CN117721203A - Composition for detecting thyroid cancer and application thereof - Google Patents

Abstract

The present application provides a composition for detecting thyroid cancer and uses thereof, the composition comprising: a nucleic acid for detecting methylation status of a target gene, wherein the target gene is one, two or three of MIR578 gene, FOXN3 gene and SPTBN1 gene. The application also provides a kit comprising the composition and application of the composition in preparing a kit for detecting thyroid cancer in vitro.

Description

Composition for detecting thyroid cancer and application thereof

Technical Field

The application belongs to the field of molecular biology, relates to gene detection, and in particular relates to a composition for detecting thyroid cancer and application thereof.

Background

Based on the national cancer center data, the new incidence of 2016 years thyroid cancer (thyroid carcinoma) is 20.3 tens of thousands and the death cases are 0.8 tens of thousands. Thyroid cancer prognosis is better, mortality is lower, but morbidity continues to increase.

Thyroid cancer includes four pathological types of papillary, follicular, undifferentiated and medullary cancers. Papillary cancers with low malignancy and better prognosis are most common, with the vast majority of thyroid cancers originating from follicular epithelial cells, except medullary cancers. The incidence rate has a certain relationship with the region, sex and the like. The female morbidity is more, and the ratio of the male to the female is 1:2-4. Onset can occur at any age, but is seen in young and strong years. Most thyroid cancers occur in the lateral thyroid gland lobes, often as a single tumor.

At present, early diagnosis of thyroid cancer mainly depends on traditional modes such as finding a nodule by an imaging means, further puncture examination of the nodule and the like, and has low examination efficiency and low detection sensitivity. For example, cervical ultrasound, while sensitive, has the ability to identify thyroid nodules is related to the clinical experience of the sonographer. CT is poorly observed for patients with nodules less than or equal to 5mm in maximum diameter and nodules combined with diffuse lesions. MRI is insensitive to calcification, and has long examination time and is easily affected by respiratory and swallowing actions. Therefore, the early thyroid cancer has small size, is not easy to find by the traditional method, has low detection efficiency at present, and is not suitable for large-scale popularization and screening.

Recent studies have shown that epigenetics play an important role in the development and progression of cancer. As an important mechanism of epigenetic science, DNA methylation regulation of various cancers has been intensively studied. Study data showed that: regulation of gene methylation is related to biological mechanisms such as chromatin structure and gene expression regulation; changes in cellular gene methylation occur early in tumor formation and are throughout the course of cancer development and progression; methylation of cancer suppressor genes is an important molecular mechanism for the transformation of precancerous lesion tissues into malignant tumor cells. But currently there is a lack of detection techniques, methods and products for the detection of thyroid cancer methylation genes. Thus, there is a current need for methylation gene markers with high sensitivity and specificity for thyroid cancer detection.

Disclosure of Invention

Based on the problems existing in the existing thyroid cancer detection, the purpose of the application is to provide a composition for detecting thyroid cancer and application thereof.

The specific technical scheme of the application is as follows:

1. a composition for detecting thyroid cancer in vitro, the composition comprising:

nucleic acid for detecting methylation status of a target gene,

wherein the methylation state of the target gene is characterized by methylation of a target sequence of the target gene,

wherein the target gene is one, two or three of MIR578 gene, FOXN3 gene and SPTBN1 gene.

2. The composition of item 1, wherein the MIR578 gene has a target sequence set forth in SEQ ID NO:1-4, or comprises SEQ ID NO: 1-4.

3. The composition of item 1, wherein the FOXN3 gene has a target sequence set forth in SEQ ID NO:5-8, or comprises SEQ ID NO: 5-8.

4. The composition of item 1, wherein the target sequence of the SPTBN1 gene is as set forth in SEQ ID NO:9-12, or comprises any one of SEQ ID NOs: 9-12.

5. The composition according to any one of items 1 to 4, wherein the nucleic acid for detecting methylation status of a target gene comprises:

A primer that is a fragment of at least 9 nucleotides in a target sequence of the target gene, the fragment comprising at least one CpG dinucleotide sequence.

6. The composition according to any one of items 1 to 5, wherein the nucleic acid for detecting methylation status of a target gene comprises:

a probe which is a fragment of at least 15 nucleotides hybridised to the target sequence of the target gene under moderately stringent or stringent conditions,

the fragment comprises at least one CpG dinucleotide sequence.

7. The composition according to any one of items 1 to 6, further comprising:

an agent that converts an unmethylated cytosine base at position 5 of a target sequence of a target gene into uracil.

8. The composition according to any one of items 1 to 7, wherein the nucleic acid for detecting a methylation state of a target gene further comprises:

blocking agents that preferentially bind to target sequences in the unmethylated state.

9. The composition according to item 8, wherein,

the at least 9 nucleotide fragment, which is SEQ ID NO:13 and SEQ ID NO:14, or it is SEQ ID NO:16 and SEQ ID NO:17, or it is SEQ ID NO:19 and SEQ ID NO: 20;

The at least 15 nucleotide fragment, which is SEQ ID NO:15, or SEQ ID NO:18, or SEQ ID NO: 21.

10. An oligonucleotide for detecting thyroid cancer in vitro, comprising:

the SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 or a fragment of at least 9 nucleotides in its complement comprising at least one CpG dinucleotide sequence; and/or

The SEQ ID NO:5 or SEQ ID NO:6 or SEQ ID NO:7 or SEQ ID NO:8 or a fragment of at least 9 nucleotides in its complement comprising at least one CpG dinucleotide sequence; and/or

The SEQ ID NO:9 or SEQ ID NO:10 or SEQ ID NO:11 or SEQ ID NO:12 or a complementary sequence thereof and comprises at least one CpG dinucleotide sequence.

11. The oligonucleotide of item 10, further comprising:

hybridization to the SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 or a fragment of at least 15 nucleotides in its complement comprising at least one CpG dinucleotide sequence; and/or

Hybridization to the SEQ ID NO:5 or SEQ ID NO:6 or SEQ ID NO:7 or SEQ ID NO:8 or a fragment of at least 15 nucleotides in its complement comprising at least one CpG dinucleotide sequence; and/or

Hybridization to the SEQ ID NO:9 or SEQ ID NO:10 or SEQ ID NO:11 or SEQ ID NO:12 or a complementary sequence thereof and comprises at least one CpG dinucleotide sequence.

12. The oligonucleotide of item 11, further comprising:

blocking agents that preferentially bind to target sequences in the unmethylated state.

13. An oligonucleotide for detecting thyroid cancer in vitro, comprising:

SEQ ID NO:13 and SEQ ID NO: 14.

14. The oligonucleotide of item 13, further comprising:

SEQ ID NO: 15.

15. An oligonucleotide for detecting thyroid cancer in vitro, comprising:

SEQ ID NO:16 and SEQ ID NO: 17.

16. The oligonucleotide of item 15, further comprising:

SEQ ID NO: 18.

17. An oligonucleotide for detecting thyroid cancer in vitro, comprising:

SEQ ID NO:19 and SEQ ID NO: 20.

18. The oligonucleotide of item 17, further comprising:

SEQ ID NO: 21.

19. A kit comprising the composition of any one of claims 1-9 or comprising the oligonucleotide of any one of claims 10-18.

20. The kit of item 19, further comprising at least one additional component selected from the group consisting of:

nucleoside triphosphates, DNA polymerase and buffers required for the function of the DNA polymerase.

21. The kit of claim 19 or 20, wherein the sample for detection of the kit comprises: cell lines, histological sections, tissue biopsies/paraffin-embedded tissues, body fluids, faeces, colonic exudates, urine, plasma, serum, whole blood, isolated blood cells, cells isolated from blood, or combinations thereof.

22. The kit of any one of claims 19-21, further comprising: and (3) a specification.

23. Use of the composition of any one of claims 1 to 9 or the oligonucleotide of any one of claims 10 to 18 in the preparation of a kit for detecting thyroid cancer in vitro.

Use of the mir578 gene, FOXN3 gene or SPTBN1 gene for the preparation of a kit for the in vitro detection of thyroid cancer.

25. The use according to item 24, wherein the target sequence of the MIR578 gene is set forth in SEQ ID NO:1-4, or comprises SEQ ID NO: 1-4.

26. The use of item 24, wherein the FOXN3 gene has a target sequence as set forth in SEQ ID NO:5-8, or comprises SEQ ID NO: 5-8.

27. The use according to item 24, wherein the target sequence of the SPTBN1 gene is as set forth in SEQ ID NO:9-12, or comprises any one of SEQ ID NOs: 9-12.

ADVANTAGEOUS EFFECTS OF INVENTION

The application has the following beneficial effects:

by detecting the target sequences of three thyroid cancer methylation genes, namely MIR578 gene, FOXN3 gene and/or SPTBN1 gene, the methylation states of the 3 genes can be sensitively and specifically detected, so that the detection of peripheral blood episomal DNA can be realized.

The detection of peripheral blood samples of thyroid cancer patients and normal control individuals shows that: the system and the method can sensitively and specifically detect thyroid cancer, thereby ensuring the correctness and the reliability of detection results. Therefore, the system and the method for detecting thyroid cancer have important clinical application value. The method utilizes the epigenomic and bioinformatic technologies to find a plurality of methylation genes related to thyroid cancer by analyzing genome methylation data of the thyroid cancer, determines a target sequence of methylation abnormality of the methylation genes of the thyroid cancer, and can sensitively and specifically detect the methylation state of the genes through the target sequence of the methylation genes, so that the method can be used for detecting free DNA of peripheral blood.

The composition is used for screening asymptomatic people in a non-invasive mode, harm caused by invasive detection is reduced, and the composition has higher sensitivity and accuracy and can realize real-time monitoring.

Detailed Description

The present application is described in detail below. While specific embodiments of the present application are shown, it should be understood that the present application may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.

Unless otherwise indicated, practice of the present application will employ conventional molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and genetics techniques, which are within the skill of the art. Such techniques are described in detail in the literature as Molecular Cloning: ALaboratory Manual, second edition (Sambrook et al, 1989); oligonucleotide Synthesis (M.J.Gait, 1984); animal Cell Culture (R.I. Freshney, 1987); methods in Enzymology Cluster books (American academic Press Co., ltd.); current Protocols in Molecular Biology (F.M. Ausubel et al, 1987 edition, and periodic updates); and (2) PCR: the Polymerase Chain Reaction (Mullis et al, 1994 edition). Primers, probes, blockers and kits useful in the present application can be prepared using standard techniques well known in the art.

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

Definition of the definition

"precancerous" in this application means a cell that is at an early stage of, or is prone to be transformed into, a cancer cell. Such cells may exhibit one or more phenotypic traits characteristic of cancer cells.

"stringent hybridization conditions" and "high stringency" in this application refer to conditions under which a probe hybridizes to its target sequence, typically in a complex mixture of nucleic acids. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. For detailed guidance on nucleic acid hybridization, reference may be made to Tijssen, biochemical and molecular biological techniques-nucleic acid probe hybridization, "review of hybridization principles and nucleic acid assay strategies. Typically, stringent conditions are those about 5-10deg.C below the melting point (Tm) for a specific nucleic acid at a defined ionic strength pH. At Tm temperatures (at defined ionic strength, pH and nucleic acid concentration), 50% of the probes complementary to the target hybridize uniformly to the target sequence. Stringent conditions can also be achieved with the addition of destabilizing agents. For selective or specific hybridization, the positive signal is twice, preferably 10 times, that of the background hybridization. Exemplary stringent hybridization conditions are as follows: hybridization was performed at 42℃in a solution of 50% formamide, 5 XSSC and 1% SDS, or at 65℃in a solution of 5 XSSC and 1% SDS, followed by washing at 65℃in a solution of 0.2XSSC and 0.1% SDS.

Also, if the polypeptides encoded by the nucleic acids are substantially similar, the nucleic acids are substantially similar even if they are not capable of hybridizing under stringent conditions. In this case, the nucleic acid is typically hybridized under moderately stringent hybridization conditions. As an example, "moderately stringent hybridization conditions" include hybridization in a solution of 40% formamide, 1M sodium chloride and 1% SDS at 37 ℃ and washing in a solution of 1xSSC at 45 ℃. It will be apparent to one of ordinary skill in the art that guidance in achieving the conditions to achieve the same stringency is available in the prior art. For PCR, temperatures around 36℃are typically suitable for low stringency amplification, while annealing temperatures range between 32℃and 48℃based on the length of the primer. For highly stringent PCR amplification, it is typically at 62℃and the annealing temperature for highly stringent hybridization ranges between 50℃and 65℃based on the length and specificity of the primers. For cycling conditions of high stringency and low stringency amplification, typically, include: the denaturation phase is continued for 30 seconds to 2 minutes at 90-95 ℃, the annealing phase is continued for 30 seconds to 2 minutes, and the extension phase is continued for 1 to 2 minutes at about 72 ℃. Tools and guidelines for low and high stringency amplification reactions are available in the prior art.

"oligonucleotide" in this application refers to a molecule consisting of two or more nucleotides, preferably three or more nucleotides, the exact size of which may depend on a number of factors, which in turn are determined by the ultimate function and use of the oligonucleotide. In certain embodiments, the oligonucleotide may comprise a length of 10 nucleotides to 100 nucleotides. In certain embodiments, the oligonucleotides may comprise a length of 10 nucleotides to 30 nucleotides, or may have lengths of 20 and 25 nucleotides. In some particular embodiments, oligonucleotides shorter than these lengths are also suitable.

"primer" in this application means an oligonucleotide capable of acting as a point of initiation of synthesis, whether it is naturally occurring in a purified restriction digest or synthetically produced, when placed under conditions that induce synthesis of a primer extension product complementary to a nucleic acid strand, i.e., in the presence of a nucleotide and an inducer such as a DNA or RNA polymerase, and at a suitable temperature and pH. The primer may be single-stranded or double-stranded and must be long enough to prime the synthesis of the desired extension product in the presence of the primer. The exact length of the primer depends on a variety of factors, including temperature, primer source and method used. For example, for diagnostic and prognostic applications, an oligonucleotide primer will typically contain at least or more than about 9, 10, or 15, or 20, or 25 or more nucleotides, depending on the complexity of the target sequence, but it may contain fewer nucleotides or more nucleotides. Factors involved in determining the appropriate length of the primer are well known to those skilled in the art.

"primer pair" in this application means a primer pair that hybridizes to the opposite strand of a target DNA molecule or to a region of the target DNA flanked by nucleotide sequences to be amplified.

"primer site" in this application refers to a region of target DNA or other nucleic acid to which a primer hybridizes.

The term "probe" as used herein, when referring to a nucleic acid sequence, is used in its ordinary sense to refer to a selected nucleic acid sequence that hybridizes to a target sequence under defined conditions and can be used to detect the presence of the target sequence. Those skilled in the art will appreciate that in some cases, probes may also be used as primers, and primers may be used as probes.

"DNA methylation" in this application refers to the addition of a methyl group to the 5-position of cytosine (C), which is typically (but not necessarily) the case with CpG (cytosine followed by guanine) dinucleotides. As used herein, "increased degree of methylation" or "substantial degree of methylation" refers to the presence of at least one methylated cytosine nucleotide in a DNA sequence, wherein the corresponding C in a normal control sample (e.g., a DNA sample extracted from a non-cancerous cell or tissue sample or a DNA sample treated for methylation of DNA residues) is unmethylated, and in certain embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more C can be methylated, wherein the C at these positions in the control DNA sample is unmethylated.

In embodiments, a variety of different methods may be used to detect DNA methylation changes. Methods for detecting DNA methylation include, for example, methylation-sensitive restriction endonuclease (MSRE) assays using southern or Polymerase Chain Reaction (PCR) assays, methylation-specific or methylation-sensitive PCR (MS-PCR), methylation-sensitive single nucleotide primer extension (MS-SnuPE), high Resolution Melting (HRM) assays, bisulfite sequencing, pyrosequencing, methylation-specific single strand conformation assays (MS-SSCA), combinatorial bisulfite restriction assays (COBRA), methylation-specific gradient gel electrophoresis (MS-DGGE), methylation-specific melting curve assays (MS-MCA), methylation-specific high performance liquid chromatography (MS-DHPLC), methylation-specific Microarray (MSO). These assays may be PCR assays, quantitative assays using fluorescent markers or southern blot assays.

"methylation determination" in this application refers to any determination that determines the methylation state of one or more CpG dinucleotide sequences within a DNA sequence.

"detecting" in this application refers to any process of observing a marker or a change in a marker (e.g., a change in the methylation state of a marker or the expression level of a nucleic acid or protein sequence) in a sample, whether or not the marker or the change in the marker is actually detected. In other words, the act of detecting the marker or a change in the marker of the sample is "detecting" even if the marker is determined to be absent or below the sensitivity level. The detection may be quantitative, semi-quantitative, or non-quantitative observation, and may be based on comparison to one or more control samples. It is to be understood that detecting thyroid cancer as disclosed herein includes detecting pre-cancerous cells that begin to develop into, or are about to develop into, thyroid cancer cells, or have an increased propensity to develop into thyroid cancer cells. Detecting thyroid cancer may also include detecting a possible probability of death or a possible prognosis of a disease condition.

"homology", "identity" and "similarity" in this application refer to sequence similarity between 2 nucleic acid molecules. The positions in each sequence can be compared to determine "homology", "identity" or "similarity", and the sequences can be aligned for comparison purposes. When an equivalent position in the compared sequences is occupied by the same base, the molecules are identical at that position; when an equivalent site is occupied by the same or a similar amino acid (e.g., similar in steric or charged properties) residue, the molecule may be said to be homologous (similar) at that position. Expression of homology/similarity or percent identity refers to a function of the number of identical or similar amino acids at positions shared by the compared sequences. "unrelated" or "non-homologous" sequences share less than 40% identity, preferably less than 25% identity, with the sequences of the present application. The absence of residues (amino acids or nucleic acids) or the presence of redundant residues also reduces identity and homology/similarity when comparing 2 sequences. In particular embodiments, two or more sequences or subsequences are considered substantially or significantly homologous, similar or identical if their sequences are about 60% identical, or about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical over a defined region when compared and aligned for maximum correspondence over a comparison window or defined region, as determined using BLAST or BLAST 2.0 sequence comparison algorithms having default parameters described below, or as determined by manual alignment and visual inspection provided on-line by, for example, the national center for biotechnology information (National Center for Biotechnology Information (NCBI)). The definition also relates to or can be used to test the complement of a sequence. Thus, for example, if a nucleotide sequence can be predicted to occur naturally in a DNA duplex, or can occur naturally in the form of one or both of the complementary strands, the nucleotide sequence that is complementary to a specified target sequence or variant thereof is itself considered "similar" to the target sequence, and when reference is made to a "similar" nucleic acid sequence, includes single-stranded sequences, their complementary sequences, double-stranded strand complexes, sequences capable of encoding the same or similar polypeptide products, and any permissible variants of any of the foregoing, to the extent permitted by the context herein. The circumstances in which similarity must be limited to analysis of a single nucleic acid strand sequence may include, for example, detection and quantification of expression of a particular RNA sequence or coding sequence in a cell. The definition also includes sequences with deletions and/or additions, as well as sequences with substitutions. In embodiments, identity or similarity may be over a region of at least about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 10, 21, 22, 23, 24, 25 or more nucleotides in length, or over a region of more than about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or more than about 100 nucleotides in length.

"amplification" in this application refers to the process of obtaining multiple copies from a particular locus of a nucleic acid, such as genomic DNA or cDNA. Amplification may be accomplished using any of a variety of known means including, but not limited to, polymerase Chain Reaction (PCR), transcription-based amplification, and Strand Displacement Amplification (SDA).

The "fluorescence-based real-time PCR" or "real-time fluorescence quantitative PCR" of the present application means such a method: and adding a fluorescent group into the PCR reaction system, monitoring the whole PCR process in real time by utilizing fluorescent signal accumulation, and finally quantitatively analyzing the unknown template through a standard curve. In this PCR technique, there is a very important concept, the cycle threshold, also called Ct value. C represents Cycle, t represents threshold, and Ct has the meaning of: the number of cycles that the fluorescent signal within each reaction tube experiences when reaching a set threshold. For example, the fluorescence threshold (threshold) is set as follows: the fluorescent signal of the first 15 cycles of the PCR reaction served as the fluorescent background signal, and the default (default) setting of the fluorescent threshold was 10 times the standard deviation of the fluorescent signal of 3-15 cycles.

The "cut off value of real-time PCR" in the present application means a critical Ct value for a biomarker that determines whether a sample is negative or positive. According to some specific real-time aspects of the present application, the "critical Ct value (Cut Off value) is derived based on statistical processing from a certain number of sample data, and may be different depending on the desired sensitivity or specificity requirements.

The "sensitivity" of the present application means the proportion of cancers detected from a certain cancer sample, and the calculation formula is: sensitivity= (detected cancer/all cancers), whereas "specificity" indicates the normal proportion detected in a certain normal human sample, the formula is specificity= (detected negative/total negative).

A "label" or "detectable moiety" in this application is a component that is detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical or other physical means. For example, useful labels include 32P, fluorescent dyes, electron dense reagents, enzymes (e.g., enzymes commonly used in ELISA), biotin, digoxin, or haptens, and proteins that can be prepared to be detectable, e.g., by incorporating a radiolabel into the peptide or for detecting antibodies that specifically react with the peptide.

Nucleic acid molecules can be detected using a variety of different methods. Nucleic acid detection methods include, for example, PCR and nucleic acid hybridization (e.g., southern blot, northern blot, or in situ hybridization). In particular, oligonucleotides (e.g., oligonucleotide primers) capable of amplifying a target nucleic acid may be used in a PCR reaction. The PCR method generally comprises the steps of: obtaining a sample, isolating nucleic acids (e.g., DNA, RNA, or both) from the sample, and contacting the nucleic acids with one or more oligonucleotide primers that specifically hybridize to a template nucleic acid under conditions that enable amplification of the template nucleic acid to occur. In the presence of a template nucleic acid, amplification products are produced. Conditions for nucleic acid amplification and detection of amplification products are known to those skilled in the art. Various improvements to the basic PCR technique have been developed, including, but not limited to, anchored PCR, RACE PCR, RT-PCR, and Ligase Chain Reaction (LCR). The primer pairs in the amplification reaction must anneal to the opposite strand of the template nucleic acid and should be kept at a suitable distance from each other so that the polymerase can efficiently polymerize across the region and so that the amplified product can be easily detected, for example, using electrophoresis. For example, oligonucleotide primers may be designed using a computer program such as OLIGO (Molecular Biology Insights inc., cascades, colo.) to aid in designing primers with similar melting temperatures. Typically, the oligonucleotide primer is 9-30 or 40 or 50 nucleotides in length (e.g., 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides in length), although the oligonucleotide primer may be longer or shorter as long as appropriate amplification conditions are used.

Detection of the amplification product or hybridization complex is typically accomplished using a detectable label. The term "label", when referring to a nucleic acid, is intended to include direct labeling of the nucleic acid by coupling (i.e., physically linking) a detectable substance to the nucleic acid, as well as indirect labeling of the nucleic acid by reaction with another reagent that directly labels the detectable substance. Detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic groups include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; examples of luminescent materials include luminol; examples of bioluminescent materials include luciferase, luciferin and aequorin. Examples of indirect labeling include end-labeling a nucleic acid with biotin such that the nucleic acid can be detected with fluorescently labeled streptavidin.

In one aspect, the present application provides a composition for detecting thyroid cancer in vitro, the composition comprising a nucleic acid for detecting methylation status within a target sequence of a target gene, wherein the target gene methylation status is characterized by methylation of the target sequence of the target gene, wherein the target gene is one, two, or three of the MIR578 gene, FOXN3 gene, and SPTBN1 gene.

The present application provides a set of target sequences for a target gene that emits aberrant methylation in thyroid cancer, including the target sequences for the MIR578 gene, FOXN3 gene, and SPTBN 1. Wherein, the target sequence of the MIR578 gene is shown as SEQ ID NO:1-4, or comprises the amino acid sequence as set forth in SEQ ID NO: 1-4. The target sequence of the FOXN3 gene is shown as SEQ ID NO:5-8, or comprises the amino acid sequence set forth in SEQ ID NO: 5-8. The target sequence of the SPTBN1 gene is shown as SEQ ID NO:9-12, or comprises the amino acid sequence set forth in any one of SEQ ID NOs: 9-12.

It will also be appreciated by those skilled in the art that the target sequences of the MIR578 gene, the FOXN3 gene and the SPTBN1 are not limited to the specific sequences listed above. The target sequence of the MIR578 gene should encompass the sequence corresponding to SEQ ID NO:1-4, comprising one or two or more nucleotide mutations, but substantially still functionally identical thereto, as well as sequences identical to those of SEQ ID NOs: 1-4, a sequence having 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in any one of claims 1-4. The target sequence of the FOXN3 gene should cover a sequence identical to SEQ ID NO:5-8, comprising one or two or more nucleotide mutations, but substantially still functionally identical thereto, as well as sequences identical to those of SEQ ID NO:5-8, a sequence having 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in any one of figures. The target sequence of SPTBN1 should encompass the sequence corresponding to SEQ ID NO:9-12, comprising one or two or more nucleotide mutations, but substantially still functionally identical thereto, as well as sequences identical to those of SEQ ID NOs: 9-12, a sequence having 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in any one of claims.

The target sequences (5 '-3') of the MIR578 gene are as follows:

SEQ ID NO：1

TACAAGAGCATAATTACTAATTATCATAATTATAAGTCTTATGTTCTTCTAAGCAAAACAAATAGCTCGTATCTCTACAGCGTTTCTCAGCTTTGATCTGGTGAAGTTTCCTTTGTGGGCTCAGGTCAGGCGGAGTGAGTGGAGCCCAGGGCGGCTGCTGCGTTAGAAATCCGGGTGCAGCAGATGGCGCCCTTTCCTCAGCTATCTCGCCCCAGAGCTGGGAGGTGGTGCCACAGGCTGCCGATTCTTCGCGGTTCTTCAGGCAAAGGAAAAGGAGCTGCTGGAGTTAAAATACCAGTGACTTTCTAGTCT

the sequence (5 '-3') of the target sequence of the MIR578 gene after bisulfite treatment is as follows:

SEQ ID NO：2

TATAAGAGTATAATTATTAATTATTATAATTATAAGTTTTATGTTTTTTTAAGTAAAATAAATAGTTCGTATTTTTATAGCGTTTTTTAGTTTTGATTTGGTGAAGTTTTTTTTGTGGGTTTAGGTTAGGCGGAGTGAGTGGAGTTTAGGGCGGTTGTTGCGTTAGAAATTCGGGTGTAGTAGATGGCGTTTTTTTTTTAGTTATTTCGTTTTAGAGTTGGGAGGTGGTGTTATAGGTTGTCGATTTTTCGCGGTTTTTTAGGTAAAGGAAAAGGAGTTGTTGGAGTTAAAATATTAGTGATTTTTTAGTTT

the reverse complement (5 '-3') of the target sequence of the MIR578 gene is as follows:

SEQ ID NO：3

AGACTAGAAAGTCACTGGTATTTTAACTCCAGCAGCTCCTTTTCCTTTGCCTGAAGAACCGCGAAGAATCGGCAGCCTGTGGCACCACCTCCCAGCTCTGGGGCGAGATAGCTGAGGAAAGGGCGCCATCTGCTGCACCCGGATTTCTAACGCAGCAGCCGCCCTGGGCTCCACTCACTCCGCCTGACCTGAGCCCACAAAGGAAACTTCACCAGATCAAAGCTGAGAAACGCTGTAGAGATACGAGCTATTTGTTTTGCTTAGAAGAACATAAGACTTATAATTATGATAATTAGTAATTATGCTCTTGTA

the sequence (5 '-3') of the reverse complement of the target sequence of the MIR578 gene after bisulfite treatment is as follows:

SEQ ID NO：4

AGATTAGAAAGTTATTGGTATTTTAATTTTAGTAGTTTTTTTTTTTTTGTTTGAAGAATCGCGAAGAATCGGTAGTTTGTGGTATTATTTTTTAGTTTTGGGGCGAGATAGTTGAGGAAAGGGCGTTATTTGTTGTATTCGGATTTTTAACGTAGTAGTCGTTTTGGGTTTTATTTATTTCGTTTGATTTGAGTTTATAAAGGAAATTTTATTAGATTAAAGTTGAGAAACGTTGTAGAGATACGAGTTATTTGTTTTGTTTAGAAGAATATAAGATTTATAATTATGATAATTAGTAATTATGTTTTTGTA

the target sequence (5 '-3') of the FOXN3 gene is as follows:

SEQ ID NO：5

TGTGGAGAGAGGGAGGCTTCCTGTGAGGCCCACTTCCTGTGAAGCGTTTGGTCTATGGACAGCCTATTTTTAAAAATGTAAGTCCTTCCTTTGAGGCCCCCCGGAACTGGCAGCAGGCCGACGGGCAATCGTTCCTCTGGCCGCACACTTTCAAGTGACCGAATGTACCCAAACTGTTTGATAAGGTCATTTGTAAATATTTACTGATTTGCAGAGAAAAGAAGTTAAATTACATAAACCCGTGCCCACCTCCTGCTTTCAGCCAACAGTAAGATT

the sequence (5 '-3') of the target sequence of the FOXN3 gene after bisulfite treatment is as follows:

SEQ ID NO：6

TGTGGAGAGAGGGAGGTTTTTTGTGAGGTTTATTTTTTGTGAAGCGTTTGGTTTATGGATAGTTTATTTTTAAAAATGTAAGTTTTTTTTTTGAGGTTTTTCGGAATTGGTAGTAGGTCGACGGGTAATCGTTTTTTTGGTCGTATATTTTTAAGTGATCGAATGTATTTAAATTGTTTGATAAGGTTATTTGTAAATATTTATTGATTTGTAGAGAAAAGAAGTTAAATTATATAAATTCGTGTTTATTTTTTGTTTTTAGTTAATAGTAAGATT

the reverse complement (5 '-3') of the target sequence of the FOXN3 gene is as follows:

SEQ ID NO：7

AATCTTACTGTTGGCTGAAAGCAGGAGGTGGGCACGGGTTTATGTAATTTAACTTCTTTTCTCTGCAAATCAGTAAATATTTACAAATGACCTTATCAAACAGTTTGGGTACATTCGGTCACTTGAAAGTGTGCGGCCAGAGGAACGATTGCCCGTCGGCCTGCTGCCAGTTCCGGGGGGCCTCAAAGGAAGGACTTACATTTTTAAAAATAGGCTGTCCATAGACCAAACGCTTCACAGGAAGTGGGCCTCACAGGAAGCCTCCCTCTCTCCACA

the sequence (5 '-3') of the reverse complement of the target sequence of the FOXN3 gene after bisulfite treatment is as follows:

SEQ ID NO：8

###

SEQ ID NO：9

AATAGAAGGTGTGAGGGAGGAGTGCACCCCTAGGCCCACCCATAACAAAAGGCTGTTATTCCGAAAGGGCTGAGGAAGGTTTTAAAACTGCTCGCCGAGAAGGGTGGAGCCTACACACAGGAAATGTCTTAACTGTCCTCTCTGGACAACGTAAAGTTTAAATTTAAAAAAAATCATGTGCCCCTGATATTTTACCTCATACGCTGTTTCTCAAGGAAATCCCTTCGAAAGGGGTAAGCTTCGTGTTTTGTGTGGTAGCTTTAAAAATAATTTTTTTTAGTGTGACCCTTGTCTCCTAATTTAGCCCCAGTGACTTTCTTATTTTTAAATATTGTGGTTTAGGAGTTGCACAAGTTTAGTGTTGGTATTTCTGTAGCAGAAAACCACCCATGTTGAGGAATTGAGAAAGGGTGAATTAACTTTCAAGTATGGTGGACCTCAG

The target sequence of the SPTBN1 gene after bisulfite treatment (5 '-3') is as follows:

SEQ ID NO：10

AATAGAAGGTGTGAGGGAGGAGTGTATTTTTAGGTTTATTTATAATAAAAGGTTGTTATTTCGAAAGGGTTGAGGAAGGTTTTAAAATTGTTCGTCGAGAAGGGTGGAGTTTATATATAGGAAATGTTTTAATTGTTTTTTTTGGATAACGTAAAGTTTAAATTTAAAAAAAATTATGTGTTTTTGATATTTTATTTTATACGTTGTTTTTTAAGGAAATTTTTTCGAAAGGGGTAAGTTTCGTGTTTTGTGTGGTAGTTTTAAAAATAATTTTTTTTAGTGTGATTTTTGTTTTTTAATTTAGTTTTAGTGATTTTTTTATTTTTAAATATTGTGGTTTAGGAGTTGTATAAGTTTAGTGTTGGTATTTTTGTAGTAGAAAATTATTTATGTTGAGGAATTGAGAAAGGGTGAATTAATTTTTAAGTATGGTGGATTTTAG

the reverse complement (5 '-3') of the target sequence of the SPTBN1 gene is as follows:

SEQ ID NO：11

CTGAGGTCCACCATACTTGAAAGTTAATTCACCCTTTCTCAATTCCTCAACATGGGTGGTTTTCTGCTACAGAAATACCAACACTAAACTTGTGCAACTCCTAAACCACAATATTTAAAAATAAGAAAGTCACTGGGGCTAAATTAGGAGACAAGGGTCACACTAAAAAAAATTATTTTTAAAGCTACCACACAAAACACGAAGCTTACCCCTTTCGAAGGGATTTCCTTGAGAAACAGCGTATGAGGTAAAATATCAGGGGCACATGATTTTTTTTAAATTTAAACTTTACGTTGTCCAGAGAGGACAGTTAAGACATTTCCTGTGTGTAGGCTCCACCCTTCTCGGCGAGCAGTTTTAAAACCTTCCTCAGCCCTTTCGGAATAACAGCCTTTTGTTATGGGTGGGCCTAGGGGTGCACTCCTCCCTCACACCTTCTATT

The sequence (5 '-3') of the reverse complement of the target sequence of the SPTBN1 gene after bisulfite treatment is as follows:

SEQ ID NO：12

TTGAGGTTTATTATATTTGAAAGTTAATTTATTTTTTTTTAATTTTTTAATATGGGTGGTTTTTTGTTATAGAAATATTAATATTAAATTTGTGTAATTTTTAAATTATAATATTTAAAAATAAGAAAGTTATTGGGGTTAAATTAGGAGATAAGGGTTATATTAAAAAAAATTATTTTTAAAGTTATTATATAAAATACGAAGTTTATTTTTTTCGAAGGGATTTTTTTGAGAAATAGCGTATGAGGTAAAATATTAGGGGTATATGATTTTTTTTAAATTTAAATTTTACGTTGTTTAGAGAGGATAGTTAAGATATTTTTTGTGTGTAGGTTTTATTTTTTTCGGCGAGTAGTTTTAAAATTTTTTTTAGTTTTTTCGGAATAATAGTTTTTTGTTATGGGTGGGTTTAGGGGTGTATTTTTTTTTTATATTTTTTATT

target sequences and related sequences of MIR578 gene, FOXN3 gene and SPTBN1 gene are shown in Table 1:

table 1: target sequences of MIR578 gene, FOXN3 gene and SPTBN1 gene and related sequences

Preferably, the nucleic acid for detecting methylation status of a gene of interest comprises a fragment of at least 9 nucleotides in a target sequence of the gene of interest, wherein the fragment comprises at least one CpG dinucleotide sequence. In certain preferred embodiments, the nucleic acid for detecting methylation status of a target gene comprises a fragment of at least 9 nucleotides, preferably a fragment of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or more nucleotides in a sequence that has been bisulfite converted from a target sequence of the target gene, such as by using bisulfite to convert a sample DNA to be tested, wherein the fragment of a nucleotide comprises at least one CpG dinucleotide sequence.

More preferably, the nucleic acid for detecting methylation status of a gene of interest comprises a fragment of at least 15 nucleotides that hybridizes under moderate stringency or stringent conditions to a target sequence of said gene of interest, wherein said fragment of nucleotides comprises at least one CpG dinucleotide sequence. In certain preferred embodiments, the nucleic acid for detecting the methylation state of a target gene, such as by converting a test sample DNA using bisulfite, comprises a fragment of at least 15 nucleotides, preferably a fragment of at least 16, 17, 18, 19, 20, 21, 22 or more nucleotides, in a sequence following bisulfite conversion of a target sequence hybridized to the target gene under moderately stringent or stringent conditions, wherein the fragment of nucleotides comprises at least one CpG dinucleotide sequence.

Preferably, the composition further comprises an agent that converts an unmethylated cytosine base at position 5 of the target sequence of the target gene to uracil. More preferably, the reagent is bisulphite.

The nucleic acid used to detect the methylation state of a gene of interest can also include a blocking agent that preferentially binds to DNA in the unmethylated state.

Preferably, the composition comprises one or more of the primers, probes as shown in table 2:

TABLE 2 primer and probe sequences used herein

Sequence numbering	Primer probe numbering	Sequence (5 '-3')
			SEQ ID NO：13	MIR578_1F	GTTTAGGGCGGTTGTTGCG
SEQ ID NO：14	MIR578_1R	CCACCTCCCAACTCTAAAACG
			SEQ ID NO：15	MIR578_1P	CGGGTGTAGTAGATGGCGT
SEQ ID NO：16	FOXN3_1F	AGGTTTTTCGGAATTGGTAGTA
			SEQ ID NO：17	FOXN3_1R	ACATTCGATCACTTAAAAATATACG
SEQ ID NO：18	FOXN3_1P	AAAAACGATTACCCGTCGACC
			SEQ ID NO：19	SPTBN1_1F	GTGTTTTTGATATTTTATTTTATACG
SEQ ID NO：20	SPTBN1_1R	CTACCACACAAAACACG
			SEQ ID NO：21	SPTBN1_1P	CGAAAAAATTTCCTTAAAAAACAACG

"F" in Table 2 represents a forward primer; "R" means the reverse primer; "P" means a probe.

Preferably, the fluorescent labeling patterns of the probe sequences used in the present application are shown in Table 3.

TABLE 3 fluorescent labelling of probe sequences for use herein

In certain embodiments, the composition further comprises an agent that converts an unmethylated cytosine base at position 5 of the gene to uracil. Preferably, the agent is bisulphite. Bisulfite modification of DNA is a known tool for assessing CpG methylation status. In eukaryotic DNA, 5-methylcytosine is the most common covalent base modification. 5-methylcytosine cannot be identified by sequencing because 5-methylcytosine has the same base pairing behavior as cytosine. Furthermore, the epigenetic information carried by 5-methylcytosine is completely lost during PCR amplification. The most commonly used method for analyzing DNA for the presence of 5-methylcytosine is based on the specific reaction of bisulfite with cytosine; after subsequent alkaline hydrolysis, unmethylated cytosines are converted to uracil which corresponds to thymine in pairing behavior; but under these conditions 5-methylcytosine remains unmodified. The original DNA is thus converted in such a way that the 5-methylcytosine, which was originally indistinguishable from cytosine in its hybridization behavior, can now be detected as the only cytosine remaining by conventional known molecular biological techniques, for example by amplification and hybridization. All of these techniques are now fully utilized based on different base pairing properties. Thus, typically, the present application provides for the use of bisulfite technology in combination with one or more methylation assays for determining the methylation status of CpG dinucleotide sequences within a target sequence of a gene of interest. Furthermore, the methods of the present application are suitable for analyzing heterogeneous samples, such as low concentrations of tumor cells in blood or stool. Thus, when analyzing the methylation status of CpG dinucleotide sequences in such samples, one skilled in the art can use quantitative assays to determine the methylation level (e.g., percentage, fraction, ratio, proportion or degree) of a particular CpG dinucleotide sequence, rather than the methylation status. Accordingly, the term methylation status or methylation status shall also be taken to mean a value reflecting the methylation status of a CpG dinucleotide sequence.

In another aspect, the present application provides an oligonucleotide for detecting thyroid cancer in vitro, comprising: SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 or a fragment of at least 9 nucleotides in its complement comprising at least one CpG dinucleotide sequence; and/or SEQ ID NO:5 or SEQ ID NO:6 or SEQ ID NO:7 or SEQ ID NO:8 or a fragment of at least 9 nucleotides in its complement comprising at least one CpG dinucleotide sequence; and/or SEQ ID NO:9 or SEQ ID NO:10 or SEQ ID NO:11 or SEQ ID NO:12 or a complementary sequence thereof and comprises at least one CpG dinucleotide sequence.

Preferably the oligonucleotide for in vitro detection of thyroid cancer comprises: for SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 or a complementary sequence thereof, a fragment of at least 9 nucleotides in the bisulfite converted sequence; and/or to SEQ ID NO:5 or SEQ ID NO:6 or SEQ ID NO:7 or SEQ ID NO:8 or a complementary sequence thereof, and comprising at least 9 nucleotides of a CpG dinucleotide sequence; and/or to SEQ ID NO:9 or SEQ ID NO:10 or SEQ ID NO:11 or SEQ ID NO:12 or a complement thereof, and comprising at least one CpG dinucleotide sequence.

The oligonucleotide for detecting thyroid cancer in vitro of the present application further comprises: hybridization to the SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 or a fragment of at least 15 nucleotides in its complement comprising at least one CpG dinucleotide sequence; and/or hybridizes to the SEQ ID NO:5 or SEQ ID NO:6 or SEQ ID NO:7 or SEQ ID NO:8 or a fragment of at least 15 nucleotides in its complement comprising at least one CpG dinucleotide sequence; and/or hybridizes to the SEQ ID NO:9 or SEQ ID NO:10 or SEQ ID NO:11 or SEQ ID NO:12 or a complementary sequence thereof and comprises at least one CpG dinucleotide sequence.

Preferably, the oligonucleotide for in vitro detection of thyroid cancer comprises: hybridization to SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 or a complementary sequence thereof, and comprising at least 15 nucleotides of a CpG dinucleotide sequence; and/or hybridizes under moderately stringent or stringent conditions to the sequence set forth in SEQ ID NO:5 or SEQ ID NO:6 or SEQ ID NO:7 or SEQ ID NO:8 or a complementary sequence thereof, and comprising at least 15 nucleotides of a CpG dinucleotide sequence; and/or hybridizes under moderately stringent or stringent conditions to the sequence set forth in SEQ ID NO:9 or SEQ ID NO:10 or SEQ ID NO:11 or SEQ ID NO:12 or a complement thereof, and comprising at least one CpG dinucleotide sequence.

The oligonucleotide for detecting thyroid cancer in vitro can further comprise: blocking agents that preferentially bind to DNA in the unmethylated state.

In a specific embodiment, an oligonucleotide for detecting thyroid cancer in vitro, comprising: SEQ ID NO:13 and SEQ ID NO: 14. It also includes: SEQ ID NO: 15.

In another specific embodiment, an oligonucleotide for detecting thyroid cancer in vitro, comprising: SEQ ID NO:16 and SEQ ID NO: 17. It also includes: SEQ ID NO: 18.

In another specific embodiment, an oligonucleotide for detecting thyroid cancer in vitro, comprising: SEQ ID NO:19 and SEQ ID NO:20, further comprising: SEQ ID NO: 21.

In another aspect, the present application provides a kit comprising the composition. The kit further comprises at least one additional component selected from the group consisting of: nucleoside triphosphates, DNA polymerase and buffers required for the function of the DNA polymerase.

Typically, the kit further comprises a container for holding a patient sample. And, the kit also includes instructions for use and interpretation of the test results.

The application also relates to the application of the composition and the oligonucleotide in preparing a kit for detecting thyroid cancer in vitro.

In yet another aspect, the present application provides a method of detecting thyroid cancer in vitro, the method comprising the steps of:

1) Separating target sequences or fragments thereof of target genes in a sample to be detected;

2) Determining the methylation status of the target sequence of the target gene;

3) Judging the state of a sample according to the detection result of the methylation state of the target sequence of the target gene, thereby realizing in-vitro detection of thyroid cancer.

According to certain preferred embodiments, the method further comprises the steps of:

1) Extracting genome DNA of a sample to be detected;

2) Treating the DNA sample obtained in step 1) with a reagent to convert the 5-unmethylated cytosine base into uracil or another base, i.e., the 5-unmethylated cytosine base of the target sequence of the target gene into uracil or another base, the converted base being different from the 5-unmethylated cytosine base in hybridization performance and being detectable;

3) Contacting the DNA sample treated in step 2) with a DNA polymerase and primers for the target sequence of the target gene such that the target sequence of the treated target gene is amplified to produce amplified products or not; the target sequence of the treated target gene produces amplified products if DNA polymerization occurs; the target sequence of the treated target gene is not amplified if DNA polymerization does not occur;

4) Detecting the amplified product with a probe; and

5) Determining the methylation status of at least one CpG dinucleotide of the target sequence of the target gene based on the presence or absence of the amplification product.

Preferably, a typical primer comprises a fragment of a target sequence of the target gene comprising a sequence that hybridizes to a sequence selected from the group consisting of SEQ ID NOs: 1-4, any one of SEQ ID NOs: 5-8 and SEQ ID NO:9-12 of any one of the fragments of at least 9 nucleotides.

Preferably, a typical probe comprises a fragment of a target sequence of said target gene comprising a sequence which hybridizes to a sequence selected from the group consisting of SEQ ID NOs: 1-4, any one of SEQ ID NOs: 5-8 and SEQ ID NO:9-12 of any one of the following.

Preferably, one or more of the primers, probes are as set forth in table 2 above.

And, the contacting or amplifying comprises using at least one of the following methods: using a thermostable DNA polymerase as the amplification enzyme, using a polymerase lacking 5'-3' exonuclease activity, using Polymerase Chain Reaction (PCR), producing an amplification product nucleic acid molecule with a detectable label.

Preferably, the methylation status is determined by means of PCR, such as "fluorescence-based real-time PCR technique", methylation sensitive single nucleotide primer extension reaction (Ms-SNuPE), methylation Specific PCR (MSP), and methylation CpG island amplification (MCA), etc., is used to determine the methylation status of at least one CpG dinucleotide of the target sequence of the gene of interest. Among these, the "fluorescence-based real-time PCR" assay is a high throughput quantitative methylation assay that uses fluorescence-based real-time PCR (TaqMan) techniques, requiring no further manipulation after the PCR step. Briefly, the "fluorescence-based real-time PCR" method starts with a mixed sample of genomic DNA that is converted into a mixed pool of methylation-dependent sequence differences in a sodium bisulfite reaction according to standard procedures. Fluorescence-based PCR was then performed in an "offset" (biased) reaction (using PCR primers overlapping known CpG dinucleotides). Sequence differences can be produced at the amplification level and at the fluorescence detection amplification level. The "fluorescence-based real-time PCR" assay can be used as a quantitative test for methylation status in genomic DNA samples, where sequence discrimination occurs at the level of probe hybridization. In this quantitative format, the PCR reaction provides methylation specific amplification in the presence of fluorescent probes that overlap specific CpG dinucleotides. An unbiased control for the amount of starting DNA is provided by the following reaction: wherein neither the primer nor the probe covers any CpG dinucleotide. The "fluorescence-based real-time PCR" method can be used with any suitable probe, such as "TaqMan", "Lightcycler", etc. The TaqMan probe is double labeled with a fluorescent reporter (RCHFRrter) and a Quencher (Quencher) and is designed to be specific for a relatively high GC content region such that it melts at a temperature about 10℃higher than the forward or reverse primer during the PCR cycle. This allows the TaqMan probe to remain fully hybridized during the PCR annealing/extension step. When Taq polymerase enzymatically synthesizes a new strand in PCR, it eventually encounters an annealed TaqMan probe. Taq polymerase 5 'to 3' endonuclease activity will then displace the TaqMan probe by digesting it, thereby releasing the fluorescent reporter molecule for quantitative detection of its now unquenched signal using a real-time fluorescent detection system. Typical reagents for "fluorescence-based real-time PCR" analysis may include, but are not limited to: target sequence PCR primers for the target gene; a non-specific amplification blocker; taqMan or Lightcycler probes; optimized PCR buffers and deoxynucleotides; taq polymerase, and the like.

In certain preferred embodiments, the methylation status of at least one CpG dinucleotide in the target sequence of the target gene is determined from the critical Ct value of the real-time PCR reaction. The method for analyzing DNA in the biological sample by utilizing the real-time PCR reaction can conveniently realize the detection of the methylation state of the target sequence of the target gene, and can rapidly and conveniently judge whether the detected sample is positive according to the critical Ct value of the PCR reaction, thereby providing a noninvasive and rapid thyroid cancer in-vitro detection method.

The sample is selected from the group consisting of a cell line, a histological section, a tissue biopsy/paraffin-embedded tissue, a body fluid, stool, a colonic outflow, urine, plasma, serum, whole blood, isolated blood cells, cells isolated from blood, or a combination thereof. Preferred samples are plasma and tissue.

The inventors found that there was a significant difference between the methylation status of the target sequences of the MIR578 gene, FOXN3 gene and SPTBN1 gene in thyroid cancer tissue, plasma and the methylation status of the target sequences of these genes in plasma of parathyroid cancer tissue, benign nodular patients: in thyroid cancer tissues, plasma, the target sequences of MIR578 gene, FOXN3 gene and SPTBN1 gene are hypermethylated, whereas in parathyroid cancer tissues, benign nodule patient plasma, the target sequences of MIR578 gene, FOXN3 gene and SPTBN1 gene are not methylated or hypomethylated.

Accordingly, the present application provides a system and method for detecting thyroid cancer by detecting the methylation status of target sequences of MIR578 gene, FOXN3 gene, and SPTBN1 gene in a sample, which enables noninvasive, rapid detection of thyroid cancer.

Examples

The materials used in the test and the test methods are generally and/or specifically described herein, and in the examples which follow,% represents wt%, i.e., weight percent, unless otherwise specified. The reagents or apparatus used were conventional reagent products commercially available without the manufacturer's knowledge.

Example 1 primer and Probe test

The screening and confirmation process of the marker in this embodiment is approximately as follows: collecting cancer tissues and tissue samples beside cancer patients, sequencing by targeted capturing methylation, screening the cancer tissues, hypomethylation differential methylation areas (DMR/MMU/MRA and the like) in benign tissues, designing a primer probe to detect the tissue samples and peripheral blood plasma of the cancer patients and benign disease patients, and comprehensively confirming candidate markers as MIR578 genes, FOXN3 genes and SPTBN1 genes according to the cancer detection sensitivity and the specificity of a control sample. Primers and probes were designed based on the target sequences of MIR578 gene, FOXN3 gene and SPTBN1 gene. The designed MIR578 gene, FOXN3 gene, SPTBN1 gene and primer probe sequences are shown in Table 2.

The cell line DNA of normal human leukocytes is normally in a low/unmethylated state and can be used as a negative control, with an amount of 15.75 ng/reaction in this embodiment; the total methylated DNA was in a high/total methylated state and served as a positive control, and the amount of DNA used in this embodiment was 200 pg/reaction. Firstly, bisulphite conversion is carried out on a DNA sample, and real-time PCR amplification is carried out by using the converted BisDNA as a template and using the primer probe. The beta Actin (ACTB) gene is used as an internal reference, a beta actin gene amplicon is created by using a primer complementary to the beta actin gene sequence, and the beta actin gene amplicon is detected with a specific probe. Each sample is subjected to at least one real-time PCR, and in some embodiments, two or three real-time PCR assays are performed. The PCR system for the primer probe test is shown in Table 4 below.

TABLE 4 PCR System for primer probe test

	Volume (mu L)	Final concentration
			Taq DNA Polymerase(Biochain)	1.2	/
4.2×buffer(Biochain)	11.9	1×
			Forward primer F (10. Mu.M)	1.0	200nM
Reverse primer R (10. Mu.M)	1.0	200nM
			Probe P (10 mu M)	1.0	200nM
ACTB_F(10μM)	0.5	100nM
			ACTB_R(10μM)	0.5	100nM
ACTB_P(10μM)	0.375	75nM
			BisDNA	4.0	/
H 2 O	19.05	/
			Total	50.0	/

Note that: "F" represents a forward primer; "R" means the reverse primer; "P" means a probe.

The PCR amplification procedure used was: 94 ℃ for 20min; (93 ℃,30s;57 ℃,35 s-read fluorescent signal) 45 cycles; 40℃for 5s.

As a result, as shown in Table 5, when the bisDNA of the total methylated DNA was used as a template, MIR578 gene, FOXN3 gene and SPTBN1 gene were amplified efficiently; when the bisDNA of WBC is used as the template, the target genes except the reference gene ACTB are not amplified.

TABLE 5 results of primer probe test for each Gene

Where "No Ct" indicates that No Ct value is detected.

Example 2

24 thyroid cancer tissue samples (10 ng/reaction) and 24 thyroid cancer paracentesis tissue samples (10 ng/reaction) were selected, genomic DNA was extracted, bisulfite-converted to BisDNA, and methylation of FOXN3, SPTBN1, MIR578 genes was detected according to the PCR reaction system shown in Table 5 and the reaction procedure in example 1. Finally, ct values of real-time PCR of 24 thyroid cancer tissues and 24 parathyroid cancer tissues on target sequences of target genes are measured.

TABLE 6 PCR System of different primer and probe combinations

Note that: "F" represents a forward primer; "R" means the reverse primer; "P" means a probe.

The test results are shown in tables 7 and 8. As shown in Table 7, the sensitivity of detection of thyroid cancer by FOXN3, SPTBN1 and MIR578 genes alone was 83.33%, 79.17% and 87.5%, respectively; the sensitivity of the combined interpretation of the three genes reaches 95.83 percent. As shown in Table 8, methylation of the target sequence of the target gene has good specificity, and the specificity of detection of parathyroid cancer tissue by FOXN3, SPTBN1 and MIR578 genes alone is 91.67%, 100% and 91.67%, respectively, so that the specificity of joint interpretation is 91.67%.

Table 7: detection positivity of FOXN3, SPTBN1 and MIR578 genes in thyroid cancer tissue and paracancerous tissue

Table 8: FOXN3, SPTBN1 and MIR578 genes are negative in detection of thyroid cancer tissue and paracancer tissue

Example 3

40 thyroid cancer plasma samples (3.5 mL) and 60 benign nodule plasma samples (3.5 mL) were selected, genomic DNA was extracted, bisulfite-converted to BisDNA, and methylation was detected in accordance with the PCR reaction system of example 1, in combination with FOXN3, SPTBN1, and MIR578 genes. Finally, ct values of real-time PCR of 40 thyroid cancer plasma samples and 60 benign nodule plasma samples for target sequences of the target genes were measured.

According to the PCR result, the critical values of Ct values of the three genes were all selected to be ct=38, and the results are shown in table 9, and the sensitivity of separately detecting thyroid cancer by using FOXN3, SPTBN1, MIR578 genes is 77.5%, 72.5% and 77.5%, respectively; the sensitivity of the combined interpretation of the three genes reaches 95 percent and the specificity reaches 96.67 percent.

Table 9: detection results of combined FOXN3, SPTBN1 and MIR578 genes in cancer plasma and benign nodule plasma

The above experimental results show that the methylated DNA of the target sequence of the target gene is a marker of thyroid cancer. Through the detection of the target gene target sequence methylation DNA, the in-vitro noninvasive detection of thyroid cancer can be realized, and the detection rate of thyroid cancer of nodular group can be improved.

Claims (10)

1. A composition for detecting thyroid cancer in vitro, the composition comprising:

nucleic acid for detecting methylation status of a target gene,

wherein the methylation state of the target gene is characterized by methylation of a target sequence of the target gene,

wherein the target gene is one, two or three of MIR578 gene, FOXN3 gene and SPTBN1 gene.

2. The composition of claim 1, wherein the MIR578 gene has a target sequence set forth in SEQ ID NO:1-4, or comprises SEQ ID NO: 1-4.

3. The composition of claim 1, wherein the FOXN3 gene has a target sequence set forth in SEQ ID NO:5-8, or comprises SEQ ID NO: 5-8.

4. The composition of claim 1, wherein the target sequence of the SPTBN1 gene is as set forth in SEQ ID NO:9-12, or comprises any one of SEQ ID NOs: 9-12.

5. The composition of any one of claims 1 to 4, wherein the nucleic acid for detecting methylation status of a target gene comprises:

a primer which is a fragment of at least 9 nucleotides in a target sequence of the target gene,

The fragment comprises at least one CpG dinucleotide sequence.

6. The composition of any one of claims 1-5, wherein the nucleic acid for detecting methylation status of a target gene comprises:

a probe which is a fragment of at least 15 nucleotides hybridised to the target sequence of the target gene under moderately stringent or stringent conditions,

the fragment comprises at least one CpG dinucleotide sequence.

7. The composition of any one of claims 1-6, further comprising:

an agent that converts an unmethylated cytosine base at position 5 of a target sequence of a target gene into uracil.

8. The composition of any one of claims 1-7, wherein the nucleic acid for detecting methylation status of a target gene further comprises:

blocking agents that preferentially bind to target sequences in the unmethylated state.

9. The composition of claim 8, wherein,

the at least 9 nucleotide fragment, which is SEQ ID NO:13 and SEQ ID NO:14, or it is SEQ ID NO:16 and SEQ ID NO:17, or it is SEQ ID NO:19 and SEQ ID NO: 20;

the at least 15 nucleotide fragment, which is SEQ ID NO:15, or SEQ ID NO:18, or SEQ ID NO: 21.

10. An oligonucleotide for detecting thyroid cancer in vitro, comprising:

the SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 or a fragment of at least 9 nucleotides in its complement comprising at least one CpG dinucleotide sequence; and/or

The SEQ ID NO:5 or SEQ ID NO:6 or SEQ ID NO:7 or SEQ ID NO:8 or a fragment of at least 9 nucleotides in its complement comprising at least one CpG dinucleotide sequence; and/or

The SEQ ID NO:9 or SEQ ID NO:10 or SEQ ID NO:11 or SEQ ID NO:12 or a complementary sequence thereof and comprises at least one CpG dinucleotide sequence.