WO2024036802A1 - 一种用于肺腺癌早期诊断的多基因甲基化试剂盒 - Google Patents

一种用于肺腺癌早期诊断的多基因甲基化试剂盒 Download PDF

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WO2024036802A1
WO2024036802A1 PCT/CN2022/134024 CN2022134024W WO2024036802A1 WO 2024036802 A1 WO2024036802 A1 WO 2024036802A1 CN 2022134024 W CN2022134024 W CN 2022134024W WO 2024036802 A1 WO2024036802 A1 WO 2024036802A1
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methylation
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王传新
杜鲁涛
李培龙
李娟�
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Shandong University
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  • the invention belongs to the field of tumor molecular biology, and specifically relates to a multi-gene methylation kit for early diagnosis of lung adenocarcinoma.
  • Lung cancer is one of the most common malignant tumors of the respiratory system and the leading cause of cancer-related deaths, seriously endangering human health.
  • lung cancer includes small cell lung cancer and non-small cell lung cancer, of which non-small cell lung cancer accounts for about 85%.
  • non-small cell lung cancers lung adenocarcinoma accounts for approximately 50%.
  • Adenocarcinoma is the most common pathological type of lung cancer.
  • the prognosis of lung adenocarcinoma is still not optimistic. Among them, a very important reason is that there are certain difficulties in the early diagnosis of lung adenocarcinoma.
  • DNA methylation plays an important role in the occurrence and development of tumors.
  • DNA methylation refers to the addition of a methyl group provided by S-adenosylmethionine to the fifth carbon atom of cytosine under the action of methyltransferase. It usually occurs in CpG islands and is important for maintaining the structure of chromosomes. It plays an important role in stabilizing and regulating gene expression.
  • hypermethylation inhibits gene expression, while hypomethylation promotes gene expression.
  • Hypomethylation of proto-oncogenes and/or hypermethylation of tumor suppressor genes can promote the occurrence and development of tumors.
  • NCOA2 is a member of the NCOA family. Studies have shown that the NCOA family, especially NCOA2 and NCOA3, are involved in normal biological processes and carcinogenesis. For example, activation of NCOA3 promotes tumor growth in pancreatic ductal adenocarcinoma. NCOA2-mediated co-activation of anti-tumor target genes inhibits MYC-induced liver cancer. Inhibition of NCOA2 severely attenuates prostate cancer survival, growth, and metastasis. In lung cancer, NCOA2 can be phosphorylated by PFKFB4 at Ser487, which changes the transcriptional activity of NCOA2, thereby promoting lung cancer cell proliferation, migration, and invasion.
  • RUNX family transcription factors are key regulators of development and are often dysregulated in cancer.
  • RUNX3 is a member of the RUNX family and has been described as a tumor promoter or suppressor, reflecting its complex role in tumorigenesis.
  • RUNX3 is a downstream effector molecule of the TGF- ⁇ signaling pathway and plays an important role in regulating cell death, angiogenesis, endothelial-mesenchymal transition, cell migration and invasion.
  • Runx3 silencing regulates YBX1 expression through miR-148a-3p and promotes the progression of non-small cell lung cancer by increasing the levels of Cyclin D1, Cyclin B1, Slug-1, MMP-2 and MMP-9.
  • RUNX3 was identified as a key factor in the propensity of pancreatic ductal adenocarcinoma cells to proliferate locally or disseminate throughout the body. Overexpression of RUNX3 promotes the growth and inhibits apoptosis of head and neck cancer cells. In addition, studies have shown that RUNX3 plays an important role in immunity and inflammation, so RUNX3 may indirectly affect the occurrence and development of tumors.
  • BTD biotinidase
  • the BTD (biotinidase) gene is located on chromosome 3p25.
  • 61 mutations in three of the four exons of BTD and 1 mutation in an intronic gene have been reported, leading to severe BTD defects.
  • BTD is an enzyme that can cleave biocytin, which is a product of proteolytic digestion by holocarboxylase.
  • Severe BTD deficiency (average normal activity in serum is less than 10%) is an autosomal recessive disorder in which patients are unable to cleave biocytin and cannot recycle biotin.
  • Biotinidase deficiency is characterized primarily by neurological, cutaneous manifestations, and metabolic abnormalities.
  • BTD genes have been rarely reported in tumors and deserve further study.
  • BRE also known as TNFRSF1A regulator and BRCC45, is an evolutionarily highly conserved protein. It is a death receptor-associated protein in the cytoplasm and a component of the BRCA1/2-containing DNA repair complex in the nucleus. Studies have found that BRE has anti-apoptotic activity. High expression of BRE can enhance the growth of esophageal squamous cell carcinoma cells by promoting cell cycle progression and resisting apoptosis. Silencing of BRE inhibits these malignant phenotypes of esophageal squamous cell carcinoma cells. Mechanistic studies showed that BRE overexpression activated the phosphorylation of AKT, and inhibition of the AKT pathway by MK2206 reduced BRE-induced growth and apoptosis resistance of esophageal squamous cell carcinoma cells.
  • ARID1B is located at 6q25.3 and is a typical nuclear tumor suppressor. However, cytoplasm-localized ARID1B can bind c-RAF (RAF1) and PPP1CA, thereby stimulating RAF-ERK signaling and ⁇ -catenin (CTNNB1) transcriptional activity. ARID1B cytoplasmic localization was associated with increased active forms of ERK1 and ERK2 (also known as MAPK3 and MAPK1) and ⁇ -catenin levels, significantly correlated with advanced tumor stage and lymph node positivity levels. In breast cancer, high expression of ARID1B is closely related to histological grade and tumor size of invasive breast cancer.
  • ARID1B expression is often increased in aggressive triple-negative breast cancer subtypes and is associated with reduced five-year disease-free survival. Compared with controls, MDA-MB-231 cells with reduced ARID1B activity showed a delay in the G1 to S phase cell cycle transition and therefore reduced cell proliferation. Medulloblastoma is a common malignant brain tumor in children. Decreased expression of ARID1B leads to a significant increase in the malignant potential of medulloblastoma cells. In bladder urothelial carcinoma, ARID1B expression was significantly associated with tumor size, TNM stage, overall survival, and progression-free survival.
  • ARID1B may be a predictive marker for selecting patients for adjuvant chemotherapy in high-risk subgroups.
  • non-small cell lung cancer 7% of patients with non-small cell lung cancer carry ARID1A mutations, and 4% of patients with non-small cell lung cancer carry ARID1B mutations. Mutations in ARID1A and ARID1B have been shown to be associated with sensitivity to immune checkpoint inhibitors in patients with non-small cell lung cancer. Patients carrying these mutations respond better to treatment and have longer progression-free survival.
  • Peripheral blood has the advantage of being less invasive and easier to obtain.
  • Peripheral blood mononuclear cells include monocytes and lymphocytes, which can be extracted and isolated by a kit, which is simple, convenient and low-cost.
  • peripheral blood mononuclear cells play an important role in tumor immunity. Studies have shown that abnormal changes in DNA methylation levels of T lymphocytes and B lymphocytes are related to cancer. This provides a basis for us to study the DNA methylation level of peripheral blood mononuclear cells in patients with lung adenocarcinoma, and provides a new idea for the early diagnosis of lung adenocarcinoma.
  • multi-gene methylation detection Compared with single-gene methylation detection, multi-gene methylation detection has a lower detection limit and higher sensitivity, and is especially suitable for the early diagnosis of lung adenocarcinoma.
  • a multi-gene methylation kit for early diagnosis of lung adenocarcinoma needs to meet the following conditions: First, the content of the marker in peripheral blood mononuclear cells must be sufficient and stable. It is easy to detect; secondly, the marker should have high sensitivity and specificity so that it can detect and distinguish the methylation status of tumor cells and that of normal cells, and also distinguish other tumors and non-tumor cells. Methylation status in neoplastic diseases. Third, the level of this marker is not affected by other tumors, non-neoplastic diseases, exercise, diet, pregnancy, etc.
  • the present invention proposes a new multi-gene methylation kit for early diagnosis of lung adenocarcinoma.
  • NCOA2, RUNX3, BTD, BRE, and ARID1B gene methylation level markers derived from peripheral blood mononuclear cells can be used as diagnostic markers for lung adenocarcinoma patients.
  • This examination method is easily accepted by subjects and is more likely to be used as a diagnostic marker for lung adenocarcinoma patients.
  • a multi-gene methylation kit for early diagnosis of lung adenocarcinoma includes the following six sets of primers and probes: methylation-specific primers for NCOA2, RUNX3, BTD, BRE, and ARID1B gene target sites; Methylation-specific TaqMan probes for NCOA2, RUNX3, BTD, BRE, and ARID1B gene target sites; internal reference gene primers; internal reference gene TaqMan probes.
  • the internal reference gene is actin.
  • the methylation-specific primers and probes of the NCOA2 gene target site include:
  • Reverse primer ATTTTACTAAAATAATAATCTTCTTTCTCCG;
  • Probe TCCTCTTCTTTCATTATCATATTTACTCATTTCTTTC.
  • the methylation-specific primers and probes of the RUNX3 gene target site include:
  • Reverse primer AAATAACAACTCTAAACCTAAAACTAAACG;
  • the methylation-specific primers and probes of the BTD gene target site include:
  • Reverse primer AAAAAACCCCAAACCACTACTTTAT;
  • the methylation-specific primers and probes of the BRE gene target site include:
  • Reverse primer AACATACCCTCAACTCACTATTCTCAA;
  • the methylation-specific primers and probes of the ARID1B gene target site include:
  • Reverse primer AATACTAATTCTAACACTCCCTAACAACG;
  • Probe ACTCTCTAAACTATAATTCTATCATCTATAAAATAACTACG.
  • the primers and probes of the internal reference gene include:
  • Reverse primer AACCAATAAAAACCTACTCCTCCCTTAAA;
  • Probe ACCACCACCCAACACACAATAACAAACACA.
  • the kit also includes: KAPA PROBE FAST qPCR Master Mix (2 ⁇ ); KAPA PROBE FAST ROX Low (50 ⁇ ); ddH 2 O.
  • the PCR amplification reaction system consists of: KAPA PROBE FAST qPCR Master Mix (2 ⁇ ): 5 ⁇ l; KAPA PROBE FAST ROX Low (50 ⁇ ): 0.2 ⁇ l; NCOA2 forward primer (F): 0.25 ⁇ l; NCOA2 reverse primer (R): 0.25 ⁇ l; NCOA2 probe: 0.1 ⁇ l; RUNX3 forward primer (F): 0.25 ⁇ l; RUNX3 reverse primer (R): 0.25 ⁇ l; RUNX3 probe: 0.1 ⁇ l; BTD Forward primer (F): 0.25 ⁇ l; BTD reverse primer (R): 0.25 ⁇ l; BTD probe: 0.1 ⁇ l; BRE forward primer (F): 0.25 ⁇ l; BRE reverse primer (R): 0.25 ⁇ l; BRE probe: 0.1 ⁇ l; ARID1B forward primer (F): 0.25 ⁇ l; ARID1B reverse primer (R): 0.25 ⁇ l; ARID1B probe: 0.1 ⁇ l; actin forward primer (F):
  • the present invention is based on the joint detection of NCOA2, RUNX3, BTD, BRE and ARID1B gene methylation, and determines the detection results by calculating the PCR amplification results of NCOA2, RUNX3, BTD, BRE, ARID1B genes and internal reference genes, which improves Sensitivity and stability of test results.
  • the PCR amplification results of NCOA2, RUNX3, BTD, BRE, ARID1B genes and internal reference genes refer to the Ct value, that is, the cycle experienced when the fluorescence signals of NCOA2, RUNX3, BTD, BRE, ARID1B genes and internal reference genes reach the set threshold. frequency.
  • NCOA2, RUNX3, BTD, BRE, ARID1B genes and internal reference genes ⁇ Ct value (NCOA2, RUNX3, BTD, BRE, ARID1B gene Ct value - internal reference gene Ct value) ⁇ -1.236, the judgment result: methylation positive; ⁇ Ct value (NCOA2, RUNX3, BTD, BRE, ARID1B gene Ct value - internal reference gene Ct value) > -1.236, the judgment result: methylation negative. If the actin Ct value is >32 or there is no amplification, all results will be invalid and need to be retested.
  • the present invention is based on multi-gene methylation joint detection, has high sensitivity, and greatly improves the detection rate of early lung adenocarcinoma.
  • the qPCR method is easy to operate and the results are easy to interpret.
  • This invention only needs to extract 2-3ml of blood from the patient, is less invasive, has good patient compliance, is relatively simple to operate, and is suitable for large-scale clinical application.
  • the present invention is derived from human peripheral blood mononuclear cells, and the detection results are more accurate.
  • Figure 1 shows the internal reference gene methylation qPCR amplification curve, the single gene methylation qPCR amplification curve of NCOA2, RUNX3, BTD, BRE, and ARID1B, and the comparison chart of multi-gene methylation qPCR amplification curve.
  • Figure 2 is a scatter plot of sample detection results.
  • Figure 3 shows the ROC curve of the sample test results.
  • peripheral blood samples were collected from 35 lung adenocarcinoma patients and 30 healthy subjects with the consent of the subjects. All peripheral blood samples were obtained from the Second Hospital of Shandong University and Qilu Hospital of Shandong University. All 35 patients with lung adenocarcinoma were admitted to the hospital for treatment for the first time. They had not received any related treatment such as surgery, radiotherapy, chemotherapy, immunotherapy, etc. before admission. Their pathological types were confirmed by postoperative pathological examination. All peripheral blood specimens were obtained before patients were admitted to the hospital for the first time and before receiving any treatment. None of the 30 healthy subjects had a history of malignant tumors, autoimmune diseases, or other major diseases such as heart, liver, and kidneys.
  • peripheral blood as follows to obtain mononuclear cells: add 3ml of human lymphocyte separation solution to a 15ml centrifuge tube, then gently transfer the peripheral blood to the separation solution, and then centrifuge at 400g for 30 minutes; take another 15ml centrifuge tube and add 10ml PBS, absorb the middle white film layer from the centrifuge tube after centrifugation for 30 minutes, transfer it to PBS, and centrifuge at 250g for 10 minutes; after centrifugation, discard the supernatant, add 5 ml of PBS, and centrifuge at 250g for 10 minutes; discard the supernatant, and the precipitate is peripheral blood Mononuclear cells were stored at -80°C until use.
  • CT Coversion Reagent centrifuge briefly (3000r ⁇ 5min), protect from light, open the lid, add 900ul double distilled water, 300ul M-Dilution Buffer, 50ul M-Dissolving Buffer, mix by inversion, shake at room temperature for 10 minutes (wrap with tin foil when shaking) , protect from light), completely dissolved. (Prepare PCR tubes while shaking).
  • the Illumina Infinium Methylation EPIC BeadChip was used to detect the DNA methylation levels of peripheral blood mononuclear cells in 35 patients with lung adenocarcinoma and 30 healthy subjects.
  • the sequencing data were mainly analyzed using the ChAMP package in R language.
  • the ⁇ value is used to represent the DNA methylation level. After probe filtering, matrix normalization, and batch effect correction, the adj.p value is calculated using the Benjamini-Hochberg method.
  • >0.06 and adj.p value ⁇ 0.05 are defined as differential methylation sites.
  • >0.06 and adj.p value ⁇ 0.05 are defined as differential methylation sites.
  • Peripheral blood samples were collected from 35 lung adenocarcinoma patients and 50 healthy subjects with consent from the subjects. All peripheral blood samples were obtained from the Second Hospital of Shandong University and Qilu Hospital of Shandong University. The inclusion criteria, specimen collection, isolation of peripheral blood mononuclear cells, extraction of peripheral blood mononuclear cell DNA, and modification of peripheral blood mononuclear cell DNA are all the same as the screening stage of the target gene.
  • the five selected hypermethylated sites were analyzed by pyrosequencing.
  • Primer design Use PyroMark Assay Design 2.0 for primer design.
  • PCR amplification system 50 ⁇ L, as shown in Table 2.
  • the PCR amplification procedure is as follows:
  • reaction binding beads Add 2 ⁇ L of reaction binding beads, 38 ⁇ L of binding buffer, and 40 ⁇ L of PCR product to the 96-well PCR reaction plate, and mix thoroughly for 10 min at room temperature.
  • Turn on the vacuum pump to absorb the binding beads and PCR product suspension, and immerse them in 70% ethanol, 0.2M NaOH and washing buffer for 5 seconds each.
  • Turn off the vacuum pump place the bound beads and PCR product on the probe into 40 ⁇ L of annealing buffer (containing 1.5 ⁇ L of sequencing primer), denature at 85°C for 2 minutes, and cool to room temperature to anneal and hybridize the primers to the template.
  • annealing buffer containing 1.5 ⁇ L of sequencing primer
  • the present invention finally identified five methylation genes for early diagnosis of lung adenocarcinoma: NCOA2, RUNX3, BTD, BRE, and ARID1B, providing a method for the early diagnosis of lung adenocarcinoma. New detection methods.
  • the kit includes: KAPA PROBE FAST qPCR Master Mix (2 ⁇ ); KAPA PROBE FAST ROX Low (50 ⁇ ); ddH 2 O; NCOA2, RUNX3, Methylation-specific primers for target sites of BTD, BRE, and ARID1B genes; methylation-specific TaqMan probes for target sites of NCOA2, RUNX3, BTD, BRE, and ARID1B genes; primers for internal reference genes; TaqMan probes for internal reference genes.
  • the internal reference gene is actin.
  • the NCOA2 gene target site is a methylation modified site located in the 5'UTR
  • the RUNX3 gene target site is a methylated modified site located in the Body
  • the BTD gene target site is a methylated modified site located in the 3'UTR.
  • the BRE gene target site is a methylation modification site located on the Body
  • the ARID1B gene target site is a methylation modification site located on the Body.
  • this kit can jointly detect the above five genes at the same time in one PCR reaction well, which improves the sensitivity of the detection and is especially suitable for the early detection of lung adenocarcinoma.
  • the methylation-specific primers and probes of the NCOA2 gene target site include:
  • Reverse primer ATTTTACTAAAATAATAATCTTCTTTCTCCG;
  • Probe TCCTCTTCTTTCATTATCATATTTACTCATTTCTTTC.
  • the methylation-specific primers and probes of the RUNX3 gene target site include:
  • Reverse primer AAATAACAACTCTAAACCTAAAACTAAACG;
  • the methylation-specific primers and probes of the BTD gene target site include:
  • Reverse primer AAAAAACCCCAAACCACTACTTTAT;
  • the methylation-specific primers and probes of the BRE gene target site include:
  • Reverse primer AACATACCCTCAACTCACTATTCTCAA;
  • the methylation-specific primers and probes of the ARID1B gene target site include:
  • Reverse primer AATACTAATTCTAACACTCCCTAACAACG;
  • Probe ACTCTCTAAACTATAATTCTATCATCTATAAAATAACTACG.
  • the primers and probes of the internal reference gene include:
  • Reverse primer AACCAATAAAAACCTACTCCTCCCTTAAA;
  • Probe ACCACCACCCAACACACAATAACAAACACA.
  • Primers The purity of all primers should reach electrophoresis grade (PAGE) or HPLC grade and contain no contaminants. Provide quality inspection certificates of synthetic products issued by the synthesis institution, such as PAGE electrophoresis results or HPLC analysis patterns, proving that after purification using PAGE or HPLC, there should be an obvious single-peak PAGE or HPLC analysis pattern, and the concentration is 10ng/ ⁇ l for later use.
  • PAGE electrophoresis grade
  • HPLC HPLC grade
  • Peripheral blood samples were collected from 84 lung adenocarcinoma patients and 84 healthy subjects with consent from the subjects. All peripheral blood samples were obtained from the Second Hospital of Shandong University and Qilu Hospital of Shandong University. The inclusion criteria are the same as the target gene screening stage.
  • the target gene screening stage is the same as in Example 1.
  • the target gene screening stage is the same as in Example 1.
  • the target gene screening stage is the same as in Example 1.
  • the target gene screening stage is the same as in Example 1.
  • this embodiment designs a multiplex methylation PCR detection method with two different fluorophore probes.
  • the FAM fluorophore was used to label five methylated genes, and the VIC fluorophore was used to label the internal reference gene. This method quantitatively detected the total methylation level of the five genes.
  • the system composition of the qPCR amplification reaction is: KAPA PROBE FAST qPCR Master Mix (2 ⁇ ): 5 ⁇ l; KAPA PROBE FAST ROX Low (50 ⁇ ): 0.2 ⁇ l; NCOA2 forward primer (F): 0.25 ⁇ l; NCOA2 reverse primer Forward primer (R): 0.25 ⁇ l; NCOA2 probe: 0.1 ⁇ l; RUNX3 forward primer (F): 0.25 ⁇ l; RUNX3 reverse primer (R): 0.25 ⁇ l; RUNX3 probe: 0.1 ⁇ l; BTD forward primer ( F): 0.25 ⁇ l; BTD reverse primer (R): 0.25 ⁇ l; BTD probe: 0.1 ⁇ l; BRE forward primer (F): 0.25 ⁇ l; BRE reverse primer (R): 0.25 ⁇ l; BRE probe: 0.1 ⁇ l; ARID1B forward primer (F): 0.25 ⁇ l; ARID1B reverse primer (R): 0.25 ⁇ l; ARID1B probe: 0.1 ⁇ l; actin
  • the detection genes are NCOA2 methylation gene, RUNX3 methylation gene, BTD methylation gene, BRE methylation gene, ARID1B methylation gene, and the internal reference gene is actin.
  • the reporter fluorescent group at the 5' end of the probes for the NCOA2, RUNX3, BTD, BRE, and ARID1B genes is FAM
  • the quenching group at the 3' end of the probes for the NCOA2, RUNX3, BTD, BRE, and ARID1B genes is BHQ1
  • the internal reference gene The reporter fluorescent group at the 5' end of the probe is VIC
  • the quenching group at the 3' end of the probe for the internal reference gene is BHQ1.
  • Step (8) Prepare the mixed solution in the qPCR tube according to step (8), and then perform the PCR reaction according to the following conditions: Pre-denaturation Reps: 1, 95°C, 3min; Cycle reaction: Reps: 40, 95°C, 3sec; 58°C, 30sec .
  • NC in the table represents healthy person samples
  • LUAD represents lung adenocarcinoma patient samples.
  • the internal reference gene actin of the sample should all have amplification signals, actin Ct value ⁇ 32, and an S-shaped amplification curve. When the above conditions are met, the experiment is proven to be valid and the analysis can continue. Otherwise, it is recommended to retest.
  • Figure 1 shows the internal reference gene methylation qPCR amplification curve, single gene methylation qPCR amplification curves of NCOA2, RUNX3, BTD, BRE, and ARID1B, and a comparison chart of multi-gene methylation qPCR amplification curves.
  • the abscissa represents the number of cycles required for qPCR amplification to reach the threshold
  • the ordinate represents the logarithmic value of the corrected fluorescence intensity. It can be seen that the amplification effect of multi-gene methylation qPCR is the superposition of the amplification effects of multiple single-gene methylation qPCR.
  • Figure 2 Draw a scatter plot based on the sample test results. Among them, the abscissa represents the sample type, and the ordinate represents the difference between the number of cycles of the polygene and the number of cycles of the internal reference gene when qPCR amplification reaches the threshold. It can be seen that there are significant differences in polygene methylation between lung adenocarcinoma patients and healthy controls (P ⁇ 0.0001).
  • Figure 3 Draw the ROC curve based on the sample test results.

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Abstract

公开了一种用于肺腺癌早期诊断的多基因甲基化试剂盒。试剂盒包括以下引物与探针：NCOA2、RUNX3、BTD、BRE、ARIDIB基因目的位点的甲基化特异性引物；NCOA2、RUNX3、BTD、BRE、ARIDIB基因目的位点的甲基化特异性Taqman探针；内参基因引物、Taqman探针。

Description

一种用于肺腺癌早期诊断的多基因甲基化试剂盒 技术领域
本发明属于肿瘤分子生物学领域，具体涉及一种用于肺腺癌早期诊断的多基因甲基化试剂盒。
背景技术
肺癌是呼吸系统最常见的恶性肿瘤之一，是癌症相关死亡最主要的原因，严重危害人类健康。从病理学分型的角度来看，肺癌包括小细胞肺癌和非小细胞肺癌，其中非小细胞肺癌约占85％。在非小细胞肺癌当中，肺腺癌约占50％。腺癌是肺癌最常见的病理类型。尽管手术、放疗、化疗、靶向治疗等治疗技术有了一定的进步，肺腺癌的预后仍不乐观。其中，很重要的一个原因是肺腺癌的早期诊断存在一定的困难，相当多的患者在初诊时已经处于中晚期，预后较差。目前，低剂量螺旋CT是筛查早期肺腺癌常用的检查手段，但是它存在特性差的弊端，许多肺部良性病变可能会被误诊为肺癌。传统的血清肿瘤标志物(NSE、SCC、Cyfra 21-1、ProGRP、CEA等)的灵敏度和特异度有限。因此，寻找一种新型非侵入性、特异性高的肺腺癌诊断标志物显得尤为重要。
DNA甲基化作为表观遗传修饰的一种形式，在肿瘤的发生发展中扮演着重要的角色。DNA甲基化是指在甲基转移酶的作用下，于胞嘧啶第五位碳原子上添加S-腺苷甲硫氨酸提供的一个甲基，通常发生在CpG岛，对维持染色体的结构稳定、调控基因表达等有重要作用。一般地，高甲基化会抑制基因的表达，低甲基化会促进基因的表达。原癌基因的低甲基化和(或)抑癌基因的高甲基化会促进肿瘤的发生发展。已有研究证明，许多恶性肿瘤，如结直肠癌、胃癌、乳腺癌、胰腺癌等，存在DNA甲基化水平的异常改变，使用基因甲基化水平作为 恶性肿瘤诊断的生物标志物已成为一个新的研究方向。
NCOA2属于NCOA家族中的一员，研究表明，NCOA家族尤其是NCOA2和NCOA3参与正常的生物过程和致癌作用。例如，NCOA3的激活促进了胰腺导管腺癌的肿瘤生长。NCOA2介导的抗肿瘤靶基因共激活抑制MYC诱导的肝癌。NCOA2的抑制严重减弱了前列腺癌的存活、生长和转移能力。在肺癌中，NCOA2可被PFKFB4在Ser487位点磷酸化，NCOA2的转录活性因此发生改变，从而促进肺癌细胞增殖、迁移和侵袭。
RUNX家族转录因子是发育的关键调节因子，并且在癌症中常常失调。RUNX3是RUNX家族中的一员，被描述为肿瘤启动子或抑制子，反映了它在肿瘤发生中的复杂作用。RUNX3是TGF-β信号通路的下游效应分子，在调节细胞死亡、血管生成、内皮间质转化、细胞迁移和侵袭等方面作用重大。Runx3沉默通过miR-148a-3p调节YBX1表达，通过增加Cyclin D1、Cyclin B1、Slug-1、MMP-2和MMP-9水平来促进非小细胞肺癌的进展。RUNX3的表达被确定为胰腺导管腺癌细胞倾向于局部增殖或在全身播散的关键因素。RUNX3的过表达促进了头颈癌细胞的生长并抑制了凋亡。此外，有研究表明，RUNX3在免疫和炎症中发挥重要作用，因此RUNX3可能间接影响肿瘤的发生发展。
BTD(生物素酶)基因位于染色体3p25。迄今为止，已报道了BTD的四个外显子中的三个中的61个突变和一个内含子基因中的1个突变，这些突变导致了严重的BTD缺陷。BTD是可以切割生物胞素的酶，生物胞素是全羧化酶蛋白水解消化的产物。严重的BTD缺乏(血清中的平均正常活性低于10％)是一种常染色体隐性遗传疾病，病人不能切割生物胞素，不能回收生物素。未经治疗的个体会继发性地缺乏生物素，这反过来会导致生物素依赖性羧化酶的活性降低，从而导致有毒代谢物的积累，进而导致相应临床症状的出现。生物素酶缺乏症的特征主要是神经、皮肤表现和代谢异常。然而，BTD基因在肿瘤中的 报道较少，值得进一步研究。
BRE，又称为TNFRSF1A调节剂和BRCC45，是一种进化上高度保守的蛋白质。它是细胞质中的一种死亡受体相关蛋白，也是细胞核中含有BRCA1/2的DNA修复复合物的组成部分。研究发现BRE具有抗凋亡活性，BRE的高表达可通过促进细胞周期进程和抗细胞凋亡来增强食管鳞癌细胞生长，BRE的沉默抑制了食管鳞癌细胞的这些恶性表型。机制研究表明，BRE过表达激活了AKT的磷酸化，并且MK2206对AKT通路的抑制降低了BRE诱导的食管鳞癌细胞生长和凋亡抗性。
ARID1B位于6q25.3，是典型的核肿瘤抑制因子。然而，细胞质定位的ARID1B可以结合c-RAF(RAF1)和PPP1CA，从而刺激RAF-ERK信号传导和β-连环蛋白(CTNNB1)转录活性。ARID1B细胞质定位与ERK1和ERK2(也称为MAPK3和MAPK1)活性形式和β-连环蛋白水平的增加，与晚期肿瘤分期和淋巴结阳性水平显著相关。在乳腺癌中，ARID1B的高表达与浸润性乳腺癌的组织学分级和肿瘤大小密切相关。在侵袭性三阴性乳腺癌亚型中，ARID1B的表达常常增加，并与五年无病生存率降低有关。与对照组相比，具有ARID1B活性降低的MDA-MB-231细胞在G1期到S期细胞周期过渡中表现出延迟，因此细胞增殖减少。髓母细胞瘤是一种常见的儿童恶性脑肿瘤，ARID1B的表达下降导致髓母细胞瘤细胞的恶性潜能显著增加。在膀胱尿路上皮癌中，ARID1B表达与肿瘤大小、TNM分期、总生存期和无进展生存期显著相关。在ARID1B低表达患者中，辅助化疗的益处优于在ARID1B高表达患者中观察到的益处，表明ARID1B可能是高风险亚组中选择辅助化疗患者的预测标志物。在非小细胞肺癌中，7％的非小细胞肺癌患者携带ARID1A突变，4％的非小细胞肺癌患者携带ARID1B突变。ARID1A和ARID1B的突变被证实与非小细胞肺癌患者对免疫检查点抑制剂的敏感性相关。携带这些突变的患者对治疗有更好的反应，并且无进展生存期延长。
外周血具有侵入性小、较易获得的优势。外周血单个核细胞包括单核细胞、淋巴细胞，可由试剂盒提取分离得到，简单方便，且成本较低。作为免疫系统的重要组成部分，外周血单个核细胞在肿瘤免疫中发挥着重要作用。已有研究表明，T淋巴细胞、B淋巴细胞的DNA甲基化水平异常改变与癌症发生有关。这为我们研究肺腺癌患者外周血单个核细胞的DNA甲基化水平提供了依据，为肺腺癌的早期诊断提供了新思路。
相比于单基因甲基化检测，多基因甲基化检测限更低，敏感性更高，尤其适用于肺腺癌的早期诊断。一种用于肺腺癌早期诊断的多基因甲基化试剂盒要成为一种诊断试剂，需要满足以下条件：第一，该标志物在外周血单个核细胞中的含量要足够、稳定存在、易被检测；第二，该标志物应具有较高的灵敏度和特异度，以便能够检出并辨别肿瘤细胞的甲基化状态和正常细胞的甲基化状态，并且还能区别其他肿瘤以及非肿瘤性疾病的甲基化状态。第三，该标志物的水平不受其他肿瘤、非肿瘤性疾病、运动、饮食、怀孕等影响。
发明内容
本发明针对传统肺腺癌早期诊断中存在的问题提出一种新型的用于肺腺癌早期诊断的多基因甲基化试剂盒。外周血单个核细胞来源的NCOA2、RUNX3、BTD、BRE、ARID1B基因甲基化水平标志物可作为肺腺癌患者的诊断标志物，这一检查方法容易被受检者接受，更可能成为肺腺癌患者的早期诊断的有效手段。
为了达到上述目的，本发明是采用下述的技术方案实现的：
一种用于肺腺癌早期诊断的多基因甲基化试剂盒，试剂盒包括以下六组引物与探针：NCOA2、RUNX3、BTD、BRE、ARID1B基因目的位点的甲基化特异性引物；NCOA2、RUNX3、BTD、BRE、ARID1B基因目的位点的甲基化特异性TaqMan探针；内参基因引物；内参基因TaqMan探 针。所述内参基因为actin。
所述NCOA2基因目的位点的甲基化特异性引物和探针包括：
正向引物：TAAAAGTTATTGTCGGTGGTAGAAGA，
反向引物：ATTTTACTAAATAATAATCTTCTTTCTCCG；
探针：TCCTCTTCTTTCATTATCATATTTACTCATTTCTTTC。
所述RUNX3基因目的位点的甲基化特异性引物和探针包括：
正向引物：GGGGTTTATAGTTAGTTTGGGTTC，
反向引物：AAATAACAACTCTAAACCTAAAACTAAACG；
探针：ACCCTAACATCTATTATAAAATCCCGATACATCTAT。
所述BTD基因目的位点的甲基化特异性引物和探针包括：
正向引物：TATATTTTTTTTTAATAAATTTTTTAGTATGCG，
反向引物：AAAAAACCCCAAACCACTACTTTAT；
探针：AACTAACATTTATTTTTATTTTAACTTAAAACCAATCG。
所述BRE基因目的位点的甲基化特异性引物和探针包括：
正向引物：GTTTTATTTAATAAGTTTTTTAGAAGTTTTTCG，
反向引物：AACATACCCTCAACTCACTATTCTCAA；
探针：AACGAAAACGAAAATTATAACAAAAATATCAACC。
所述ARID1B基因目的位点的甲基化特异性引物和探针包括：
正向引物：TTTGTTAGGTAGTGCGGTGTTTG，
反向引物：AATACTAATTCTAACACTCCCTAACAACG；
探针：ACCTCTCTAAACTATAATTCTATCATCTATAAAATAACTACG。
所述内参基因的引物和探针包括：
正向引物：TGGTGATGGAGGAGGTTTAGTAAGT，
反向引物：AACCAATAAAACCTACTCCTCCCTTAAA；
探针：ACCACCACCCAACACACAATAACAAACACA。
所述试剂盒中，还包括：KAPA PROBE FAST qPCR Master Mix(2 ×)；KAPA PROBE FAST ROX Low(50×)；ddH 2O。
所述试剂盒中，PCR扩增反应的体系组成为：KAPA PROBE FAST qPCR Master Mix(2×)：5μl；KAPA PROBE FAST ROX Low(50×)：0.2μl；NCOA2正向引物(F)：0.25μl；NCOA2反向引物(R)：0.25μl；NCOA2探针：0.1μl；RUNX3正向引物(F)：0.25μl；RUNX3反向引物(R)：0.25μl；RUNX3探针：0.1μl；BTD正向引物(F)：0.25μl；BTD反向引物(R)：0.25μl；BTD探针：0.1μl；BRE正向引物(F)：0.25μl；BRE反向引物(R)：0.25μl；BRE探针：0.1μl；ARID1B正向引物(F)：0.25μl；ARID1B反向引物(R)：0.25μl；ARID1B探针：0.1μl；actin正向引物(F)：0.06μl；actin反向引物(R)：0.06μl；actin探针：0.05μl；DNA：1μl；ddH 2O：0.63μl。
本发明基于NCOA2、RUNX3、BTD、BRE、ARID1B基因甲基化联合检测，通过NCOA2、RUNX3、BTD、BRE、ARID1B基因与内参基因PCR扩增结果的计算，来对检测结果给出判定，提高了检测结果的敏感性和稳定性。NCOA2、RUNX3、BTD、BRE、ARID1B基因与内参基因的PCR扩增结果是指Ct值，即NCOA2、RUNX3、BTD、BRE、ARID1B基因与内参基因的荧光信号达到设定的阈值时所经历的循环次数。通过NCOA2、RUNX3、BTD、BRE、ARID1B基因与内参基因PCR扩增结果的计算来对检测结果给出判定的标准为：ΔCt值(NCOA2、RUNX3、BTD、BRE、ARID1B基因Ct值-内参基因Ct值)≤-1.236，判定结果：甲基化阳性；ΔCt值(NCOA2、RUNX3、BTD、BRE、ARID1B基因Ct值-内参基因Ct值)＞-1.236，判定结果：甲基化阴性。若actin Ct值＞32或无扩增，所有结果均无效，需重新检测。
与现有技术相比，本发明的优点和积极效果在于：
1.本发明基于多基因甲基化联合检测，敏感性高，大大提高了 早期肺腺癌的检出率。采用qPCR方法，操作简便，且结果判读较简单。
2.本发明只需抽取患者2-3ml血液，创伤性小、患者依从性好、操作相对简便，适合临床大规模应用。
3.本发明从肿瘤免疫的角度出发，取材自人外周血单个核细胞，检测结果更为准确。
附图说明
图1为内参基因甲基化qPCR扩增曲线，NCOA2、RUNX3、BTD、BRE、ARID1B单个基因甲基化qPCR扩增曲线，以及多基因甲基化qPCR扩增曲线对比图。
图2为样本检测结果的散点图。
图3为样本检测结果的ROC曲线。
具体实施方式
为了能够更清楚地理解本发明的上述目的、特征和优点，下面结合具体实施例对本发明做进一步说明。需要说明的是，在不冲突的情况下，本申请的实施例及实施例中的特征可以相互组合。
在下面的描述中阐述了很多具体细节以便于充分理解本发明，但是，本发明还可以采用不同于在此描述的其他方式来实施，因此，本发明并不限于下面公开说明书的具体实施例的限制。
实施例1
目的基因的筛选与验证
1.目的基因的筛选
1.1研究对象
本实施例中，外周血标本共来自经受试者同意的35例肺腺癌患者和30例健康体检者。所有外周血标本来自山东大学第二医院和山东大学齐鲁医院。35例肺腺癌患者均为首次入院接受治疗的患者，且入院 前未曾接受手术、放疗、化疗、免疫治疗等任何相关治疗，其病理类型均经术后病理检查确认。所有外周血标本均在患者首次入院且未接受任何治疗前取得。30例健康体检者均无恶性肿瘤病史、自身免疫病以及心、肝、肾等其他重大疾病。
1.2标本采集
每位研究对象采集外周血一次(2-3ml/管)后，轻轻颠倒混匀5-10次，立即提取单个核细胞。
1.3外周血单个核细胞的分离
将外周血进行如下处理获得单个核细胞：在15ml离心管加3ml人淋巴细胞分离液，然后将外周血轻轻转移到分离液上面，随后400g离心30min；另取15ml离心管1支，加入10ml PBS，从离心30min结束后离心管中吸取中间白膜层，转移至PBS中，250g离心10min；离心结束后，弃上清，加入5ml PBS，250g离心10min；弃上清，沉淀即为外周血单个核细胞，放-80℃待用。
1.4外周血单个核细胞DNA的提取
(1)取外周血单个核细胞，10000rpm(～11200×g)离心1min，倒尽上清，加200μl缓冲液GA，振荡至彻底悬浮。
(2)加入20μl Proteinase K溶液，混匀。
(3)加入200μl缓冲液GB，充分颠倒混匀，70℃放置10min，溶液应变清亮，简短离心以除去管盖内壁的水珠。
(4)加入200μl无水乙醇，充分振荡混匀15sec，此时可能会出现絮状沉淀，简短离心以除去管盖内壁的水珠。
(5)将上一步所得溶液和絮状沉淀都加入一个吸附柱CB3中(吸附柱CB3放入收集管中)，12000rpm(～13400×g)离心30sec，倒掉废液，将吸附柱CB3放回收集管中。
(6)向吸附柱CB3中加入500μl缓冲液GD(使用前请先检查是 否已加入无水乙醇)，12000rpm(～13400×g)离心30sec，倒掉废液，将吸附柱CB3放回收集管中。
(7)向吸附柱CB3中加入600μl漂洗液PW(使用前请先检查是否已加入无水乙醇)，12000rpm(～13400×g)离心30sec，倒掉废液，将吸附柱CB3放回收集管中。
(8)重复操作步骤(7)。
(9)将吸附柱CB3放回收集管中，12000rpm(～13400×g)离心2min，倒掉废液。将吸附柱CB3置于室温放置数分钟，以彻底晾干吸附材料中残余的漂洗液。
(10)将吸附柱CB3转入一个干净的1.5ml离心管中，向吸附膜中间部位悬空滴加50-200μl洗脱缓冲液TB，室温放置2-5min，12000rpm(～13400×g)离心2min，将溶液收集到离心管中。
1.5外周血单个核细胞DNA的修饰
(1)CT Coversion Reagent简短离心(3000r×5min)，避光开盖，加900ul双蒸水、300ul M-Dilution Buffer、50ul M-Dissolving Buffer，颠倒混匀，室温震荡10min(震荡时包上锡纸，避光)，完全溶解。(震荡时准备PCR管)。
(2)20ul DNA样本加入PCR管中(依据DNA浓度计算DNA的量，1000/DNA浓度，若≥20，取20，若为3.1，加4，最少2ul(即向上进1)，一共20ul，不够的用双蒸水补齐)，在各PCR管中再加130ul完全溶解的CT Coversion Regent。
(3)将PCR管中液体漩涡混匀，简短离心后加入PCR仪中，设置程序：98℃，10min，64℃，2.5h，4℃，hold。
(4)将收集管套入Zymo-spin Tm Ic Column，加入600ul M-Binding Buffer。
(5)将PCR内液体加入(约150ul)含有600ul M-Binding Buffer 的Zymo-spin Tm Ic Column中，关盖，颠倒混匀1min(用手来回摇晃就行)。
(6)匀速离心(13400r/min)30s，弃废液。
(7)向Colum中加入100ul M-wash Buffer，匀速离心30s，弃废液。
(8)向Colum中加入200ul M-Desulphonation Buffer，室温放置15-20min，匀速离心30s，弃废液。
(9)向Colum中加入200ul M-wash Buffer，匀速离心30s，弃废液。
(10)重复步骤9。
(11)将吸附栓置于1.5ml EP管中，加入20ul M-Elution Buffer到核区(柱底部)，全速离心30s，洗脱修饰后的DNA(只取清液)。
(12)将1.5ml EP管做标记，放入-20℃备用。
1.6 Illumina Infinium Methylation EPIC BeadChip测试
(1)采用Illumina Infinium Methylation EPIC BeadChip对35例肺腺癌患者和30例健康体检者外周血单个核细胞DNA甲基化水平进行检测，测序数据主要用R语言中的ChAMP包进行分析。
(2)用β值表示DNA甲基化水平，经过探针过滤、矩阵归一化、校正批次效应后，采用Benjamini-Hochberg法计算adj.p值。
(3)将|Δβ|＞0.06且adj.p值＜0.05的CpG定义为差异甲基化位点，通过比较肺腺癌与健康对照组甲基化水平，我们初步筛选了1415个差异甲基化位点，其中包括1178个高甲基化位点和237个低甲基化位点。进一步，我们将筛选条件调整为|Δβ|＞0.08且adj.p值＜1.0E-05。结果显示，共有29个差异甲基化位点，其中包括26个高甲基化位点和3个低甲基化位点。最后，我们综合考虑基因与位点的位置关系、引物设计因素等，选出5个高甲基化位点用于后 续验证，这5个高甲基化位点对应的基因分别为NCOA2、RUNX3、BTD、BRE、ARID1B。
2.目的基因的验证
外周血标本共来自经受试者同意的35例肺腺癌患者和50例健康体检者。所有外周血标本来自山东大学第二医院和山东大学齐鲁医院。其入组标准、标本采集、外周血单个核细胞的分离、外周血单个核细胞DNA的提取、外周血单个核细胞DNA的修饰，均同目的基因的筛选阶段。
将筛选出的5个高甲基化位点进行焦磷酸测序分析。
2.1引物设计：使用PyroMark Assay Design 2.0进行引物设计，
引物序列信息，如下表1所示。
表1引物序列
Figure PCTCN2022134024-appb-000001
2.2PCR扩增：PCR扩增体系(50μL)，如表2所示。
表2 PCR扩增体系
Figure PCTCN2022134024-appb-000002
PCR扩增程序如下：
Figure PCTCN2022134024-appb-000003
2.3Pyrosequencing检测
在96孔PCR反应板中加入反应结合珠2μL，结合缓冲液38μL，PCR产物40μL，室温下充分混匀10min。开启真空泵吸取结合珠及PCR产物混悬液，依次浸入70％乙醇，0.2M NaOH和冲洗缓冲液中各5s。关闭真空泵，将探头上的结合珠及PCR产物置于40μL退火缓冲液(含测序引物1.5μL)中，85℃变性2min，冷却至室温，使引物与模板退火杂交。根据Pyrosequencing软件序列设计信息，计算出剂量，在试剂舱中依次加入底物混合物、酶混合物及四种dNTP(QIAGEN)。将试剂舱及96孔反应板放入Pyrosequencing检测仪(PyroMark Q96 ID，QIAGEN)中进行反应。
2.4数据分析
分析焦磷酸测序数据，5个高甲基化位点得到了验证，可用于肺腺癌的早期诊断。这5个高甲基化位点对应的基因分别为NCOA2、RUNX3、BTD、BRE、ARID1B。
综上，本发明经过上述筛选与验证，最终确定了用于肺腺癌早期 诊断的5个甲基化基因：NCOA2、RUNX3、BTD、BRE、ARID1B，为肺腺癌的早期诊断提供了一种新型检测方法。
实施例2
用于肺腺癌早期诊断的多基因甲基化试剂盒，其中试剂盒包括：KAPA PROBE FAST qPCR Master Mix(2×)；KAPA PROBE FAST ROX Low(50×)；ddH 2O；NCOA2、RUNX3、BTD、BRE、ARID1B基因目的位点的甲基化特异性引物；NCOA2、RUNX3、BTD、BRE、ARID1B基因目的位点的甲基化特异性TaqMan探针；内参基因引物；内参基因TaqMan探针。所述内参基因为actin。NCOA2基因目的位点为位于5’UTR的一个甲基化修饰的位点，RUNX3基因目的位点为位于Body的一个甲基化修饰的位点，BTD基因目的位点为位于3’UTR的一个甲基化修饰的位点，BRE基因目的位点为位于Body的一个甲基化修饰的位点，ARID1B基因目的位点为位于Body的一个甲基化修饰的位点。
该试剂盒的优势在于在一个PCR反应孔中，对上述5个基因同时进行联合检测，提高了检测的灵敏度，尤其适用于肺腺癌的早期检测。
所述NCOA2基因目的位点的甲基化特异性引物和探针包括：
正向引物：TAAAAGTTATTGTCGGTGGTAGAAGA，
反向引物：ATTTTACTAAATAATAATCTTCTTTCTCCG；
探针：TCCTCTTCTTTCATTATCATATTTACTCATTTCTTTC。
所述RUNX3基因目的位点的甲基化特异性引物和探针包括：
正向引物：GGGGTTTATAGTTAGTTTGGGTTC，
反向引物：AAATAACAACTCTAAACCTAAAACTAAACG；
探针：ACCCTAACATCTATTATAAAATCCCGATACATCTAT。
所述BTD基因目的位点的甲基化特异性引物和探针包括：
正向引物：TATATTTTTTTTTAATAAATTTTTTAGTATGCG，
反向引物：AAAAAACCCCAAACCACTACTTTAT；
探针：AACTAACATTTATTTTTATTTTAACTTAAAACCAATCG。
所述BRE基因目的位点的甲基化特异性引物和探针包括：
正向引物：GTTTTATTTAATAAGTTTTTTAGAAGTTTTTCG，
反向引物：AACATACCCTCAACTCACTATTCTCAA；
探针：AACGAAAACGAAAATTATAACAAAAATATCAACC。
所述ARID1B基因目的位点的甲基化特异性引物和探针包括：
正向引物：TTTGTTAGGTAGTGCGGTGTTTG，
反向引物：AATACTAATTCTAACACTCCCTAACAACG；
探针：ACCTCTCTAAACTATAATTCTATCATCTATAAAATAACTACG。
所述内参基因的引物和探针包括：
正向引物：TGGTGATGGAGGAGGTTTAGTAAGT，
反向引物：AACCAATAAAACCTACTCCTCCCTTAAA；
探针：ACCACCACCCAACACACAATAACAAACACA。
(1)仪器、试剂、引物：
仪器：NANNODROP 2000、ABI QuantstudioDX、离心机、水浴锅、旋涡震荡仪、冰箱。
试剂：Histopaque-1077人淋巴细胞分离液(SIGMA)、DNA提取试剂盒(TIANGEN)、EZ DNA Methylation-Gold Kit(Zymo)、KAPA PROBE FAST qPCR Master Mix(2×)；KAPA PROBE FAST ROX Low(50×)、ddH 2O。
引物：所有引物纯度应达到电泳级(PAGE)或HPLC级，不含杂带。提供合成机构出具的合成产物的质检证明，如PAGE电泳结果或HPLC分析图谱，证明使用PAGE或者HPLC纯化后应有明显单峰PAGE或HPLC分析图谱，浓度为10ng/μl备用。
(2)研究对象
外周血标本共来自经受试者同意的84例肺腺癌患者和84例健康体检者。所有外周血标本来自山东大学第二医院和山东大学齐鲁医院。 入组标准同目的基因筛选阶段。
(3)标本采集
同实施例1目的基因筛选阶段。
(4)外周血单个核细胞的分离
同实施例1目的基因筛选阶段。
(5)外周血单个核细胞DNA的提取
同实施例1目的基因筛选阶段。
(6)外周血单个核细胞DNA的修饰
同实施例1目的基因筛选阶段。
(7)多基因甲基化联合检测方法的建立
基于qPCR方法，在一个反应孔中，同时定量检测多个基因的甲基化水平。因此，本实施例设计一种带有两种不同荧光团探针的多重甲基化PCR检测方法。FAM荧光团用于标记5个甲基化基因，VIC荧光团用于标记内参基因，该方法定量检测5个基因的总甲基化水平。
(8)qPCR反应的体系和条件
所述qPCR扩增反应的体系组成为：KAPA PROBE FAST qPCR Master Mix(2×)：5μl；KAPA PROBE FAST ROX Low(50×)：0.2μl；NCOA2正向引物(F)：0.25μl；NCOA2反向引物(R)：0.25μl；NCOA2探针：0.1μl；RUNX3正向引物(F)：0.25μl；RUNX3反向引物(R)：0.25μl；RUNX3探针：0.1μl；BTD正向引物(F)：0.25μl；BTD反向引物(R)：0.25μl；BTD探针：0.1μl；BRE正向引物(F)：0.25μl；BRE反向引物(R)：0.25μl；BRE探针：0.1μl；ARID1B正向引物(F)：0.25μl；ARID1B反向引物(R)：0.25μl；ARID1B探针：0.1μl；actin正向引物(F)：0.06μl；actin反向引物(R)：0.06μl；actin探针：0.05μl；DNA：1μl；ddH 2O：0.63μl。
所述检测基因为NCOA2甲基化基因、RUNX3甲基化基因、BTD甲基 化基因、BRE甲基化基因、ARID1B甲基化基因,所述内参基因为actin。所述NCOA2、RUNX3、BTD、BRE、ARID1B基因的探针5’端报告荧光基团为FAM,NCOA2、RUNX3、BTD、BRE、ARID1B基因的探针3’端猝灭基团为BHQ1，内参基因的探针5’端报告荧光基团为VIC，内参基因的探针3’端猝灭基团为BHQ1。
按照步骤(8)在qPCR管中配制下混合液，然后按照下述条件进行PCR反应：预变性Reps：1，95℃，3min；循环反应：Reps：40，95℃，3sec；58℃，30sec。
(9)PCR反应的加样布局(见表3,表4)
将待测样品于PCR仪96孔板中加样，布局见下表。表中NC代表健康人样本，LUAD代表肺腺癌患者样本。
表3 PCR反应的加样布局
Figure PCTCN2022134024-appb-000004
表4 PCR反应的加样布局
Figure PCTCN2022134024-appb-000005
(10)检测结果以及结果分析
样本的内参基因actin均应有扩增信号,actin Ct值≤32，且呈S型扩增曲线,满足以上条件时，证明实验有效,可继续分析。否则，建议重新检测。
图1为内参基因甲基化qPCR扩增曲线，NCOA2、RUNX3、BTD、BRE、ARID1B单个基因甲基化qPCR扩增曲线，以及多基因甲基化qPCR扩增曲线对比图。其中，横坐标表示qPCR扩增达到阈值所需的循环次数，纵坐标表示校正荧光强度的对数值。可以看出，多基因甲基化qPCR扩增效果是多个单基因甲基化qPCR扩增效果的叠加。
图2：依据样本检测结果，绘制散点图。其中，横坐标表示样本类型，纵坐标表示qPCR扩增达到阈值时，多基因的循环次数与内参基因循环次数的差值。可以看出，肺腺癌患者与健康对照者的多基因甲基化差异明显(P＜0.0001)。
图3：依据样本检测结果，绘制ROC曲线。诊断临界值为-1.236，AUC＝0.893，敏感性为88.1％，特异性为82.1％。
以上所述，仅是本发明的较佳实施例而已，并非是对本发明作其它形式的限制，任何熟悉本专业的技术人员可能利用上述揭示的技术内容加以变更或改型为等同变化的等效实施例应用于其它领域，但是凡是未脱离本发明技术方案内容，依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与改型，仍属于本发明技术方案的保护范围。

Claims (4)

  1. 外周血单个核细胞中的NCOA2、RUNX3、BTD、BRE、ARID1B基因甲基化水平标志物肺腺癌早期诊断试剂盒中的应用。
  2. 一种用于肺腺癌早期诊断的多基因甲基化试剂盒，其特征在于，包括NCOA2、RUNX3、BTD、BRE、ARID1B基因目的位点的甲基化特异性引物和甲基化特异性TaqMan探针，以及内参actin基因引物和内参actin基因TaqMan探针，
    NCOA2基因目的位点的甲基化特异性引物序列如序列表SEQ ID NO.1和SEQ ID NO.2所示，探针序列如序列表SEQ ID NO.3所示；
    RUNX3基因目的位点的甲基化特异性引物序列如序列表SEQ ID NO.4和SEQ ID NO.5所示，探针序列如序列表SEQ ID NO.6所示；
    BTD基因目的位点的甲基化特异性引物序列如序列表SEQ ID NO.7和SEQ ID NO.8所示，探针序列如序列表SEQ ID NO.9所示；
    BRE基因目的位点的甲基化特异性引物序列如序列表SEQ ID NO.10和SEQ ID NO.11所示，探针序列如序列表SEQ ID NO.12所示；
    ARID1B基因目的位点的甲基化特异性引物序列如序列表SEQ ID NO.13和SEQ ID NO.14所示，探针序列如序列表SEQ ID NO.15所示；
    内参act in基因目的位点的甲基化特异性引物序列如序列表SEQ ID NO.16和SEQ ID NO.17所示，探针序列如序列表SEQ ID NO.18所示。
  3. 根据权利要求2所示用于肺腺癌早期诊断的多基因甲基化试剂盒，其特征在于，NCOA2、RUNX3、BTD、BRE、ARID1B基因的探针5’端报告荧光基团为FAM,NCOA2、RUNX3、BTD、BRE、ARID1B基因的探针3’端猝灭基团为BHQ1，内参actin基因的探针5’端报告荧光基团为VIC，内参actin基因的探针3’端猝灭基团为BHQ1。
  4. 根据权利要求2所示用于肺腺癌早期诊断的多基因甲基化试剂盒，其特征在于，还包括一个诊断模型，公式为：
    ΔCt值＝(NCOA2、RUNX3、BTD、BRE、ARID1B基因总Ct值)-内参基因Ct值；
    若ΔCt值≤-1.236，则表示甲基化阳性；
    若ΔCt值＞-1.236，则表示甲基化阴性；
    actin Ct值＞32或无扩增，为无效结果，需重新检测。
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