WO2024036785A1 - 胃癌早期筛查的dna甲基化标志物组合及试剂盒 - Google Patents

胃癌早期筛查的dna甲基化标志物组合及试剂盒 Download PDF

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WO2024036785A1
WO2024036785A1 PCT/CN2022/132108 CN2022132108W WO2024036785A1 WO 2024036785 A1 WO2024036785 A1 WO 2024036785A1 CN 2022132108 W CN2022132108 W CN 2022132108W WO 2024036785 A1 WO2024036785 A1 WO 2024036785A1
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王传新
杜鲁涛
李培龙
李娟�
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山东大学
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    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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  • the invention belongs to the technical field of tumor molecular biology and relates to a DNA methylation marker combination and a kit for early screening of gastric cancer.
  • Gastric cancer is one of the most common malignant tumors in the digestive system and the fifth most common malignant tumor in the world. Its case fatality rate ranks fourth among malignant tumors, seriously threatening human life and health.
  • the occurrence of gastric cancer is a complex process involving multiple factors. Its incidence is closely related to Helicobacter pylori infection, diet, lifestyle, genetics and other factors. It is especially prone to occur in the elderly, and the incidence rate in men is significantly higher than that in women. .
  • Early detection and treatment of gastric cancer are crucial to improving the prognosis of patients. The 5-year survival rate of early gastric cancer can reach more than 95%.
  • DNA methylation is an important epigenetic factor, which refers to the covalent bonding of a methyl group at the 5' carbon position of cytosine in genomic CpG dinucleotides under the action of DNA methyltransferase.
  • a large number of studies have confirmed that DNA methylation can cause changes in chromatin structure, DNA conformation, DNA stability and the interaction between DNA and proteins, thereby controlling gene expression.
  • Abnormal methylation in the promoter region can lead to the activation or activation of oncogenes. Silencing of tumor suppressor genes. Since DNA methylation often occurs early in the cancer process, it is increasingly used in tumor diagnosis and can be used as a reliable way to screen for early gastric cancer.
  • PBMC Peripheral blood mononuclear cells
  • T/B lymphocytes
  • monocytes monocytes
  • phagocytes phagocytes
  • dendritic cells dendritic cells
  • the present invention proposes a novel DNA methylation marker combination and kit for early screening of gastric cancer, aiming at the problems existing in traditional gastric cancer diagnosis such as invasiveness, time-consuming and labor-intensive, and low accuracy.
  • the invention provides a methylation site combination for early screening of gastric cancer.
  • the combination includes chr12_10541790 in the KLRK1 gene promoter region, chr14_22927264 and chr14_22926867 in the TRDJ3 gene promoter region, and chr14_22931150 in the TRDC gene promoter region.
  • the present invention also provides a methylation detection primer and probe combination for early screening of gastric cancer, which combination includes specific primers and probes for detecting the methylation level of the site.
  • the methylation detection primer pair sequence of the KLRK1 gene promoter region chr12_10541790 is 5'-GATAATAATATATTTAGTTTTTTGTTTATAGTCG-3' and 5'-AACTAAATTATTACCAAATAAAATAAACAACT-3';
  • the nucleotide sequence of the methylation probe is
  • the chr14_22927264 methylation detection primer pair sequence in the TRDJ3 gene promoter region is 5'-GATGTGAAGTTTAGGTAGGGGATGT-3' and 5'-TACCTCATCACCAAAATCAAAATCG-3';
  • the nucleotide sequence of the methylation probe in the TRDJ3 gene promoter region chr14_22927264 is 5'-FAM-CTAAAACCAAACAAAAAAAAATAACAATTATATAAAACC-BHQ1-3';
  • the methylation detection primer pair sequences of chr14_22926867 in the TRDJ3 gene promoter region are 5'-GGGAAATGGTAGAATTTGGTGAG-3' and 5'-ACATCAATCAAACTACAATCAACTACG-3';
  • the nucleotide sequence of chr14_22926867 methylation probe in the promoter region of the TRDJ3 gene is 5'-FAM-TTCTATTTCCCTCCTTTTCAATACTCTAATCTC-BHQ1-3';
  • the methylation detection primer pair sequences of chr14_22931150 in the TRDC gene promoter region are 5'-GGAAGGGATGGTATTTTTTCG-3' and 5'-CATTCTATCACTTAAAAAATCGAAAATC-3';
  • the nucleotide sequence of the methylation probe in the TRDC gene promoter region chr14_22931150 is 5'-FAM-AAAACTACATAACTAACTCAAAAATTCTAAAAATAACAAT-BHQ1-3'.
  • the combination also includes methylation detection primers and probes of the internal reference gene ACTB gene.
  • the methylation detection primer of the internal reference gene ACTB gene includes a forward primer and a reverse primer.
  • the nucleotide sequences of the forward primer and the reaction primer are respectively 5'-TGGTGATGGAGGAGGTTTAGTAAGT-3' and 5'-AACCAATAAAAACCTACTCCTCCCTTAAA-3'. ;
  • nucleotide sequence of the methylation probe of the internal reference gene ACTB gene is 5'-VIC-ACCACCACCACCCAACACACAATAACAAACACA-BHQ1-3'.
  • the 5' end of the methylation probe of the methylation site provided by the invention is labeled with the fluorescent group FAM, and the 3' end is labeled with the quenching group BHQ1; the 5' end of the internal reference gene ACTB gene It is labeled with the fluorescent group VIC, and the 3' end is labeled with the quenching group BHQ1.
  • the present invention provides the application of the above primer and probe combination in preparing a kit for early screening of gastric cancer.
  • the methylation detection primer-probe combination provided by the invention can be used to prepare a kit for early screening of gastric cancer.
  • kits for early screening of gastric cancer includes the primer and probe combination, a PCR reaction solution, a positive quality control product and a negative quality control product.
  • step (3) Perform sulfite conversion on the DNA extracted in step (2) to obtain converted DNA;
  • the Ct value of the VIC fluorescence channel is >35 or there is no amplification. If ⁇ Ct is any result, the results are invalid and the transformed sample needs to be re-extracted and tested.
  • the Ct value of the FAM fluorescence channel and the Ct value of the VIC fluorescence channel respectively refer to the number of cycles experienced by the fluorescence signals of the FAM and VIC fluorescence channels during the PCR amplification process to reach the set threshold.
  • the smaller the Ct value the PCR amplification reaches the plateau.
  • the fewer the number of cycles required for PCR amplification the higher the initial content of the target gene; conversely, the larger the Ct value, the more cycles required for PCR amplification to reach the plateau phase, and the lower the initial content of the target gene.
  • the invention provides a kit for detecting the methylation status of KLRK1, TRDJ3 and TRDC genes in human peripheral blood mononuclear cells for early screening of gastric cancer. It has the advantages of non-invasiveness, high sensitivity and high accuracy, and is easy to use. It is accepted by the subjects, is easy to operate and has strong applicability.
  • the present invention provides a primer-probe combination for early screening of gastric cancer, which can specifically detect the methylation status of the KLRK1 gene promoter region chr12_10541790, the TRDJ3 gene promoter region chr14_22927264 and chr14_22926867, and the TRDC gene promoter region chr14_22931150. .
  • the primer-probe combination for early screening of gastric cancer provided by the present invention also includes methylation primers and probes of the internal reference gene ACTB gene.
  • the present invention prefers the ACTB gene as the internal reference gene for real-time monitoring of sample quality and sample The sulfite conversion process of DNA ensures the validity of the test results.
  • the present invention provides a kit for early screening of gastric cancer, which can detect the genomic DNA of peripheral blood mononuclear cells of patients with suspected gastric cancer to screen for gastric cancer.
  • the detection sensitivity can reach 81.65% and the specificity can reach 81.35%. .
  • the detection method provided by the present invention is simple, fast, safe and non-invasive. It only requires 3 mL of blood to complete the detection. It can obtain accurate detection results on the basis of reducing the patient's pain and has high clinical application value.
  • Figure 1 is a comparison chart between a single marker signal and the internal reference methylation qPCR amplification curve and the multi-marker signal superposition and the internal reference methylation qPCR amplification curve.
  • the ordinate represents the logarithmic value of the corrected fluorescence intensity
  • the abscissa represents fluorescence quantification.
  • the number of cycles of qPCR amplification, and the Ct value is the number of cycles required to reach the threshold line.
  • Figure 2 is a scatter plot of ⁇ Ct values for gastric cancer patients and healthy controls provided in Example 2.
  • Figure 3 is the receiver operating characteristic curve provided in Example 2.
  • This embodiment provides a methylation marker combination for early screening of gastric cancer, including KLRK1 gene chr12_10541790, TRDJ3 gene chr14_22927264 and chr14_22926867, and TRDC gene chr14_22931150.
  • the MethPrimer online website was used to design the sulfite modified sequence.
  • the specific primers and probes used to detect the methylation status of the above markers are based on the following principles: (1) In order to obtain the best performance, the methyl photopolymerase chain reaction amplicon is preferably 80-100 base pairs. Short amplicons; (2) Each primer and probe oligo should contain at least two CpGs.
  • CpG should be located at or near the 3' end of the primer sequence, and the last five bases at the 3' end of the primer oligomer should not contain more than three guanine (G) and/or cytosine (C);
  • G guanine
  • C cytosine
  • the CpG should be located in the middle part of the probe oligomer sequence; (5) Avoid single nucleotide repeats in the primer and probe sequences ; (6)
  • the melting temperature (Tm) of the probe oligomer should be 5-10°C higher than the melting temperature of the primer.
  • the Tm of each primer should be 59°C ⁇ 2°C, and in one detection, the Tm of each primer should be The difference between them should not exceed 1°C; (7) Avoid using probe sequences that start with guanine (G) at the 5' end, and the probe sequence should contain less guanine (G) than cytosine (C); (8) )The length of the probe oligomer does not exceed 30bp.
  • the forward primer sequence of KLRK1 gene chr12_10541790 is shown in SEQ ID No.1, the reverse primer sequence is shown in SEQ ID No.2, and the fluorescent probe sequence is shown in SEQ ID No.3. The 5' end of the probe is labeled with FAM. Label the 3' end of the needle BHQ1.
  • the forward primer sequence of TRDJ3 gene chr14_22926867 is shown in SEQ ID No.4, the reverse primer sequence is shown in SEQ ID No.5, and the fluorescent probe sequence is shown in SEQ ID No.6. The 5' end of the probe is labeled with FAM. Label the 3' end of the needle BHQ1.
  • the forward primer sequence of TRDJ3 gene chr14_22927264 is shown in SEQ ID No.7
  • the reverse primer sequence is shown in SEQ ID No.8
  • the fluorescent probe sequence is shown in SEQ ID No.9.
  • the 5' end of the probe is labeled with FAM.
  • the forward primer sequence of chr14_22931150 in the TRDC gene promoter region is as shown in SEQ ID No.10
  • the reverse primer sequence is as shown in SEQ ID No.11
  • the fluorescent probe sequence is as shown in SEQ ID No.12
  • the 5' end of the probe is labeled with FAM.
  • the 3' end of the probe is labeled with BHQ1.
  • the forward primer sequence of the internal reference gene ACTB gene is as shown in SEQ ID No.13
  • the reverse primer sequence is as shown in SEQ ID No.14
  • the fluorescent probe sequence is as shown in SEQ ID No.15
  • the 5' end of the probe is labeled with VIC.
  • the 3' end of the probe is labeled with BHQ1.
  • the present invention also provides a kit for detecting the methylation status of KLRK1 gene chr12_10541790, TRDJ3 gene chr14_22927264 and chr14_22926867, and TRDC gene chr14_22931150.
  • the kit also includes a PCR reaction solution and a positive substance. Control materials and negative control materials.
  • the PCR reaction solution includes PCR reaction buffer, dNTPs, Mg 2+ , Taq DNA polymerase, etc., the positive quality control product is bisulfite-modified fully methylated human genomic DNA, and the negative quality control product Bisulfite modified unmethylated human genomic DNA.
  • the method of using the kit of the present invention is as follows:
  • OD260/280 is between 1.8 and 2.0.
  • the amplification program settings are as shown in Table 3.
  • ⁇ Ct Ct value of VIC fluorescence channel-Ct value of FAM fluorescence channel.
  • Example 1 The samples of the research subjects were tested according to the method described in Example 1. 3 mL of blood was taken from 218 gastric cancer patients confirmed by histopathology and 193 healthy controls who had gastric diseases excluded by gastroscopy, and peripheral blood mononuclear nuclei were isolated. cells, extract genomic DNA and perform sulfite conversion, followed by single-tube multi-channel PCR amplification and detection.
  • test results are shown in Table 5 and Figure 3. It can be seen that the PCR results of peripheral blood mononuclear cell genomic DNA samples from 178 gastric cancer patients and 157 healthy controls were valid. Among the 218 gastric cancer patients, 178 tested positive. , the sensitivity was 81.65%; among 193 healthy control samples, 157 cases were tested positive, and the specificity was 81.35%. The accuracy rate of the kit for early detection of gastric cancer is 81.51%, and the area under the curve is 0.897.
  • Sensitivity (true positive rate) number of true positives/(number of true positives + number of false negatives)*100%.
  • Accuracy (number of true positives + number of true negatives)/(number of true positives + number of true negatives + number of false positives + number of false negatives) * 100%.
  • the amplification curve of methylation detection on peripheral blood mononuclear cell genomic DNA samples of patients clinically diagnosed with gastric cancer provided in this example is shown in Figure 1. It can be seen that the Ct value of the VIC channel is ⁇ 35, ⁇ Ct>2.0157 and The FAM channel has an "S" amplification curve, indicating that the test result is positive; the amplification curve of methylation detection of genomic DNA samples of peripheral blood mononuclear cells from clinically diagnosed healthy controls provided in this example is shown in Figure 2 It can be seen that the Ct value of the VIC channel is ⁇ 35 and ⁇ Ct ⁇ 2.0157, indicating that the test result is negative; the receiver operating characteristic curve provided in this embodiment is shown in Figure 3. It can be seen that the ROC curve is close to the upper left corner, This shows that the accuracy of the obtained prediction results is high.
  • the primer and probe combination provided by the present invention for detecting the methylation levels of KLRK1 gene chr12_10541790, TRDJ3 gene chr14_22927264 and chr14_22926867, and TRDC gene chr14_22931150 can specifically detect the methylation level of the site, and ACTB is preferred.
  • the gene is used as an internal reference gene to monitor sample quality in real time.
  • the kit prepared using the primer-probe combination can be used for early screening of gastric cancer. It has the advantages of high sensitivity and strong specificity. It is also easy to operate, convenient, fast, safe and non-invasive. , is conducive to clinical application and promotion, and has very important practical significance for non-invasive gastric cancer screening and early diagnosis.

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Abstract

本发明属于肿瘤分子生物学技术领域，涉及一种检测人外周血样本单个核细胞中DNA甲基化位点的试剂在胃癌早期筛查试剂或试剂盒中的应用，所述位点包括KLRK1基因chr12_10541790位点、TRDJ3基因chr14_22927264位点和chr14_22926867 位点、TRDC 基因 chr14_22931150位点。

Description

胃癌早期筛查的DNA甲基化标志物组合及试剂盒 技术领域
本发明属于肿瘤分子生物学技术领域，涉及一种用于胃癌早期筛查的DNA甲基化标志物组合及试剂盒。
背景技术
胃癌(gastric cancer)是消化系统最为常见的恶性肿瘤之一，也是全球第五常见的恶性肿瘤，其病死率在恶性肿瘤中排第四位，严重威胁人类的生命健康。胃癌的发生是一个多因素共同参与的复杂过程，其发病主要与幽门螺杆菌感染、饮食、生活方式及遗传等因素密切相关，尤其易发生在老年人之中，且男性发病率显著高于女性。胃癌的早期发现和治疗对于改善患者的预后至关重要，早期胃癌的5年生存率可以达到95％以上。然而，由于胃癌起病隐匿，早期通常没有临床症状或仅表现出非特异性临床症状，因此在患者初诊时可能已经达到晚期，这使得胃癌的早期诊断和治疗受到极大的限制，总体预后不良。目前常用来诊断胃癌的几种诊断方法在临床应用方面存在着不同程度的局限性：胃镜检查价格昂贵、创伤性大、病人依从性差；血清肿瘤标志物(如胃蛋白酶原、幽门螺杆菌抗体、CEA、CA19-9等)检测及灵敏度低、特异性差；CT检查对早期诊断有限，对细小肿块较易出现漏诊。因此，寻找非侵入性、高灵敏度和高特异度的新型标志物用于无创性胃癌筛查早期诊断，是目前临床上亟需解决的问题。
表观遗传学(epigenetics)是一门新兴学科，其定义为不涉及基因组的碱基序列改变的情况下，基因的表达发生改变。DNA甲基化是一个重要的表观遗传因素，指在DNA甲基化转移酶的作用下，在基因组CpG二核苷酸的胞嘧啶5'碳位共价键结合一个甲基基团。大量研究证实，DNA甲基化能引起染色质结构、DNA构象、DNA稳定性及DNA与 蛋白质相互作用方式的改变，从而控制基因表达，启动子区域的异常甲基化可导致癌基因的激活或抑癌基因的沉默。由于DNA甲基化往往发生在癌症发生过程的早期，所以在肿瘤诊断中应用越来越广泛，可作为胃癌早期筛查的可靠途径。
外周血单个核细胞(peripheral blood mononuclear cell,PBMC)，即外周血中具有单个核的细胞，主要包括淋巴细胞(T/B)、单核细胞、吞噬细胞、树突状细胞和其他少量细胞类型，其中淋巴细胞约占70％～90％。已有研究发现，在肿瘤发生发展中，DNA甲基化通过调控免疫细胞功能，如介导细胞分化、成熟、浸润等，参与机体对肿瘤的免疫应答，PBMC的甲基化研究能够为肿瘤的早期无创性诊断和免疫治疗监测提供新的方法。
发明内容
本发明针对传统胃癌诊断中存在的具有侵入性、费时费力、准确性低等问题提出一种新型的用于胃癌早期筛查的DNA甲基化标志物组合及试剂盒。
为了达到上述目的，本发明是采用下述的技术方案实现的：
本发明提供一种用于胃癌早期筛查的甲基化位点组合，所述组合包括KLRK1基因启动子区的chr12_10541790、TRDJ3基因启动子区的chr14_22927264和chr14_22926867、TRDC基因启动子区的chr14_22931150。
本发明还提供了用于胃癌早期筛查的甲基化检测引物及探针组合，所述组合包括用于检测所述位点甲基化水平的特异性引物和探针。
所述KLRK1基因启动子区chr12_10541790的甲基化检测引物对序列为5'-GATAATAATATATTTAGTTTTTTGTTTATAGTCG-3'和 5'-AACTAAATTATTACCAAATAAAATAAACAACT-3'；
甲基化探针的核苷酸序列为
5'-FAM-AAACACTTAATCTTAACTAAAAAACCAAAAAACAA-BHQ1-3'；
所述TRDJ3基因启动子区的chr14_22927264甲基化检测引物对序列为5'-GATGTGAAGTTTAGGTAGGGGATGT-3'和5'-TACCTCATCACCAAAAATCAAAATCG-3'；
所述TRDJ3基因启动子区chr14_22927264的甲基化探针的核苷酸序列为5'-FAM-CTAAAACCAAACAAAAAAAAATAACAATTATATAAAACC-BHQ1-3'；
所述TRDJ3基因启动子区chr14_22926867的甲基化检测引物对序列为5'-GGGAAATGGTAGAATTTGGTGAG-3'和5'-ACATCAATCAAACTACAATCAACTACG-3'；
所述TRDJ3基因启动子区的chr14_22926867甲基化探针的核苷酸序列为5'-FAM-TTCTATTTCCCTCCTTTTCAATACTCTAATCTC-BHQ1-3'；
所述TRDC基因启动子区chr14_22931150的甲基化检测引物对序列为5'-GGAAGGGATGGTATTTTTTCG-3'和5'-CATTCTATCACTTAAAAAATCGAAAATC-3'；
所述TRDC基因启动子区chr14_22931150的甲基化探针的核苷酸序列为5'-FAM-AAAACTACATAACTAACTCAAAAATTCTAAAAATAACAAT-BHQ1-3'。
所述组合还包括内参基因ACTB基因的甲基化检测引物和探针。所述内参基因ACTB基因的甲基化检测引物包括正向引物和反向引物，所述正向引物和反应引物的核苷酸序列分别 5'-TGGTGATGGAGGAGGTTTAGTAAGT-3'和5'-AACCAATAAAACCTACTCCTCCCTTAAA-3'；
所述内参基因ACTB基因的甲基化探针的核苷酸序列5'-VIC-ACCACCACCCAACACACAATAACAAACACA-BHQ1-3'。
本发明所提供的甲基化位点的甲基化探针的5'端均标记有荧光基团FAM，3'端均标记有淬灭基团BHQ1；所述内参基因ACTB基因的5'端标记有荧光基团VIC，3'端标记有淬灭基团BHQ1。
本发明提供上述的引物及探针组合在制备用于胃癌早期筛查试剂盒中的应用。
本发明提供的甲基化检测引物探针组合能够用于制备胃癌早期筛查的试剂盒。
一种用于胃癌早期筛查的试剂盒，所述试剂盒包括所述的引物及探针组合，还包括PCR反应液、阳性质控品和阴性质控品。
试剂盒的使用方法包括以下步骤：
(1)分离人外周血样本中的单个核细胞；
(2)提取人外周血单个核样本中的基因组DNA；
(3)对步骤(2)中所述提取得到的DNA进行亚硫酸盐转化，得到转化后的DNA；
(4)将步骤(3)所述转化后的DNA与权利要求3、4所述的引物探针组合混合，制备得到引物探针预混液，随后与PCR反应液混合，并进行单管多通道荧光定量PCR扩增检测，根据所述PCR扩增的结果得到检测结果。
所述检测结果的判读标准为：
ΔCt＝VIC荧光通道的Ct值-FAM荧光通道的Ct值
VIC荧光通道的Ct值≤35，ΔCt＞2.0157，则结果为阳性；
VIC荧光通道的Ct值≤35，ΔCt≤2.0157，则结果为阴性；
VIC荧光通道的Ct值>35或无扩增，ΔCt为任何结果，结果皆为无效，需要重新提取转化样本后检测。
所述FAM荧光通道的Ct值和VIC荧光通道的Ct值分别指PCR扩增过程中FAM和VIC荧光通道的荧光信号达到设定阈值所经历的循环数，Ct值越小，PCR扩增到达平台期所需循环数越少，则目的基因起始含量越高；反之，Ct值越大，PCR扩增到达平台期所需循环数越多，则目的基因起始含量越低。
本发明提供了一种检测人外周血单个核细胞中KLRK1、TRDJ3和TRDC基因甲基化状态的试剂盒用于胃癌的早期筛查，具有非侵入性、高灵敏度、高准确度的优点，容易被受检者接受，且操作简便，可应用性强。
与现有技术相比，本发明的优点和积极效果在于：
1.本发明提供了用于胃癌早期筛查的引物探针组合，能够特异性检测出KLRK1基因启动子区chr12_10541790、TRDJ3基因启动子区chr14_22927264和chr14_22926867、TRDC基因启动子区chr14_22931150的甲基化状态。
2.本发明提供的用于胃癌早期筛查的引物探针组合，还包括内参基因ACTB基因的甲基化引物和探针，本发明优选ACTB基因作为内参基因，用于实时监控样本质量和样本DNA的亚硫酸盐转化过程，保证检测结果的有效性。
3.本发明提供了一种用于胃癌早期筛查的试剂盒，能够对疑似胃癌病人的外周血单个核细胞基因组DNA进行检测以筛查胃癌，检测灵敏度能够达到81.65％，特异性能达到81.35％。
4.本发明提供的检测方法操作简便、快速，安全无创，仅需要3mL血也便可完成检测，在减少患者痛苦的基础上能够得到准确的检测结 果，具有很高的临床应用价值。
附图说明
图1为单个标志物信号与内参甲基化qPCR扩增曲线及多标志物信号叠加与内参甲基化qPCR扩增曲线对比图，其中纵坐标表示校正荧光强度的对数值，横坐标表示荧光定量qPCR扩增的循环数，Ct值为达到阈值线所需要的循环数。
图2是实施例2中提供的胃癌患者和健康对照者的ΔCt值的散点图。
图3是实施例2中提供的受试者工作特征曲线。
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
本实施例提供了一种用于胃癌早期筛查的甲基化标志物组合，包括KLRK1基因chr12_10541790、TRDJ3基因chr14_22927264和chr14_22926867、TRDC基因chr14_22931150。
根据美国国立生物技术信息中心(National Biotechnology Information Center，NCBI)公开的人类全基因组序列KLRK1基因、TRDJ3基因、TRDC基因和内参基因ACTB基因序列，针对亚硫酸盐修饰后的序列，利用MethPrimer在线网站设计用于检测上述标志物甲基化状态的特异性引物及探针，原则如下：(1)为了获得最佳性能，甲基光聚合酶链反应扩增子优选80-100个碱基对的较短扩增子；(2)每 个引物和探针寡聚物应包含至少两个CpG。(3)CpG应位于引物序列的3’端或其附近，引物寡聚物3’端的最后五个碱基不应含有三个以上的鸟嘌呤(G)和/或胞嘧啶(C)；(4)为最大化甲基化和未甲基化模板分子之间的熔点差异，CpG应该位于探针寡聚体序列的中间部分；(5)避免引物和探针序列中的单核苷酸重复；(6)探针低聚物的熔化温度(Tm)应比引物的熔化温度高5-10℃，每个引物的Tm应在59℃±2℃，且在一次检测中，每个引物之间的差异不应超过1℃；(7)避免使用5’端以鸟嘌呤(G)开始的探针序列，且探针序列含有鸟嘌呤(G)应该比胞嘧啶(C)少；(8)探针寡聚体的长度不超过30bp。
KLRK1基因chr12_10541790的正向引物序列如SEQ ID No.1，反向引物序列如SEQ ID No.2所示，荧光探针序列如SEQ ID No.3所示，探针5'端标记FAM，探针3'端标记BHQ1。TRDJ3基因chr14_22926867的正向引物序列如SEQ ID No.4，反向引物序列如SEQ ID No.5所示，荧光探针序列如SEQ ID No.6所示，探针5'端标记FAM，探针3'端标记BHQ1。TRDJ3基因chr14_22927264的正向引物序列如SEQ ID No.7，反向引物序列如SEQ ID No.8所示，荧光探针序列如SEQ ID No.9所示，探针5'端标记FAM，探针3'端标记BHQ1。TRDC基因启动子区chr14_22931150的正向引物序列如SEQ ID No.10，反向引物序列如SEQ ID No.11，荧光探针序列如SEQ ID No.12所示，探针5'端标记FAM，探针3'端标记BHQ1。内参基因ACTB基因的正向引物序列如SEQ ID No.13，反向引物序列如SEQ ID No.14所示，荧光探针序列如SEQ ID No.15所示，探针5'端标记VIC，探针3'端标记BHQ1。
基于上述引物探针组合，本发明还提供了一种用于检测KLRK1基因chr12_10541790、TRDJ3基因chr14_22927264和chr14_22926867、TRDC基因chr14_22931150甲基化状态的试剂盒，所述试剂盒还包括PCR反应液、阳性质控品和阴性质控品。所述PCR反应液包括PCR反 应缓冲液、dNTP、Mg 2+、Taq DNA聚合酶等，所述阳性质控品为亚硫酸氢盐修饰的全甲基化人类基因组DNA，所述阴性质控品为亚硫酸氢盐修饰的非甲基化人类基因组DNA。
本发明所述试剂盒的使用方法如下：
1、分离人外周血单个核细胞
(1)每位研究对象采血一次(3mL/管，EDTA抗凝全血)后，2小时内使用
Figure PCTCN2022132108-appb-000001
-1077溶液分离单个核细胞。
(2)在15mL离心管中预先加入淋巴细胞分离液，使分离液：血液＝1:1，小心的将3mL血液加到分离液的上面，400×g室温离心30min(制动和加速最低)。
(3)用一次性吸管小心地将白膜层(单个核细胞层)重悬于10mL PBS中。
(4)250×g室温离心10min。吸弃上清，重复洗涤细胞沉淀两次，吸弃上清，将细胞沉淀保存于-80℃待用。
2、提取样本的基因组DNA
(1)将外周血单个核细胞悬液10,000rpm离心1min，倒尽上清，加200μL缓冲液GA，振荡至彻底悬浮。
(2)加20μL Proteinase K溶液，混匀。
(3)加200μL缓冲液GB，充分颠倒混匀，70℃处理10min。
(4)加200μL无水乙醇，充分振荡混匀15s。
(5)将(4)所得溶液和絮状沉淀都加入一个吸附柱CB3中(吸附柱放入收集管中)，12000rpm离心30s，弃废液。
(6)加500μL缓冲液GD，12000rpm离心30s，弃废液。
(7)加600μL漂洗液PW，12000rpm离心30s，弃废液，重复一次。
(8)12000rpm空管离心2min，弃废液，将吸附柱放入新的1.5mL 离心管中，开盖静置2min，彻底挥发乙醇。
(9)向吸附膜的中间部位悬空滴加50μL洗脱缓冲液TE，室温放置2-5min，12000rpm离心2min，将溶液收集到离心管中。
(10)使用Nanodrop 2000对DNA浓度和纯度进行测定，OD260/280在1.8～2.0之间。
3、人外周血单个核细胞基因组DNA的亚硫酸盐转化
(1)取0.2mL离心管，加入20μL的DNA溶液(1000ng DNA，用双蒸水补齐)，130μL完全溶解的CT Coversion Regent振荡混匀，短暂离心。
(2)将(1)所得混合液98℃处理10min，64℃处理2.5h，4℃无限循环。
(3)将收集管套入吸附柱，加入600μL M-Binding Buffer。
(4)将(2)所得产物加入含有600μL M-Binding Buffer的吸附柱中，关盖，颠倒混匀1min，13400rpm离心30s，弃废液。
(5)加100μL M-wash Buffer，13400rpm离心30s，弃废液。
(6)加200μL M-Desulphonation Buffer，室温放置15-20min，13400rpm离心30s，弃废液。
(7)加200μL M-wash Buffer，13400rpm离心30s，弃废液，重复一次。
(8)将吸附柱置于新的1.5mL离心管中，向吸附膜的中间部位滴加20μL M-Elution Buffer，静置2min，13400rpm离心30s，洗脱修饰后的DNA。
4、PCR扩增与检测
按照下表1配制20μL的PCR反应液。
表1 PCR反应液
组分 一人份加入 终浓度
  量  
2×AceQ qPCR Probe Master Mix 10μL 1×
KLRK1 chr12_10541790的前向引物 0.5μL 0.25μM
KLRK1 chr12_10541790的后向引物 0.5μL 0.25μM
KLRK1 chr12_10541790的探针 0.2μL 0.10μM
TRDJ3 chr14_22927264的前向引物 0.5μL 0.25μM
TRDJ3 chr14_22927264的后向引物 0.5μL 0.25μM
TRDJ3 chr14_22927264的探针 0.2μL 0.10μM
TRDJ3 chr14_22926867的前向引物 0.5μL 0.25μM
TRDJ3 chr14_22926867的后向引物 0.5μL 0.25μM
TRDJ3 chr14_22926867的探针 0.2μL 0.10μM
TRDC chr14_22931150的前向引物 0.5μL 0.25μM
TRDC chr14_22931150的后向引物 0.5μL 0.25μM
TRDC chr14_22931150的探针 0.2μL 0.10μM
ACTB前向引物 0.12μL 0.06μM
ACTB后向引物 0.12μL 0.06μM
ACTB探针 0.1μL 0.05μM
50×ROX Reference Dye 1 0.4μL 0.4μL
模板DNA 2μL -
ddH2O 2.46μL -
于PCR八连管中配制好体系，做好标记，盖紧管盖，瞬时低速离心，置于ABI 7500中进行反应。
仪器荧光通道设置如下表2。
表2仪器荧光通道设置
目的分子 荧光基因 淬灭集团
KLRK1 chr12_10541790 FAM BHQ1
TRDJ3 chr14_22927264 FAM BHQ1
TRDJ3 chr14_22926867 FAM BHQ1
TRDC chr14_22931150 FAM BHQ1
ACTB VIC BHQ1
扩增程序设定如下表3。
表3扩增程序
Figure PCTCN2022132108-appb-000002
PCR结果判读如下表4所示。
若判读结果为阴性，患胃癌的风险较低，建议定期随访；若判读结果为阳性，患胃癌的风险较高，建议进行胃镜检查。
表4 PCR结果判读
Figure PCTCN2022132108-appb-000003
其中，ΔCt＝VIC荧光通道的Ct值-FAM荧光通道的Ct值。
实施例2
按照实施例1所述使用方法对研究对象的样本进行检测，取218位经过组织病理学确认的胃癌患者和193位经胃镜检查排除胃部疾病的健康对照者的血液3mL，分离外周血单个核细胞，提取基因组DNA并进行亚硫酸盐转化，后续进行单管多通道PCR扩增和检测。
检测结果如表5和图3所示，可以看到178例胃癌患者和157例健康对照者的外周血单个核细胞基因组DNA样本的PCR结果有效，在218例胃癌患者中，检测阳性为178例，灵敏度为81.65％；在193例健康对照者样本中，有157例检测为阳性，特异性为81.35％。本发明试剂盒用于早期检测胃癌的准确率为81.51％，曲线下面积为0.897。
表5检测结果
Figure PCTCN2022132108-appb-000004
注：灵敏度(真阳性率)＝真阳性人数/(真阳性人数+假阴性人数)*100％。
指正确判断病人的程度，即实际有病而被正确诊断的百分比。
注：特异性(真阴性率)＝真阴性人数/(真阴性人数+假阳性人数)*100％。
指正确判断非病人的程度，即实际无病而被正确诊断为无病的百分比。
注：准确率＝(真阳性人数+真阴性人数)/(真阳性人数+真阴性人数+假阳性人数+假阴性人数)*100％。
指正确判断非病人的程度，即实际无病而被正确诊断为无病的百分比。
本实施例中提供的临床确诊为胃癌患者的外周血单个核细胞基因组DNA样本进行甲基化检测的扩增曲线如图1所示，可以看出VIC通道的Ct值≤35，ΔCt＞2.0157且FAM通道有“S”扩增曲线，表明检测结果为阳性；本实施例中提供的临床确诊为健康对照者的外周血单个核细胞基因组DNA样本进行甲基化检测的扩增曲线如图2所示，可以看出VIC通道的Ct值≤35，ΔCt≤2.0157，表明检测结果为阴性；本实施例中提供的受试者工作特征曲线如图3所示，可以看出ROC曲线接近左上角，说明得到的预测结果准确率高。
综上所述，本发明提供的检测KLRK1基因chr12_10541790、TRDJ3基因chr14_22927264和chr14_22926867、TRDC基因chr14_22931150甲基化水平的引物及探针组合，能够特异性检测出位点的甲基化水平，同时优选ACTB基因作为内参基因，实时监控样本质量，利用所述引物探针组合制备的试剂盒，可以用于胃癌的早期筛查，具有灵敏度高、特异性强的优点，同时操作简单、方便快捷、安全无创，有利于临床应用和推广，对无创性胃癌筛查和早期诊断，具有非常重要的现实意义。
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 (6)

  1. 检测人外周血样本单个核细胞中KLRK1基因的chr 12_10541790位点、TRDJ3基因的chr14_22927264位点和chr14_22926867位点、TRDC基因的chr14_22931150位点甲基化状态的物质在胃癌早期筛查试剂或试剂盒中的应用。
  2. 一种胃癌早期筛查试剂盒，其特征在于，包括KLRK1基因chr12_10541790位点、TRDJ3基因chr14_22927264位点和chr14_22926867位点、TRDC基因chr14_22931150位点的甲基化检测引物对及探针，其中，
    所述KLRK1基因chr12_10541790位点的甲基化检测引物对包括正向引物和反向引物，所述正向引物和反应引物的核苷酸序列分别如SEQ ID No.1和SEQ ID No.2所示；
    所述KLRK1基因chr12_10541790位点的甲基化探针的核苷酸序列如SEQ ID No.3所示；
    所述TRDJ3基因chr14_22927264位点的甲基化检测引物对包括正向引物和反向引物，所述正向引物和反应引物的核苷酸序列分别如SEQ ID No.4和SEQ ID No.5所示；
    所述TRDJ3基因chr14_22927264位点的甲基化探针的核苷酸序列如SEQ ID No.6所示；
    所述TRDJ3基因chr14_22926867位点的甲基化检测引物对包括正向引物和反向引物，所述正向引物和反应引物的核苷酸序列分别如SEQ ID No.7和SEQ ID No.8所示；
    所述TRDJ3基因chr14_22926867位点的甲基化探针的核苷酸序列如SEQ ID No.9所示；
    所述TRDC基因chr14_22931150位点的甲基化检测引物对包括正向引物和反向引物，所述正向引物和反应引物的核苷酸序列分别 如SEQ ID No.10和SEQ ID No.11所示；
    所述TRDC基因chr14_22931150位点的甲基化探针的核苷酸序列如SEQ ID No.12所示。
  3. 根据权利要求2所述胃癌早期筛查试剂盒，其特征在于，还包括内参基因ACTB的甲基化检测引物对和探针，正向引物和反应引物的核苷酸序列分别如SEQ ID No.13和SEQ ID No.14所示；探针的核苷酸序列如SEQ ID No.15所示。
  4. 根据权利要求3所述胃癌早期筛查试剂盒，其特征在于，KLRK1基因chr12_10541790位点、TRDJ3基因chr14_22927264位点和chr14_22926867位点、TRDC基因chr14_22931150位点甲基化探针的5'端均标记有荧光基团FAM，3'端均标记有淬灭基团BHQ1；内参基因ACTB基因的5'端标记有荧光基团VIC，3'端标记有淬灭基团BHQ1。
  5. 根据权利要求4所述胃癌早期筛查试剂盒，其特征在于，还包括阳性质控品和阴性质控品，所述阳性质控品为亚硫酸氢盐修饰的全甲基化人类基因组DNA，所述阴性质控品为亚硫酸氢盐修饰的非甲基化人类基因组DNA。
  6. 根据权利要求4所述胃癌早期筛查试剂盒，其特征在于，所述试剂盒还包括一个诊断模型，公式如下：
    ΔCt＝VIC荧光通道的Ct值-FAM荧光通道的Ct值；
    VIC荧光通道的Ct值≤35，ΔCt＞2.0157，则结果为阳性；
    VIC荧光通道的Ct值≤35，ΔCt≤2.0157，则结果为阴性；
    VIC荧光通道的Ct值>35或无扩增，ΔCt为任何结果，结果皆为无效，需要重新提取转化样本后检测；
    若判读结果为阴性，代表患胃癌的风险低；若判读结果为阳性，代表患胃癌的风险高。
PCT/CN2022/132108 2022-08-16 2022-11-16 胃癌早期筛查的dna甲基化标志物组合及试剂盒 WO2024036785A1 (zh)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105624279A (zh) * 2015-01-22 2016-06-01 香港中文大学深圳研究院 胃癌标记物、其表达和甲基化检测方法、试剂盒及应用
CN109196359A (zh) * 2016-02-29 2019-01-11 基础医疗股份有限公司 用于评估肿瘤突变负荷的方法和系统

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3443001A4 (en) * 2016-04-11 2020-04-29 Obsidian Therapeutics, Inc. REGULATED BIOCIRCUIT SYSTEMS

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105624279A (zh) * 2015-01-22 2016-06-01 香港中文大学深圳研究院 胃癌标记物、其表达和甲基化检测方法、试剂盒及应用
CN109196359A (zh) * 2016-02-29 2019-01-11 基础医疗股份有限公司 用于评估肿瘤突变负荷的方法和系统

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
LIN FENGJUAN; DAI CONGQI; GE XIAOXIAO; TANG WENBO; LIN YING; WANG YAN; LI JIN: "Prognostic significance and functional implication of immune activating receptor NKG2D in gastric cancer", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, ELSEVIER, AMSTERDAM NL, vol. 487, no. 3, 20 April 2017 (2017-04-20), Amsterdam NL , pages 619 - 624, XP085001185, ISSN: 0006-291X, DOI: 10.1016/j.bbrc.2017.04.104 *
WANG JUN, YU SHAOJUN, CHEN GUOFENG, KANG MUXING, JIN XIAOLI, HUANG YI, LIN LELE, WU DAN, WANG LIE, CHEN JIAN: "A novel prognostic signature of immune‐related genes for patients with colorectal cancer", JOURNAL OF CELLULAR AND MOLECULAR MEDICINE, UNIVERSITY PRESS CAROL DAVILA, BUCHAREST, RO, vol. 24, no. 15, 1 August 2020 (2020-08-01), RO , pages 8491 - 8504, XP093140639, ISSN: 1582-1838, DOI: 10.1111/jcmm.15443 *
WANG KAI, GAO SHUAI: "Research progress of molecular diagnostic markers for primary hepatocellular carcinoma", JOURNAL OF MOLECULAR DIAGNOSTICS AND THERAPY., vol. 12, no. 1, 1 January 2020 (2020-01-01), pages 1 - 5, 38, XP093140640 *
XU GUANGRU; ZHANG MINGHUI; ZHU HONGXING; XU JINHUA: "A 15-gene signature for prediction of colon cancer recurrence and prognosis based on SVM", GENE, ELSEVIER AMSTERDAM, NL, vol. 604, 18 December 2016 (2016-12-18), NL , pages 33 - 40, XP029875976, ISSN: 0378-1119, DOI: 10.1016/j.gene.2016.12.016 *

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