CN117778572A - Nucleic acid combination for detecting thyroid cancer, detection kit and application - Google Patents
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Abstract
The invention belongs to the field of biological medicine, and in particular relates to a nucleic acid combination for detecting thyroid cancer, a detection kit and application. The detection kit provided by the invention can effectively distinguish thyroid cancer patients from healthy people by detecting the methylation level of the thyroid cancer methylation biomarker in the sample, effectively improve the detection rate of thyroid cancer, is beneficial to improving the life quality and survival rate of patients, has high detection accuracy, can reduce the detection of false negative results, and avoids excessive diagnosis and treatment.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a nucleic acid combination for detecting thyroid cancer, a detection kit and application.
Background
Thyroid cancer (thyroid carcinoma, TC) is one of the most common tumors in the endocrine system, accounting for 2.59% of the incidence of cancer. In terms of diagnosis, thyroid cancer often has painless thyroid nodules as the major clinical manifestation. Clinically, diagnosis is mainly carried out by methods such as palpation, color Doppler ultrasound, nuclide imaging, fine needle puncture cytology examination (fine needle aspiration biopsy, FNAB) and the like, but the properties of thyroid nodules are difficult to determine, and even if pathological biopsy is carried out, thyroid adenoma and nodular hyperplasia, benign tumors and malignant tumors are not easy to clearly identify. The main problem to be solved is currently urgent to avoid both extremes of under-or over-treatment.
DNA methylation changes are one of the earliest molecular changes that occur during the course of cancer and are tissue specific throughout the course of cancer, and the high methylation levels of tumor suppressor genes have been identified as important mechanisms to suppress gene expression and promote cancer cell growth and expansion. Studies have shown that multiple oncogenes such as CDKN2A, RASSF1, TSHR, PTEN, and the like can be diagnostic markers for thyroid cancer.
Although many studies on tumor markers are performed, the methods are different, but single tumor markers cannot be widely used because of low sensitivity and specificity.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a nucleic acid combination, a detection kit and application for detecting thyroid cancer, so as to solve the technical problems of poor sensitivity and specificity, low accuracy and the like of detecting thyroid cancer by using a thyroid cancer methylation biomarker in the prior art.
To achieve the above object, the present invention provides a use of a nucleic acid combination for detecting the methylation level of a thyroid cancer methylation biomarker in a sample in the preparation of a thyroid cancer diagnostic product;
taking GRCh38.p14 as a reference genome, wherein the thyroid cancer methylation biomarker is selected from at least one of (a) - (c):
(a) A full length region or a partial region of chr3:98733897-98734297, said partial region comprising at least one CpG dinucleotide site;
(b) A polynucleotide molecule in which the CpG dinucleotide site in (a) is partially or fully methylated;
(c) A polynucleotide molecule having more than 85% sequence identity to (a) wherein the CpG dinucleotide site is identical to (a).
Preferably, the thyroid cancer diagnostic product comprises at least one of a primer, a probe, a kit, a chip, a sequencing library, a membrane strip, and a protein array.
The invention also provides a nucleic acid combination for detecting thyroid cancer for detecting the methylation level of a thyroid cancer methylation biomarker in a sample;
taking GRCh38.p14 as a reference genome, wherein the thyroid cancer methylation biomarker is selected from at least one of (a) - (c):
(a) A full length region or a partial region of chr3:98733897-98734297, said partial region comprising at least one CpG dinucleotide site;
(b) A polynucleotide molecule in which the CpG dinucleotide site in (a) is partially or fully methylated;
(c) A polynucleotide molecule having more than 85% sequence identity to (a) wherein the CpG dinucleotide site is identical to (a).
Preferably, the thyroid cancer methylation biomarker is selected from at least one of SEQ ID No.2 and SEQ ID No. 5.
Preferably, the nucleic acid combination comprises a methylation primer pair for detecting the methylation level of the thyroid cancer methylation biomarker.
Further preferably, the nucleotides of the methylation primer pairs are as shown in SEQ ID NO. 11-12,
preferably, the nucleic acid combination further comprises a pair of unmethylated primers for detecting the methylation level of the thyroid cancer methylation biomarker;
the nucleotide of the unmethylated primer pair is shown as SEQ ID NO. 13-14.
Preferably, the nucleic acid combination further comprises a detection probe; further preferably, the nucleotide sequence of the detection probe is shown as SEQ ID NO. 17.
Preferably, the 5 'end of the detection probe comprises a fluorescence reporting group, and the 3' end comprises a fluorescence quenching group.
The invention also provides a detection kit for detecting thyroid cancer, which comprises the nucleic acid combination.
Preferably, the detection kit further comprises one or more of an upstream primer and a downstream primer of the reference gene, a detection probe, a DNA extraction reagent, a DNA purification reagent, a methylation conversion reagent, an amplification reagent, a positive control and a negative control.
In general, the above technical solutions conceived by the present invention have the following beneficial effects compared with the prior art:
(1) The nucleic acid combination and detection kit for detecting thyroid cancer provided by the invention can effectively distinguish thyroid cancer patients from healthy people by detecting the methylation level of the thyroid cancer methylation biomarker in a sample, and realizes detection of thyroid cancer with low invasiveness, low cost, high sensitivity and high specificity.
(2) In a preferred embodiment, the sensitivity of the detection kit provided by the invention for detecting thyroid cancer blood samples by detecting the methylation level of a target region (chr 3: 98733897-98734297) of the ST3GAL6 gene can reach 87.8%, and the specificity of the detection kit for detecting healthy human blood samples can reach 93.4%. The detection kit provided by the invention can effectively improve the detection rate of thyroid cancer, is beneficial to improving the life quality and survival rate of patients, has high detection accuracy, can reduce the detection of false negative results, and avoids excessive diagnosis and treatment.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the following examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
The term "diagnosis" refers to determining the health status of a subject, and encompasses detecting the presence or absence of a disease, responding to a therapeutic regimen, assessing risk of recurrence, assessing risk and extent of cancerous lesions, prognostic assays, and the like. In some cases, the term "diagnosis" refers to the use of a single factor in determining, validating or confirming a clinical state of a patient. In some embodiments, "detecting" thyroid cancer refers to detecting the presence or absence of a disease, i.e., determining whether a subject has thyroid cancer.
The term "subject" refers to a subject receiving observation, detection or experiment. In some embodiments, the subject may be a mammal. Mammals include, but are not limited to, primates (including humans and non-human primates) and rodents (e.g., mice and rats). In some embodiments, the mammal may be a human.
The term "sample" or "specimen" includes substances obtained from any individual (preferably a human) or isolated tissue, cell or body fluid (e.g., plasma) suitable for detection of the methylation state of DNA. For example, the samples may include, but are not limited to: blood samples, tissue samples (e.g., paraffin embedded samples), cell samples; preferably, the sample includes, but is not limited to: a blood sample, a plasma sample, a serum sample, or a combination thereof.
The term "methylation biomarker" refers to a biochemical marker that includes at least one methylation locus and/or the methylation state of at least one methylation locus, e.g., a hypermethylation locus. In particular, a methylation biomarker is one or more nucleic acid loci whose methylation status differs between a cancer individual and a non-cancer individual. Wherein "methylation locus" refers to a region or fragment of DNA comprising at least one region of differential methylation. Under selected conditions, such as cancer states, the methylation loci are hypermethylated, i.e., include a greater number or frequency of methylation sites than in non-cancer states; alternatively, in a cancerous state, the methylation locus may be hypomethylated, i.e., include fewer numbers or frequencies of methylation sites than in a non-cancerous state. Methylation biomarkers can be used in disease diagnosis, in determining disease stage, or to evaluate the safety or efficacy of new drugs or therapies in a target population. Screening methylation biomarkers for disease screening and early diagnosis can greatly improve the clinical treatment effect of patients.
The term "oligonucleotide" or "polynucleotide" or "nucleotide" or "nucleic acid" refers to a molecule having two or more deoxyribonucleotides or ribonucleotides, preferably more than three, and typically more than ten. The exact size will depend on many factors, which in turn depend on the ultimate function or use of the oligonucleotide. The oligonucleotides may be produced by any means, including chemical synthesis, DNA replication, reverse transcription, or a combination thereof. Typical deoxyribonucleotides of DNA are thymine, adenine, cytosine and guanine. Typical ribonucleotides of RNA are uracil, adenine, cytosine and guanine.
The term "methylation" is a form of chemical modification of DNA that can alter genetic manifestations without altering the DNA sequence. DNA methylation refers to covalent binding of a methyl group at the 5 th carbon position of cytosine of a genomic CpG dinucleotide under the action of a DNA methyltransferase. DNA methylation can cause alterations in chromatin structure, DNA conformation, DNA stability, and the manner in which DNA interacts with proteins, thereby regulating gene expression.
The term "methylation level" refers to whether or not cytosine in one or more CpG dinucleotides in a DNA sequence is methylated, or the frequency/proportion/percentage of methylation, representing both qualitative and quantitative concepts. In practical application, different detection indexes can be adopted to compare the DNA methylation level according to practical conditions. As in some cases, the comparison may be made based on Ct values detected by the sample; in some cases, the ratio of gene methylation in the sample, i.e., number of methylated molecules/(number of methylated molecules+number of unmethylated molecules). Times.100, can be calculated and then compared; in some cases, statistical analysis and integration of each index is also required to obtain a final decision index.
The term "primer" refers to an oligonucleotide that can be used in an amplification method (e.g., polymerase chain reaction, PCR) to amplify a sequence of interest based on a polynucleotide sequence corresponding to a gene of interest or a portion thereof. Typically, at least one of the PCR primers used to amplify a polynucleotide sequence is sequence specific for that polynucleotide sequence. The exact length of the primer will depend on many factors, including temperature, source of primer, and method used. For example, for diagnostic and prognostic applications, the oligonucleotide primers will typically contain at least 10, 15, 20, 25 or more nucleotides, but may also contain fewer nucleotides, depending on the complexity of the target sequence. In the present disclosure, the term "primer" refers to a pair of primers that hybridize to the double strand of a target DNA molecule or to regions of the target DNA molecule that flank the nucleotide sequence to be amplified. "primer pair" refers to a group of an upstream primer and a downstream primer.
The term "sequencing by sulphide PCR (Bisulfite Sequencing PCR, BSP)" is the conversion of unmethylated cytosines to uracil by bisulphite treatment of genomic DNA, uracil is converted to thymine in subsequent PCR reactions, whereas methylated cytosines cannot be deaminated and remain at the completion of the reaction; and designing primers in a non-methylation region for PCR amplification, cloning and sequencing the amplified PCR product, comparing the measured sequence with the original sequence, counting methylation sites and the number, and analyzing the methylation degree.
The term "methylation-specific PCR" is one of the most sensitive experimental techniques currently studied for methylation, and a minimum of about 50pg of DNA methylation can be found. After the single-stranded DNA is subjected to bisulfite conversion, all unmethylated cytosines are deaminated to uracil, and methylated cytosines in CpG sites are kept unchanged, so that two pairs of primers aiming at methylated and unmethylated sequences are respectively designed, and the methylated and unmethylated DNA sequences can be distinguished through PCR amplification. In the present disclosure, methylation primers are added when performing real-time quantitative methylation-specific PCR, and if the Ct value meets the requirement (e.g., ct.ltoreq.45 in a plasma sample), it indicates that the target sequence is methylated.
The term "methylation specific fluorescent quantitative PCR (qMSP)" is an experimental technique combining fluorescent quantitative PCR technology and methylation specific PCR technology. The technology designs a proper primer pair based on the sequence difference of the DNA with different methylation states after being converted by bisulfite, so that the methylated sequence and the unmethylated sequence are distinguished, and the final detection index of the qMSP is a fluorescent signal, so that a fluorescent probe or a fluorescent dye is required to be added in addition to the methylation detection primer in the qMSP reaction system. Compared with the traditional methylation specific PCR technology, the qMSP detection DNA methylation level has higher sensitivity and specificity, is more suitable for detecting trace amounts of DNA fragments with abnormal methylation mixed in the DNA of patients in early cancer, does not need gel electrophoresis detection, and is simpler and more convenient to operate.
The term "TaqMan probe" refers to a stretch of oligonucleotide sequences comprising a 5 'fluorescent group and a 3' quenching group. When the probe binds to the corresponding site on the DNA, the probe does not fluoresce because of the presence of a quenching group near the fluorescent group. During amplification, if the probe binds to the amplified strand, the 5'-3' exonuclease activity of the DNA polymerase (e.g., taq enzyme) digests the probe and the fluorescent group is far from the quenching group, its energy is not absorbed, i.e., a fluorescent signal is generated. The fluorescence signal is also identical to the target fragment with a synchronous exponential increase per PCR cycle.
The invention provides an application of a nucleic acid combination in preparing a thyroid cancer diagnosis product, wherein the nucleic acid combination is used for detecting the methylation level of a thyroid cancer methylation biomarker in a sample;
taking GRCh38.p14 as a reference genome, wherein the thyroid cancer methylation biomarker is selected from at least one of (a) - (c):
(a) A full length region or a partial region of chr3:98733897-98734297, said partial region comprising at least one CpG dinucleotide site;
(b) A polynucleotide molecule in which the CpG dinucleotide site in (a) is partially or fully methylated;
(c) A polynucleotide molecule having more than 85% sequence identity to (a) wherein the CpG dinucleotide site is identical to (a).
In some embodiments, the thyroid cancer methylation biomarker is selected from at least one of SEQ ID No.2 and SEQ ID No. 5.
In some embodiments, the diagnosis includes, but is not limited to, differential diagnosis of thyroid cancer, evaluation and dynamic monitoring of residual microcosmic lesions, assisted determination of recurrence and prognosis of thyroid cancer, evaluation of therapeutic effect of drugs, and monitoring of drug resistance. Wherein, the differential diagnosis of the thyroid cancer can be specifically used for confirming whether an individual suffers from the thyroid cancer or is more likely to suffer from the thyroid cancer; the foregoing evaluation of the residual of the micro-lesion and the dynamic monitoring may be specifically used for confirming whether the micro-lesion remains in the individual; the auxiliary judgment of the recurrence and prognosis of the thyroid cancer refers to the prediction of the risk and prognosis of the recurrence of the thyroid cancer, and can be specifically used for confirming the possibility or the exacerbation tendency of the recurrence of the thyroid cancer of an individual, and can be used for guiding clinical diagnosis and treatment; the drug efficacy evaluation and drug resistance monitoring can be specifically used for confirming whether a certain drug or treatment means is effective for an individual.
In some embodiments, the thyroid cancer diagnostic product comprises at least one of a primer, a probe, a kit, a chip, a sequencing library, a membrane strip, and a protein array.
The invention also provides a nucleic acid combination for detecting thyroid cancer for detecting the methylation level of a thyroid cancer methylation biomarker in a sample;
taking GRCh38.p14 as a reference genome, wherein the thyroid cancer methylation biomarker is selected from at least one of (a) - (c):
(a) A full length region or a partial region of chr3:98733897-98734297, said partial region comprising at least one CpG dinucleotide site;
(b) A polynucleotide molecule in which the CpG dinucleotide site in (a) is partially or fully methylated;
(c) A polynucleotide molecule having more than 85% sequence identity to (a) wherein the CpG dinucleotide site is identical to (a).
In some embodiments, the thyroid cancer methylation biomarker is selected from at least one of SEQ ID No.2 and SEQ ID No. 5.
In some embodiments, the nucleic acid combinations described above include methylation primer pairs for detecting the methylation level of the thyroid cancer methylation biomarker described above.
In some embodiments, the above nucleic acid combinations include methylation primer pairs for detecting the methylation level of a full length region or a partial region of chr3: 98733897-98734297. It will be appreciated that in detecting the methylation level of chr3:98733897-98734297, it is possible to detect the entire length of the region, and also a portion of the region.
In a preferred embodiment, the nucleotides of the methylation primer pairs are shown as SEQ ID NOS.11-12,
in some embodiments, the above nucleic acid combinations further comprise a pair of unmethylated primers for detecting the methylation level of the above thyroid cancer methylation biomarker.
In some embodiments, the above nucleic acid combinations further comprise a pair of unmethylated primers for detecting the methylation level of the full length region or partial region of chr3: 98733897-98734297.
In a preferred embodiment, the nucleotides of the unmethylated primer pair are shown as SEQ ID NOS.13-14.
It is also within the scope of the present invention if one primer set has at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% etc.) or more sequence identity with the nucleotide sequences shown in the methylated primer set and the unmethylated primer set, and the primer set has a certain thyroid cancer diagnosis function (specificity or sensitivity is equivalent to or slightly reduced or slightly increased or greatly increased as compared with the primer set of the present application).
In some embodiments, the above nucleic acid combinations further comprise a detection probe.
In a preferred embodiment, the nucleotide sequence of the detection probe is shown as SEQ ID NO. 17.
The invention also provides a detection kit for detecting thyroid cancer, which comprises the nucleic acid combination.
In some embodiments, the above-described detection kit further comprises one or more of an upstream primer and a downstream primer of the reference gene, a detection probe, a DNA extraction reagent, a DNA purification reagent, a methylation conversion reagent, an amplification reagent, a positive control, and a negative control.
In some embodiments, the reference gene may be, but is not limited to ACTB. In an alternative specific example, the nucleotide sequences of the upstream and downstream primers of the reference gene ACTB are shown in SEQ ID NO. 15-16, and the nucleotide sequence of the detection probe is shown in SEQ ID NO.18. It will be appreciated that in other embodiments, other reference genes may be selected, and that the internal reference primer pairs may be designed accordingly.
In some embodiments, the detection probes each comprise a fluorescent reporter group at the 5 'end and a fluorescent quenching group at the 3' end. The fluorescent reporter group of each probe is independently selected from any one of FAM, VIC, HEX, NED, ROX, TET, JOE, CY and CY 5; the fluorescence quenching group of each detection probe is independently selected from any one of TAMRA, MGB, BHQ, BHQ, BHQ2 and BHQ 3. When more than two detection probes are arranged in the same reaction system, the fluorescent groups connected to different detection probes are different. Examples of the fluorescent reporter group and the fluorescent quenching group of each detection probe independently include, but are not limited to, those listed above.
In some embodiments, the methylation conversion reagent is used to deaminate unmethylated cytosines in DNA to uracil while methylated cytosines remain unchanged. The methylation converting reagent of the present invention is not particularly limited, and any reagent reported in the prior art for achieving cytosine to uracil conversion may be used, such as one or more of hydrazine salt, bisulfite (e.g., sodium bisulfite, potassium bisulfite, ammonium bisulfite) and bisulfite (e.g., sodium bisulfite, potassium bisulfite, calcium bisulfite, magnesium bisulfite, aluminum bisulfite, cesium bisulfite, ammonium bisulfite, etc.).
In some embodiments, the amplification reagents include, but are not limited to, amplification buffers, dNTPs, DNA polymerase, and Mg 2+ One or more of the following. In some embodiments, the above positiveControls refer to thyroid cancer methylation biomarkers comprising methylation therein, which are used to monitor the detection performance of reagents in the kit. The negative control mentioned above refers to a thyroid cancer methylation biomarker that does not contain methylation therein, and is used to monitor whether the experiment is contaminated.
In some embodiments, the above-described test kit further comprises a container for holding a biological sample of the subject. In some embodiments, the above-described kits further comprise instructions for use and interpretation of the test results.
In some embodiments, the sample includes, but is not limited to, cells, tissues, organs, and/or biological fluids of an individual obtained by any method known to those of skill in the art. In some embodiments, the sample is selected from the group consisting of: histological sections, tissue biopsies, paraffin-embedded tissues, body fluids, surgical resection samples, isolated blood cells, cells isolated from blood, and any combination thereof. In some embodiments, the bodily fluid is selected from the group consisting of: whole blood, serum, plasma, and any combination thereof.
Based on the present disclosure, one of ordinary skill in the art can detect the methylation level of the methylation biomarker of thyroid cancer by any technique known in the art, and diagnosis of thyroid cancer is within the scope of the present invention. The above methods of detecting the methylation level of thyroid cancer methylation biomarkers in a sample include, but are not limited to, bisulfite Sequencing (BSP), methylation-specific PCR (qMSP), bisulfite microarray analysis, methylation-sensitive random primer polymerase chain reaction (MS AP-PCR), methylation-sensitive single nucleotide primer extension (MS-SNuPE), methylation-sensitive DNA restriction enzyme analysis, restriction enzyme-based sequencing, restriction enzyme-based microarray analysis, joint bisulfite restriction analysis (COBRA), methylation CpG island amplification (MCA), methylation CpG island amplification and microarray (MCAM), hpaII small fragment enrichment (HELP) by ligation-mediated PCR, methylation-specific pyrosequencing (HELP-Seq), TET-assisted pyridine borane sequencing (TAPS), gal hydrolysis and ligation-adaptor-dependent PCR (GLAD-PCR), methylation DNA immunoprecipitation sequencing (meiip-Seq) or methylation DNA immunoprecipitation-microarray analysis (meip-p-mp), and magneto-restriction enzyme-based magneto-sensitive magnetic resonance analysis using a ligation-mediated PCR.
The invention also provides a method for detecting thyroid cancer by detecting the methylation level of a methylation biomarker of thyroid cancer in a sample, which comprises the following steps: collecting peripheral blood of a subject, separating plasma or serum, extracting free DNA in a plasma sample, converting the free DNA by using a methylation conversion reagent, purifying the converted DNA, performing qPCR amplification by using the detection kit provided by the invention, and detecting the methylation level of a thyroid cancer methylation biomarker in the sample by a qMSP method so as to judge whether the plasma sample to be detected is thyroid cancer.
The following describes the above technical scheme in detail with reference to specific embodiments.
Example 1 determination of thyroid cancer methylation biomarkers and detection regions
And collecting 50 cancer tissue samples, 50 beside cancer tissue samples and 50 peripheral blood samples of healthy people which are clinically diagnosed as thyroid cancer as training sets, screening specific methylated areas, selecting areas with sensitivity and specificity of the detection tissue samples being more than or equal to 80% and detecting the areas with specificity of the peripheral blood samples being more than or equal to 90% for further verification. The collection process of all samples has been approved by the ethics committee.
The invention takes GRCh38.p14 as a reference genome, and provides a thyroid cancer methylation biomarker which is a polynucleotide molecule SEQ ID NO.1 and SEQ ID NO.2 separated based on SFRS1 genes and ST3GAL6 genes, wherein the SEQ ID NO.1 is positioned at a base fragment of 58005728-58006128 th site of a 17 th chromosome positive strand of the SFRS1 genes; SEQ ID NO.2 is a fragment of the ST3GAL6 gene at the base 98733897-98734297 of the positive strand of chromosome 3. The specific sequence is as follows:
the DNA sequence of SEQ ID NO.1 is (5 '-3'):
TTACCTTGAAATTCCACTGTTAAGACCACTGTATCCAATTCTGGTCAAAGAAAAGAATACGTGTATAACCTACCTCATGAGATCTAAACTTAGTGTTATCCAGTTTTCGAACTGCATAGGTCATATCTTCTTTCCGTACAAACTCCACGACACCAGTGCCATCTCGGTAAACATCAGCATAACATACATCACCTGCTTCACGCATGTGATCCTTTAAATCCTGCCAACTTCCACTTGGAGGCAGTCCTGAAAAAGTGATTTTTTTTTTCTTAGTACCAATTATCTTAAATTTCACGTTAAGCTGGTAAGGTACATTAGGACAAAGAGTACTTTAAAGTACTTAATAGGTGTACTTAAGTGATACCAGGTAAAGAGAGCTGGTAAGATTCTGGAATCCAGAG。
the DNA sequence of SEQ ID NO.2 is (5 '-3'):
AAAATAACGTGGTGACTGCTAGTGTATCAGAAATCACACCGAGTCTAAAGTCGTAAGTCCCATTTGCCTTACGAGGACCAGCTGGGTGCTGTGAGTAGGTGGATAGTTAAGGAAACTGTAGATCTGCTGAAAGCTCCTCGTGGTCTGTGGTCTCGTGTCCACGCTGACCTTTGAACCTCGTAACTCTTCGGGTCTCCAGCGGCCACCTCTTTTAGGCTAGCCCGAATGTTTGACTTTCCGGTCCTCAATCTTCCTCACCTAGCTGCAGACCATAAAACTGTCTTTTTATTTACAGGGAGATGTTAAATATTTGGGAACATCTGCTCAGGCTGCCAGGTTATTTTCCGTTTGAAAGCATGCCGATTTGACATTTGCAAGTTGGTTTATGTTGCTTCCTTTTT。
after the above polynucleotide molecules are subjected to bisulfite or bisulfite treatment, "C" in the double strand of DNA is converted to "U", which is then converted to "T" by the subsequent PCR, but bisulfite does not allow the above conversion of "C" of DNA which has been methylated. The sequences of SEQ ID NO.1 and SEQ ID NO.2 after bisulphite treatment are shown in Table 1.
TABLE 1 bisulphite treated sequences of SEQ ID NO.1, SEQ ID NO.2
Designing primers according to the above bisulfite-treated polynucleotide molecules, two pairs of primers are required for the sequencing by sulfide PCR (bisulfite sequencing PCR, BSP), one pair being for the bisulfite-treated methylated sequence (fully methylated and bisulfite-converted sequence); the other pair is for unmethylated sequences after bisulfite treatment (sequences that are completely unmethylated and after bisulfite conversion). According to the primer design principle that the methylation primer pair only specifically amplifies the methylation sequence and the unmethylation primer pair only specifically amplifies the unmethylation sequence, the methylation primer pair and the unmethylation primer pair designed in the embodiment are shown in table 2.
TABLE 2 nucleotide sequences of methylated primer pairs and unmethylated primer pairs
The methylation state of the target region of the SFRS1 gene and the ST3GAL6 gene in thyroid cancer tissue samples, paracancerous normal tissue samples and peripheral blood samples is detected based on the primer pair and the pyrosequencing method, and the specific steps are as follows:
1) Extraction of sample DNA
When the DNA samples are thyroid cancer tissues, paracancerous normal tissues and peripheral blood, the blood/cell/tissue genome DNA extraction kit (catalog number: DP 304) of Tiangen biochemical technology (Beijing) limited company is adopted to extract the cell genome DNA of each sample, and the specific operation is described in the kit specification.
2) Bisulphite conversion and purification treatments
The extracted genome DNA of each sample is respectively subjected to bisulphite conversion, and the nucleic acid conversion kit is a nucleic acid purification reagent (Huhan mechanical preparation 20200843) of the life technology Co., ltd., of Wuhan Ai Misen, and the specific operation is described in the specification of the kit.
3) PCR reaction
Adding SYBR Green PCR Mix methylation primer pair and unmethylation primer pair of methylation biomarker (target region of SFRS1 gene and target region of ST3GAL6 gene) of thyroid cancer by using the bisulfite converted polynucleotide molecule as template, and adding upstream and downstream primers of internal reference gene ACTB, wherein the upstream primers are: 5'-AAGGTGGTTGGGTGGTTGTTTTG-3' (SEQ ID NO. 15), the downstream primer is: 5'-AATAACACCCCCACCCTGC-3' (SEQ ID NO. 16). The PCR reaction system and the reaction conditions are shown in tables 3 and 4.
TABLE 3 SYBR Green PCR reaction System
| Component (A) | Dosage (mu L) |
| SYBR Green PCR Mix | 17.5 |
| Methylation (unmethylation) upstream primer (10. Mu.M) | 0.5 |
| Methylation (unmethylation) downstream primer (10. Mu.M) | 0.5 |
| ACTB upstream primer (10 mu M) | 0.5 |
| ACTB downstream primer (10. Mu.M) | 0.5 |
| Template DNA | 4 |
| Ultrapure water | Supplement to 25 |
TABLE 4 SYBR Green PCR reaction procedure
4) Pyrosequencing
And sending the PCR product to a sequencing company for pyrosequencing, and analyzing a sequencing peak diagram based on the methylation state of the key CpG sites of pyrosequencing. Specifically, methylation of cytosine in a CpG nucleotide is classified into two types: i.e., methylated and unmethylated, where methylation is in turn divided into fully methylated and partially methylated, a CpG dinucleotide site is considered partially methylated if the sequencing result of the cytosine at that site reveals both a C and a T at the position of the cytosine. If more than 95% of the C's in CpG dinucleotide sites in an amplicon are methylated, the sample is considered methylated in this region.
5) Analysis of results
After the PCR reaction is finished, comparing the sequencing result of the amplified product with the pathological result, and calculating the methylation state of CpG sites of the amplified product to calculate the sensitivity and the specificity of the target region. Sensitivity = methylation positive sample/sample with positive pathological result; specificity = methylation negative samples/samples with negative pathological results. The detection results are shown in Table 5.
TABLE 5 sensitivity and specificity of different target regions on training set
As can be seen from Table 5, the SFRS1 gene and the ST3GAL6 gene were used as thyroid cancer methylation biomarkers, and the methylation level in thyroid cancer tissue samples was significantly higher than that in paracancestral tissue samples. On the training set samples, the specificity of the peripheral blood sample is 88.0% by detecting the methylation level of the target region (SEQ ID NO. 1) of the SFRS1 gene, the preliminary screening standard for detecting the specificity of the peripheral blood sample is not met, the specificity is not less than 90%, the methylation level of the target region (SEQ ID NO. 2) of the ST3GAL6 gene is detected, the detection sensitivity and the specificity are both met, and the preliminary screening standard for detecting the specificity of the peripheral blood sample is not less than 90%, so that the target region (SEQ ID NO. 2) of the ST3GAL6 gene is selected to enter the next step of verification.
Example 2 detection of methylation status of target region of ST3GAL6 Gene on blood sample
The differential methylation sites obtained by screening are evaluated in the test set of 48 thyroid cancer patients and 48 healthy human blood samples by using a pyrosequencing method, and the specific process is as follows:
1) Extraction of DNA samples
When the DNA sample is a blood sample, the blood plasma cfDNA is extracted by using a magnetic bead method serum/blood plasma free DNA extraction kit (catalog number: DP 709) of Tiangen biochemical technology (Beijing) limited company, the volume of the blood plasma is 1.5mL, the specific operation is shown in the kit instruction, and 50 mu L of purified water is used for eluting after the extraction is completed.
2) Bisulphite conversion and purification treatments
The extracted genome DNA of each sample is respectively subjected to bisulphite conversion, the nucleic acid conversion kit is a nucleic acid purification reagent (Huhan mechanical equipment 20200843) of the life technology Co., ltd., wuhan Ai Misen, specific experimental operation is described in the kit instruction, and 30 mu L of purified water is used for eluting after the conversion is completed.
3) PCR reaction, pyrosequencing and analysis of the results were performed as in example 1.
4) Detection result
Thyroid cancer samples and normal samples can be distinguished by detecting the methylation status of the target region (SEQ ID NO. 2) of the ST3GAL6 gene in the test set samples. The sensitivity of the target region (SEQ ID NO. 2) of the ST3GAL6 gene for detecting the thyroid cancer blood sample was 85.4%, and the specificity for detecting the healthy human blood sample was 93.5%. Thus, selection of the target region of the ST3GAL6 gene (SEQ ID NO. 2) was chosen for validation into subsequent clinical samples.
Example 3 methylation detection kit for detection of thyroid cancer blood samples based on qMSP method
In the embodiment, after blinding an acquired sample in the test process, a test operator performs the test, and a result interpreter compares the obtained detection result with a pathological result (gold standard) according to the interpretation standard to examine the clinical effectiveness of the thyroid cancer methylation biomarker.
In order to realize noninvasive detection and further improve the sensitivity of the kit for diagnosing thyroid cancer blood samples, the embodiment incorporates 151 healthy human blood samples and 49 thyroid cancer patient blood samples. The study was approved by the institutional ethics committee to meet the development criteria, and patients all signed an informed consent document. The present implementation diagnoses thyroid cancer patients by detecting the methylation level of a thyroid cancer methylation biomarker (i.e., a target region of the ST3GAL6 gene) in a blood sample of the subject. The specific process is as follows:
1) DNA sample extraction, bisulfite conversion and purification treatments were as in example 2.
2) qPCR reaction
The detection probe is designed by taking the polynucleotide sequence after bisulphite conversion as a template, and the TaqMan probe method is used for carrying out PCR amplification on the target region of the ST3GAL6 gene on the clinical sample. Each PCR tube was added with a reaction component, a template, a methylation primer pair (SEQ ID NO. 11-12) and a detection probe (SEQ ID NO.17: 5'-TCAAAAATCAACGTAAACAC-3') of the target region of the ST3GAL6 gene, and an upstream primer and a downstream primer (SEQ ID NO. 15-16) and a detection probe (SEQ ID NO.18: 5'-GGAGTGGTTTTTGGGTTTG-3') of the internal reference gene ACTB. The 5 'end of the detection probe is a fluorescence report group, such as FAM, ROX, VIC, CY5, and the 3' end is a fluorescence quenching group, such as TAMRA, BHQ, BHQ, MGB, and the like. In this example, the fluorescent reporter group at the 5 '-end of the detection probe of the target region of the ST3GAL6 gene is ROX, the fluorescent quenching group at the 3' -end is BHQ or BHQ1, the fluorescent reporter group at the 5 '-end of the detection probe of the ACTB gene is FAM, and the fluorescent quenching group at the 3' -end is MGB. Negative and positive controls also need to be provided: the template of the negative control PCR tube is TE buffer solution, and other components are the same as those of the experimental tube; template of positive control PCR tube 10 3 copies/. Mu.L of plasmid containing the fully methylated and bisulfite converted ACTB sequence and 10 3 The other components were identical to the experimental tubes as the copies/. Mu.L of the plasmid-equivalent volume mixture containing the fully methylated and bisulfite converted target region. The qPCR reaction system and the reaction conditions are shown in Table 6 and Table 7.
TABLE 6 qPCR reaction System
TABLE 7 qPCR procedure
After qPCR reaction is completed, the baseline, which is typically a fluorescent signal of 3-15 cycles, is manually adjusted and a suitable threshold is set, which is placed in the exponential amplification phase.
3) Analysis of results
Analyzing the result of qPCR reaction, requiring (1) no amplification of negative control PCR tube; (2) the positive control PCR tube has obvious index increasing period, and the Ct value of the target area of the positive control PCR tube is between 26 and 30; (3) the Ct value of the reference gene of the sample to be detected is less than or equal to 34.
If the positive control, the negative control and the reference gene all meet the requirements, the detection result of the sample to be detected can be analyzed and the result can be interpreted, otherwise, the detection must be carried out again when the experiment is invalid. For a blood sample, the positive judgment value is 45, and if the Ct value obtained by amplifying a methylation primer pair and a detection probe of a target region of the ST3GAL6 gene is less than or equal to 45, the sample is judged to be methylation positive in the target region; if Ct value > 45 amplified using the methylation primer pair and the detection probe of the target region of the ST3GAL6 gene, the sample is judged to be methylation negative in the target region.
4) Detection result
The sensitivity of diagnosing thyroid cancer by detecting the methylation level of the target region of the ST3GAL6 gene in the blood sample by the qMSP method was 87.8%, and the specificity of detecting the blood sample of a healthy person was 93.4%. Therefore, the detection kit prepared by the invention can effectively distinguish healthy people from thyroid cancer patients, can effectively improve the detection rate of thyroid cancer, and has high detection accuracy.
It will be readily appreciated by those skilled in the art that the foregoing description is merely a preferred embodiment of the invention and is not intended to limit the invention, but any modifications, equivalents, improvements or alternatives falling within the spirit and principles of the invention are intended to be included within the scope of the invention.
Claims (10)
1. Use of a nucleic acid combination for detecting the methylation level of a thyroid cancer methylation biomarker in a sample in the preparation of a thyroid cancer diagnostic product;
taking GRCh38.p14 as a reference genome, wherein the thyroid cancer methylation biomarker is selected from at least one of (a) - (c):
(a) A full length region or a partial region of chr3:98733897-98734297, said partial region comprising at least one CpG dinucleotide site;
(b) A polynucleotide molecule in which the CpG dinucleotide site in (a) is partially or fully methylated;
(c) A polynucleotide molecule having more than 85% sequence identity to (a) wherein the CpG dinucleotide site is identical to (a).
2. The use of claim 1, wherein the thyroid cancer diagnostic product comprises at least one of a primer, a probe, a kit, a chip, a sequencing library, a membrane strip, and a protein array.
3. A nucleic acid combination for detecting thyroid cancer, characterized in that it is used to detect the methylation level of a thyroid cancer methylation biomarker in a sample;
taking GRCh38.p14 as a reference genome, wherein the thyroid cancer methylation biomarker is selected from at least one of (a) - (c):
(a) A full length region or a partial region of chr3:98733897-98734297, said partial region comprising at least one CpG dinucleotide site;
(b) A polynucleotide molecule in which the CpG dinucleotide site in (a) is partially or fully methylated;
(c) A polynucleotide molecule having more than 85% sequence identity to (a) wherein the CpG dinucleotide site is identical to (a).
4. The nucleic acid combination of claim 3, wherein the thyroid cancer methylation biomarker is selected from at least one of SEQ ID No.2 and SEQ ID No. 5.
5. The nucleic acid combination of claim 3 or 4, wherein the nucleic acid combination comprises a methylation primer pair for detecting the methylation level of the thyroid cancer methylation biomarker;
the nucleotide of the methylation primer pair is shown as SEQ ID NO. 11-12.
6. The nucleic acid combination of claim 5, further comprising a pair of unmethylated primers for detecting the methylation level of the thyroid cancer methylation biomarker;
the nucleotide of the unmethylated primer pair is shown as SEQ ID NO. 13-14.
7. The nucleic acid combination of claim 5, further comprising a detection probe; the nucleotide sequence of the detection probe is shown as SEQ ID NO. 17.
8. The nucleic acid assembly of claim 7, wherein the detection probe comprises a fluorescent reporter group at the 5 'end and a fluorescent quenching group at the 3' end.
9. A test kit for the detection of thyroid cancer, characterized in that it comprises a nucleic acid combination according to any one of claims 3 to 8.
10. The test kit of claim 9, further comprising one or more of an upstream primer and a downstream primer of the internal reference gene, a detection probe, a DNA extraction reagent, a DNA purification reagent, a methylation conversion reagent, an amplification reagent, a positive control, and a negative control.
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