up-down-region of the said region 365.

In one or more embodiments, the agent for detecting the methylation level of DNA detects the methylation level of a fragment of one or more groups of genes selected from the following (a) to (d): (a) one or more genes selected from: COL23A1, ILDR2, DHRS3, KIF1A, GDNF, TBX18, (b) COL23A1, ILDR2, DHRS3, (c) COL23A1, ILDR2, DHRS3 and KIF1A, (d) COL23A1, ILDR2, DHRS3 and one or two selected from GDNF and TBX18, and (e) a nucleic acid region within 5Kb or 10Kb upstream and downstream of any one of the genes of any one of (a) - (d).

In one or more embodiments, the fragment of each gene comprises a corresponding one or more sites selected from the following sites or a nucleic acid region within 500bp upstream and downstream thereof:

ZMIZ 1: 81001968, 81001996, 81002041, 81002052, 81002054, 81002056, 81002062, 81002083, 81002110, 81002116, 81002123, 81002129, 81002133, 81002137, 81002139, 81002164, 81002168, 81002223, 81002241, 81002253 of chromosome 10,

c15orf 52: 40626309, 40626312, 40626386 of chromosome 15,

SLC16a 3: 80189165, 80189174, 80189177, 80189197, 80189225, 80189230, 80189239, 80189645, 80189671, 80189674, 80189684, 80189687, 80189698, 80189709, 80189719, 80189726, 80189728, 80189739, 80189757, 80189787, 80189792, 80189811, 80189817, 80189832, 80189841 of chromosome 17,

ZNF 512B: 62588634, 62588638, 62588672 of chromosome 20,

SLC17a 5: 74290205, 74290207, 74290220, 74290225 and 74290228 of chromosome 6,

LIMK 1: 73508994, 73509017, 73509055, 73509062, 73509073, 73509075, 73509112, 73509133, 73509138, 73509148 and 73509160 of chromosome 7,

and (2) PLEC: 145013661, 145013673 of chromosome 8,

TOR 4A: 140172787, 140172790, 140172812 of chromosome 9,

TMEM 131L: 154409945, 154409963, 154409972, 154409978, 154409997, 154410003 and 154410006 of chromosome 4,

DNM 2: 10870373, 10870377, 10870427, 10870429, 10870441 and 10870448 of chromosome 19,

IL 17C: 88700818, 88700826, 88700844, 88700849, 88700857, 88700869, 88700875, 88700891, 88700897, 88700916, 88700920, 88700937, 88700943, 88700948, 88700967, 88700970, 88700993, 88701004, 88701021, 88701029, 88701036, 88701043, 88701051, 88701060, 88701074, 88701081, 88701090, 88701099, 88701111, 88701115, 88701133, 88701140, 88701148, 88701159, 88701161, 88701176, 88701178, 88701180, 88701183, 88701190, 88701201, 88701204, 88701210, 88701212, 88701236, 88701240, 88701266, 88701278, 88701281, 88701285, 88701305, 88701421, 88701442, 88701451 of chromosome 16,

PRDM 16: 3229914, 3229921, 3229950, 3229968, 3229973, 3310213, 3310229, 3310235, 3310238, 3310240, 3310268, 3310287, 3310312, 3310314, 3310317, 3310329 of chromosome 1,

TSHR: 81421983, 81421989, 81422010, 81422017, 81422032, 81422035, 81422063, 81422084 of chromosome 14,

KIF 1A: 241759696, 241759701, 241759714 and 241759716 of chromosome 2,

DAPK: 90112842, 90112853, 90112861 and 90112866 of chromosome 9,

CDH 1: 68771035, 68771037, 68771045, 68771051, 68771059, 68771064 and 68771073 of chromosome 16,

TPO: 1481013, 1481015, 1481022 and 1481039 of chromosome 2,

RARG: 53613176, 53613182, 53613190, 53613202, 53613210 and 53613218 of chromosome 12,

MT1 JP: 56669271, 56669292, 56669295, 56669300, 56669318, 56669322, 56669324, 56669327, 56669344, 56669351, 56669353, 56669402, 56669414, 56669423, 56669430, 56669433, 56669437, 56669451, 56669453, 56669455, 56669463, 56669474, 56669480, 56669482, 56669485, 56669487, 56669490, 56669519, 56669533, 56669553, 56669564, 56669573, 56669578, 56669588, 56669590, 56669606, 56669610 of chromosome 16,

TBX 3: 115174750, 115174773, 115174780 of chromosome 12,

BIN 1: 127822478, 127822492, 127822495, 127822514, 127822551, 127822568, 127822582, 127822593, 127822616, 127822644 of chromosome 2,

TIMP 2: 76921845, 76921853, 76921860 of chromosome 17,

CFAP 65: 219866132, 219866139, 219866148, 219866158, 219866165, 219866168, 219866199, 219866218 of chromosome 2,

PRR 15: 29605992, 29606026, 29606040, 29606047, 29606056, 29606062, 29606073, 29606179, 29606191, 29606201, 29606204, 29606220, 29606222, 29606227, 29606231, 29606255, 29606257, 29606262, 29606271, 29606277, 29606289, 29606320 of chromosome 7,

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

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

TBX 15: 119535725, 119535730, 119535740, 119535742, 119535750, 119535759, 119535766, 119535812, 119535817, 119535821, 119535823, 119535876, 119535879, 119535884, 119535891 of chromosome 1,

COL23a 1: 178003785, 178003798, 178003803, 178003814, 178003823, 178003825, 178003834, 178003841, 178003844 of chromosome 5,

ILDR 2: 166890429, 166890436, 166890440, 166890442, 166890448, 166890452, 166890456, 166890461, 166890468, 166890473, 166890475, 166890480, 166890492, 166890500, 166890503, 166890509, 166890516, 166890528, 166890535, 166890543, 166890555, 166890559, 166890568, 166890573, 166890584, 166890586 of chromosome 1,

DHRS 3: 12656091, 12656114, 12656132, 12656152, 12656170, 12656175, 12656182, 12656187, 12656197, 12656200, 12656211, 12656315, 12656323, 12656340, 12656355, 12656367 of chromosome 1,

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

TBX 18: 85477032, 85477035, 85477070, 85477083, 85477106, 85477124, 85477151, 85477153, 85477166 of chromosome 6,

SIM 2: 38069563, 38069579, 38069619, 38069625, 38069638, 38069650, 38069662, 38069664, 38069676, 38069681 of chromosome 21,

HOXA 9: 27204848, 27204854, 27204858, 27204861, 27204863, 27204879, 27204884, 27204894, 27204897, 27204918, 27204929, 27204938, 27204945, 27204948, 27204951, 27204958, 27204981, 27204984 of chromosome 7,

EHBP1L 1: 65352612, 65352621, 65352635, 65352639, 65352642, 65352651, 65352654, 65352665, 65352670 of chromosome 11,

GJC 2: 228345954, 228345957, 228345965, 228345978, 228345980, 228345989 of chromosome 1,

RCOR 2: 63687223, 63687238, 63687247, 63687250, 63687259, 63687282, 63687288, 63687299, 63687318, 63687325 of chromosome 11,

PRDM 1: 106429711, 106429722, 106429731, 106429747, 106429750, 106429761, 106429769, 106429771 of chromosome 6,

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

RPS7P 5: 240161502, 240161507, 240161511, 240161516, 240161523, 240161527, 240161530, 240161535, 240161546, 240161558 and 240161560 of chromosome 1,

FOXI 2: 129534843, 129534853, 129534866, 129534879, 129534891, 129534910, 129534912, 129534924 of chromosome 10,

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

GAS 6: 114524043, 114524062, 114524068, 114524084, 114524095, 114524131, 114524138, 114524142, 114524150, 114524158 of chromosome 13,

MCRIP 2: 698072, 698142, 698153, 698168, 698208, 698218, 698222, 698230 of chromosome 16,

LINC 01977: 77789596, 77789601, 77789612, 77789620, 77789628, 77789632, 77789635, 77789640 of chromosome 17,

EGR 3: 22548250, 22548260, 22548269, 22548279, 22548283, 22548287, 22548296, 22548299 of chromosome 8,

SOX 17: 55379566, 55379568, 55379573, 55379579, 55379583, 55379591, 55379599, 55379602, 55379608, 55379617 and 55379620 of chromosome 8,

PAX 5: 36986087, 36986093, 36986098, 36986101, 36986103, 36986117, 36986131, 36986138, 36986141, 36986143, 36986147, 36986149, 36986156 of chromosome 9,

NECRL 1: 105344464, 105344482, 105344493, 105344495, 105344497, 105344503, 105344506, 105344513, 105344516, 105344519 and 105344526 of chromosome 10,

IRX 4: 1876386, 1876395, 1876397, 1876403, 1876420, 1876424, 1876432, 1876436, 1876449, 1876456, 1876459, 1876463 of chromosome 5,

RUSC 1: 155295135, 155295171, 155295181, 155295192, 155295196, 155295212, 155295229, 155295236 of chromosome 1.

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

a fragment of the C15orf52 gene comprises one or more of positions 40626309, 40626312 of the C15orf52 gene,

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

a fragment of ZNF512B gene comprises one or more of positions 62588634, 62588638 and 62588672 of ZNF512B gene,

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

fragments of the LIMK1 gene comprise one or more of positions 73509112, 73509133, 73509138, 73509148, 73509160 of the LIMK1 gene,

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

fragments of the TOR4A gene comprise one or more of positions 140172787, 140172790, 140172812 of the TOR4A gene,

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

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

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

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

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

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

fragments of the BIN1 gene comprise one or more of positions 127822478, 127822492, 127822495, 127822514, 127822551, 127822568, 127822582, 127822593, 127822616 of the BIN1 gene,

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

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

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

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

fragments of the DAPK gene comprise one or more of positions 90112842, 90112853, 90112861, 90112866 of the DAPK gene,

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

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

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

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

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

fragments of the MCC gene comprise one or more of positions 112538999, 112539011, 112539018, 112539022, 112539061 of the MCC gene,

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

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

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

the fragment of DHRS3 gene comprises the sites of DHRS3 gene: 12656340, 12656355, 12656367,

fragments of the GDNF gene contain the site of the GDNF gene: 37834770, 37834772, 37834774, 37834777, 37834780, 37834784, 37834792, 37834799, 37834802, 37834806, 37834811,

a fragment of the TBX18 gene contains the sites of the TBX18 gene: 85477035, 85477070, 85477083, 85477106,

the fragment of the SIM2 gene contains the position of the SIM2 gene: 38069638, 38069650, 38069662, 38069664, 38069676, 38069681,

a fragment of the HOXA9 gene comprises the position of the HOXA9 gene: 27204854, 27204858, 27204861, 27204863, 27204879,

the fragment of EHBP1L1 gene comprises the sites of EHBP1L1 gene: 65352621, 65352635, 65352639, 65352642, 65352651, 65352654, 65352665, 65352670,

a fragment of the GJC2 gene contains the sites of the GJC2 gene: 228345965, 228345978, 228345980, 228345989,

a fragment of the RCOR2 gene contains the sites of the RCOR2 gene: 63687223, 63687238, 63687247, 63687250, 63687259,

a fragment of the PRDM1 gene comprises the sites of the PRDM1 gene: 106429722, 106429731, 106429747, 106429750, 106429761, 106429769, 106429771,

fragments of the UNCX gene contain the sites of the UNCX gene: 1263643, 1263655, 1263659, 1263664, 1263676,

the fragment of the RPS7P5 gene comprises the locus of the RPS7P5 gene: 240161511, 240161516, 240161523, 240161527, 240161530,

a fragment of FOXI2 gene comprises the loci of FOXI2 gene: 129534910, 129534912, 129534924,

the fragment of the ACRBP gene contains the sites of the ACRBP gene: 6756182, 6756187, 6756191, 6756195, 6756211,

a fragment of the GAS6 gene comprises the position of the GAS6 gene: 114524062, 114524068, 114524084, 114524095, 114524131, 114524138,

the fragment of the MCRIP2 gene comprises the site of the MCRIP2 gene: 698072, 698142, 698153, 698168, 698208,

the fragment of the LINC01977 gene comprises the site of the LINC01977 gene: 77789596, 77789601, 77789612, 77789620,

the fragment of EGR3 gene comprises the position of EGR3 gene: 22548269, 22548279, 22548283, 22548287, 22548296, 22548299,

a fragment of the SOX17 gene comprises the position of SOX17 gene: 55379602, 55379608, 55379617, 55379620,

a fragment of the PAX5 gene comprises the sites of the PAX5 gene: 36986087, 36986093, 36986098, 36986101, 36986103,

the fragment of the nell 1 gene contains the site of the nell 1 gene: 105344493, 105344495, 105344497,

a fragment of IRX4 gene comprises the sites of IRX4 gene: 1876386, 1876395, 1876397, 1876403,

a fragment of the RUSC1 gene comprises the position of the RUSC1 gene: 155295192, 155295196, 155295212.

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

In a preferred embodiment of the second aspect, the agent for detecting DNA methylation level detects DNA methylation level of a fragment of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 genes selected from the group consisting of: SLC16A3, CDH1, TSHR, RARG, PRR15, MCC, TBX15, DPYS, COL23A1, ILDR2, NEURL1, BIN1, DNM2, IL 17C. In one or more embodiments, the agent for detecting DNA methylation level detects DNA methylation level of a fragment of two genes selected from: 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 1 and BIN1, SLC16A 1 and DNM 1, SLC16A 1 and 1, CDH1 and RARG, CDH1 and PRR1, CDTBHR 1 and 1, CDTBRARG 72 and CDRARG, CDTBRARG 72 and 1, CDRARG 1 and 1, CDRARG 1 and 1, CDRARG 1 and 1, and 1, CDRARG 1 and 1, CDROL3672 and 1, and 1, and 1, and 1, and 1, and 1 and, TBX15 and ILDR2, TBX15 and nell 1, TBX15 and BIN1, TBX15 and DNM2, TBX 2 and IL17 2, DPYS and COL23 a2, DPYS and ILDR2, DPYS and nell 2, DPYS and BIN 2, DPYS and DNM2, DPYS and IL17 2, COL23 a2 and IL dr2, COL23 a2 and ILDR2, COL23 a2 and nell 2, COL23 a2 and BIN 2, COL23 a2 and IL17 2, ILDR2 and ilurl 2, ILDR2 and BIN 2, ILDR2 and ILDR2, ILDR2 and DNM2, ILDR2 and IL17 2, nell 2 and BIN 2, dnd 2 and BIN 2. In one or more embodiments, the agent for detecting DNA methylation level detects DNA methylation level of a fragment of a gene selected from the group consisting of: SLC16A3 and CDH1 and TSHR, CDH1 and TSHR and RARG, TSHR and RARG and PRR15, RARG and PRR15 and MCC, PRR15 and MCC and TBX15, MCC and TBX15 and DPYS, TBX15 and DPYS and COL23a1, DPYS and COL23a1 and ILDR2, COL23a1 and ILDR2 and nell 1, ILDR2 and nell 1 and BIN1, nell 1 and BIN1 and DNM2, or BIN1 and DNM2 and IL 17C. In one or more embodiments, the agent for detecting DNA methylation level detects DNA methylation level of a fragment of a gene selected from the group consisting of: SLC16A3 and CDH1 and TSHR and RARG, SLC16A3 and CDH1 and TSHR and PRR15, SLC16A3 and CDH1 and TSHR and MCC, SLC16A3 and CDH1 and TSHR and TBX15, SLC16A3 and CDH1 and TSHR and DPYS, SLC16A3 and CDH1 and TSHR and COL23a1, SLC16A3 and CDH1 and TSHR and ILDR2, SLC16A3 and CDH1 and TSHR and nell 1, SLC16A3 and CDH1 and CDH1, SLC16A3 and CDH1 and TSHR and DNM2, or SLC16A3 and CDH1 and TSHR 17 hr 17C. In one or more embodiments, the agent for detecting DNA methylation level detects DNA methylation level of a fragment of five genes selected from: SLC16A and CDH and TSHR and RARG and PRR, SLC16A and CDH and TSHR and PRR and MCC, SLC16A and CDH and TSHR and MCC and TBX, SLC16A and CDH and TSHR and TBX and DPYS, SLC16A and CDH and TSHR and DPYS and COL23A, SLC16A and CDH and TSHR and COL23A and ILDR, SLC16A and CDH and TSHR and ILDR and nell, SLC16A and CDH and TSHR and nell and BIN, SLC16A and CDH and TSHR and BIN and DNM, or SLC16A and CDH and TSHR and DNM and IL 17. In one or more embodiments, the agent for detecting DNA methylation level detects DNA methylation level of a fragment of a gene selected from the group consisting of: SLC16A3 and CDH1 and TSHR and RARG and PRR15 and MCC, SLC16A3 and CDH1 and TSHR and PRR15 and MCC and TBX15, SLC16A3 and CDH1 and TSHR and MCC and TBX15 and DPYS, SLC16A3 and CDH1 and TSHR and TBX15 and DPYS and COL23a1, SLC16A3 and CDH1 and TSHR and DPYS and COL23a1 and ILDR2, SLC16A3 and CDH1 and TSHR and COL23a1 and ILDR2 and nell 1, SLC16A3 and CDH 3 and TSHR and ILDR 3 and nell 3 and nen 3, SLC16A3 and CDH 3 and cdhr 3 and NEURL 3 and SLC 3 and cdhr and ndr 3 and ndh 3 and SLC 3 and nd3672. In one or more embodiments, the reagent for detecting DNA methylation level detects DNA methylation level of fragments of 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 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 dpr 867 and coldr 361 and ILDR2 and nedr 1 and binn 1 and dny 1, and DPYS 1 and dny 1 and nddr 1 and nedr 1 and 1. In one or more embodiments, the agent for detecting DNA methylation levels detects DNA methylation levels of fragments of 8, 9, 10, 11, 12, 13, 14 or all 15 genes selected from the following genes: SLC16A3, CDH1, TSHR, RARG, PRR15, MCC, TBX15, DPYS, COL23A1, ILDR2, NEURL1, BIN1, DNM2 and IL 17C. In one or more embodiments, the agent: (1) detecting the DNA methylation level of the sites of one or more groups of (a1) - (a6) below: (a1) site of COL23a1 gene: 178003785, 178003798, 178003803, 178003814, 178003823, 178003825, 178003834, 178003841, 178003844, (a2) position of ILDR2 gene: 166890429, 166890436, 166890440, 166890442, 166890448, 166890452, 166890456, 166890461, 166890468, 166890473, 166890475, 166890480, 166890492, 166890500, 166890503, 166890509, 166890516, 166890528, 166890535, 166890543, 166890555, 166890559, 166890568, 166890573, 166890584, 166890586, or any combination thereof, (a3) the site of the DHRS3 gene: 12656091, 12656114, 12656132, 12656152, 12656170, 12656175, 12656182, 12656187, 12656197, 12656200, 12656211, 12656315, 12656323, 12656340, 12656355, 12656367, (a4) the position of KIF1A gene: 241759696, 241759701, 241759714, 241759716, (a5) the position of the GDNF gene: 37834763, 37834770, 37834772, 37834774, 37834777, 37834780, 37834784, 37834792, 37834799, 37834802, 37834806, 37834811, (a6) the position of the TBX18 gene: 85477032, 85477035, 85477070, 85477083, 85477106, 85477124, 85477151, 85477153, 85477166, or (2) detecting the level of DNA methylation of a nucleic acid fragment of one or more genes selected from the group consisting of 50-1000 bp: COL23a1, ILDR2, DHRS3, KIF1A, GDNF, TBX18, wherein the fragment of COL23a1 gene comprises the position of COL23a1 gene: 178003785, 178003798, 178003803, 178003814, 178003823, 178003825, 178003834, 178003841, 178003844, the fragment of ILDR2 gene comprises the site of ILDR2 gene: 166890429, 166890436, 166890440, 166890442, 166890448, 166890452, 166890456, 166890461, 166890468, 166890473, 166890475, 166890480, 166890492, 166890500, 166890503, 166890509, 166890516, 166890528, 166890535, 166890543, 166890555, 166890559, 166890568, 166890573, 166890584, 166890586, or a fragment of the DHRS3 gene comprising the site of the DHRS3 gene: 12656091, 12656114, 12656132, 12656152, 12656170, 12656175, 12656182, 12656187, 12656197, 12656200, 12656211, 12656315, 12656323, 12656340, 12656355, 12656367, a fragment of KIF1A gene comprising the site of KIF1A gene: 241759696, 241759701, 241759714, 241759716, the segment of the GDNF gene comprising the site of the GDNF gene: 37834763, 37834770, 37834772, 37834774, 37834777, 37834780, 37834784, 37834792, 37834799, 37834802, 37834806, 37834811, a fragment of the TBX18 gene comprising the position of the TBX18 gene: 85477032, 85477035, 85477070, 85477083, 85477106, 85477124, 85477151, 85477153, 85477166, or (3) detecting the DNA methylation level of a nucleic acid region within 10Kb upstream and downstream of the gene in (2). The sites are referenced to the human reference genome hg19 version.

In one or more embodiments, (a1) - (a6) are: site of COL23a1 gene: 178003798, 178003803, 178003814, 178003823, 178003825, 178003834, 178003841, 178003844, at a position of ILDR2 gene: 166890516, 166890528, 166890535, 166890543, 166890555, 166890559, 166890568, 166890573, 166890584, 166890586, the site of the DHRS3 gene: 12656091, 12656114, 12656132, 12656152, 12656170, 12656175, 12656182, 12656187, 12656197, 12656200, 12656211, 12656315, 12656323, 12656340, 12656355, 12656367, one or more or all of the sites of the KIF1A gene: 241759696, 241759701, 241759714, 241759716, the site of the GDNF gene: 37834770, 37834772, 37834774, 37834777, 37834780, 37834784, 37834792, 37834799, 37834802, 37834806, 37834811, at the position of TBX18 gene: 85477035, 85477070, 85477083, 85477106.

In one or more embodiments, (a1) - (a6) are: site of COL23a1 gene: 178003798, 178003803, 178003814, 178003823, 178003825, 178003834, 178003841, 178003844, at a position of ILDR2 gene: 166890516, 166890528, 166890535, 166890543, 166890555, 166890559, 166890568, 166890573, 166890584, 166890586, the site of the DHRS3 gene: 12656170, 12656175, 12656182, 12656197, 12656200, 12656211, 12656315, 12656323, at a position of KIF1A gene: 241759696, 241759701, 241759714, 241759716, the site of the GDNF gene: 37834770, 37834772, 37834774, 37834777, 37834780, 37834784, 37834792, 37834799, 37834802, 37834806, 37834811, at the position of TBX18 gene: 85477035, 85477070, 85477083, 85477106.

In one or more embodiments, the reagent for detecting DNA methylation level detects DNA methylation level at the following sites: (a1) one or more of (a) to (a3), and optionally (a4) to (a6), preferably said site comprises: (a1) one or more of (a3) and optionally (a4), or (a1) - (a3) and optionally (a5) - (a 6); more preferably, the site comprises: (a1) - (a3) and optionally (a4) or optionally (a5) - (a 6). In one or more embodiments, the reagent for detecting DNA methylation level detects DNA methylation level at the following sites: (a1) - (a4), or, (a1) - (a3) and (a5) - (a 6).

In one or more embodiments, the reagent is a primer that can amplify one or more fragments selected from the group consisting of: (b1) a fragment of COL23A1 gene amplified by SEQ ID NOS.4 and 5 as primers, (b2) a fragment of ILDR2 gene amplified by SEQ ID NOS.6 and 7 as primers, (b3) a fragment of DHRS3 gene amplified by SEQ ID NOS.8 and 9 as primers, (b4) a fragment of KIF1A gene amplified by SEQ ID NOS.10 and 11 as primers, (b5) a fragment of GDNF gene amplified by SEQ ID NOS.12 and 13 as primers, and (b6) a fragment of TBX18 gene amplified by SEQ ID NOS.14 and 15 as primers. Preferably, the primers are capable of amplifying: (b1) one or more sets of (a) - (b3), and optionally (b4) - (b6), more preferably, the primers are capable of amplifying: (b1) one or more of (b3) and optionally (b4), or (b1) - (b3) and optionally (b5) - (b 6); more preferably, the primers are capable of amplifying: (b1) - (b3) and optionally (b4) or optionally (b5) - (b 6). In one or more embodiments, the primer is any one of SEQ ID NOS 4-15 or a sequence that is 90% identical thereto. Preferably, the primer is selected from (1) one or more or all of SEQ ID NOS: 4-9, (2) one or more or all of SEQ ID NOS: 4-11, (3) one or more or all of SEQ ID NOS: 4-9, 12-15, or (4) a sequence that is 90% identical to any of (1) - (3).

In one or more embodiments, the agent is a probe that is capable of hybridizing to one or more fragments selected from the group consisting of: (b1) a fragment of COL23A1 gene amplified by SEQ ID NOS.4 and 5 as primers, (b2) a fragment of ILDR2 gene amplified by SEQ ID NOS.6 and 7 as primers, (b3) a fragment of DHRS3 gene amplified by SEQ ID NOS.8 and 9 as primers, (b4) a fragment of KIF1A gene amplified by SEQ ID NOS.10 and 11 as primers, (b5) a fragment of GDNF gene amplified by SEQ ID NOS.12 and 13 as primers, and (b6) a fragment of TBX18 gene amplified by SEQ ID NOS.14 and 15 as primers. Preferably, the primer is capable of hybridizing to: (b1) one or more of (a) - (b3), and optionally one or more of (b4) - (b 6); alternatively, the primers hybridize to the following fragments: (b1) one or more of (b3) and optionally (b4), or (b1) - (b3) and optionally (b5) - (b 6); alternatively, the primers hybridize to the following fragments: (b1) - (b3) and optionally (b4) or optionally (b5) - (b 6). In one or more embodiments, the probe is any one of SEQ ID NOs 16-21 or a sequence 90% identical thereto. Preferably, the probe is selected from (1) one or more or all of SEQ ID NOS: 16-18, (2) one or more or all of SEQ ID NOS: 16-19, (3) one or more or all of SEQ ID NOS: 16-18, 20-21, or (4) a sequence that is 90% identical to any of (1) - (3).

In one or more embodiments, the fragments have a length of 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 60-180 bp.

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

In one or more embodiments of the above aspects, the gene or site comprises a sense strand or an antisense strand of DNA.

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

In one or more embodiments of the above aspects, the reagent for detecting DNA methylation is a reagent used in one or more methods selected from the group consisting of: bisulfite conversion based PCR (e.g., methylation specific PCR), DNA sequencing (e.g., bisulfite sequencing, whole genome methylation sequencing, simplified methylation sequencing), methylation sensitive restriction enzyme analysis, fluorometry, methylation sensitive high resolution melting curve, chip-based methylation profile analysis, mass spectrometry (e.g., flight mass spectrometry). Preferably, the agent is selected from one or more of: bisulfite and its derivatives, PCR buffer solution, polymerase, dNTP, primer, probe, restriction enzyme sensitive or insensitive to methylation, enzyme digestion buffer solution, fluorescent dye, fluorescence quencher, fluorescence reporter, exonuclease, alkaline phosphatase, internal standard, and reference substance.

Preferably, the reagent comprises a primer. The primer sequences are methylation specific or non-specific. Preferably, the sequence of the primer comprises a non-methylation specific blocking sequence (Blocker). Preferably, the primer is any one of SEQ ID NOs 4-15 or a sequence 90% identical thereto.

Preferably, the reagent comprises a probe. The 5 'end of the sequence of the probe is marked with a fluorescent reporter group, and the 3' end is marked with a quenching group. Preferably, the sequence of the probe comprises mgb (minor groove binder) or lna (packed nucleic acid). Preferably, the probe is any one of SEQ ID NOs 16-21 or a sequence 90% identical thereto.

In a further aspect of the invention there is provided a kit for identifying the nature of a thyroid nodule comprising an agent according to the second aspect of the invention and optionally a nucleic acid molecule according to the first aspect of the invention. In one or more embodiments, the reagent for detecting DNA methylation is a reagent used in one or more methods selected from the group consisting of: bisulfite conversion based PCR (e.g., methylation specific PCR), DNA sequencing (e.g., bisulfite sequencing, whole genome methylation sequencing, simplified methylation sequencing), methylation sensitive restriction enzyme analysis, fluorometry, methylation sensitive high resolution melting curve, chip-based methylation profile analysis, mass spectrometry (e.g., flight mass spectrometry). Preferably, the agent is selected from one or more of: bisulfite and its derivatives, PCR buffer solution, polymerase, dNTP, primer, probe, restriction enzyme sensitive or insensitive to methylation, enzyme digestion buffer solution, fluorescent dye, fluorescence quencher, fluorescence reporter, exonuclease, alkaline phosphatase, internal standard, and reference substance. In one or more embodiments, the kit further comprises reagents for detecting gene mutations. In one or more embodiments, the agent for detecting a mutation in a gene is an agent used in one or more methods selected from the group consisting of: PCR-single strand conformation polymorphism, heteroduplex analysis, mutation enrichment PCR, mutation gradient gel electrophoresis, chemical cleavage mismatch, allele-specific oligonucleotide analysis, ligase chain reaction, allele-specific amplification, RNase A cleavage, in situ chromosome hybridization, fluorescence in situ hybridization techniques, DNA sequence analysis, enzymatic cleavage mismatch, cut fragment length polymorphism, dideoxy fingerprinting, mismatch-junction protein truncation testing, primer extension, oligonucleotide ligation detection, capillary electrophoresis, chip-based methods, and the like. Preferably, the reagent for detecting gene mutation comprises: primer, probe, buffer solution, polymerase, dNTP, restriction enzyme, enzyme digestion buffer solution, fluorescent dye, fluorescence quencher, fluorescence reporter, exonuclease, alkaline phosphatase, internal standard and reference substance.

In one or more embodiments, the kit further comprises a reagent for detecting the mutation level at the V600E site of BRAF gene and/or the mutation level at the C228T/C250T site of TERT gene.

In a further aspect the invention provides the use of a nucleic acid molecule as described herein and/or an agent as described herein in the manufacture of a kit for identifying a thyroid nodule property in a sample. The reagents include a reagent for detecting DNA methylation and optionally a reagent for detecting a mutation in a gene as described in any of the embodiments herein. The gene mutation is selected from: mutation at V600E site of BRAF gene and mutation at C228T/C250T site of TERT gene. The reagent for detecting DNA methylation is as described in the second to fourth aspects herein.

In a further aspect, the present invention provides the use of an agent for detecting DNA methylation, which agent detects the level of DNA methylation in a sample selected from the regions described in (1) and (2) below, and optionally a nucleic acid molecule described herein, in the manufacture of a kit for identifying the nature of a thyroid nodule: (1) a fragment of one or more genes selected from the group consisting of: ZMIZ1, C15orf52, SLC16A3, ZNF512B, SLC17A5, LIMK1, PLEC, TOR4A, TMEM131L, DNM2, IL17C, PRDM16, MT1JP, TBX3, BIN1, TIMP2, CFAP65, TSHR, KIF1A, DAPK, CDH1, TPO, RARG, PRR15, DPYS, MCC, TBX15, COL23A 15, ILDR 15, DHRS 15, GDNF, TBX15, SIM 15, HOXA 15, EHBP1L 15, GJC 15, RCOR 15, PRDM 15, UNCX, RPS7P 15, FOXI 15, ACRBP, GAS 15, MCRIP 15, SORIC 019C 3677, SOIRRIC 15, SOJC 15, RUX 15, PRDM 15, and up-down-stream nucleic acid region of the gene (up-and down-stream region of the gene, the gene of the SLC 15, the gene of the. Preferably, the agent detects the level of methylation of one or more genes selected from the group consisting of: COL23A1, ILDR2, DHRS3, KIF1A, GDNF, TBX 18.

In one or more embodiments, the reagent detects the methylation level of fragments of one or more sets of genes in (1) - (4) below in a 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) a nucleic acid region within 5Kb or 10Kb upstream and downstream of any one of the genes in (1) to (3).

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

KIF 1A: 241759696, 241759701, 241759714 and 241759716 of chromosome 2,

preferably, the detection site of each gene is selected from one or more of the following sites or a nucleic acid region within 500bp upstream and downstream thereof:

KIF 1A: 241759696, 241759701, 241759714 and 241759716 of chromosome 2,

COL23a 1: 178003798, 178003803, 178003814, 178003823, 178003825, 178003834, 178003841, 178003844 of chromosome 5,

ILDR 2: 166890516, 166890528, 166890535, 166890543, 166890555, 166890559, 166890568, 166890573, 166890584, 166890586 of chromosome 1,

DHRS 3: 12656340, 12656355, 12656367 of chromosome 1,

GDNF: 37834770, 37834772, 37834774, 37834777, 37834780, 37834784, 37834792, 37834799, 37834802, 37834806 and 37834811 of chromosome 5,

TBX 18: 85477035, 85477070, 85477083, 85477106 of chromosome 6.

In one or more embodiments, the agent for detecting DNA methylation detects the level of methylation at one or more of the following (a1) - (a6) sets of sites: (a1) site of COL23a1 gene: 178003785, 178003798, 178003803, 178003814, 178003823, 178003825, 178003834, 178003841, 178003844, (a2) position of ILDR2 gene: 166890429, 166890436, 166890440, 166890442, 166890448, 166890452, 166890456, 166890461, 166890468, 166890473, 166890475, 166890480, 166890492, 166890500, 166890503, 166890509, 166890516, 166890528, 166890535, 166890543, 166890555, 166890559, 166890568, 166890573, 166890584, 166890586, or any combination thereof, (a3) the site of the DHRS3 gene: 12656091, 12656114, 12656132, 12656152, 12656170, 12656175, 12656182, 12656187, 12656197, 12656200, 12656211, 12656315, 12656323, 12656340, 12656355, 12656367, (a4) the position of KIF1A gene: 241759696, 241759701, 241759714, 241759716, (a5) the position of the GDNF gene: 37834763, 37834770, 37834772, 37834774, 37834777, 37834780, 37834784, 37834792, 37834799, 37834802, 37834806, 37834811, (a6) the position of the TBX18 gene: 85477032, 85477035, 85477070, 85477083, 85477106, 85477124, 85477151, 85477153, 85477166. The reagents for detecting DNA methylation are as described in other embodiments herein.

In one or more embodiments, the agent for detecting DNA methylation level detects DNA methylation levels at the following sites: (a1) one or more of (a) to (a3), and optionally one or more of (a4) to (a 6); preferably, the site comprises: (a1) one or more of (a3) and optionally (a4), or (a1) - (a3) and optionally (a5) - (a 6); more preferably, the site comprises: (a1) - (a3) and optionally (a4) or optionally (a5) - (a 6). In one or more embodiments, the reagent for detecting DNA methylation level detects DNA methylation level at the following sites: (a1) - (a4), or, (a1) - (a3) and (a5) - (a 6).

In one or more embodiments, the kit further comprises a reagent that detects the level of mutation at the V600E site of the BRAF gene.

In one or more embodiments, the kit further comprises reagents to detect the level of mutation at the C228T/C250T site of the TERT gene.

In one or more embodiments of use, the gene or site includes a sense strand or an antisense strand of DNA.

In one or more embodiments of use, the locus is referenced to the human reference genome hg19 version.

In one or more embodiments of use, the kit further comprises a reagent 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.

In one or more embodiments of use, identifying thyroid nodule properties comprises: and comparing with a control sample, or obtaining a score according to the methylation level and/or the mutation level, and identifying the thyroid nodule property according to the comparison result or the score.

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

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

In one or more embodiments of use, the agent for detecting DNA methylation is an agent used in one or more methods selected from the group consisting of: bisulfite conversion based PCR (e.g., methylation specific PCR), DNA sequencing (e.g., bisulfite sequencing, whole genome methylation sequencing, simplified methylation sequencing), methylation sensitive restriction enzyme analysis, fluorometry, methylation sensitive high resolution melting curve, chip-based methylation profile analysis, mass spectrometry (e.g., flight mass spectrometry). Preferably, the agent is selected from one or more of: bisulfite and its derivatives, PCR buffer solution, polymerase, dNTP, primer, probe, restriction enzyme sensitive or insensitive to methylation, enzyme digestion buffer solution, fluorescent dye, fluorescence quencher, fluorescence reporter, exonuclease, alkaline phosphatase, internal standard, and reference substance.

Preferably, the primer sequence is methylation specific or non-specific. Preferably, the sequence of the primer comprises a non-methylation specific blocking sequence (Blocker). Preferably, the primer is any one of SEQ ID NOs 4-15 or a sequence 90% identical thereto.

Preferably, the probe sequence is labeled at the 5 'end with a fluorescent reporter group and at the 3' end with a quencher group. Preferably, the sequence of the probe comprises mgb (minor groove binder) or lna (packed nucleic acid). Preferably, the probe is any one of SEQ ID NOs 16-21 or a sequence 90% identical thereto.

The present invention also provides a primer that detects the level of DNA methylation in a region selected from the following regions (1) and (2): (1) a fragment of one or more genes selected from the group consisting of: ZMIZ1, C15orf52, SLC16A3, ZNF512B, SLC17A5, LIMK1, PLEC, TOR4A, TMEM131L, DNM2, IL17C, PRDM16, MT1JP, TBX3, BIN1, TIMP2, CFAP65, TSHR, KIF1A, DAPK, CDH1, TPO, RARG, PRR15, DPYS, MCC, TBX15, COL23A 15, ILDR 15, DHRS 15, GDNF, TBX15, SIM 15, HOXA 15, EHBP1L 15, GJC 15, RCOR 15, PRDM 15, UNCX, RPS7P 15, FOXI 15, ACRBP, GAS 15, MCRIP 15, SORIC 019C 3677, SOIRRIC 15, SOJC 15, RUX 15, PRDM 15, and up-down-stream nucleic acid region of the gene (up-and down-stream region of the gene, the gene of the SLC 15, the gene of the. Preferably, the primers detect the level of methylation at the sites described herein.

The present invention also provides a probe which detects the level of DNA methylation in a region selected from the following regions (1) and (2): (1) a fragment of one or more genes selected from the group consisting of: ZMIZ1, C15orf52, SLC16A3, ZNF512B, SLC17A5, LIMK1, PLEC, TOR4A, TMEM131L, DNM2, IL17C, PRDM16, MT1JP, TBX3, BIN1, TIMP2, CFAP65, TSHR, KIF1A, DAPK, CDH1, TPO, RARG, PRR15, DPYS, MCC, TBX15, COL23A 15, ILDR 15, DHRS 15, GDNF, TBX15, SIM 15, HOXA 15, EHBP1L 15, GJC 15, RCOR 15, PRDM 15, UNCX, RPS7P 15, FOXI 15, ACRBP, GAS 15, MCRIP 15, SORIC 019C 3677, SOIRRIC 15, SOJC 15, RUX 15, PRDM 15, and up-down-stream nucleic acid region of the gene (up-and down-stream region of the gene, the gene of the SLC 15, the gene of the. Preferably, the probe detects the methylation level of a site described herein.

The invention also provides a method for screening thyroid nodule benign and malignant, which comprises the following steps: (1) detecting the level of methylation of a gene, site, or nucleic acid region described herein in a sample of a subject; optionally (2) detecting the mutation level of V600E site of BRAF gene and/or the mutation level of C228T/C250T site of TERT gene; (3) comparing to a control sample, or obtaining a score based on the methylation level and/or mutation level, e.g., by calculation; (4) and (4) identifying the thyroid nodule property according to the comparison result or the score of the step (3).

The invention also provides a method for screening thyroid nodule benign and malignant, which comprises the following steps: (1) detecting the mutation level of V600E site of BRAF gene and/or the mutation level of C228T/C250T site of TERT gene; optionally (2) detecting the methylation level of a gene, site or nucleic acid region described herein in a sample from the subject; (3) comparing to a control sample, or obtaining a score based on the mutation level and/or methylation level, e.g., by calculation; (4) and (4) identifying the thyroid nodule property according to the comparison result or the score of the step (3).

In one or more embodiments, the method further comprises, prior to step (1): extraction of sample DNA, quality inspection, and/or conversion of unmethylated cytosines on DNA to bases that do not bind guanine.

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

In one or more embodiments, the detection includes, but is not limited to: bisulfite conversion based PCR (e.g., methylation specific PCR), DNA sequencing (e.g., bisulfite sequencing, whole genome methylation sequencing, simplified methylation sequencing), methylation sensitive restriction enzyme analysis, fluorometry, methylation sensitive high resolution melting curve, chip-based methylation profile analysis, mass spectrometry (e.g., flight mass spectrometry).

In one or more embodiments, step (4) comprises: the methylation level and/or mutation level of the subject sample is altered compared to the control sample, and when the methylation level and/or mutation level meets a threshold, the thyroid nodule is identified as a benign or malignant nodule.

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

In one or more embodiments, the sample is from a human, preferably from a tissue, cell, or bodily fluid, such as thyroid tissue or blood. In one or more embodiments, the sample is a thyroid nodule biopsy, preferably a fine needle biopsy. In one or more embodiments, the sample is plasma.

In one or more embodiments, the sample is from a subject having benign or malignant nodules of the thyroid. In one or more embodiments, the sample is from a patient with goiter.

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

The invention also provides a kit for identifying the nature of thyroid nodules, comprising primers and/or probes for detecting the methylation level of genes, sites, nucleic acid regions as described herein.

Drawings

FIG. 1 is a LabChip test single library fragment profile of the present invention.

FIGS. 2A-C are ROC curve analyses of the invention for the detection of 10 thyroid cancers and 10 benign nodules of the thyroid. A: a tissue sample; b, C: plasma samples.

FIG. 3 is a ROC curve analysis of plasma samples from 20 cases of thyroid cancer and 20 cases of benign nodules of the thyroid in one embodiment of the present invention.

FIG. 4 is a ROC curve analysis of plasma samples from 20 cases of thyroid cancer and 20 cases of benign nodules of the thyroid in one embodiment of the present invention.

Detailed Description

The inventors have found that specific chromosomes, genes or methylation sites are associated with malignant thyroid nodules.

Referring to thyroid nodules, the terms "benign" and "malignant" as used herein refer to the nature of thyroid nodules. Generally, benign is manifested by slow growth of nodules, uniform texture, good mobility, smooth surface, cystic changes, no enlargement of lymph nodes, no calcification, etc. Malignancy is characterized by uncontrolled malignant cell growth, spread and tissue infiltration. Ultrasound signs that suggest that thyroid nodules are malignant include: the height of the nodule is greater than the width, lack of acoustic halo, micro-calcification, irregular boundaries, reduced echo, solid nodules, abundant blood flow within the nodule, and the like. In some embodiments, the malignant thyroid nodule comprises thyroid cancer.

The inventors found that the nature of thyroid nodules is correlated with the methylation level of fragments of one or more genes selected from the group consisting of: ZMIZ1, C15orf52, SLC16A3, ZNF512B, SLC17A5, LIMK1, PLEC, TOR4A, TMEM131L, DNM2, IL17C, PRDM16, MT1JP, TBX3, BIN1, TIMP2, CFAP65, TSHR, KIF1A, DAPK, CDH1, TPO, RARG, PRR15, DPYS, MCC, TBX15, COL23A 15, ILDR 15, DHRS 15, GDNF, TBX15, SIM 15, HOXA 15, EHBP1L 15, GJC 15, RCOR 15, PRDM 15, UNCX, RPS7P 15, FOXI 15, ACRBP, GAS 15, MCEGR 15, SORIC 019RIC 01972, SORIC 15, SOIRX 15, RUX 15, RUSC 15, RUX 15, and NEIRRS 15. Preferably, the gene is selected from the group consisting of: (1) LIMK1 and SLC17a5, (2) BIN1 and DNM2, (3) BIN1 and SLC16A3, (4) SLC16A3, DNM2 and IL17C, (5) SLC16A3, DNM2, IL17C, CDH1 and TSHR, (6) COL23a1, ILDR2 and DHRS3, (7) COL23a1, ILDR2, DHRS3 and KIF1A, (8), COL23a1, ILDR2, DHRS3, GDNF and TBX 18.

The inventors have also found that the properties of thyroid nodules are correlated with methylation levels at one or more sites selected from the following, the numbering of which refers to the human reference genome hg 19:

c15orf 52: 40626309, 40626312, 40626386 of chromosome 15, SLC16 A3: 80189165, 80189174, 80189177, 80189197, 80189225, 80189230, 80189239, 80189645, 80189671, 80189674, 80189684, 80189687, 80189698, 80189709, 80189719, 80189726, 80189728, 80189739, 80189757, 80189787, 80189792, 80189811, 80189817, 80189832, 80189841 of chromosome 17,

ZNF 512B: 62588634, 62588638, 62588672 of chromosome 20,

SLC17a 5: 74290205, 74290207, 74290220, 74290225 and 74290228 of chromosome 6,

and (2) PLEC: 145013661, 145013673 of chromosome 8,

TOR 4A: 140172787, 140172790, 140172812 of chromosome 9,

KIF 1A: 241759696, 241759701, 241759714 and 241759716 of chromosome 2,

DAPK: 90112842, 90112853, 90112861 and 90112866 of chromosome 9,

TPO: 1481013, 1481015, 1481022 and 1481039 of chromosome 2,

TBX 3: 115174750, 115174773, 115174780 of chromosome 12,

TIMP 2: 76921845, 76921853, 76921860 of chromosome 17,

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

Herein, methods for detecting DNA Methylation are well known in the art, such as Bisulfite conversion based PCR (e.g., Methylation-specific PCR (MSP)), DNA Sequencing (e.g., Bisulfite Sequencing, BS), Whole genome Methylation Sequencing (WGBS), Reduced Methylation Sequencing (RRBS)), Methylation-Sensitive Restriction enzyme assay (Methylation-Sensitive Dependent Restriction Enzymes), fluorescence quantification, Methylation-Sensitive High resolution Melting curve (MS-HRM), chip-based Methylation mass spectrometry, mass spectrometry (e.g., flight spectroscopy). In one or more embodiments, detecting comprises detecting either strand at the gene or site. Detecting the methylation level of the site comprises detecting that the methylation level of a nucleic acid region within 500bp upstream and downstream of the site is correlated.

Thus, the present invention relates to a reagent for detecting DNA methylation. Reagents used in the above-described methods for detecting DNA methylation are well known in the art. Illustratively, the reagent for detecting DNA methylation may be a reagent used in one or more methods selected from the group consisting of: bisulfite conversion based PCR (e.g., methylation specific PCR), DNA sequencing (e.g., bisulfite sequencing, whole genome methylation sequencing, simplified methylation sequencing), methylation sensitive restriction enzyme analysis, fluorometry, methylation sensitive high resolution melting curve, chip-based methylation profile analysis, mass spectrometry (e.g., flight mass spectrometry).

The reagent for detecting DNA methylation may comprise one or more of: bisulfite and its derivatives, PCR buffer solution, polymerase, dNTP, primer, probe, restriction enzyme sensitive or insensitive to methylation, enzyme digestion buffer solution, fluorescent dye, fluorescence quencher, fluorescence reporter, exonuclease, alkaline phosphatase, internal standard, and reference substance. In detection methods involving DNA amplification, the reagents for detecting DNA methylation include primers. The primer sequences are methylation specific or non-specific. Preferably, the sequence of the primer comprises a non-methylation specific blocking sequence (Blocker). Blocking sequences may enhance the specificity of methylation detection. The reagent for detecting DNA methylation may further comprise a probe. Typically, the sequence of the probe is labeled at the 5 'end with a fluorescent reporter group and at the 3' end with a quencher group. Preferably, the sequence of the probe comprises MGB or LNA.

Herein, methods for detecting gene mutations and reagents therefor 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 cleavage mismatch, allele-specific oligonucleotide analysis, ligase chain reaction, allele-specific amplification, RNase A cleavage, chromosome in situ hybridization, fluorescence in situ hybridization techniques, DNA sequence analysis, enzymatic cleavage mismatch, cleavage fragment length polymorphism, dideoxy fingerprinting, mismatch-junction protein truncation assays, primer extension, oligonucleotide-ligation assays, capillary electrophoresis, chip-based methods, and the like. In one or more embodiments, detecting comprises detecting either strand at the gene or site.

Accordingly, the present invention relates to a reagent for detecting a gene mutation. Reagents used in the above-described methods for detecting gene mutations are well known in the art. Exemplary reagents for detecting gene mutations include: primer, probe, buffer solution, polymerase, dNTP, restriction enzyme, enzyme digestion buffer solution, fluorescent dye, fluorescence quencher, fluorescence reporter, exonuclease, alkaline phosphatase, internal standard and reference substance.

The invention also relates to a kit for identifying the nature of a thyroid nodule comprising an agent as described herein, in particular an agent as described in the second and/or third aspect of the invention. The kit may further comprise a nucleic acid molecule as described herein, in particular a nucleic acid molecule as described in the first aspect, as an internal standard or positive control. The kit may comprise reagents for detecting gene mutations. As used herein, a "primer" refers to a nucleic acid molecule having a specific nucleotide sequence that directs the synthesis at the initiation of nucleotide polymerization. The primers are typically two oligonucleotide sequences synthesized by man, one primer complementary to one DNA template strand at one end of the target region and the other primer complementary to the other DNA template strand at the other end of the target region, which functions as the initiation point for nucleotide polymerization. Primers designed artificially in vitro are widely used in Polymerase Chain Reaction (PCR), qPCR, sequencing, probe synthesis, and the like. Generally, the primers are designed such that the amplified products are 50-150 bp, 60-140, 70-130, 80-120bp in length. Preferably, the product is 80-100bp in length.

In one or more embodiments, the reagent for detecting DNA methylation comprises a probe. The 5 'end of the sequence of the probe is marked with a fluorescent reporter group, and the 3' end is marked with a quenching group. Preferably, the sequence of the probe comprises mgb (minor groove binder) or lna (packed nucleic acid). MGB and LNA are used to increase the Tm (long temperature) value, increase the specificity of the assay, and increase the flexibility of probe design.

The term "variant" or "mutant" as used herein refers to a polynucleotide that has a nucleic acid sequence altered by insertion, deletion or substitution of one or more nucleotides compared to a reference sequence, while retaining its ability to hybridize to other nucleic acids. A mutant according to any of the embodiments herein 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 to a reference sequence and retaining the biological activity of the reference sequence. Sequence identity between two aligned sequences can be calculated using, for example, BLASTn from NCBI. Mutants also include nucleotide sequences that have one or more mutations (insertions, deletions, or substitutions) in the reference sequence and in the nucleotide sequence, while still retaining the biological activity of the reference sequence. The plurality of mutations typically refers to within 1-10, such as 1-8, 1-5, or 1-3. The substitution may be a substitution between purine nucleotides and pyrimidine nucleotides, or a substitution between purine nucleotides or between pyrimidine nucleotides. The substitution is preferably a conservative substitution. For example, conservative substitutions with nucleotides of similar or analogous properties are not typically made in the art to alter the stability and function of the polynucleotide. Conservative substitutions are, for example, exchanges between purine nucleotides (A and G), exchanges between pyrimidine nucleotides (T or U and C). Thus, substitution of one or more sites with residues from the same in the polynucleotides of the invention will not substantially affect their activity. In particular, the positions described herein contained in the variants of the invention are not mutated. That is, the method of the present invention detects methylation at the sites in the corresponding sequences, and mutations may occur at bases other than these sites.

The invention also provides a method for screening thyroid nodule benign and malignant, which comprises the following steps: (1) detecting the level of methylation of a gene, site, or nucleic acid region described herein in a sample of a subject; optionally (2) detecting the mutation level of V600E site of BRAF gene and/or the mutation level of C228T/C250T site of TERT gene; (3) methylation levels are measured in comparison to control samples, or by calculating a score; (4) when the interpretation criteria are met, the subject is identified as a benign or malignant nodule. Typically, the method further comprises, prior to step (1): extraction of sample DNA, quality inspection, and/or conversion of unmethylated cytosines on DNA to bases that do not bind guanine.

As used herein, a "DNA" or "DNA molecule" is a deoxyribonucleic acid. The basic unit of DNA is deoxyribonucleotide, which is condensed by phosphodiester bond to form a long chain molecule. Each deoxyribonucleotide consists of a phosphate, a deoxyribose, and a base. Bases (bp) of DNA are mainly adenine (A), guanine (G), cytosine (C) and thymine (T). In the double-helix structure of double-stranded DNA, A is hydrogen-bonded to T, and G is hydrogen-bonded to C. The form of DNA includes cDNA, genomic DNA, fragmented DNA, or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be of any length, such as 50-500bp, 100-400bp, 150-300bp or 200-250 bp.

As used herein, "uracil" or "U" is a component of RNA. An "RNA" or "RNA molecule" is a ribonucleic acid. RNA is a long chain molecule formed by the condensation of ribonucleotides via phosphodiester bonds. Each ribonucleotide molecule consists of a phosphate, a ribose and a base. RNA has 4 main bases, namely adenine (A), guanine (G), cytosine (C) and uracil (U). In base pairing of RNA, U replaces the position of T in DNA, i.e. a is hydrogen-bonded to U and G is hydrogen-bonded to C.

Transformation can occur between bases of DNA or RNA. "transformation", "cytosine transformation" or "CT transformation" as used herein is the process of converting an unmodified cytosine base (C) to a base that does not bind guanine, such as a uracil base (U), by treating the DNA using non-enzymatic or enzymatic methods. Non-enzymatic or enzymatic methods of performing cytosine conversion are well known in the art. Illustratively, non-enzymatic methods include bisulfite or bisulfate treatments, such as calcium bisulfite, sodium bisulfite, potassium bisulfite, ammonium bisulfite, sodium bisulfate, potassium bisulfate, ammonium bisulfate, and the like. Illustratively, the enzymatic method includes a deaminase treatment. The transformed DNA is optionally purified. DNA purification methods suitable for use herein are well known in the art.

In reference to cytosine, "modification" refers to the introduction or removal of a chemical group on the cytosine base. During cytosine conversion, the modified cytosine base is more stable than the unmodified cytosine base and is less susceptible or unaffected by the conversion process to become U. In one or more embodiments, the modification refers to methylation. As used herein, "methylation" or "DNA methylation" refers to the covalent attachment of a methyl group at the cytosine 5' carbon position of a CpG dinucleotide in genomic DNA to form a 5-methylcytosine (5 mC).

Optionally, the modified cytosine can be protected from downstream transformation or deamination by non-enzymatic or enzymatic means prior to transformation of the cytosine as described herein. Non-enzymatic or enzymatic methods suitable for protecting modified cytosines are well known in the art. For example, TET2(ten-eleven transition 2) and/or an oxidation enhancer may protect the modified cytosine. TET2 can oxidize 5mC and 5hmC to 5caC by a cascade reaction. The oxidation enhancer may convert 5hmC to 5ghmC by glycosylation. Oxidation enhancers suitable for performing such glycosylation are well known in the art.

In one or more embodiments, the interpretation criteria are: the methylation level and/or the mutation level of the subject sample is increased or decreased when compared to the control sample. When the methylation level and/or mutation level meet a certain threshold, a malignant nodule is identified. And (4) carrying out mathematical analysis on the methylation level of the detected gene to obtain a fitting equation of the score. And for the detected sample, when the score is larger than the threshold value, judging that the result is positive, namely the malignant nodule, otherwise, judging that the result is negative, namely the benign nodule. Methods of conventional mathematical analysis and processes of determining thresholds are known in the art, an exemplary method being binary Logistic regression analysis. Typically, the threshold is 0.

For example, when the properties of the nodule are identified by the methylation levels of BIN1 and SLC16A3 genes, a binary Logistic regression analysis is performed on the methylation levels of BIN1 gene and SLC16A3 gene, with the fitting equation:

score 3.45-0.08 × BIN1 +0.01 × SLC16A3 methylation level.

Therefore, if the score of BIN1 and SLC16A3 genes in the sample is greater than 0, the result is determined to be positive, i.e., a malignant nodule.

Herein, the sample is from a mammal, preferably a human. The sample may be from any organ (e.g., thyroid), tissue (e.g., epithelial tissue, connective tissue, muscle tissue, and neural tissue), cell (e.g., thyroid nodule biopsy), or body fluid (e.g., blood, plasma, serum, interstitial fluid, urine). In general, it is sufficient that the sample contains genomic DNA or cfdna (circulating free DNA or Cell free DNA). cfDNA is called circulating free DNA or cell free DNA, and is a degraded DNA fragment that is released into plasma. Illustratively, the sample is a thyroid nodule biopsy, preferably a fine needle biopsy. Alternatively, the sample is plasma.

Exemplary embodiments:

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

A fragment of chromosome 7 comprising one or more of positions 73508994, 73509017, 73509055, 73509062, 73509073, 73509075, 73509112, 73509133, 73509138, 73509148, 73509160 on chromosome 7,

a fragment of chromosome 6 comprising one or more of sites 74290205, 74290207, 74290220, 74290225, 74290228 on chromosome 6;

a fragment of chromosome 2 comprising one or more of positions 127822478, 127822492, 127822495, 127822514, 127822551, 127822568, 127822582, 127822593, 127822616, 127822644 on chromosome 2,

a fragment of chromosome 19 comprising one or more of sites 10870373, 10870377, 10870427, 10870429, 10870441, 10870448 on chromosome 19;

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

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

the above-mentioned sites in the variant are not mutated.

2. The nucleic acid molecule of embodiment 1, wherein the nucleic acid molecule further comprises a fragment of chromosome 14 comprising one or more of positions 81421983, 81421989, 81422010, 81422017, 81422032, 81422035, 81422063, 81422084 on chromosome 14, said fragment being 50-5000bp, preferably 50-1000bp long, or a variant having at least 70% identity thereto, and the fragment of chromosome 16 further comprises one or more of positions 68771035, 68771037, 68771045, 68771051, 68771059, 68771064, 68771073 on chromosome 16,

the above-mentioned sites in the variant are not mutated.

3. A reagent for detecting DNA, which comprises a reagent for detecting the methylation level of DNA in a region selected from the following regions (1) and (2):

(1) a fragment of one or more genes selected from the group consisting of: ZMIZ1, C15orf52, SLC16A3, ZNF512B, SLC17A5, LIMK1, PLEC, TOR4A, TMEM131L, DNM2, IL17C, PRDM16, MT1JP, TBX3, BIN1, TIMP2, CFAP65, TSHR, KIF1A, DAPK, CDH1, TPO, RARG, PRR15, DPYS, MCC, TBX15, COL23A 15, ILDR 15, DHRS 15, GDNF, TBX15, SIM 15, HOXA 15, EHBP1L 15, GJC 15, RCOR 15, PRDM 15, UNCX, RPS7P 15, FOXI 15, ACRBP, GAS 15, MCEGR 15, SORIC 019RIC 01972, SORIC 15, SOIRX 15, RUX 15, and NEX 15, wherein the length of the fragment 3650-1000 is the fragment 15,

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

wherein the fragment of the ZMIZ1 gene comprises the positions of the ZMIZ1 gene: 81001968, 81001996, 81002041, 81002052, 81002054, 81002056, 81002062, 81002083, 81002110, 81002116, 81002123, 81002129, 81002133, 81002137, 81002139, 81002164, 81002168, 81002223, 81002241, 81002253,

a fragment of the C15orf52 gene comprises the sites of the C15orf52 gene: 40626309, 40626312, 40626386,

the fragment of the SLC16A3 gene contains the SLC16A3 gene at the position: 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,

fragments of ZNF512B gene comprise the sites of ZNF512B gene: 62588634, 62588638, 62588672,

the fragment of the SLC17A5 gene contains the SLC17A5 gene at the position: 74290205, 74290207, 74290220, 74290225, 74290228,

fragments of the LIMK1 gene include the sites of LIMK1 gene: 73508994, 73509017, 73509055, 73509062, 73509073, 73509075, 73509112, 73509133, 73509138, 73509148, 73509160,

the fragments of the PLEC gene comprise the sites of the PLEC gene: 145013661, 145013673,

the fragment of TOR4A gene comprises the sites of TOR4A gene: 140172787, 140172790, 140172812,

a fragment of the TMEM131L gene contains the sites of the TMEM131L gene: 154409945, 154409963, 154409972, 154409978, 154409997, 154410003, 154410006,

a fragment of the DNM2 gene comprises the site of the DNM2 gene: 10870373, 10870377, 10870427, 10870429, 10870441, 10870448,

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

a fragment of the PRDM16 gene comprises the sites of the PRDM16 gene: 3229914, 3229921, 3229950, 3229968, 3229973, 3310213, 3310229, 3310235, 3310238, 3310240, 3310268, 3310287, 3310312, 3310314, 3310317, 3310329,

fragments of the TSHR gene contain sites of the TSHR gene: 81421983, 81421989, 81422010, 81422017, 81422032, 81422035, 81422063, 81422084,

fragments of the KIF1A gene comprise the sites of the KIF1A gene: 241759696, 241759701, 241759714, 241759716,

fragments of the DAPK gene contain the site of the DAPK gene: 90112842, 90112853, 90112861, 90112866,

a fragment of the CDH1 gene comprises the sites of the CDH1 gene: 68771035, 68771037, 68771045, 68771051, 68771059, 68771064, 68771073,

the fragment of the TPO gene contains the sites of the TPO gene: 1481013, 1481015, 1481022, 1481039,

the fragment of the RARG gene contains the site of the RARG gene: 53613176, 53613182, 53613190, 53613202, 53613210, 53613218,

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

a fragment of the TBX3 gene contains the sites of the TBX3 gene: 115174750, 115174773, 115174780,

the fragment of BIN1 gene contains the sites of BIN1 gene: 127822478, 127822492, 127822495, 127822514, 127822551, 127822568, 127822582, 127822593, 127822616, 127822644,

a fragment of TIMP2 gene comprises the sites of TIMP2 gene: 76921845, 76921853, 76921860,

the fragment of CFAP65 gene contains the sites of CFAP65 gene: 219866132, 219866139, 219866148, 219866158, 219866165, 219866168, 219866199, 219866218,

a fragment of the PRR15 gene comprises the sites of the PRR15 gene: 29605992, 29606026, 29606040, 29606047, 29606056, 29606062, 29606073, 29606179, 29606191, 29606201, 29606204, 29606220, 29606222, 29606227, 29606231, 29606255, 29606257, 29606262, 29606271, 29606277, 29606289, 29606320,

the DPYS gene fragment contains the locus of the DPYS gene: 105478870, 105478873, 105478878, 105478905, 105478908, 105478916, 105478918, 105478945, 105478956, 105478965, 105478974, 105478983, 105478986, 105478989,

fragments of the MCC gene contain the sites of the MCC gene: 112538999, 112539011, 112539018, 112539022, 112539061, 112539084, 112539104, 112539128,

a fragment of the TBX15 gene contains the sites of the TBX15 gene: 119535725, 119535730, 119535740, 119535742, 119535750, 119535759, 119535766, 119535812, 119535817, 119535821, 119535823, 119535876, 119535879, 119535884, 119535891,

the fragment of COL23a1 gene comprises the sites of COL23a1 gene: 178003785, 178003798, 178003803, 178003814, 178003823, 178003825, 178003834, 178003841, 178003844,

the fragment of ILDR2 gene contains the site of ILDR2 gene: 166890429, 166890436, 166890440, 166890442, 166890448, 166890452, 166890456, 166890461, 166890468, 166890473, 166890475, 166890480, 166890492, 166890500, 166890503, 166890509, 166890516, 166890528, 166890535, 166890543, 166890555, 166890559, 166890568, 166890573, 166890584, 166890586,

the fragment of DHRS3 gene comprises the sites of DHRS3 gene: 12656091, 12656114, 12656132, 12656152, 12656170, 12656175, 12656182, 12656187, 12656197, 12656200, 12656211, 12656315, 12656323, 12656340, 12656355, 12656367,

fragments of the GDNF gene contain the site of the GDNF gene: 37834763, 37834770, 37834772, 37834774, 37834777, 37834780, 37834784, 37834792, 37834799, 37834802, 37834806, 37834811,

a fragment of the TBX18 gene contains the sites of the TBX18 gene: 85477032, 85477035, 85477070, 85477083, 85477106, 85477124, 85477151, 85477153, 85477166,

the fragment of the SIM2 gene contains the position of the SIM2 gene: 38069563, 38069579, 38069619, 38069625, 38069638, 38069650, 38069662, 38069664, 38069676, 38069681,

a fragment of the HOXA9 gene comprises the position of the HOXA9 gene: 27204848, 27204854, 27204858, 27204861, 27204863, 27204879, 27204884, 27204894, 27204897, 27204918, 27204929, 27204938, 27204945, 27204948, 27204951, 27204958, 27204981, 27204984,

the fragment of EHBP1L1 gene comprises the sites of EHBP1L1 gene: 65352612, 65352621, 65352635, 65352639, 65352642, 65352651, 65352654, 65352665, 65352670,

a fragment of the GJC2 gene contains the sites of the GJC2 gene: 228345954, 228345957, 228345965, 228345978, 228345980, 228345989,

a fragment of the RCOR2 gene contains the sites of the RCOR2 gene: 63687223, 63687238, 63687247, 63687250, 63687259, 63687282, 63687288, 63687299, 63687318, 63687325,

a fragment of the PRDM1 gene comprises the sites of the PRDM1 gene: 106429711, 106429722, 106429731, 106429747, 106429750, 106429761, 106429769, 106429771,

fragments of the UNCX gene contain the sites of the UNCX gene: 1263643, 1263655, 1263659, 1263664, 1263676, 1263694, 1263716, 1263723,

the fragment of the RPS7P5 gene comprises the locus of the RPS7P5 gene: 240161502, 240161507, 240161511, 240161516, 240161523, 240161527, 240161530, 240161535, 240161546, 240161558, 240161560,

a fragment of FOXI2 gene comprises the loci of FOXI2 gene: 129534843, 129534853, 129534866, 129534879, 129534891, 129534910, 129534912, 129534924,

the fragment of the ACRBP gene contains the sites of the ACRBP gene: 6756182, 6756187, 6756191, 6756195, 6756211, 6756225, 6756230, 6756270,

a fragment of the GAS6 gene comprises the position of the GAS6 gene: 114524043, 114524062, 114524068, 114524084, 114524095, 114524131, 114524138, 114524142, 114524150, 114524158,

the fragment of the MCRIP2 gene comprises the site of the MCRIP2 gene: 698072, 698142, 698153, 698168, 698208, 698218, 698222, 698230,

the fragment of the LINC01977 gene comprises the site of the LINC01977 gene: 77789596, 77789601, 77789612, 77789620, 77789628, 77789632, 77789635, 77789640,

the fragment of EGR3 gene comprises the position of EGR3 gene: 22548250, 22548260, 22548269, 22548279, 22548283, 22548287, 22548296, 22548299,

a fragment of the SOX17 gene comprises the position of SOX17 gene: 55379566, 55379568, 55379573, 55379579, 55379583, 55379591, 55379599, 55379602, 55379608, 55379617, 55379620,

a fragment of the PAX5 gene comprises the sites of the PAX5 gene: 36986087, 36986093, 36986098, 36986101, 36986103, 36986117, 36986131, 36986138, 36986141, 36986143, 36986147, 36986149, 36986156,

the fragment of the nell 1 gene contains the site of the nell 1 gene: 105344464, 105344482, 105344493, 105344495, 105344497, 105344503, 105344506, 105344513, 105344516, 105344519, 105344526,

a fragment of IRX4 gene comprises the sites of IRX4 gene: 1876386, 1876395, 1876397, 1876403, 1876420, 1876424, 1876432, 1876436, 1876449, 1876456, 1876459, 1876463,

a fragment of the RUSC1 gene comprises the position of the RUSC1 gene: 155295135, 155295171, 155295181, 155295192, 155295196, 155295212, 155295229, 155295236.

4. The reagent for detecting DNA according to embodiment 3,

a fragment of the ZMIZ1 gene comprises one or more of positions 81002041, 81002052, 81002054, 81002056, 81002062, 81002083 of the ZMIZ1 gene,

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

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

7. A reagent for detecting DNA methylation which detects the level of methylation at one or more of the following (a) - (d):

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

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

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

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

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

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

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

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

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

8. The reagent of embodiment 7, wherein the reagent further detects the methylation level at:

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

9. The agent according to any one of embodiments 2 to 8, further comprising one or more characteristics selected from the group consisting of:

the fragment includes a sense strand or an antisense strand of DNA,

the reagent for detecting DNA methylation is a reagent used in one or more methods selected from the group consisting of: PCR based on bisulfite conversion, DNA sequencing, methylation sensitive restriction enzyme analysis, fluorescence quantification, methylation sensitive high resolution melting curve method, chip-based methylation map analysis, mass spectrometry,

preferably, the reagent for detecting DNA methylation is selected from one or more of: bisulfite and its derivatives, PCR buffer solution, polymerase, dNTP, primer, probe, restriction endonuclease sensitive or insensitive to methylation, enzyme digestion buffer solution, fluorescent dye, fluorescence quencher, fluorescence reporter, exonuclease, alkaline phosphatase, internal standard, and reference substance,

preferably, the primer is a methylation specific or non-specific primer, preferably the sequence of the primer comprises a non-methylation specific blocking sequence (Blocker), preferably the primer is SEQ ID NO:1, 2, 4, 5, 7, 8 or a sequence 90% identical thereto,

preferably, the probe has a reporter sequence, preferably the probe is SEQ ID NO 3, 6, 9 or a sequence having 90% identity thereto,

the reagent for detecting gene mutation is a reagent used in one or more methods selected from the group consisting of: PCR-single strand conformation polymorphism, heteroduplex analysis, mutation enrichment PCR, mutation gradient gel electrophoresis, chemical cleavage mismatch, allele-specific oligonucleotide analysis, ligase chain reaction, allele-specific amplification, RNase A cleavage, in situ chromosome hybridization, fluorescence in situ hybridization, DNA sequence analysis, enzymatic cleavage mismatch, cut fragment length polymorphism, dideoxy fingerprinting, mismatch-junction protein truncation testing, primer extension, oligonucleotide ligation detection, capillary electrophoresis, chip-based methods,

preferably, the reagent for detecting gene mutation comprises: primer, probe, buffer solution, polymerase, dNTP, restriction enzyme, enzyme digestion buffer solution, fluorescent dye, fluorescence quencher, fluorescence reporter, exonuclease, alkaline phosphatase, internal standard and reference substance.

10. A kit for identifying a thyroid nodule property comprising an agent according to any one of embodiments 2 to 9 and optionally a nucleic acid molecule according to embodiment 1.

11. Use of a reagent for detecting DNA, which detects the level of methylation at one or more of the following (a) - (d), and optionally a nucleic acid molecule of embodiment 1, in the preparation of a kit for identifying a thyroid nodule property in a sample, wherein:

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

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

preferably, the agent is as described in any one of embodiments 8-9.

12. The use according to embodiment 11, wherein said use has one or more characteristics selected from the group consisting of:

the kit also comprises a reagent for detecting the mutation level of the V600E site of the BRAF gene and/or the mutation level of the C228T/C250T site of the TERT gene,

the identifying thyroid nodule properties comprises: comparing with a control sample, or obtaining a score based on the methylation level and/or mutation level, identifying a thyroid nodule property based on the comparison or score,

the sample is from a human, preferably from a tissue, cell or body fluid, such as thyroid tissue or blood,

the sample contains genomic DNA or cfDNA.

13. A method of identifying a thyroid nodule property comprising:

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

optionally (2) detecting the mutation level of V600E site of BRAF gene and/or the mutation level of C228T/C250T site of TERT gene;

(3) (ii) comparing to a control sample, or obtaining a score based on the methylation level and/or mutation level;

(4) identifying the nature of the thyroid nodule according to the comparison result or score of the step (3),

preferably, step (4) comprises:

a change in the methylation level and/or the mutation level of the subject sample as compared to the control sample, and identifying the thyroid nodule as a benign or malignant nodule when the methylation level and/or the mutation level meets a threshold value, or

When the score meets a threshold, the thyroid nodule is identified as a benign or malignant nodule.

Examples

The present invention will be described in further detail with reference to the following drawings and specific examples. In the following examples, the experimental methods without specifying specific conditions were generally carried out in the same manner as described in the conventional conditions.

Example 1: simplified methylation Sequencing (RRBS) screening for methylation sites of benign and malignant differences in thyroid nodules

1) Sample preparation

DNA extraction of the tissues of 37 thyroid cancers and 37 benign nodules of the thyroid gland was carried out using the QIAamp DNA Mini Kit (QIAGEN, cat # 51304); the concentration of DNA was measured using the QubitTM dsDNA HS Assay Kit (Thermo, cat # Q32854); quality control was performed using 1% agarose gel electrophoresis.

2) MspI enzyme digestion

The reaction system is prepared as follows:

components	Volume (μ l)
		10×Buffer Tango	2.0
MspI(10U/μl)	1.0
		Nuclease-free Water + DNA	17.0
Total of	20.0

The reaction procedure is as follows: stored at 37 ℃ for 2 hours and 4 ℃.

3) End repair and addition of A

The reaction system is prepared as follows:

components	Volume (μ l)
		Enzyme-digested DNA product	20.0
End Repair&A-Tailing Buffer	4.0
		End Repair&A-Tailing Enzyme Mix	2.0
Nuclease-free water	14.0
		Total of	40.0

The reaction procedure is as follows: 30 minutes at 20 ℃, 30 minutes at 65 ℃ and 4 ℃.

4) Joint connection

The reaction system is prepared as follows:

components	Volume (μ l)
		End repair and addition of A product	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 of	50.0

The reaction procedure is as follows: 16 ℃ overnight, 65 ℃ for 10 minutes, 4 ℃.

5) Post-ligation purification

i. After ligation the solution was transferred to 50. mu.l of AMPure beads, vortexed, incubated at room temperature for 5 minutes and centrifuged briefly at low speed. Placing the centrifuge tube on a magnetic frame until the solution is clear;

washing twice with 80% ethanol solution;

drying the magnetic beads at room temperature;

add 32. mu.l ddH₂O, incubation for 2 minutes at room temperature, placing the tube on a magnetic rack until the solution is clear, and pipetting 30 μ l of the supernatant solution to transfer to a new tube.

6) Bisulfite conversion

Using Methycode^TMBisulfite Conversion of the DNA from step 5 with the bisufite Conversion Kit (Thermo, cat # MECV 50) to convert unmethylated cytosine (cytosine, C) to uracil (uracil, U); methylated cytosines are not altered after conversion.

The transformation reagent was formulated as follows:

components	Volume (μ l)
		Nuclease-free water	800.0
Dilution Buffer	300.0
		Resuspension Buffer	50.0

Mu.l of the prepared transformation reagent was added to 30. mu.l of the ligation purified product obtained in step 5, and mixed well. The reaction procedure is as follows: the mixture was stored at 98 ℃ for 10 minutes, at 64 ℃ for 2.5 hours, and at 4 ℃.

The treated DNA was recovered according to the instructions, and finally, 43. mu.l of the eluate was used to elute the DNA, and 41.6. mu.l was transferred to the next reaction.

7) Library amplification

The reaction system is prepared as follows:

components	Volume (mu 1)
		10×PfuTurbo Cx reaction buffer	5.0
dNTPs(25mM each dNTP)	0.4
		Primer mix	2.0
PfuTurbo Cx hotstart DNA polymerase(2.5U/μl)	1.0
		Bisulfite conversion of DNA	41.6
Total of	50.0

The reaction procedure is as follows: 2 minutes at 95 ℃; 30 seconds at 95 ℃, 30 seconds at 65 ℃,1 minute at 72 ℃ and 15 cycles; 5 minutes at 72 ℃ and 4 ℃.

8) Library purification

i. Library amplification products were added to 50. mu.l of AMPure beads, vortexed, incubated at room temperature for 5 minutes, and centrifuged briefly at low speed. Placing the centrifuge tube on a magnetic frame until the solution is clear;

washing twice with 80% ethanol solution;

drying the magnetic beads at room temperature;

add 40. mu.l ddH₂O, incubation for 2 minutes at room temperature, placing the tube on a magnetic rack until the solution is clear, and pipetting 38 μ l of the supernatant solution to transfer to a new tube.

9) Library quality control

The Qubit measures the library concentration and the labchip (perkinelmer) measures the distribution of the library fragments as shown in figure 1.

10) Sequencing

Sequencing was performed using the Illumina platform HiSeq X Ten with PE 150.

11) Data analysis

After bioinformatics analysis, the CpG sites with methylation differences between benign and malignant thyroid nodules as shown in table 1 were obtained, including the chromosomes where CpG is located, the CpG initiation sites, the corresponding genes, the P values for statistical comparison, and the ratios between the methylated CpG sites of the malignant thyroid nodules and the benign thyroid nodules.

TABLE 1 566 CpG sites of benign and malignant methylation differences of thyroid nodules and the corresponding 51 genes

Chromosome	CpG initiation site	Name of Gene	P value	Malignant/benign
					chr10	81001968	ZMIZ1	1.2E-125	0.47
chr10	81001996	ZMIZ1	1.80E-13	0.35

Chromosome	CpG initiation site	Name of Gene	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

Chromosome	CpG initiation site	Name of Gene	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

Chromosome	CpG initiation site	Name of Gene	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

Chromosome	CpG initiation site	Name of Gene	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
					chr16	56669324	MT1JP	8.78E-26	0.07
chr16	56669327	MT1JP	5.7E-19	0.40
					chr16	56669344	MT1JP	5.42E-08	0.48
chr16	56669351	MT1JP	1.3E-28	0.44
					chr16	56669353	MT1JP	1.4E-50	0.33
chr16	56669402	MT1JP	1.2E-125	0.47
					chr16	56669414	MT1JP	9.8E-86	0.28
chr16	56669423	MT1JP	1.4E-50	0.33
					chr16	56669430	MT1JP	1.2E-125	0.47
chr16	56669433	MT1JP	4.8E-14	0.45
					chr16	56669437	MT1JP	9.8E-86	0.28
chr16	56669451	MT1JP	9.8E-86	0.28
					chr16	56669453	MT1JP	2.6E-37	0.49
chr16	56669455	MT1JP	1.4E-50	0.33
					chr16	56669463	MT1JP	1.3E-28	0.44
chr16	56669474	MT1JP	9.8E-86	0.28
					chr16	56669480	MT1JP	1.2E-125	0.47
chr16	56669482	MT1JP	1.2E-125	0.47
					chr16	56669485	MT1JP	1.2E-125	0.47
chr16	56669487	MT1JP	5.68E-47	0.30
					chr16	56669490	MT1JP	0.000069	0.47
chr16	56669519	MT1JP	1.77E-36	0.23
					chr16	56669533	MT1JP	1.4E-50	0.33
chr16	56669553	MT1JP	1.2E-125	0.47
					chr16	56669564	MT1JP	1.6E-87	0.24
chr16	56669573	MT1JP	9.8E-86	0.28
					chr16	56669578	MT1JP	1.4E-50	0.33
chr16	56669588	MT1JP	4.8E-14	0.45
					chr16	56669590	MT1JP	3.05E-06	0.50
chr16	56669606	MT1JP	6.7E-37	0.40
					chr16	56669610	MT1JP	5.54E-67	0.25
chr12	115174750	TBX3	1.80E-13	0.35
					chr12	115174773	TBX3	1.32E-27	0.05
chr12	115174780	TBX3	5.7E-19	0.40
					chr2	127822447	BIN1	1.2E-10	0.47
chr2	127822478	BIN1	1.1E-08	0.18
					chr2	127822492	BIN1	1.3E-06	0.43
chr2	127822495	BIN1	1.9E-09	0.27
					chr2	127822514	BIN1	5.9E-09	0.25
chr2	127822551	BIN1	1.4E-09	0.26
					chr2	127822568	BIN1	4.3E-08	0.33
chr2	127822582	BIN1	5.5E-10	0.24
					chr2	127822593	BIN1	2.5E-08	0.42
chr2	127822616	BIN1	9.8E-10	0.30
					chr2	127822644	BIN1	8.6E-07	0.37
chr17	76921845	TIMP2	9.8E-86	0.28
					chr17	76921853	TIMP2	9.8E-86	0.28
chr17	76921860	TIMP2	1.2E-125	0.47
					chr2	219866132	CFAP65	9.8E-86	0.28
chr2	219866139	CFAP65	1.4E-50	0.33
					chr2	219866148	CFAP65	1.4E-50	0.33
chr2	219866158	CFAP65	1.4E-50	0.33
					chr2	219866165	CFAP65	1.2E-125	0.47
chr2	219866168	CFAP65	0.029973178	0.39
					chr2	219866199	CFAP65	1.2E-125	0.47
chr2	219866218	CFAP65	7.40E-75	0.20

Chromosome	CpG initiation site	Name of Gene	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

Chromosome	CpG initiation site	Name of Gene	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

Chromosome	CpG initiation site	Name of Gene	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

Chromosome	CpG initiation site	Name of Gene	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

Chromosome	CpG initiation site	Name of Gene	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

Example 2 verification of differential Methylation sites by Methylation-specific PCR (MSP) and Quantitative Methylation-specific PCR (Q-MSP)

1) Sample preparation

DNA extraction of tissues or plasma of 10 thyroid cancers and 10 benign nodules of thyroid using QIAamp DNA Mini Kit (QIAGEN, cat # 51304); using qubits^TMThe concentration of the DNA was measured by the dsDNA HS Assay Kit (Thermo, cat # Q32854); quality control was performed using 1% agarose gel electrophoresis.

2) DNA transformation

Using Methycode^TMBisulfite Conversion of the DNA obtained in step 1 with the Bisulfit Conversion Kit (Thermo, cat # MECV 50), unmethylated cytosine (cytosine, C) was passed throughConversion to uracil (uracil, U); methylated cytosines are not altered after conversion.

3) Preparation of PCR mixture

The method comprises the following steps of preparing a PCR reaction solution, a primer mixture and a probe mixture, wherein a single sample is prepared as follows:

MSP reaction system composition

Components	Volume (μ l)
		Platinum^TMII Hot-Start PCR Master Mix(2×)	10.00
Water (W)	7.44
		Forward primer F of target gene, 100. mu.M	0.12
Target Gene reverse primer R, 100. mu.M	0.12
		Internal reference gene forward primer F, 100. mu.M	0.12
Internal reference gene reverse primer R, 100. mu.M	0.12
		Gene of interest Probe P, 100. mu.M (FAM/BHQ1)	0.04
Internal reference gene probe P, 100. mu.M (HEX/BHQ1)	0.04
		Sample DNA (10.0 ng)/Positive control/negative control	2.00
In all	20.00

Q-MSP reaction system composition

Note: the gradient diluted standard was a bisulfite converted 30ng 4-fold gradient diluted 6 gradient fully methylated positive standard.

4) PCR reaction

Setting PCR program as pre-denaturation at 94 deg.c for 2 min; denaturation at 94 ℃ for 30s, annealing and extension at 60 ℃ for 1min, and 45 cycles. The fluorescence signal was collected during the 60 ℃ annealing extension phase.

5) Analysis of detection results

ROC curve analysis of methylation levels for each gene as shown in FIGS. 2A-C, the AUC for each gene was greater than 0.6.

Example 3 multiplex Pre-amplification methylation-specific PCR (preaMP-MSP) for discrimination of benign and malignant thyroid nodules

1) Sample preparation

cfDNA extraction of plasma from 20 cases of thyroid cancer and 20 cases of benign thyroid nodules was performed using the QIAamp Circulating Nucleic Acid Kit (QIAGEN, cat # 55114); using qubits^TM dsDNA HS Assay Kit(Thermo, cargo number: q32854) detecting the concentration of cfDNA; quality testing was performed using LabChip 3K Assay.

2) DNA transformation

Using Methycode^TMBisulfite Conversion of cfDNA from step 1 by Bisulfit Conversion Kit (Thermo, cat # MECOV50), unmethylated cytosine (cytosine, C) being converted to uracil (uracil, U); methylated cytosines are not altered after conversion.

3) Pre-amplification PCR reaction

The pre-amplification PCR mixture comprises PCR reaction liquid and primer mixture. The primer mixture contained a pair of primers each for COL23A1(SEQ ID NOS: 4 and 5), ILDR2(SEQ ID NOS: 6 and 7), DHRS3(SEQ ID NOS: 8 and 9), GDNF (SEQ ID NOS: 12 and 13), TBX18(SEQ ID NOS: 14 and 15), and the reference genes (SEQ ID NOS: 1 and 2).

Pre-amplification PCR reaction system composition

Setting PCR program as pre-denaturation at 94 deg.c for 2 min; denaturation at 94 ℃ for 15s, annealing at 60 ℃ for 45s, extension at 68 ℃ for 1min, and 15 cycles; extending for 1min at 72 ℃, and storing at 4 ℃.

4) MSP reaction

The reaction system, which contains Platinum, was prepared according to the manufacturer's kit instructions^TMII Hot-Start PCR Master Mix (2X), water, primer Mix (same above), probe Mix (probe sequence 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)), 1:100 diluted pre-amplified PCR product. Wherein, the COL23A1 gene comprises positions 178003798, 178003803, 178003814, 178003823, 178003825, 178003834, 178003841 and 178003844, and the ILDR2 gene comprises positions 166890516, 166890528, 166890535, 166890543, 166890555 and 166890559. 166890568, 166890573, 166890584, 166890586, DHRS3 gene includes positions 166890516, 166890528, 166890535, 166890543, 166890555, 166890559, 166890568, 166890573, 166890584, 166890586, GDNF gene includes positions 37834770, 37834772, 37834774, 37834777, 37834780, 37834784, 37834792, 37834799, 37834802, 37834806, 37834811, TBX18 gene includes positions 85477035, 85477070, 85477083, 85477106. Setting PCR program as pre-denaturation at 94 deg.c for 2 min; denaturation at 94 ℃ for 30s, annealing and extension at 60 ℃ for 1min, and 45 cycles. The fluorescence signal was collected during the 60 ℃ annealing extension phase.

5) Analysis of detection results

Methylation level (methylation level) of 2^{-delta Ct sample to be detected}/2^{- Δ Ct positive standard} X 100. Wherein Δ Ct ═ Ct_{Target gene}–Ct_{Internal reference gene}。

Performing binary Logistic regression analysis on methylation levels of the COL23A1 gene, the ILDR2 gene, the DHRS3 gene, the GDNF gene and the TBX18 gene, wherein a fitting equation is a Score (Score) of-2.53 +1.78 multiplied by the methylation level of the COL23A1 +1.64 multiplied by the ILDR2 methylation level +7.28 multiplied by the DHRS3 methylation level-2.47 multiplied by the GDNF methylation level-1.35 multiplied by the TBX18 methylation level, and judging that the results are positive if the scores of the detected COL23A1, ILDR2, DHRS3, GDNF and TBX18 gene are more than 0, namely judging that the results are malignant nodules.

The scores are shown in Table 2 and the ROC analysis is shown in FIG. 3. According to the interpretation standard, 5 cases of 20 cases of thyroid benign nodules are positive, 13 cases of 20 cases of thyroid cancer are positive, the specificity reaches 80.0%, and the sensitivity is 65.0%.

TABLE 2

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

Example 4 multiplex Pre-amplification methylation-specific PCR (preaMP-MSP) for discrimination of benign and malignant thyroid nodules

Steps 1) -4) As in example 3, except that, in step 3), the primer mixture contained a pair of primers each for COL23A1(SEQ ID NOS: 4 and 5), ILDR2(SEQ ID NOS: 6 and 7), DHRS3(SEQ ID NOS: 8 and 9), KIF1A (SEQ ID NOS: 10 and 11), and the internal reference genes (SEQ ID NOS: 1 and 2). The probes of the respective genes 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 locus comprises 178003798, 178003803, 178003814, 178003823, 178003825, 178003834, 178003841 and 178003844, the KIF1A gene locus comprises 241759696, 241759701, 241759714 and 241759716, the ILDR2 gene locus comprises 166890516, 166890528, 166890535, 166890543, 166890555, 166890559, 166890568, 166890573, 166890584 and 166890586, and the DHRS3 gene locus comprises 12656170, 12656175, 12656182, 12656197, 12656200, 12656211, 12656315 and 12656323.

5) Analysis of detection results

Performing binary Logistic regression analysis on the methylation levels of the COL23A1 gene, the KIF1A gene, the ILDR2 gene and the DHRS3 gene, wherein the fitting equation is that the Score (Score) is-3.37 +2.03 multiplied by the COLA23A1 methylation level +15.86 multiplied by the KIF1A methylation level +1.32 multiplied by the ILDR2 methylation level +5.74 multiplied by the DHRS3 methylation level, and the judgment method is that the scores of the COL23A1, the KIF1A, the ILDR2 and the DHRS3 gene are detected to be more than 0, and judging that the result is positive, namely, the malignant nodule.

The scores are shown in Table 3 and the ROC analysis is shown in FIG. 4. According to the interpretation standard, 7 cases of 20 cases of thyroid benign nodules are positive, 12 cases of 20 cases of thyroid cancer are positive, the specificity reaches 90.0%, and the sensitivity is 60.0%.

TABLE 3

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

The above-mentioned embodiments only express the embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

<110> Shanghai Kun Yuanzhi Co., Ltd

<120> reagent for detecting DNA methylation and use thereof

<130> 20A600

<150> 202010038550.8

<151> 2020-01-14

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Claims

1. An isolated mammalian-derived nucleic acid molecule having a nucleic acid sequence selected from the group consisting of (1) and (2) below, or a variant thereof having at least 70% identity thereto:

(1) a nucleic acid fragment of one or more genes selected from the group consisting of: COL23A1, ILDR2, DHRS3, KIF1A, GDNF, TBX18, said fragments being 50-1000bp in length, wherein,

wherein the above-mentioned site in the variant is not mutated;

preferably, the fragment of COL23a1 gene comprises one or more of positions 178003798, 178003803, 178003814, 178003823, 178003825, 178003834, 178003841, 178003844 of COL23a1 gene, the fragment of ILDR2 gene comprises one or more of positions 166890516, 166890528, 166890535, 166890543, 166890555, 166890559, 166890568, 166890573, 166890584, 166890586 of ILDR2 gene, the fragment of DHRS3 gene comprises positions of DHRS3 gene: 12656340, 12656355, 12656367, a fragment of the KIF1A gene comprising the site of the KIF1A gene: 241759696, 241759701, 241759714, 241759716, the segment of the GDNF gene comprising the site of the GDNF gene: 37834770, 37834772, 37834774, 37834777, 37834780, 37834784, 37834792, 37834799, 37834802, 37834806, 37834811, a fragment of the TBX18 gene comprises the site of the TBX18 gene: 85477035, 85477070, 85477083, 85477106,

more preferably, the nucleic acid molecule comprises one or more fragments selected from the group consisting of:

the fragment of COL23A1 gene amplified by using SEQ ID NO. 4 and 5 as primers,

the fragment of ILDR2 gene amplified by using SEQ ID NOS.6 and 7 as primers,

the fragment of DHRS3 gene amplified by using SEQ ID NO 8 and 9 as primers,

the fragment of KIF1A gene amplified by using SEQ ID NO. 10 and 11 as primers,

fragments of the GDNF gene amplified by using SEQ ID NO 12 and 13 as primers,

fragments of the TBX18 gene amplified by SEQ ID NO. 14 and 15 as primers.

2. A reagent for detecting the level of DNA methylation, said reagent:

(1) detecting the DNA methylation level of the sites of one or more groups of (a1) - (a6) below:

(a1) site of COL23a1 gene: 178003785, 178003798, 178003803, 178003814, 178003823, 178003825, 178003834, 178003841, 178003844,

(a2) site of ILDR2 gene: 166890429, 166890436, 166890440, 166890442, 166890448, 166890452, 166890456, 166890461, 166890468, 166890473, 166890475, 166890480, 166890492, 166890500, 166890503, 166890509, 166890516, 166890528, 166890535, 166890543, 166890555, 166890559, 166890568, 166890573, 166890584, 166890586,

(a3) site of DHRS3 gene: 12656091, 12656114, 12656132, 12656152, 12656170, 12656175, 12656182, 12656187, 12656197, 12656200, 12656211, 12656315, 12656323, 12656340, 12656355, 12656367,

(a4) site of KIF1A gene: 241759696, 241759701, 241759714, 241759716,

(a5) site of GDNF Gene: 37834763, 37834770, 37834772, 37834774, 37834777, 37834780, 37834784, 37834792, 37834799, 37834802, 37834806, 37834811,

(a6) site of TBX18 gene: 85477032, 85477035, 85477070, 85477083, 85477106, 85477124, 85477151, 85477153, 85477166, or

(2) Detecting the DNA methylation level of a nucleic acid fragment of one or more genes selected from the group consisting of 50-1000bp long: COL23A1, ILDR2, DHRS3, KIF1A, GDNF, TBX18, wherein,

a fragment of the TBX18 gene contains the sites of the TBX18 gene: 85477032, 85477035, 85477070, 85477083, 85477106, 85477124, 85477151, 85477153, 85477166, or

(3) Detecting the DNA methylation level of the nucleic acid region within 10Kb of the upstream and downstream of the gene in (2).

3. The reagent of claim 2, wherein the site comprises:

(a1) one or more of (a3), and optionally one or more of (a4) - (a6),

preferably, the site comprises:

(a1) - (a3) and optionally (a4) or

(a1) One or more of (a3) and optionally (a5) - (a 6);

more preferably, the site comprises:

(a1) - (a3) and optionally (a4) or optionally (a5) - (a 6).

4. The reagent of claim 2, wherein the reagent is a primer that can amplify one or more fragments selected from the group consisting of:

(b1) the fragment of COL23A1 gene amplified by using SEQ ID NO. 4 and 5 as primers,

(b2) the fragment of ILDR2 gene amplified by using SEQ ID NOS.6 and 7 as primers,

(b3) the fragment of DHRS3 gene amplified by using SEQ ID NO 8 and 9 as primers,

(b4) the fragment of KIF1A gene amplified by using SEQ ID NO. 10 and 11 as primers,

(b5) fragments of the GDNF Gene amplified by SEQ ID NOS 12 and 13 as primers, and

(b6) the fragment of TBX18 gene amplified by using SEQ ID NO. 14 and 15 as primers,

preferably, the primers are capable of amplifying:

(b1) one or more of (b3), and optionally one or more of (b4) - (b6),

more preferably, the primers are capable of amplifying:

(b1) - (b3) and optionally (b4) or

(b1) One or more of (b3) and optionally (b5) - (b 6);

more preferably, the primers are capable of amplifying:

(b1) - (b3) and optionally (b4) or optionally (b5) - (b 6).

5. The reagent of claim 2, wherein the reagent is a probe that hybridizes to one or more fragments selected from the group consisting of:

preferably, the probe is capable of hybridizing to:

(b1) one or more of (b3), and optionally one or more of (b4) - (b6),

more preferably, the primers hybridize to the following fragments:

(b1) - (b3) and optionally (b4) or

(b1) One or more of (b3) and optionally (b5) - (b 6);

more preferably, the primers hybridize to the following fragments:

(b1) - (b3) and optionally (b4) or optionally (b5) - (b 6).

6. The agent according to any one of claims 2 to 5, wherein the agent further has one or more characteristics selected from the group consisting of:

the fragment includes a sense strand or an antisense strand of DNA,

reagents for detecting DNA methylation also include reagents used in one or more of the following methods: PCR based on bisulfite conversion, DNA sequencing, methylation sensitive restriction enzyme analysis, fluorescence quantification, methylation sensitive high resolution melting curve method, chip-based methylation map analysis, mass spectrometry,

preferably, the reagent for detecting DNA methylation further comprises a reagent selected from one or more of: bisulfite and its derivatives, PCR buffer, polymerase, dNTP, methylation sensitive or insensitive restriction enzyme, enzyme digestion buffer, fluorescent dye, fluorescence quencher, fluorescence reporter, exonuclease, alkaline phosphatase, internal standard, and reference substance.

7. A kit for identifying the nature of thyroid nodules comprising the reagent of any one of claims 2-7 and optionally the nucleic acid molecule of claim 1.

8. The kit of claim 7, further comprising a reagent for detecting the level of mutation at the V600E site of the BRAF gene and/or the level of mutation at the C228T/C250T site of the TERT gene.

9. Use of an agent for detecting DNA methylation, which agent detects the level of methylation of one or more genes selected from the group consisting of: COL23A1, ILDR2, DHRS3, KIF1A, GDNF, TBX18,

preferably, the reagent detects the methylation level of one or more of the following (a1) - (a6) groups of sites:

(a4) site of KIF1A gene: 241759696, 241759701, 241759714, 241759716,

(a6) site of TBX18 gene: 85477032, 85477035, 85477070, 85477083, 85477106, 85477124, 85477151, 85477153, 85477166.

10. Use according to claim 9, characterized in that it has one or more characteristics selected from:

the sample contains genomic DNA and/or cfDNA.