A probe primer set and a kit for evaluating the sensitivity of 5-FU in treating gastric cancer

Abstract

The present invention discloses a probe primer set and a kit for evaluating the sensitivity of gastric cancer to 5-FU treatment. The kit evaluates the sensitivity of gastric cancer patients to 5-fluorouracil (5-FU) drugs by detecting the expression levels of AGPS, FAR1, GNPAT, and Peds1 gene mRNAs in tumor tissue samples of gastric cancer patients, and determines whether the patients are suitable for 5-FU drug treatment. The present invention designs primer sets and probes for amplifying AGPS, FAR1, GNPAT, and Peds1 genes, uses the GAPDH gene as an internal reference gene, detects the sample to be tested by fluorescence PCR, establishes a comprehensive evaluation model, determines the regression coefficients of AGPS, FAR1, GNPAT, and Peds1 genes, and uses a comprehensive evaluation value of 0.5 as the positive judgment value. This kit is easy to operate, has a short detection time, strong specificity, and high sensitivity.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a probe primer set and a kit for evaluating the sensitivity of 5-FU in treating gastric cancer. Background Art

[0002] Gastric cancer is a malignant tumor originating from gastric mucosal epithelium, ranking first in the incidence of malignant tumors in China. Most patients with early gastric cancer have no obvious symptoms, resulting in a low early diagnosis rate and thus affecting the treatment effect. The occurrence and development mechanism of gastric cancer has not been fully clarified yet. Although the current medical technology level is constantly developing, there is an endless stream of methods for the early diagnosis, treatment and prognosis judgment of gastric cancer, but the clinical treatment effect of advanced gastric cancer with rapid growth and high metastasis is still poor, with only about 20% 5-year survival rate. Exploring potential therapeutic targets of gastric cancer from the perspective of molecular mechanisms is of great significance for clinical treatment. Therefore, finding relevant biomarkers of gastric cancer tissues is of great significance for the diagnosis and treatment of gastric cancer and has important value for the diagnosis and treatment of early gastric cancer. Clinically, early gastric cancer is mainly treated by surgery, and advanced and late gastric cancer is mainly treated by chemotherapy.

[0003] 5-Fluorouracil (5-FU) is one of the most commonly used drugs in current combination chemotherapy, but the generation of drug resistance of cancer cells to 5-FU during the treatment process seriously hinders the development of its clinical application. Therefore, exploring the mechanism of drug resistance generation and finding countermeasures to overcome drug resistance have become the key to improving the treatment effect and prognosis of 5-FU in treating gastric cancer. The expressions of AGPS, FAR1, GNPAT and Peds1 genes or related proteins are abnormal in gastric cancer tissues, but there is no research or report on the correlation between the combined detection of these 4 genes and gastric cancer drug resistance. Summary of the Invention

[0004] In view of this, in order to overcome the deficiencies in the prior art, the technical problem to be solved by the present invention is to provide a probe primer set and a kit for evaluating the sensitivity of 5-FU in treating gastric cancer.

[0005] In order to achieve the above object, the present invention is implemented through the following solutions:

[0006] Use of reagents for detecting AGPS gene, FAR1 gene, GNPAT gene and Peds1 gene in the preparation of a kit for evaluating the sensitivity of 5-fluorouracil in treating gastric cancer, wherein the accession number of the AGPS gene on GenBank is NM_003659.4, the accession number of the FAR1 gene on GenBank is NM_032228.6, the accession number of the GNPAT gene on GenBank is NM_001316350, and the accession number of the Peds1 gene on GenBank is NM_001162505.

[0007] A detection reagent, the detection reagent comprising: a combination of upstream and downstream primers and probes for detecting one or several of the AGPS gene, FAR1 gene, GNPAT gene, and Peds1 gene respectively;

[0008] The nucleotide sequences of the upstream and downstream primers for amplifying the AGPS gene are shown in SEQ ID NO: 1 and SEQ ID NO: 2, the nucleotide sequence of the probe for detecting the AGPS gene is shown in SEQ ID NO: 11, and the accession number of the AGPS gene on GenBank is NM_003659.4;

[0009] The nucleotide sequences of the upstream and downstream primers for amplifying the FAR1 gene are shown in SEQ ID NO: 3 and SEQ ID NO: 4, the nucleotide sequence of the probe for detecting the FAR1 gene is shown in SEQ ID NO: 12, and the accession number of the FAR1 gene on GenBank is NM_032228.6;

[0010] The nucleotide sequences of the upstream and downstream primers for amplifying the GNPAT gene are shown in SEQ ID NO: 5 and SEQ ID NO: 6, the nucleotide sequence of the probe for detecting the GNPAT gene is shown in SEQ ID NO: 13, and the accession number of the GNPAT gene on GenBank is NM_001316350;

[0011] The nucleotide sequences of the upstream and downstream primers for amplifying the Peds1 gene are shown in SEQ ID NO: 7 and SEQ ID NO: 8, the nucleotide sequence of the probe for detecting the Peds1 gene is shown in SEQ ID NO: 14, and the accession number of the Peds1 gene on GenBank is NM_001162505.

[0012] Preferably, the detection reagent comprises: the combination of upstream and downstream primers and probes for detecting the AGPS gene, FAR1 gene, GNPAT gene, and Peds1 gene as described in claim 2.

[0013] Preferably, the detection reagent further contains a primer set and a probe for amplifying the GAPDH gene, the nucleotide sequences of the primer set for amplifying the GAPDH gene are shown in SEQ ID NO: 9 and SEQ ID NO: 10, the nucleotide sequence of the probe for detecting the GAPDH gene is shown in SEQ ID NO: 15, and the accession number of the GAPDH gene on GenBank is NM_001256799.3.

[0014] Preferably, the probes are respectively labeled with fluorescent groups.

[0015] More preferably, the 5'-end of the probe is labeled with any one of the fluorescent groups FAM, VIC or HEX.

[0016] More preferably, the 5'-end of the probe for the AGPS gene, FAR1 gene, GNPAT gene and / or Peds1 gene is labeled with the FAM fluorescent group, and the 5'-end of the probe for the GAPDH gene is labeled with the VIC fluorescent group.

[0017] Preferably, the probes are respectively labeled with quenching groups.

[0018] Preferably, the 3'-end of the probe is labeled with any one of the quenching groups BHQ1, BHQ2 or Tamara.

[0019] More preferably, the 3'-end of the probe is labeled with the BHQ1 quenching group.

[0020] Use of the detection reagent in the preparation of a kit for evaluating the sensitivity of 5-fluorouracil in the treatment of gastric cancer.

[0021] A kit for evaluating the sensitivity of 5-fluorouracil in the treatment of gastric cancer, the kit containing the detection reagent.

[0022] Preferably, the kit further contains a primer set and a probe for amplifying an internal reference gene, the internal reference gene being the GAPDH gene, the nucleotide sequences of the primer set for amplifying the GAPDH gene being as shown in SEQ ID NO: 9 and SEQ ID NO: 10, the nucleotide sequence of the probe for detecting the GAPDH gene being as shown in SEQ ID NO: 15, and the accession number of the GAPDH gene on GenBank being NM_001256799.3.

[0023] More preferably, the kit evaluates the sensitivity of 5-fluorouracil in the treatment of gastric cancer through a comprehensive evaluation value, and the calculation formula of the comprehensive evaluation value is Logit(P) = -4.971 - 2.878×ΔCt (Ct value of AGPS - Ct value of internal reference) - 6.24×ΔCt (Ct value of FAR1 - Ct value of internal reference) - 0.73×ΔCt (Ct value of GNPAT - Ct value of internal reference) - 9.024×ΔCt (Ct value of Peds1 - Ct value of internal reference),

[0024] The Ct value of AGPS is the Ct value of the PCR fluorescence signal of the AGPS gene, the Ct value of FAR1 is the Ct value of the PCR fluorescence signal of the FAR1 gene, the Ct value of GNPAT is the Ct value of the PCR fluorescence signal of the GNPAT gene, the Ct value of Peds1 is the Ct value of the PCR fluorescence signal of the Peds1 gene, the Ct value of the internal reference is the Ct value of the PCR fluorescence signal of the GAPDH gene, and the Logit(P) is the comprehensive evaluation value;

[0025] A comprehensive evaluation value less than 0.5 indicates that gastric cancer is insensitive to 5-fluorouracil treatment, and a comprehensive evaluation value greater than or equal to 0.5 indicates that gastric cancer is sensitive to 5-fluorouracil treatment.

[0026] More preferably, the probes are respectively labeled with fluorescent groups.

[0027] More preferably, the 5'-end of the probe is labeled with any one of FAM, VIC or HEX fluorescent groups.

[0028] More preferably, the 5'-end of the probe for the AGPS gene, FAR1 gene, GNPAT gene and / or Peds1 gene is labeled with a FAM fluorescent group, and the 5'-end of the probe for the GAPDH gene is labeled with a VIC fluorescent group.

[0029] Preferably, the 3'-end of the probe is labeled with any one of BHQ1, BHQ2 or Tamara quenching groups.

[0030] More preferably, the 3'-end of the probe is labeled with a BHQ1 quenching group.

[0031] Preferably, the kit further contains an enzyme mixture, and the enzyme mixture contains reverse transcriptase, RNase inhibitor, hot start Taq enzyme and dNTPs.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The present invention discloses a kit for evaluating the sensitivity of 5-FU in the treatment of gastric cancer. The kit evaluates the sensitivity of gastric cancer patients to 5-fluorouracil (5-FU) drugs by detecting the expression levels of AGPS, FAR1, GNPAT and Peds1 gene mRNAs in tumor tissue samples of gastric cancer patients, and determines whether the patients are suitable for 5-FU drug treatment. The present invention designs primer sets and probes for amplifying the AGPS, FAR1, GNPAT and Peds1 genes, uses the GAPDH gene as an internal reference gene, detects the sample to be tested by fluorescence PCR, establishes a comprehensive evaluation model, determines the regression coefficients of Fatty Acyl-CoA Reductase 1 and CAD genes, and uses a comprehensive evaluation value of 0.5 as a positive judgment value. This kit is easy to operate, has a short detection time, strong specificity, high sensitivity, and low price, and is used to guide clinicians in the use of 5-fluorouracil. Description of the Drawings

[0034] Figure 1It is the amplification curve graph of the negative control product. Among them, A is the curve of the FAR1 gene and the internal reference gene, B is the curve of the AGPS gene and the internal reference gene, C is the curve of the GNPAT gene and the internal reference gene, and D is the curve of the Peds1 gene and the internal reference gene.

[0035] Figure 2 It is the amplification curve graph of the positive control product. Among them, A is the curve of the AGPS gene and the internal reference gene, B is the curve of the FAR1 gene and the internal reference gene, C is the curve of the GNPAT gene and the internal reference gene, and D is the curve of the Peds1 gene and the internal reference gene. Specific implementation manners

[0036] The present invention will be further elaborated in detail below in conjunction with the accompanying drawings of the specification and specific embodiments. The embodiments are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following embodiments are all conventional methods unless otherwise specified; the materials, reagents, etc. used are all reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0037] Example 1 Primer and probe design

[0038] There are many genes with abnormal expression in gastric cancer tissues. For example, the MUC5AC gene is one of the mucin genes that make up the gastric mucosa and is down-regulated in gastric cancer and precancerous lesions; the abnormality of the FHIT transcript is a common and tumor-specific change in gastric cancer, and the abnormality of the FHIT gene is related to the occurrence of some gastric cancers in China. There are abnormal amplifications of the c-met gene and overexpressions of c-met and PCNA proteins in gastric cancer tissues. Compared with normal gastric tissues, the expression rates of PDCD5 protein and mRNA are decreased in gastric cancer tissues, indicating that the pro-apoptotic effect caused by the abnormal expression of the PDCD5 gene is of great significance in the process of tumor formation. PDCD5 is a positive regulator of apoptosis. The decrease in the expression rate of PDCD5 in tissues has a certain warning effect on the early diagnosis of gastric cancer. Survivin is not expressed in normal gastric mucosa tissues, is lowly expressed in adjacent tissues, and is strongly positively expressed in gastric cancer tissues, indicating that the inhibition of apoptosis caused by the abnormal expression of the Survivin gene plays a certain role in the occurrence of gastric cancer.

[0039] According to the nucleotide sequences of the AGPS gene (NM_003659.4), FAR1 gene (NM_032228.6), GNPAT gene (NM_001316350), Peds1 gene (NM_001162505), and GAPDH gene (NM_001256799.3) registered in GenBank, specific primers and probes were designed, and their specificities were identified by the BLAST function of NCBI. The GAPDH gene is the internal reference gene, and the primer and probe sequences of each gene are shown in Table 1.

[0040] Table 1 Primer and Probe Sequence Table for AGPS, FAR1, GNPAT, and Peds1 Genes

[0041]

[0042]

[0043] Example 3 Reaction System Research

[0044] I. Test Scheme

[0045] The composition of the real-time fluorescence PCR reaction system is as follows: the final reaction concentration of reverse transcriptase is 2.0 U, the final reaction concentration of RNase inhibitor is 0.5 U, the final reaction concentration of hot-start Taq enzyme is 2.0 U, the final reaction concentration of dNTPs is 0.4 mM, and the solvent is water.

[0046] The reaction conditions are: 50°C for 10 min; 95°C for 2 min; 95°C for 15 s, 58°C for 35 s, for 45 cycles.

[0047] According to the above basic reaction and amplification conditions, using their respective positive nucleic acids as templates, the primer concentration range (0.05 - 0.5 μM), probe concentration range (0.05 - 0.3 μM), and annealing temperature (55°C - 60°C) were optimized respectively.

[0048] II. Experimental Results

[0049] Using positive nucleic acid as a template for fluorescence quantitative RT-PCR amplification, the best primer and probe concentrations were optimized and screened. The final optimal reaction system was determined as follows: the primer and probe concentrations were respectively: for PCR reaction solution A, the upstream and downstream of AGPS and GAPDH genes were each 0.2 μM, and the probe concentration of AGPS and GAPDH genes was 0.1 μmol / L; for PCR reaction solution B, the upstream and downstream of FAR1 and GAPDH genes were each 0.2 μM, and the probe concentration of FAR1 and GAPDH genes was each 0.1 μmol / L; for PCR reaction solution C, the upstream and downstream of GNPAT1 and GAPDH genes were each 0.2 μM, and the probe concentration of GNPAT and GAPDH genes was each 0.1 μmol / L; for PCR reaction solution D, the upstream and downstream of Peds1 and GAPDH genes were each 0.2 μM, and the probe concentration of Peds1 and GAPDH genes was each 0.1 μmol / L; 5 μL of template, and finally made up to 25 μL with H20.

[0050] The best amplification conditions were determined as: 50°C for 10 min; 95°C for 2 min; 95°C for 15 s, 58°C for 45 s, for 45 cycles.

[0051] Example 3 A Kit for Evaluating the Sensitivity of 5-FU in Treating Gastric Cancer

[0052] I. Composition

[0053] One tube each of PCR reaction solutions A, B, C, and D. Each tube is composed of primers, probes, and PCR buffer.

[0054] The primers in PCR reaction solution A are the upstream primer of the AGPS gene, the downstream primer of the AGPS gene, the upstream primer of the GAPDH gene, and the downstream primer of the GAPDH gene in Table 1 of Example 1; the probes are the AGPS gene probe and the GAPDH gene probe in Table 1 of Example 1. The final concentration of the primers in the amplification system is 0.2 μmol / L, the final concentration of the probes in the amplification system is 0.1 μmol / L, and the final concentration of the PCR buffer is 1×.

[0055] The primers in PCR reaction solution B are the upstream primer of the FAR1 gene, the downstream primer of the FAR1 gene, the upstream primer of the GAPDH gene, and the downstream primer of the GAPDH gene in Table 1 of Example 1; the probes are the FAR1 gene probe and the GAPDH gene probe in Table 1 of Example 1. The final concentration of the primers in the amplification system is 0.2 μmol / L, the final concentration of the probes in the amplification system is 0.1 μmol / L, and the final concentration of the PCR buffer is 1×.

[0056] The primers in PCR reaction solution C are the upstream primer of the GNPAT gene, the downstream primer of the GNPAT gene, the upstream primer of the GAPDH gene, and the downstream primer of the GAPDH gene in Table 1 of Example 1; the probes are the GNPAT gene probe and the GAPDH gene probe in Table 1 of Example 1. The final concentration of the primers in the amplification system is 0.2 μmol / L, the final concentration of the probes in the amplification system is 0.1 μmol / L, and the final concentration of the PCR buffer is 1×.

[0057] The primers in PCR reaction solution D are the upstream primer of the Peds1 gene, the downstream primer of the Peds1 gene, the upstream primer of the GAPDH gene, and the downstream primer of the GAPDH gene in Table 1 of Example 1; the probes are the Peds1 gene probe and the GAPDH gene probe in Table 1 of Example 1. The final concentration of the primers in the amplification system is 0.2 μmol / L, the final concentration of the probes in the amplification system is 0.1 μmol / L, and the final concentration of the PCR buffer is 1×.

[0058] The GAPDH gene is an internal reference gene.

[0059] One tube of enzyme mixture, which is composed of reverse transcriptase, RNase inhibitor, hot-start Taq enzyme, and dNTPs. Among them, the final reaction concentration of reverse transcriptase is 2.0 U, the final reaction concentration of RNase inhibitor is 0.5 U, the final reaction concentration of hot-start Taq enzyme is 2.0 U, the final reaction concentration of dNTPs is 0.4 mM, and the solvent is water.

[0060] The present invention determines the regression coefficients of AGPS, FAR1, GNPAT, and Peds1 genes by establishing a comprehensive evaluation model, and uses a comprehensive evaluation value of 0.5 as the positive judgment value.

[0061] One tube of positive control product: It is the RNA of gastric cancer tissue sample with a comprehensive evaluation value (P value) less than 0.5.

[0062] One tube of negative control product: It is the RNA of gastric cancer tissue sample with a comprehensive evaluation value (P value) greater than 0.5.

[0063] The calculation method of the P value is shown in the usage method of this embodiment.

[0064] II. Usage method

[0065] 1. Prepare reagents: Take 4 PCR reaction tubes, label the reaction tubes as A, B, C, and D respectively. Add 18 μL of PCR reaction solution A and 2 μL of enzyme mixture to tube A, add 18 μL of PCR reaction solution B and 2 μL of enzyme mixture to tube B, add 18 μL of PCR reaction solution C and 2 μL of enzyme mixture to tube C, and add 18 μL of PCR reaction solution D and 2 μL of enzyme mixture to tube D. After thoroughly mixing the reagents in the above reaction tubes, set aside for later use.

[0066] 2. Add samples: Add 5 μL of negative control product, 5 μL of positive control product, and 5 μL of RNA of the sample to be tested (gastric cancer tissue) to each PCR reaction tube, and tighten the tube caps.

[0067] 3. PCR detection: Place the prepared PCR reaction tubes in a fluorescence quantitative PCR instrument, and set the reaction program as shown in Table 2.

[0068] Table 2 PCR reaction program

[0069]

[0070] Note: * indicates collecting fluorescence signals

[0071] 4. Result analysis

[0072] Open the Amplification Plot window under Results. Select the location of the target sample to be analyzed. Change the Baseline value to start: 3, stop: 10, and open the manual setting Threshold: 2 - 6e+3. Double-click the value on the Rn coordinate to open the Graph settings window, change Log to Linear in the Post Run Settings, click OK, then open the Analysis preferences window, and select Analyze in the Analysis menu to automatically analyze the results.

[0073] Save the Ct values of the fluorescence signals of each measured gene as a data analysis file.

[0074] In the present invention, regression coefficients of AGPS, FAR1, GNPAT, and Peds1 genes are determined by establishing a comprehensive evaluation model, and a comprehensive evaluation value of 0.5 is used as a positive judgment value.

[0075] The ΔCt value of each target gene = Ct value of the target gene - Ct value of the internal reference gene.

[0076] Comprehensive evaluation value (P), that is, Logit(P) = -4.971 - 2.878×ΔCt (Ct value of AGPS - Ct value of the internal reference) - 6.24×ΔCt (Ct value of FAR1 - Ct value of the internal reference) - 0.73×ΔCt (Ct value of GNPAT - Ct value of the internal reference) - 9.024×ΔCt (Ct value of Peds1 - Ct value of the internal reference).

[0077] III. Result Interpretation

[0078] As Figure 1 shown in A, B, C, and D of Figure 2 the amplification curves of the FAM and VIC channels of the negative control product in the 4 PCR reaction tubes are all obvious S-shaped curves, and the calculated comprehensive evaluation value (P value) of the negative control product is less than 0.5; as

[0079] shown in A, B, C, and D of

[0080] the amplification curves of the FAM and VIC channels of the positive control product in the 4 PCR reaction tubes are all obvious S-shaped curves, and the calculated comprehensive evaluation value (P value) of the positive control product is greater than 0.5.

[0081] Example 4 Detection of Samples of Gastric Cancer Insensitive to 5-FU Drug Treatment

[0082] I. Experimental Method

[0083] Select 10 gastric cancer tissue samples that are insensitive to 5-FU treatment clinically, extract RNA from all samples as the test samples. Use the kit of Example 2 to detect the test samples, and at the same time use negative and positive control products for quality control.

[0084] II. Experimental Results

[0085] The amplification curves of the FAM and VIC channels of the negative control product are both obvious S-shaped curves, and the comprehensive evaluation value (P value) of the negative control product is less than 0.5; the amplification curves of the FAM and VIC channels of the positive control product are both obvious S-shaped curves, and the comprehensive evaluation value (P value) of the positive control product is greater than 0.5. Both the negative and positive control products meet the quality control requirements of the kit, so the test results of the specimens to be tested are valid.

[0086] From the test results of 10 samples to be tested, it can be seen that the comprehensive evaluation values (P values) of all gastric cancer tissue samples insensitive to 5-FU treatment are less than 0.5. The detailed results are shown in Table 3.

[0087] Table 3 Detection of gastric cancer tissue samples insensitive to 5-FU treatment

[0088]

[0089]

[0090] The results show that the comprehensive evaluation values (P values) of 10 gastric cancer tissues insensitive to 5-FU treatment are all less than 0.5, and the test results are completely consistent with the clinical results, indicating that the kit of Example 2 of the present invention has good specificity.

[0091] Example 5 Detection of gastric cancer samples sensitive to 5-FU treatment

[0092] I. Experimental method

[0093] Select 10 gastric cancer tissue samples clinically sensitive to 5-FU treatment, extract RNA from all samples as samples to be tested. Use the kit of Example 2 to detect the samples to be tested, and at the same time use negative and positive control products for quality control.

[0094] II. Experimental results

[0095] Both the negative and positive control products meet the quality control requirements of the kit of Example 2, so the test results of the specimens to be tested are valid. The comprehensive evaluation values (P values) of 10 gastric cancer positive tissue samples sensitive to 5-FU treatment are all greater than 0.5. The test results are shown in Table 4, and the test results are completely consistent with the clinical results, indicating that the kit of Example 2 has good specificity.

[0096] Table 4 Detection of gastric cancer tissue samples sensitive to 5-FU treatment

[0097]

[0098]

[0099] The results showed that the comprehensive evaluation values (P values) of 10 specimens sensitive to 5-FU treatment were all greater than 0.5, and the test results were completely consistent with the clinical results, indicating that the kit of Example 2 had good sensitivity.

[0100] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. What is described in the above embodiments and the specification only illustrates the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements. The scope of protection required by the present invention is defined by the appended claims, the specification and their equivalents.

Claims

1. A probe primer set for evaluating the sensitivity of 5-FU in treating gastric cancer, characterized in that, The probe primer set includes: combinations of upstream and downstream primers and probes for detecting the AGPS gene, FAR1 gene, GNPAT gene, and Peds1 gene respectively; The nucleotide sequences of the upstream and downstream primers for amplifying the AGPS gene are shown in SEQ ID NO: 1 and SEQ ID NO: 2, the nucleotide sequence of the probe for detecting the AGPS gene is shown in SEQ ID NO: 11, and the accession number of the AGPS gene on GenBank is NM_003659.4; The nucleotide sequences of the upstream and downstream primers for amplifying the FAR1 gene are shown in SEQ ID NO: 3 and SEQ ID NO: 4, the nucleotide sequence of the probe for detecting the FAR1 gene is shown in SEQ ID NO: 12, and the accession number of the FAR1 gene on GenBank is NM_032228.6; The nucleotide sequences of the upstream and downstream primers for amplifying the GNPAT gene are shown in SEQ ID NO: 5 and SEQ ID NO: 6, the nucleotide sequence of the probe for detecting the GNPAT gene is shown in SEQ ID NO: 13, and the accession number of the GNPAT gene on GenBank is NM_001316350; The nucleotide sequences of the upstream and downstream primers for amplifying the Peds1 gene are shown in SEQ ID NO: 7 and SEQ ID NO: 8, the nucleotide sequence of the probe for detecting the Peds1 gene is shown in SEQ ID NO: 14, and the accession number of the Peds1 gene on GenBank is NM_001162505.

2. The probe primer set according to claim 1, wherein The detection reagent also contains a primer set and a probe for amplifying the GAPDH gene. The nucleotide sequences of the primer set for amplifying the GAPDH gene are shown in SEQ ID NO: 9 and SEQ ID NO: 10, the nucleotide sequence of the probe for detecting the GAPDH gene is shown in SEQ ID NO: 15, and the accession number of the GAPDH gene on GenBank is NM_001256799.

3.

3. The probe primer set according to claim 1 or 2, characterized in that, The 3' end of the probe is labeled with a quenching group, and the 5' end of the probe is labeled with a fluorescent group.

4. Use of reagents for detecting AGPS gene, FAR1 gene, GNPAT gene and Peds1 gene in the preparation of a kit for evaluating the sensitivity of 5-fluorouracil in the treatment of gastric cancer, characterized in that, The reagent for detecting the AGPS gene, FAR1 gene, GNPAT gene, and Peds1 gene contains the probe primer set described in claim 1.

5. A kit for evaluating the sensitivity of gastric cancer to 5-FU, characterized in that, The kit contains the probe primer set described in claim 1.

6. The kit according to claim 5, characterized in that, The kit also contains a primer set and a probe for amplifying an internal reference gene. The internal reference gene is the GAPDH gene. The nucleotide sequences of the primer set for amplifying the GAPDH gene are shown in SEQ ID NO: 9 and SEQ ID NO: 10, the nucleotide sequence of the probe for detecting the GAPDH gene is shown in SEQ ID NO: 15, and the accession number of the GAPDH gene on GenBank is NM_001256799.

3.

7. The kit according to claim 6, wherein, The kit evaluates the sensitivity of 5-fluorouracil in the treatment of gastric cancer through a comprehensive evaluation value. The formula for the comprehensive evaluation value is Logit(P) = -4.971 - 2.878×ΔCt (Ct value AGPS - Ct value 内参 ) - 6.24×ΔCt (Ct value FAR1 - Ct value 内参 ) - 0.73×ΔCt (Ct value GNPAT- Ct value 内参 ) - 9.024×ΔCt (Ct value Peds1 - Ct value 内参 ); The Ct value AGPS is the Ct value of the PCR fluorescence signal of the AGPS gene, and the Ct value FAR1 is the Ct value of the PCR fluorescence signal of the FAR1 gene, and the Ct value GNPAT is the Ct value of the PCR fluorescence signal of the GNPAT gene, and the Ct value Peds1 is the Ct value of the PCR fluorescence signal of the Peds1 gene, and the Ct value 内参 is the Ct value of the PCR fluorescence signal of the GAPDH gene, and the Logit(P) is the comprehensive evaluation value; A comprehensive evaluation value less than 0.5 indicates that gastric cancer is insensitive to 5-fluorouracil treatment, and a comprehensive evaluation value greater than or equal to 0.5 indicates that gastric cancer is sensitive to 5-fluorouracil treatment.

8. Use of the kit according to any one of claims 5-7 in the preparation of a kit for evaluating the sensitivity of 5-fluorouracil in the treatment of gastric cancer.

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