Kit for detecting drug sensitivity of pancreatic cancer cells based on gene variation analysis

Abstract

The invention provides a kit for detecting pancreatic cancer cell drug sensitivity based on gene variation analysis. Whole genome sequencing analysis finds a gene variation site rs1562535735 which can be potentially used as pancreatic cancer cell drug sensitivity, survival curve analysis is carried out according to the site, it is found that the gene variation site is closely related to pancreatic cancer, the gene variation site can be used for prognosis analysis of patients, and clinical application is facilitated. And reference is provided for accurate treatment and prognosis diagnosis of pancreatic cancer patients.

Description

Technical Field

[0001] The present invention relates to the technical field of pancreatic cancer auxiliary diagnosis, with an international classification number of G01N, and in particular to a kit for detecting drug sensitivity of pancreatic cancer cells based on gene variation analysis. Background Art

[0002] Pancreatic cancer is a malignant tumor that originates in the pancreatic ductal epithelium and acinar cells, known in the medical community as the "king of cancers." While its cause remains unclear, studies suggest that factors such as long-term smoking, poor diet, elevated body mass index, and chronic pancreatic damage may increase the risk of pancreatic cancer. Symptoms of pancreatic cancer typically develop in the late stages of the disease and include jaundice, weight loss, and abdominal discomfort or pain.

[0003] Pancreatic cancer is a highly malignant tumor that is difficult to treat and has limited chemotherapy effectiveness. Due to the large individual differences among patients and their individual conditions, each patient's response to chemotherapy drugs varies, with some patients exhibiting resistance to the drugs. Therefore, screening patients for drug sensitivity is necessary.

[0004] A Chinese invention with publication number CN104111254A and classification number G01N21 / 78 discloses a tumor chemotherapy drug sensitivity detection kit, which records that the kit includes tissue cell dispase, antibiotic solution, amphotericin solution, EGTA-Trypsin solution, serum-containing culture medium DF10, serum-free culture medium Kertin-SFM, specimen preservation solution, specimen cleaning solution, gel-coated culture flask, cell fixative, cell dispase, cell filter membrane, neutral red staining solution and collagen gel preparation reagent.

[0005] A Chinese invention with publication number CN110196327A and classification number G01N33 / 574 discloses a kit for detecting drug sensitivity of rectal cancer cells based on gene mutation analysis. The kit is described as consisting of the following substances: (1) a rectal cancer cell drug reagent; (2) an indicator solution A containing magnetic nanoparticles surface-modified with antibodies to wild-type P53 protein labeled with green fluorescent protein; (3) an indicator solution B containing magnetic nanoparticles surface-modified with antibodies to anti-mutated P53 protein labeled with red fluorescent protein; and (4) a substrate culture medium and a dilution buffer.

[0006] The Chinese invention with publication number CN101329342A and classification number G01N33 / 569 discloses a method and a kit for detecting tuberculosis bacteria and anti-tuberculosis drug sensitivity; the method for detecting tuberculosis bacteria provided therein includes the preparation of antibodies, antigens, and enzyme-labeled antibodies, and the detection of the presence of phages by combining phage amplification and enzyme-linked immunosorbent assay technology.

[0007] Currently, there is no reliable gene mutation site that can serve as a diagnostic marker for pancreatic cancer drug sensitivity. Summary of the Invention

[0008] To address the technical issues existing in the prior art, the present invention provides a kit for detecting drug sensitivity in pancreatic cancer cells based on gene mutation analysis. This invention uses whole-genome sequencing to identify gene mutation sites that potentially indicate drug sensitivity in pancreatic cancer cells. Survival curve analysis based on these sites reveals a strong correlation between these gene mutation sites and pancreatic cancer. This can be used for patient prognosis analysis, providing a reference for precise treatment and prognostic diagnosis of pancreatic cancer patients.

[0009] One of the objectives of the present invention is to provide the use of rs1562535735 as a diagnostic marker in the preparation of a reagent for auxiliary diagnosis and / or evaluation of drug sensitivity of pancreatic cancer cells. The nucleotide sequence of rs1562535735 is shown in SEQ ID No. 1.

[0010] Preferably, the genotype of rs1562535735 is detected to assist in the diagnosis and / or assessment of drug sensitivity of pancreatic cancer cells.

[0011] Preferably, the drug is lapatinib.

[0012] Preferably, when the genotype is TT or TC, the patient is sensitive to lapatinib treatment.

[0013] Preferably, when the genotype is CC, the patient is insensitive to lapatinib treatment.

[0014] One of the purposes of the present invention is to provide a kit for auxiliary diagnosis and / or evaluation of drug sensitivity of pancreatic cancer cells, wherein the kit contains primers for detecting the rs1562535735 genotype.

[0015] Preferably, the primers for specifically amplifying rs1562535735 are: the upstream primer sequence is SEQ ID No. 2, and the downstream primer sequence is SEQ ID No. 3.

[0016] Further preferably, the test sample in the kit is blood.

[0017] One of the purposes of the present invention is to use rs1562535735 in preparing a kit for auxiliary diagnosis and / or evaluation of drug sensitivity of pancreatic cancer cells.

[0018] The advantages of the present invention are as follows: The present invention discovered the gene variation site rs1562535735, which has the potential to be used as a drug sensitivity for pancreatic cancer cells, through whole genome sequencing analysis, and performed survival curve analysis based on this site. It was found that this gene variation site is closely associated with pancreatic cancer, which can be used for patient prognosis analysis and provide a reference for the precise treatment and prognostic diagnosis of pancreatic cancer patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 .Prognostic analysis. DETAILED DESCRIPTION

[0020] The present invention will be further described in detail below with reference to specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0021] The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Based on the specific embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work are within the scope of protection of the present invention.

[0022] In the examples of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the examples of the present invention, unless otherwise specified, the technical means used are conventional means well known to those skilled in the art.

[0023] Example 1 Screening of drug sensitivity gene mutation sites in pancreatic cancer cells 1. Preparation of experimental materials: All trial subjects were patients diagnosed with pancreatic cancer by clinicians from January 2022 to December 2022. Among them, 20 patients were sensitive to lapatinib treatment and 20 patients were insensitive to lapatinib treatment. Blood samples of the above patients were collected for future use.

[0024] 2. Test methods 1. Genomic DNA was extracted from the patient's blood sample using a blood genomic DNA extraction kit (DP348, purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.). For detailed procedures, please refer to the kit instructions.

[0025] 2. A DNA sequencing library was prepared using the second-generation sequencing DNA rapid library construction kit (WE0229, purchased from Beijing Biolab Technology Co., Ltd.), and then subjected to Illumina sequencing analysis.

[0026] 3. GATK was used to analyze the targeted sequence capture data of each sample to obtain SNP sites, and annovar was used to annotate the corresponding sites. GenABEL and R packages were used to evaluate the SNPs and the sensitivity of pancreatic cancer patients to lapatinib treatment, and potential gene mutation sites sensitive to lapatinib treatment were screened based on the ranking.

[0027] According to the statistical results (Table 1-2), the rs1562535735 locus ranks first. Different genetic mutations exist in pancreatic cancer patients. Among them, the genotype in patients who are sensitive to lapatinib treatment is TT or TC, and the genotype in patients who are insensitive to lapatinib treatment is CC. Based on the genotype, it can be determined that the detection sensitivity of this locus can reach 100%, which has good diagnostic value and can be used to determine the drug sensitivity of pancreatic cancer.

[0028] Table 1 Analysis of gene locus variation ranking

[0029] Table 2 Genotype analysis of patients at rs1562535735

[0030] Example 2 Analysis of rs1562535735 locus and pancreatic cancer prognosis 1. Preparation of experimental materials: All trial subjects were patients diagnosed with pancreatic cancer by clinicians between April 2023 and August 2023. They were all treated with lapatinib, and blood samples were collected from the above patients for future use.

[0031] 2. Test methods 1. Use the blood genomic DNA extraction kit (DP348) to extract genomic DNA from the patient's blood sample. For detailed procedures, please refer to the kit instructions.

[0032] 2. Design PCR primers based on the upstream and downstream sequences of rs1562535735. The specific primer sequences are as follows: Forward primer 5'-AATATAAAAATCCAGCCCAGTTCATGAC-3' (SEQ ID NO. 2), Reverse primer 5'-TTGAGAATAACACGGCCTTTTGAC-3' (SEQ ID NO. 3) The 20 μL PCR reaction system included: 10 μL 10× PCR Buffer, 1 μL SYBR Green I fluorescent dye, 1 μL dNTP, 1 μL each of upstream and downstream primers, 2 μL Taq DNA polymerase, and 4 μL DNA. The PCR reaction procedure was: 94°C pre-denaturation for 5 min; 94°C for 45 s, 57°C for 60 s, and 72°C extension for 45 s, for a total of 30 cycles; 72°C extension for 5 min; and storage at 4°C.

[0033] 3. The PCR amplification products were sequenced by Illumina to analyze the gene variation site information, and GenABEL and R packages were used to evaluate the correlation between the gene variation site and the prognosis of pancreatic cancer patients.

[0034] According to the Kaplan-Meier survival curve Figure 1 The results showed that patients with genotype TT or TC had a good prognosis, while patients with genotype CC had a poor prognosis (P<0.05), further confirming that the gene mutation sites described in the present invention can be used to detect pancreatic cancer drug sensitivity and its prognostic analysis.

[0035] The above embodiments are intended to illustrate the essential content of the present invention, but are not intended to limit the scope of protection of the present invention. Those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the essence and scope of protection of the technical solutions of the present invention.

Claims

1. A kit for assisting in the diagnosis and / or evaluation of drug sensitivity of pancreatic cancer cells, characterized in that: The kit contains primers for detecting the rs1562535735 genotype.

2. The kit according to claim 1, wherein The upstream primer sequence is SEQ ID No. 2, and the downstream primer sequence is SEQ ID No.

3.

3. The kit according to claim 1, wherein The test sample in the kit is blood.

4. Use of rs1562535735 as a diagnostic marker in the preparation of a reagent for auxiliary diagnosis and / or assessment of drug sensitivity of pancreatic cancer cells.

5. The use according to claim 4, characterized in that The nucleotide sequence of rs1562535735 is shown in SEQ ID No.

1.

6. The use according to claim 4, characterized in that The rs1562535735 genotype can be detected to assist in the diagnosis and / or assessment of pancreatic cancer cell drug sensitivity.

7. The use according to claim 6, characterized in that The pancreatic cancer cell drug is lapatinib.

8. The use according to claim 6, characterized in that When the genotype is TT or TC, it is sensitive to lapatinib treatment.

9. The use according to claim 6, characterized in that When the genotype is CC, it is insensitive to lapatinib treatment.

10. Use of rs1562535735 in the preparation of a kit for auxiliary diagnosis and / or assessment of drug sensitivity of pancreatic cancer cells.

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