Digital PCR (Polymerase Chain Reaction) detection kit for gastric cancer

Abstract

The invention discloses a stomach cancer digital PCR detection kit, and belongs to the technical field of stomach cancer in-vitro detection. The invention prepares the stomach cancer digital PCR detection kit for simultaneously detecting related genes of pepsinogen I (PGI), pepsinogen II (PGII) and gastrin 17 (G-17), the kit simultaneously detects and analyzes the related genes of the stomach cancer through a digital PCR method, the detection result is accurate, the omission ratio of detection is reduced, the sensitivity, specificity and sensitivity are relatively high, and the kit can be used for detecting and analyzing the related genes of the stomach cancer. Meanwhile, the quantitative analysis effect is achieved, data support can be provided in the screening process or the subsequent treatment process, and potential clinical application value is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of in vitro detection of gastric cancer, and in particular to a digital PCR detection kit for gastric cancer. Background Art

[0002] Gastric cancer has always been one of the hot topics that people pay attention to. According to statistics, the incidence and mortality of gastric cancer are high worldwide, and early detection and treatment of gastric cancer are the key to improving the survival rate of gastric cancer patients.

[0003] Early screening for gastric cancer can be carried out through a series of examination methods, such as color Doppler ultrasound, biochemical immunoassays such as pepsinogen (PG) and gastrin 17, puncture biopsy, gastroscopy, gastric MRI and CT. In view of the fact that color Doppler ultrasound imaging, MRI, and CT technologies need to rely on large medical equipment, the high cost is not conducive to analysis and promotion; and puncture biopsy and gastroscopy technology will not only cause harm to the tester, but also require professional physicians to operate, which is not conducive to large-scale screening applications. Therefore, at this stage, gastric cancer screening mainly adopts the method of detecting pepsinogen (PG) and gastrin 17. The methods for detecting pepsinogen (PG) and gastrin 17 include biochemical reaction method, enzyme-linked immunosorbent assay, chemiluminescence method, and fluorescence analysis method. However, the above methods still have the following disadvantages: they are easily affected by nonspecific reactions, resulting in relatively low sensitivity and specificity, and need to rely on standard products and standard curves for quantification. Therefore, the development of new detection methods has become a top priority. Summary of the invention

[0004] In view of the above-mentioned prior art, the purpose of the present invention is to provide a digital PCR detection kit for gastric cancer.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] In a first aspect of the present invention, a primer-probe composition for detecting gastric cancer is provided, wherein the primer-probe composition comprises a primer pair and a probe, and the primer pair and the probe are as follows:

[0007] (1) The nucleotide sequences of the primer pair and probe designed for pepsinogen I are shown in SEQ ID NO: 1-SEQ ID NO: 2, the nucleotide sequence of the probe is shown in SEQ ID NO: 3-SEQ ID NO: 4, and the 5' end of the probe is connected to a FAM fluorescent group;

[0008] (2) The nucleotide sequences of the primer pair and probe designed for pepsinogen II are shown in SEQ ID NO:5-SEQ ID NO:6, the nucleotide sequence of the probe is shown in SEQ ID NO:7-SEQ ID NO:8, and the 5' end of the probe is connected to a HEX fluorescent group;

[0009] (3) The nucleotide sequences of the primer pair and probe designed for gastrin 17 are shown in SEQ ID NO:9-SEQ ID NO:10, and the nucleotide sequence of the probe is shown in SEQ ID NO:11-SEQ ID NO:12. The 5' end of the probe is connected to a ROX fluorescent group.

[0010] The second aspect of the present invention provides a digital PCR detection kit containing the primer-probe combination for detecting gastric cancer, wherein the digital PCR detection kit includes droplet generation oil, PCR premix and primer-probe solution.

[0011] Furthermore, in the droplet generating oil, the volume ratio of EM90 to mineral oil is 5:95 or 7:93.

[0012] Furthermore, in the PCR premix, the concentration of Taq DNA polymerase is 0.01U / μL-0.11U / μL, the concentration of dNTPs is 0.1mmol / L-0.6mmol / L, and the concentration of PCR buffer is 1×-3×.

[0013] Furthermore, the primer probe solution includes a PGI primer probe solution, a PGII primer probe solution and a G-17 primer probe solution.

[0014] Furthermore, in the primer-probe solution, the concentration of each primer or probe is 1 μmol / L-2 μmol / L.

[0015] Furthermore, the method for using the digital PCR detection kit includes the following steps:

[0016] (1) Preparation of digital PCR reaction system: The total volume of the digital PCR system reaction system is 50 μL, including 15 μL of PCR premix, 1 μL of PGI primer probe solution, 1 μL of PGII primer probe solution, 1 μL of G-17 primer probe solution, 5 μL of sample, and 27 μL of pure water;

[0017] (2) Digital PCR amplification: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 25 s, annealing at 56°C for 15 s, extension at 72°C for 25 s, 40 cycles; high temperature at 95°C for 10 min; cooling and holding at 4°C.

[0018] Beneficial effects of the present invention:

[0019] The present invention prepares a gastric cancer digital PCR detection kit for simultaneously detecting genes related to pepsinogen I (PGI), pepsinogen II (PGII) and gastrin 17 (G-17). The kit uses a digital PCR method to simultaneously detect and analyze genes related to gastric cancer, thereby solving the problem of a single detection index. Not only is the detection result accurate, but the missed detection rate is also reduced, and the kit has high sensitivity, specificity and sensitivity. At the same time, the effect of quantitative analysis is achieved. Data support can be provided both in screening and in subsequent treatment processes, thereby ensuring the sustainable applicability of the detection result, and having potential clinical application value. DETAILED DESCRIPTION

[0020] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0021] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0022] The test materials used in the examples of the present invention that are not specifically described are all conventional test materials in the art and can be purchased through commercial channels. The CAS number of the mineral oil used in the present invention is 8042-47-5, and the CAS number of the EM90 used in the present invention is 54392-26-6.

[0023] The primers and probes used in the present invention are as follows:

[0024] a) The primer sequences designed for the pepsinogen I (PGI) gene are as follows:

[0025] Forward sequence (SEQ ID NO: 1): 5'-CCTTGAAGCACACCATTACA-3',

[0026] Reverse sequence (SEQ ID NO: 2): 5′-TACCCACTGCATCAGGAACA-3′;

[0027] The probe is the following sequence labeled with FAM fluorescent group:

[0028] Forward sequence (SEQ ID NO: 3): 5'-GTCCAGCGTCCAGCACACAG-3',

[0029] Reverse sequence (SEQ ID NO:4): 5'-ACACAGGCCAGGTATTTCAG-3'.

[0030] b) The primer sequences designed for the pepsinogen II (PGII) gene are as follows:

[0031] Forward sequence (SEQ ID NO: 5): 5'-TGGTGTCCTTGATCCACTTCCG-3',

[0032] Reverse sequence (SEQ ID NO:6): 5′-GATTGTGGGAGGCTGGGAGTG-3′;

[0033] The probe is the following sequence labeled with a HEX fluorescent group:

[0034] Forward sequence (SEQ ID NO:7): 5'-GAATCACCCCCACAAGTGTC-3',

[0035] Reverse sequence (SEQ ID NO:8): 5'-ACACAGGCCAGAGGGTCCCT-3'.

[0036] c) The primer sequences designed for gastrin 17 (G-17) gene are as follows:

[0037] Forward sequence (SEQ ID NO:9): 5'-AACACCATCCCTCCTCGAACC-3',

[0038] Reverse sequence (SEQ ID NO: 10): 5′-CAGATATGTCATTCTGGGAGGTC-3′;

[0039] The probe is the following sequence labeled with ROX fluorescent group:

[0040] Forward sequence (SEQ ID NO: 11): 5'-CTGTGATACAGTGGATAGCCGCT-3',

[0041] Reverse sequence (SEQ ID NO: 12): 5'-CCTAACAAAAGAGCTGAAAAGCCC-3'.

[0042] Embodiment 1:

[0043] A gastric cancer digital PCR detection kit, comprising:

[0044] Microdroplet generation oil, PCR premix, primer probe solution. The primer probe solution includes PGI primer probe solution (hereinafter referred to as solution A), PGII primer probe solution (hereinafter referred to as solution B), G-17 primer probe solution (hereinafter referred to as solution C).

[0045] 1.1 Preparation of droplet-generating oil

[0046] EM90 and mineral oil were mixed at a volume ratio of 5:95 to obtain droplet-generating oil.

[0047] 1.2 Preparation of PCR premix

[0048] Each component raw material was diluted with purified water to obtain a PCR premix solution, the composition of which is shown in Table 1:

[0049] Table 1 Composition of PCR premix

[0050]

[0051]

[0052] 1.3 Preparation of primer-probe solution

[0053] (1) Prepare a 1 μmol / L solution of the designed and synthesized PGI primers and probes as solution A;

[0054] (2) preparing a 1 μmol / L solution of the designed and synthesized PGII primers and probe as solution B;

[0055] (3) Prepare a 1 μmol / L solution of the designed and synthesized G-17 primer and probe as solution C.

[0056] 1.4 Preparation of PCR amplification reagents

[0057] Prepare PCR amplification reagents according to Table 2, mix thoroughly and centrifuge for later use.

[0058] Table 2 PCR amplification reagent composition

[0059] Reagents Volume(uL) PCR master mix 15 Solution A 1 Solution B 1 Solution C 1 sample 5 Pure water 27

[0060] Example 2

[0061] A gastric cancer digital PCR detection kit, comprising:

[0062] Microdroplet generation oil, PCR premix, primer probe solution. The primer probe solution includes PGI primer probe solution (hereinafter referred to as solution A), PGII primer probe solution (hereinafter referred to as solution B), G-17 primer probe solution (hereinafter referred to as solution C).

[0063] 2.1 Preparation of droplet-generating oil:

[0064] EM90 and mineral oil were mixed at a volume ratio of 7:93 to obtain droplet-generating oil.

[0065] 2.2 Preparation of PCR premix

[0066] Each component raw material was diluted with purified water to obtain a PCR premix solution, the composition of which is shown in Table 3:

[0067] Table 3 PCR premix composition

[0068]

[0069]

[0070] 2.3 Preparation of primer-probe solution

[0071] (1) preparing a 2 μmol / L solution of the designed and synthesized PGO primers and probes as solution A;

[0072] (2) preparing a 2 μmol / L solution of the designed and synthesized PGII primers and probe as solution B;

[0073] (3) Prepare a 2 μmol / L solution of the designed and synthesized G-17 primer and probe as solution C.

[0074] 2.4 Preparation of PCR amplification reagents

[0075] Prepare PCR amplification reagents according to Table 3, mix thoroughly and centrifuge for later use.

[0076] Example 3

[0077] A gastric cancer digital PCR detection kit, comprising:

[0078] Microdroplet generation oil, PCR premix, primer probe solution. The primer probe solution includes PGI primer probe solution (hereinafter referred to as solution A), PGII primer probe solution (hereinafter referred to as solution B), G-17 primer probe solution (hereinafter referred to as solution C).

[0079] 3.1. Preparation of droplet-generating oil:

[0080] EM90 and mineral oil were mixed at a volume ratio of 5:95 to obtain droplet-generating oil.

[0081] 3.2. Preparation of PCR premix:

[0082] Each component raw material was diluted with purified water to obtain a PCR premix solution, the composition of which is shown in Table 4:

[0083] Table 4 PCR premix composition

[0084] Components Final concentration Taq DNA polymerase 0.05U / uL dNTPs 0.3mmoL / L PCR buffer 2×

[0085] 3.3 Preparation of primer probe solution:

[0086] (1) Prepare a 2 μmol / L solution of the designed and synthesized PGI primers and probes as solution A;

[0087] (2) preparing a 2 μmol / L solution of the designed and synthesized PGI primers and probe as solution B;

[0088] (3) Prepare a 2 μmol / L solution of the designed and synthesized G-17 primer and probe as solution C.

[0089] 3.4 Preparation of PCR amplification reagents

[0090] Prepare PCR amplification reagents according to Table 2, mix thoroughly and centrifuge for later use.

[0091] Test Example 1

[0092] The test kits prepared in Examples 1 to 3 were used to test blood samples of 50 patients with clinically confirmed gastric cancer and 60 random patients without gastric cancer, and the steps were as follows:

[0093] 1. Droplet Generation and Amplification

[0094] Use the digital PCR chip, add 80 μL of generated oil to the oil well, add 30 μL of PCR amplification reagent to the sample well, cover with a seal, put into the droplet generator, generate 50 μL of droplets, transfer to the PCR reaction tube for amplification. Put the PCR reaction tube on a common PCR instrument, set the amplification parameters according to Table 5, and run the program for amplification.

[0095] Table 5 PCR reaction program

[0096]

[0097] 2. Signal Detection

[0098] The amplified reaction tube was transferred to the digital PCR analysis system for signal detection. The fluorescence detection channel settings were: FAM was selected to detect the PGI gene, HEX was selected to detect the PGII gene, and G-17 was selected to detect the PSMA gene. The test results are shown in Tables 6 to 8:

[0099] Table 6 Comparison of Example 1 and clinical results

[0100]

[0101] Table 7 Comparison of Example 2 and Clinical Results

[0102]

[0103] Table 8 Comparison of Example 3 and Clinical Results

[0104]

[0105] The results in Tables 6 to 8 show that the positive and negative test results of the test kits prepared in Examples 1 to 3 are consistent with the results of clinical diagnosis of gastric cancer, and have high sensitivity and specificity.

[0106] Test Example 2

[0107] Using the kit prepared in Example 3, a blood sample with clinically confirmed gastric cancer was obtained, and the total RNA of the sample was extracted using a commercially available total RNA extraction and purification kit. The RNA was prepared to a concentration of 50,000 copies / mL and diluted step by step to a concentration gradient of 5,000 copies / mL, 500 copies / mL, 50 copies / mL, and 5 copies / mL. The cDNA was reverse transcribed and synthesized using a cDNA synthesis kit for detection.

[0108] According to the results of gradient dilution, the kit prepared in Example 3 detects samples with a concentration of 5 copies / mL to 50,000 copies / mL, and the correlation coefficient r=0.9999, indicating that the kit can detect samples with a concentration range of 5 copies / mL to 50,000 copies / mL when detecting clinical samples, and the results are accurate, and the sensitivity can reach the lower limit of 5 copies / mL.

[0109] The present invention constructs a droplet digital PCR reaction system to form a kit product, which mainly includes droplet generation oil, PCR premix, and primer probe solution. The present invention uses a digital PCR chip, adds droplet generation oil to the oil hole, adds amplification reagent to the sample hole, and after covering the sealing strip, uses a droplet generator to disperse the gene and amplification reaction solution of the sample into the droplet to form an oil-in-water structure, and then amplifies. The corresponding fluorescent color droplets are counted by a digital PCR instrument, and the number of genes tested can be absolutely quantified without a standard curve. The kit of the present invention is designed for pepsinogen I (PGI), pepsinogen II (PGII) and gastrin 17 (G-17) related genes. Primers and probes can be detected for three gene indicators at the same time, and gastric cancer is analyzed and screened comprehensively, and the screening rate of positive samples is improved, with high sensitivity and specificity. The test results of the kit of the present invention are accurate, and the detection sensitivity is high, and the lower limit of the concentration reaches 5 copies / mL. The present invention can easily realize the screening analysis of gastric cancer using blood samples, and has a high potential value for clinical detection of gastric cancer.

[0110] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A primer-probe combination for detecting gastric cancer, characterized in that: The primer-probe composition comprises a primer pair and a probe, wherein the primer pair and the probe are as follows: the nucleotide sequences of the primer pair and the probe designed for pepsinogen I are shown in SEQ ID NO:1-SEQ ID NO:4, and the 5' end of the probe is connected to a FAM fluorescent group; the nucleotide sequences of the primer pair and the probe designed for pepsinogen II are shown in SEQ ID NO:5-SEQ ID NO:8, and the 5' end of the probe is connected to a HEX fluorescent group; the nucleotide sequences of the primer pair and the probe designed for gastrin 17 are shown in SEQ ID NO:9-SEQ ID NO:12, and the 5' end of the probe is connected to a ROX fluorescent group.

2. A digital PCR detection kit comprising the primer-probe combination for detecting gastric cancer according to claim 1, characterized in that: The digital PCR detection kit comprises droplet generation oil, PCR premix and primer probe solution.

3. The digital PCR detection kit according to claim 2, characterized in that: In the droplet-generating oil, the volume ratio of EM90 to mineral oil is 5:95 or 7:

93.

4. The digital PCR detection kit according to claim 2, characterized in that: In the PCR premix, the concentration of Taq DNA polymerase is 0.01U / μL-0.11U / μL, the concentration of dNTPs is 0.1mmol / L-0.6mmol / L, and the concentration of PCR buffer is 1×-3×.

5. The digital PCR detection kit according to claim 2, characterized in that: The primer probe solution includes a PGI primer probe solution, a PGII primer probe solution and a G-17 primer probe solution.

6. The digital PCR detection kit according to claim 5, characterized in that: In the primer-probe solution, the concentration of each primer or probe is 1 μmol / L-2 μmol / L.

7. The digital PCR detection kit according to any one of claims 2 to 6, characterized in that: The method of using the digital PCR detection kit includes the following steps: (1) Preparation of digital PCR reaction system: The total volume of the digital PCR system reaction system is 50 μL, including 15 μL of PCR premix, 1 μL of PGI primer probe solution, 1 μL of PGII primer probe solution, 1 μL of G-17 primer probe solution, 5 μL of sample, and 27 μL of pure water; (2) Digital PCR amplification: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 25 s, annealing at 56°C for 15 s, extension at 72°C for 25 s, 40 cycles; high temperature at 95°C for 10 min; cooling and holding at 4°C.

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