Kit for detecting TP53 gene mutation

Abstract

The invention provides a kit for detecting TP53 gene mutation. The kit for detecting TP53 gene mutation provided by the invention can accurately detect six TP53 gene mutation sites, namely R175H, R249S, R282W, R248Q, R273H and G245S, in genome DNA to be detected at the same time through a multiple fluorescent PCR reaction system and a multi-channel fluorescent signal, can accurately detect the six mutation sites, has ultrahigh amplification efficiency, detection efficiency, sensitivity and specificity, and can be widely applied to detection of TP53 gene mutation. The genotype of a sample with the genome DNA content of 1 ng can be accurately detected, the operation is simple and convenient, and the cost is low.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to a kit for detecting TP53 gene mutations. Background Technology

[0002] As one of the most important tumor suppressor genes in humans, the TP53 gene participates in biological processes such as regulating cell growth cycle, repairing damaged DNA, and inducing apoptosis. The p53 protein it encodes plays a core role in maintaining genome stability and regulating cell cycle and apoptosis.

[0003] Mutations in the DNA-binding region of the TP53 gene are closely related to the development and progression of more than 50% of human malignancies, including ovarian cancer, lung cancer, liver cancer, breast cancer, colorectal cancer, and other common cancers. TP53 gene mutations are mainly concentrated in its highly conserved functional domains, and the vast majority are missense mutations, leading to the loss of normal tumor-suppressive function and even the acquisition of tumor-promoting activity in the p53 protein. TP53 gene mutations may be one of the major pathogenic factors for human tumors. Accurate and efficient detection of common TP53 gene mutation sites is crucial for assessing tumor risk, strengthening screening in high-risk groups, achieving early detection and treatment, improving treatment outcomes, and significantly impacting patient survival.

[0004] Currently, TP53 gene mutation detection mainly relies on various molecular biology techniques. Sanger sequencing, as the traditional gold standard, offers high accuracy and can detect unknown mutations, but its low sensitivity (typically requiring a mutation frequency >15-20% for reliable detection), cumbersome operation, and long processing time make it unsuitable for large-scale clinical sample or low-frequency mutation detection needs. Next-generation sequencing (NGS) technology, while enabling high-throughput, multi-gene parallel analysis, is limited in its widespread application in routine clinical diagnosis due to its expensive equipment, complex library construction process, lengthy testing cycle, and demanding bioinformatics analysis capabilities. Traditional qPCR technology cannot distinguish high-frequency mutation subtypes and is susceptible to wild-type interference.

[0005] Detection methods based on specific primers, such as amplification arrest mutation system PCR (ARMS-PCR), have become a common choice for targeted mutation detection in clinical practice due to their simplicity, low cost, and high sensitivity (detecting 1-5% mutation frequency). However, existing ARMS-PCR technology still has significant limitations: First, it can usually only design detection systems for single or a small number of mutation sites. For scenarios requiring simultaneous screening of multiple mutation sites, multiple independent reactions are necessary, resulting in high sample consumption, low detection efficiency, and a significant increase in cost. Second, the TaqMan probes used in conventional ARMS-PCR still lack specificity in distinguishing between highly homologous wild-type and mutant sequences, especially in regions rich in GC or with complex secondary structures, which are prone to false positives or false negatives. In addition, primer interactions in multiplex PCR reaction systems can easily lead to non-specific amplification or primer dimers, severely affecting amplification efficiency and detection accuracy.

[0006] Therefore, there is an urgent need to develop a kit that can accurately detect multiple TP53 gene mutation sites simultaneously, and has high detection efficiency, high sensitivity, high specificity, and low cost. Summary of the Invention

[0007] To address the problems of low sensitivity, specificity, detection efficiency, and high cost in existing technologies for detecting multiple TP53 gene mutation sites, this invention provides a kit for detecting TP53 gene mutations.

[0008] According to a first aspect of the present invention, a kit for detecting TP53 gene mutations is provided, the kit comprising reagent A, reagent B, reagent C, reagent D, reagent E and reagent F; Reagent A includes R282W wild-type primers, R248Q wild-type primers, R282W detection probes, R248Q detection probes, R282W universal primers, and R248Q universal primers. The nucleotide sequence of the R282W wild-type primers is shown in SEQ ID NO:1, and the nucleotide sequence of the R248Q wild-type primers is shown in SEQ ID NO:2. Reagent B includes R282W mutant primers, R248Q mutant primers, R282W detection probes, R248Q detection probes, R282W universal primers, and R248Q universal primers. The nucleotide sequence of the R282W mutant primers is shown in SEQ ID NO:3, and the nucleotide sequence of the R248Q mutant primers is shown in SEQ ID NO:4. In reagents A and B, the nucleotide sequence of the R282W detection probe is shown in SEQ ID NO:5, the nucleotide sequence of the R248Q detection probe is shown in SEQ ID NO:6, the nucleotide sequence of the R282W universal primer is shown in SEQ ID NO:7, and the nucleotide sequence of the R248Q universal primer is shown in SEQ ID NO:8. Reagent C includes R273H wild-type primers, G245S wild-type primers, R273H detection probes, G245S detection probes, R273H universal primers, and G245S universal primers. The nucleotide sequence of the R273H wild-type primers is shown in SEQ ID NO:9, and the nucleotide sequence of the G245S wild-type primers is shown in SEQ ID NO:10. Reagent D includes R273H mutant primers, G245S mutant primers, R273H detection probes, G245S detection probes, R273H universal primers, and G245S universal primers. The nucleotide sequence of the R273H mutant primers is shown in SEQ ID NO:11, and the nucleotide sequence of the G245S mutant primers is shown in SEQ ID NO:12. In reagents C and D, the nucleotide sequence of the R273H detection probe is shown in SEQ ID NO:13, the nucleotide sequence of the G245S detection probe is shown in SEQ ID NO:14, the nucleotide sequence of the R273H universal primer is shown in SEQ ID NO:15, and the nucleotide sequence of the G245S universal primer is shown in SEQ ID NO:16. Reagent E includes R175H wild-type primer, R249S wild-type primer, R175H detection probe, R249S detection probe, R175H universal primer and R249S universal primer. The nucleotide sequence of the R175H wild-type primer is shown in SEQ ID NO:17, and the nucleotide sequence of the R249S wild-type primer is shown in SEQ ID NO:18. Reagent F includes R175H mutant primers, R249S mutant primers, R175H detection probes, R249S detection probes, R175H universal primers, and R249S universal primers. The nucleotide sequence of the R175H mutant primers is shown in SEQ ID NO:19, and the nucleotide sequence of the R249S mutant primers is shown in SEQ ID NO:20. In reagents E and F, the nucleotide sequence of the R175H detection probe is shown in SEQ ID NO:21, the nucleotide sequence of the R249S detection probe is shown in SEQ ID NO:22, the nucleotide sequence of the R175H universal primer is shown in SEQ ID NO:23, and the nucleotide sequence of the R249S universal primer is shown in SEQ ID NO:24.

[0009] The R175H mutation changes the amino acid at position 175 of the p53 protein encoded by the TP53 gene from arginine (R) to histidine (H); the R249S mutation changes the amino acid at position 249 of the p53 protein encoded by the P53 gene from arginine (R) to serine (S); the R282W mutation changes the amino acid at position 282 of the p53 protein encoded by the P53 gene from arginine (R) to tryptophan (W); the R248Q mutation changes the amino acid at position 248 of the p53 protein encoded by the P53 gene from arginine (R) to glutamine (Q); the R273H mutation changes the amino acid at position 273 of the p53 protein encoded by the P53 gene from arginine (R) to histidine (H); and the G245S mutation changes the amino acid at position 245 of the p53 protein encoded by the P53 gene from glycine (G) to serine (S).

[0010] This invention is based on Amplification Refractory Mutation System PCR (ARMS-PCR) technology. Targeting six TP53 gene mutation sites—R175H, R249S, R282W, R248Q, R273H, and G245S—wild-type and mutant primers were designed and screened to obtain primers for each of these six target mutation sites. Simultaneously, detection probes and universal primers were designed for detecting these six mutation sites. The universal primers can form primer sets (equivalent to upstream and downstream primers) with the wild-type and mutant primers for amplifying the DNA to be tested. Multiple wild-type primers, mutant primers, universal primers, and detection probes are used as components of the kit, resulting in the kit provided by this invention for detecting TP53 gene mutations.

[0011] The kit provided by this invention uses ARMS-PCR to design specific primers and probes for six TP53 gene mutation sites: R175H, R249S, R282W, R248Q, R273H, and G245S. These primers and probes can specifically bind to the target gene. During the PCR extension reaction, the exonuclease activity of Taq enzyme can cleave the fluorescent group at the 5' end of the detection probe from the probe, making it free in the reaction system. This removes the shielding of the 3' end fluorescence quencher group, allowing it to receive light stimulation and emit fluorescence that can be detected by the instrument. This enables the detection of the target gene in a completely closed reaction system.

[0012] Using the kit for detecting TP53 gene mutations provided by this invention, six TP53 gene mutation sites (R175H, R249S, R282W, R248Q, R273H, and G245S) in the genomic DNA to be tested can be accurately detected simultaneously through a multiplex fluorescent PCR reaction system and multi-channel fluorescence signals. The kit can accurately detect the above six mutation sites and has extremely high amplification efficiency, detection efficiency, sensitivity, and specificity. It can accurately detect the genotype of samples with a genomic DNA content of 1 ng. The kit is simple to operate and low in cost.

[0013] The kit provided by this invention has broad application prospects in the fields of molecular diagnosis, prognostic assessment, efficacy monitoring and personalized medicine for cancer. It provides clinicians and researchers with an efficient and stable detection tool, and solves the problems of low throughput, insufficient sensitivity, cumbersome operation or imperfect quality control in the existing technology. It is of great significance in the detection of TP53 gene mutations.

[0014] Preferably, the R282W detection probe, R248Q detection probe, R273H detection probe, G245S detection probe, R175H detection probe, and R249S detection probe all contain a quenching group and a fluorescent group, wherein the quenching group is MGB and the fluorescent group is FAM or ROX.

[0015] Preferably, the quenching group is located at the 3' end of the R282W, R248Q, R273H, G245S, R175H, and R249S detection probes; and the fluorescent group is located at the 5' end of the R282W, R248Q, R273H, G245S, R175H, and R249S detection probes.

[0016] By labeling the quencher group MGB at the 3' end and the fluorescent group FAM or ROX at the 5' end of the detection probe, the MGB group greatly enhances the binding ability of the probe to the target sequence (increased Tm value), significantly improves the ability to distinguish single base mismatches, and ensures that low-frequency mutations can be detected with high specificity and high sensitivity in complex samples (such as tumor samples with a large amount of wild-type background), effectively reducing false positive and false negative results.

[0017] Preferably, the kit further includes an internal control primer set, which includes a first internal control primer and a second internal control primer. The nucleotide sequence of the first internal control primer is shown in SEQ ID NO:25, and the nucleotide sequence of the second internal control primer is shown in SEQ ID NO:26.

[0018] Preferably, the kit further includes an internal control probe, the nucleotide sequence of which is shown in SEQ ID NO:27.

[0019] Preferably, the 3' end of the internal control probe is labeled with a quenching group MGB, and the 5' end of the internal control probe is labeled with a fluorescent group CY5.

[0020] The housekeeping gene (RPPH1) is stably expressed in various human tissues. The first and second internal control primers in the internal control primer set of the kit provided in this protocol are both designed for the housekeeping gene (RPPH1). If the internal control primers do not work during the detection process using the kit for detecting TP53 gene mutations, it indicates that no DNA template has been added or the amplification system is not working properly, and the experiment fails. Therefore, by introducing the internal control primer set and internal control probe into the kit for detecting TP53 gene mutations, this protocol can be used to detect whether the amplification system can amplify normally, thereby further improving the accuracy of the detection results.

[0021] The internal standard in the kit provided in this protocol uses a pair of housekeeping gene-specific primers (i.e., the first and second internal control primers in the internal control primer set), which, combined with a specific probe, can specifically bind to a segment of DNA template in the middle of the primer amplification region. The internal standard uses the CY5 channel, thereby enabling monitoring of the detection process in a fully closed reaction system and effectively monitoring the occurrence of false negatives.

[0022] Preferably, the kit further includes an external control plasmid set, which includes a first external control plasmid and a second external control plasmid. The nucleotide sequence of the first external control plasmid is shown in SEQ ID NO:28, and the nucleotide sequence of the second external control plasmid is shown in SEQ ID NO:29.

[0023] The first and second external control plasmids in the external control plasmid group of the kit provided in this protocol both contain the wild-type gene sequence of the conserved region of TP53.

[0024] This method introduces a set of external control plasmids containing a first and a second external control plasmid into a kit for detecting TP53 gene mutations. This allows for the detection of whether the overall PCR amplification system is functioning correctly, acting as a positive control. If the external control plasmid detection fails, it indicates that the kit is faulty or the experimental instrument is malfunctioning, and the results are unreliable. Therefore, introducing a set of external control plasmids containing a first and a second external control plasmid into a kit for detecting TP53 gene mutations improves the reliability of the final detection results.

[0025] Preferably, the above-mentioned kit also includes a negative control, which is physiological saline.

[0026] This approach introduces a negative control into the kit used to detect TP53 gene mutations to detect contamination in the system, providing a stable and controllable chemical environment for the entire detection process and ensuring the specificity, sensitivity, and reliability of the reaction.

[0027] Preferably, the kit further includes a PCR premix, which comprises the following components: hot-start Taq DNA polymerase, 10-1000 μM dNTPs, 2-10 mM MgCl2, 0.01-0.5 wt% dimethyl sulfoxide (DMSO), and 0.01-0.5 wt% formamide.

[0028] Preferably, the PCR premix further includes a PCR buffer containing Tris, NaCl, ethyl phenyl polyethylene glycol (NP-40), and MgCl2.

[0029] Preferably, the kit further includes a nucleic acid release agent comprising the following components: 0.01-0.1 MHCl, 0.01-0.05 wt% sodium dodecyl sulfate (SDS), 1-10 mM Surfactin, and 0.05-3 wt% trehalose.

[0030] Surfactin is a surfactant with an amphiphilic structure produced by fermentation of the microorganism Bacillus subtilis. Its molecular formula is C0. 53 H 93 N7O 13 .

[0031] By introducing PCR premix and nucleic acid release agent into a kit for detecting TP53 gene mutations and regulating their components and concentrations to meet the aforementioned ranges, the PCR premix containing these components ensures efficient, specific, and stable multiplex PCR amplification targeting specific mutation sites in the TP53 gene. The nucleic acid release agent containing these components efficiently lyses cells, releases nucleic acids, and to some extent protects the integrity of the nucleic acids. Furthermore, the addition of Surfactin and trehalose helps reduce inhibitor interference and stabilize nucleic acids. In addition, the PCR premix and nucleic acid release agent can simplify or replace traditional DNA extraction and purification steps, greatly simplifying the nucleic acid extraction steps and the preparation steps of the PCR amplification system. This enables rapid release and direct detection of the sample, significantly shortening the overall detection time, resulting in high detection efficiency, simple operation, and low cost, making it particularly suitable for rapid clinical testing scenarios. Attached Figure Description

[0032] Figure 1 The amplification curves for testing R175H wild-type and mutant samples were obtained using the R175H wild-type primers and R175H mutant primers provided in the kits for detecting TP53 gene mutations provided in Example 1 and Comparative Examples 1-4.

[0033] Figure 2 The amplification curves for testing R249 wild-type and mutant samples were obtained using the R249S wild-type primers and R249S mutant primers from the kits provided in Example 1 and Comparative Examples 5-8 for detecting TP53 gene mutations.

[0034] Figure 3 The amplification curves for detecting R282W wild-type and mutant samples were obtained using the R282W wild-type primers and R282W mutant primers provided in the kits for detecting TP53 gene mutations provided in Example 1 and Comparative Examples 9-12.

[0035] Figure 4 The amplification curves for detecting R248Q wild-type and mutant samples were obtained using the R248Q wild-type primers and R248Q mutant primers provided in the kits for detecting TP53 gene mutations provided in Example 1 and Comparative Examples 13-16 for Test Example 1.

[0036] Figure 5 The amplification curves for detecting R273H wild-type and mutant samples were obtained using the R273H wild-type primers and R273H mutant primers provided in the kits for detecting TP53 gene mutations provided in Example 1 and Comparative Examples 17-20 for Test Example 1.

[0037] Figure 6 The amplification curves for testing G245S wild-type and mutant samples were obtained using the G245S wild-type primers and G245S mutant primers provided in the kits for detecting TP53 gene mutations provided in Example 1 and Comparative Examples 21-24.

[0038] Figure 7 The amplification curve results for clinical specimen 1 and clinical specimen 2 were obtained by using the kit for detecting TP53 gene mutations provided in Examples 1-3, and referring to the detection method in Example 1.

[0039] Figure 8 The amplification curve of the internal control when testing wild-type and mutant samples using the kit for detecting TP53 gene mutations provided in Example 1 in Test Example 3.

[0040] Figure 9 The amplification curves of wild-type and mutant R282W and R248Q samples were detected using the kit for detecting TP53 gene mutations provided in Example 1 for Test Example 3.

[0041] Figure 10 The amplification curves of wild-type and mutant R273H and G245S samples were detected using the kit for detecting TP53 gene mutations provided in Example 1 for Test Example 3.

[0042] Figure 11 The amplification curves of wild-type and mutant R175H and R249S samples were detected using the kit for detecting TP53 gene mutations provided in Example 1 for Test Example 3. Detailed Implementation

[0043] 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.

[0044] Example 1 A kit for detecting TP53 gene mutations, comprising reagent A, reagent B, reagent C, reagent D, reagent E, reagent F, external control plasmid set (i.e., positive control), physiological saline (i.e., negative control), PCR premix, and nucleic acid release agent; The PCR premix includes the following components: hot-start Taq DNA polymerase, 10–1000 μM dNTPs, 2–10 mM MgCl2, 0.01–0.5 wt% DMSO, 0.01–0.5 wt% formamide, and PCR buffer (containing Tris, NaCl, NP-40, and MgCl2). The nucleic acid release agent comprises the following components: 0.01~0.1 M HCl, 0.01~0.05 wt% SDS, 1~10 mM Surfactin, and 0.05~3 wt% Trehalose; Reagent A includes R282W wild-type primers, R248Q wild-type primers, R282W detection probes, R248Q detection probes, R282W universal primers, and R248Q universal primers. The nucleotide sequence of the R282W wild-type primers is shown in SEQ ID NO:1, and the nucleotide sequence of the R248Q wild-type primers is shown in SEQ ID NO:2. Reagent B includes R282W mutant primers, R248Q mutant primers, R282W detection probes, R248Q detection probes, R282W universal primers, and R248Q universal primers. The nucleotide sequence of the R282W mutant primers is shown in SEQ ID NO:3, and the nucleotide sequence of the R248Q mutant primers is shown in SEQ ID NO:4. In reagents A and B, the nucleotide sequence of the R282W detection probe is shown in SEQ ID NO:5, the nucleotide sequence of the R248Q detection probe is shown in SEQ ID NO:6, the nucleotide sequence of the R282W universal primer is shown in SEQ ID NO:7, and the nucleotide sequence of the R248Q universal primer is shown in SEQ ID NO:8. Reagent C includes R273H wild-type primers, G245S wild-type primers, R273H detection probes, G245S detection probes, R273H universal primers, and G245S universal primers. The nucleotide sequence of the R273H wild-type primers is shown in SEQ ID NO:9, and the nucleotide sequence of the G245S wild-type primers is shown in SEQ ID NO:10. Reagent D includes R273H mutant primers, G245S mutant primers, R273H detection probes, G245S detection probes, R273H universal primers, and G245S universal primers. The nucleotide sequence of the R273H mutant primers is shown in SEQ ID NO:11, and the nucleotide sequence of the G245S mutant primers is shown in SEQ ID NO:12. In reagents C and D, the nucleotide sequence of the R273H detection probe is shown in SEQ ID NO:13, the nucleotide sequence of the G245S detection probe is shown in SEQ ID NO:14, the nucleotide sequence of the R273H universal primer is shown in SEQ ID NO:15, and the nucleotide sequence of the G245S universal primer is shown in SEQ ID NO:16. Reagent E includes R175H wild-type primer, R249S wild-type primer, R175H detection probe, R249S detection probe, R175H universal primer and R249S universal primer. The nucleotide sequence of the R175H wild-type primer is shown in SEQ ID NO:17, and the nucleotide sequence of the R249S wild-type primer is shown in SEQ ID NO:18. Reagent F includes R175H mutant primers, R249S mutant primers, R175H detection probes, R249S detection probes, R175H universal primers, and R249S universal primers. The nucleotide sequence of the R175H mutant primers is shown in SEQ ID NO:19, and the nucleotide sequence of the R249S mutant primers is shown in SEQ ID NO:20. In reagents E and F, the nucleotide sequence of the R175H detection probe is shown in SEQ ID NO:21, the nucleotide sequence of the R249S detection probe is shown in SEQ ID NO:22, the nucleotide sequence of the R175H universal primer is shown in SEQ ID NO:23, and the nucleotide sequence of the R249S universal primer is shown in SEQ ID NO:24. Reagents A, B, C, D, E, and F all include internal control primer sets and internal control probes.

[0045] The nucleotide sequences of the primers and probes contained in the kit for detecting TP53 gene mutations provided in this embodiment are shown in Table 1.

[0046] Table 1. Nucleotide sequences of primers and probes contained in the kit for detecting TP53 gene mutations.

[0047] The specific steps for using the above-mentioned kit for detecting TP53 gene mutations to detect DNA extracted from tissues or blood are as follows: (1) DNA extraction Extract the DNA to be tested from tissue or blood at a concentration ≥1 ng / μL; or treat EDTA-anticoagulated whole blood samples with the nucleic acid release agent in the kit to obtain the DNA to be tested. (2) qPCR amplification Add the DNA to be tested to the PCR premix (the PCR premix includes hot-start Taq DNA polymerase, 200 μM dNTPs, 5 mM MgCl2, 0.2 wt% DMSO, 0.2 wt% formamide, and PCR buffer containing Tris, NaCl, NP40, and MgCl2), mix well, and amplify the DNA using qPCR (real-time quantitative polymerase chain reaction) according to the following reaction program: pre-denaturation at 95℃ for 5 minutes; cycling phase: 95℃ for 15 seconds → 60℃ for 30 seconds (36 cycles); collect fluorescence signals at 60℃: FAM, CY5, and ROX.

[0048] In the above experiments, to detect the genotype of the target mutation site in the TP53 gene, and to avoid the difficulty in distinguishing between wild-type and mutant target sites during detection by adding both wild-type and mutant primers targeting the same mutation site in the same system, two systems were set up: System 1 and System 2, System 3 and System 4, and System 5 and System 6. Systems 1, 3, and 5 used wild-type primers for detecting the wild-type TP53 gene target site, while systems 2 and 4 used wild-type primers. 6. Mutant primers are added for detecting the target site of the mutant TP53 gene. Specifically, in addition to the DNA to be tested, system 1 also includes reagent A (R282W wild-type primers, R282W universal primers, R282W detection probe, R248Q wild-type primers, R248Q universal primers, R248Q detection probe, internal control primer set, internal control probe), and system 2 also includes reagent B (R282W mutant primers, R282W universal primers, R282W detection probe, R248Q mutant primers, R2...). System 3 contains 48Q universal primers, R248Q detection probes, internal control primer sets, and internal control probes. System 4 also contains reagent C (R273H wild-type primers, R273H universal primers, R273H detection probes, G245S wild-type primers, G245S universal primers, G245S detection probes, internal control primer sets, and internal control probes). System 4 also contains reagent D (R273H mutant primers, R273H universal primers, R273H detection probes, G245S mutant primers, G245S universal primers, and G245S detection probes). System 5 also includes reagent E (R175H wild-type primers, R175H universal primers, R175H detection probe, R249S wild-type primers, R249S universal primers, R249S detection probe, internal control primer set, internal control probe), and system 6 also includes reagent F (R175H mutant primers, R175H universal primers, R175H detection probe, R249S mutant primers, R249S universal primers, R249S detection probe, internal control primer set, internal control probe).

[0049] (3) Result interpretation Table 2. Result interpretation criteria for detecting the genotype of the target site of the mutant TP53 gene using a kit for detecting TP53 gene mutations.

[0050] The final test results (genotype of TP53 gene mutation site) are judged according to the result interpretation criteria shown in Table 2. If the Ct value of the same gene to be tested is ≤36 in both wells (i.e., two systems) and |ΔCt|<5, it is judged as a heterozygous genotype. However, if 3.5<|ΔCt|<5, it is considered to be a heterozygous result. This result belongs to the gray area and can be output as heterozygous.

[0051] Simultaneously, internal control primer sets (internal reference), internal control probes, external control plasmid sets (positive control), and physiological saline (negative control) were added to each system to determine whether the detection results were valid. For positive control Ct≤33 and negative control Ct>36, the experimental results were valid and analyzed. For the detection sample reaction wells, if Ct≤34 for the internal control housekeeping gene RPPH1 (CY5 channel), the result of that well was valid; if Ct>34 for the internal control housekeeping gene RPPH1 (CY5 channel), the result of that well was invalid in this experiment. For the internal control |ΔCT|≤3 between two wells of the same gene, the results of the target site in that sample were analyzed; if |ΔCT|>3, the difference in sample loading was considered too large, and the detection result of that gene was invalid.

[0052] Example 2 This embodiment provides a kit for detecting TP53 gene mutations. Compared with Example 1, the difference in composition is that the nucleotide sequence of the internal control probe is as shown in SE ID NO:30, and the specific nucleotide sequence is 5'-CY5-AGCTTGGAACAGACTCACGGC-MGB-3'.

[0053] Apart from the differences mentioned above, the materials, formulation ratios, and preparation operations used in this embodiment are strictly consistent with those in Example 1.

[0054] Example 3 This embodiment provides a kit for detecting TP53 gene mutations. Compared with Example 1, the difference in composition is that the nucleotide sequence of the first internal control primer in the internal control primer set is shown in SEQ ID NO:31, and the nucleotide sequence of the second internal control primer is shown in SEQ ID NO:32. The specific nucleotide sequence of the first internal control primer is 5'-ATTACCACTACTCAGGATA-3', and the specific nucleotide sequence of the second internal control primer is 5'-CAGATAGCGATGGTGAGCA-3'.

[0055] Apart from the differences mentioned above, the materials, formulation ratios, and preparation operations used in this embodiment are strictly consistent with those in Example 1.

[0056] Comparative Example 1 This comparative example provides a kit for detecting TP53 gene mutations. Compared with Example 1, the difference in composition is that the nucleotide sequence of the R175H wild-type primer in reagent E is as shown in SEQ ID NO:33, and the specific nucleotide sequence is 5'-GACGGAGGTTGTGAGACG-3'.

[0057] 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.

[0058] Comparative Example 2 This comparative example provides a kit for detecting TP53 gene mutations. Compared with Example 1, the difference in composition is that the nucleotide sequence of the R175H wild-type primer in reagent E is as shown in SEQ ID NO:34, and the specific nucleotide sequence is 5'-GACGGAGGTTGTGAAGCG-3'.

[0059] 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.

[0060] Comparative Example 3 This comparative example provides a kit for detecting TP53 gene mutations. Compared with Example 1, the difference in composition is that the nucleotide sequence of the R175H mutant primer in reagent F is as shown in SEQ ID NO:35, and the specific nucleotide sequence is 5'-GACGGAGGTTGTGAGACA-3'.

[0061] 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.

[0062] Comparative Example 4 This comparative example provides a kit for detecting TP53 gene mutations. Compared with Example 1, the difference in composition is that the nucleotide sequence of the R175H mutant primer in reagent F is as shown in SEQ ID NO:36, and the specific nucleotide sequence is 5'-GACGGAGGTTGTGAAGCA-3'.

[0063] 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.

[0064] Comparative Example 5 This comparative example provides a kit for detecting TP53 gene mutations. Compared with Example 1, the difference in composition is that the nucleotide sequence of the R249S wild-type primer in reagent E is as shown in SEQ ID NO:37, and the specific nucleotide sequence is 5'-GATGATGGTGAGGATGAGC-3'.

[0065] 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.

[0066] Comparative Example 6 This comparative example provides a kit for detecting TP53 gene mutations. Compared with Example 1, the difference in composition is that the nucleotide sequence of the R249S wild-type primer in reagent E is as shown in SEQ ID NO:38, and the specific nucleotide sequence is 5'-GATGATGGTGAGGATAGGC-3'.

[0067] 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.

[0068] Comparative Example 7 This comparative example provides a kit for detecting TP53 gene mutations. Compared with Example 1, the difference in composition is that the nucleotide sequence of the R249S mutant primer in reagent F is as shown in SEQ ID NO:39, and the specific nucleotide sequence is 5'-GATGATGGTGAGGATGAGT-3'.

[0069] 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.

[0070] Comparative Example 8 This comparative example provides a kit for detecting TP53 gene mutations. Compared with Example 1, the difference in composition is that the nucleotide sequence of the R249S mutant primer in reagent F is as shown in SEQ ID NO:40, and the specific nucleotide sequence is 5'-GATGATGGTGAGGATAGGT-3'.

[0071] 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.

[0072] Comparative Example 9 This comparative example provides a kit for detecting TP53 gene mutations. Compared with Example 1, the difference in composition is that the nucleotide sequence of the R282W wild-type primer in reagent A is as shown in SEQ ID NO:41, and the specific nucleotide sequence is 5'-TCTCTTCCTCTGTGCGTCG-3'.

[0073] 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.

[0074] Comparative Example 10 This comparative example provides a kit for detecting TP53 gene mutations. Compared with Example 1, the difference in composition is that the nucleotide sequence of the R282W wild-type primer in reagent A is as shown in SEQ ID NO:42, and the specific nucleotide sequence is 5'-TCTCTTCCTCTGTGCACCG-3'.

[0075] 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.

[0076] Comparative Example 11 This comparative example provides a kit for detecting TP53 gene mutations. Compared with Example 1, the difference in composition is that the nucleotide sequence of the R282W mutant primer in Reagent B is as shown in SEQ ID NO:43, and the specific nucleotide sequence is 5'-TCTCTTCCTCTGTGCGTCA-3'.

[0077] 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.

[0078] Comparative Example 12 This comparative example provides a kit for detecting TP53 gene mutations. Compared with Example 1, the difference in composition is that the nucleotide sequence of the R282W mutant primer in Reagent B is as shown in SEQ ID NO:44, and the specific nucleotide sequence is 5'-TCTCTTCCTCTGTGCACCA-3'.

[0079] 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.

[0080] Comparative Example 13 This comparative example provides a kit for detecting TP53 gene mutations. Compared with Example 1, the difference in composition is that the nucleotide sequence of the R248Q wild-type primer in reagent A is as shown in SEQ ID NO:45, and the specific nucleotide sequence is 5'-ATGGGCGGCATGAATCG-3'.

[0081] 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.

[0082] Comparative Example 14 This comparative example provides a kit for detecting TP53 gene mutations. Compared with Example 1, the difference in composition is that the nucleotide sequence of the R248Q wild-type primer in reagent A is as shown in SEQ ID NO:46, and the specific nucleotide sequence is 5'-ATGGGCGGCATGAGCCG-3'.

[0083] 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.

[0084] Comparative Example 15 This comparative example provides a kit for detecting TP53 gene mutations. Compared with Example 1, the difference in composition is that the nucleotide sequence of the R248Q mutant primer in Reagent B is as shown in SEQ ID NO:47, and the specific nucleotide sequence is 5'-ATGGGCGGCATGAATCA-3'.

[0085] 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.

[0086] Comparative Example 16 This comparative example provides a kit for detecting TP53 gene mutations. Compared with Example 1, the difference in composition is that the nucleotide sequence of the R248Q mutant primer in Reagent B is as shown in SEQ ID NO:48, and the specific nucleotide sequence is 5'-ATGGGCGGCATGAGCCA-3'.

[0087] 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.

[0088] Comparative Example 17 This comparative example provides a kit for detecting TP53 gene mutations. Compared with Example 1, the difference in composition is that the nucleotide sequence of the R273H wild-type primer in reagent C is as shown in SEQ ID NO:49, and the specific nucleotide sequence is 5'-CAGGACAGGCACAAATAC-3'.

[0089] 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.

[0090] Comparative Example 18 This comparative example provides a kit for detecting TP53 gene mutations. Compared with Example 1, the difference in composition is that the nucleotide sequence of the R273H wild-type primer in reagent C is as shown in SEQ ID NO:50, and the specific nucleotide sequence is 5'-CAGGACAGGCACAAGCAC-3'.

[0091] 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.

[0092] Comparative Example 19 This comparative example provides a kit for detecting TP53 gene mutations. Compared with Example 1, the difference in composition is that the nucleotide sequence of the R273H mutant primer in reagent D is as shown in SEQ ID NO:51, and the specific nucleotide sequence is 5'-CAGGACAGGCACAAATAA-3'.

[0093] 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.

[0094] Comparative Example 20 This comparative example provides a kit for detecting TP53 gene mutations. Compared with Example 1, the difference in composition is that the nucleotide sequence of the R273H mutant primer in reagent D is as shown in SEQ ID NO:52, and the specific nucleotide sequence is 5'-CAGGACAGGCACAAGCAA-3'.

[0095] 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.

[0096] Comparative Example 21 This comparative example provides a kit for detecting TP53 gene mutations. Compared with Example 1, the difference in composition is that the nucleotide sequence of the G245S wild-type primer in reagent C is as shown in SEQ ID NO:53, and the specific nucleotide sequence is 5'-GTAACAGTTCCTGCATGGGGC-3'.

[0097] 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.

[0098] Comparative Example 22 This comparative example provides a kit for detecting TP53 gene mutations. Compared with Example 1, the difference in composition is that the nucleotide sequence of the G245S wild-type primer in reagent C is as shown in SEQ ID NO:54, and the specific nucleotide sequence is 5'-GTAACAGTTCCTGCATGGAAC-3'.

[0099] 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.

[0100] Comparative Example 23 This comparative example provides a kit for detecting TP53 gene mutations. Compared with Example 1, the difference in composition is that the nucleotide sequence of the G245S mutant primer in reagent D is as shown in SEQ ID NO:55, and the specific nucleotide sequence is 5'-GTAACAGTTCCTGCATGGAAA-3'.

[0101] 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.

[0102] Comparative Example 24 This comparative example provides a kit for detecting TP53 gene mutations. Compared with Example 1, the difference in composition is that the nucleotide sequence of the G245S mutant primer in reagent D is as shown in SEQ ID NO:56, and the specific nucleotide sequence is 5'-GTAACAGTTCCTGCATGAGAA-3'.

[0103] 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.

[0104] Test Example 1 This test case aims to investigate the detection efficacy of wild-type and mutant primers targeting six target mutation sites in the kits provided in Example 1 and Comparative Examples 1-24 for detecting TP53 gene mutations.

[0105] 1. Wild-type and mutant primers targeting the target mutation site R175H in Example 1 and Comparative Examples 1-4 For ease of comparison, the nucleotide sequences of the R175H wild-type primers and R175H mutant primers for the target mutation site R175H provided in the kits for detecting TP53 gene mutations provided in Example 1 and Comparative Examples 1-4 are integrated into Table 3.

[0106] Table 3. Nucleotide sequences of the R175H wild-type primers and R175H mutant primers targeting the target mutation site R175H in the kits provided in Examples 1 and Comparative Examples 1-4 for detecting TP53 gene mutations.

[0107] The R175H wild-type and R175H mutant samples were detected using the R175H wild-type primers and R175H mutant primers provided in the kits for detecting TP53 gene mutations provided in Example 1 and Comparative Examples 1-4, respectively. The Ct values ​​are shown in Table 4, and the amplification curves are shown in Table 4. Figure 1 As shown, where, Figure 1Figures A and B show the amplification curve detection results of R175H wild-type and mutant samples, respectively, using the R175H wild-type primers from the kit provided in Example 1. Figure 1 CD represent the amplification curve detection results of R175H wild-type and mutant samples using the R175H wild-type primers provided in Comparative Example 1, respectively. Figure 1 E and F represent the amplification curve detection results of R175H wild-type and mutant samples using the R175H wild-type primers provided in Comparative Example 2, respectively. Figure 1 GH represent the amplification curve detection results of R175H wild-type and mutant samples using the R175H mutant primers provided in Example 1, respectively. Figure 1 IJ represent the amplification curve detection results of R175H wild-type and mutant samples using the R175H mutant primers provided in Comparative Example 3, respectively. Figure 1 KL represents the amplification curve detection results of R175H wild-type and mutant samples using the R175H mutant primers provided in Comparative Example 4.

[0108] Table 4. Detection results of R175H wild-type primers and R175H mutant primers in the kits provided in Examples 1 and Comparative Examples 1-4 for detecting TP53 gene mutations on R175H wild-type and mutant samples.

[0109] Depend on Figure 1As shown in Table 4, when using the R175H wild-type primers in the kit provided in Example 1 to detect R175H wild-type and mutant samples, the R175H wild-type primers with nucleotide sequences as shown in SEQ ID NO:9 had the required efficiency in amplifying R175H wild-type samples, and no R175H mutant samples were detected. However, when using the R175H wild-type primers in the kits provided in Comparative Examples 1-2 to detect R175H wild-type and mutant samples, the R175H wild-type primers with nucleotide sequences as shown in SEQ ID NO:33 and 34 had the required efficiency in amplifying R175H wild-type samples, but R175H mutant samples were detected. When using the R175H mutant primers in the kit provided in Example 1 to detect R175H wild-type and mutant samples, the nucleotide sequences as shown in SEQ ID NO:9... The R175H mutant primer shown in NO:11 amplifies the R175H mutant specimen with the required efficiency and no R175H wild-type specimen is detected. However, when the R175H mutant primers in the kits provided in Comparative Examples 3-4 are used to detect both R175H wild-type and mutant specimens, the R175H mutant primers with nucleotide sequences as shown in SEQ ID NO:35 and 36 amplify the R175H mutant specimen with the required efficiency, but R175H wild-type specimens are detected.

[0110] The above results indicate that, compared with Comparative Examples 1-4, the R175H wild-type primer with nucleotide sequence as shown in SEQ ID NO:9 and the R175H mutant primer with nucleotide sequence as shown in SEQ ID NO:11 in the kit provided in Example 1 are more suitable for detecting the target site R175H of the TP53 gene, and can effectively distinguish between the R175H wild-type and mutant sites.

[0111] 2. Wild-type and mutant primers for R249S targeting the target mutation site R249S in Example 1 and Comparative Examples 5-8 For ease of comparison, the nucleotide sequences of the R249S wild-type primers and R249S mutant primers for detecting TP53 gene mutations provided in Example 1 and Comparative Examples 5-8 are integrated into Table 5.

[0112] Table 5. Nucleotide sequences of the R249S wild-type primers and R249S mutant primers targeting the target mutation site R249S in the kits provided in Examples 1 and Comparative Examples 5-8 for detecting TP53 gene mutations.

[0113] The R249S wild-type and mutant samples were detected using the R249S wild-type and R249S mutant primers provided in the kits for detecting TP53 gene mutations provided in Example 1 and Comparative Examples 5-8, respectively. The Ct values ​​are shown in Table 6, and the amplification curves are shown in... Figure 2 As shown, where, Figure 2 Figures A and B show the amplification curve detection results of R249S wild-type and mutant samples using the R249S wild-type primers provided in Example 1, respectively. Figure 2 CD represent the amplification curve detection results of R249S wild-type and mutant samples using the R249S wild-type primers provided in Comparative Example 5, respectively. Figure 2 E and F represent the amplification curve detection results of R249S wild-type and mutant samples using the R249S wild-type primers provided in Comparative Example 6, respectively. Figure 2 GH represent the amplification curve detection results of R249S wild-type and mutant samples using the R249S mutant primers provided in the kit of Example 1, respectively. Figure 2 IJ represent the amplification curve detection results of R249S wild-type and mutant samples using the R249S mutant primers provided in Comparative Example 7. Figure 2 KL represents the amplification curve detection results of R249S wild-type and mutant specimens using the R249S mutant primers provided in Comparative Example 8.

[0114] Table 6. Detection results of R249S wild-type and R249S mutant primers in the kits provided in Examples 1 and Comparative Examples 5-8 for detecting TP53 gene mutations on R249S wild-type and mutant specimens.

[0115] Depend on Figure 2As shown in Table 6, when using the R249S wild-type primers in the kit provided in Example 1 to detect R249S wild-type and mutant samples, the R249S wild-type primers with nucleotide sequences as shown in SEQ ID NO:18 had the required efficiency in amplifying R249S wild-type samples, and no R249S mutant samples were detected. However, when using the R249S wild-type primers in the kits provided in Comparative Examples 5-6 to detect R249S wild-type and mutant samples, the R249S wild-type primers with nucleotide sequences as shown in SEQ ID NO:37 and 38 had the required efficiency in amplifying R249S wild-type samples, but R249S mutant samples were detected. When using the R249S mutant primers in the kit provided in Example 1 to detect R249S wild-type and mutant samples, the nucleotide sequences as shown in SEQ ID NO:18... The R249S mutant primers shown in NO:20 amplified R249S mutant samples with the required efficiency, and no R249S wild-type samples were detected. However, when using the R249S mutant primers in the kits provided in Comparative Examples 7-8 to detect both R249S wild-type and mutant samples, the R249S mutant primers with nucleotide sequences as shown in SEQ ID NO:39 and 40 amplified R249S mutant samples with the required efficiency, but both were detected when amplifying R249S wild-type samples.

[0116] The above results indicate that, compared with comparative examples 5-8, the R249S wild-type primer with nucleotide sequence as shown in SEQ ID NO:18 and the R249S mutant primer with nucleotide sequence as shown in SEQ ID NO:20 in the kit provided in Example 1 are more suitable for detecting the target site R249 of the TP53 gene, and can effectively distinguish between the wild-type and mutant sites of R249.

[0117] 3. Wild-type and mutant primers for R282W targeting the target mutation site R282W in Example 1 and Comparative Examples 9-12 For ease of comparison, the nucleotide sequences of the R282W wild-type primers and R282W mutant primers for the target mutation site R282W provided in the kits for detecting TP53 gene mutations provided in Example 1 and Comparative Examples 9-12 are integrated into Table 7.

[0118] Table 7. Nucleotide sequences of the R282W wild-type primers and R282W mutant primers targeting the target mutation site R282W in the kits provided in Examples 1 and Comparative Examples 9-12 for detecting TP53 gene mutations.

[0119] The R282W wild-type and mutant samples were detected using the R282W wild-type and R282W mutant primers provided in the kits for detecting TP53 gene mutations provided in Example 1 and Comparative Examples 9-12, respectively. The Ct values ​​are shown in Table 8, and the amplification curves are shown in... Figure 3 As shown, where, Figure 3 Figures A and B show the amplification curve detection results of R282W wild-type and mutant samples using the R282W wild-type primers provided in Example 1, respectively. Figure 3 CD represent the amplification curve detection results of R282W wild-type and mutant samples using the R282W wild-type primers provided in Comparative Example 9, respectively. Figure 3 E and F represent the amplification curve detection results of R282W wild-type and mutant samples using the R282W wild-type primers provided in Comparative Example 10, respectively. Figure 3 GH represent the amplification curve detection results of R282W wild-type and mutant samples using the R282W mutant primers provided in Example 1, respectively. Figure 3 IJ represent the amplification curve detection results of R282W wild-type and mutant samples using the R282W mutant primers provided in Comparative Example 11. Figure 3 KL represents the amplification curve detection results of R282W wild-type and mutant specimens using the R282W mutant primers provided in Comparative Example 12.

[0120] Table 8. Detection results of R282W wild-type and R282W mutant primers in the kits provided in Examples 1 and Comparative Examples 9-12 for detecting TP53 gene mutations on R282W wild-type and mutant samples.

[0121] Depend on Figure 3As shown in Table 8, when using the R282W wild-type primers in the kit provided in Example 1 to detect both wild-type and mutant R282W samples, the R282W wild-type primers with nucleotide sequences as shown in SEQ ID NO:1 achieved the required efficiency in amplifying wild-type R282W samples, and no R282W mutant samples were detected. However, when using the R282W wild-type primers in the kits provided in Comparative Examples 9-10 to detect both wild-type and mutant R282W samples, the R282W wild-type primers with nucleotide sequences as shown in SEQ ID NO:41 and 42 achieved the required efficiency in amplifying wild-type R282W samples, but all R282W mutant samples were detected. When using the R282W mutant primers in the kit provided in Example 1 to detect both wild-type and mutant R282W samples, the nucleotide sequences as shown in SEQ ID NO:1... The R282W mutant primers shown in NO:3 amplified the R282W mutant specimens with the required efficiency, and no R282W wild-type specimens were detected. However, when the R282W mutant primers in the kits provided in Comparative Examples 11-12 were used to detect both R282W wild-type and mutant specimens, the R282W mutant primers with nucleotide sequences as shown in SEQ ID NO:43 and 44 amplified the R282W mutant specimens with the required efficiency, but both were detected when amplifying the R282W wild-type specimens.

[0122] The above results indicate that, compared with comparative examples 9-12, the R282W wild-type primer with nucleotide sequence as shown in SEQ ID NO:1 and the R282W mutant primer with nucleotide sequence as shown in SEQ ID NO:3 in the kit provided in Example 1 are more suitable for detecting the target site R282W of the TP53 gene, and can effectively distinguish between the R282W wild-type and mutant sites.

[0123] 4. Wild-type and mutant primers for R248Q targeting the target mutation site R248Q in Example 1 and Comparative Examples 13-16 For ease of comparison, the nucleotide sequences of the R248Q wild-type primers and R248Q mutant primers for the target mutation site R248Q provided in the kits for detecting TP53 gene mutations provided in Example 1 and Comparative Examples 13-16 are integrated into Table 9.

[0124] Table 9. Nucleotide sequences of the R248Q wild-type primers and R248Q mutant primers targeting the target mutation site R248Q in the kits for detecting TP53 gene mutations provided in Examples 1 and Comparative Examples 13-16.

[0125] R248Q wild-type and mutant samples were detected using the R248Q wild-type and R248Q mutant primers provided in the kits for detecting TP53 gene mutations provided in Example 1 and Comparative Examples 13-16, respectively. The Ct values ​​are shown in Table 10, and the amplification curves are shown in... Figure 4 As shown, where, Figure 4 Figures A and B show the amplification curve detection results of R248Q wild-type and mutant samples, respectively, using the R248Q wild-type primers from the kit provided in Example 1. Figure 4 CD represent the amplification curve detection results of R248Q wild-type and mutant samples using the R248Q wild-type primers provided in Comparative Example 13, respectively. Figure 4 E and F represent the amplification curve detection results of R248Q wild-type and mutant samples using the R248Q wild-type primers provided in Comparative Example 14, respectively. Figure 4 GH represent the amplification curve detection results of R248Q wild-type and mutant samples using the R248Q mutant primers provided in the kit of Example 1, respectively. Figure 4 IJ represent the amplification curve detection results of R248Q wild-type and mutant samples using the R248Q mutant primers provided in Comparative Example 15, respectively. Figure 4 KL represents the amplification curve detection results of R248Q wild-type and mutant specimens using the R248Q mutant primers provided in Comparative Example 16.

[0126] Table 10. Detection results of R248Q wild-type and R248Q mutant primers in the kits provided in Examples 1 and Comparative Examples 13-16 for detecting TP53 gene mutations on R248Q wild-type and mutant specimens.

[0127] Depend on Figure 4As shown in Table 10, when using the R248Q wild-type primers in the kit provided in Example 1 to detect R248Q wild-type and mutant samples, the R248Q wild-type primers with nucleotide sequences as shown in SEQ ID NO:2 had the required efficiency in amplifying R248Q wild-type samples, and no R248Q mutant samples were detected. However, when using the R248Q wild-type primers in the kits provided in Comparative Examples 13-14 to detect R248Q wild-type and mutant samples, the R248Q wild-type primers with nucleotide sequences as shown in SEQ ID NO:45 and 46 had the required efficiency in amplifying R248Q wild-type samples, but R248Q mutant samples were detected. When using the R248Q mutant primers in the kit provided in Example 1 to detect R248Q wild-type and mutant samples, the nucleotide sequences as shown in SEQ ID NO:2... The R248Q mutant primers shown in NO:4 amplified R248Q mutant samples with the required efficiency, and no R248Q wild-type samples were detected. However, when using the R248Q mutant primers in the kits provided in Comparative Examples 15-16 to detect both R248Q wild-type and mutant samples, the R248Q mutant primers with nucleotide sequences as shown in SEQ ID NO:47 and 48 amplified R248Q mutant samples with the required efficiency, but both were detected when amplifying R248Q wild-type samples.

[0128] The above results indicate that, compared with comparative examples 13-16, the R248Q wild-type primers with nucleotide sequences as shown in SEQ ID NO:2 and the R248Q mutant primers with nucleotide sequences as shown in SEQ ID NO:4 in the kit provided in Example 1 are more suitable for detecting the target site R248Q of the TP53 gene, and can effectively distinguish between the R248Q wild-type and mutant sites.

[0129] 5. Wild-type and mutant primers for R273H targeting the target mutation site R273H in Example 1 and Comparative Examples 17-20 For ease of comparison, the nucleotide sequences of the wild-type and mutant primers for the target mutation site R273H provided in the kits for detecting TP53 gene mutations in Examples 1 and Comparative Examples 17-20 are integrated into Table 11.

[0130] Table 11 Nucleotide sequences of the R273H wild-type primers and R273H mutant primers targeting the target mutation site R273H in the kits for detecting TP53 gene mutations provided in Examples 1 and Comparative Examples 17-20.

[0131] The R273H wild-type and mutant samples were detected using the R273H wild-type primers and R273H mutant primers provided in the kits for detecting TP53 gene mutations provided in Example 1 and Comparative Examples 17-20, respectively. The Ct values ​​are shown in Table 12, and the amplification curves are shown in... Figure 5 As shown, where, Figure 5 Figures A and B show the amplification curve detection results of R273H wild-type and mutant samples using the R273H wild-type primers provided in Example 1, respectively. Figure 5 CD represent the amplification curve detection results of R273H wild-type and mutant samples using the R273H wild-type primers provided in Comparative Example 17, respectively. Figure 5 E and F represent the amplification curve detection results of R273H wild-type and mutant samples using the R273H wild-type primers provided in Comparative Example 18, respectively. Figure 5 GH represent the amplification curve detection results of R273H wild-type and mutant samples using the R273H mutant primers provided in Example 1, respectively. Figure 5 IJ represent the amplification curve detection results of R273H wild-type and mutant samples using the R273H mutant primers provided in Comparative Example 19, respectively. Figure 5 KL represents the amplification curve detection results of R273H wild-type and mutant samples using the R273H mutant primers provided in Comparative Example 20.

[0132] Table 12. Detection results of R273H wild-type primers and R273H mutant primers in the kits provided in Examples 1 and Comparative Examples 17-20 for detecting TP53 gene mutations on R273H wild-type and mutant samples.

[0133] Depend on Figure 5As shown in Table 12, when using the R273H wild-type primers in the kit provided in Example 1 to detect R273H wild-type and mutant samples, the R273H wild-type primers with nucleotide sequences as shown in SEQ ID NO:9 had the required efficiency in amplifying R273H wild-type samples, and no R273H mutant samples were detected. However, when using the R273H wild-type primers in the kits provided in Comparative Examples 17-18 to detect R273H wild-type and mutant samples, the R273H wild-type primers with nucleotide sequences as shown in SEQ ID NO:49 and 50 had the required efficiency in amplifying R273H wild-type samples, but R248Q mutant samples were detected. When using the R273H mutant primers in the kit provided in Example 1 to detect R273H wild-type and mutant samples, the nucleotide sequences as shown in SEQ ID NO:9... The R273H mutant primers shown in NO:11 amplified R273H mutant samples with the required efficiency, and no R273H wild-type samples were detected. However, when using the R273H mutant primers in the kits provided in Comparative Examples 19-20 to detect both R273H wild-type and mutant samples, the R273H mutant primers with nucleotide sequences as shown in SEQ ID NO:51 and 52 amplified R273H mutant samples with the required efficiency, but R273H wild-type samples were detected.

[0134] The above results indicate that, compared with comparative examples 17-20, the R273H wild-type primer with nucleotide sequence as shown in SEQ ID NO:9 and the R273H mutant primer with nucleotide sequence as shown in SEQ ID NO:11 in the kit provided in Example 1 are more suitable for detecting the target site R273H of the TP53 gene, and can effectively distinguish between the R273H wild-type and mutant sites.

[0135] 6. Wild-type and mutant primers for the target mutation site G245S in Example 1 and Comparative Examples 21-24 For ease of comparison, the nucleotide sequences of the G245S wild-type primers and G245S mutant primers for detecting TP53 gene mutations provided in Example 1 and Comparative Examples 21-24 are integrated into Table 13.

[0136] Table 13 Nucleotide sequences of the G245S wild-type primers and G245S mutant primers targeting the G245S mutation site in the kits provided in Examples 1 and Comparative Examples 21-24 for detecting TP53 gene mutations.

[0137] The G245S wild-type and mutant samples were tested using the G245S wild-type and G245S mutant primers provided in the kits for detecting TP53 gene mutations provided in Example 1 and Comparative Examples 21-24, respectively. The Ct values ​​are shown in Table 14, and the amplification curves are shown in Table 14. Figure 6 As shown, where, Figure 6 Figures A and B show the amplification curve detection results of G245S wild-type and mutant samples using the G245S wild-type primers provided in Example 1, respectively. Figure 6 CD represent the amplification curve detection results of G245S wild-type and mutant samples using the G245S wild-type primers provided in Comparative Example 21, respectively. Figure 6 E and F represent the amplification curve detection results of G245S wild-type and mutant samples using the G245S wild-type primers provided in Comparative Example 22, respectively. Figure 6 GH represent the amplification curve detection results of G245S wild-type and mutant samples using the G245S mutant primers provided in Example 1, respectively. Figure 6 IJ represent the amplification curve detection results of G245S wild-type and mutant samples using the G245S mutant primers provided in Comparative Example 23, respectively. Figure 6 KL represents the amplification curve detection results of G245S wild-type and mutant specimens using the G245S mutant primers provided in Comparative Example 24.

[0138] Table 14. Detection results of G245S wild-type and G245S mutant primers in the kits provided in Examples 1 and Comparative Examples 21-24 for detecting TP53 gene mutations on G245S wild-type and mutant specimens.

[0139] Depend on Figure 6As shown in Table 14, when using the G245S wild-type primers in the kit provided in Example 1 to detect G245S wild-type and mutant samples, the G245S wild-type primers with nucleotide sequences as shown in SEQ ID NO:10 had the required efficiency in amplifying G245S wild-type samples, and no G245S mutant samples were detected. However, when using the G245S wild-type primers in the kits provided in Comparative Examples 21-22 to detect G245S wild-type and mutant samples, the G245S wild-type primers with nucleotide sequences as shown in SEQ ID NO:53 and 54 had the required efficiency in amplifying G245S wild-type samples, but R248Q mutant samples were detected. When using the G245S mutant primers in the kit provided in Example 1 to detect G245S wild-type and mutant samples, the nucleotide sequences as shown in SEQ ID NO:10... The G245S mutant primer shown in NO:12 amplifies G245S mutant samples with the required efficiency and no G245S wild-type samples are detected. However, when using the G245S mutant primers in the kits provided in Comparative Examples 23-24 to detect G245S wild-type and mutant samples, the G245S mutant primers with nucleotide sequences as shown in SEQ ID NO:55 and 56 amplify G245S mutant samples with the required efficiency, but G245S wild-type samples are detected.

[0140] The results above indicate that, compared with comparative examples 21-24, the G245S wild-type primer with nucleotide sequence as shown in SEQ ID NO:10 and the G245S mutant primer with nucleotide sequence as shown in SEQ ID NO:12 in the kit provided in Example 1 are more suitable for detecting the target site G245S of the TP53 gene, and can effectively distinguish between the G245S wild-type and mutant sites.

[0141] Test Example 2 This test case aims to study the detection efficacy of the internal control probes in the kits for detecting TP53 gene mutations provided in Examples 1 and 2, and the internal control primer set in the kit for detecting TP53 gene mutations provided in Example 3, on different clinical specimens (i.e., clinical specimen 1 and clinical specimen 2).

[0142] For ease of comparison, the nucleotide sequences of the internal control probes and internal control primer sets from the kits provided in Examples 1-3 for detecting TP53 gene mutations are integrated into Table 15.

[0143] Table 15 Nucleotide sequences of the internal control probes and internal control primer sets in the kits provided in Examples 1-3 for detecting TP53 gene mutations.

[0144] Using the kits for detecting TP53 gene mutations provided in Examples 1-3, clinical specimen 1 and clinical specimen 2 were tested according to the detection method in Test Example 1. The Ct value results are shown in Table 16, and the amplification curve results are shown in Table 16. Figure 7 As shown, where, Figure 7 A and B are the amplification curve results of clinical specimen 1 and clinical specimen 2 detected using the kit provided in Example 1, respectively. Figure 7 CD represents the amplification curve results of clinical specimen 1 and clinical specimen 2 detected using the kit provided in Example 2, respectively. Figure 7 EF represents the amplification curve results of clinical specimen 1 and clinical specimen 2 detected using the kit provided in Example 3.

[0145] Table 16. Detection results of the kits provided in Examples 1-3 for detecting TP53 gene mutations on different clinical specimens.

[0146] From Table 16 and Figure 7 It can be seen that when using the kit provided in Example 1 to detect clinical specimen 1 and clinical specimen 2, the amplification efficiency of both clinical specimens 1 and 2 meets the requirements. However, when using the kits provided in Examples 2 and 3 to detect clinical specimens 1 and 2, the amplification efficiency of both clinical specimens 1 and 2 does not meet the requirements. The above results indicate that, compared with Examples 2 and 3, the kit provided in Example 1 is suitable for detecting TP53 gene mutations, and its amplification or detection efficiency is higher.

[0147] Test Example 3 This test example aims to test the sensitivity (FAM signal, ROX signal, CY5 signal) of the kit provided in Example 1 for detecting TP53 gene mutations, referring to the detection method in Test Example 1. Simultaneously, the housekeeping gene RPPH1 is used as an internal control (its role in the detection is to monitor whether the PCR reaction system has been added and whether the PCR reaction process at the test wells is normal). The amplification curve obtained by amplifying the internal control using the internal control primer set is shown below. Figure 8 As shown, the amplification curves of wild-type and mutant primers targeting the two target mutation sites R282W and R248Q for wild-type and mutant samples are as follows. Figure 9 As shown, the amplification curves of wild-type and mutant primers targeting the two target mutation sites R273H and G245S for wild-type and mutant samples are as follows. Figure 10As shown, the amplification curves of wild-type and mutant primers targeting the two target mutation sites R175H and R249S for wild-type and mutant samples are as follows. Figure 11 As shown, where, Figure 9 Figures A and B show the amplification curve detection results of R282W and R248Q wild-type samples using the kit for detecting TP53 gene mutations provided in Example 1, respectively. Figure 9 CD represent the amplification curve detection results of R282W and R248Q mutant samples using the kit for detecting TP53 gene mutations provided in Example 1. Figure 10 Figures A and B show the amplification curves of R273H and G245S wild-type samples obtained using the kit provided in Example 1 for detecting TP53 gene mutations, respectively. Figure 10 CD represent the amplification curve detection results of R273H and G245S mutant samples using the kit for detecting TP53 gene mutations provided in Example 1. Figure 11 Figures A and B show the amplification curves of R175H and R249S wild-type samples obtained using the kit for detecting TP53 gene mutations provided in Example 1, respectively. Figure 11 CD represent the amplification curve detection results of R175H and R249S mutant samples using the kit for detecting TP53 gene mutations provided in Example 1.

[0148] Depend on Figures 9-11 It can be seen that the detection limit of the kit provided in Example 1 is 50 copies (FAM Ct value = 34.5 ± 0.3), there is no crossover of wild-type background, and only 1 ng of genomic DNA (i.e. the DNA to be tested) needs to be added to the PCR system to detect it normally.

[0149] Test Example 4 This test case aims to study the consistency of the results of detecting TP53 gene mutations in clinical samples using the kit provided in Example 1 and Sanger sequencing (first-generation sequencing), referring to the detection method of Test Case 1. The results are shown in Table 17. A total of 70 clinical samples were tested, including 20 from healthy individuals (Sample 1-20#) and 50 from cancer patients (Ca1-50#).

[0150] Table 17 Comparison of test results for different clinical samples using the kit for detecting TP53 gene mutations provided in Example 1 and Sanger sequencing, including 20 healthy human samples (Sample 1#-Sample 20#) and 50 cancer patient samples (Ca 1#-Ca 50#):

[0151] As shown in Table 17, when the clinical samples were tested using the kit for detecting TP53 gene mutations provided in Example 1, the test results showed good consistency with the test results using Sanger sequencing (first-generation sequencing), with an accuracy of 100%.

[0152] 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 kit for detecting TP53 gene mutations, characterized in that: The kit includes reagent A, reagent B, reagent C, reagent D, reagent E, and reagent F; The reagent A includes R282W wild-type primers, R248Q wild-type primers, R282W detection probes, R248Q detection probes, R282W universal primers, and R248Q universal primers. The nucleotide sequence of the R282W wild-type primers is shown in SEQ ID NO:1, and the nucleotide sequence of the R248Q wild-type primers is shown in SEQ ID NO:

2. The reagent B includes R282W mutant primers, R248Q mutant primers, R282W detection probes, R248Q detection probes, R282W universal primers, and R248Q universal primers. The nucleotide sequence of the R282W mutant primers is shown in SEQ ID NO:3, and the nucleotide sequence of the R248Q mutant primers is shown in SEQ ID NO:

4. In reagent A and reagent B, the nucleotide sequence of the R282W detection probe is shown in SEQ ID NO:5, the nucleotide sequence of the R248Q detection probe is shown in SEQ ID NO:6, the nucleotide sequence of the R282W universal primer is shown in SEQ ID NO:7, and the nucleotide sequence of the R248Q universal primer is shown in SEQ ID NO:

8. The reagent C includes an R273H wild-type primer, a G245S wild-type primer, an R273H detection probe, a G245S detection probe, an R273H universal primer, and a G245S universal primer. The nucleotide sequence of the R273H wild-type primer is shown in SEQ ID NO:9, and the nucleotide sequence of the G245S wild-type primer is shown in SEQ ID NO:

10. The reagent D includes an R273H mutant primer, a G245S mutant primer, an R273H detection probe, a G245S detection probe, a universal R273H primer, and a universal G245S primer. The nucleotide sequence of the R273H mutant primer is shown in SEQ ID NO:11, and the nucleotide sequence of the G245S mutant primer is shown in SEQ ID NO:

12. In reagent C and reagent D, the nucleotide sequence of the R273H detection probe is shown in SEQ ID NO:13, the nucleotide sequence of the G245S detection probe is shown in SEQ ID NO:14, the nucleotide sequence of the R273H universal primer is shown in SEQ ID NO:15, and the nucleotide sequence of the G245S universal primer is shown in SEQ ID NO:

16. The reagent E includes an R175H wild-type primer, an R249S wild-type primer, an R175H detection probe, an R249S detection probe, a R175H universal primer, and an R249S universal primer. The nucleotide sequence of the R175H wild-type primer is shown in SEQ ID NO:17, and the nucleotide sequence of the R249S wild-type primer is shown in SEQ ID NO:

18. The reagent F includes an R175H mutant primer, an R249S mutant primer, an R175H detection probe, an R249S detection probe, a universal R175H primer, and a universal R249S primer. The nucleotide sequence of the R175H mutant primer is shown in SEQ ID NO:19, and the nucleotide sequence of the R249S mutant primer is shown in SEQ ID NO:

20. In reagent E and reagent F, the nucleotide sequence of the R175H detection probe is shown in SEQ ID NO:21, the nucleotide sequence of the R249S detection probe is shown in SEQ ID NO:22, the nucleotide sequence of the R175H universal primer is shown in SEQ ID NO:23, and the nucleotide sequence of the R249S universal primer is shown in SEQ ID NO:

24.

2. The kit for detecting TP53 gene mutations as described in claim 1, characterized in that: The R282W detection probe, the R248Q detection probe, the R273H detection probe, the G245S detection probe, the R175H detection probe, and the R249S detection probe each contain a quenching group and a fluorescent group. The quenching group is MGB, and the fluorescent group is FAM or ROX.

3. The kit for detecting TP53 gene mutations as described in claim 2, characterized in that: The quenching group is located at the 3' end of the R282W detection probe, the R248Q detection probe, the R273H detection probe, the G245S detection probe, the R175H detection probe, and the R249S detection probe; The fluorescent group is located at the 5' end of the R282W detection probe, the R248Q detection probe, the R273H detection probe, the G245S detection probe, the R175H detection probe, and the R249S detection probe.

4. The kit for detecting TP53 gene mutations as described in claim 1, characterized in that: The kit also includes an internal control primer set, which includes a first internal control primer and a second internal control primer. The nucleotide sequence of the first internal control primer is shown in SEQ ID NO:25, and the nucleotide sequence of the second internal control primer is shown in SEQ ID NO:

26.

5. The kit for detecting TP53 gene mutations as described in claim 4, characterized in that: The kit also includes an internal control probe, the nucleotide sequence of which is shown in SEQ ID NO:

27.

6. The kit for detecting TP53 gene mutations as described in claim 5, characterized in that: The 3' end of the internal control probe is labeled with a quenching group MGB, and the 5' end of the internal control probe is labeled with a fluorescent group CY5.

7. The kit for detecting TP53 gene mutations as described in claim 1, characterized in that: The kit also includes an external control plasmid set, which includes a first external control plasmid and a second external control plasmid. The nucleotide sequence of the first external control plasmid is shown in SEQ ID NO:28, and the nucleotide sequence of the second external control plasmid is shown in SEQ ID NO:

29.

8. The kit for detecting TP53 gene mutations as described in claim 1, characterized in that, The kit also includes a PCR premix, which comprises the following components: hot-start Taq DNA polymerase, 10–1000 μM dNTPs, 2–10 mM MgCl2, 0.01–0.5 wt% DMSO, and 0.01–0.5 wt% formamide.

9. The kit for detecting TP53 gene mutations as described in claim 8, characterized in that: The PCR premix also includes a PCR buffer containing Tris, NaCl, NP-40, and MgCl2.

10. The kit for detecting TP53 gene mutations as described in claim 1, characterized in that, The kit also includes a nucleic acid release agent comprising the following components: 0.01-0.1 M HCl, 0.01-0.05 wt% SDS, 1-10 mM Surfactin, and 0.05-3 wt% trehalose.

Images