Composition and kit for detecting methylation of human plasma RPS2 gene and DCDC2 gene of liver cancer

Abstract

The invention discloses a composition for detecting methylation of a human plasma RPS2 gene and a DCDC2 gene of liver cancer, which can be used for realizing early screening and early diagnosis of liver cancer through detection of plasma DNA (Deoxyribonucleic Acid). Meanwhile, the invention further provides a kit for methylation of the RPS2 gene and the DCDC2 gene, the sensitivity of detection of liver cancer patients reaches 85.88%, and the specificity is 92.23%.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a composition for detecting methylation of human plasma RPS2 gene and DCDC2 gene of liver cancer. Background Art

[0002] With the increasing incidence of hepatocellular carcinoma (HCC) every year, a method that can accurately detect liver cancer in its early stages is needed. Among current detection technologies, ultrasound examination is often used in combination with serum alpha-fetoprotein (AFP) testing. However, AFP is ineffective in detecting early liver cancer and often produces false positive results in patients with hepatitis. Ultrasound examination has limitations in detecting early lesions smaller than 2 cm. Therefore, there is an urgent need for a highly sensitive and specific biomarker to facilitate the detection of early liver cancer, which can effectively improve the survival rate of liver cancer patients. Summary of the invention

[0003] The purpose of the present invention is to provide a composition and a kit for detecting methylation of the human plasma RPS2 gene and DCDC2 gene of liver cancer, which is used for early screening and diagnosis of liver cancer. The detection targets the CpG sites in the promoter region of the RPS2 and DCDC2 genes, has high accuracy, convenient sampling, and low trauma.

[0004] To achieve the above object, a technical solution adopted by the present invention is: a composition for detecting methylation of RPS2 gene and DCDC2 gene in human plasma of liver cancer, wherein the composition is RPS2 gene and DCDC2 gene.

[0005] Furthermore, the primer sequence of the RPS2 gene is SEQ ID No: 1; and the primer sequence of the DCDC2 gene is SEQ ID No: 2.

[0006] Furthermore, the probe sequence of the RPS2 gene is SEQ ID No: 4; and the probe sequence of the DCDC2 gene is SEQ ID No: 5.

[0007] Furthermore, the internal reference of the fluorescent quantitative PCR detection is the ACTB gene, the primer sequence of the ACTB gene is SEQ ID No: 3, and the probe primer sequence is SEQ ID No: 6.

[0008] A kit for detecting early liver cancer comprises RPS2 gene primers and probes, DCDC2 gene primers and probes, ACTB internal reference gene primers and probes and PCR amplification enzyme reaction solution.

[0009] Beneficial effects of the present invention:

[0010] The present invention provides a RPS2 and DCDC2 gene dual methylation combined detection kit, which can be used to achieve early screening and early diagnosis of liver cancer through the detection of plasma DNA; specifically:

[0011] First, we designed primers and probes for the CG-rich regions of the RPS2 and DCDC2 genes to establish a methylation amplification system, and tested the sensitivity and specificity of the PCR system for liver cancer using FFPE samples;

[0012] The performance of the kit was then verified through a large number of clinical plasma sample tests, including a full range of tests on benign liver lesion samples and other tumor samples;

[0013] The present invention has developed a new dual-gene methylation detection kit, which detects RPS2 and DCDC2 genes with a sensitivity of 85.88% and a specificity of 92.23% for gastric cancer detection. The method is simple and non-invasive and can achieve early screening and diagnosis of liver cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is a ROC curve diagram of the PCR system detection of the RPS2 gene and the DCDC2 gene of the present invention on the sample. DETAILED DESCRIPTION

[0015] The preferred embodiments of the present invention are described in detail below in conjunction with the accompanying drawings so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making a clearer and more definite definition of the protection scope of the present invention.

[0016] Embodiment 1

[0017] The specific preparation method of the detection kit provided by the present invention is as follows:

[0018] Establish a target segment amplification system. For the RPS2 gene (Gene ID: 6187) and the DCDC2 gene (Gene ID: 51473), we screened two CpG-rich segments from each target, designed a pair of primers for each segment, and screened out the best amplification system through whole genome methylation quality control products.

[0019] The amplification reaction system is as shown in Table 1-1:

[0020] Table 1-1

[0021] Components Specification volume PCR Mix 2× 10μL RPS2 upstream amplification primers 20μM 0.4μl RPS2 downstream amplification primers 20μM 0.4μl RPS2 fluorescent probe 20μM 0.2μl DCDC2 upstream amplification primers 20μM 0.4μl DCDC2 downstream amplification primers 20μM 0.4μl DCDC2 fluorescent probe 20μM 0.2μl Internal reference upstream amplification primer 20μM 0.4μl Internal reference downstream amplification primers 20μM 0.4μl Internal reference fluorescent probe 20μM 0.2μl Nuclease-free water 2μl DNA 5μl Overall system 20μl

[0022] The upstream primer and downstream primer sequences of RPS2 gene, DCDC2 gene and internal reference gene are shown in Table 1-2:

[0023] Table 1-2

[0024]

[0025]

[0026] The amplification reaction procedure is as follows: Table 1-3:

[0027] Table 1-3

[0028]

[0029] Embodiment 2

[0030] To verify the detection performance of the target for liver cancer, we collected tissue sections (including cancer and adjacent tissues) from 58 liver cancer patients and 32 other cancer samples (including colorectal cancer, gastric cancer, lung cancer, and esophageal cancer).

[0031] We first obtained DNA from tissue sections by extraction. The DNA extraction from tissue sections was performed using a commercial kit, the Blood / Cell / Tissue Gene DNA Extraction Kit (centrifugal column type) (Cat. No. YDP304) produced by Tiangen Biochemical Technology (Beijing) Co., Ltd. After the extraction, the extracted DNA was subjected to methylation and sulfurization treatment. The DNA methylation treatment was performed using a commercial kit, the EZ DNA Methylation Kit (Cat. No. D5005) produced by ZYMO Research. The specific kit conversion process was performed according to the kit instructions. The converted DNA was tested for methylation PCR.

[0032] After being treated with the methylation treatment kit, the methylated RPS2 or DCDC2 gene will generate a fluorescent signal during the fluorescence quantitative PCR detection process, while the unmethylated gene will not generate a fluorescent signal.

[0033] The results of RPS2 or DCDC2 gene methylation detection in 90 biopsy samples are shown in Table 2-1:

[0034] Table 2-1

[0035]

[0036]

[0037]

[0038]

[0039] From the above results, it can be seen that RPS2 and DCDC2 genes were methylated positive in all 40 liver cancer tissue samples, while they were methylated negative in adjacent cancer tissues and other cancer samples, indicating that the two target genes can effectively detect liver cancer. The following experiment uses plasma samples to test the sensitivity and specificity of the detection.

[0040] Embodiment 3

[0041] In order to verify the clinical performance of the dual-gene methylation joint detection kit, we collected plasma samples from several patients clinically diagnosed with liver cancer, normal people, and patients with benign liver lesions, and used the RPS2 and DCDC2 gene dual-gene methylation system for detection and verification.

[0042] A total of 85 plasma samples from liver cancer, 18 plasma samples from intrahepatic cholangiocarcinoma, 20 plasma samples from hepatitis, 15 plasma samples from cirrhosis, and 50 plasma samples from healthy people were tested.

[0043] First, the DNA of the plasma sample was extracted. The plasma DNA extraction used a commercial kit, the magnetic bead serum / plasma free DNA extraction kit (Cat. No. DP709) produced by Tiangen Biochemical Technology (Beijing) Co., Ltd.; after the extraction, the extracted DNA was subjected to methylation and sulfurization treatment. The DNA methylation treatment used a commercial kit, the EZ DNA Methylation Kit (Cat. No. D5005) produced by ZYMOResearch. The specific kit conversion process was operated according to the kit instructions. The converted DNA was tested by methylation PCR.

[0044] After being treated with the methylation treatment kit, the methylated RPS2 or DCDC2 gene will produce a fluorescent signal during the fluorescence quantitative PCR test, while the unmethylated gene will not produce a fluorescent signal. The methylation test results of the RPS2 or DCDC2 gene of all plasma samples are shown in Table 3-1:

[0045] Table 3-1

[0046]

[0047]

[0048]

[0049]

[0050]

[0051]

[0052] Using dual-target combined detection of plasma samples, the optimal positive judgment value of the kit based on the above data analysis is as follows:

[0053] 1. DCDC2 gene CT value ≤ 38, judged as DCDC2 methylation positive; CT value > 38 or Unde, judged as DCDC2 methylation negative;

[0054] 2. If the CT value of RPS2 gene is ≤38, it is judged as RPS2 methylation positive; if the CT value is greater than 38 or Unde, it is judged as RPS2 gene methylation negative;

[0055] 3. The method for determining sample test results is shown in Table 3-2 below:

[0056] Table 3-2

[0057]

[0058] Using the above-mentioned positive and negative judgment method, 73 of the 85 liver cancer plasma samples were positive for methylation detection, with a sensitivity of 85.88%; 8 of the 103 non-liver cancer samples were positive for methylation detection, with a specificity of 92.23%.

[0059] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structural transformations made using the contents of the present invention's specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A composition for detecting methylation of RPS2 gene and DCDC2 gene in human plasma of liver cancer, characterized in that: The composition is RPS2 gene and DCDC2 gene.

2. A composition for detecting methylation of RPS2 gene and DCDC2 gene in human plasma of liver cancer according to claim 1, characterized in that: The primer sequence of the RPS2 gene is SEQ ID No: 1; the primer sequence of the DCDC2 gene is SEQ ID No:

2.

3. A composition for detecting methylation of RPS2 gene and DCDC2 gene in human plasma of liver cancer according to claim 2, characterized in that: The probe sequence of the RPS2 gene is SEQ ID No: 4; the probe sequence of the DCDC2 gene is SEQ ID No:

5.

4. A composition for detecting methylation of RPS2 gene and DCDC2 gene in human plasma of liver cancer according to claim 3, characterized in that: The internal reference of the fluorescence quantitative PCR detection is the ACTB gene, the primer sequence of the ACTB gene is SEQ ID No: 3, and the probe primer sequence is SEQ ID No:

6.

5. A kit for detecting early gastric cancer, characterized in that: It includes RPS2 gene primers and probes, DCDC2 gene primers and probes, ACTB internal reference gene primers and probes, and PCR amplification enzyme reaction solution.

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