CN116377060A - Probe primer group for evaluating drug resistance of gastric cancer and/or colorectal cancer to 5-fluorouracil and kit thereof - Google Patents

Abstract

The invention discloses a probe primer group and a kit for evaluating drug resistance of gastric cancer and/or colorectal cancer to 5-fluorouracil. The kit comprises primer groups and probes for detecting Col10a1, DDR2 and ACTB genes, wherein the ACTB genes are taken as reference genes, fluorescent PCR is used for detecting the expression conditions of mRNA of the Col10a1 genes, the DDR1 genes and the DDR2 genes of a sample to be detected, a comprehensive evaluation model is established, regression coefficients of the Col10a1, the DDR1 and the DDR2 genes are determined, and a comprehensive evaluation value of-0.85 is taken as a positive judgment value to judge whether a patient is suitable for 5-fluorouracil drug treatment. The kit has the advantages of strong specificity, high sensitivity, simple and convenient operation, short detection time, high-flux detection realization and low price.

Description

Probe primer set for evaluating gastric cancer and or colorectal cancer resistance to 5-fluorouracil and its kit

technical field

The invention relates to the technical field of molecular detection, in particular to a probe primer set and kit for evaluating gastric cancer and/or colorectal cancer resistance to 5-fluorouracil.

Background technique

Gastric cancer and colorectal cancer are the most common malignant tumors of the digestive tract with a high incidence worldwide, and their morbidity and mortality are increasing year by year. At present, gastric cancer and colorectal cancer in my country present the characteristics of high incidence and mortality risks. Due to the lack of large-scale screening, the early diagnosis rate of gastric cancer and colorectal cancer in my country is relatively low, and many patients are diagnosed as advanced when they are discovered. . Most patients with advanced gastric cancer and colorectal cancer have missed the best treatment period. Although there are various treatment methods such as surgery, radiotherapy and chemotherapy, and biological targeted therapy, their 5-year survival rate is still very low.

5-Fluorouracil (5-FU) belongs to anti-metabolism drugs. It can not only affect the replication of DNA through the action of intracellular enzymes, but also can be converted into fluorouracil nucleoside and incorporated into cellular RNA to interfere with protein synthesis, thereby exerting cytotoxicity and inhibiting Tumor cell function is often used as a standard first-line drug in the treatment of gastric cancer and colorectal cancer, but in recent years, it is easy to cause chemotherapy resistance or even multi-drug resistance, which has seriously affected the efficacy of gastric cancer and colorectal cancer chemotherapy.

Chemotherapy resistance is mainly caused by drug abuse, gene expression heterogeneity and instability of tumor cells, etc., which can lead to tumor chemotherapy failure and tumor recurrence. Compared with continuously upgrading chemotherapy regimens, it is necessary to find a suitable drug route and formulate a personalized regimen. Treatment options have important clinical implications. At present, many genes and proteins are abnormally expressed in gastric cancer tissue and colorectal cancer tissue, but not every gene is related to chemotherapy resistance, and it is not clear which gene can overcome 5-FU resistance, thereby Treat stomach and colorectal cancer.

Contents of the invention

In view of this, in order to overcome the deficiencies in the prior art, the technical problem solved by the present invention is to provide a probe primer set and kit for evaluating gastric cancer and or colorectal cancer resistance to 5-fluorouracil .

In order to achieve the above object, the present invention is achieved through the following schemes:

A probe primer set for evaluating the resistance of gastric cancer and or colorectal cancer to 5-fluorouracil, the probe primer set includes: a probe for respectively detecting one or more of the Col10a1 gene, DDR1 gene or DDR2 gene and the above Combination of downstream primers;

The accession number of the Col10a1 gene on GenBank is XM_011535432.4, the nucleotide sequence of the probe for detecting the Col10a1 gene is shown in SEQ ID NO: 9, and the nucleosides of the upstream and downstream primers for amplifying the Col10a1 gene The acid sequence is shown in SEQ ID NO: 1 and SEQ ID NO: 2;

The accession number of the DDR1 gene on GenBank is NM_001202521.1, the nucleotide sequence of the probe for detecting the DDR1 gene is shown in SEQ ID NO: 10, and the nucleosides of the upstream and downstream primers of the amplified DDR1 gene The acid sequence is shown in SEQ ID NO: 3 and SEQ ID NO: 4;

The accession number of the DDR2 gene on GenBank is NM_001014796.3, the nucleotide sequence of the probe for detecting the DDR2 gene is shown in SEQ ID NO: 11, and the nucleosides of the upstream and downstream primers of the amplified DDR2 gene The acid sequences are shown in SEQ ID NO:5 and SEQ ID NO:6.

Preferably, the probe primer set includes: a combination of the probe for detecting the Col10a1 gene, the DDR1 gene and the DDR2 gene and upstream and downstream primers.

Preferably, the probe primer set also contains a probe and a primer set for amplifying the ACTB gene, the accession number of the ACTB gene on GenBank is NM_001101.5, and the nucleotide sequence of the probe for detecting the ACTB gene As shown in SEQ ID NO: 12, the nucleotide sequences of the primer set for amplifying the ACTB gene are shown in SEQ ID NO: 7 and SEQ ID NO: 8.

Preferably, the 3' end of the probe is labeled with a quencher group, and the 5' end of the probe is labeled with a fluorescent group.

More preferably, the 5' end of the probe is labeled with any one of FAM, VIC, Texas Red or Cy5 fluorophores.

More preferably, the 5' end of the Col10a1 gene is labeled with a FAM fluorophore, the 5' end of the DDR1 gene is labeled with a VIC fluorophore, the 5' end of the DDR2 gene is labeled with a Texas Red fluorophore, and the ACTB The 5' end of the gene is labeled with a Cy5 fluorophore.

More preferably, the 3' end of the probe is labeled with any of BHQ1, BHQ2 or Tamara quenching groups.

More preferably, the 3' end of the col10a1 gene, DDR1 gene and/or DDR2 gene probe is labeled with a BHQ1 quenching group, and the 3' end of the ACTB gene probe is labeled with a BHQ2 quenching group.

The probe primer set is used to prepare a kit for evaluating the drug resistance of gastric cancer and/or colorectal cancer to 5-fluorouracil.

The application of reagents for detecting Col10a1 gene, DDR1 gene and/or DDR2 gene in the preparation of a kit for evaluating gastric cancer and/or colorectal cancer resistance to 5-fluorouracil, the accession number of the Col10a1 gene on GenBank is XM_011535432. 4. The accession number of the DDR1 gene on GenBank is NM_001202521.1, and the accession number of the DDR2 gene on GenBank is NM_001014796.3.

Preferably, the reagent for detecting the Col10a1 gene, the DDR1 gene and/or the DDR2 gene comprises the probe primer set.

A kit for evaluating the resistance of gastric cancer and/or colorectal cancer to 5-fluorouracil, the kit contains reagents for detecting Col10a1 gene, DDR1 gene and/or DDR2 gene.

Preferably, the kit contains reagents for detecting Col10a1 gene, DDR1 gene and DDR2 gene.

More preferably, the reagent is a probe primer set: the accession number of the Col10a1 gene on GenBank is XM_011535432.4, and the nucleotide sequence of the probe for detecting the Col10a1 gene is shown in SEQ ID NO: 9, The nucleotide sequences of the upstream and downstream primers for amplifying the Col10a1 gene are shown in SEQ ID NO: 1 and SEQ ID NO: 2;

The accession number of the DDR1 gene on GenBank is NM_001202521.1, the nucleotide sequence of the probe for detecting the DDR1 gene is shown in SEQ ID NO: 10, and the nucleosides of the upstream and downstream primers of the amplified DDR1 gene The acid sequence is shown in SEQ ID NO: 3 and SEQ ID NO: 4;

The accession number of the DDR2 gene on GenBank is NM_001014796.3, the nucleotide sequence of the probe for detecting the DDR2 gene is shown in SEQ ID NO: 11, and the nucleosides of the upstream and downstream primers of the amplified DDR2 gene The acid sequences are shown in SEQ ID NO:5 and SEQ ID NO:6.

More preferably, the kit also contains a probe and a primer set for amplifying the ACTB gene, the accession number of the ACTB gene on GenBank is NM_001101.5, and the nucleotide sequence of the probe for detecting the ACTB gene As shown in SEQ ID NO: 12, the nucleotide sequences of the primer set for amplifying the ACTB gene are shown in SEQ ID NO: 7 and SEQ ID NO: 8.

Preferably, the 3' end of the probe is labeled with a quencher group, and the 5' end of the probe is labeled with a fluorescent group.

More preferably, the 5' end of the probe is labeled with any one of FAM, VIC, Texas Red or Cy5 fluorophores.

More preferably, the 5' end of the Col10a1 gene is labeled with a FAM fluorophore, the 5' end of the DDR1 gene is labeled with a VIC fluorophore, the 5' end of the DDR2 gene is labeled with a Texas Red fluorophore, and the ACTB The 5' end of the gene is labeled with a Cy5 fluorophore.

More preferably, the 3' end of the probe is labeled with any of BHQ1, BHQ2 or Tamara quenching groups.

More preferably, the 3' end of the col10a1 gene, DDR1 gene and/or DDR2 gene probe is labeled with a BHQ1 quenching group, and the 3' end of the ACTB gene probe is labeled with a BHQ2 quenching group.

Preferably, the kit also includes PCR buffer, dNTPs, hot start Taq enzyme, RNase inhibitor, reverse transcriptase, negative quality control and/or positive quality control.

Preferably, the kit is based on the comprehensive evaluation value of gastric cancer and/or colorectal cancer resistance to 5-fluorouracil, and the calculation formula of the comprehensive evaluation value is Logit (P value) = 5.148-0.065 × ΔCt (Ct value Col10a1- Ct value ACTB)-2.249×ΔCt(Ct value DDR1-Ct value ACTB)-1.341×ΔCt(Ct value DDR2-Ct value ACTB),

Described Ct value Col10a1 is the PCR fluorescence signal Ct value of col10a1 gene, and described Ct value DDR1 is the PCR fluorescence signal Ct value of DDR1 gene, and described Ct value DDR2 is the PCR fluorescence signal Ct value of DDR2 gene, and described Ct value ACTB Be the PCR fluorescent signal Ct value of ACTB gene, and described Logit (P) is comprehensive evaluation value;

A comprehensive evaluation value greater than -0.85 indicates that gastric cancer and/or colorectal cancer is resistant to 5-fluorouracil treatment, which is judged as negative, and a comprehensive evaluation value less than or equal to -0.85 indicates that gastric cancer and/or colorectal cancer is resistant to 5-fluorouracil treatment. Drug resistance was judged as positive.

Preferably, the positive quality control product is a gastric cancer and/or colorectal cancer tissue sample with a comprehensive evaluation value less than -0.85, and the negative quality control product is a gastric cancer and/or colorectal cancer tissue sample with a comprehensive evaluation value greater than -0.85 .

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

The invention discloses a kit for evaluating drug resistance of gastric cancer and/or colorectal cancer to 5-fluorouracil. The kit contains primer sets and probes for detecting col10a1, DDR1, DDR2, and ACTB genes, uses ACTB gene as an internal reference gene, and uses fluorescent PCR to detect the expression of col10a1 gene, DDR1 gene, and DDR2 gene mRNA in the sample to be tested, and establishes a comprehensive Evaluate the model, determine the regression coefficient of Col10a1, DDR1 and DDR2 genes, and use the comprehensive evaluation value -0.85 as the positive judgment value to judge whether the patient is suitable for 5-fluorouracil drug treatment, so as to formulate a personalized treatment plan. The expression of ACTB gene mRNA is relatively stable in various tissues and cells, and it is used as a reference for gene detection to correct loading errors and operational errors and improve the accuracy of results. The kit has strong specificity, high sensitivity, simple operation, short detection time, can realize high-throughput detection, and is low in price.

Description of drawings

Figure 1 is the amplification curve of the negative quality control.

Figure 2 is the amplification curve of the positive quality control.

Detailed ways

The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, which are only used to explain the present invention, and are not intended to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available reagents and materials unless otherwise specified.

Example 1 Design of primers and probes for detection of Col10a1, DDR1, DDR2 and ACTB genes

In GenBank, the nucleotide sequences of Col10a1 (XM_011535432.4) gene, DDR1 (NM_001202521.1) gene, DDR2 (NM_001014796.3) gene and internal reference gene ACTB (NM_001101.5) were determined. Nucleotide sequence, respectively design specific primers and probes, use the BLAST function of NCBI to identify the specificity of primers and probes.

Specifically, the upstream primer of the col10a1 gene is: 5'-gctaagggtgaaaggggttc-3' (SEQ ID NO: 1);

The downstream primer of col10a1 gene is: 5'-gtcctccaactccaggatca-3' (SEQ ID NO: 2);

DDR1 gene upstream primer is: 5'-atcagctacccaatgctgct-3' (SEQ ID NO: 3);

DDR1 gene downstream primer is: 5'-gccctgcacacggtaatagt-3' (SEQ ID NO: 4);

DDR2 gene upstream primer is: 5'-cgccacagcataagcataga-3' (SEQ ID NO: 5);

DDR2 gene downstream primer is: 5'taaagctcccaaaccaatgc-3' (SEQ ID NO: 6);

The upstream primer of ACTB gene is: 5'-agagctacgagctgcctgac-3' (SEQ ID NO: 7);

The downstream primer of ACTB gene is: 5'-agcactgtgttggcgtacag-3' (SEQ ID NO: 8);

The col10a1 gene probe is: 5'-FAM-ccagggtacccaggaaaaccagg-BHQ1-3' (SEQ ID NO: 9);

The DDR1 gene probe is: 5'-VIC-ccagggtacccaggaaaaccagg-BHQ1-3' (SEQ ID NO: 10);

The DDR2 gene probe is: 5'-Texas Red-ccacaggctccattttgcaggtc-BHQ1-3' (SEQ ID NO: 11);

The ACTB gene probe is: 5'-Cy5-ttccgctgccctgaggcact-BHQ2-3' (SEQ ID NO: 12).

Example 2 A kit for evaluating the drug resistance of gastric cancer and colorectal cancer to 5-fluorouracil

1. The composition of the kit

1. 1 tube of FCPCR reaction solution, consisting of primers, probes and PCR buffer. The final concentration of the primer amplification system is 0.2-0.5 μmol/L, and the final concentration of the probe amplification system is 0.1-0.3 μmol/L. The final concentration of PCR buffer is 1×.

The final concentrations of primers SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 are 0.3 μmol/L, and the final concentrations of primers SEQ ID NO: 5 and SEQ ID NO: 6 The final concentration of primers SEQ ID NO: 7 and SEQ ID NO: 8 is 0.2 μmol/L, and the probes are SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12 The final concentrations were 0.2μmol/L, 0.3μmol/L, 0.1μmol/L and 0.2μmol/L, respectively.

The primers are the upstream and downstream primers of the col10a1 gene, DDR1 gene, DDR2 gene and ACTB gene described in Example 1, and their nucleotide sequences are shown in SEQ ID NO: 1-8.

The probes are the probes of the col10a1 gene, DDR1 gene, DDR2 gene and ACTB gene described in Example 1, and their nucleotide sequences are shown in SEQ ID NO: 9-12.

The ACTB gene is an internal reference gene.

2. 1 tube of FC enzyme mixture, consisting of dNTPs, hot start Taq enzyme, RNase inhibitor and reverse transcriptase.

The final reaction concentration of dNTPs was 10 mM, the hot start Taq enzyme was 2.0 U, the RNase inhibitor was 0.5 U, the reverse transcriptase was 2.0 U, and the solvent was water.

3. Negative quality control 1 tube: RNA from gastric cancer or colorectal cancer tissue samples with comprehensive evaluation value (P value) greater than -0.85.

4. Positive quality control 1 tube: RNA from gastric cancer or colorectal cancer tissue samples with comprehensive evaluation value (P value) less than -0.85.

For the calculation method of P value, see the method used in this example. The regression coefficients of col10a1 gene, DDR1 gene and DDR2 gene were determined by establishing a comprehensive evaluation model, and the comprehensive evaluation value -0.85 was used as the positive judgment value.

2. How to use the kit

1. Prepare reagents: Take 18 μL of the FCPCR reaction solution of this example (containing 5 μL of 5×PCR buffer, 0.75 μL each of primers SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, primers 1.25 μL each of SEQ ID NO: 5 and SEQ ID NO: 6, 0.5 μL each of primers SEQ ID NO: 7 and SEQ ID NO: 8, 0.5 μL each of probes SEQ ID NO: 9 and SEQ ID NO: 12, NO: 10 0.75μL, SEQ ID NO: 11 0.25μL, and finally make up to 18μL with nuclease-free water), FC enzyme mixture 2μL (containing 25mM dNTPs 0.4μL, 5U/μL hot-start Taq enzyme 0.4μL, 1U/μL 0.5 μL of RNase inhibitor and 0.4 μL of 5U/μL reverse transcriptase, make up to 2 μL with water), mix well and set aside.

2. Adding samples: Take 5 μL each of the RNA of the sample to be tested (stomach cancer tissue or colorectal cancer tissue), the positive quality control substance, and the negative quality control substance, add it to the PCR reaction tube, and tightly cap the tube.

3. PCR detection: Use a fluorescent quantitative PCR instrument to detect the PCR reaction tube after adding the above sample, and set the reaction program:

50°C, 10min; 95°C, 2min; 95°C, 15s, 58°C*, 35s, 45 cycles. Where * indicates the collection of fluorescent signals.

4. Result analysis

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

Save the measured fluorescent signal Ct value of each gene as a data analysis file.

The present invention determines the regression coefficients of the col10a1 gene, the DDR1 gene and the DDR2 gene by establishing a comprehensive evaluation model, and adopts a comprehensive evaluation value of -0.85 as a positive judgment value.

ΔCt value of each target gene = Ct value target gene - Ct value internal reference gene.

Comprehensive evaluation value (P), that is, Logit (P value) = 5.148-0.065×ΔCt (Ct value Col10a1-Ct value internal reference)-2.249×ΔCt (Ct value DDR1-Ct value internal reference)-1.341×ΔCt (Ct value DDR2- Ct value internal reference).

3. Interpretation of results

As shown in A, B, C and D in Figure 1, the amplification curves of FAM, VIC, Texas Red, and CY5 channels of negative quality control products are all obvious S-shaped curves, and the comprehensive evaluation value of negative quality control products (P value) greater than -0.85.

As shown in A, B, C and D in Figure 2, the amplification curves of the FAM, VIC, Texas Red, and CY5 channels of the positive quality control are all obvious S-shaped curves, and the comprehensive evaluation value of the positive quality control ( P value) is less than -0.85;

When the comprehensive evaluation value (P value) of the sample to be tested is less than or equal to -0.85, it shows that the sample to be tested has low drug resistance to 5-fluorouracil, indicating that the sample patient has high sensitivity to 5-FU drug treatment of gastric cancer, and the judgment is positive.

When the comprehensive evaluation value (P value) of the sample to be tested is greater than -0.85, it indicates that the sample to be tested is highly resistant to 5-fluorouracil drugs, indicating that the sample patient has low sensitivity to 5-fluorouracil drugs in the treatment of gastric cancer, and it is judged as negative .

Example 3 Detection of Gastric Cancer Samples Without Drug Resistance to 5-Fluorouracil Treatment

1. Experimental method

The RNA of 15 cases of gastric cancer tissue samples that were not resistant to 5-fluorouracil therapy were extracted as samples to be tested. Use the kit of Example 2 to detect the samples to be tested, and use positive and negative quality controls for quality control.

2. Experimental results

Both negative and positive quality control products meet the quality control requirements of the kit, so the test results of the samples to be tested are valid. The comprehensive evaluation values (P value) of 15 cases of gastric cancer positive tissue samples without drug resistance to 5-fluorouracil treatment are all less than -0.85, and its test results are shown in Table 1. It is feasible for the kit to detect gastric cancer samples that are not resistant to 5-fluorouracil therapy and has good detection performance.

Table 1 Detection of gastric cancer tissue samples without resistance to 5-fluorouracil therapy

Example 4 Detection of colorectal cancer samples without drug resistance to 5-fluorouracil treatment

1. Experimental method

The RNA of 15 cases of colorectal cancer tissue samples that were not resistant to 5-fluorouracil therapy were extracted as samples to be tested. Use the kit of Example 2 to detect the samples to be tested, and use positive and negative quality controls for quality control.

2. Experimental results

Both negative and positive quality control products meet the quality control requirements of the kit, so the test results of the samples to be tested are valid. The comprehensive evaluation value (P value) of 15 cases of positive tissue samples of colorectal cancer with no resistance to 5-fluorouracil treatment was less than -0.85, and the test results were shown in Table 2. The test results were completely consistent with the clinical results, indicating that the use of The kit in Example 2 is feasible to detect colorectal cancer samples that are not resistant to 5-fluorouracil treatment, and has good detection performance.

Table 2 Detection of colorectal cancer tissue samples without resistance to 5-fluorouracil therapy

Example 5 Detection of gastric cancer samples resistant to 5-fluorouracil treatment

1. Experimental method

The RNA of 10 cases of gastric cancer tissue samples resistant to 5-fluorouracil therapy were extracted as samples to be tested. Use the kit of Example 2 to detect the samples to be tested, and use positive and negative quality controls for quality control.

2. Experimental results

Both negative and positive quality control products meet the quality control requirements of the kit, so the test results of the samples to be tested are valid. The comprehensive evaluation value (P value) of 10 cases to the gastric cancer positive tissue sample of 5-fluorouracil treatment drug resistance is all greater than-0.85, and its detection result is as shown in table 3, and detection result is completely consistent with clinical result, illustrates that utilizes embodiment 2 It is feasible for the kit to detect gastric cancer samples that are resistant to 5-fluorouracil therapy and has good detection performance.

Table 3 Detection of gastric cancer tissue samples resistant to 5-fluorouracil therapy

sample number Sample classification sample type Comprehensive evaluation value (P value) Test results 1 Resistant to 5-fluorouracil therapy stomach cancer 42.02 Negative 2 Resistant to 5-fluorouracil therapy stomach cancer 24.41 Negative 3 Resistant to 5-fluorouracil therapy stomach cancer 30.82 Negative 4 Resistant to 5-fluorouracil therapy stomach cancer 7.26 Negative 5 Resistant to 5-fluorouracil therapy stomach cancer 1.75 Negative 6 Resistant to 5-fluorouracil therapy stomach cancer 22.05 Negative 7 Resistant to 5-fluorouracil therapy stomach cancer 41.8 Negative 8 Resistant to 5-fluorouracil therapy stomach cancer 34.54 Negative 9 Resistant to 5-fluorouracil therapy stomach cancer 1.15 Negative 10 Resistant to 5-fluorouracil therapy stomach cancer 6.93 Negative

Example 6 Detection of colorectal cancer samples resistant to 5-fluorouracil treatment

1. Experimental method

The RNA of 10 cases of colorectal cancer tissue samples resistant to 5-fluorouracil therapy were extracted as samples to be tested. Use the kit of Example 2 to detect the samples to be tested, and use positive and negative quality controls for quality control.

2. Experimental results

Both negative and positive quality control products meet the quality control requirements of the kit, so the test results of the samples to be tested are valid. The comprehensive evaluation value (P value) of 10 cases to the colorectal cancer positive tissue sample of 5-fluorouracil treatment drug resistance is all greater than-0.85, and its detection result is as shown in table 4, and detection result is in full agreement with clinical result, illustrates that utilize embodiment The kit of 2 is feasible for the detection of colorectal cancer samples resistant to 5-fluorouracil therapy, and has good detection performance.

Table 4 Detection of colorectal cancer tissue samples resistant to 5-fluorouracil therapy

sample number Sample classification sample type Comprehensive evaluation value (P value) Test results 1 Resistant to 5-fluorouracil therapy colorectal cancer 44.83 Negative 2 Resistant to 5-fluorouracil therapy colorectal cancer 46.8 Negative 3 Resistant to 5-fluorouracil therapy colorectal cancer 14.66 Negative 4 Resistant to 5-fluorouracil therapy colorectal cancer 22.4 Negative 5 Resistant to 5-fluorouracil therapy colorectal cancer 4.72 Negative 6 Resistant to 5-fluorouracil therapy colorectal cancer 5.14 Negative 7 Resistant to 5-fluorouracil therapy colorectal cancer 32.52 Negative 8 Resistant to 5-fluorouracil therapy colorectal cancer 9.53 Negative 9 Resistant to 5-fluorouracil therapy colorectal cancer 35.56 Negative 10 Resistant to 5-fluorouracil therapy colorectal cancer 25.2 Negative

Example 7 Kit Sensitivity Verification

1. Experimental method

One case of gastric cancer tissue sample with no resistance to 5-fluorouracil treatment and RNA from one case of gastric cancer tissue sample with no resistance to 5-fluorouracil treatment were selected as samples to be tested, RNA was extracted, and then quantified by Qubit3.0 fluorescence The concentration of RNA was determined by the instrument, and then the RNA was diluted to 100ng/μL, 50ng/μL, 25ng/μL and 10ng/μL as the sample to be tested to verify the sensitivity of the kit. The kit in Example 2 was used to detect the sample to be tested, and Positive and negative quality controls were used for quality control.

2. Experimental results

Both negative and positive quality control products meet the quality control requirements of the kit, so the test results of the samples to be tested are valid. The comprehensive evaluation values (P values) of the four gradient samples were all less than -0.85, and the test results were all positive, indicating that the sensitivity of this kit can reach 10 μg/μL. The test results are shown in Table 5.

Table 5 Sensitivity sample detection situation

Example 8 Kit Specificity Verification

1. Experimental method

Specific samples of pancreatic cancer, lung cancer, liver cancer, and polyps were selected as samples to be tested, RNA was extracted, and the samples to be tested were detected using the kit in Example 2, and quality control was performed with positive and negative quality controls.

2. Experimental results

Both negative and positive quality control products meet the quality control requirements of the kit, so the test results of the samples to be tested are valid. The comprehensive evaluation values (P value) of pancreatic cancer samples, lung cancer samples, liver cancer samples, polyp samples, and hemorrhoid samples were all greater than -0.85, and the test results were all negative, indicating that this kit has good specificity. Pancreatic cancer samples, lung cancer samples Samples, liver cancer samples, and polyp samples will not affect the test results of the kit, and the test results are shown in Table 6.

Table 6 Specific sample detection results

sample number Sample classification Comprehensive evaluation value (P value) Test results 1 pancreatic cancer 25.24 Negative 2 lung cancer 12.25 Negative 3 liver cancer 19.32 Negative 4 polyp 2.38 Negative 5 hemorrhoid 22.21 Negative

The basic principles, main features and advantages of the present invention have been shown and described above. Those skilled in the art should understand that the present invention is not limited by the above-mentioned embodiments, and that described in the above-mentioned embodiments and the specification only illustrates the principles of the present invention, and the present invention will also have other functions without departing from the spirit and scope of the present invention. For various changes and improvements, the protection scope of the present invention is defined by the appended claims, description and their equivalents.

Claims (10)

1. A probe primer set for evaluating gastric cancer and/or colorectal cancer drug resistance to 5-fluorouracil, characterized in that, the probe primer set comprises: respectively detecting one or A combination of several probes and upstream and downstream primers; The accession number of the Col10a1 gene on GenBank is XM_011535432.4, the nucleotide sequence of the probe for detecting the Col10a1 gene is shown in SEQ ID NO: 9, and the nucleosides of the upstream and downstream primers for amplifying the Col10a1 gene The acid sequence is shown in SEQ ID NO: 1 and SEQ ID NO: 2; The accession number of the DDR1 gene on GenBank is NM_001202521.1, the nucleotide sequence of the probe for detecting the DDR1 gene is shown in SEQ ID NO: 10, and the nucleosides of the upstream and downstream primers of the amplified DDR1 gene The acid sequence is shown in SEQ ID NO: 3 and SEQ ID NO: 4; The accession number of the DDR2 gene on GenBank is NM_001014796.3, the nucleotide sequence of the probe for detecting the DDR2 gene is shown in SEQ ID NO: 11, and the nucleosides of the upstream and downstream primers of the amplified DDR2 gene The acid sequences are shown in SEQ ID NO:5 and SEQ ID NO:6. 2. probe primer set according to claim 1, is characterized in that, described probe primer set comprises: the combination of the probe of detection Col10a1 gene, DDR1 gene and DDR2 gene described in claim 1 and upstream and downstream primer . 3. probe primer set according to claim 1, is characterized in that, described probe primer set also contains the probe and primer set of amplification ACTB gene, and the accession number of described ACTB gene on GenBank is NM_001101. 5. The nucleotide sequence of the probe for detecting the ACTB gene is shown in SEQ ID NO: 12, and the nucleotide sequence of the primer set for amplifying the ACTB gene is shown in SEQ ID NO: 7 and SEQ ID NO: 8 shown. 4. The probe primer set according to any one of claims 1 to 3, wherein the 3' end of the probe is marked with a quencher group, and the 5' end of the probe is marked with a fluorescent group. 5. A kit for preparing and evaluating the drug resistance of gastric cancer and/or colorectal cancer to 5-fluorouracil according to any one of claims 1 to 4. 6. Application of reagents for detecting Col10a1 gene, DDR1 gene and/or DDR2 gene in the preparation of a test kit for evaluating gastric cancer and/or colorectal cancer resistance to 5-fluorouracil, characterized in that the Col10a1 gene is on GenBank The accession number of the DDR1 gene is XM_011535432.4, the accession number of the DDR1 gene on GenBank is NM_001202521.1, and the accession number of the DDR2 gene on GenBank is NM_001014796.3. 7. A kit for evaluating the drug resistance of gastric cancer and/or colorectal cancer to 5-fluorouracil, characterized in that the kit contains reagents for detecting Col10a1 gene, DDR1 gene and/or DDR2 gene. 8. The kit according to claim 7, characterized in that the kit contains reagents for detecting the Col10a1 gene, the DDR1 gene and the DDR2 gene. 9. The kit according to claim 7 or 8, wherein the reagent is a probe primer set: the accession number of the Col10a1 gene on GenBank is XM_011535432.4, and the probe for detecting the Col10a1 gene The nucleotide sequence of the gene is shown in SEQ ID NO: 9, and the nucleotide sequences of the upstream and downstream primers for amplifying the Col10a1 gene are shown in SEQ ID NO: 1 and SEQ ID NO: 2; The accession number of the DDR1 gene on GenBank is NM_001202521.1, the nucleotide sequence of the probe for detecting the DDR1 gene is shown in SEQ ID NO: 10, and the nucleosides of the upstream and downstream primers of the amplified DDR1 gene The acid sequence is shown in SEQ ID NO: 3 and SEQ ID NO: 4; The accession number of the DDR2 gene on GenBank is NM_001014796.3, the nucleotide sequence of the probe for detecting the DDR2 gene is shown in SEQ ID NO: 11, and the nucleosides of the upstream and downstream primers of the amplified DDR2 gene The acid sequences are shown in SEQ ID NO:5 and SEQ ID NO:6. 10. test kit according to claim 9, is characterized in that, also contains the probe and the primer set of amplifying ACTB gene in the described test kit, and the accession number of described ACTB gene on GenBank is NM_001101.5, so The nucleotide sequence of the probe for detecting the ACTB gene is shown in SEQ ID NO: 12, and the nucleotide sequence of the primer set for amplifying the ACTB gene is shown in SEQ ID NO: 7 and SEQ ID NO: 8.

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