CN113186293A

CN113186293A - Nucleic acid composition, kit and detection method for detecting lung cancer related gene methylation

Info

Publication number: CN113186293A
Application number: CN202110626219.2A
Authority: CN
Inventors: 谷红仓; 钱飞箭; 王云飞; 车仙荣
Original assignee: Hangzhou Shengting Medical Technology Co ltd
Current assignee: Hangzhou Shengting Medical Technology Co ltd
Priority date: 2021-06-04
Filing date: 2021-06-04
Publication date: 2021-07-30

Abstract

The invention discloses a nucleic acid composition, a kit and a detection method for detecting methylation of a lung cancer related gene; a nucleic acid composition comprising: methylation specific primers and probes of target sites of RASSF1A gene, methylation specific primers and probes of target sites of CDKN2A gene, methylation specific primers and probes of target sites of PTGER4 gene, methylation specific primers and probes of target sites of SHOX2 gene, and specific primers and probes of ACTB reference gene; the kit prepared from the nucleic acid composition disclosed by the invention has the advantages of accurate detection result of lung cancer related gene methylation, high timeliness, high detection flux, good specificity, good sensitivity and the like.

Description

Nucleic acid composition, kit and detection method for detecting lung cancer related gene methylation

Technical Field

The invention relates to the technical field of biological detection, in particular to a nucleic acid composition, a kit and a detection method for detecting lung cancer related gene methylation.

Background

Lung cancer has become one of the leading causes of cancer death in humans. Lung cancer is the most prevalent cancer in china, and morbidity and mortality have increased year by year. The incidence and mortality of lung cancer is the highest of all tumors, but lung cancer is not the most diagnosed, breast and prostate tumors have higher diagnostic rates in the united states, and early diagnosis enables early treatment, greatly improving 5-year survival (89 and 99%, respectively); the corresponding 5-year survival rate of lung cancer is 15%. At present, the 5-year overall survival rate of the stage 1 lung cancer can reach 80 percent, and if early treatment is discovered as soon as possible, the 5-year survival rate of a lung cancer patient can be greatly improved.

Unfortunately, early diagnosis of lung cancer has been difficult in clinical practice. At present, the diagnosis of cancer is mainly based on clinical symptoms, imaging detection, histopathological examination and the like, but many cancers have late clinical symptoms, and biopsy detection is difficult, so that early diagnosis of cancer and prognosis of patients are seriously influenced. The latest research shows that: abnormal methylation of DNA is prevalent in cancer cells, and a portion of small fragments of DNA (ctdna) from cancer cells enter the peripheral circulation. These small, abnormally methylated fragments of DNA from cancer cells can be used for early screening and detection of tumors. In fact, early screening and detection of tumors by abnormal methylation of ctDNA has become a hot point of research and development in international and domestic countries.

The advantages of DNA methylation as a novel molecular marker in tumor diagnosis are mainly as follows: (1) compared with normal cells of the same tissue, the tumor cells have more changed DNA methylation sites; (2) abnormal methylation of tumor DNA occurs earlier in the process of tumorigenesis; (3) DNA methylation is relatively stable. Therefore, methylation detection has great application value in early screening and diagnosis of lung cancer.

Smoking is one of the causes of lung cancer, and several genetic abnormalities which are frequently found in human lung cancer are found in the respiratory tract of normal smoking people. Among them, abnormal methylation of the CDKN2Ap16 gene has been studied in many cases. When the exfoliated cells in the sputum specimen of the normal smoking population are detected, the abnormal methylation frequency of the p16 gene is found to be higher, but the change is not found in the sputum specimen of the normal non-smoking population, which indicates that the gene change is possibly closely related to the generation process of the early respiratory tract tumor. While the degree of methylation of the promoter of the related gene in the sputum increases with the increased risk of tumor, the CDKN2A/p16 and/or CDKN2A gene can be detected in the sputum 3 years before the clear diagnosis of squamous cell lung carcinoma. Currently, there are many studies to detect the cellular or DNA methylation state in blood, sputum, alveolar lavage fluid in order to find markers for early diagnosis of lung cancer. .

SHOX2(Short status Homeobox 2), translated into a Short Homeobox gene, is a member of Homeobox gene family, is a transcription regulatory factor, and the gene expression regulation is closely related to organ development. It was found that the SHOX2 gene is expressed in mesoderm and ectoderm at embryonic stage, and plays an important role in the development of bone, heart and nervous system. In addition, SHOX2 gene methylation is associated with malignant diseases such as lung cancer, neuroblastoma, breast cancer and ovarian cancer; at present, the methylation of the SHOX2 gene is applied to clinical detection, and the role of the methylation in the diagnosis and identification of lung cancer is increasingly clear. SHOX2 can be used not only as a marker for early detection of lung cancer, but also its methylation can be used as an independent predictor of non-small cell lung cancer (NSCLC) prognosis.

RASSF1A (Ras Association Domain Family Member 1) gene encodes a protein similar to RAS effector protein, and is a potential tumor suppressor. The target gene regulated by RASSF1A relates to gene transcription, signal transduction, cytoskeleton, cell cycle, cell adhesion, apoptosis and other aspects, and has wide biological effects. RASSF1A is a novel tumor suppressor gene, and its role in the process of tumor development and development is particularly important. It is presently believed that inactivation of expression of the RASSF1A gene is primarily associated with aberrant hypermethylation, heterozygous deletions and chromosomal deletions of the promoter region. The frequency of inactivation of the RASSF1A gene in human tumors is very high. For example, the RASSF1A gene is hypermethylated and inactivated in 70% or more of small cell lung cancer, 91% of renal cell carcinoma, 62% of bladder cancer, 71% of thyroid cancer, 84% of nasopharyngeal carcinoma, 70% or more of prostate cancer, and the like.

The PTGER4(Prostaglandin E Receptor 4) gene encodes a member of the G-protein coupled Receptor family, which is one of the four receptors identified for Prostaglandin E2(PGE 2). The receptor can activate T-cytokine signaling. It has been shown to mediate PGE 2-induced expression of early growth response 1(EGR1), regulate the level and stability of cyclooxygenase 2mRNA, and lead to phosphorylation of glycogen synthase kinase 3. Related pathways include eicosanoid ligand binding to receptors and GPCR signaling. The activity of PTGER4 is mediated by g(s) proteins that stimulate adenylate cyclase. Has relaxing effect on smooth muscle. May play an important role in regulating renal hemodynamics, intestinal epithelial transport, adrenal aldosterone secretion and uterine function. Diseases associated with PTGER4 include malignant epithelial mesothelioma and basal cell carcinoma of the penis. Knockout studies in mice have shown that this receptor may be involved in neonatal adaptation of the circulation, osteoporosis and the initiation of cutaneous immune responses.

The large regulation system composed of the gene family of CDKN2A (Cyclin Dependent Kinase Inhibitor 2A, P16) and its related factors is one of the most critical regulation pathways of the cell cycle, and is coordinated with other positive and negative regulation factors to determine the normal operation of the cell cycle and cell proliferation, differentiation, aging and apoptosis, and is closely related to the occurrence, development, treatment and prognosis of various human tumors. In most cases CDKN2A was inactivated by homozygous deletion. One of the mechanisms by which CDKN2A may be inactivated is methylation of the promoter region of the gene.

At present, a nucleic acid composition and a kit which are accurate in result, fast in timeliness, high in detection flux, good in specificity and good in sensitivity cannot be found in early detection of lung cancer methylation, and the invention solves the problems, can indirectly provide effective information for early diagnosis of human lung cancer, and provides a simple, convenient and feasible method for early detection and early treatment and treatment effect monitoring of lung cancer.

Disclosure of Invention

In order to solve the defects of the prior art, the invention aims to provide the nucleic acid composition, the kit and the detection method for detecting the methylation of the lung cancer related gene.

In order to achieve the above object, the present invention adopts the following technical solutions:

a nucleic acid composition for detecting lung cancer-associated gene methylation, comprising: methylation specific primers and probes of target sites of RASSF1A gene, methylation specific primers and probes of target sites of CDKN2A gene, methylation specific primers and probes of target sites of PTGER4 gene, methylation specific primers and probes of target sites of SHOX2 gene, and specific primers and probes of ACTB reference gene;

methylation specific primers and probes for target sites of RASSF1A gene include:

a forward primer: 5 '-CGGGGTCGTTTTTGGTTTC-3' SEQ ID 01,

reverse primer: 5 '-CCGATAAATCCGTATTCGC-3' SEQ ID 02,

detecting a probe: 5 'tag-TCGCTTTGTTACGTTTAAAGT-3' tag SEQ ID 03;

methylation specific primers and probes for the target site of the CDKN2A gene comprise:

a forward primer: 5 '-ACGTCTGAGCGATGTTC-3' SEQ ID 04,

reverse primer: 5 '-TACCACGCTAACTCTACGAA-3' SEQ ID 05,

detecting a probe: 5 'tag-CTTCGACTAATACCCCGAAA-3' tag SEQ ID 06;

methylation specific primers and probes for the PTGER4 gene target site include:

a forward primer: 5 '-TGAGACGGTTTTGGAAGTC-3' SEQ ID 07,

reverse primer: 5 '-ATCTCACCATAACGACAAAACG-3' SEQ ID 08,

detecting a probe: 5 'tag-ACCCCCAAATCAACCTCAAATATCCTAA-3' tag SEQ ID 09;

methylation specific primers and probes for the target site of the SHOX2 gene include:

a forward primer: 5 '-TCGTCGCGTTTTCGTTTTTTC-3' SEQ ID10,

reverse primer: 5 '-CGACCGCTACACCAAACGA-3' SEQ ID 11,

detecting a probe: 5 'tag-CGTCGTTTTGTCGGTCGTCGGATTGTC-3' tag SEQ ID 12;

ACTB reference gene specific primers and probes include:

a forward primer: 5 '-GTGAGGAGGAGGTTAGTAAGTT-3' SEQ ID 13,

reverse primer: 5 '-GCACTTTCCGAAAACGAACG-3' SEQ ID 14,

detecting a probe: 5 'tag-ACCACCCCCAACACACAATACAAACACA-3' tag SEQ ID 15.

A kit for detecting lung cancer-associated gene methylation, comprising: PCR reaction solution, negative quality control product, positive quality control product and no-template control; the PCR reaction solution comprises: methylation specific primers and probes of target sites of RASSF1A gene, methylation specific primers and probes of target sites of CDKN2A gene, methylation specific primers and probes of target sites of PTGER4 gene, methylation specific primers and probes of target sites of SHOX2 gene, specific primers and probes of ACTB reference gene, 2X PCR buffer, dNTP, nuclease-free water, genomic DNA sample, Taq enzyme;

a forward primer: 5 '-CGGGGTCGTTTTTGGTTTC-3' SEQ ID 01,

reverse primer: 5 '-CCGATAAATCCGTATTCGC-3' SEQ ID 02,

detecting a probe: 5 'tag-TCGCTTTGTTACGTTTAAAGT-3' tag SEQ ID 03;

a forward primer: 5 '-ACGTCTGAGCGATGTTC-3' SEQ ID 04,

reverse primer: 5 '-TACCACGCTAACTCTACGAA-3' SEQ ID 05,

detecting a probe: 5 'tag-CTTCGACTAATACCCCGAAA-3' tag SEQ ID 06;

a forward primer: 5 '-TGAGACGGTTTTGGAAGTC-3' SEQ ID 07,

reverse primer: 5 '-ATCTCACCATAACGACAAAACG-3' SEQ ID 08,

detecting a probe: 5 'tag-ACCCCCAAATCAACCTCAAATATCCTAA-3' tag SEQ ID 09;

a forward primer: 5 '-TCGTCGCGTTTTCGTTTTTTC-3' SEQ ID10,

reverse primer: 5 '-CGACCGCTACACCAAACGA-3' SEQ ID 11,

detecting a probe: 5 'tag-CGTCGTTTTGTCGGTCGTCGGATTGTC-3' tag SEQ ID 12;

ACTB reference gene specific primers and probes include:

a forward primer: 5 '-GTGAGGAGGAGGTTAGTAAGTT-3' SEQ ID 13,

reverse primer: 5 '-GCACTTTCCGAAAACGAACG-3' SEQ ID 14,

detecting a probe: 5 'tag-ACCACCCCCAACACACAATACAAACACA-3' tag SEQ ID 15.

In the foregoing kit for detecting methylation of a lung cancer-related gene, the final concentration of the components of the PCR reaction solution is: 1X PCR buffer, 0.4-0.6. mu.M RASSF1A forward primer, 0.4-0.6. mu.M RASSF1A reverse primer, 0.2-0.3. mu.M MRASSF1A detection probe, 0.4-0.6. mu.M CDKN2A forward primer, 0.4-0.6. mu.M CDKN2A reverse primer, 0.2-0.3. mu.M CDKN2A detection probe, 0.4-0.6. mu.M PTGER4 forward primer, 0.4-0.6. mu.M PTGER4 reverse primer, 0.2-0.3. mu.M PTGER4 detection probe, 0.4-0.6. mu.M SHOX2 forward primer, 0.4-0.6. mu.M SH 2 reverse primer, 0.2-0.3. mu.M SH 2 detection probe, 0.1. mu.0.0.6. mu.0. mu.0.0. mu.0.0.0.0.0. mu.0.0.0.0.0.5. mu.M Taq ATPasteB/0.05-0.0.05 mM genome DNA polymerase enzyme/water/cDNA.

In the foregoing kit for detecting methylation of a lung cancer-related gene, the positive quality control substance is genomic DNA of the Hct116 cell line treated with SssI methylase.

In the kit for detecting methylation of a lung cancer related gene, the negative quality control product is genomic DNA of a Hct116 cell line with genes DNMT1 and DNMT3b knocked out.

In the kit for detecting methylation of the lung cancer related gene, the no-template control does not contain human genome DNA.

A detection method for detecting methylation of a lung cancer related gene, comprising the steps of:

the method comprises the following steps: extracting DNA of a sample to be detected, and taking the DNA after conversion treatment as a genome DNA sample of a PCR template;

step two: carrying out PCR amplification on a genome DNA sample by using a kit for detecting methylation detection of the lung cancer related gene;

the kit comprises: PCR reaction solution, negative quality control product, positive quality control product and no-template control; the PCR reaction solution comprises: methylation specific primers and probes of target sites of RASSF1A gene, methylation specific primers and probes of target sites of CDKN2A gene, methylation specific primers and probes of target sites of PTGER4 gene, methylation specific primers and probes of target sites of SHOX2 gene, specific primers and probes of ACTB reference gene, 2X PCR buffer, dNTP, nuclease-free water, genomic DNA sample, Taq enzyme;

a forward primer: 5 '-CGGGGTCGTTTTTGGTTTC-3' SEQ ID 01,

reverse primer: 5 '-CCGATAAATCCGTATTCGC-3' SEQ ID 02,

detecting a probe: 5 'tag-TCGCTTTGTTACGTTTAAAGT-3' tag SEQ ID 03;

a forward primer: 5 '-ACGTCTGAGCGATGTTC-3' SEQ ID 04,

reverse primer: 5 '-TACCACGCTAACTCTACGAA-3' SEQ ID 05,

detecting a probe: 5 'tag-CTTCGACTAATACCCCGAAA-3' tag SEQ ID 06;

a forward primer: 5 '-TGAGACGGTTTTGGAAGTC-3' SEQ ID 07,

reverse primer: 5 '-ATCTCACCATAACGACAAAACG-3' SEQ ID 08,

detecting a probe: 5 'tag-ACCCCCAAATCAACCTCAAATATCCTAA-3' tag SEQ ID 09;

a forward primer: 5 '-TCGTCGCGTTTTCGTTTTTTC-3' SEQ ID10,

reverse primer: 5 '-CGACCGCTACACCAAACGA-3' SEQ ID 11,

detecting a probe: 5 'tag-CGTCGTTTTGTCGGTCGTCGGATTGTC-3' tag SEQ ID 12;

ACTB reference gene specific primers and probes include:

a forward primer: 5 '-GTGAGGAGGAGGTTAGTAAGTT-3' SEQ ID 13,

reverse primer: 5 '-GCACTTTCCGAAAACGAACG-3' SEQ ID 14,

detecting a probe: 5 'tag-ACCACCCCCAACACACAATACAAACACA-3' tag SEQ ID 15;

step three: and determining whether the sample to be detected is methylated or not according to the fluorescence Ct values of the RASSF1A, CDKN2A, PTGER4, SHOX2 and ACTB gene amplification results.

In the aforementioned detection method for detecting methylation of a lung cancer-related gene, in the first step, the reagent used in the conversion treatment is bisulfite or bisulfite.

In the second step of the detection method for detecting methylation of a lung cancer-related gene, the reaction procedure of PCR amplification is: denaturation at 95 deg.C for 10 min; cycles 45cycles, 95 ℃ 15sec, 60 ℃ 30 sec.

The invention has the advantages that:

the invention finds that the nucleic acid composition of the specific forward primers, the specific reverse primers and the specific probes of RASSF1A, CDKN2A, SHOX2 and PTGER4 has synergistic effect on the sensitivity and the specificity of detecting the methylation of the lung cancer related genes;

the gene ACTB is used as an internal reference gene to control the quality of a sample, the situation that housekeeping genes are possibly methylated is considered, the position without CpG sites is selected when an internal reference primer and a probe are designed, and the sequence after bisulfite treatment is designed, so that the quality control of the housekeeping genes on the sample is ensured;

the invention designs the primer and the probe with high specificity, configures the kit with convenient use and reliable detection result, and combines the scientific and reasonable PCR reaction system, so that the invention has the characteristics of high speed, high flux, sensitivity and good specificity, when the kit is used for screening lung cancer, the positive detection rate (sensitivity) is 100 percent, and the negative detection rate (specificity) is 90 percent, thereby realizing the quick and accurate measurement of the methylation degree of the genes related to the lung cancer, so as to indirectly and effectively diagnose and treat the lung cancer in time, reduce the medical cost, save social resources and improve the life quality.

Drawings

FIG. 1 is a graph showing the amplification curve of a methylation positive sample of RASSF1A gene according to the present invention;

FIG. 2 is a graph showing the amplification curve of a methylation positive sample of the CDKN2A gene according to the present invention;

FIG. 3 is a graph showing the amplification curve of a methylation positive sample of PTGER4 gene according to the present invention;

FIG. 4 is a graph showing the amplification curve of a methylation positive sample of the SHOX2 gene of the present invention;

FIG. 5 is a graph of the amplification curve of a negative sample according to the present invention;

FIG. 6 is a graph showing that neither the target gene nor the reference gene of the present invention was amplified.

Detailed Description

The invention is described in detail below with reference to the figures and the embodiments.

the reagent adopted in the conversion treatment is bisulfite or bisulfite, and the sequence after the bisulfite treatment is adopted for design, so that the quality control of housekeeping genes on the sample can be ensured; the examples herein are not exhaustive, and any examples are applicable to the present invention as long as the sequences can be pre-processed.

Step two: performing PCR amplification on a genomic DNA sample by using the kit for detecting the methylation detection of the lung cancer related gene, wherein the reaction procedure of the PCR amplification is as follows: denaturation at 95 deg.C for 10 min; cycle 45cycles, 95 ℃ 15sec, 60 ℃ 30sec, it being noted that: the amplification procedure of PCR is not limited, and any procedure capable of amplifying a genomic DNA sample is applicable to the present invention;

the kit comprises: PCR reaction solution, negative quality control product, positive quality control product and no-template control; the PCR reaction solution comprises: methylation specific primers and probes of target sites of RASSF1A gene, methylation specific primers and probes of target sites of CDKN2A gene, methylation specific primers and probes of target sites of PTGER4 gene, methylation specific primers and probes of target sites of SHOX2 gene, specific primers and probes of ACTB reference gene, 2X PCR buffer, dNTP, nuclease-free water, genomic DNA sample, Taq enzyme.

Preferably, the positive control is genomic DNA of the Hct116 cell line treated with SssI methylase, the negative control is genomic DNA of the Hct116 cell line with the genes DNMT1 and DNMT3b deleted, and the no-template control indicates that no human genomic DNA is contained.

As an example, the final concentrations of the components of the PCR reaction solution were: 1 XPCR buffer, 0.4-0.6 μ M RASSF1A forward primer, 0.4-0.6 μ M RASSF1A reverse primer, 0.2-0.3 μ M RASSF1A detection probe, 0.4-0.6 μ M CDDKN 2A forward primer, 0.4-0.6 μ M CDKN2A reverse primer, 0.2-0.3 μ M CDKN2A detection probe, 0.4-0.6 μ M PTGER4 forward primer, 0.4-0.6 μ M PTGER4 reverse primer, 0.2-0.3 μ M PTGER4 detection probe, 0.4-0.6 μ M SHOX2 forward primer, 0.4-0.6 μ M SH 2 reverse primer, 0.2-0.3 μ M SHGER 2 detection probe, 0.1.1 μ M SHOX2 forward primer, 0.4-0.6 μ M SH 2 reverse primer, 0.05-0.05 μ M Taq ATP DNA polymerase, 0.05-0.05 μ M DNA polymerase enzyme/DNA: the reagent formulation of the reaction solution is not limited, and is provided as a preferred embodiment, and other reaction solution formulations capable of being matched with the amplification of the sample DNA are also applicable to the present invention.

a forward primer: 5 '-CGGGGTCGTTTTTGGTTTC-3' SEQ ID 01,

reverse primer: 5 '-CCGATAAATCCGTATTCGC-3' SEQ ID 02,

detecting a probe: 5 'tag-TCGCTTTGTTACGTTTAAAGT-3' tag SEQ ID 03, preferably, the 5 'tag is FAM and the 3' tag is BHQ 1;

a forward primer: 5 '-ACGTCTGAGCGATGTTC-3' SEQ ID 04,

reverse primer: 5 '-TACCACGCTAACTCTACGAA-3' SEQ ID 05,

detecting a probe: 5 'tag-CTTCGACTAATACCCCGAAA-3' tag SEQ ID 06, preferably, the 5 'tag is ROX and the 3' tag is BHQ 2;

a forward primer: 5 '-TGAGACGGTTTTGGAAGTC-3' SEQ ID 07,

reverse primer: 5 '-ATCTCACCATAACGACAAAACG-3' SEQ ID 08,

detecting a probe: 5 'tag-ACCCCCAAATCAACCTCAAATATCCTAA-3' tag SEQ ID 09, as a preferred, 5 'tag is TAmRA and 3' tag is BHQ 2;

a forward primer: 5 '-TCGTCGCGTTTTCGTTTTTTC-3' SEQ ID10,

reverse primer: 5 '-CGACCGCTACACCAAACGA-3' SEQ ID 11,

detecting a probe: 5 'tag-CGTCGTTTTGTCGGTCGTCGGATTGTC-3' tag SEQ ID 12, preferably, the 5 'tag is VIC and the 3' tag is BHQ 1;

ACTB reference gene specific primers and probes include:

a forward primer: 5 '-GTGAGGAGGAGGTTAGTAAGTT-3' SEQ ID 13,

reverse primer: 5 '-GCACTTTCCGAAAACGAACG-3' SEQ ID 14,

detecting a probe: 5 ' CY5-ACCACCCCCAACACACAATACAAACACA-3 ' marker SEQ ID 15, preferably, the 5 ' marker is CY5 and the marker is BHQ 2;

remarking: the purity of the primers should be PAGE or HPLC grade.

BHQ1 and BHQ2 are preferable as the quenching marker group, and a fluorescent marker is also preferable, and any product that can be used for labeling can be applied to the present invention.

Here, it is to be emphasized that: the detection method of the present invention is not limited, and any detection method using the nucleic acid composition of the present invention is not specifically exemplified herein, as long as it is within the scope of the present invention.

The technical effects of the invention are verified through experiments as follows:

first, a kit for an experiment was prepared using the following procedure.

Firstly, materials: a kit for detecting methylation of a lung cancer-associated gene, comprising: methylation specific primers and hydrolysis probes of target sites of RASSF1A gene; methylation specific primers and hydrolysis probes of target sites of the CDKN2A gene; methylation specific primers and hydrolysis probes of the PTGER4 gene target site; methylation specific primers and hydrolysis probes of the target site of the SHOX2 gene; primers and hydrolysis probes of ACTB reference genes;

a forward primer: 5 '-CGGGGTTCGTTTTGTGGTTTC-3' (SEQ ID NO.1)

Reverse primer: 5 '-CCGATTAAATCCGTACTTCGC-3' (SEQ ID NO.2)

Detecting a probe: 5 'FAM-TCGCGTTTGTTAGCGTTTAAAGT-3' BHQ1(SEQ ID NO.3)

a forward primer: 5 '-ACGTCGTGAGCGAGTGTTC-3' (SEQ ID NO.4)

Reverse primer: 5 '-TACCAACGCTAACTCTAACGAA-3' (SEQ ID NO.5)

Detecting a probe: 5 'ROX-CTTCCGACTAATACCCCCGAAA-3' BHQ2(SEQ ID NO.6)

a forward primer: 5 '-TGAGATCGGTTTTGAGAAGTC-3' (SEQ ID NO.7)

Reverse primer: 5 '-ATCTCACCTATAACGACAACAACG-3' (SEQ ID NO.8)

Detecting a probe: 5 'TAMRA-ACCCCCAAAATCAACCCTCAAATATCCTAA-3' BHQ2(SEQ ID NO.9)

a forward primer: 5 '-TCGTCGTCGTTTTCGTTTTTTTC-3' (SEQ ID NO.10)

Reverse primer: 5 '-CGACCGACTACACCGAAACGA-3' (SEQ ID NO.11)

Detecting a probe: 5 'VIC-CGTCGTTTTCGTCGGTCGTTCGGATTGTC-3' BHQ1(SEQ ID NO.12)

ACTB reference gene specific primers and probes include:

a forward primer: 5 '-GTGATGGAGGAGGTTTAGTAAGTT-3' (SEQ ID NO.13)

Reverse primer: 5 '-GCACTCTTCCGAAAACGAAACG-3' (SEQ ID NO.14)

Detecting a probe: 5 'CY 5-ACCACCACCCAACACACAATAACAAACACA-3' BHQ2(SEQ ID NO.15)

Selecting a positive quality control product and a negative quality control product:

positive quality control product and negative quality control product. The positive quality control product is the genome DNA of the Hct116 cell line treated by SssI methylase, and can ensure that C in all CpG sequences in the genome is methylated at the position of C5; the negative quality control product is the genome DNA of the Hct116 cell line with genes DNMT1 and DNMT3b knocked out;

thirdly, PCR reaction solution composition:

the kit comprises a PCR reaction solution containing the specific primer and the probe, wherein the PCR reaction solution comprises:

RASSF1A forward primer: 5' -CGGGGTTCGTTTTGTGGTTTC-3

RASSF1A reverse primer: 5' -CCGATTAAATCCGTACTTCGC-3

RASSF1A detection probe: 5 'FAM-TCGCGTTTGTTAGCGTTTAAAGT-3' BHQ1

CDKN2A forward primer: 5' -ACGTCGTGAGCGAGTGTTC-3

CDKN2A reverse primer: 5' -TACCAACGCTAACTCTAACGAA-3

CDKN2A detection probe: 5 'ROX-CTTCCGACTAATACCCCCGAAA-3' BHQ2

PTGER4 forward primer: 5' -TGAGATCGGTTTTGAGAAGTC-3

PTGER4 reverse primer: 5' -ATCTCACCTATAACGACAACAACG-3

PTGER4 detection probe: 5 'TAMRA-ACCCCCAAAATCAACCCTCAAATATCCTAA-3' BHQ2

SHOX2 forward primer: 5' -TCGTCGTCGTTTTCGTTTTTTTC-3

SHOX2 reverse primer: 5' -CGACCGACTACACCGAAACGA-3

SHOX2 detection probe: 5 'VIC-CGTCGTTTTCGTCGGTCGTTCGGATTGTC-3' BHQ1

ACTB forward primer: 5' -GTGATGGAGGAGGTTTAGTAAGTT-3

ACTB reverse primer: 5' -GCACTCTTCCGAAAACGAAACG-3

ACTB detection probe: 5 'CY 5-ACCACCACCCAACACACAATAACAAACACA-3' BHQ2

2X PCR buffer, Taq enzyme, dNTP and nuclease-free water. Were purchased from biotechnology, inc.

The final concentration of the PCR reaction solution components was:

1 XPCR buffer, 0.6. mu.M (. mu.mol/L) RASSF1A forward primer, 0.6. mu.M (. mu.mol/L) RASSF1A reverse primer, 0.3. mu.M (. mu.mol/L) RASSF1A detection probe, 0.6. mu.M (. mu.mol/L) SHOX2 forward primer, 0.6. mu.M (. mu.mol/L) SHOX2 reverse primer, 0.3. mu.M (. mu.mol/L) SHOX2 detection probe, 0.6. mu.M (. mu.mol/L) CDKN2A forward primer, 0.6. mu.M (. mu.mol/L) CDKN2 reverse primer, 0.3. mu.M (. mu.mol/L) CDKN2 detection probe, 0.6. mu.M (. mu.mol/L) GERR 4 forward primer, 0.6. mu.M (. mu.mol/L) GER 6. mu.4834 reverse primer, 0.M ACT/L detection probe, 0.0.25. mu.M GER 2 detection probe, 0.25. mu.25. mu.M ACTB detection probe, 0.25mM (mmol/L) dNTP;

(II) detecting the methylation of the lung cancer related gene by using the kit sample prepared by the method:

first, technical principle

A pair of specific primers and probes are designed in the promoter regions of the lung cancer related gene and the reference gene in the human genome. Then, the primers and the probes are used for amplifying the sample DNA converted by the bisulfite, whether the sample to be detected is methylated or not is determined according to the Ct value of the relative fluorescence value of the PCR amplification result of the related genes, and the risk of the lung cancer is indirectly determined according to the methylation.

Second, detection method

The method comprises the following steps: and extracting DNA of a sample to be detected, carrying out bisulfite conversion treatment on the DNA, and carrying out qPCR amplification by using the converted DNA as a PCR template.

Wherein, the reagent used for the Conversion treatment is Bisulfite or Bisulfite, and other auxiliary reagents (corresponding reagents are purchased from epiect Fast bisulfate Conversion Kit of QIAGEN, Germany);

step two: providing the kit for detecting the methylation of the lung cancer related genes, and carrying out PCR amplification on the template;

step three: determining whether the sample to be detected is methylated or not according to the relative fluorescence value Ct of the PCR amplification result of the related gene;

the specific detection method comprises the following steps:

one) plasma separation

1. 10ml of peripheral blood was drawn using a free DNA sampling tube and plasma separation was performed the first time after arrival at the laboratory.

2. Centrifuging at 1600rpm for 20min at 4 deg.C, and packaging the plasma in sterile centrifuge tubes.

3. The plasma after the primary separation was centrifuged at 16,000rpm for 10min at 4 ℃ to remove residual cells. After centrifugation, the plasma was dispensed into sterile centrifuge tubes.

II) extracting plasma DNA:

1. 2ml of plasma samples were taken and the corresponding amounts of reagents were added as shown in Table 1 according to the following system:

TABLE 1

Volume of plasma	Proteinase K	Buffer GHP	FineMag Particles K
				2ml	200μl	3.0ml	30μl

2. After the sample and the reagent are fully mixed, the mixture is incubated at room temperature for 20min, and the mixture is inverted and mixed for 10sec every 3-5min, so that the magnetic beads and the nucleic acids are fully combined. After incubation, removing liquid drops on the inner wall of the tube wall by brief centrifugation;

3. placing the centrifugal tube in a magnetic force and standing for 2min, carefully removing liquid when the magnetic beads are completely adsorbed, and taking down the centrifugal tube;

4. adding 1ml Buffer RBP, shaking and mixing for 1min to make the magnetic beads fully suspended, and centrifuging briefly to remove the dropping liquid on the inner wall;

5. placing the centrifugal tube in a magnetic force and standing for 1min, carefully removing liquid when the magnetic beads are completely adsorbed, and taking down the centrifugal tube;

6. adding 1ml 80% ethanol (ready for use), shaking and mixing for 1min each time to suspend the magnetic beads thoroughly, and centrifuging briefly to remove the inner wall dripping liquid.

7. Placing the centrifugal tube on magnetic force and standing for 1min, carefully removing liquid when the magnetic beads are completely adsorbed, and taking down the centrifugal tube.

8. Repeat step 6-7 once, total 2 ethanol elution.

9. Placing the centrifugal tube on magnetic force, and air drying at room temperature for 5-10 min.

10. And (3) taking down the centrifugal tube from the magnetic force, adding 45 mu l of Buffer EB, shaking and uniformly mixing to suspend the magnetic beads, incubating at 56 ℃ for 5min, slightly shaking the centrifugal tube every 2min during the incubation period to fully absorb and strip the nucleic acid, and then centrifuging for a short time to remove the liquid on the tube wall and the tube cover.

11. The centrifuge tube was placed on a magnetic rack and allowed to stand for 2min, and when the magnetic beads were completely adsorbed, the DNA solution was carefully transferred to a 1.5ml collection tube and stored under appropriate conditions.

12. Taking 1 mul of DNA eluent to carry out the concentration determination of the Qubit HS, recording the data result, and storing the rest samples in a refrigerator at the temperature of-20 ℃ if the experiment is not continued.

Third) DNA transformation procedure:

1. the extracted DNA was formulated into a bisulfite conversion system according to the following formulation as shown in Table 2;

TABLE 2

Reagent composition	Reaction volume (μ l)
		Extracted DNA	40μl
Bisulfite Solution	85
		DNA protect Buffer	15
Total of	140

2. After the transformation system is configured, placing the obtained product in a PCR instrument for reaction according to the following reaction conditions, wherein the reaction program is shown in Table 3;

TABLE 3

3. DNA purification after bisulfite conversion:

4. the PCR tube was transiently detached and the bisulfite converted product was transferred to a 1.5ml EP tube.

5. To each sample was added 310. mu.l of Buffer BL reagent

6. To each sample was added 250. mu.l of absolute ethanol, the solution was vortexed for 15 seconds and briefly centrifuged.

7. Transferring the mixture to a spin column, and centrifuging at 14000rpm for 1min

8. The filtrate was discarded, 500. mu.l of Buffer BW was added, and the mixture was centrifuged at 14000rpm for 1min

9. Discarding the filtrate, adding 500. mu.l Buffer BD, incubating for 15min, and centrifuging at 14000rpm for 1min

10. The filtrate was discarded, 500. mu.l of Buffer BW was added, and the mixture was centrifuged at 14000rpm for 1min

11. Repeat step 10

12. Discarding the filtrate, adding 250 μ l of anhydrous ethanol, centrifuging at 14000rpm for 1min

13. Discarding the filtrate, leaving at 14000rpm for 1min

14. Drying at room temperature for 3-5min to remove residual ethanol

15. Add 15. mu.l Buffer EB, incubate and elute at room temperature for 1min, centrifuge at 14000rpm for 1min, collect DNA and store at-20 ℃.

Four) qPCR reaction configuration

1. The qPCR instrument used was a real-time fluorescent quantitative PCR system (available from life technology, Inc.) model ABI 7500, USA, with a reaction system of 20. mu.l

2. qPCR reaction system configuration and conditions, as shown in table 4 below:

TABLE 4

Composition (I)	Reaction volume (μ l)
		DNA after bisulfite conversion	2.0
Primers	5.6
		Probe	1.4
RNase-free Water	1.0
		2X PCR Mix	10.0
Total of	20

3. PCR reaction sample addition layout:

3 parallel detections are carried out on the DNA sample to be detected, the positive quality control product, the negative quality control product and the template-free control, and the 96-hole sample adding layout of the PCR instrument is shown in the following table 5. In the table, PC represents a Positive Control (Positive Control), NC represents a Negative Control (Negative Control), NTC represents a No-Template Control (No Template Control), and S represents a test sample (sample)

TABLE 5

1

2

3

4

5

6

7

8

9

10

11

12

A

PC

S7

B

NC

S8

C

S1

S9

D

S2

S10

E

S3

S1..

F

S4

NTC

G

S5

H

S6

4. The PCR reaction procedure is shown in table 6:

TABLE 6

5. The results are shown in Table 7 and analyzed as follows

1) Running amplification curve analysis without template control (NTC), wherein no amplification curve is required, which indicates that the experiment is pollution-free and can continue to analyze;

2) the internal reference genes (ACTB) of the quality control product should have amplification signals and present an S-shaped amplification curve, and the internal reference Ct values should accord with the results of the following table 7; the target gene of the negative quality control product rises due to no amplification curve, the Ct value of the gene of the positive quality control product accords with the following table, and when the quality control product detection meets the following conditions, the experiment is proved to be effective, and the analysis can be continued;

TABLE 7

3) The internal reference genes of the sample have amplification signals and are in an S-shaped amplification curve, and the PCR detection result of the sample is interpreted according to the following table 8;

TABLE 8

Results of samples	Target gene	ACTB
			Positive for methylation	Ct≤40	Ct≤40
Methylation negative	No amplification, or Ct > 40	Ct≤40
			Failure of the experiment	Any result	Ct＞40

Referring to fig. 1-6, fig. 1 is a graph of amplification of methylation positive samples of RASSF1A gene, fig. 2 is a graph of amplification of methylation positive samples of CDKN2A gene, fig. 3 is a graph of amplification of methylation positive samples of PTGER4 gene, fig. 4 is a graph of amplification of methylation positive samples of SHOX2 gene, fig. 5 is a graph of amplification of negative samples, it can be seen that the relationship between the amplification curves of lung cancer related methylation genes and ACTB internal reference genes in different methylation samples, and if there is no amplification curve in both target genes and internal reference genes, fig. 6 shows that the samples need to be repeatedly detected or re-sampled if they are disqualified.

(III) the kit samples are used for carrying out sensitivity and specificity detection on lung cancer and normal human plasma:

plasma samples from 20 normal persons and 20 lung cancer patients were used as the subjects to be monitored. Free DNA was extracted from each sample. The extraction of DNA can be carried out by any standard means known in the art, and specifically, in this case, the sample DNA used is extracted by using a specific extraction protocol according to the detection system of example 2.

The DNA sample is then pretreated to convert the 5' unmethylated cytosine to uracil. In this experimental case, the pretreatment was achieved by bisulfite reagent treatment. The modification of bisulfite DNA was pretreated by conversion using the detection system in example 2.

Then, the pretreated DNA samples of 20 normal persons and 20 cases of lung cancer were added to the test system of the above case 2 to test the RASSF1A, SHOX2, PTGER4, CDKN2A and the reference gene ACTB in a multiplex manner. Real-time PCR was performed on bisulfite converted DNA.

Wherein, the PCR conditions adopted in the experimental case are carried out according to the qPCR conditions in the step (II) above for detecting the methylation of the lung cancer related gene by using the kit sample prepared above.

Finally, tables 1 and 2 below show the results of testing 20 normal human and 20 lung cancer patient samples using the multiplex assay of the present invention. As can be seen from tables 9 and 10, the positive detection rate and the negative detection rate of this protocol were high.

TABLE 9 results of plasma sample detection of lung cancer patients using the kit of the present invention

Sample positive determination method	Number of samples to be tested	Number of positive samples	Positive rate
				At least one target positive	20	20	100％

TABLE 10 results of the measurement of a plasma sample of a normal human by using the kit of the present invention

Sample positive determination method	Number of samples to be tested	Number of negative samples	Negative rate
				At least one target positive	20	18	90％

According to the detection results in the table, when the kit is used for lung cancer screening, the positive detection rate (sensitivity) is 100%, and the negative detection rate (specificity) is 90%.

From the above verification experiments, it can be seen that: the nucleic acid composition of the RASSF1A, CDKN2A, SDC and PTGER4 specific forward primers, the specific reverse primers and the specific probe have a synergistic effect on the sensitivity and specificity of detecting the methylation of the lung cancer related genes, and the detection of the methylation of the lung cancer by combining the RASSF1A, CDKN2A, SDC and PTGER4 specific forward primers, the specific reverse primers and the specific probe is an innovative finding, and is not a result obtained by mechanization experiments.

The invention is not aimed at diagnosing diseases, and the obtained detection can not directly judge whether the diseases exist, indirectly provides effective information for early diagnosis of the human lung cancer, and provides a simple, convenient and feasible method for early detection and treatment of the human lung cancer.

The foregoing illustrates and describes the principles, general features, and advantages of the present invention. It should be understood by those skilled in the art that the above embodiments do not limit the present invention in any way, and all technical solutions obtained by using equivalent alternatives or equivalent variations fall within the scope of the present invention.

Sequence listing

<110> Hangzhou shengting medical technology Co Ltd

<120> nucleic acid composition, kit and detection method for detecting methylation of lung cancer related genes

<141> 2021-06-04

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Claims

1. A nucleic acid composition for detecting methylation of a lung cancer-associated gene, comprising: methylation specific primers and probes of target sites of RASSF1A gene, methylation specific primers and probes of target sites of CDKN2A gene, methylation specific primers and probes of target sites of PTGER4 gene, methylation specific primers and probes of target sites of SHOX2 gene, and specific primers and probes of ACTB reference gene;

the methylation specific primers and probes of the RASSF1A gene target site comprise:

a forward primer: 5 '-CGGGGTCGTTTTTGGTTTC-3' SEQ ID 01,

reverse primer: 5 '-CCGATAAATCCGTATTCGC-3' SEQ ID 02,

detecting a probe: 5 'tag-TCGCTTTGTTACGTTTAAAGT-3' tag SEQ ID 03;

the methylation specific primers and probes for the CDKN2A gene target site comprise:

a forward primer: 5 '-ACGTCTGAGCGATGTTC-3' SEQ ID 04,

reverse primer: 5 '-TACCACGCTAACTCTACGAA-3' SEQ ID 05,

detecting a probe: 5 'tag-CTTCGACTAATACCCCGAAA-3' tag SEQ ID 06;

the methylation specific primers and probes of the PTGER4 gene target site comprise:

a forward primer: 5 '-TGAGACGGTTTTGGAAGTC-3' SEQ ID 07,

reverse primer: 5 '-ATCTCACCATAACGACAAAACG-3' SEQ ID 08,

detecting a probe: 5 'tag-ACCCCCAAATCAACCTCAAATATCCTAA-3' tag SEQ ID 09; the methylation specific primers and probes of the target site of the SHOX2 gene comprise:

a forward primer: 5 '-TCGTCGCGTTTTCGTTTTTTC-3' SEQ ID10,

reverse primer: 5 '-CGACCGCTACACCAAACGA-3' SEQ ID 11,

detecting a probe: 5 'tag-CGTCGTTTTGTCGGTCGTCGGATTGTC-3' tag SEQ ID 12;

the ACTB reference gene specific primers and probes comprise:

a forward primer: 5 '-GTGAGGAGGAGGTTAGTAAGTT-3' SEQ ID 13,

reverse primer: 5 '-GCACTTTCCGAAAACGAACG-3' SEQ ID 14,

detecting a probe: 5 'tag-ACCACCCCCAACACACAATACAAACACA-3' tag SEQ ID 15.

2. A kit for detecting methylation of a lung cancer-associated gene, comprising: PCR reaction solution, negative quality control product, positive quality control product and no-template control; the PCR reaction solution comprises: methylation specific primers and probes of target sites of RASSF1A gene, methylation specific primers and probes of target sites of CDKN2A gene, methylation specific primers and probes of target sites of PTGER4 gene, methylation specific primers and probes of target sites of SHOX2 gene, specific primers and probes of ACTB reference gene, 2X PCR buffer, dNTP, nuclease-free water, genomic DNA sample, Taq enzyme;

a forward primer: 5 '-CGGGGTCGTTTTTGGTTTC-3' SEQ ID 01,

reverse primer: 5 '-CCGATAAATCCGTATTCGC-3' SEQ ID 02,

detecting a probe: 5 'tag-TCGCTTTGTTACGTTTAAAGT-3' tag SEQ ID 03;

a forward primer: 5 '-ACGTCTGAGCGATGTTC-3' SEQ ID 04,

reverse primer: 5 '-TACCACGCTAACTCTACGAA-3' SEQ ID 05,

detecting a probe: 5 'tag-CTTCGACTAATACCCCGAAA-3' tag SEQ ID 06;

a forward primer: 5 '-TGAGACGGTTTTGGAAGTC-3' SEQ ID 07,

reverse primer: 5 '-ATCTCACCATAACGACAAAACG-3' SEQ ID 08,

detecting a probe: 5 'tag-ACCCCCAAATCAACCTCAAATATCCTAA-3' tag SEQ ID 09;

the methylation specific primers and probes of the target site of the SHOX2 gene comprise:

a forward primer: 5 '-TCGTCGCGTTTTCGTTTTTTC-3' SEQ ID10,

reverse primer: 5 '-CGACCGCTACACCAAACGA-3' SEQ ID 11,

detecting a probe: 5 'tag-CGTCGTTTTGTCGGTCGTCGGATTGTC-3' tag SEQ ID 12;

the ACTB reference gene specific primers and probes comprise:

a forward primer: 5 '-GTGAGGAGGAGGTTAGTAAGTT-3' SEQ ID 13,

reverse primer: 5 '-GCACTTTCCGAAAACGAACG-3' SEQ ID 14,

detecting a probe: 5 'tag-ACCACCCCCAACACACAATACAAACACA-3' tag SEQ ID 15.

3. The kit for detecting methylation of a lung cancer-related gene according to claim 2, wherein the PCR reaction solution comprises the following components in final concentration: 1 XPCR buffer, 0.4-0.6 mu M RASSF1A forward primer, 0.4-0.6 mu M RASSF1A reverse primer, 0.2-0.3 mu M RASSF1A detection probe, 0.4-0.6 mu M CDKN2A forward primer, 0.4-0.6 mu M CDKN2A reverse primer, 0.2-0.3 mu M CDKN2A detection probe, 0.4-0.6 mu M PTGER4 forward primer, 0.4-0.6 mu M PTGER4 reverse primer, 0.2-0.3 mu M PTGER4 detection probe, 0.4-0.6 mu M SHOX2 forward primer, 0.4-0.6 mu M SH 2 reverse primer, 0.2-0.3 mu M SH 2 detection probe, 0.4-0.6 mu M SHOX2 forward primer, 0.0.0-0.05 mu M Taq DNA polymerase chain terminator, 0.05-0.05 mM DNA polymerase chain terminator, 0.05-0.05 DNA polymerase chain terminator, and MACOX DNA.

4. The kit of claim 2, wherein the positive quality control product is genomic DNA of Hct116 cell line treated with SssI methylase.

5. The kit of claim 2, wherein the negative quality control product is genomic DNA of Hct116 cell line after knocking out genes DNMT1 and DNMT3 b.

6. A detection method for detecting methylation of a lung cancer-associated gene, comprising the steps of:

a forward primer: 5 '-CGGGGTCGTTTTTGGTTTC-3' SEQ ID 01,

reverse primer: 5 '-CCGATAAATCCGTATTCGC-3' SEQ ID 02,

detecting a probe: 5 'tag-TCGCTTTGTTACGTTTAAAGT-3' tag SEQ ID 03;

a forward primer: 5 '-ACGTCTGAGCGATGTTC-3' SEQ ID 04,

reverse primer: 5 '-TACCACGCTAACTCTACGAA-3' SEQ ID 05,

detecting a probe: 5 'tag-CTTCGACTAATACCCCGAAA-3' tag SEQ ID 06;

a forward primer: 5 '-TGAGACGGTTTTGGAAGTC-3' SEQ ID 07,

reverse primer: 5 '-ATCTCACCATAACGACAAAACG-3' SEQ ID 08,

detecting a probe: 5 'tag-ACCCCCAAATCAACCTCAAATATCCTAA-3' tag SEQ ID 09;

a forward primer: 5 '-TCGTCGCGTTTTCGTTTTTTC-3' SEQ ID10,

reverse primer: 5 '-CGACCGCTACACCAAACGA-3' SEQ ID 11,

detecting a probe: 5 'tag-CGTCGTTTTGTCGGTCGTCGGATTGTC-3' tag SEQ ID 12;

the ACTB reference gene specific primers and probes comprise:

a forward primer: 5 '-GTGAGGAGGAGGTTAGTAAGTT-3' SEQ ID 13,

reverse primer: 5 '-GCACTTTCCGAAAACGAACG-3' SEQ ID 14,

detecting a probe: 5 'tag-ACCACCCCCAACACACAATACAAACACA-3' tag SEQ ID 15;

7. The method as claimed in claim 6, wherein in the first step, the reagent used in the conversion treatment is bisulfite or bisulfite.

8. The method according to claim 6, wherein in the second step, the PCR amplification reaction procedure is as follows: denaturation at 95 deg.C for 10 min; cycles 45cycles, 95 ℃ 15sec, 60 ℃ 30 sec.