WO2024031860A1 - 一种用于结直肠癌诊断的多基因dna甲基化联合检测试剂盒及应用 - Google Patents

一种用于结直肠癌诊断的多基因dna甲基化联合检测试剂盒及应用 Download PDF

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WO2024031860A1
WO2024031860A1 PCT/CN2022/132111 CN2022132111W WO2024031860A1 WO 2024031860 A1 WO2024031860 A1 WO 2024031860A1 CN 2022132111 W CN2022132111 W CN 2022132111W WO 2024031860 A1 WO2024031860 A1 WO 2024031860A1
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colorectal cancer
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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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    • C12Q1/6858—Allele-specific amplification
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
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  • the invention belongs to the technical field of genetic engineering technology and relates to a diagnostic kit, detection method and clinical application based on colorectal cancer TRGV9, TRDJ3, TRDC, FAM174B, ZMIZ2 multi-gene DNA methylation detection.
  • CRC colorectal cancer
  • CCA carcinoembryonic antigen
  • PBMC peripheral blood mononuclear cells
  • the present invention proposes a non-invasive technical solution based on the DNA methylation levels of TRGV9, TRDJ3, TRDC, FAM174B, and ZMIZ2 multi-gene DNA methylation levels in order to solve the technical problems of early colorectal cancer diagnosis technology such as invasiveness, high price, and low accuracy.
  • Colorectal cancer diagnostic kit which has high detection sensitivity and specificity, in order to achieve accurate and rapid diagnosis of early colorectal cancer.
  • a primer and probe for colorectal cancer diagnostic detection based on peripheral blood mononuclear cell DNA are used to detect colorectal cancer-specific DNA methylation markers.
  • the colorectal cancer-specific Markers include: TRGV9, TRDJ3, TRDC, FAM174B, and ZMIZ2 genes.
  • the detection primer is used to amplify a sequence of the colorectal cancer specific marker.
  • TRGV9 amplified region sequence
  • the TRGV9 gene detection primer includes a forward primer and a reverse primer.
  • the nucleotide sequence of the forward primer is shown in SEQ ID NO.1
  • the nucleotide sequence of the reverse primer is shown in SEQ ID NO.2.
  • the nucleotide sequence of the probe is shown in SEQ ID NO. 3, and both ends of the probe are labeled with a fluorescent reporter group and a fluorescent quenching group respectively.
  • the TRDJ3 gene detection primer includes a forward primer and a reverse primer.
  • the nucleotide sequence of the forward primer is shown in SEQ ID NO.4, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.5. .
  • the nucleotide sequence of the probe is shown in SEQ ID NO. 6, and both ends of the probe are labeled with a fluorescent reporter group and a fluorescent quenching group respectively.
  • the TRDC gene detection primer includes a forward primer and a reverse primer.
  • the nucleotide sequence of the forward primer is shown in SEQ ID NO.7
  • the nucleotide sequence of the reverse primer is shown in SEQ ID NO.8 .
  • the nucleotide sequence of the probe is shown in SEQ ID NO. 9, and both ends of the probe are labeled with a fluorescent reporter group and a fluorescent quenching group respectively.
  • the FAM174B gene detection primer includes a forward primer and a reverse primer.
  • the nucleotide sequence of the forward primer is shown in SEQ ID NO.10
  • the nucleotide sequence of the reverse primer is shown in SEQ ID NO.11.
  • the nucleotide sequence of the probe is shown in SEQ ID NO. 12, and both ends of the probe are labeled with a fluorescent reporter group and a fluorescent quenching group respectively.
  • the ZMIZ2 gene detection primer includes a forward primer and a reverse primer.
  • the nucleotide sequence of the forward primer is shown in SEQ ID NO.13
  • the nucleotide sequence of the reverse primer is shown in SEQ ID NO.14.
  • the nucleotide sequence of the probe is shown in SEQ ID NO. 15, and both ends of the probe are labeled with a fluorescent reporter group and a fluorescent quenching group respectively.
  • the fluorescent reporter group/fluorescence quenching group is selected from fluorescent reporter groups such as FAM, VIC, HEX, ROX, NED, CY3, CY5, etc./fluorescence quenchers such as BHQ1, BHQ2, BHQ3, Dabyc 1, Tamra, etc. group, and the fluorescent reporter groups on the TRGV9, TRDJ3, and TRDC gene probes in the kit are different from those on the FAM174B and ZMIZ2 gene probes.
  • the fluorescent reporter group at the 5' end of TRGV9, TRDJ3, and TRDC genes is FAM; the fluorescent reporter group at the 5' end of FAM174B and ZMIZ2 genes is CY5, the fluorescent reporter group at the 5' end of ⁇ -actin is VIC, and the quenching group is BHQ1;
  • the diagnostic model is as follows:
  • ⁇ CT 1 CT VIC -CT FAM ,
  • CT 2 CT VIC -CT CY5 ,
  • Multi-PCR Score 1/[1+exp(10.73266+11.57596* ⁇ CT 1 -15.31471* ⁇ CT 2 )].
  • VIC When VIC reaches the set threshold, it means that the DNA loading amount is within the allowable range, and the FAM and CY5 results are credible.
  • VIC if the Multi-PCR Score is greater than 0.5, the result is negative; if the Multi-PCR Score is less than 0.5, the result is positive.
  • the present invention also proposes a colorectal cancer diagnostic kit, which at least includes detection reagents that specifically identify TRGV9, TRDJ3, TRDC, FAM174B, and ZMIZ2 genes.
  • colorectal cancer detection reagent is not limited to liquid form.
  • the kit also includes specific primers and probes for PCR amplification of the ⁇ -actin gene.
  • the ⁇ -actin gene detection primers include a forward primer and a reverse primer.
  • the nucleotide sequence of the forward primer is such as SEQ. ID NO.16 is shown, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.17.
  • the nucleotide sequence of the probe is shown in SEQ ID NO. 18, and both ends of the probe are labeled with a fluorescent reporter group and a fluorescent quenching group respectively.
  • the fluorescent reporter group/fluorescence quenching group is selected from fluorescent reporter groups such as FAM, VIC, HEX, ROX, NED, CY3, CY5, etc./fluorescence quenching groups such as BHQ1, BHQ2, BHQ3, Dabyc1, Tamra, etc., and In the kit, the fluorescent reporter groups on the TRGV9, TRDJ3, and TRDC gene probes, the fluorescent reporter groups on the FAM174B, ZMIZ2 gene probes, and the ⁇ -actin gene probe are different from each other.
  • the kit also includes one or more of the following components: PCR reaction solution, primers, probes, ROX, positive quality control products, and negative quality control products.
  • the PCR reaction solution includes hot-start Taq enzyme, PCR buffer, MgCl 2 , dUTP, and dNTPs.
  • Such commonly used methylation quantitative PCR reagents can be purchased separately from the market or configured by yourself.
  • the positive quality control product is prepared from human genomic DNA with methylated ⁇ -actin gene.
  • the negative quality control product is prepared from human genomic DNA with unmethylated ⁇ -actin gene.
  • the method of using the aforementioned colorectal cancer detection kit includes the following steps:
  • the present invention provides a method for diagnosing colorectal cancer, which includes relative quantification of hypermethylated genes (TRGV9, ZMIZ2, TRDC) and hypomethylated genes (FAM174B, TRDJ3) in samples, and judging colorectal cancer through Multi-PCR Score. The occurrence of cancer.
  • peripheral blood mononuclear cells are used as the detection sample.
  • the PCR probe primer master mix used to specifically identify TRGV9 gene DNA methylation includes: 0.1-1uM probe, 0.1-1uM forward primer and 0.1-1uM reverse primer;
  • the PCR probe primer master mix used to specifically identify TRDJ3 gene DNA methylation includes: 0.1-1uM probe, 0.1-1uM forward primer and 0.1-1uM reverse primer;
  • the PCR probe primer master mix used to specifically identify TRDC gene DNA methylation includes: 0.1-1uM probe, 0.1-1uM forward primer and 0.1-1uM reverse primer;
  • the PCR probe primer premix used to specifically identify DNA methylation of the FAM174B gene includes: 0.1-1uM probe, 0.1-1uM forward primer and 0.1-1uM reverse primer;
  • the PCR probe primer master mix used to specifically identify ZMIZ2 gene DNA methylation includes: 0.1-1uM probe, 0.1-1uM forward primer and 0.1-1uM reverse primer.
  • the type of detection specimen is peripheral blood, the sample is easy to obtain, non-invasive, and the patient's compliance is good.
  • the detection kit provided by the present invention designs specific primers and probes for TRGV9, TRDJ3, TRDC, FAM174B, and ZMIZ2 genes, and selects ⁇ -actin as the internal reference gene to ensure high sensitivity and good specificity of the detection results.
  • the detection kit provided contains negative quality control materials and positive quality control materials, which can prevent the occurrence of false negative results and false positive results to a large extent. It has a broad application in the field of in vitro diagnosis of colorectal cancer peripheral blood PBMC methylation. prospect.
  • the detection kit provided by the present invention is easy to operate, greatly reduces detection costs, and improves detection efficiency.
  • PCR fluorescence detection is a fully closed operation, which does not require electrophoresis, hybridization and other operations, and can effectively prevent contamination.
  • the detection kit provided by the present invention is suitable for a variety of fluorescence quantitative PCR instruments and can be widely used in clinical applications.
  • the methylation detection method provided by the present invention can be used to achieve early diagnosis of colorectal cancer and effectively improve the five-year survival rate of colorectal cancer patients.
  • Figure 1 is a schematic diagram of the fluorescence quantitative detection results of TRGV9, TRDJ3, TRDC genes, FAM174B, ZMIZ2 genes and ⁇ -actin genes in Example 1: wherein, the ordinate represents the fluorescence intensity, the abscissa represents the cycle number of fluorescence quantitative PCR amplification, FAM Indicates the multiple methylation detection results of the three genes TRGV9, TRDJ3, and TRDC in the tested specimen, CY5 indicates the multiple methylation detection results of the two genes FAM174B and TRDJ3 in the tested specimen, and VIC indicates the detection of the ⁇ -actin gene in the tested specimen. result, the ordinate represents the fluorescence intensity, the abscissa represents the cycle number of fluorescence quantitative PCR amplification, FAM Indicates the multiple methylation detection results of the three genes TRGV9, TRDJ3, and TRDC in the tested specimen, CY5 indicates the multiple methylation detection results of the two genes FAM174B and TRDJ3
  • Figure 2 is the ROC curve of the combined diagnosis of colorectal cancer by TRGV9, TRDJ3, TRDC, FAM174B, and ZMIZ2 genes in Example 2.
  • Figure 3 is the ROC curve of the combined diagnosis of early colorectal cancer by TRGV9, TRDJ3, TRDC, FAM174B, and ZMIZ2 genes in Example 2.
  • the kit includes the following components:
  • PCR reaction solution primers, probes, ROX, positive quality control materials, negative quality control materials.
  • the PCR reaction solution includes hot-start Taq enzyme, PCR buffer, MgCl 2 , dUTP, and dNTPs;
  • Primers include the forward primer and reverse primer of the methylation genes TRGV9, TRDJ3, TRDC, FAM174B, ZMIZ2 and the internal reference gene ⁇ -actin;
  • the probes include detection probes for the methylated genes TRGV9, TRDJ3, TRDC, FAM174B, ZMIZ2 and the internal reference gene ⁇ -actin.
  • nucleotide sequences of the primers and probes of the colorectal cancer peripheral blood mononuclear cell methylation genes TRGV9, TRDJ3, TRDC, FAM174B, ZMIZ2 and the internal reference gene ⁇ -actin used in this example can be found in Table 1.
  • X1, X2, and X3 are fluorescent reporter groups, and Y is a fluorescence quenching group.
  • X1 is FAM
  • X2 is CY5
  • X3 is VIC
  • Y is BHQ1.
  • Negative quality control product prepared from unmethylated human genomic DNA of ⁇ -actin gene.
  • Positive quality control product prepared from human genomic DNA methylated by ⁇ -actin gene.
  • Tiangen blood/cell/tissue genomic DNA extraction kit (DP304): add 200ul buffer GA to the above cell pellet, shake until completely suspended, add 20ul Proteinase K solution, mix; add 200ul buffer GB , mix thoroughly by inverting, and place at 70°C for 10 minutes; add 200ul of absolute ethanol, shake thoroughly and mix for 15 seconds; add the solution and flocculent precipitate obtained in the previous step to an adsorption column CB3 (the adsorption column is placed in the collection tube), Centrifuge (12,000rpm, 30s) and discard the waste liquid; add 500ul of buffer GD to adsorption column CB3, centrifuge (12,000rpm, 30s) and discard the waste liquid; add 600ul of rinse solution PW to adsorption column CB3 and centrifuge (12,000rpm , 30s), discard the waste liquid; repeat the above steps; put the adsorption column CB3 back into the collection tube, centrifuge (12,000rpm, 2min), discard the waste liquid; place
  • the DNA samples extracted above were subjected to bisulfite conversion using Zymo Research according to the manufacturer's instructions. Please refer to the following instructions for specific operating steps: Add 130 ⁇ L CT Conversion Reagent (ready for use, avoid light during operation) to the 1000ng DNA sample extracted above, vortex and mix, according to 98°C, 10min; 64°C, 2.5 h; 4°C, ⁇ reaction conditions for conversion; add 600 ⁇ L M-Binding Buffer to the adsorption column, then add the converted sample, mix, centrifuge (10000rpm, 30s), discard the waste liquid; add 100 ⁇ L to the adsorption column M-Wash Buffer, centrifuge (10000rpm, 30s), discard the waste liquid; add 200 ⁇ L M-Des ⁇ Lphonation Buffer to the adsorption column, let stand at room temperature for 20 minutes, centrifuge (10000rpm, 1min), discard the waste liquid; add 200 ⁇ L M to the adsorption column -Wash Buffer, centrifuge (10000rpm
  • Samples should be tested as soon as possible after processing.
  • the storage time at -20°C is generally no more than 4 months. If storage for a longer period of time is required, please store the sample at -70°C to avoid repeated freezing and thawing.
  • the final concentration of the PCR amplification reaction system is composed of: 1xPCR reaction solution; 0.1-1uM TRGV9 gene methylation primer, 0.1-1uM TRDJ3 gene methylation primer, 0.1-1uM TRDC gene methylation primer, 0.1-1uM FAM174B gene methylation primer, 0.1-1uM ZMIZ2 gene methylation primer, 0.1-1uM ⁇ -actin primer; 0.1-1uM TRGV9 Taqman hydrolysis probe, 0.1-1uM TRDJ3 Taqman hydrolysis probe, 0.1-1uM TRDC Taqman hydrolysis probe , 0.1-1uM FAM174B Taqman hydrolysis probe, 0.1-1uM ZMIZ2 Taqman hydrolysis probe, 0.1-1uM ⁇ -actin Taqman hydrolysis probe; ROX Low Reference Dye (50X).
  • Reaction solution components volume PCR reaction solution 5 ⁇ L TRGV9 gene methylation forward primer 0.25 ⁇ L TRGV9 gene methylation backward primer 0.25 ⁇ L TRGV9 gene hydrolysis probe 0.1 ⁇ L TRDJ3 gene methylation forward primer 0.25 ⁇ L TRDJ3 gene methylation backward primer 0.25 ⁇ L TRDJ3 gene hydrolysis probe 0.1 ⁇ L TRDC gene methylation forward primer 0.25 ⁇ L TRDC gene methylation backward primer 0.25 ⁇ L TRDC gene hydrolysis probe 0.1 ⁇ L FAM174B gene methylation forward primer 0.25 ⁇ L FAM174B gene methylation backward primer 0.25 ⁇ L FAM174B gene hydrolysis probe 0.1 ⁇ L
  • the results are automatically saved, and the results are automatically analyzed using the instrument's supporting software.
  • the negative quality control product should have no obvious amplification curve change or the Ct value should be ⁇ 35, and the Ct value of the positive quality control product should be ⁇ 10. When the quality control product meets the above conditions, it indicates that the experiment is valid and the analysis can continue.
  • VIC When VIC reaches the set threshold (i.e. CT value ⁇ 35), it means that the DNA loading amount is within the allowable range, and the FAM and CY5 results are credible.
  • the test results are interpreted according to the following formula: a Multi-PCR Score greater than 0.5 indicates a negative result; a Multi-PCR Score less than 0.5 indicates a positive result.
  • ⁇ CT 1 CT VIC -CT FAM ,
  • Multi-PCR Score 1/[1+exp(10.73266+11.57596* ⁇ CT 1 -15.31471* ⁇ CT 2 )].
  • This embodiment provides a test of the detection accuracy of the kit. Experimental testing was performed on 94 clinical samples using the kit and experimental method provided in Example 1. The test results are shown in Table 4.
  • test results of 94 clinical samples tested were compared with the gold standard method for colorectal cancer detection (colonoscopy combined with pathological testing). There were 48 samples with normal pathological diagnosis and 46 samples with colorectal cancer. The implementation method provided in Example 1 was used for detection. The specific comparison results are shown in Table 5.
  • This example provides receiver operating characteristic curves (ROC curves) of healthy controls and colorectal cancer patients. Experimental detection was performed on healthy controls and colorectal cancer patients using the kit and experimental method provided in Example 1. The results are shown in Figure 2. This experimental example provides colorectal cancer DNA methylation detection technology that can clearly distinguish healthy controls and colorectal cancer patients, with an area under the curve (AUC) of 0.971.
  • AUC area under the curve
  • this experimental example provides colorectal cancer DNA methylation detection technology that can clearly distinguish healthy controls and early-stage colorectal cancer patients, with an area under the curve (AUC) of 0.994.

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Abstract

提供了基于荧光定量PCR技术的结直肠癌多基因甲基化联合检测试剂盒及其临床应用。通过联合检测外周血单个核细胞中TRGV9、TRDJ3、TRDC、FAM174B、ZMIZ2基因甲基化水平，能够有效判断结直肠癌的发生情况。

Description

一种用于结直肠癌诊断的多基因DNA甲基化联合检测试剂盒及应用 技术领域
本发明属于基因工程技术技术领域，涉及一种基于结直肠癌TRGV9、TRDJ3、TRDC、FAM174B、ZMIZ2多基因DNA甲基化检测的诊断试剂盒、检测方法及其临床应用。
背景技术
2018年全球癌症统计数据显示，结直肠癌(CRC)在全球发病率排名第三，死亡率排名第二，估计新发病例约200万/年，死亡人数超过88万/年。研究人员普遍认为，提高CRC的诊断水平是进一步降低CRC发病率和死亡率的最有效策略。目前，根据CRC筛查指南，结肠镜检查和粪便免疫化学试验(FIT)已被推荐为CRC筛查的一线选择。然而，结肠镜检查的是侵入性操作、肠道准备繁琐、并发症较难避免且费用昂贵，患者依从性较差。FIT是一种非侵入性测试，可以提高参与率，但检测的敏感性较低。定期检测癌胚抗原(CEA)能够为CRC提供可靠的指征，特异性和敏感性仍不满足临床需求。因此，仍然亟需开发一种基于血液的用于CRC诊断的无创、准确的检测方法。
越来越多的证据表明，在表观遗传改变中，DNA甲基化的异常改变被认为是包括CRC在内的许多癌症类型发生和发展过程中的最主要现象，并且它们可以在早期肿瘤中癌症患者非侵入性基质中(如血清、血浆或外周血单个核细胞(PBMC)被检测到。而PBMC可从常规采集的血液中轻松获取，且能够提供与癌症进展相关的潜在生物分子(如DNA、RNA和蛋白质等)，从而识别肿瘤的存在。学者能够从PBMCs基因表达模式中进一步检测到这种信号，使得差异DNA甲基化位点的检测可作为癌症诊断的标志物也成为一个新的研究方向。
近年来已有文献报道包括CRC在内的不同类型癌症中鉴定癌症 特异性DNA甲基化生物标志物的研究，但多停留在初期研究阶段，因此，进一步深入探索和开发便捷、特异有效、能够在临床推广应用的甲基化诊断试剂盒具有重要的临床意义。
发明内容
本发明针对早期结直肠癌诊断技术存在的侵入性、价格昂贵、准确性低等技术问题提出一种无创的技术方案，该方案基于TRGV9、TRDJ3、TRDC、FAM174B、ZMIZ2多基因DNA甲基化水平的结直肠癌诊断试剂盒，该试剂盒具有较高的检测灵敏度和特异度，以期实现准确、快捷的诊断早期结直肠癌。
为了达到上述目的，本发明是采用下述的技术方案实现的：
一种基于外周血单个核细胞DNA的结直肠癌诊断检测的引物和探针，所述检测引物和探针用于检测结直肠癌特异性DNA甲基化标志物，所述结直肠癌特异性标志物包括：TRGV9、TRDJ3、TRDC、FAM174B、ZMIZ2基因。检测引物用于扩增所述结直肠癌特异性标志物的一段序列。
TRGV9扩增区域序列：
Figure PCTCN2022132111-appb-000001
TRDJ3扩增区域序列：
Figure PCTCN2022132111-appb-000002
Figure PCTCN2022132111-appb-000003
TRDC扩增区域序列：
Figure PCTCN2022132111-appb-000004
FAM174B扩增区域序列：
Figure PCTCN2022132111-appb-000005
ZMIZ2扩增区域序列：
Figure PCTCN2022132111-appb-000006
Figure PCTCN2022132111-appb-000007
TRGV9基因检测引物包括正向引物和反向引物，所述正向引物的核苷酸序列如SEQ ID NO.1所示，所述反向引物的核苷酸序列如SEQ ID NO.2所示。所述探针的核苷酸序列如SEQ ID NO.3所示，所述探针的两端分别标记有荧光报告基团和荧光淬灭基团。
TRDJ3基因检测引物包括正向引物和反向引物，所述正向引物的核苷酸序列如SEQ ID NO.4所示，所述反向引物的核苷酸序列如SEQ ID NO.5所示。所述探针的核苷酸序列如SEQ ID NO.6所示，所述探针的两端分别标记有荧光报告基团和荧光淬灭基团。
TRDC基因检测引物包括正向引物和反向引物，所述正向引物的核苷酸序列如SEQ ID NO.7所示，所述反向引物的核苷酸序列如SEQ ID NO.8所示。所述探针的核苷酸序列如SEQ ID NO.9所示，所述探针的两端分别标记有荧光报告基团和荧光淬灭基团。
FAM174B基因检测引物包括正向引物和反向引物，所述正向引物的核苷酸序列如SEQ ID NO.10所示，所述反向引物的核苷酸序列如SEQ ID NO.11所示。所述探针的核苷酸序列如SEQ ID NO.12所示，所述探针的两端分别标记有荧光报告基团和荧光淬灭基团。
ZMIZ2基因检测引物包括正向引物和反向引物，所述正向引物的核苷酸序列如SEQ ID NO.13所示，所述反向引物的核苷酸序列如SEQ ID NO.14所示。所述探针的核苷酸序列如SEQ ID NO.15所示，所述探针的两端分别标记有荧光报告基团和荧光淬灭基团。
进一步地，所述荧光报告基团/荧光淬灭基团选自FAM、VIC、HEX、ROX、NED、CY3、CY5等荧光报告基团/BHQ1、BHQ2、BHQ3、Dabyc 1、Tamra等荧光淬灭基团，且试剂盒中TRGV9、TRDJ3、TRDC基因探针上的荧光报告基团和FAM174B、ZMIZ2基因探针上的荧光报告基团互不相同。
作为优选，TRGV9、TRDJ3、TRDC基因5'端荧光报告基团为FAM；FAM174B、ZMIZ2基因5'端荧光报告基团为CY5，β-actin5'端荧光报告基团为VIC，淬灭基团为BHQ1；
诊断模型如下：
△CT 1＝CT VIC-CT FAM,
△CT 2＝CT VIC-CT CY5，
Multi-PCR Score＝1/[1+exp(10.73266+11.57596*△CT 1-15.31471*△CT 2)]。
以VIC达到设定阈值表示DNA上样量在允许范围内，FAM、CY5结果可信。根据以上公式对检测结果进行判读，即Multi-PCR Score大于0.5，表明结果阴性；Multi-PCR Score小于0.5，表明结果阳性。
若VIC荧光通道的Ct值>35或无扩增，ΔCt为任何结果皆为无效，需要重新提取转化样本后检测。
本发明还提出一种结直肠癌诊断试剂盒，至少包括特异性识别TRGV9、TRDJ3、TRDC、FAM174B、ZMIZ2基因的检测试剂。
需要说明的是，所述结直肠癌检测试剂并不限定为液体形式。
所述试剂盒还包括β-actin基因的PCR扩增特异性引物和探针，所述β-actin基因检测引物包括正向引物和反向引物，所述正向引物的核苷酸序列如SEQ ID NO.16所示，所述反向引物的核苷酸序列如SEQ ID NO.17所示。所述探针的核苷酸序列如SEQ ID NO.18所示，所述探针的两端分别标记有荧光报告基团和荧光淬灭基团。
所述荧光报告基团/荧光淬灭基团选自FAM、VIC、HEX、ROX、NED、CY3、CY5等荧光报告基团/BHQ1、BHQ2、BHQ3、Dabyc1、Tamra等荧光淬灭基团，且试剂盒中TRGV9、TRDJ3、TRDC基因探针上的荧光报告基团和FAM174B、ZMIZ2基因探针上的荧光报告基团以及β-actin基因探针上的荧光报告基团互不相同。
进一步地，所述试剂盒还包括一下组分中的一种或几种：PCR反应液、引物、探针、ROX、阳性质控品、阴性质控品。所述PCR反应液包括热启动Taq酶、PCR缓冲液、MgCl 2、dUTP、dNTPs，此类甲基化定量PCR常用试剂可经市场途径单独购买或自行配置。
进一步地，所述阳性质控品由β-actin基因甲基化的人基因组DNA配制而成。
进一步地，所述阴性质控品由β-actin基因非甲基化的人基因组DNA配制而成。
前述结直肠癌检测试剂盒的使用方法，包括如下步骤：
(1)分离并提取外周血单个核细胞；
(2)提取外周血单个核细胞基因组DNA；
(3)将所提取单个核细胞基因组DNA进行亚硫酸盐转化；
(4)采用前述结直肠癌检测试剂盒中的试剂对亚硫酸盐转化后的待测样本进行甲基化定量PCR检测；
(5)对检测结果进行分析。
本发明提供一种结直肠癌诊断方法，包括对样本中高甲基化基因(TRGV9、ZMIZ2、TRDC)及低甲基化基因(FAM174B、TRDJ3)的相对定量，并通过Multi-PCR Score判断结直肠癌的发生情况。
作为优选，外周血单个核细胞作为检测样本。
用于特异性识别TRGV9基因DNA甲基化的PCR探针引物预混液包括：0.1-1uM的探针、0.1-1uM的正向引物和0.1-1uM的反向引物；
用于特异性识别TRDJ3基因DNA甲基化的PCR探针引物预混液包括：0.1-1uM的探针、0.1-1uM的正向引物和0.1-1uM的反向引物；
用于特异性识别TRDC基因DNA甲基化的PCR探针引物预混液包括：0.1-1uM的探针、0.1-1uM的正向引物和0.1-1uM的反向引物；
用于特异性识别FAM174B基因DNA甲基化的PCR探针引物预混液包括：0.1-1uM的探针、0.1-1uM的正向引物和0.1-1uM的反向引物；
用于特异性识别ZMIZ2基因DNA甲基化的PCR探针引物预混液包括：0.1-1uM的探针、0.1-1uM的正向引物和0.1-1uM的反向引物。
与现有技术相比，本发明的优点和积极效果在于：
1.本发明所提供的检测试剂盒，检测标本类型为外周血，样本较易获得、无创，且患者依从性较好。
2.本发明所提供的检测试剂盒针对TRGV9、TRDJ3、TRDC、FAM174B、ZMIZ2基因设计特异性引物和探针，并选用β-actin作为内参基因，保证检测结果的灵敏度高、特异性好，所提供的检测试剂盒包含阴性质控品和阳性质控品，能够较大程度防止假阴性结果和假阳性结果的产生，在结直肠癌外周血PBMC甲基化的体外诊断领域具有较为广阔的应用前景。
3.与焦磷酸测序等技术相比，本发明所提供的检测试剂盒操作简便，大大降低检测成本，提高检测效率。PCR荧光检测是全封闭式操作，无需进行电泳、杂交等操作，能够有效防止污染。
4.本发明所提供的检测试剂盒适用于多种荧光定量PCR仪，能够在临床推广应用。
5.肿瘤早期进展过程中多存在甲基化的异常改变，利用本发明所提供的甲基化检测方法可实现结直肠癌的早期诊断，有效提高结直肠癌患者的五年生存率。
附图说明
图1为实施例1中TRGV9、TRDJ3、TRDC基因，FAM174B、ZMIZ2基因以及β-actin基因荧光定量检测结果示意图：其中，纵坐标表示荧光强度，横坐标表示荧光定量PCR扩增的循环数，FAM表示受试标本中TRGV9、TRDJ3、TRDC三个基因多重甲基化检测结果，CY5表示受试标本中FAM174B、TRDJ3两个基因多重甲基化检测结果，VIC表示受试标本中β-actin基因检测结果，
图2为实施例2中TRGV9、TRDJ3、TRDC、FAM174B、ZMIZ2基因联合诊断结直肠癌的ROC曲线。
图3为实施例2中TRGV9、TRDJ3、TRDC、FAM174B、ZMIZ2基因联合诊断早期结直肠癌的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
1.试剂盒的制备及其使用方法
试剂盒包括以下组分：
PCR反应液、引物、探针、ROX、阳性质控品、阴性质控品。
(1)PCR反应液包括热启动Taq酶、PCR缓冲液、MgCl 2、dUTP、dNTPs；
(2)引物包括甲基化基因TRGV9、TRDJ3、TRDC、FAM174B、ZMIZ2及内参基因β-actin的正向引物和反向引物；
(3)探针包括甲基化基因TRGV9、TRDJ3、TRDC、FAM174B、ZMIZ2 及内参基因β-actin的检测探针。
本实施例中使用的结直肠癌外周血单个核细胞甲基化基因TRGV9、TRDJ3、TRDC、FAM174B、ZMIZ2以及内参基因β-actin的引物和探针的核苷酸序列可参见表1。
表1引物和探针的核苷酸序列表
Figure PCTCN2022132111-appb-000008
Figure PCTCN2022132111-appb-000009
注：X1、X2、X3为荧光报告基团，Y为荧光淬灭基团。在本发明的一种优选方式中，X1为FAM，X2为CY5，X3为VIC，Y为BHQ1。
(4)阴性质控品：由β-actin基因非甲基化的人基因组DNA配制而成。
(5)阳性质控品：由β-actin基因甲基化的人基因组DNA配制而成。
2.实验过程
2.1外周血单个核细胞的分离
采血前一晚空腹，采血当天使用EDTA-K2抗凝管采集受试者外周静脉血3ml，室温静置30min后进行如下操作：取一支15ml无菌离心管，加入3ml淋巴细胞分离液Histopaque-1077(Sigma)；用塑料吸管轻轻混匀外周血后，沿管壁呈45°将外周血轻柔、缓慢的加入淋巴细胞分离液上层；将离心管小心置于水平离心机中，室温450x g离心30分钟；小心吸取中间云雾状的白膜层细胞，置于另一离心管中，并加入10ml的PBS，450x g离心10分钟，以洗掉多余的Ficoll；弃上清，用1ml PBS吹打混匀后加入10ml的PBS，250x g离心10分钟；重复上述步骤再次洗涤细胞，弃上清后获得细胞沉淀。
2.2外周血单个核细胞DNA提取
按照天根血液/细胞/组织基因组DNA提取试剂盒(DP304)标准操作流程：向上述细胞沉淀中加入200ul缓冲液GA，振荡至彻底悬浮，加入20ul Proteinase K溶液，混匀；加入200ul缓冲液GB，充分颠倒混匀，70℃放置10min；加人200ul无水乙醇，充分振荡混匀15sec；将上一步所得溶液和絮状沉淀都加入一个吸附柱CB3中(吸 附柱放入收集管中)，离心(12,000rpm，30s)，弃废液；向吸附柱CB3中加入500ul缓冲液GD，离心(12,000rpm，30s)，弃废液；向吸附柱CB3中加入600ul漂洗液PW，离心(12,000rpm，30s)，弃废液；重复上述操作步骤；将吸附柱CB3放回收集管中，离心(12,000rpm，2min)，弃废液；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；将吸附柱CB3转入一个干净的离心管中，向吸附膜的中间部位悬空滴加50-200ul洗脱缓冲液TE，室温放置2-5min，离心(12,000rpm，2min)，将溶液收集到离心管中。并通过NanoDrop ND-1000(Thermo Scientific)分光光度计测量浓度。
2.3亚硫酸氢盐转化
按制造商说明书要求使用Zymo Research的要求对上述提取获得的DNA样品进行亚硫酸氢盐转化。具体操作步骤可参考如下说明：向上述提取好的1000ng DNA样本中加入130μL CT Conversion Reagent(现用现配，操作过程注意避光)，涡旋混匀，根据98℃，10min；64℃，2.5h；4℃，∞的反应条件进行转化；向吸附柱中加入600μL M-Binding Buffer，随后加入转化后的样本，混匀，离心(10000rpm，30s)，弃废液；向吸附柱中加入100μL M-Wash Buffer，离心(10000rpm，30s)，弃废液；向吸附柱中加入200μL M-DesμLphonation Buffer，室温静置20min，离心(10000rpm，1min)，弃废液；向吸附柱中加入200μL M-Wash Buffer，离心(10000rpm，1min)，弃废液；向吸附柱中加入200μL M-Wash Buffer，离心(10000rpm，1min)，弃废液；将吸附柱转移至新的收集管中，，加入20μL M-Elution Buffer,室温静置5min，离心(10000rpm，1min)，获得亚硫酸盐转化后的DNA样本。
样本处理后应尽快进行检测，-20℃保存时间一般不超过4个月，如需更长时间的保存请将样本置于-70℃，避免反复冻融。
2.4 PCR反应的体系和反应条件
所述PCR扩增反应体系的终浓度组成为：1xPCR反应液；0.1-1uM TRGV9基因甲基化引物、0.1-1uM TRDJ3基因甲基化引物、0.1-1uM TRDC基因甲基化引物、0.1-1uM FAM174B基因甲基化引物、0.1-1uM ZMIZ2基因甲基化引物、0.1-1uMβ-actin引物；0.1-1uM TRGV9 Taqman水解探针、0.1-1uM TRDJ3 Taqman水解探针、0.1-1uM TRDC Taqman水解探针、0.1-1uM FAM174B Taqman水解探针、0.1-1uM ZMIZ2 Taqman水解探针、0.1-1uMβ-actin Taqman水解探针；ROX Low Reference Dye(50X)。
2.4.1试剂准备
按照表2中提供的试剂配制待用。
表2反应液配置(样本检测/阴性质控/阳性质控)
反应液组分 体积
PCR反应液 5μL
TRGV9基因甲基化前向引物 0.25μL
TRGV9基因甲基化后向引物 0.25μL
TRGV9基因水解探针 0.1μL
TRDJ3基因甲基化前向引物 0.25μL
TRDJ3基因甲基化后向引物 0.25μL
TRDJ3基因水解探针 0.1μL
TRDC基因甲基化前向引物 0.25μL
TRDC基因甲基化后向引物 0.25μL
TRDC基因水解探针 0.1μL
FAM174B基因甲基化前向引物 0.25μL
FAM174B基因甲基化后向引物 0.25μL
FAM174B基因水解探针 0.1μL
ZMIZ2基因甲基化前向引物 0.25μL
ZMIZ2基因甲基化后向引物 0.25μL
ZMIZ2基因水解探针 0.1μL
β-actin前向引物 0.06μL
β-actin后向引物 0.06μL
β-actin Taqman水解探针 0.05μL
ROX Low Reference Dye(50X) 0.02μL
无菌无酶水 补足体积至9μL
2.4.2加样
向PCR扩增管中加入1μL待测核酸样品/阴性质控品/阳性质控品/无模板对照，终体积为10μL/管，盖紧管盖，瞬时低速离心，等待上机。
注：待测样本、阴性质控品、阳性质控品、无模板对照均设置三个复孔进行检测。
2.4.3 PCR扩增
PCR扩增的程序如表3所示。
表3荧光通道选择及扩增循环参数设定
Figure PCTCN2022132111-appb-000010
注：选ROX校正，淬灭基团选None.
设置完毕，保存文件，运行反应程序。
2.4.4 PCR结果分析
反应结束后自动保存结果,使用仪器配套软件自动分析结果,
首先进行无模板对照的扩增曲线分析，FAM、CY5、VIC通道无明显扩增信号，表明实验无污染，可以继续分析。
阴性质控品应无明显扩增曲线变化或Ct值应≥35，阳性质控品的Ct值应≤10。质控品满足上述条件时，表明实验有效，可继续分析。
2.4.5检测结果判读
以VIC达到设定阈值(即CT值<35),表示DNA上样量在允许范围内，FAM、CY5结果可信。根据如下公式对检测结果进行判读，即Multi-PCR Score大于0.5，表明结果阴性；Multi-PCR Score小于0.5，表明结果阳性。
△CT 1＝CT VIC-CT FAM,
△CT 2＝CT VIC-CT CY5
Multi-PCR Score＝1/[1+exp(10.73266+11.57596*△CT 1-15.31471*△CT 2)]。
3.准确性检验
本实施例提供试剂盒的检测准确性的检验，通过实施例1提供的试剂盒和实验方法对94例临床样本进行实验检测，检测结果如表4所示。
表4 94例临床样本检测结果
Figure PCTCN2022132111-appb-000011
Figure PCTCN2022132111-appb-000012
Figure PCTCN2022132111-appb-000013
将检测的94例临床样本与结直肠癌检测金标准方法(结直肠镜联合病理检测)的检测结果进行对比分析，病理诊断正常的样本48例，结直肠癌样本46例。采用实施例1提供的实施方法进行检测，具体比较结果见表5。
表5结直肠癌甲基化检测技术对结直肠癌患者及健康对照者样本的检测结果对比
Figure PCTCN2022132111-appb-000014
4.健康对照者与结直肠癌患者的受试者工作特征曲线
本实施例提供健康对照者与结直肠癌患者的受试者工作特征曲线(ROC曲线)。通过实施例1提供的试剂盒和实验方法对健康对照者和结直肠癌患者进行实验检测。结果如图2所示，本实验例提供结直 肠癌DNA甲基化检测技术可以明显区分健康对照者和结直肠癌患者，曲线下面积(AUC)为0.971。
5.健康对照者与早期结直肠癌患者的受试者工作特征曲线
通过实施例1提供的试剂盒和实验方法对健康对照者和早期结直肠癌患者进行实验检测。
如图3所示，本实验例提供结直肠癌DNA甲基化检测技术可以明显区分健康对照者和早期结直肠癌患者，曲线下面积(AUC)为0.994。
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 (5)

  1. 外周血单个核细胞中TRGV9、TRDJ3、TRDC、FAM174B、ZMIZ2基因在制备结直肠癌诊断试剂盒中的应用。
  2. 一种用于结直肠癌诊断的多基因DNA甲基化联合检测试剂盒，其特征在于，所述试剂盒包括用于检测TRGV9、TRDJ3、TRDC、FAM174B、ZMIZ2基因甲基化的引物对和基因特异的水解探针，
    所述TRGV9基因检测正向引物的核苷酸序列如SEQ ID NO.1所示，反向引物的核苷酸序列如SEQ ID NO.2所示，探针的核苷酸序列如SEQ ID NO.3所示；
    TRDJ3基因检测正向引物的核苷酸序列如SEQ ID NO.4所示，反向引物的核苷酸序列如SEQ ID NO.5所示，探针的核苷酸序列如SEQ ID NO.6所示；
    TRDC基因检测正向引物的核苷酸序列如SEQ ID NO.7所示，反向引物的核苷酸序列如SEQ ID NO.8所示；探针的核苷酸序列如SEQ ID NO.9所示；
    FAM174B基因检测正向引物的核苷酸序列如SEQ ID NO.10所示，反向引物的核苷酸序列如SEQ ID NO.11所示，探针的核苷酸序列如SEQ ID NO.12所示；
    ZMIZ2基因检测正向引物的核苷酸序列如SEQ ID NO.13所示，反向引物的核苷酸序列如SEQ ID NO.14所示；探针的核苷酸序列如SEQ ID NO.15所示。
  3. 根据权利要求2所述用于结直肠癌诊断的多基因DNA甲基化联合检测试剂盒，其特征在于，所述试剂盒还包括β-actin基因的PCR扩增特异性引物和探针，所述β-actin基因检测正向引物的核苷酸序列如SEQ ID NO.16所示，反向引物的核苷酸序列如SEQ ID NO.17所示，探针的核苷酸序列如SEQ ID NO.18所示。
  4. 根据权利要求2所述用于结直肠癌诊断的多基因DNA甲基化联合检测试剂盒，其特征在于，所述特异性识别TRGV9、TRDJ3、TRDC、FAM174B、ZMIZ2基因甲基化的探针5’端标记有荧光报告基团，3’端标记有荧光淬灭基团；TRGV9、TRDJ3、TRDC基因探针5’端的荧光报告基团为FAM，FAM174B、ZMIZ2基因探针5’端的荧光报告基团为CY5，β-actin基因探针5’端的荧光报告基团为VIC，TRGV9、TRDJ3、TRDC、FAM174B、ZMIZ2及β-actin基因3’端荧光淬灭基团均为BHQ1。
  5. 根据权利要求2所述用于结直肠癌诊断的多基因DNA甲基化联合检测试剂盒，其特征在于，
    试剂盒中的阴性质控品由β-actin基因非甲基化的人基因组DNA配制而成；阳性质控品由β-actin基因甲基化的人基因组DNA配制而成；阴性质控品无明显扩增曲线变化或Ct值≥35，阳性质控品的Ct值≤10，为有效实验；
    还包括一个诊断模型，诊断模型公式如下：
    △CT 1＝CT VIC-CT FAM,
    △CT 2＝CT VIC-CT CY5，
    Multi-PCR Score＝1/[1+exp(10.73266+11.57596*△CT 1-15.31471*△CT 2)]；
    Multi-PCR Score大于0.5，结果为阴性；
    Multi-PCR Score小于0.5，结果为阳性；
    若VIC荧光通道的Ct值>35或无扩增，ΔCt为任何结果皆为无效，需要重新提取转化样本后检测。
PCT/CN2022/132111 2022-08-11 2022-11-16 一种用于结直肠癌诊断的多基因dna甲基化联合检测试剂盒及应用 Ceased WO2024031860A1 (zh)

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