Probe, primer group, kit and application

Abstract

The invention provides a probe, a primer group, a kit and application, the primer group and the probe comprise a probe T, a probe C, an upstream primer and a downstream primer, and the nucleotide sequence of the probe T is as shown in SEQ ID NO.1; the nucleotide sequence of the probe C is as shown in SEQ ID NO. 2; the nucleotide sequence of the upstream primer is as shown in SEQ ID NO.3; and the nucleotide sequence of the downstream primer is as shown in SEQ ID NO. 4. According to the invention, through the design of double probes, the yield of the FBXW7 gene chr4: 152326101 Cgt is effectively increased; and the technical reliability of T mutation detection is improved.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a probe, primer set, reagent kit, and its application. Background Technology

[0002] The FBXW7 gene is located at 4q31.3, spanning 215.5 kb on chromosome 4. It contains 20 exons (GeneID: 55294) and 21 transcripts, 15 of which encode proteins (http: / / asia.ensembl.org / Homo_sapiens / Gene / Summary?db=core;g=ENSG00000109670;r=4:152320544-152536092). The full-length FBXW7 protein contains 707 amino acid residues and plays a crucial role in protein ubiquitination and proteasome-mediated degradation. It is involved in regulating the degradation of various oncoproteins and is a tumor suppressor. Inactivation / loss of FBXW7 protein is a significant cause of tumor development and progression. Studies have confirmed that FBXW7 gene heterogeneity, such as downregulation of expression, affects tumor differentiation, invasion, and metastasis; furthermore, it exhibits a high mutation frequency in various human cancers. However, how to efficiently detect the heterogeneity characteristics of the FBXW7 gene, including mutations, in tumors and its related clinical significance remains an important scientific problem to be solved. Summary of the Invention

[0003] In view of this, the present invention provides a probe, primer set, kit and application. The dual probe design effectively improves the technical reliability of detecting the FBXW7 gene chr4:152326101 C>T mutation.

[0004] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a primer set and probe comprising probe T, probe C, an upstream primer and a downstream primer, wherein the nucleotide sequence of probe T is shown in SEQ ID NO.1; the nucleotide sequence of probe C is shown in SEQ ID NO.2; the nucleotide sequence of the upstream primer is shown in SEQ ID NO.3; and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.4.

[0005] Specifically, the sequence SEQ ID NO.1: 5'-FAM-AGGAGGGTTGTTAGTAGA-MGB-3'; the sequence SEQ ID NO.2: 5'-VIC-AGGAGGGTTGTTAGTGGA-MGB-3'; the sequence SEQ ID NO.3: 5'-CAGCAGTCCGCTGTGTTCAA-3'; the sequence SEQ ID NO.4: 5'-TCCCACACCTTTACCATAAAATCA-3'.

[0006] In the above technical solution, the probe pair for detecting the human FBXW7 gene chr4:152326101 C>T mutation consists of probe C and probe T, wherein probe T is used to detect the variant FBXW7 gene fragment; probe C is used to detect the wild-type FBXW7 gene fragment.

[0007] Preferably, the nucleotide sequence of the probe T has a FAM label at the 5' end and an MGB label at the 3' end.

[0008] Preferably, the nucleotide sequence of probe C has a VIC tag at the 5' end and an MGB tag at the 3' end.

[0009] Secondly, the present invention provides the application of the primer set and probe described above in the preparation of a preparation for detecting the mutant FBXW7 gene.

[0010] Preferably, the mutated FBXW7 gene is the FBXW7 gene chr4:152326101 C>T mutation.

[0011] Thirdly, the present invention provides a kit comprising the aforementioned primer set and probe.

[0012] Preferably, it also includes PCR amplification reagents, positive controls, negative controls, and standards.

[0013] Preferably, the PCR amplification reagents include Taq deoxyribonucleic acid polymerase, deoxyribonucleic acid triphosphate, magnesium chloride, buffer solution, and double-distilled water.

[0014] In the above technical solution, the probe T positive control is a DNA sequence containing the target fragment of the coding region of the human FBXW7 gene and having a C→T mutation (single nucleotide T) at the chr4:152326101 site of the FBXW7 gene; the probe C positive control is a DNA sequence containing the same target fragment as the probe T positive control, but without a C→T mutation (single nucleotide C) at the chr4:152326101 site of the FBXW7 gene; the double positive control consists of probe T positive control and probe C positive control in the same proportion; the negative control is double-distilled water.

[0015] The standards are in vitro recombinant plasmids containing a partial DNA sequence of FBXW7, including probe T-positive standards, probe C-positive standards, and double-positive standards. The probe T-positive standard is a recombinant of plasmid pcDNA3.0 containing a DNA sequence with a C→T mutation (single nucleotide T) at the FBXW7 gene chr4:152326101 site. The probe C-positive standard is a recombinant of plasmid pcDNA3.0 containing a DNA sequence without a C→T mutation (single nucleotide C) at the FBXW7 gene chr4:152326101 site. The double-positive standards consist of probe T-positive standards and probe C-positive standards in equal proportions.

[0016] Specifically, the nucleotide sequence of the probe T positive control is shown in SEQ ID NO.5: 5'-CATGTTGCAG CAGTC CGCTG TGTTC AATAT GATGG CAGGA GGGTT GTTAG TAGAG CATAT GATTT TATGGTAAAG GTGTG GGAT-3'; the nucleotide sequence of the probe C positive control is shown in SEQ ID NO.6: 5'-CATGTTGCAG CAGTC CGCTG TGTTC AATAT GATGG CAGGA GGGTT GTTAG TGGAG CATAT GATTT TATGGTAAAG GTGTG GGAT-3'; the FBXW7 nucleotide sequence (circular double-stranded DNA) of the probe T positive standard is shown in SEQ ID NO.7: pcDNA3-TTAAA AATTC TAAAC GTGGG TTTTT TTGTT TTGTT TTGTT TTTCT GTTTCTCCCT CTGCA GAGTT GTTAG CGGTT CTCGA GATGC CACTC TTAGG GTTTG GGATA TTGAG ACAGGCCAGT GTTTA CATGT TTTGA TGGGT CATGT TGCAG CAGTC CGCTG TGTTC AATAT GATGG CAGGAGGGTT GTTAG TAGAG CATAT GATTT TATGG TAAAG GTGTG GGATC CAGAG ACTGA AACCT GTCTACACAC-pcDNA3; The FBXW7 nucleotide sequence of the probe C positive standard is shown in SEQ ID NO.8: pcDNA3-TTAAA AATTC TAAAC GTGGG TTTTT TTGTT TTGTT TTGTT TTTCT GTTTC TCCCT CTGCA GAGTTGTTAG CGGTT CTCGA GATGC CACTC TTAGG GTTTG GGATA TTGAG ACAGG CCAGT GTTTA CATGTTTTGA TGGGT CATGT TGCAG CAGTC CGCTG TGTTC AATAT GATGG CAGGA GGGTT GTTAG TGGAGCATAT GATTT TATGG TAAAG GTGTG GGATC CAGAG ACTGA AACCT GTCTA CACAC-pcDNA3.

[0017] Fourthly, the present invention provides the application of the kit described herein in the preparation of a kit for detecting the mutant FBXW7 gene.

[0018] It should be noted that the DNA sample to be tested by the kit is derived from human tumor tissue. The specific extraction method is as follows: take an appropriate amount of tumor tissue, separate the cells using Proteinase K, and extract the DNA from the cells using the phenol-chloroform extraction method as the DNA to be tested.

[0019] Fifthly, the present invention provides the application of the kit described herein in the detection of aflatoxin-associated liver cancer.

[0020] In the above technical solution, the present invention also provides performance verification (including its sensitivity, specificity and accuracy), clarifying the technical reliability of detecting the chr4:152326101 C>T mutation in the FBXW7 gene; it also analyzes the sensitivity and specificity of the detection technology for the molecular diagnosis of aflatoxin-related liver cancer by detecting the chr4:152326101 C>T mutation in the FBXW7 gene in patients with early aflatoxin-related liver cancer and patients with liver cancer without aflatoxin exposure, thereby confirming that the probe pairs, primers and kits provided by the present invention can be applied to the molecular subtyping diagnosis of aflatoxin-related liver cancer.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The present invention adopts a dual probe design, which effectively improves the technical reliability of the detection of FBXW7 gene chr4:152326101 C>T mutation.

[0022] (2) This invention can simultaneously evaluate three cases: wild-type homozygotes, mutant homozygotes, and heterozygotes, thus improving the application value of molecular typing diagnosis.

[0023] (3) This invention establishes the association between the FBXW7 gene chr4:152326101 C>T mutation and the gene heterogeneity characteristics of aflatoxin-related liver cancer, thereby enabling early molecular subtyping diagnosis of aflatoxin-related liver cancer. Attached Figure Description

[0024] Figure 1 This is a sensitivity analysis chromatogram of the reagent kit provided in Example 3 of the present invention; Figure 2 This is a gel electrophoresis image of the amplification product provided in Example 4 of the present invention; Figure 3 The image shows the detection results of the FBXW7 gene chr4:152326101 C>T mutation provided in Example 5 of this invention.

[0025] Note: In Figure 1 In the diagram, A represents the detection rate of positive samples for probe T; B represents the detection rate of positive samples for probe C; and C represents the detection rate of positive samples for both probes T and C. Figure 3 In the diagram, Figure A shows a positive result for probe T, Figure B shows a positive result for probe C, Figure C shows a double positive result for both probe T and probe C, and Figure D shows a negative result. Detailed Implementation

[0026] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention.

[0027] Example 1 This embodiment provides a specific method for "probe and primer design": After sequencing the human FBXW7 gene, probe sequences were designed based on its nucleotide sequence and specific detection site (chr4:152326101C>T) as follows: 5'-FAM-AGGAGGGTTGTTAGTAGA-MGB-3' and 5'-VIC-AGGAGGGTTGTTAGTGGA-MGB-3', named "Probe T" and "Probe C", respectively, to detect variant and wild-type FBXW7 gene fragments.

[0028] The corresponding PCR amplification primers for the probe pairs were designed as follows: Upstream primer: 5'-CAGCAGTCCGCTGTGTTCAA-3' Downstream primer: 5'-TCCCACACCTTTACCATAAAATCA-3' Using artificially synthesized mutant FBXW7 gene fragments (nucleotide sequence SEQ ID No. 5), wild-type FBXW7 gene fragments (nucleotide sequence SEQ ID No. 6), and DNA-free double-distilled water as a control, the probe pair was validated using real-time PCR. The results showed that the mutant FBXW7 gene fragment reacted with the specific probe T and emitted fluorescence, but did not produce fluorescence when reacting with probe C; the wild-type FBXW7 gene fragment reacted with the specific probe C and emitted fluorescence, but did not produce fluorescence when reacting with probe T; DNA-free double-distilled water did not emit fluorescence when reacting with either probe. Therefore, this probe pair is validated to be suitable for detecting the human FBXW7 gene chr4:152326101 C>T mutation.

[0029] Example 2 This embodiment provides a kit for detecting the chr4:152326101 C>T mutation in the human FBXW7 gene. Its main components are shown in Table 1. Table 1

[0030] The PCR amplification reagent is a mixture containing Taq deoxyribonucleic acid polymerase, deoxyribonucleic acid triphosphate, magnesium chloride, and buffer solution. The initial unit is "2x", and it is diluted to a final concentration of "1x" before use.

[0031] The FBXW7 upstream primer solution is a solution containing the FBXW7 upstream primer (sequence: 5'-CAGCAGTCCGCTGTGTTCAA-3'), with an initial concentration of 100 μM, which is diluted to a final concentration of 200 nM before use.

[0032] The FBXW7 downstream primer solution is a solution containing the FBXW7 downstream primer (sequence: 5'-TCCCACACCTTTACCATAAAATCA-3'), with an initial concentration of 100 μM, which should be diluted to a final concentration of 200 nM before use.

[0033] The probe T solution is a solution containing probe T (sequence: 5'-FAM-AGGAGGGTTGTTAGTAGA-MGB-3'), with an initial concentration of 100 μM, which is diluted to a final concentration of 200 nM before use.

[0034] The probe C solution is a solution containing probe C (sequence: 5'-VIC-AGGAGGGTTGTTAGTGGA-MGB-3'), with an initial concentration of 100 μM, which is diluted to a final concentration of 200 nM before use.

[0035] The probe T positive control solution is a solution containing the probe T positive control (sequence: 5'-CATGT TGCAG CAGTCCGCTG TGTTC AATAT GATGG CAGGA GGGTT GTTAG TAGAG CATAT GATTT TATGG TAAAG GTGTGGGAT-3'), with an initial concentration of 100 μM, which is diluted to a final concentration of 200 nM before use.

[0036] The probe C positive control solution is a solution containing the probe C positive control (sequence: 5'-CATGT TGCAG CAGTCCGCTG TGTTC AATAT GATGG CAGGA GGGTT GTTAG TGGAG CATAT GATTT TATGG TAAAG GTGTGGGAT-3'), with an initial concentration of 100 μM, which is diluted to a final concentration of 200 nM before use.

[0037] The probe T-positive standard solution is a solution containing the DNA sequence of the FBXW7 gene with a C→T mutation (single nucleotide T) at the chr4:152326101 site, and the plasmid pcDNA3.0 recombinant (sequence: pcDNA3-TTAAA AATTC TAAAC GTGGGTTTTT TTGTT TTGTT TTGTT TTTCT GTTTC TCCCT CTGCA GAGTT GTTAG CGGTT CTCGA GATGCCACTC TTAGG GTTTG GGATA TTGAG ACAGG CCAGT GTTTA CATGT TTTGA TGGGT CATGT TGCAGCAGTC CGCTG TGTTC AATAT GATGG CAGGA GGGTT GTTAG TAGAG CATAT GATTT TATGG TAAAGGTGTG GGATC CAGAG ACTGA AACCT GTCTA CACAC-pcDNA3), with an initial concentration of 1.00 x 10⁻⁶. 10 c / μL, dilute according to the ratio before use.

[0038] The probe C positive standard solution is a solution containing the DNA sequence of the FBXW7 gene at chr4:152326101 site without C→T mutation (single nucleotide C) and the plasmid pcDNA3.0 recombinant (sequence: pcDNA3-TTAAA AATTC TAAACGTGGG TTTTT TTGTT TTGTT TTGTT TTTCT GTTTC TCCCT CTGCA GAGTT GTTAG CGGTT CTCGAGATGC CACTC TTAGG GTTTG GGATA TTGAG ACAGG CCAGT GTTTA CATGT TTTGA TGGGT CATGTTGCAG CAGTC CGCTG TGTTC AATAT GATGG CAGGA GGGTT GTTAG TGGAG CATAT GATTT TATGGTAAAG GTGTG GGATC CAGAG ACTGA AACCT GTCTA CACAC-pcDNA3), with an initial concentration of 1.00 x 10⁻⁶. 10 c / μL, dilute according to the ratio before use.

[0039] DNA-free double-distilled water is water free of DNA and any other impurities, and has undergone double distillation disinfection.

[0040] Example 3 This embodiment provides a specific method for sensitivity analysis of the FBXW7 gene chr4:152326101 C>T mutation detection kit: Step 1: Preparation and dilution of standard products The probe T positive standard solution was diluted with DNA-free double-distilled water to a final concentration of 2.00 x 10⁻⁶. 7 c / μL, then according to 10 1 The standard was diluted in eight stages, resulting in concentrations of 2.00 x 10⁻⁶. 7 c / μL, 2.00x10 6 c / μL, 2.00x10 5 c / μL, 2.00x10 4 c / μL, 2.00x10 3 c / μL, 2.00x10 2 c / μL, 2.00x10 1 c / μL, 2.00x10 0 c / μL.

[0041] The probe C positive standard solution was diluted with DNA-free double-distilled water to a final concentration of 2.00 x 10⁻⁶. 7 c / μL, then according to 10 1 The standard was diluted in seven stages, resulting in concentrations of 2.00 x 10⁻⁶. 7 c / μL, 2.00x10 6 c / μL, 2.00x10 5 c / μL, 2.00x10 4 c / μL, 2.00x10 3 c / μL, 2.00x10 2 c / μL, 2.00x10 1 c / μL, 2.00x10 0 c / μL.

[0042] Double-positive standards are prepared by mixing equal volumes of the above-mentioned probe T-positive standard solution and probe C-positive standard solution. They are also divided into 8 grades, each with a final concentration of 2.00 x 10⁻⁶. 7 c / μL, 2.00x10 6 c / μL, 2.00x10 5 c / μL, 2.00x10 4 c / μL, 2.00x10 3 c / μL, 2.00x10 2 c / μL, 2.00x10 1 c / μL, 2.00x10 0 c / μL.

[0043] Step 2: PCR reaction system and condition settings Using the kit from Example 2, each diluted standard was used as a detection template to perform an amplification reaction in a single well of a 96-well plate with a total volume of 25 μL. The system composition is shown in Table 2. Table 2 PCR reaction system

[0044] After pre-denaturation at 94°C for 5 minutes, the reaction was carried out in the following repeated cycle: denaturation at 94°C for 45 seconds, annealing at 60°C for 60 seconds, repeated 50 times and then stopped.

[0045] Each concentration of standard solution was repeated 10 times; meanwhile, 0.05 μL of DNA-free double-distilled water was used as a negative control in place of the test standard solution, and PCR amplification was performed under the same conditions.

[0046] The above experiment was repeated once.

[0047] Step 3: Experimental Results and Data Analysis Experimental results are as follows Figure 1 As shown, for probe T, when the template amount is 2.00x10 2 When the reaction system is c / or higher, the positive detection rate is 100%; when the template amount is 2.00 x 10⁻⁶. 1 When the c / reaction system was used, the positive detection rate was 96.67%, corresponding to a CT value of 24.35; when the template amount was 2.00x10 0 When the reaction system was in c / , the positive rate was 63.33%, corresponding to a CT value of 27.36.

[0048] For probe C, when the template amount is 2.00x10 2 When the reaction system is c / or higher, the positive detection rate is 100%; when the template amount is 2.00 x 10⁻⁶. 1 When the c / reaction system was used, the positive detection rate was 96.67%, corresponding to a CT value of 24.43; when the template amount was 2.00x10 0 When the reaction system was in c / , the positive rate was 53.33%, corresponding to a CT value of 27.89.

[0049] When the amount of double-positive template is 2.00x10 2 When the reaction system is c / or higher, the detection positive rate of probes T and C is 100%; when the amount of double-positive template is 2.00 x 10⁻⁶. 1 In the c / reaction system, the positive detection rates of probes T and C were 96.67% and 96.67%, respectively, corresponding to CT values ​​of 24.37 and 24.41; when the template amount was 2.00x10 0In the c / reaction system, the positive detection rates of probes T and C were 63.33% and 56.67%, respectively, with corresponding CT values ​​of 27.42 and 28.21.

[0050] Based on the above results, it can be concluded that in a 25 μL reaction system, the analytical sensitivity (LOD, Limit of Detection) for detecting the FBXW7 gene chr4:152326101 C>T mutation of this kit is 20 copies, i.e., LOD=0.8c / μL.

[0051] Example 4 This case study provides a specific method for the specificity analysis of the FBXW7 gene chr4:152326101 C>T mutation detection kit.

[0052] Step 1: Preparing the verification template In this embodiment, the probes T and C positive controls included in the kit of Example 2 are used as the target template; pcDNA3.0 is used as a non-target template.

[0053] Step 2: PCR reaction system and condition settings Using the kit from Example 2 and a pre-prepared non-target template, an amplification reaction with a total volume of 25 μL was performed in a single well of a 96-well plate. The system composition is shown in Table 3. Table 3 PCR reaction system

[0054] After pre-denaturation at 94°C for 5 minutes, the reaction was carried out in the following repeated cycle: denaturation at 94°C for 45 seconds, annealing at 60°C for 60 seconds, repeated 50 times and then stopped.

[0055] Each target template and non-target template were tested three times; meanwhile, 1.00 μL of DNA-free double-distilled water was used as a negative control instead of the test standard solution, and PCR amplification was performed under the same conditions.

[0056] Step 3: Verification of amplification products The amplification products were subjected to agarose gel electrophoresis. The results of this experiment showed that the target template obtained a single specific band. Figure 2 lane#2), rather than the target template ( Figure 2 lane #3) and negative control ( Figure 2 No bands were generated in lane #4, indicating that this kit has good specificity.

[0057] Example 5 This embodiment provides an association study between aflatoxin-related hepatocellular carcinoma gene heterogeneity characteristics and the FBXW7 gene chr4:152326101 C>T mutation: Step 1: Collect aflatoxin-associated liver cancer and control tumor samples. A total of 528 tumor tissue specimens from sporadic aflatoxin-associated hepatocellular carcinoma (HCC) patients were collected from the Affiliated Hospital of Youjiang Medical University for Nationalities, with a mean age of 48.8 ± 10.1 years (standard deviation), including 172 female specimens. 364 tumor tissue specimens from sporadic HCC patients without aflatoxin exposure were also collected from the same hospital, with a mean age of 48.2 ± 10.3 years (standard deviation), including 113 female specimens. The two groups (aflatoxin-associated HCC and non-aflatoxin-associated HCC) were well-comparable in terms of baseline characteristics such as age, sex, and ethnicity.

[0058] Step 2: DNA extraction from tumor tissue samples Tumor cells were isolated from the collected tumor tissue samples using proteinase K lysis, and sample DNA was extracted from the tumor cells using phenol-chloroform extraction. The sample DNA was then uniformly diluted to 50 ng / μL and used as the DNA template for testing.

[0059] Step 3: Detection of FBXW7 gene chr4:152326101 C>T mutation Using the kit from Example 2, each DNA template was amplified in a single well of a 96-well plate with a total volume of 25 μL. The composition of the system is shown in Table 4. Table 4 Detection Kits

[0060] After pre-denaturation at 94°C for 5 minutes, the reaction was carried out in the following repeated cycle: denaturation at 94°C for 45 seconds, annealing at 60°C for 60 seconds, repeated 50 times and then stopped.

[0061] Simultaneously, 0.05 μL of probe T positive control solution was used to replace the DNA sample to be tested as probe T positive control, 0.05 μL of probe C positive control solution was used to replace the DNA sample to be tested as probe C positive control, 0.05 μL of probe T positive control solution and 0.05 μL of probe C positive control solution were used to replace the DNA sample to be tested as double positive control, and 1.00 μL of DNA-free double-distilled water was used to replace the DNA sample to be tested as negative control. PCR amplification reaction was performed under the same conditions.

[0062] The PCR reaction results of the DNA samples to be tested and the control standards were detected and analyzed using a real-time PCR instrument. The results are as follows: Figure 3As shown, because DNA has a double-stranded structure, there are four possible test results, as follows: Result 1 ( Figure 3 A): When the bases at the chr4:152326101 site of the FBXW7 gene on both strands are both T, only the FAM fluorescence of probe T can be detected, but the VIC fluorescence of probe C cannot be detected, which corresponds to the result of the positive control of probe T. Result 2 ( Figure 3 B): When the bases at the chr4:152326101 site of the FBXW7 gene on both strands are both C, only the VIC fluorescence of probe C can be detected, but the FAM fluorescence of probe T cannot be detected, which corresponds to the result of the positive control of probe C. Result 3 ( Figure 3 C): When one of the two strands is C and the other is T, both the VIC fluorescence of probe C and the FAM fluorescence of probe T can be detected, which corresponds to the results of the double positive control of probe C and probe T. Result 4 ( Figure 3 D): If the DNA is ineffective, the results will correspond to those of the negative control.

[0063] Result 1 and Result 3 both showed the FBXW7 gene chr4:152326101 C>T mutation, indicating a homozygous and heterozygous mutant of the FBXW7 gene chr4:152326101. Result 2 showed no FBXW7 gene chr4:152326101 C>T mutation and was a wild-type homozygote of the FBXW7 gene chr4:152326101. Result 4 was the negative control.

[0064] The DNA sample from step two was tested, and the results were as follows: Among 528 DNA samples from aflatoxin-associated hepatocellular carcinoma (HCC) tumor tissues, 101 samples showed the FBXW7 gene chr4:152326101 C>T mutation; while in 364 DNA samples from non-aflatoxin-associated HCC tumor tissues, the FBXW7 gene chr4:152326101 C>T mutation was not detected.

[0065] Step 4: Correlation analysis between the FBXW7 gene chr4:152326101 C>T mutation and aflatoxin exposure levels First, determine the aflatoxin exposure level in the DNA sample. Based on our team's earlier research, we used aflatoxin-DNA adducts (ADA) for evaluation. The specific steps are as follows: (1) Place the tumor tissue sample DNA in a 1.5 mL centrifuge tube, add an appropriate amount of anhydrous ethanol to promote precipitation, centrifuge at 10,000 rpm for 5 min, and discard the supernatant. (2) Add 200 µL of Na2CO3-NaHCO3 mixture, mix for 5 min, add 1 mL of anhydrous ethanol to promote precipitation, centrifuge at 10,000 rpm for 5 min, and discard the supernatant. After air drying, add 100 µL of 0.01 M PBS (pH 7.4) to dissolve the DNA. (3) Prepare an appropriate ratio of AFB1 and fetal bovine DNA, place in a sealed bag, react for 1 hour, and use an enzyme-linked immunosorbent assay (ELISA) reader to determine the nucleic acid concentration to prepare a standard. (4) Prepare an enzyme-labeled titration plate for ADA detection: Take a 96-well titration plate, add 50 ng of ADA standard, ensure even distribution during addition, seal with plastic wrap, and incubate at 37℃ for 3 hours or 4℃ overnight. (5) Take the enzyme-labeled titration plate prepared in step (4) and prepare a standard curve of adduct concentration versus inhibition rate. (6) Continue to add 100 μL of Washing Buffer per well to the enzyme-labeled titration plate, wash for 5 min, centrifuge at 500 rpm for 1 min, discard the supernatant, and repeat this operation twice. (7) Adjust the sample DNA concentration and denature it: Add 25 μg of DNA sample to a 200 μL PCR tube, add 1xPBS to 50 μL, place in a PCR instrument at 98℃ for 3 min, and immediately place on ice for 5 min after the reaction. (8) Add 100 μL of freshly prepared 1% FCS to each well in the microplate. After 5 min, add 50 μL of the denatured sample DNA from step (7) above. (9) Add 25 μL of 10A1 (diluted at 1:2500) to each well and incubate at 37°C for 90 min. Discard the supernatant. (10) Add 100 μL of Washing Buffer to each well and wash for 5 min. Discard the supernatant. Repeat this operation twice. (11) Add 50 μL of Mouse IgG antibody (diluted at 1:1000) to each well and incubate at 37°C for 90 min. Discard the supernatant. (12) Add 100 μL of Washing Buffer to each well and wash for 5 min. Repeat this operation twice. (13) Add 100 μL of 10 μM diethanolamine to each well (make sure to cover the bottom of the well), wash for 5 min, discard the supernatant, and repeat this operation once. (14) Add 50 μL of 1 mg / mL sigma104 (prepared fresh with 10 M diethanolamine) to each well, incubate at 37°C for 30 min, and then measure the absorbance at 405 nm using an enzyme-linked immunosorbent assay (ELISA) reader and calculate the ADA concentration.

[0066] Based on the average concentration of ADA in AHCC tumor tissue (2.90±1.54µmol / mol DNA), aflatoxin exposure levels were divided into two groups: a low-exposure group and a high-exposure group. Patients with ADA concentrations less than or equal to 2.90µmol / mol DNA in AHCC tumor tissue were classified into the low-exposure group, while patients with ADA concentrations greater than 2.90µmol / mol DNA were classified into the high-exposure group.

[0067] Next, a multivariate logistic regression analysis model was applied to analyze the association between aflatoxin exposure level and the FBXW7 gene chr4:152326101 C>T mutation. The results showed a positive correlation between the two. The effect of this correlation was that with the increase of aflatoxin exposure, the risk of FBXW7 gene chr4:152326101 C>T mutation increased by 9.7 times, indicating that FBXW7 gene chr4:152326101 C>T mutation is an important genetic heterogeneity feature of aflatoxin-related hepatocellular carcinoma (Table 5).

[0068] Table 5. Effects of aflatoxin exposure on the FBXW7 gene chr4:152326101 C>T mutation.

[0069] Note: * indicates P<0.01. Abbreviations: OR, odds ratio; CI, confidence interval.

[0070] Unless otherwise specified, all raw materials used in this invention are existing substances that can be purchased directly from the market.

[0071] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A primer set and probe, characterized in that, include: Probe T, nucleotide sequence as shown in SEQ ID NO.1; Probe C, nucleotide sequence as shown in SEQ ID NO.2; The upstream primer, with its nucleotide sequence shown in SEQ ID NO.3; and The downstream primer has the nucleotide sequence shown in SEQ ID NO.

4.

2. The primer set and probe according to claim 1, characterized in that, The nucleotide sequence of the probe T has a FAM label at the 5' end and an MGB label at the 3' end.

3. The primer set and probe according to claim 1, characterized in that, The nucleotide sequence of probe C has a VIC tag at the 5' end and an MGB tag at the 3' end.

4. The use of the primer set and probe according to any one of claims 1-3 in the preparation of a preparation for detecting the mutant FBXW7 gene.

5. The application according to claim 4, characterized in that, The mutated FBXW7 gene is a C>T mutation at chr4:152326101.

6. A reagent kit, characterized in that, Includes the primer set and probe as described in any one of claims 1-3.

7. The reagent kit according to claim 6, characterized in that, It also includes PCR amplification reagents, positive controls, negative controls, and standards.

8. The reagent kit according to claim 7, characterized in that, PCR amplification reagents include Taq deoxyribonucleic acid polymerase, deoxyribonucleic acid triphosphate, magnesium chloride, buffer solution, and double-distilled water.

9. The use of the kit according to any one of claims 6-8 in detecting the mutant FBXW7 gene.

10. The use of the kit according to claims 6-8 in the detection of aflatoxin-associated liver cancer.

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