A gastric cancer prognosis assessment composition, kit and system

Abstract

The present invention discloses a composition, a kit and a system for evaluating the prognosis of gastric cancer, and relates to the field of biotechnology. The composition is composed of cg00984694, cg09046939, cg13946520 and cg01466678. The gastric cancer prognosis evaluation kit includes a detection reagent for detecting the methylation level of a methylation site for evaluating the prognosis of gastric cancer, and the detection reagent includes a methylation detection primer pair and a probe, and the sequence is shown in SEQ ID NO: 1-12. The kit of the present invention has good sensitivity and specificity in evaluating the prognosis of gastric cancer.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a gastric cancer prognosis evaluation composition, a kit and a system. Background Art

[0002] Gastric cancer is a digestive tract tumor associated with multiple factors. The occurrence of gastric cancer stems from the complex interaction between the environment, bacteria and the host. The risk factors for gastric cancer mainly include the following aspects: (1) age, gender and family history; (2) unhealthy lifestyle habits: smoking, drinking and unhealthy eating habits are modifiable risk factors. The dose and duration of smoking are also related to the risk of gastric cancer. Multidirectional studies have also observed a correlation between drinking and the risk of gastric cancer. (3) Helicobacter pylori infection: There may be two mechanisms for the association between Helicobacter pylori and gastric cancer risk: the indirect inflammatory response of Helicobacter pylori infection to the gastric mucosa and the direct epigenetic changes of Helicobacter pylori to gastric epithelial cells.

[0003] The prognosis of gastric cancer is also related to a variety of risk factors, which are mainly divided into patient-related factors, tumor-related factors and treatment-related factors. Patient-related risk factors include gender, age, and nutritional status; tumor-related risk factors include tumor diameter, degree of differentiation, and stage; treatment-related factors include the choice of surgical method, degree of surgical radicality, postoperative standardized case reporting, perioperative adjuvant therapy, etc.

[0004] Chinese patent CN110656178A discloses the application and detection kit of the gastric cancer prognostic diagnostic marker TREM1, and the application is to evaluate the prognosis of gastric cancer patients by detecting the relative expression level of the marker TREM1. The invention also discloses a detection kit including primers and internal references of the marker TREM1. The gastric cancer prognostic diagnostic marker TERM1 provided by the invention has high specificity and sensitivity, and its relative expression level can be detected by a fast and simple method, thereby accurately judging and evaluating the prognosis of gastric cancer patients, realizing the grading and stratification of gastric cancer risk levels at the molecular level, quickly and accurately predicting the prognosis of gastric cancer, and classifying and treating patients with different prognostic levels, thereby guiding the individualized precision treatment of gastric cancer patients, saving medical costs, and having extremely high clinical application value.

[0005] Chinese patent CN114966060A discloses a gastric cancer prognosis biomarker and its application, wherein the biomarker includes SR-BI, and high expression of SR-BI indicates that the prognosis of gastric cancer patients is poor. The biomarker includes SR-BI and CD4+Tcells, and high expression of SR-BI and low expression of CD4+Tcells indicate that the prognosis of gastric cancer patients is poor; low expression of SR-BI and high expression of CD4+Tcells indicate that the prognosis of gastric cancer patients is good. The application of the biomarker includes evaluating or predicting the prognostic risk, predicting the suitability of immunotherapy, predicting the survival rate, formulating treatment / medication plans, constructing a model for predicting the prognostic risk of gastric cancer, constructing a model for the suitability of immunotherapy, constructing a model for predicting the survival rate of gastric cancer, preparing a detection reagent or kit for predicting the prognostic risk of gastric cancer, and preparing any one or a combination of detection reagents or kits for predicting the survival rate of gastric cancer.

[0006] Prognosis assessment for cancer remains an important topic of research, and providing a gastric cancer prognosis assessment kit with high predictive sensitivity and specificity is a technical problem that needs to be solved urgently. Summary of the invention

[0007] The purpose of the present invention is to provide a gastric cancer prognosis assessment kit, system and method.

[0008] To achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows:

[0009] In one aspect, the present invention provides a composition for evaluating the prognosis of gastric cancer, wherein the composition consists of cg00984694, cg09046939, cg13946520 and cg01466678.

[0010] In another aspect, the present invention provides a gastric cancer prognosis evaluation kit, wherein the gastric cancer prognosis evaluation kit comprises a detection reagent for detecting the methylation level of a methylation site for gastric cancer prognosis evaluation, wherein the detection reagent comprises a methylation detection primer pair, and the methylation detection primer pair comprises:

[0011] The primer pair used to detect cg00984694, the sequence is shown in SEQ ID NO: 1-2;

[0012] The primer pair used to detect cg09046939, the sequence is shown in SEQ ID NO:3-4;

[0013] The primer pair used to detect cg13946520, the sequence is shown in SEQ ID NO:5-6;

[0014] The primer pair used to detect cg01466678 has the sequences shown in SEQ ID NOs: 7-8.

[0015] Specifically, the detection reagent further includes a methylation detection probe, and the methylation detection probe includes:

[0016] The probe for detecting cg00984694, the sequence of which is shown in SEQ ID NO:9;

[0017] The probe for detecting cg09046939, the sequence of which is shown in SEQ ID NO: 10;

[0018] The probe for detecting cg13946520 has a sequence as shown in SEQ ID NO: 11;

[0019] The probe used to detect cg01466678 has a sequence as shown in SEQ ID NO:12.

[0020] Specifically, the detection reagent also includes a primer pair and a probe for detecting an internal reference gene.

[0021] Furthermore, the internal reference gene is selected from any one of GAPDH, ACTB, β-actin, and HPRT1.

[0022] Furthermore, the internal reference gene is ACTB.

[0023] Furthermore, the sequence of the primer pair of the internal reference gene is shown in SEQ ID NO:13-14, and the sequence of the probe is shown in SEQ ID NO:15.

[0024] Specifically, the final concentration of the primers in the gastric cancer prognosis assessment kit is 10-40 mM;

[0025] Furthermore, the final concentration of the primers in the gastric cancer prognosis evaluation kit is 20 mM.

[0026] Specifically, the final concentration of the primers in the gastric cancer prognosis assessment kit is 40-60 mM;

[0027] Furthermore, the final concentration of the primers in the gastric cancer prognosis assessment kit is 50 mM.

[0028] Specifically, the 5' end of the probe is labeled with a fluorescent reporter group, and the 3' end of the probe is labeled with a fluorescent quencher group.

[0029] Further, the fluorescent reporter group is selected from any one of FAM, ROX, HEX, CY5, VIC, TET, JOE, Cy3, Cy7, RED610, Texas Red, RED670, NED, AMCA, Pacific Blue, Atto 425, BODIPY FL, Atto 590, and Cy5.5;

[0030] Furthermore, the fluorescent reporter group is selected from any one of FAM, ROX, HEX, and CY5.

[0031] Furthermore, the fluorescence quenching group is selected from any one of 6-TAMRA, BHQ-1, BHQ-2, BHQ-3, Dabcyl, Eclipse, MGB, and QYS-7;

[0032] Furthermore, the fluorescence quenching group is selected from any one of BHQ-1 and BHQ-2.

[0033] Specifically, the 5' end of the probe is labeled with FAM, and the 3' end of the probe is labeled with BHQ-1.

[0034] Specifically, the kit also includes a positive quality control product and a negative quality control product.

[0035] Furthermore, the positive quality control product is human methylated standard DNA, and the negative quality control product is human unmethylated standard DNA.

[0036] Specifically, the kit also includes a PCR reaction system, which includes 2×HotTaqMaster Mix, 1 μL upstream primer, 1 μL downstream primer, 1 μL probe and 5 μL sample template.

[0037] According to some embodiments of the present invention, the PCR reaction system includes 10 μL 2×HotTaq MasterMix, 1 upstream primer, 1 downstream primer, a probe and a sample template.

[0038] Specifically, the PCR reaction program of the kit is: 95°C, 5 min; 95°C, 15 s, 60°C, 30 s, 45 cycles; 40°C, 5 min.

[0039] In another aspect, the present invention provides a gastric cancer prognosis evaluation system, wherein the gastric cancer prognosis evaluation system comprises:

[0040] 1) Detection components for detecting the methylation degree of cg00984694, cg09046939, cg13946520 and cg01466678;

[0041] 2) Result judgment component.

[0042] The beneficial effects of the present invention are:

[0043] The kit provided by the present invention for detecting the methylation sites of cg00984694, cg09046939, cg13946520 and cg01466678 has good sensitivity and specificity when used for gastric cancer prognosis assessment, and is of great value for the prognosis assessment of gastric cancer patients. DETAILED DESCRIPTION

[0044] In order to make the technical means, creative features, purpose and efficacy of the present invention easy to understand, the present invention is further explained in conjunction with specific embodiments below, but the following embodiments are only preferred embodiments of the present invention, not all. Based on the embodiments in the implementation mode, other embodiments obtained by those skilled in the art without creative work all belong to the protection scope of the present invention. In the following embodiments, unless otherwise specified, the operating methods used are conventional operating methods, the equipment used are conventional equipment, and the equipment materials used in each embodiment are the same.

[0045] Example 1 Gastric cancer prognosis assessment composition and primers and probes thereof

[0046] The gastric cancer prognosis evaluation composition of the present invention is composed of cg00984694, cg09046939, cg13946520 and cg01466678. The primer and probe sequences of the composition are specifically shown in Table 1 below.

[0047] Table 1 Primer and probe sequences

[0048]

[0049]

[0050] Example 2 Preparation of Gastric Cancer Prognosis Assessment Kit

[0051] The gastric cancer prognosis evaluation kit of the present invention includes the primers and probes in Table 1 above, PCR reaction solution, positive quality control and negative quality control, the positive quality control is human methylated standard DNA, and the negative quality control is human non-methylated standard DNA. The gastric cancer prognosis evaluation kit prepared by the present invention is used to detect the sample, and the specific steps of the detection are:

[0052] Step 1. DNA extraction

[0053] The QIAamp DNA Mini Kit was used to extract the whole genome DNA of the tissue, and the DNA concentration was detected by NanoDrop2000 ultra-micro spectrophotometer.

[0054] Step 2: Methylation modification

[0055] Using EZ DNAMethylation Gold TM Kit methylation conversion kit (purchased from Zymo Research) converts unmethylated cytosine in DNA sequences into uracil.

[0056] Step 3: qPCR amplification

[0057] (1) Preparation of primer and probe combination

[0058] Prepare according to Table 2 below:

[0059] Table 2 Primer probe composition

[0060]

[0061]

[0062] (2) Preparation of qPCR reaction solution

[0063] Prepare the PCR reaction solution according to Table 3 below, mix and centrifuge the prepared PCR reaction solution, and then put it into the PCR reaction tank.

[0064] Table 3 qPCR reaction system

[0065] Components Dosage (μL) 2×HotTaq Master Mix 10 Upstream primer 1 Downstream primer 1 Probe 1 <![CDATA[ddH2O]]> 2 total 20

[0066] (3) Sample addition: Add 5 μL of DNA sample into the PCR reaction tube. The sample volume for both the positive quality control and the negative quality control is 5 μL. After sample addition, cover the tube and centrifuge to mix.

[0067] (4) PCR reaction procedure:

[0068] PCR amplification was performed according to Table 4 below, with 3 replicate wells for each sample and water as a blank control.

[0069] Table 4 PCR amplification program

[0070]

[0071] (5) Interpretation of test results

[0072] The internal reference gene ACTB channel of the sample to be tested has an obvious amplification curve and the Ct value is less than 35, which means that the test sample is valid. On the premise that the sample to be tested is valid, the detection channel has an obvious amplification curve, and the Ct value is less than 35, which can be judged as positive, and the Ct value is greater than 35, which can be judged as negative.

[0073] Comparative Example 1

[0074] The difference between Comparative Example 1 and Example 2 is that the preparation of the primer-probe combination is different. The preparation of the primer-probe in Comparative Example 1 is shown in Table 5 below.

[0075] Table 5 Primer probe composition

[0076]

[0077]

[0078] Comparative Example 2

[0079] The difference between Comparative Example 2 and Example 2 is that the preparation of the primer-probe combination is different. The preparation of the primer-probe in Comparative Example 2 is shown in Table 6 below.

[0080] Table 6 Primer probe composition

[0081]

[0082] Experimental Example 1

[0083] The test samples were 60 samples with pathological diagnosis (diagnostic criteria were in accordance with the Standards for Diagnosis and Treatment of Gastric Cancer (2018 edition), typing and grading were in accordance with the WHC and Lauren typing, and gastric cancer staging was in accordance with the staging standards established by the International Union Against Cancer) confirmed gastric cancer with a survival rate of less than 5 years and 40 samples with pathological diagnosis confirmed gastric cancer with a survival rate of greater than 5 years. The tissue samples were tested using the kits of Example 2 and Comparative Examples 1-2, respectively.

[0084] Step 1: DNA extraction

[0085] The QIAamp DNA Mini Kit was used to extract the whole genome DNA of the tissue, and the DNA concentration was detected by NanoDrop2000 ultra-micro spectrophotometer.

[0086] Step 2: Methylation modification

[0087] Using EZ DNA Methylation Gold TM Kit methylation conversion kit (purchased from Zymo Research) converts unmethylated cytosine in DNA sequences into uracil.

[0088] Step 3: qPCR amplification

[0089] (1) The preparation of primer and probe combination is as in Example 2 and Comparative Examples 1-2.

[0090] (2) Preparation of qPCR reaction solution

[0091] Prepare the PCR reaction solution according to the table below, mix and centrifuge the prepared PCR reaction solution, and then put it into the PCR reaction tank.

[0092] Table 7 qPCR reaction system

[0093] Components Dosage (μL) 2×HotTaq Master Mix 10 Upstream primer 1 Downstream primer 1 Probe 1 <![CDATA[ddH2O]]> 2 total 20

[0094] (3) Sample addition: Add 5 μL of DNA sample into the PCR reaction tube. The sample volume for both the positive quality control and the negative quality control is 5 μL. After sample addition, cover the tube and centrifuge to mix.

[0095] (4) PCR reaction procedure:

[0096] Perform PCR amplification according to the table below, with 3 replicate wells for each sample and water as a blank control.

[0097] Table 8 PCR amplification program

[0098]

[0099] (5) Interpretation of test results

[0100] The internal reference gene ACTB channel of the sample to be tested has an obvious amplification curve and the Ct value is less than 35, which means that the test sample is valid. On the premise that the sample to be tested is valid, the detection channel has an obvious amplification curve, and a Ct value of less than 35 can be judged as a poor prognosis, and a Ct value of more than 35 can be judged as a good prognosis.

[0101] The test results are shown in the following table:

[0102] Table 9 Tissue sample test results

[0103]

[0104] Table 10 Sensitivity and specificity

[0105] Sensitivity Specificity Example 2 96.67％ 100％ Comparative Example 1 88.33％ 87.5％ Comparative Example 2 83.33％ 87.5％

[0106] It can be seen from the above table that when the method of Example 2 is used to evaluate the prognosis of gastric cancer, the sensitivity is 96.67% and the specificity is 100%, indicating that the kit of the present invention can be well applied to the prognosis evaluation of gastric cancer.

[0107] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A composition for evaluating gastric cancer prognosis, characterized in that: The composition consists of cg00984694, cg09046939, cg13946520 and cg01466678.

2. A gastric cancer prognosis assessment kit, characterized in that: The gastric cancer prognosis evaluation kit includes a detection reagent for detecting the methylation level of a methylation site for gastric cancer prognosis evaluation, and the detection reagent includes a methylation detection primer pair, and the methylation detection primer pair includes: The primer pair used to detect cg00984694, the sequence is shown in SEQ ID NO: 1-2; The primer pair used to detect cg09046939, the sequence is shown in SEQ ID NO:3-4; The primer pair used to detect cg13946520, the sequence is shown in SEQ ID NO:5-6; The primer pair used to detect cg01466678 has the sequences shown in SEQ ID NOs: 7-8.

3. The gastric cancer prognosis evaluation kit according to claim 2, characterized in that: The detection reagent also includes a methylation detection probe, and the methylation detection probe includes: The probe for detecting cg00984694, the sequence of which is shown in SEQ ID NO:9; The probe for detecting cg09046939, the sequence of which is shown in SEQ ID NO: 10; The probe for detecting cg13946520 has a sequence as shown in SEQ ID NO: 11; The probe used to detect cg01466678 has a sequence as shown in SEQ ID NO:

12.

4. The gastric cancer prognosis evaluation kit according to claim 2, characterized in that: The detection reagent also includes a primer pair and a probe for detecting an internal reference gene. The sequence of the primer pair is shown in SEQ ID NO:13-14, and the sequence of the probe is shown in SEQ ID NO:

15.

5. The gastric cancer prognosis evaluation kit according to claim 1, characterized in that: The 5' end of the probe is labeled with a fluorescent reporter group, and the 3' end of the probe is labeled with a fluorescent quencher group.

6. The gastric cancer prognosis evaluation kit according to claim 5, characterized in that: The fluorescent reporter group is selected from any one of FAM, ROX, HEX, CY5, VIC, TET, JOE, Cy3, Cy7, RED610, Texas Red, RED670, NED, AMCA, Pacific Blue, Atto 425, BODIPYFL, Atto 590, and Cy5.

5.

7. The gastric cancer prognosis evaluation kit according to claim 5, characterized in that: The fluorescence quenching group is selected from any one of 6-TAMRA, BHQ-1, BHQ-2, BHQ-3, Dabcyl, Eclipse, MGB, and QYS-7.

8. The gastric cancer prognosis evaluation kit according to any one of claims 1 to 7, characterized in that: The final concentration of the primers in the gastric cancer prognosis evaluation kit is 10-40mM.

9. The gastric cancer prognosis evaluation kit according to any one of claims 1 to 7, characterized in that: The final concentration of the primers in the gastric cancer prognosis evaluation kit is 40-60mM.

10. A gastric cancer prognosis assessment system, characterized in that: The gastric cancer prognosis evaluation system comprises: 1) Detection components for detecting the methylation degree of cg00984694, cg09046939, cg13946520 and cg01466678; 2) Result judgment component.

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