WO2017124854A1 - 一组用于肠癌早期诊断的ndrg4基因甲基化检测引物和探针及其试剂盒 - Google Patents

一组用于肠癌早期诊断的ndrg4基因甲基化检测引物和探针及其试剂盒 Download PDF

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WO2017124854A1
WO2017124854A1 PCT/CN2016/109629 CN2016109629W WO2017124854A1 WO 2017124854 A1 WO2017124854 A1 WO 2017124854A1 CN 2016109629 W CN2016109629 W CN 2016109629W WO 2017124854 A1 WO2017124854 A1 WO 2017124854A1
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probe
kit
ndrg4
intestinal cancer
primer
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邹鸿志
吴珊
牛智通
赵荣淞
余浩
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广州市康立明生物科技有限责任公司
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    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers

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  • the invention belongs to the field of biomedical technology. More specifically, it relates to a group of NDRG4 gene methylation detection primers and probes for early diagnosis of intestinal cancer and a detection kit thereof.
  • CRC Colorectal cancer
  • NDRG4 N-mycdownstream-reguiatedgene4
  • the NDRG4 gene is 32 kb in length and consists of 17 exons and 16 introns. A large number of studies have shown that this gene and tumor cells grow and differentiate. It has a certain relationship with metastasis.
  • the 5' regulatory region of the gene contains CpG island, which is often methylated during the development of colorectal cancer.
  • NDRG4 gene The methylation of NDRG4 gene is considered to be an important biological feature of colorectal cancer (Veerie et al. ). Therefore, the NDRG4 gene is a potential biomarker for candidate tumor suppressor genes and rectal cancer, and methylation of the NDRG4 promoter can be used as a biomarker for early diagnosis of colorectal cancer.
  • Detection sensitivity and specificity are particularly important factors, and it is of great significance for the accurate diagnosis of early diagnosis of intestinal cancer. Detection sensitivity and specificity are affected by many factors, therefore, to ensure and improve the NDRG4 gene.
  • the related research on sensitivity and specificity of methylation detection is the focus and difficulty of using this gene for early diagnosis of intestinal cancer.
  • the technical problem to be solved by the present invention is to overcome the shortcomings and defects of the existing NDRG4 gene detection for early diagnosis of intestinal cancer, and provide a highly sensitive and highly specific NDRG4 gene methylation detection primer and probe, and detection
  • the sample can be easily sampled and used for clinical screening, which is of great significance for the early diagnosis of intestinal cancer.
  • the present invention provides the following technical solutions:
  • the present invention provides a combination of a primer and a probe, the sequence of which is shown in SEQ ID No. 1 and/or SEQ ID No. 2; the sequence of the probe is shown in SEQ ID No. 3. .
  • the invention also provides the use of the primer and probe composition for the preparation of a NDRG4 gene methylation detection reagent.
  • the present invention also provides the use of the composition of the primer and the probe for preparing a colon cancer diagnostic reagent; preferably, the intestinal cancer is early intestinal cancer.
  • the invention also provides a kit comprising the composition of the primer and probe.
  • the kit further comprises a capture probe; the sequence of the capture probe is set forth in SEQ ID No. 4.
  • the primer, the probe or the capture probe are used in the kit at a concentration of 50 to 500 ⁇ M, respectively.
  • the kit further comprises an amplification system; the amplification system comprising dNTPs, magnesium ions, reaction buffer, enzyme or nuclease-free water.
  • the amplification system in the kit comprises 5-20 mM dNTPs, 10-40 mM magnesium ions, 1 ⁇ 10 ⁇ reaction buffer, enzyme, nuclease-free water. .
  • the final concentration of each component of the amplification system in the kit is:
  • the enzyme in the kit is a hot start polymerase.
  • the amplification procedure of the kit is:
  • the test sample of the kit is feces; the kit further comprises a fecal pretreatment reagent, the fecal pretreatment reagent being a grinding buffer.
  • the invention also provides the use of the kit in the detection of methylation of the NDRG4 gene.
  • the invention also provides the use of the kit for the diagnosis of intestinal cancer; the intestinal cancer is early stage cancer.
  • the present invention also provides a method for detecting intestinal cancer, wherein the sample is mixed with the primer or probe composition or the detection reagent in the kit, and then amplified, and the detection result is obtained according to the amplification result.
  • the target of amplification is a marker of intestinal cancer; the marker of intestinal cancer is the NDRG4 gene.
  • the obtaining the detection result is comparing the amplification result of the sample to be tested with the normal sample, and when the amplification result of the sample to be tested is significantly different or significantly different from the amplification result of the normal sample, The donor of the sample to be tested is positive.
  • the invention discloses a group of NDRG4 gene methylation detection primers and probes for early diagnosis of intestinal cancer and a detection kit thereof.
  • the primers are NDRG4-1FP and NDRG4-1RP, the nucleotide sequences thereof are shown in SEQ ID NO. 1 and 2, respectively; the probe is NDRG4-2Pb, and the nucleotide sequence thereof is set forth in SEQ ID NO. Show.
  • the kit is comprising the above primers and probes Kit.
  • the invention utilizes the above primers and probes to construct a set of NDRG4 gene methylation detection system and program, and builds a kit, which has high sensitivity and specificity for the detection of NDRG4 gene methylation, and can accurately and specifically Diagnosis of early colorectal cancer, and the detection method is simple and rapid, and has very important significance and application prospects for early diagnosis and screening of intestinal cancer.
  • Figure 1 shows the amplification results of the primer set NDRG4-0FP/NDRG4-0RP
  • Figure 2 shows the amplification results of the primer set NDRG4-1FP/NDRG4-1RP
  • Figure 3 shows the amplification results of the primer set NDRG4-2FP/NDRG4-2RP
  • Figure 4 shows the amplification results of the primer set NDRG4-3FP/NDRG4-3RP
  • Figure 5 shows the amplification results of the primer set NDRG4-4FP/NDRG4-4RP
  • Figure 6 is an amplification result of the probe NDRG4-1Pb
  • Figure 7 is a result of amplification of the probe NDRG4-2Pb
  • Figure 8 is an amplification result of the probe NDRG4-3Pb
  • Figure 9 shows the results of the test of the stool sample.
  • the invention discloses an NDRG4 gene methylation detection primer and probe and a kit thereof for early diagnosis of intestinal cancer, and those skilled in the art can learn from the contents of the paper and appropriately improve the process parameters. It is to be understood that all such alternatives and modifications are obvious to those skilled in the art and are considered to be included in the present invention.
  • the method and the application of the present invention have been described by the preferred embodiments, and it is obvious that the method and application described herein may be modified or appropriately modified and combined without departing from the scope of the present invention. The technique of the present invention is applied.
  • the object of the present invention is to provide a set of NDRG4 gene methylation detection primers and probes for early diagnosis of intestinal cancer
  • Another object of the present invention is to provide a NDRG4 gene methylation detection kit prepared by using the above primers and probes.
  • a set of NDRG4 gene methylation detection primers and probes for early diagnosis of intestinal cancer the primers being NDRG4-1FP and NDRG4-1RP, the nucleotide sequences of which are shown in SEQ ID NO. 1 and 2, respectively;
  • the needle is NDRG4-2Pb, and its nucleotide sequence is shown in SEQ ID NO.
  • a kit for detecting methylation of NDRG4 gene for early diagnosis of intestinal cancer comprising the above primers NDRG4-1FP, NDRG4-1RP and probe NDRG4-2Pb.
  • the kit further includes a reagent for obtaining a PCR template when the PCR is amplified by using the primers NDRG4-1FP, NDRG4-1RP and the probe NDRG4-2Pb, and the required reagent includes capturing the target fragment when extracting the DNA of the sample to be tested.
  • the capture probe NDRG4-CP, the nucleotide sequence of the capture probe NDRG4-CP is shown in SEQ ID NO.
  • the above primers NDRG4-1FP, NDRG4-1RP and probe NDRG4-2Pb are used at a concentration of 50 to 500 ⁇ M.
  • the above primers NDRG4-1FP, NDRG4-1RP and probe NDRG4-2Pb are each used at a concentration of 100 ⁇ M.
  • the detection kit further comprises a PCR amplification system reagent, including dNTP, magnesium ion, reaction buffer (buffer), when performing PCR amplification using primers NDRG4-1FP, NDRG4-1RP and probe NDRG4-2Pb. Enzyme, nuclease-free water.
  • the system reagent comprises 5-20 mM dNTPs, 10-40 mM magnesium ions, 1 x 10 x reaction buffer, enzyme, nuclease-free water.
  • the system reagent comprises 5-15 mM dNTPs, 15 to 35 mM magnesium ions, 1 x 10 x reaction buffer, enzyme, nuclease-free water.
  • the system reagent comprises 10 mM dNTPs, 25 mM magnesium ions, 5 x reaction buffer (5 x buffer), enzyme, nuclease-free water.
  • the final concentration of each component in the PCR system is as follows:
  • the PCR system is carried out in the following ratios:
  • the enzyme is a hot start polymerase.
  • a hot start polymerase such as Promega's hot start enzyme or SIGMA's hot start enzyme.
  • the PCR procedure is as follows:
  • the PCR procedure is as follows:
  • the sample to be tested is feces
  • the kit further includes fecal pretreatment.
  • the reagent and the pretreatment reagent are grinding buffers.
  • the grinding buffer is specifically one or more of nuclease-free water, physiological saline, TE, or PBS.
  • the above primers and probes and kits thereof have good application prospects in early diagnosis of intestinal cancer.
  • the invention aims at early diagnosis of intestinal cancer, and has designed a set of primers and probes which have very good detection effect on methylation of NDRG4 gene, and successfully constructed a set of NDRG4 gene methylation detection system by using the above primers and probes. And procedures, and set up a kit, for the detection of NDRG4 gene methylation, with high sensitivity and specificity, can accurately and specifically diagnose early intestinal cancer, has important significance and application prospects for early diagnosis and screening of intestinal cancer .
  • the primers, probes and detection kits of the present invention can use feces as a sample, the sample processing is simple and easy to take, and the detection method is a common PCR method, and the operation is simple and rapid, and only takes about 75 to 90 minutes. It can be tested without the need for expensive instrumentation and testing costs, making it ideal for large sample testing and grassroots screening.
  • NDRG4-1Pb SEQ ID No.1 CGGAAGTTACGCGCGAGG NDRG4-2Pb SEQ ID No. 3 TCGTTTATCGGGTATTTTAGTCGCGT NDRG4-3Pb SEQ ID No. 12 TCGTTCGTTTATCGGGTATTTT
  • the target fragment can be grasped by the following capture probe.
  • Capture probe 1 (SEQ ID No. 4): TCCCTCGCGCGTGGCTTCCGCCTTCTGCGCGGCTGGGGTGCCCGGTGG
  • the DNA fragment in the sixth step was methylated with EZmethylationkit (product of Zymo Research), and the final sample was subjected to PCR detection.
  • EZmethylationkit product of Zymo Research
  • each set of primers and probes designed in Example 1 were used for detection, and primers and probes were optimized.
  • the results are as follows:
  • the probe is shown in Table 7 below:
  • the test kit includes the following components:
  • the capture probe NDRG4-CP the nucleotide sequence of which is shown in SEQ ID NO.
  • the function of the capture probe NDRG4-CP is to capture the target fragment when extracting the DNA of the sample to be tested.
  • PCR amplification system reagent 10 mM dNTP, 25 mM magnesium ion, 5 x reaction buffer (5 x buffer), enzyme, nuclease-free water.
  • Sample (such as stool) pretreatment reagent grinding buffer.
  • the grinding buffer is specifically one or more of nuclease-free water, physiological saline, TE, or PBS.
  • FIG. 9A shows the ROC curve of colorectal cancer detected by the NDRG4 gene
  • FIG. 9B shows the ROC curve of the NDRG4 gene for detecting precancerous adenoma (polyp diameter ⁇ 1 cm).
  • the above kit of the present invention has a sensitivity to detection of NDRG4 gene of 45.7%, a specificity of 97.8%, and an area under the subject curve of 0.694.
  • the primers and probes of the present invention and the detection kit thereof can not only detect and diagnose colorectal cancer more sensitively and accurately, especially early colorectal cancer, but also have good detection for pre-cancerous adenomas. Sensitivity and high specificity can be used for detection and diagnosis in the pre-cancerous adenoma stage.
  • NDRG4 gene methylation detection primers and probes and kits thereof for early diagnosis of intestinal cancer provided by the present invention are described in detail above.
  • the principles and embodiments of the present invention have been described with reference to specific examples, and the description of the above embodiments is only to assist in understanding the method of the present invention and its core idea. It should be noted that those skilled in the art can make various modifications and changes to the present invention without departing from the spirit and scope of the invention.

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Abstract

一组用于肠癌早期诊断的NDRG4基因甲基化检测引物和探针及其检测试剂盒。所述引物为NDRG4-1FP和NDRG4-1RP，其核苷酸序列分别如SEQ ID NO.1和2所示；所述探针为NDRG4-2Pb，其核苷酸序列如SEQ ID NO.3所示。所述试剂盒为包含有上述引物和探针的试剂盒。利用上述引物和探针，构建了一套NDRG4基因甲基化检测体系和程序，并组建成试剂盒，对于NDRG4基因甲基化的检测，具有高敏感性和特异性，能够准确特异地诊断早期肠癌，而且检测方法操作简便、快速，对肠癌的早期诊断和筛查具有非常重要的意义和应用前景。

Description

一组用于肠癌早期诊断的NDRG4基因甲基化检测引物和探针及其试剂盒
本申请要求于2016年01月23日提交中国专利局、申请号为201610048731.2、发明名称为“一组用于肠癌早期诊断的NDRG4基因甲基化检测引物和探针及其试剂盒”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于生物医药技术领域。更具体地，涉及一组用于肠癌早期诊断的NDRG4基因甲基化检测引物和探针及其检测试剂盒。
背景技术
大肠癌(colorectalcancer，CRC)是临床最常见的恶性肿瘤之一，近些年随着经济的发展，人们的生活方式尤其是饮食结构的变化，肠癌已经成为我国发病率上升最快的恶性肿瘤之一，严重威胁着国人的生命和健康。而且，晚期患者预后很差，目前，对肠癌进行早期诊断早期治疗以提高肠癌的治愈率，降低死亡率，已经成为迫切的需要。
DNA甲基化常常是肿瘤发生发展过程中的早期事件，因此，特异基因的甲基化可作为肿瘤早期诊断的分子标志物。NDRG4(N-mycdownstream-reguiatedgene4)为抑癌基因NDRG基因家族成员之一，NDRG4基因长32kb，由17个外显子及16个内含子组成，大量研究表明该基因与肿瘤细胞的生长、分化和转移有一定关系，其基因5’端调控区域含有CpG岛，在结直肠癌发生发展过程中常常被甲基化，NDRG4基因甲基化被认为是结直肠癌的重要生物学特征(Veerie等)。因此，NDRG4基因是候选肿瘤抑制基因和直肠癌的潜在生物标志物，NDRG4启动子的甲基化可作为生物标志物，用于早期诊断结直肠癌。
而对于NDRG4基因甲基化的检测而言，检测敏感性和特异性是尤其至关重要的因素，对肠癌早期诊断的准确判断具有重要的意义。而检测敏感性和特异性受多方面因素的影响，因此，对于保证和提高NDRG4基因 甲基化检测敏感性和特异性的相关研究，是利用该基因进行肠癌早期诊断的重点和难点。
发明内容
本发明要解决的技术问题是克服现有利用NDRG4基因检测进行肠癌早期诊断技术的不足和缺陷，提供一种高敏感性、高特异性的NDRG4基因甲基化检测引物和探针，而且检测样本可使用方便获取处理的粪便样品，非常适用于临床筛查，对肠癌的早期诊断具有重要的意义。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了一种引物和探针的组合物，所述引物的序列如SEQ ID No.1和/或SEQ ID No.2所示；所述探针的序列如SEQ ID No.3所示。
本发明还提供了所述引物和探针的组合物在制备NDRG4基因甲基化检测试剂中的应用。
本发明还提供了所述引物和探针的组合物在制备肠癌诊断试剂中的应用；优选的，所述肠癌为早期肠癌。
本发明还提供了一种试剂盒，包括所述引物和探针的组合物。
在本发明的一些具体实施方案中，所述试剂盒还包括捕获探针；所述捕获探针的序列如SEQ ID No.4所示。
在本发明的一些具体实施方案中，所述试剂盒中所述引物、所述探针或所述捕获探针的使用浓度分别为50～500μM。
在本发明的一些具体实施方案中，所述试剂盒还包括扩增体系；所述扩增体系包括dNTP、镁离子、反应缓冲液、酶或无核酸酶的水。
在本发明的一些具体实施方案中，所述试剂盒中所述扩增体系包括5～20mM的dNTP、10～40mM的镁离子、1×～10×反应缓冲液、酶、无核酸酶的水。
在本发明的一些具体实施方案中，所述试剂盒中扩增体系中各组分的反应终浓度为：
Figure PCTCN2016109629-appb-000001
Figure PCTCN2016109629-appb-000002
在本发明的一些具体实施方案中，所述试剂盒中酶为热启动聚合酶。
在本发明的一些具体实施方案中，所述试剂盒的扩增程序为：
95℃300s；1个循环；
95℃20s，62℃30s，70℃30s；10个循环；
95℃20s，58℃60s，72℃30s；30～40个循环；
37℃30s；1个循环。
在本发明的一些具体实施方案中，所述试剂盒的检测样本为粪便；所述试剂盒还包括粪便预处理试剂，所述粪便预处理试剂为研磨缓冲液。
本发明还提供了所述试剂盒在NDRG4基因甲基化检测中的应用。
本发明还提供了所述试剂盒在肠癌诊断中的应用；所述肠癌为早期肠癌。
本发明还提供了一种肠癌的检测方法，取样本与所述的引物和探针的组合物或所述的试剂盒中的检测试剂混合后扩增，根据扩增结果获得检测结果。
在本发明的一些具体实施方案中，所述扩增的目的物为肠癌标志物；所述肠癌标志物为NDRG4基因。
在本发明的一些具体实施方案中，所述获得检测结果为比较待测样本与正常样本的扩增结果，当所述待测样本与正常样本的扩增结果具有显著差异或极显著差异时，所述待测样本的供体呈阳性。
本发明公开了一组用于肠癌早期诊断的NDRG4基因甲基化检测引物和探针及其检测试剂盒。所述引物为NDRG4-1FP和NDRG4-1RP，其核苷酸序列分别如SEQ ID NO.1和2所示；所述探针为NDRG4-2Pb，其核苷酸序列如SEQ ID NO.3所示。所述试剂盒为包含有上述引物和探针 的试剂盒。本发明利用上述引物和探针，构建了一套NDRG4基因甲基化检测体系和程序，并组建成试剂盒，对于NDRG4基因甲基化的检测，具有高敏感性和特异性，能够准确特异地诊断早期肠癌，而且检测方法操作简便、快速，对肠癌的早期诊断和筛查具有非常重要的意义和应用前景。
附图说明
图1为引物组NDRG4-0FP/NDRG4-0RP的扩增结果；
图2为引物组NDRG4-1FP/NDRG4-1RP的扩增结果；
图3为引物组NDRG4-2FP/NDRG4-2RP的扩增结果；
图4为引物组NDRG4-3FP/NDRG4-3RP的扩增结果；
图5为引物组NDRG4-4FP/NDRG4-4RP的扩增结果；
图6为探针NDRG4-1Pb的扩增结果；
图7为探针NDRG4-2Pb的扩增结果；
图8为探针NDRG4-3Pb的扩增结果；
图9为粪便样品的检测结果。
具体实施方式
本发明公开了一种用于肠癌早期诊断的NDRG4基因甲基化检测引物和探针及其试剂盒，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明的目的是提供一组用于肠癌早期诊断的NDRG4基因甲基化检测引物和探针
本发明的另一目的是提供一种利用上述引物和探针制备出的NDRG4基因甲基化检测试剂盒。
本发明上述目的通过以下技术方案实现：
一组用于肠癌早期诊断的NDRG4基因甲基化检测引物和探针，所述引物为NDRG4-1FP和NDRG4-1RP，其核苷酸序列分别如SEQIDNO.1和2所示；所述探针为NDRG4-2Pb，其核苷酸序列如SEQIDNO.3所示。
一种用于肠癌早期诊断的NDRG4基因甲基化检测试剂盒，包含有上述引物NDRG4-1FP、NDRG4-1RP和探针NDRG4-2Pb。
进一步地，上述试剂盒还包括利用引物NDRG4-1FP、NDRG4-1RP和探针NDRG4-2Pb进行PCR扩增时，获得PCR模板所需试剂，所需试剂包括提取待测样品DNA时捕获目的片段的捕获探针NDRG4-CP，捕获探针NDRG4-CP的核苷酸序列如SEQIDNO.4所示。
优选地，上述引物NDRG4-1FP、NDRG4-1RP和探针NDRG4-2Pb的使用浓度均为50～500μM。
更优选地，上述引物NDRG4-1FP、NDRG4-1RP和探针NDRG4-2Pb的使用浓度均为100μM。
进一步优选地，上述检测试剂盒还包括利用引物NDRG4-1FP、NDRG4-1RP和探针NDRG4-2Pb进行PCR扩增时，PCR扩增体系试剂，包括dNTP、镁离子、反应缓冲液(buffer)、酶、无核酸酶的水。
优选地，所述体系试剂包括5～20mM的dNTP、10～40mM的镁离子、1×～10×反应缓冲液、酶、无核酸酶的水。
更优选地，所述体系试剂包括5～15mM的dNTP、15～35mM的镁离子、1×～10×反应缓冲液、酶、无核酸酶的水。
最优选地，所述体系试剂包括10mM的dNTP、25mM的镁离子、5×反应缓冲液(5×buffer)、酶、无核酸酶的水。
优选地，利用上述试剂盒进行NDRG4基因甲基化检测时，PCR体系中各组分的反应终浓度如下：
Figure PCTCN2016109629-appb-000003
Figure PCTCN2016109629-appb-000004
更优选地，利用上述试剂盒进行NDRG4基因甲基化检测时，PCR体系按照如下比例进行：
Figure PCTCN2016109629-appb-000005
更优选地，所述酶为热启动聚合酶。如Promega公司的热启动酶或者SIGMA公司的热启动酶。
另外，优选地，利用上述试剂盒进行NDRG4基因甲基化检测时，PCR程序如下：
95℃300s；1个循环；
95℃20s，62℃30s，70℃30s；10个循环；
95℃20s，58℃60s，72℃30s；30～40个循环；
37℃30s；1个循环。
更优选地，利用上述试剂盒进行NDRG4基因甲基化检测时，PCR程序如下：
95℃300s；1个循环；
95℃20s，62℃30s，70℃30s；10个循环；
95℃20s，58℃60s，72℃30s；35～40个循环；
37℃30s；1个循环。
另外，优选地，上述待测样品为粪便，上述试剂盒还包括粪便预处理 试剂，预处理试剂为研磨缓冲液。
进一步优选地，所述研磨缓冲液具体为：无核酸酶的水、生理盐水、TE、或PBS中的一种或多种。
本发明上述引物和探针及其试剂盒在肠癌的早期诊断方面具有很好的应用前景。
本发明具有以下有益效果：
本发明针对肠癌的早期诊断，设计获得了一组对NDRG4基因甲基化检测效果非常好的引物和探针，并且利用上述引物和探针，成功构建了一套NDRG4基因甲基化检测体系和程序，并组建成试剂盒，对于NDRG4基因甲基化的检测，具有高敏感性和特异性，能够准确特异诊断早期肠癌，对肠癌的早期诊断和筛查具有重要的意义和应用前景。
另外，本发明上述的引物、探针及检测试剂盒，可以使用粪便作为样本，样本处理简单、易取，并且检测方法为普通PCR法，操作简便、快速，只需75～90min左右的时间即可完成检测，无需高昂的仪器和检测费用，非常适用于大量样品的检测以及基层筛查。
以下结合说明书附图和具体实施例来进一步说明本发明，但实施例并不对本发明做任何形式的限定。除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明，本发明所用试剂和材料均为市购。
实施例1引物探针设计
1、针对NDRG4基因甲基化的检测，设计了大量引物，经过筛选后获得如下表1所示5组引物。
表1引物序列
Figure PCTCN2016109629-appb-000006
2、探针设计
针对NDRG4基因甲基化的检测，设计了大量探针，经过筛选后获得如下表2所示3条探针。
表2
探针名称 序列号 序列(5’→3’)
NDRG4-1Pb SEQ ID No.1 CGGAAGTTACGCGCGAGG
NDRG4-2Pb SEQ ID No.3 TCGTTTATCGGGTATTTTAGTCGCGT
NDRG4-3Pb SEQ ID No.12 TCGTTCGTTTATCGGGTATTTT
实施例2样品检测方法
1、样本处理和DNA提取：
(1)粪便样本和缓冲液按照1g粪便：4ml缓冲液的比例混合研磨后，离心留取上清液，丢弃沉淀物。
(2)取8ml上清4000rpm离心5min后转移上清5ml到事先装有3ml细胞裂解液的新的15ml离心管中。
(3)向各离心管中加入100μl捕获磁珠，水浴锅92℃孵育10min；室温摇床100rpm孵育1h，简短离心后置于磁力架5min，丢弃上清。
(4)利用下述捕获探针，可以抓到目的片段。
捕获探针1(SEQ ID No.4)：TCCCTCGCGCGTGGCTTCCGCCTTCTGCGCGGCTGGGGTGCCCGGTGG
(5)向15ml离心管中加入500μl洗涤液，振荡摇匀，使管壁磁珠完全悬下来，简短离心后转移到新的2ml离心管中。室温干浴器900rpm孵育1min，置于磁力架上1min，弃上清。重复4次。
(6)加入55μl洗脱液，简短离心，干浴器92℃900rpm孵育10min。简短离心，置于磁力架上，3min以内转移50μl洗脱液到新的EP管中。
(7)用EZmethylationkit(ZymoResearch公司产品)对第6步中的DNA片段进行甲基化处理，最后的样品进行PCR检测。
2、PCR体系和程序分别如表3、表4所示。
表3PCR体系
成分 加入量(μl)
正向引物(FP)(100μM) 0.125
反向引物(RP)(100μM) 0.125
探针(100μM) 0.05
dNTP(10mM) 1
镁离子(25mM) 5
5*buffer 5
反应酶 0.5
无核酸酶的水 8.2
待测DNA 5
合计 25
表4 PCR程序
Figure PCTCN2016109629-appb-000007
实施例3引物探针优化
按照实施例2的方法，分别利用实施例1设计的各组引物和探针进行检测，优化引物和探针。结果如下：
1、引物优化
如表5和附图1～5所示，5组引物组的扩增结果显示，最优引物组为NDRG4-1FP/NDRG4-1RP。
表5
Figure PCTCN2016109629-appb-000008
2、探针优化
如表6和附图6～8所示，3条探针组的扩增结果显示，最优探针为NDRG4-2Pb。
表6
Figure PCTCN2016109629-appb-000009
实施例4试剂盒组装
1、经过上述研究分析，最终得到最优的检测引物、探针，以及DNA捕获
探针如下表7所示：
表7
Figure PCTCN2016109629-appb-000010
2、组装试剂盒
检测试剂盒包括以下组分：
(1)引物NDRG4-1FP和NDRG4-1RP，其核苷酸序列分别如SEQIDNO.1和2所示。
(2)探针NDRG4-2Pb，其核苷酸序列如SEQIDNO.3所示。
(3)捕获探针NDRG4-CP，其核苷酸序列如SEQIDNO.4所示。捕获探针NDRG4-CP的作用是提取待测样品DNA时捕获目的片段。
(4)PCR扩增体系试剂，10mM的dNTP、25mM的镁离子、5×反应缓冲液(5×buffer)、酶、无核酸酶的水。
(5)样品(如粪便)预处理试剂：研磨缓冲液。所述研磨缓冲液具体为：无核酸酶的水、生理盐水、TE、或PBS中的一种或多种。
3、试剂盒使用方法
(1)经过大量的实验和研究显示，利用上述试剂盒进行NDRG4基因甲基化检测时，最佳的PCR体系按照如下比例进行：
Figure PCTCN2016109629-appb-000011
(2)经过大量的实验和研究显示，利用上述试剂盒进行NDRG4基因甲基化检测时，最佳的PCR程序如下：
95℃300s；1个循环；
95℃20s，62℃30s，70℃30s；10个循环；
95℃20s，58℃60s，72℃30s；35～40个循环；
37℃30s；1个循环。
实施例5样品检测
1、临床收集以下三种粪便样本：
(1)46例肠镜及病理检查确诊为肠癌患者的粪便样本(肠癌，Ca)；
(2)46例肠镜及病理检查确诊为癌前腺瘤(息肉直径≥1cm)的患者的粪便样本(癌前腺瘤，LA)；
(3)46例肠镜检查确诊为正常患者的粪便样本(正常，Norm)。
2、利用实施例4所组建的试剂盒对以上粪便样品进行检测，使用MedCalc软件对数据进行处理分析。
3、NDRG4基因检测结直肠癌和癌前腺瘤的结果如图9所示。其中，图9A为NDRG4基因检测结直肠癌的ROC曲线；图9B为NDRG4基因检测癌前腺瘤(息肉直径≥1cm)的ROC曲线。
结果显示，对于结直肠癌，本发明上述试剂盒对NDRG4基因的检测敏感性为65.2％，特异性为97.8％，受试者曲线下面积为0.826。
对于癌前腺瘤(息肉直径≥1cm)，本发明上述试剂盒对NDRG4基因检测敏感性为45.7％，特异性为97.8％，受试者曲线下面积为0.694。
综上所述，本发明的引物和探针及其检测试剂盒不仅能够更灵敏、更准确的检测诊断结直肠癌，尤其是早期结直肠癌，而且对于癌前腺瘤也具有很好的检测灵敏性和很高的特异性，可在癌前腺瘤阶段就实现检测诊断的目的。
以上对本发明所提供的用于肠癌早期诊断的NDRG4基因甲基化检测引物和探针及其试剂盒进行了详细介绍。本文应用了具体个例对本发明的原理及实施方式进行了阐述，以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。
Figure PCTCN2016109629-appb-000012
Figure PCTCN2016109629-appb-000013
Figure PCTCN2016109629-appb-000014

Claims (17)

  1. 引物和探针的组合物，其特征在于，所述引物的序列如SEQ ID No.1和/或SEQ ID No.2所示；所述探针的序列如SEQ ID No.3所示。
  2. 根据权利要求1所述的引物和探针的组合物在制备NDRG4基因甲基化检测试剂中的应用。
  3. 根据权利要求1所述的引物和探针的组合物在制备肠癌诊断试剂中的应用；优选的，所述肠癌为早期肠癌。
  4. 一种试剂盒，其特征在于，包括如权利要求1所述的引物和探针的组合物。
  5. 根据权利要求4所述的试剂盒，其特征在于，还包括捕获探针；所述捕获探针的序列如SEQ ID No.4所示。
  6. 根据权利要求4或5所述的试剂盒，其特征在于，所述引物、所述探针或所述捕获探针的使用浓度分别为50～500μM。
  7. 根据权利要求4至6任一项所述的试剂盒，其特征在于，还包括扩增体系；所述扩增体系包括dNTP、镁离子、反应缓冲液、酶或无核酸酶的水。
  8. 根据权利要求7所述的试剂盒，其特征在于，所述扩增体系包括5～20mM的dNTP、10～40mM的镁离子、1×～10×反应缓冲液、酶、无核酸酶的水。
  9. 根据权利要求7所述的试剂盒，其特征在于，所述扩增体系中各组分的反应终浓度为：
    Figure PCTCN2016109629-appb-100001
  10. 根据权利要求4至9任一项所述的试剂盒，其特征在于，所述酶为热启动聚合酶。
  11. 根据权利要求4至10任一项所述的试剂盒，其特征在于，所述试剂盒的扩增程序为：
    95℃300s；1个循环；
    95℃20s，62℃30s，70℃30s；10个循环；
    95℃20s，58℃60s，72℃30s；30～40个循环；
    37℃30s；1个循环。
  12. 根据权利要求4至11任一项所述的试剂盒，其特征在于，其检测样本为粪便；所述试剂盒还包括粪便预处理试剂，所述粪便预处理试剂为研磨缓冲液。
  13. 根据权利要求4至12任一项所述的试剂盒在NDRG4基因甲基化检测中的应用。
  14. 根据权利要求4至12任一项所述的试剂盒在肠癌诊断中的应用；所述肠癌为早期肠癌。
  15. 一种肠癌的检测方法，其特征在于，取样本与如权利要求1所述的引物和探针的组合物或如4至12任一项所述的试剂盒中的检测试剂混合后扩增，根据扩增结果获得检测结果。
  16. 根据权利要求15所述的检测方法，其特征在于，所述扩增的目的物为肠癌标志物；所述肠癌标志物为NDRG4基因。
  17. 根据权利要求15或16所述的检测方法，其特征在于，所述获得检测结果为比较待测样本与正常样本的扩增结果，当所述待测样本与正常样本的扩增结果具有显著差异或极显著差异时，所述待测样本的供体呈阳性。
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