Primer pair for detecting TP53 gene exon region mutation and kit applying same

Abstract

The invention provides a primer pair for detecting TP53 gene exon region mutation and a kit applying the primer pair. The primer pair comprises a first forward primer and a first reverse primer, the nucleotide sequence of the first forward primer is shown as SEQ ID NO: 1, and the nucleotide sequence of the first reverse primer is shown as SEQ ID NO: 2. The primer pair for detecting TP53 gene exon region mutation provided by the invention can be used for performing PCR (Polymerase Chain Reaction) amplification on cDNA (Complementary Deoxyribose Nucleic Acid) obtained by performing reverse transcription on RNA (Ribonucleic Acid) extracted from fresh EDTA (Ethylene Diamine Tetraacetic Acid) anticoagulant whole blood and identifying through Sanger sequencing to find that a PCR amplification product consistent with a theoretical sequence can be obtained; therefore, the specific amplification and mutation detection of the TP53 gene exon coding region with reliable results can be simply, conveniently and quickly completed at one time.

Description

Technical Field

[0001] This invention relates to the field of molecular diagnostics and in vitro detection technology, specifically to a primer pair for detecting mutations in the exon region of the TP53 gene and a kit for using the same. Background Technology

[0002] The occurrence and development of tumors are closely related to mutations in a variety of genes. Among them, the TP53 gene, as one of the most important tumor suppressor genes in the human body, plays a core role in key biological processes such as cell cycle regulation, DNA repair, apoptosis, and maintaining genome stability.

[0003] The TP53 gene is located on the short arm of chromosome 17 (17p13.1) and consists of 11 exons. A portion of exons 2 through 11 form a continuous coding sequence of 1182 bp, constituting the almost complete translational framework of the p53 protein. This definition is widely used in clinical molecular assays, reflecting the functional coding range from the transcription start-related region to the vicinity of the termination signal. Specifically, the exon regions encoded by the TP53 gene include: a portion of exon 2 (containing the start codon ATG and the N-terminal initiation sequence), exons 3-10 (complete coding), and a portion of exon 11 (containing the C-terminal termination signal and its surrounding sequence). Compared to the introns and promoter regions of the non-coding region, mutations in the exons encoded by the TP53 gene have the most direct impact on protein structure and function. From the perspective of transcription and translation, this region encodes all 393 amino acids of the p53 protein. In particular, the key structural units that determine the protein's three-dimensional conformation, DNA recognition, protein-protein interaction, and functional activity are all located within this range. Thus, the 1182 bp continuous coding sequence length not only ensures coverage of all key functional domains of the p53 protein, becoming the core coding region of TP53 function, but also achieves a balance between technical feasibility and cost-effectiveness in targeted sequencing design—high sensitivity can be achieved during deep sequencing, and standardized sequencing report generation is convenient.

[0004] TP53 gene mutations are among the most common and widespread genetic alterations in human cancer to date, occurring in over 50% of malignant tumor cases. The TP53 gene exhibits high mutation frequencies across various cancers, with mutation types including missense, nonsense, frameshift, and splicing aberrations. These mutations are distributed throughout its entire exon coding region, and the impact of mutations at different sites on protein function and their clinical significance varies significantly. Therefore, accurate and comprehensive mutation screening of the exon region encoded by the TP53 gene is of crucial clinical value for early tumor diagnosis, molecular subtyping, prognostic assessment, treatment strategy selection, and efficacy monitoring.

[0005] Traditional gDNA-based strategies suffer from problems such as pseudogene interference, difficulties in interpreting splicing effects, and high process time costs, which significantly limit their application in TP53 gene mutation detection. Current research on TP53 gene mutation detection typically relies on molecular biology methods, such as polymerase chain reaction (PCR) amplification combined with Sanger sequencing, which remains considered one of the "gold standards" for gene mutation detection due to its high accuracy and reliability. However, using PCR amplification combined with Sanger sequencing to cover multiple exons of the TP53 gene faces significant technical challenges and efficiency bottlenecks. Because the exon regions of the TP53 gene are relatively dispersed, and its flanking intron sequences may have highly repetitive or complex structures, current techniques typically employ multiple primer pairs to perform PCR amplification on different exons or gene fragments to achieve specific amplification of the coding region's exons. This "segmented amplification" strategy has the following limitations: First, the experimental procedures for segmented amplification are cumbersome and time-consuming, significantly increasing labor and reagent costs. Second, multiple independent PCR reactions require a large quantity of clinical samples (especially valuable, small-quantity samples), which limits the detection of limited samples. Third, there may be cross-reactions or inconsistent amplification efficiencies between multiple primer pairs, leading to amplification failure or low efficiency in some regions, creating detection blind spots and increasing the risk of missing certain key mutation sites. In addition, the complex experimental procedures also increase the possibility of contamination and operational errors, affecting the stability and reproducibility of the detection results.

[0006] While some studies have attempted to address these challenges by optimizing primers for individual exons or employing other detection technologies (such as next-generation sequencing), next-generation sequencing is relatively expensive and involves complex data analysis, making it unsuitable for all clinical scenarios. Therefore, there is an urgent need to develop a method that can efficiently and specifically amplify mutations in the exon regions encoded by the TP53 gene using a single PCR reaction. This method would ensure that the amplified products completely cover all coding exons, exhibit no non-specific bands, and meet the stringent sequence fidelity requirements of subsequent Sanger sequencing. Summary of the Invention

[0007] To address the problem of existing technologies that use multiple primer pairs to amplify different exons or gene fragments using PCR, this invention provides a primer pair for detecting mutations in the exon region of the TP53 gene and a kit for using it.

[0008] According to a first aspect of the present invention, a primer pair for detecting mutations in the exon region of the TP53 gene is provided, the primer pair comprising a first forward primer and a first reverse primer, the nucleotide sequence of the first forward primer being shown in SEQ ID NO:1, and the nucleotide sequence of the first reverse primer being shown in SEQ ID NO:2; the exon region of the TP53 gene includes a portion of the second exon region, all regions of the third to tenth exons, and a portion of the eleventh exon region.

[0009] The TP53 gene comprises 11 exons, with a portion of exon 2, all regions of exons 3-10, and a portion of exon 11 forming the coding region. The continuous coding sequence of this region is 1182 bp in length. Mutations in the TP53 gene exon coding regions encompass multiple dimensions, including tumorigenesis, progression, invasion, and treatment response. Their correspondence with the p53 protein structure, the functional stratification of mutation types, and their multidimensional association with tumor invasiveness and treatment response collectively constitute an indispensable molecular basis in modern precision oncology. Current technologies for detecting mutations in multiple exon coding regions of the TP53 gene lack a systematic process and corresponding primer / quality control strategies that support "whole blood initiation—RNA level—multiple exon coding region coverage—low-cost and scalable implementation."

[0010] The inventors of this application designed highly conserved primers based on the most conserved 5' and 3' ends of the TP53 gene exon coding region. Through experimental screening and verification, they obtained primer pairs specific to the TP53 gene exon coding region, which are the primer pairs provided by this invention for detecting mutations in the TP53 gene exon coding region. These primer pairs can detect mutations in the TP53 gene exon coding region (i.e., part of exon 2, all of exons 3 to 10, and part of exon 11). Using the primer pairs provided by this invention for detecting TP53 gene exon region mutations, PCR amplification of cDNA obtained by reverse transcription of RNA extracted from fresh EDTA-anticoagulated whole blood was performed. Sanger sequencing confirmed that the PCR amplification product was consistent with the theoretical sequence, thus achieving the purpose of detecting mutations in multiple exon coding regions of the TP53 gene. The primer pairs provided by this invention enable simple, rapid, and reliable specific amplification and mutation detection of the coding regions of the TP53 gene exons (part of exon 2, all regions of exons 3 to 10, and part of exon 11). Furthermore, the primer pairs provided by this invention can specifically amplify multiple coding regions of the TP53 gene, solving problems such as RNA instability in whole blood, false negatives / positives caused by gDNA contamination, interference from pseudogenes / homologous sequences, and limitations in splicing abnormalities, rare variants, insufficient coverage of exon coding regions, and constraints related to equipment and consumable costs.

[0011] According to a second aspect of the present invention, the use of the primer pair described above for detecting mutations in the exon region of the TP53 gene is provided in the preparation of a kit for detecting mutations in the exon region of the TP53 gene.

[0012] According to a third aspect of the present invention, a kit for detecting mutations in the exon region of the TP53 gene is provided. The kit includes component A, which includes an amplification primer pair, the amplification primer pair being the aforementioned primer pair for detecting mutations in the exon region of the TP53 gene.

[0013] The primer pairs provided by this invention for detecting mutations in the exon regions of the TP53 gene are applied to the preparation of a kit for detecting mutations in the exon regions of the TP53 gene. The resulting kit can be used to detect mutations in the coding regions of the TP53 gene exons (part of exon 2, all of exons 3 to 10, and part of exon 11). The kit is a standardized method and kit that starts with whole blood, completes RNA extraction and cDNA synthesis without separating PBMCs, and covers the TP53 exon coding region using Sanger sequencing. It solves problems such as RNA instability in whole blood, false negatives / positives caused by gDNA contamination, interference from pseudogenes / homologous sequences, and limitations in coverage of splicing abnormalities, rare variants, and exon coding regions, as well as constraints related to equipment and consumable costs.

[0014] Preferably, component A further includes an internal sequencing primer pair, which includes a second forward primer and a second reverse primer. The nucleotide sequence of the second forward primer is shown in SEQ ID NO:3, and the nucleotide sequence of the second reverse primer is shown in SEQ ID NO:4.

[0015] The kit provided in this protocol for detecting mutations in the exon regions of the TP53 gene contains not only amplification primer pairs but also the aforementioned internal sequencing primer pairs. After PCR amplification of cDNA obtained by reverse transcription of RNA extracted from fresh EDTA-anticoagulated whole blood using the amplification primer pairs, and during the sequencing of the PCR amplification products using Sanger sequencing technology, the aforementioned internal sequencing primer pairs are used to perform segmented sequencing reads of the PCR amplification products. Test results show that the theoretical reads corresponding to the internal sequencing primer pairs are consistent with the provided theoretical sequences. Therefore, the aforementioned internal sequencing primers can be used to sequence PCR amplification products obtained using the amplification primer pairs. The internal sequencing primer pairs and the amplification primer pairs can work synergistically to detect mutations in the exon coding regions of the TP53 gene (part of exon 2, all regions of exons 3 to 10, and part of exon 11) in the test samples.

[0016] Preferably, the kit further includes component B, which includes a total RNA extraction reagent, and the total RNA extraction reagent includes Trizol.

[0017] Preferably, component B further includes chloroform, isopropanol, ethanol, and diethyl pyrocarbonate (DEPC).

[0018] Preferably, the kit further includes component C, which includes oligo(dT), dNTP, reverse transcriptase, RNase inhibitor and reverse transcription buffer; the reverse transcription buffer includes the following components: 40-60 mM Tris-HCl, 60-80 mM KCl, 2-4 mM MgCl2, and 5-15 mM dithiothreitol (DTT).

[0019] Preferably, the reverse transcription buffer comprises the following components: 50 mM Tris-HCl, 75 mM KCl, 3 mM MgCl2, and 10 mM DTT.

[0020] Preferably, the pH value of Tris-HCl is 8 to 8.5.

[0021] Preferably, the pH value of Tris-HCl is 8.3.

[0022] Preferably, the kit further includes component D, which includes PCR reagents, specifically 2×PrimerStar Max Premix.

[0023] In component D above, the 2×Primer Star Max Premix in the PCR reagent refers to a PCR premix solution prepared to a 2× concentration, consisting of DNA polymerase, PCR buffer, and dNTP mixture. In actual use, it is ready to use after mixing with primers, template, and water to a 1× concentration. The 2×Primer Star Max Premix (Takara Bio, R045A / R045B) contains 2 mM Mg in a 1× system. 2+ It contains 0.4 mM dNTPs, along with a high-fidelity hot-start DNA polymerase and optimized buffer.

[0024] Preferably, the above kit further includes component E, which includes PCR product purification reagent and PCR product sequencing reagent; the PCR product purification reagent includes the following components: 5~20 mg / mL magnetic bead suspension, purification buffer, washing buffer, and elution buffer; the purification buffer contains 15~25% (w / v) PEG 8000, 1~2 M NaCl, and 5~20 mM Tris-HCl; the washing buffer is a 70~85% (v / v) ethanol solution; the elution buffer is nuclease-free water or 10 mM Tris-HCl with a pH of 8.0~8.8.

[0025] Preferably, the magnetic bead suspension is prepared by the following steps: carboxyl-modified paramagnetic magnetic beads are suspended in Tris-HCl with a concentration of 5-20 mM and a pH of 7.5-8.5, and ethylenediaminetetraacetic acid (EDTA) and a nonionic surfactant are added to the system, with the concentration of EDTA in the system being 0-2 mM and the mass fraction of the nonionic surfactant being 0-0.1%, to obtain the magnetic bead suspension.

[0026] In addition to component A (amplification primer pairs and internal sequencing primer pairs), the kit for detecting mutations in the exon regions of the TP53 gene in this protocol also includes components B, C, D, and E. Component B is used to extract RNA from whole blood samples, component C is used for the reverse transcription of the extracted RNA, component D is used for PCR amplification of the cDNA obtained from reverse transcription, and component E is used for purification and sequencing of the PCR amplification products. The synergistic effect of the above components in the kit enables the kit to detect mutations in the exon coding regions of the TP53 gene (part of exon 2, all regions of exons 3 to 10, and part of exon 11) in whole blood samples.

[0027] The kits involved in this protocol use the Trizol method to extract RNA, perform two-step reverse transcription, amplify with the Primer Star enzyme system, and complete bidirectional sequencing coverage through internal primers. It is low-cost, has a simplified process, and has good identification capabilities for splicing abnormalities and rare variants, making it suitable for widespread use in routine molecular laboratories.

[0028] According to a fourth aspect of the present invention, the above-described kit for detecting mutations in the exon region of the TP53 gene is provided for use in the preparation of products for tumor molecular subtyping, efficacy assessment, and prognosis.

[0029] The above-mentioned kit for detecting mutations in the exon regions of the TP53 gene has good clinical application value in tumor molecular subtyping, efficacy evaluation, and prognosis. Attached Figure Description

[0030] Figure 1 This is a schematic diagram of the TP53 gene structure.

[0031] Figure 2 The image shows the electrophoresis results of PCR amplification products obtained during the detection of TP53 gene exon region mutations using the kits provided in Examples 2, 3, and 4. Detailed Implementation

[0032] The technical features of the technical solution provided by the present invention will be further clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] The structural diagrams of the TP53 gene involved in the following examples or comparative examples are shown below. Figure 1 As shown, where: P1(p53, Δ40p53) indicates that the transcription of the full-length p53 protein (p53α) is driven by the P1 promoter, which is the main promoter of the TP53 gene and controls the transcription of the full-length p53 protein. It mainly produces the full-length p53 and Δ40p53 isoforms. P2 (Δ133p53, Δ160p53) indicates that the transcription of the N-terminal truncated isoform is driven by the P2 internal promoter, which is located in the region of exon 4 and controls the transcription of the N-terminal truncated isoform, producing truncated isoforms such as Δ133p53 and Δ160p53. 5'UTR stands for 5' Untranslated Region, which refers to the entire region of exon 1 plus the front region of exon 2. It is the 5' end of mature mRNA that does not encode proteins, but it is involved in translation regulation. 3'UTR stands for 3' Untranslated Region, which refers to the sequence on mature mRNA from the stop codon to the poly(A) tail. Similar to 5'UTR, it does not encode proteins, but plays a key role in the posttranscriptional regulation of gene expression.

[0034] ATG1 represents start codon 1, located in exon 2, and is the translation initiation site for the full-length p53 protein (p53α). ATG40 indicates start codon 40. Translation begins from the 40th codon, producing the Δ40p53 (i.e. p47) isomer, which deletes the first 39 amino acids. ATG133 indicates start codon 133. Translation begins from codon 133, producing the Δ133p53 isoform, which lacks the N-terminal transcription activation domain. ATG160 indicates start codon 160. Translation begins from the 160th codon, producing the Δ160p53 isomer. TAD1 (Transactivation Domain 1) represents transcription activation domain 1, corresponding to amino acids 1-40 at the N-terminus of the p53 protein. It interacts with transcription factors and activates the transcription of downstream genes. TAD2 (Transactivation Domain 2) represents transcription activation domain 2, corresponding to amino acids 41-60 at the N-terminus of the p53 protein, which assists in transcription activation. PPXP (Proline-rich domain) refers to a proline-rich domain that corresponds to amino acids 61-94 at the N-terminus of the p53 protein and is involved in signal transduction and apoptosis regulation. DNABD (DNA Binding Domain) refers to the DNA binding domain, corresponding to amino acids 100-292 at the N-terminus of the p53 protein. It is the core functional domain that recognizes and binds to the DNA sequence of target genes; this region is a mutation hotspot. NLS (Nuclear Localization Signal) refers to the nuclear localization signal that guides the p53 protein into the cell nucleus to perform transcriptional regulation functions; OD (Oligomerization Domain) represents the oligomerization domain, amino acids 307-355, which mediates the formation of a tetramer from p53; NEG (Negative Regulation Domain) represents the negative regulatory domain, amino acids 356-393, which contain phosphorylation sites that regulate the stability and activity of p53; β, γ, and α represent alternative splicing isomers at the C-terminus of the TP53 gene; Boxes 1-11 represent exon regions, with numbers 1-11 representing exons 1-11 respectively. The connecting lines between exons represent introns. The TP53 gene exon coding regions mentioned in this invention (i.e., part of exon 2, all of exons 3 to 10, and part of exon 11) refer to the regions in boxes 1-11 other than the 5'UTR and 3'UTR (the gray diagonal lines in the figure).

[0035] Example 1 A primer pair for detecting mutations in the exon region of the TP53 gene, the specific nucleotide sequence of which is shown in Table 1, includes a first forward primer and a first reverse primer. The nucleotide sequence of the first forward primer is shown in SEQ ID NO:1, and the nucleotide sequence of the first reverse primer is shown in SEQ ID NO:2.

[0036] Table 1 Primer pairs used in Example 1 for detecting mutations in the exon region of the TP53 gene

[0037] Example 2 A kit for detecting mutations in the exon region of the TP53 gene, comprising components A, B, C, D, and E; Table 2. Amplification primer pairs and internal sequencing primer pairs in the kit of Example 2

[0038] Component A includes amplification primer pairs and internal sequencing primer pairs. The specific nucleotide sequences are shown in Table 2. The amplification primer pairs include a first forward primer and a first reverse primer. The nucleotide sequence of the first forward primer is shown in SEQ ID NO:1, and the nucleotide sequence of the first reverse primer is shown in SEQ ID NO:2. The internal sequencing primer pairs include a second forward primer and a second reverse primer. The nucleotide sequence of the second forward primer is shown in SEQ ID NO:3, and the nucleotide sequence of the second reverse primer is shown in SEQ ID NO:4. Component B includes total RNA extraction reagent (Trizol), chloroform, isopropanol, ethanol, and DEPC; Component C includes oligo(dT), dNTPs, reverse transcriptase, RNase inhibitor, and reverse transcription buffer. The reverse transcription buffer consists of the following components: 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, and 10 mM DTT. Component D includes PCR reagents (2×Primer Star Max Premix); Component E includes PCR product purification reagents and PCR product sequencing reagents. The PCR product purification reagent includes the following components: 15 mg / mL magnetic bead suspension, purification buffer, washing buffer, and elution buffer. The magnetic bead suspension was prepared by the following steps: carboxyl-modified paramagnetic magnetic beads were suspended in Tris-HCl with a concentration of 15 mM and a pH of 8.3, and EDTA and nonionic surfactant were added to the system to make the concentration of EDTA 1 mM and the mass fraction of nonionic surfactant 0.05% to obtain the magnetic bead suspension. The purification buffer contains 20% (w / v) PEG 8000, 1.5 M NaCl, and 15 mM Tris-HCl; The washing solution is an 80% (v / v) ethanol solution; The elution buffer was nuclease-free water or Tris-HCl with a concentration of 10 mM and a pH of 8.3.

[0039] Comparative Example 1 A primer pair for detecting mutations in the exon region of the TP53 gene, the specific nucleotide sequences of which are shown in Table 3, includes a third forward primer and a third reverse primer. The nucleotide sequence of the third forward primer is shown in SEQ ID NO:5, and the nucleotide sequence of the third reverse primer is shown in SEQ ID NO:6.

[0040] Table 3 Primer pairs used in Comparative Example 1 for detecting mutations in the exon regions of the TP53 gene.

[0041] Comparative Example 2 A primer pair for detecting mutations in the exon region of the TP53 gene, the specific nucleotide sequences of which are shown in Table 4, includes a fourth forward primer and a fourth reverse primer. The nucleotide sequence of the fourth forward primer is shown in SEQ ID NO:7, and the nucleotide sequence of the fourth reverse primer is shown in SEQ ID NO:8.

[0042] Table 4 Primer pairs used in Comparative Example 2 for detecting mutations in the exon regions of the TP53 gene.

[0043] Comparative Example 3 This comparative example provides a kit for detecting mutations in the exon region of the TP53 gene. Compared with Example 2, the difference in composition is that the amplification primer pairs in component A are different. The specific nucleotide sequences of the amplification primer pairs and internal sequencing primer pairs in component A are shown in Table 5.

[0044] Table 5. Amplification primer pairs and internal sequencing primer pairs in the kit for Comparative Example 3

[0045] Apart from the differences mentioned above, the materials, formulation ratios, and preparation procedures used in this comparative example are strictly consistent with those in Example 1.

[0046] Comparative Example 4 This comparative example provides a kit for detecting mutations in the exon region of the TP53 gene. Compared with Example 1, the difference in composition is that the amplification primer pairs in component A are different. The specific nucleotide sequences of the amplification primer pairs and internal sequencing primer pairs in component A are shown in Table 6.

[0047] Table 6. Amplification primer pairs and internal sequencing primer pairs in the kit for Comparative Example 4

[0048] Apart from the differences mentioned above, the materials, formulation ratios, and preparation procedures used in this comparative example are strictly consistent with those in Example 1.

[0049] Test case This test example uses whole blood samples as the starting material and employs the kits for detecting mutations in the exon region of the TP53 gene provided in Example 2, Comparative Example 3, and Comparative Example 4, respectively. The specific experimental procedures are as follows: 1. Sample and RNA extraction (1) Collect fresh EDTA-anticoagulated whole blood, let it stand for 1 hour until the blood cells separate into layers, and then take 200 μL from the white blood cell layer between the plasma and the red blood cell sediment, or mix the fresh EDTA-anticoagulated whole blood and take 200 μL (in this test case, the white blood cell layer taken from the plasma and the red blood cell sediment is used for subsequent RNA extraction). (2) Add 1 mL of Trizol to the 200 μL of white blood cell layer obtained above, mix well, add 200 μL of chloroform, shake vigorously for 15 s, let stand at room temperature for 2-3 min, and then centrifuge at 12000 rpm and 4℃ for 15 min to obtain the supernatant. (3) Add 0.5 mL of isopropanol to the supernatant above, mix well and let stand at room temperature for 10 min, then centrifuge at 12000 rpm and 4℃ for 10 min, discard the supernatant and obtain the first precipitate; (4) Add 1 mL of 75% ethanol solution to the first precipitate obtained above, wash it, and centrifuge at 12000 rpm and 4℃ for 10 min. Discard the supernatant to obtain the second precipitate. Dry the second precipitate at room temperature for 5~10 min (avoid residual ethanol and avoid excessive drying) to obtain RNA. The extracted RNA can be used immediately (for subsequent reverse transcription) or stored at -80℃ for ≤1 week. (5) Add 30 μL of DEPC water to the RNA extracted above to dissolve the RNA and measure the concentration. The quality index of the extracted RNA should meet the requirement that the ratio between the absorbance value A260 at a wavelength of 260 nm and the absorbance value A280 at a wavelength of 280 nm is A260 / A280 = 1.9~2.1.

[0050] 2. Reverse transcription (1) Mix 1 μL oligo(dT), 1 μL dNTP, and 2 μg RNA (about 2-8 μL of RNA dissolved in DEPC water), then add DEPC water to make up to a total volume of 10 μL. Incubate at 65°C for 5 min, then place on an ice bath to obtain the mixture. (2) Add 4 μL of 5× reverse transcription buffer, 0.5 μL of reverse transcriptase, and 0.5 μL of RNase inhibitor to the above mixture, and then add DEPC water to make up to a total volume of 20 μL. React at 30℃ for 10 min, at 42℃ for 30 min, and at 70℃ for 15 min to obtain cDNA. The cDNA obtained by the above reverse transcription can be stored at -20℃ or used immediately in PCR reaction. The 5× reverse transcription buffer contains 200–300 mM Tris-HCl (pH 8.0–8.5), 300–400 mM KCl, 10–20 mM MgCl2, and 25–75 mM DTT. When using the 5× reverse transcription buffer, it is diluted to 1×. The final concentration of each component in the 1× reverse transcription buffer is 40–60 mM Tris-HCl, 60–80 mM KCl, 2–4 mM MgCl2, and 5–15 mM DTT.

[0051] 3. PCR amplification (1) Preparation of PCR reaction system: 25 μL of 2×Primer Star Max Premix, 0.5 μL of 10 μM forward primer, 0.5 μL of 10 μM reverse primer, 5 μL of cDNA and 19 μL of water were mixed to obtain PCR mixture. In this test example, when the whole blood sample was tested using the kit for detecting mutations in the exon region of the TP53 gene provided in Example 2, the forward primer used was the first forward primer as shown in SEQ ID NO:1, and the reverse primer used was the first reverse primer as shown in SEQ ID NO:2. In this test case, when the whole blood sample was tested using the kit for detecting mutations in the exon region of the TP53 gene provided in Comparative Example 3, the forward primer used was the third forward primer as shown in SEQ ID NO:5, and the reverse primer used was the third reverse primer as shown in SEQ ID NO:6. In this test case, when the whole blood sample was tested using the kit provided in Comparative Example 4 for detecting mutations in the exon region of the TP53 gene, the forward primer used was the fourth forward primer as shown in SEQ ID NO:7, and the reverse primer used was the fourth reverse primer as shown in SEQ ID NO:8. (2) The PCR mixture prepared above was subjected to PCR amplification according to the following PCR reaction program to obtain PCR amplification products: 95℃ 3 min; 98℃ 10 s, 58℃ 15 s, 72℃ 90 s, 30~33 cycles; 72℃ 5 min.

[0052] 4. Identification of PCR amplification products (1) The PCR amplification products obtained above were identified by agarose gel electrophoresis, and the results are as follows: Figure 1 As shown, where, Figure 1 In Figure A, the electrophoresis results of PCR amplification products obtained during the detection of TP53 gene exon region mutations using the kits provided in Example 2 and Comparative Example 3 are shown. Lanes 1-2 show the electrophoresis results of PCR amplification products obtained during the detection of TP53 gene exon region mutations using the kits provided in Example 2. Lanes 3-4 show the electrophoresis results of PCR amplification products obtained during the detection of TP53 gene exon region mutations using the kits provided in Comparative Example 3. Lane 5 shows the electrophoresis results of PCR amplification products obtained by reverse transcription using -RT negative control (sterile double-distilled water) as a template, followed by PCR amplification of the cDNA obtained from the reverse transcription. Figure 1 Image B shows the electrophoresis results of PCR amplification products obtained during the detection of TP53 gene exon region mutations using the kits provided in Example 2 and Comparative Example 4 for detecting TP53 gene exon region mutations in whole blood samples. Lanes 1-7 show the electrophoresis results of PCR amplification products obtained during the detection of TP53 gene exon region mutations using the kits provided in Comparative Example 4 for detecting TP53 gene exon region mutations in whole blood samples. Lane 8 shows the electrophoresis results of PCR amplification products obtained by reverse transcription using -RT negative control (sterile double-distilled water) as a template, followed by PCR amplification of the cDNA obtained from the reverse transcription. Lane 9 shows the electrophoresis results of PCR amplification products obtained during the detection of TP53 gene exon region mutations using the kits provided in Example 2 for detecting TP53 gene exon region mutations in whole blood samples.

[0053] Depend on Figure 1It can be seen that, in the process of detecting TP53 gene exon region mutations in whole blood samples using the kit provided in Comparative Example 3, the amplification primer pair (the third forward primer and the third reverse primer shown in SEQ ID NO:5) in the kit provided in Comparative Example 3 had poor performance and low amplification efficiency when performing PCR amplification of cDNA obtained by reverse transcription of RNA extracted from fresh EDTA anticoagulated whole blood (i.e., amplifying the TP53 gene exon coding region). In the process of detecting TP53 gene exon region mutations in whole blood samples using the kit provided in Comparative Example 4, the amplification primer pair (the fourth forward primer and the third reverse primer shown in SEQ ID NO:6) in the kit provided in Comparative Example 4 had poor performance and low amplification efficiency when performing PCR amplification of cDNA obtained by reverse transcription of RNA extracted from fresh EDTA anticoagulated whole blood (i.e., amplifying the TP53 gene exon coding region). The fourth reverse primer shown in NO:8 had poor amplification effect when performing PCR amplification (i.e., amplifying the TP53 gene exon coding region) on cDNA obtained from reverse transcription of RNA extracted from fresh EDTA-anticoagulated whole blood, making it difficult to amplify the TP53 gene exon coding region. However, when using the kit provided in Example 2 to detect mutations in the TP53 gene exon region of whole blood samples, the amplification primer pair (the first forward primer with nucleotide sequence as shown in SEQ ID NO:1 and the first reverse primer with nucleotide sequence as shown in SEQ ID NO:2) provided in Example 2 showed better amplification effect and higher amplification efficiency when performing PCR amplification (i.e., amplifying the TP53 gene exon coding region) on cDNA obtained from reverse transcription of RNA extracted from fresh EDTA-anticoagulated whole blood.

[0054] (2) Based on Sanger sequencing technology, the internal sequencing primer pairs (the second forward primer with nucleotide sequence as shown in SEQ ID NO:3 and the second reverse primer with nucleotide sequence as shown in SEQ ID NO:4) in the kit provided in Example 2 were used to perform segmented sequencing reads of the PCR amplification products obtained above (Q20≥95%, where Q20 value refers to the error probability given for the identified bases during the sequencing process. If the quality value is Q20, then the probability of misidentification is 1%, that is, the error rate is 1%, or the accuracy rate is 99%). The sequencing reads were aligned to the reference transcript (such as NM_000546.6). The sequencing results and theoretical sequences are shown in Table 7.

[0055] Table 7 Sequencing results and theoretical sequences

[0056] Based on the theoretical sequences shown in Table 7 and according to the following methods / rules, the sequencing results shown in Table 7 are evaluated: a. After quality shearing (Q20 ≥ 95%) and removal of primer sequences from the forward and reverse Sanger reads, local alignment was performed with the corresponding theoretical read sequence fragments (SEQ ID NO: 9, SEQ ID NO: 10) to confirm the read locations and coverage. Subsequently, all valid reads were incorporated, and a full alignment was performed with the theoretical sequence (SEQ ID NO: 11) to generate a consensus sequence for the sample. If both the -RT negative control and the blank control are negative, and the target region is fully covered (bidirectional Q20 coverage of key sites, overlap ≥ 50 bp, and consistency ≥ 99%), the test result is determined based on the consistency between the consensus sequence and the theoretical sequence. Specifically, if the consensus sequence and the theoretical sequence are completely identical within the coverage area, it is judged as negative (no variation detected). If there are stable and reproducible base differences, which are confirmed in the bidirectional reads, the variation names at the cDNA and protein levels are given according to the HGVS standard. b. For insertions / deletions, at least one side must be read through ≥30~50 bp on both sides. For mixed peaks, the secondary peak / main peak must be ≥20~25% and consistent in both directions before it can be reported as a suspected low-frequency variant. c. For cases with exon cascade deletion, partial exon end deletion / insertion, or intron sequence inclusion, the splicing is considered abnormal and should be verified using specific primers across the new splice junction or internal primers for long fragment products. Among them, splicing aberrations refer to abnormal splicing events that occur in mRNA transcripts, including exon skipping and aberrant splicing. Exon skipping refers to the complete skipping of one or more exons, resulting in sequence deletion. Aberrant splicing, in a broad sense, includes alternative 5' / 3' splicesites (leading to partial deletion / insertion of exon ends), intron retention, pseudoexon inclusion, etc. d. For cases with only unidirectional low quality or bidirectional conflict but which cannot be determined based on quality values, the conclusion is marked as "uncertain," and it is recommended to reamplify and retest or use cloning sequencing for confirmation.

[0057] The above-described determination process is used to determine the correspondence between the PCR amplification of the TP53 gene exon coding region using the amplification primer pair (the first forward primer with nucleotide sequence as shown in SEQ ID NO:1 and the first reverse primer with nucleotide sequence as shown in SEQ ID NO:2) in the kit provided in Example 2, and the obtained PCR amplification products are sequenced in segments using the internal sequencing primer pair (the second forward primer with nucleotide sequence as shown in SEQ ID NO:3 and the second reverse primer with nucleotide sequence as shown in SEQ ID NO:4) in the kit provided in Example 2, and the theoretical sequence in SEQ ID NO:11. This ensures that the reported conclusions are reproducible and traceable under the premise that Q20 ≥ 95%.

[0058] Referring to the above judgment process and based on the final sequencing and agarose gel electrophoresis results, the -RT negative control showed no amplification. However, the PCR amplification product obtained by using the amplification primer pair in the kit provided in Example 2 to detect whole blood samples was approximately 1182 bp in length. The theoretical reads corresponding to the internal sequencing primer pair were consistent with the provided theoretical sequence (the specific nucleotide sequence is shown in SEQ ID NO:11). The above results prove that the amplification primer pair in the kit provided in Example 2 (the first forward primer with nucleotide sequence as shown in SEQ ID NO:1 and the first reverse primer with nucleotide sequence as shown in SEQ ID NO:2) is specific to the exon coding region of the TP53 gene. It can amplify the cDNA obtained by reverse transcription of RNA extracted from fresh EDTA anticoagulated whole blood to obtain PCR amplification products consistent with the theoretical sequence. The PCR amplification products were sequenced using Sanger sequencing technology, thereby achieving the purpose of detecting mutations in the exon region of the TP53 gene.

[0059] Based on the agarose gel electrophoresis and sequencing results of the PCR amplification products, it can be seen that, compared with the kits provided in Comparative Examples 3-4, the kit provided in Example 2 can detect mutations in the exon regions of the TP53 gene.

[0060] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention, but such modifications or substitutions are all within the scope of protection of the present invention.

Claims

1. A primer pair for detecting mutations in the exon region of the TP53 gene, characterized in that: The primer pair includes a first forward primer and a first reverse primer, the nucleotide sequence of the first forward primer is shown in SEQ ID NO:1, and the nucleotide sequence of the first reverse primer is shown in SEQ ID NO:2; The TP53 gene exon region includes part of exon 2, all of exons 3 to 10, and part of exon 11.

2. The use of the primer pair for detecting exon region mutations of the TP53 gene as described in claim 1 in the preparation of a kit for detecting exon region mutations of the TP53 gene.

3. A kit for detecting mutations in the exon region of the TP53 gene, characterized in that: The kit includes component A, which includes an amplification primer pair, wherein the amplification primer pair is the primer pair for detecting exon region mutations of the TP53 gene as described in claim 1.

4. The kit for detecting mutations in the exon region of the TP53 gene as described in claim 3, characterized in that: Component A further includes an internal sequencing primer pair, which includes a second forward primer and a second reverse primer. The nucleotide sequence of the second forward primer is shown in SEQ ID NO:3, and the nucleotide sequence of the second reverse primer is shown in SEQ ID NO:

4.

5. The kit for detecting mutations in the exon region of the TP53 gene as described in claim 3, characterized in that: The kit also includes component B, which comprises a total RNA extraction reagent, including Trizol.

6. The kit for detecting mutations in the exon region of the TP53 gene as described in claim 5, characterized in that: Component B also includes chloroform, isopropanol, ethanol and DEPC.

7. The kit for detecting mutations in the exon region of the TP53 gene as described in claim 3, characterized in that: The kit also includes component C, which comprises oligo(dT), dNTPs, reverse transcriptase, RNase inhibitor, and reverse transcription buffer. The reverse transcription buffer comprises the following components: 40-60 mM Tris-HCl, 60-80 mM KCl, 2-4 mM MgCl2, and 5-15 mM DTT.

8. The kit for detecting mutations in the exon region of the TP53 gene as described in claim 3, characterized in that: The kit also includes component D, which includes PCR reagents, specifically 2×Primer Star MaxPremix.

9. The kit for detecting mutations in the exon region of the TP53 gene as described in claim 3, characterized in that: The kit also includes component E, which comprises PCR product purification reagent and PCR product sequencing reagent; The PCR product purification reagent comprises the following components: 5~20 mg / mL magnetic bead suspension, purification buffer, washing buffer, and elution buffer; The purification buffer contains 15-25% (w / v) PEG 8000, 1-2 M NaCl, and 5-20 mM Tris-HCl; The washing solution is an ethanol solution with a volume fraction of 70-85%; The eluent is nuclease-free water or Tris-HCl with a concentration of 10 mM and a pH of 8.0-8.

8.

10. The use of the kit for detecting mutations in the exon region of the TP53 gene as described in any one of claims 3 to 9 in the preparation of products for tumor molecular subtyping, efficacy evaluation, and prognosis.

Images