A kit for detecting a mutation in the TP53 gene

Abstract

The application provides a kit for detecting TP53 gene mutation. The kit for detecting TP53 gene mutation can accurately detect six TP53 gene mutation sites (R175H, R249S, R282W, R248Q, R273H and G245S) in the to-be-detected genomic DNA through a multiplex fluorescence PCR reaction system and a multi-channel fluorescence signal, and can accurately detect the six mutation sites, has ultrahigh amplification efficiency, detection efficiency, sensitivity and specificity, can accurately detect the genotype of a 1 ng sample of genomic DNA, and is simple to operate and low in cost.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, and particularly relates to a kit for detecting TP53 gene mutation. BACKGROUND

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

[0003] Mutations in the DNA binding region of TP53 gene are closely related to the occurrence and development of more than 50% of human malignancies, including ovarian cancer, lung cancer, liver cancer, breast cancer, colorectal cancer and other high-incidence cancers. TP53 gene mutations are mainly concentrated in its highly conserved functional domains, and most of them are missense mutations, which lead to the loss of normal tumor suppressor function of p53 protein or even the acquisition of pro-cancer activity. TP53 gene mutation may be one of the main pathogenic factors of human tumors. Through accurate and efficient detection of common TP53 gene mutation sites, the risk of tumor occurrence can be assessed, and the detection of high-risk tumor population can be strengthened to achieve early detection and early treatment, improve treatment effect, and have important clinical significance for patient survival.

[0004] Currently, TP53 gene mutation detection mainly relies on various molecular biology techniques. Although Sanger sequencing, as the traditional gold standard, has high accuracy and can find unknown mutations, its sensitivity is low (usually > 15~20% mutation frequency can be reliably detected), the operation is complicated and time-consuming, and it is difficult to meet the detection needs of large number of samples or low frequency mutations in clinical practice. Although the new generation sequencing (NGS) technology can realize high-throughput and multi-gene parallel analysis, its expensive equipment investment, complex library construction process, long detection period and high requirement of bioinformatics analysis ability limit its popularization and application in routine clinical diagnosis. Traditional qPCR technology cannot distinguish high-frequency mutation subtypes and is easily interfered by wild type.

[0005] The detection method based on specific primers, such as the amplification refractory mutation system PCR (ARMS-PCR), has become a common choice for clinical targeted mutation detection due to its simple operation, low cost and high sensitivity (can detect 1-5% mutation frequency). However, the existing ARMS-PCR technology still has significant limitations: first, it can usually only design a detection system for a single or a small number of mutation sites, and for the scene that needs to screen multiple mutation sites at the same time, multiple independent reactions need to be carried out, resulting in large sample consumption, low detection efficiency and cost multiplied; second, the TaqMan probe used in conventional ARMS-PCR is still insufficient in specificity when distinguishing between highly homologous wild-type and mutant sequences, especially in regions rich in GC or complex secondary structure, false positive or false negative results are prone to occur; in addition, the interaction between primers in the multiplex PCR reaction system easily causes non-specific amplification or primer dimer, which seriously affects the 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 at the same time and has high detection efficiency, high sensitivity, high specificity and low cost. SUMMARY

[0007] In order to solve the problems of low sensitivity, specificity and detection efficiency and high cost in detecting multiple TP53 gene mutation sites in the prior art, the present application provides a kit for detecting TP53 gene mutation.

[0008] According to a first aspect of the present application, a kit for detecting TP53 gene mutation is provided, which comprises reagent A, reagent B, reagent C, reagent D, reagent E and reagent F;

[0009] The reagent A comprises R282W wild-type primer, R248Q wild-type primer, R282W detection probe, R248Q detection probe, R282W universal primer and R248Q universal primer, the nucleotide sequence of the R282W wild-type primer is shown as SEQ ID NO: 1, and the nucleotide sequence of the R248Q wild-type primer is shown as SEQ ID NO: 2;

[0010] The reagent B comprises R282W mutant primer, R248Q mutant primer, R282W detection probe, R248Q detection probe, R282W universal primer and R248Q universal primer, the nucleotide sequence of the R282W mutant primer is shown as SEQ ID NO: 3, and the nucleotide sequence of the R248Q mutant primer is shown as SEQ ID NO: 4;

[0011] 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 in the reagent A and the reagent B;

[0012] The reagent C includes a R273H wild type primer, a G245S wild type primer, a R273H detection probe, a G245S detection probe, a 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;

[0013] The reagent D includes a R273H mutant primer, a G245S mutant primer, a R273H detection probe, a G245S detection probe, a R273H universal primer and a G245S universal 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;

[0014] 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 in the reagent C and the reagent D;

[0015] The reagent E includes a R175H wild type primer, a R249S wild type primer, a R175H detection probe, a R249S detection probe, a R175H universal primer and a 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;

[0016] The reagent F includes a R175H mutant primer, a R249S mutant primer, a R175H detection probe, a R249S detection probe, a R175H universal primer and a R249S universal 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;

[0017] The nucleotide sequence of the R175H detection probe in reagent E and reagent F is shown as SEQ ID NO: 21, the nucleotide sequence of the R249S detection probe is shown as SEQ ID NO: 22, the nucleotide sequence of the R175H universal primer is shown as SEQ ID NO: 23, and the nucleotide sequence of the R249S universal primer is shown as SEQ ID NO: 24.

[0018] The R175H mutation is that the 175th amino acid of the p53 protein encoded by the TP53 gene is changed from arginine (R) to histidine (H); the R249S mutation is that the 249th amino acid of the p53 protein encoded by the TP53 gene is changed from arginine (R) to serine (S); the R282W mutation is that the 282th amino acid of the p53 protein encoded by the TP53 gene is changed from arginine (R) to tryptophan (W); the R248Q mutation is that the 248th amino acid of the p53 protein encoded by the TP53 gene is changed from arginine (R) to glutamine (Q); the R273H mutation is that the 273th amino acid of the p53 protein encoded by the TP53 gene is changed from arginine (R) to histidine (H); and the G245S mutation is that the 245th amino acid of the p53 protein encoded by the TP53 gene is changed from glycine (G) to serine (S).

[0019] The present application is based on the amplification refractory mutation system PCR (ARMS-PCR) technology, and is directed to the six TP53 gene mutation sites of R175H, R249S, R282W, R248Q, R273H and G245S. The wild type primers and the mutant primers respectively corresponding to the above six target mutation sites are obtained by design and screening, and the detection probes and the universal primers respectively used for detecting the six mutation sites are designed. The universal primers can form primer groups (equivalent to upstream primers and downstream primers) with the wild type primers and the mutant primers respectively for amplification of the DNA to be detected. The wild type primers, the mutant primers, the universal primers and the detection probes are used as components of a kit, and the kit for detecting TP53 gene mutations is obtained.

[0020] The kit provided by the application is selected by ARMS-PCR method, and specific primers and specific probes are designed for six TP53 gene mutation sites R175H, R249S, R282W, R248Q, R273H and G245S. The primers and probes can specifically bind to the genes to be detected. In the PCR extension reaction process, the exonuclease activity of Taq enzyme can cut the fluorescent group at the 5' end of the detection probe from the probe, so that it is free in the reaction system, thereby the shielding of the 3' end fluorescent quenching group is removed, that is, it can accept light stimulation to emit fluorescence that can be detected by the instrument, so that the detection of the genes to be detected in the fully closed reaction system is realized.

[0021] The kit for detecting TP53 gene mutations provided by the application can simultaneously and accurately detect the six TP53 gene mutation sites R175H, R249S, R282W, R248Q, R273H and G245S in the genomic DNA to be detected through a multiplex fluorescence PCR reaction system and a multi-channel fluorescence signal, and can accurately detect the above six mutation sites with ultra-high amplification efficiency, detection efficiency, sensitivity and specificity, can accurately detect the genotype of a 1 ng sample of genomic DNA, is simple to operate, and has low cost.

[0022] The kit provided by the application has a broad application prospect in the fields of molecular diagnosis, prognosis evaluation, efficacy monitoring and individualized medical treatment of cancer, provides an efficient and stable detection tool for clinical and scientific researchers, solves the problems of low throughput, insufficient sensitivity, complicated operation or imperfect quality control in the prior art, and has great significance in TP53 gene mutation detection.

[0023] Preferably, 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 one quenching group and one fluorescent group, the quenching group is MGB, and the fluorescent group is FAM or ROX.

[0024] Preferably, 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; and 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.

[0025] By labeling a quencher group MGB at the 3' end of the detection probe and a fluorescent group FAM or ROX at the 5' end, the MGB group greatly enhances the binding ability of the probe to the target sequence (Tm value increases), significantly improves the discrimination ability for single base mismatch, ensures that low-frequency mutations can also be detected with high specificity and high sensitivity in complex samples (such as tumor samples containing a large amount of wild-type background), and effectively reduces false positive and false negative results.

[0026] Preferably, the kit further comprises an internal control primer set, the internal control primer set comprising a first internal control primer and a second internal control primer, the nucleotide sequence of the first internal control primer being as shown in SEQ ID NO: 25, and the nucleotide sequence of the second internal control primer being as shown in SEQ ID NO: 26.

[0027] Preferably, the kit further comprises an internal control probe, the nucleotide sequence of the internal control probe being as shown in SEQ ID NO: 27.

[0028] Preferably, the 3' end of the internal control probe is labeled with a quencher group MGB, and the 5' end of the internal control probe is labeled with a fluorescent group CY5.

[0029] The housekeeping gene (RPPH1) is stably expressed in various tissues of humans, and both the first internal control primer and the second internal control primer in the internal control primer set of the kit are primers designed for the housekeeping gene (RPPH1). During detection using the kit for detecting TP53 gene mutations, if the internal control primer does not work during the detection process, it indicates that no DNA template is added or the amplification system does not work normally, and the experiment fails. Therefore, by introducing the internal control primer set and the internal control probe into the kit for detecting TP53 gene mutations, the kit can be used to detect whether the amplification system can normally perform amplification, thereby further improving the accuracy of the detection results.

[0030] The internal standard in the kit provided in the present application uses a pair of housekeeping gene specific primers (i.e., the first internal control primer and the second internal control primer in the internal control primer set), combined with a specific probe that can specifically bind to a segment of DNA template in the middle of the primer amplification region, and the internal standard uses the CY5 channel, thereby achieving monitoring of the detection process in a fully closed reaction system, which can effectively monitor the occurrence of false negatives.

[0031] Preferably, the kit further comprises an external control plasmid set, the external control plasmid set comprising a first external control plasmid and a second external control plasmid, the nucleotide sequence of the first external control plasmid being as shown in SEQ ID NO: 28, and the nucleotide sequence of the second external control plasmid being as shown in SEQ ID NO: 29.

[0032] The first external control plasmid and the second external control plasmid in the external control plasmid group of the kit provided in the scheme are both wild-type gene sequences containing a TP53 conserved region.

[0033] The scheme can detect whether the whole PCR amplification system is working normally by introducing the external control plasmid group containing the first external control plasmid and the second external control plasmid in the kit for detecting TP53 gene mutation, which is equivalent to a positive quality control product. If the external control plasmid detection result fails, it proves that the kit is invalid or the instrument is not working, and the result is not reliable. Therefore, by introducing the external control plasmid group containing the first external control plasmid and the second external control plasmid in the kit for detecting TP53 gene mutation, the reliability of the final detection result can be improved.

[0034] Preferably, the kit further comprises a negative quality control product, and the negative quality control product is physiological saline.

[0035] The scheme introduces a negative quality control product in the kit for detecting TP53 gene mutation to detect whether there is contamination in the detection system, provides a stable and controllable chemical environment for the whole detection process, and ensures the specificity, sensitivity and reliability of the reaction.

[0036] Preferably, the kit further comprises a PCR premix, and the PCR premix 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.

[0037] Preferably, the PCR premix further comprises a PCR buffer, and the PCR buffer contains Tris, NaCl, ethyl phenyl polyethylene glycol (NP-40), and MgCl2.

[0038] Preferably, the kit further comprises a nucleic acid releasing agent, and the nucleic acid releasing agent comprises the following components: 0.01-0.1 M HCl, 0.01-0.05 wt% sodium dodecyl sulfate (SDS), 1-10 mM Surfactin, and 0.05-3 wt% trehalose.

[0039] Surfactin is a surfactant with amphiphilic structure characteristics produced by fermentation of microorganism Bacillus subtilis, and its molecular formula is C 53 H 93 N7O 13 .

[0040] By introducing the PCR premix and the nucleic acid releasing agent into the kit for detecting TP53 gene mutation and regulating the components and their contents to meet the above range, the PCR premix containing the above components can ensure that the multiplex PCR amplification for specific mutation sites of TP53 gene can be efficiently, specifically and stably performed, and the nucleic acid releasing agent containing the above components can efficiently lyse cells, release nucleic acids, and to some extent protect the integrity of nucleic acids, and the addition of Surfactin and trehalose helps to reduce the interference of inhibitors and plays a role in stabilizing nucleic acids, and the PCR premix and the nucleic acid releasing agent can simplify or replace the traditional DNA extraction and purification steps, greatly simplifying the nucleic acid extraction step and the preparation step of the PCR amplification system, realizing the rapid release and direct detection of the sample to be detected, which can significantly shorten the overall detection time, has high detection efficiency, is easy to operate and low in cost, and is especially suitable for clinical rapid detection scenes. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 The amplification curve results of the R175H wild type primer and the R175H mutant primer in the kit for detecting TP53 gene mutation provided by Test Example 1 and Examples 1 and Comparative Examples 1-4 were used to detect the R175H wild type sample and the mutant sample.

[0042] Figure 2 The amplification curve results of the R249S wild type primer and the R249S mutant primer in the kit for detecting TP53 gene mutation provided by Test Example 1 and Examples 1 and Comparative Examples 5-8 were used to detect the R249 wild type sample and the mutant sample.

[0043] Figure 3 The amplification curve results of the R282W wild type primer and the R282W mutant primer in the kit for detecting TP53 gene mutation provided by Test Example 1 and Examples 1 and Comparative Examples 9-12 were used to detect the R282W wild type sample and the mutant sample.

[0044] Figure 4 The amplification curve results of the R248Q wild type primer and the R248Q mutant primer in the kit for detecting TP53 gene mutation provided by Test Example 1 and Examples 1 and Comparative Examples 13-16 were used to detect the R248Q wild type sample and the mutant sample.

[0045] Figure 5 The amplification curve results of the R273H wild type primer and the R273H mutant primer in the kit for detecting TP53 gene mutation provided by Test Example 1 and Examples 1 and Comparative Examples 17-20 were used to detect the R273H wild type sample and the mutant sample.

[0046] Figure 6 The amplification curve results of the detection of the G245S wild type specimen and the mutant specimen by the G245S wild type primer and the G245S mutant primer in the kit for detecting TP53 gene mutation provided by Test Example 1 and Examples 1 and Comparative Examples 21-24.

[0047] Figure 7 The amplification curve results of the detection of clinical specimen 1 and clinical specimen 2 by the kit for detecting TP53 gene mutation provided by Test Example 2 and Examples 1-3 according to the detection method of Test Example 1.

[0048] Figure 8 The amplification curve chart of the internal reference when the wild type and mutant specimens are detected by the kit for detecting TP53 gene mutation provided by Test Example 3 and Example 1.

[0049] Figure 9 The amplification curve detection results of the R282W and R248Q wild type and mutant specimens by the kit for detecting TP53 gene mutation provided by Test Example 3 and Example 1.

[0050] Figure 10 The amplification curve detection results of the R273H and G245S wild type and mutant specimens by the kit for detecting TP53 gene mutation provided by Test Example 3 and Example 1.

[0051] Figure 11 The amplification curve detection results of the R175H and R249S wild type and mutant specimens by the kit for detecting TP53 gene mutation provided by Test Example 3 and Example 1. DETAILED DESCRIPTION

[0052] The technical features of the technical solutions provided by the present application will be further described clearly and completely in combination with the specific embodiments. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative labor are within the protection scope of the present application.

[0053] Example 1

[0054] A kit for detecting TP53 gene mutation, the kit comprising reagent A, reagent B, reagent C, reagent D, reagent E, reagent F, external control plasmid group (i.e. positive quality control), physiological saline (i.e. negative quality control), PCR premix, nucleic acid releasing agent;

[0055] 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, PCR buffer (containing Tris, NaCl, NP-40, MgCl2);

[0056] The nucleic acid releasing agent includes the following components: 0.01-0.1 M HCl, 0.01-0.05 wt% SDS, 1-10 mM Surfactin, 0.05-3 wt% trehalose;

[0057] The reagent A includes R282W wild type primer, R248Q wild type primer, R282W detection probe, R248Q detection probe, R282W universal primer and R248Q universal primer, the nucleotide sequence of the R282W wild type primer is shown as SEQ ID NO: 1, and the nucleotide sequence of the R248Q wild type primer is shown as SEQ ID NO: 2;

[0058] The reagent B includes R282W mutant primer, R248Q mutant primer, R282W detection probe, R248Q detection probe, R282W universal primer and R248Q universal primer, the nucleotide sequence of the R282W mutant primer is shown as SEQ ID NO: 3, and the nucleotide sequence of the R248Q mutant primer is shown as SEQ ID NO: 4;

[0059] In the reagent A and the reagent B, the nucleotide sequence of the R282W detection probe is shown as SEQ ID NO: 5, the nucleotide sequence of the R248Q detection probe is shown as SEQ ID NO: 6, the nucleotide sequence of the R282W universal primer is shown as SEQ ID NO: 7, and the nucleotide sequence of the R248Q universal primer is shown as SEQ ID NO: 8;

[0060] The reagent C includes R273H wild type primer, G245S wild type primer, R273H detection probe, G245S detection probe, R273H universal primer and G245S universal primer, the nucleotide sequence of the R273H wild type primer is shown as SEQ ID NO: 9, and the nucleotide sequence of the G245S wild type primer is shown as SEQ ID NO: 10;

[0061] The reagent D comprises a R273H mutant primer, a G245S mutant primer, a R273H detection probe, a G245S detection probe, a R273H universal primer and a G245S universal primer, the nucleotide sequence of the R273H mutant primer is shown as SEQ ID NO: 11, and the nucleotide sequence of the G245S mutant primer is shown as SEQ ID NO: 12;

[0062] In the reagent C and the reagent D, the nucleotide sequence of the R273H detection probe is shown as SEQ ID NO: 13, the nucleotide sequence of the G245S detection probe is shown as SEQ ID NO: 14, the nucleotide sequence of the R273H universal primer is shown as SEQ ID NO: 15, and the nucleotide sequence of the G245S universal primer is shown as SEQ ID NO: 16;

[0063] The reagent E comprises a R175H wild-type primer, a R249S wild-type primer, a R175H detection probe, a R249S detection probe, a R175H universal primer and a R249S universal primer, the nucleotide sequence of the R175H wild-type primer is shown as SEQ ID NO: 17, and the nucleotide sequence of the R249S wild-type primer is shown as SEQ ID NO: 18;

[0064] The reagent F comprises a R175H mutant primer, a R249S mutant primer, a R175H detection probe, a R249S detection probe, a R175H universal primer and a R249S universal primer, the nucleotide sequence of the R175H mutant primer is shown as SEQ ID NO: 19, and the nucleotide sequence of the R249S mutant primer is shown as SEQ ID NO: 20;

[0065] In the reagent E and the reagent F, the nucleotide sequence of the R175H detection probe is shown as SEQ ID NO: 21, the nucleotide sequence of the R249S detection probe is shown as SEQ ID NO: 22, the nucleotide sequence of the R175H universal primer is shown as SEQ ID NO: 23, and the nucleotide sequence of the R249S universal primer is shown as SEQ ID NO: 24;

[0066] The reagent A, the reagent B, the reagent C, the reagent D, the reagent E and the reagent F all comprise an internal control primer set and an internal control probe.

[0067] The nucleotide sequences of the primers and probes contained in the kit for detecting the TP53 gene mutation provided in the embodiment are shown in Table 1.

[0068] Table 1 Nucleotide sequences of primers and probes contained in a kit for detecting TP53 gene mutation

[0069]

[0070] The specific operation steps of detecting the DNA to be tested extracted from tissue or blood by using the above-mentioned kit for detecting TP53 gene mutation are as follows:

[0071] (1) DNA extraction

[0072] The DNA to be tested is extracted from tissue or blood, and the concentration is ≥1 ng / μL; or the EDTA anticoagulation whole blood sample is treated by using the nucleic acid releasing agent in the kit to obtain the DNA to be tested;

[0073] (2) qPCR amplification

[0074] The DNA to be tested is added into the PCR premix liquid (the PCR premix liquid includes hot-start Taq DNA polymerase, 200 μM dNTPs, 5 mM MgCl2, 0.2 wt% DMSO, 0.2 wt% formamide and PCR buffer, and the PCR buffer contains Tris, NaCl, NP40 and MgCl2), and is uniformly mixed, and the DNA to be tested is amplified by using the qPCR amplification technology (Real-Time Quantitative Polymerase Chain Reaction) and according to the following reaction program: pre-denaturation at 95℃ for 5 minutes; cycle stage: 95℃ for 15 seconds→60℃ for 30 seconds (36 cycles); the fluorescence signal is collected at the 60℃ stage: FAM, CY5 and ROX.

[0075] During the above experiment, in order to detect the genotype of the target mutation site of the TP53 gene, and to avoid the problem that it is difficult to distinguish between the wild type and the mutant target site in the process of detecting the wild type and the mutant site by adding wild type primers and mutant primers for the same target mutation site in the same system, two systems, i.e. system 1 and system 2, system 3 and system 4, system 5 and system 6, are set up. Among them, the wild type primers are added in system 1, 3 and 5 for detecting the wild type TP53 gene target site, and the mutant primers are added in system 2, 4 and 6 for detecting the mutant TP53 gene target site. Specifically, in addition to the DNA to be tested, reagent A (R282W wild type primer, R282W universal primer, R282W detection probe, R248Q wild type primer, R248Q universal primer, R248Q detection probe, internal control primer set, internal control probe) is added in system 1, reagent B (R282W mutant primer, R282W universal primer, R282W detection probe, R248Q mutant primer, R248Q universal primer, R248Q detection probe, internal control primer set, internal control probe) is added in system 2, reagent C (R273H wild type primer, R273H universal primer, R273H detection probe, G245S wild type primer, G245S universal primer, G245S detection probe, internal control primer set, internal control probe) is added in system 3, reagent D (R273H mutant primer, R273H universal primer, R273H detection probe, G245S mutant primer, G245S universal primer, G245S detection probe, internal control primer set, internal control probe) is added in system 4, reagent E (R175H wild type primer, R175H universal primer, R175H detection probe, R249S wild type primer, R249S universal primer, R249S detection probe, internal control primer set, internal control probe) is added in system 5, and reagent F (R175H mutant primer, R175H universal primer, R175H detection probe, R249S mutant primer, R249S universal primer, R249S detection probe, internal control primer set, internal control probe) is added in system 6.

[0076] (3) Result interpretation

[0077] Table 2 Result interpretation standard when detecting the genotype of the target mutation site of the mutant TP53 gene by using the kit for detecting TP53 gene mutation

[0078]

[0079] The final detection result (genotype of TP53 gene mutation site) is judged according to the result judgment standard as shown in Table 2. The Ct value of the detection result of the same gene in two holes (i.e. two systems) is ≤36, and |ΔCt|<5, which is judged as a heterozygous genotype. However, when 3.5<|ΔCt|<5, it is considered to belong to the heterozygous type, which belongs to the gray area, and the heterozygous type can be output.

[0080] At the same time, the internal control primer group (internal reference), internal control probe, external control plasmid group (positive control) and physiological saline (negative control) are added in each system to judge whether the detection result is correct. The Ct of the positive control is ≤33, the Ct of the negative control is >36, the experimental result is effective, and the analysis is carried out; the Ct of the internal reference housekeeping gene RPPH1 (CY5 channel) in the detection sample reaction hole is ≤34, then the detection hole result is effective, if the Ct of the internal reference housekeeping gene RPPH1 (CY5 channel) is >34, then the experiment of this detection hole is invalid; the internal reference |ΔCT| between the two holes of the same gene is ≤3, then the result of the sample to be tested site is analyzed, and |ΔCT|>3, it is considered that the sample loading amount is too large, and the gene detection result is invalid.

[0081] Example 2

[0082] The embodiment provides a kit for detecting TP53 gene mutation. Compared with example 1, the difference is that the nucleotide sequence of the internal control probe is shown in SEQ ID NO: 30, and the specific nucleotide sequence is 5'-CY5-AGCTTGGAACAGACTCACGGC-MGB-3'.

[0083] In addition to the above differences, the materials, formula ratio and preparation operation adopted in the embodiment are strictly consistent with those in example 1.

[0084] Example 3

[0085] The embodiment provides a kit for detecting TP53 gene mutation. Compared with example 1, the difference is that the nucleotide sequence of the internal control primer group in the first internal control primer 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'.

[0086] In addition to the above differences, the materials, formula ratio and preparation operation adopted in the embodiment are strictly consistent with those in example 1.

[0087] Comparative Example 1

[0088] The comparative example provides a kit for detecting TP53 gene mutation, compared with example 1, the difference is that the nucleotide sequence of the R175H wild type primer in reagent E is shown as SEQ ID NO: 33, and the specific nucleotide sequence is 5'-GACGGAGGTTGTGAGACG-3'.

[0089] Except for the above-mentioned difference, the materials, formula ratio and preparation operation adopted by the comparative example are strictly consistent with example 1.

[0090] Comparative example 2

[0091] The comparative example provides a kit for detecting TP53 gene mutation, compared with example 1, the difference is that the nucleotide sequence of the R175H wild type primer in reagent E is shown as SEQ ID NO: 34, and the specific nucleotide sequence is 5'-GACGGAGGTTGTGAAGCG-3'.

[0092] Except for the above-mentioned difference, the materials, formula ratio and preparation operation adopted by the comparative example are strictly consistent with example 1.

[0093] Comparative example 3

[0094] The comparative example provides a kit for detecting TP53 gene mutation, compared with example 1, the difference is that the nucleotide sequence of the R175H wild type primer in reagent E is shown as SEQ ID NO: 35, and the specific nucleotide sequence is 5'-GACGGAGGTTGTGAGACA-3'.

[0095] Except for the above-mentioned difference, the materials, formula ratio and preparation operation adopted by the comparative example are strictly consistent with example 1.

[0096] Comparative example 4

[0097] The comparative example provides a kit for detecting TP53 gene mutation, compared with example 1, the difference is that the nucleotide sequence of the R175H wild type primer in reagent E is shown as SEQ ID NO: 36, and the specific nucleotide sequence is 5'-GACGGAGGTTGTGAAGCA-3'.

[0098] Except for the above-mentioned difference, the materials, formula ratio and preparation operation adopted by the comparative example are strictly consistent with example 1.

[0099] Comparative example 5

[0100] The comparative example provides a kit for detecting TP53 gene mutation, compared with example 1, the difference is that the nucleotide sequence of the R249S wild type primer in reagent E is shown as SEQ ID NO: 37, and the specific nucleotide sequence is 5'-GATGATGGTGAGGATGAGC-3'.

[0101] Except for the above-mentioned difference, the materials, formula ratio and preparation operation adopted by the comparative example are strictly consistent with example 1.

[0102] Comparative example 6

[0103] The comparative example provides a kit for detecting TP53 gene mutation, compared with example 1, the difference is that the nucleotide sequence of the R249S wild type primer in reagent E is shown as SEQ ID NO: 38, and the specific nucleotide sequence is 5'-GATGATGGTGAGGATAGGC-3'.

[0104] Except for the above-mentioned difference, the materials, formula ratio and preparation operation adopted by the comparative example are strictly consistent with example 1.

[0105] Comparative example 7

[0106] The comparative example provides a kit for detecting TP53 gene mutation, compared with example 1, the difference is that the nucleotide sequence of the R249S wild type primer in reagent E is shown as SEQ ID NO: 38, and the specific nucleotide sequence is 5'-GATGATGGTGAGGATAGGC-3'.

[0107] Except for the above-mentioned difference, the materials, formula ratio and preparation operation adopted by the comparative example are strictly consistent with example 1.

[0108] Comparative example 8

[0109] The comparative example provides a kit for detecting TP53 gene mutation, compared with example 1, the difference is that the nucleotide sequence of the R249S wild type primer in reagent E is shown as SEQ ID NO: 38, and the specific nucleotide sequence is 5'-GATGATGGTGAGGATAGGC-3'.

[0110] Except for the above-mentioned difference, the materials, formula ratio and preparation operation adopted by the comparative example are strictly consistent with example 1.

[0111] Comparative example 9

[0112] The comparative example provides a kit for detecting TP53 gene mutation, compared with example 1, the difference is that the nucleotide sequence of the R282W wild type primer in reagent A is shown as SEQ ID NO: 41, and the specific nucleotide sequence is 5'-TCTCTTCCTCTGTGCGTCG-3'.

[0113] Except for the above-mentioned difference, the materials, formula ratio and preparation operation adopted by the comparative example are strictly consistent with example 1.

[0114] Comparative example 10

[0115] The comparative example provides a kit for detecting TP53 gene mutation, compared with example 1, the difference is that the nucleotide sequence of the R282W wild type primer in reagent A is shown as SEQ ID NO: 41, and the specific nucleotide sequence is 5'-TCTCTTCCTCTGTGCGTCG-3'.

[0116] Except for the above-mentioned difference, the materials, formula ratio and preparation operation adopted by the comparative example are strictly consistent with example 1.

[0117] Comparative example 11

[0118] The comparative example provides a kit for detecting TP53 gene mutation, compared with example 1, the difference is that the nucleotide sequence of the R282W wild type primer in reagent A is shown as SEQ ID NO: 41, and the specific nucleotide sequence is 5'-TCTCTTCCTCTGTGCGTCG-3'.

[0119] Except for the above-mentioned difference, the materials, formula ratio and preparation operation adopted by the comparative example are strictly consistent with example 1.

[0120] Comparative example 12

[0121] The comparative example provides a kit for detecting TP53 gene mutation, compared with example 1, the difference is that the nucleotide sequence of the R282W wild type primer in reagent A is shown as SEQ ID NO: 41, and the specific nucleotide sequence is 5'-TCTCTTCCTCTGTGCGTCG-3'.

[0122] Except for the above-mentioned difference, the materials, formula ratio and preparation operation adopted by the comparative example are strictly consistent with example 1.

[0123] Comparative example 13

[0124] The comparative example provides a kit for detecting TP53 gene mutation, compared with example 1, the difference is that the nucleotide sequence of the R248Q wild type primer in reagent A is shown as SEQ ID NO: 45, and the specific nucleotide sequence is 5'-ATGGGCGGCATGAATCG-3'.

[0125] Except for the above-mentioned difference, the materials, formula ratio and preparation operation adopted by the comparative example are strictly consistent with example 1.

[0126] Comparative example 14

[0127] The comparative example provides a kit for detecting TP53 gene mutation, compared with example 1, the difference is that the nucleotide sequence of the R248Q wild type primer in reagent A is shown as SEQ ID NO: 46, and the specific nucleotide sequence is 5'-ATGGGCGGCATGAGCCG-3'.

[0128] Except for the above-mentioned difference, the materials, formula ratio and preparation operation adopted by the comparative example are strictly consistent with example 1.

[0129] Comparative example 15

[0130] The comparative example provides a kit for detecting TP53 gene mutation, compared with example 1, the difference is that the nucleotide sequence of the R248Q wild type primer in reagent A is shown as SEQ ID NO: 46, and the specific nucleotide sequence is 5'-ATGGGCGGCATGAGCCG-3'.

[0131] Except for the above-mentioned difference, the materials, formula ratio and preparation operation adopted by the comparative example are strictly consistent with example 1.

[0132] Comparative example 16

[0133] The comparative example provides a kit for detecting TP53 gene mutation, compared with example 1, the difference is that the nucleotide sequence of the R248Q wild type primer in reagent A is shown as SEQ ID NO: 46, and the specific nucleotide sequence is 5'-ATGGGCGGCATGAGCCG-3'.

[0134] Except for the above-mentioned difference, the materials, formula ratio and preparation operation adopted by the comparative example are strictly consistent with example 1.

[0135] Comparative example 17

[0136] The comparative example provides a kit for detecting TP53 gene mutation, compared with example 1, the difference is that the nucleotide sequence of the R273H wild type primer in reagent C is shown as SEQ ID NO: 49, and the specific nucleotide sequence is 5'-CAGGACAGGCACAAATAC-3'.

[0137] Except for the above-mentioned difference, the materials, formula ratio and preparation operation adopted by the comparative example are strictly consistent with example 1.

[0138] Comparative example 18

[0139] The comparative example provides a kit for detecting TP53 gene mutation, compared with example 1, the difference is that the nucleotide sequence of the R273H wild type primer in reagent C is shown as SEQ ID NO: 50, and the specific nucleotide sequence is 5'-CAGGACAGGCACAAGCAC-3'.

[0140] Except for the above-mentioned difference, the materials, formula ratio and preparation operation adopted by the comparative example are strictly consistent with example 1.

[0141] Comparative example 19

[0142] The comparative example provides a kit for detecting TP53 gene mutation, compared with example 1, the difference is that the nucleotide sequence of the R273H wild type primer in reagent C is shown as SEQ ID NO: 50, and the specific nucleotide sequence is 5'-CAGGACAGGCACAAGCAC-3'.

[0143] Except for the above-mentioned difference, the materials, formula ratio and preparation operation adopted by the comparative example are strictly consistent with example 1.

[0144] Comparative example 20

[0145] The comparative example provides a kit for detecting TP53 gene mutation, compared with example 1, the difference is that the nucleotide sequence of the R273H wild type primer in reagent C is shown as SEQ ID NO: 50, and the specific nucleotide sequence is 5'-CAGGACAGGCACAAGCAC-3'.

[0146] Except for the above-mentioned difference, the materials, formula ratio and preparation operation adopted by the comparative example are strictly consistent with example 1.

[0147] Comparative example 21

[0148] The comparative example provides a kit for detecting TP53 gene mutation, compared with example 1, the difference is that the nucleotide sequence of the G245S wild type primer in reagent C is shown as SEQ ID NO: 53, and the specific nucleotide sequence is 5'-GTAACAGTTCCTGCATGGGGC-3'.

[0149] Except for the above-mentioned difference, the materials, formula ratio and preparation operation adopted by the comparative example are strictly consistent with example 1.

[0150] Comparative example 22

[0151] The comparative example provides a kit for detecting TP53 gene mutation, compared with example 1, the difference is that the nucleotide sequence of the G245S wild type primer in reagent C is shown as SEQ ID NO: 54, and the specific nucleotide sequence is 5'-GTAACAGTTCCTGCATGGAAC-3'.

[0152] Except for the above-mentioned difference, the materials, formula ratio and preparation operation adopted by the comparative example are strictly consistent with example 1.

[0153] Comparative example 23

[0154] The comparative example provides a kit for detecting TP53 gene mutation, compared with example 1, the difference is that the nucleotide sequence of the G245S wild type primer in reagent C is shown as SEQ ID NO: 54, and the specific nucleotide sequence is 5'-GTAACAGTTCCTGCATGGAAC-3'.

[0155] Except for the above-mentioned difference, the materials, formula ratio and preparation operation adopted by the comparative example are strictly consistent with example 1.

[0156] Comparative example 24

[0157] The comparative example provides a kit for detecting TP53 gene mutation, compared with example 1, the difference is that the nucleotide sequence of the G245S wild type primer in reagent C is shown as SEQ ID NO: 54, and the specific nucleotide sequence is 5'-GTAACAGTTCCTGCATGGAAC-3'.

[0158] Except for the above-mentioned difference, the materials, formula ratio and preparation operation adopted by the comparative example are strictly consistent with example 1.

[0159] Test example 1

[0160] The present test example aims to study the detection effect of the wild type primer and the mutant primer for 6 target mutation sites in the kit for detecting TP53 gene mutation provided by Example 1 and Comparative Examples 1-24.

[0161] 1. Wild type primer and mutant primer for target mutation site R175H in Example 1 and Comparative Examples 1-4

[0162] For the purpose of comparison, the nucleotide sequences of the R175H wild type primer and the R175H mutant primer for the target mutation site R175H in the kit for detecting TP53 gene mutation provided by Example 1 and Comparative Examples 1-4 are integrated into Table 3.

[0163] Table 3 Nucleotide sequences of R175H wild type primer and R175H mutant primer for target mutation site R175H in kit for detecting TP53 gene mutation provided by Example 1 and Comparative Examples 1-4

[0164]

[0165] The R175H wild type primer and the R175H mutant primer in the kit for detecting TP53 gene mutation provided by Example 1 and Comparative Examples 1-4 were used to detect the R175H wild type specimen and the mutant specimen, respectively, and the Ct value detection results are shown in Table 4, and the amplification curve results are shown in Figure 1 , wherein, Figure 1 A-B are the amplification curve detection results of the R175H wild type primer in the kit provided by Example 1 for the R175H wild type specimen and the mutant specimen, respectively, Figure 1 C-D are the amplification curve detection results of the R175H wild type primer in the kit provided by Comparative Example 1 for the R175H wild type specimen and the mutant specimen, respectively, Figure 1 E-F are the amplification curve detection results of the R175H wild type primer in the kit provided by Comparative Example 2 for the R175H wild type specimen and the mutant specimen, respectively, Figure 1 G-H are the amplification curve detection results of the R175H mutant primer in the kit provided by Example 1 for the R175H wild type specimen and the mutant specimen, respectively, Figure 1 I-J are the amplification curve detection results of the R175H mutant primer in the kit provided by Comparative Example 3 for the R175H wild type specimen and the mutant specimen, respectively, Figure 1 K-L are the amplification curve detection results of the R175H mutant primer in the kit provided by Comparative Example 4 for the R175H wild type specimen and the mutant specimen, respectively.

[0166] Table 4 Detection results of R175H wild type primer and R175H mutant primer in the kit for detecting TP53 gene mutation provided by Example 1 and Comparative Examples 1-4 on R175H wild type sample and mutant sample

[0167]

[0168] From Figure 1 As can be seen from Table 4, when the R175H wild type primer in the kit provided by Example 1 is used to detect the R175H wild type sample and the mutant sample, the R175H wild type primer with the nucleotide sequence shown in SEQ ID NO: 9 has a satisfactory amplification efficiency on the R175H wild type sample and no detection on the R175H mutant sample, while when the R175H wild type primer in the kit provided by Comparative Examples 1-2 is used to detect the R175H wild type sample and the mutant sample, the R175H wild type primer with the nucleotide sequence shown in SEQ ID NO: 33, 34 has a satisfactory amplification efficiency on the R175H wild type sample but has detection on the R175H mutant sample; when the R175H mutant primer in the kit provided by Example 1 is used to detect the R175H wild type sample and the mutant sample, the R175H mutant primer with the nucleotide sequence shown in SEQ ID NO: 11 has a satisfactory amplification efficiency on the R175H mutant sample and no detection on the R175H wild type sample, while when the R175H mutant primer in the kit provided by Comparative Examples 3-4 is used to detect the R175H wild type sample and the mutant sample, the R175H mutant primer with the nucleotide sequence shown in SEQ ID NO: 35, 36 has a satisfactory amplification efficiency on the R175H mutant sample but has detection on the R175H wild type sample.

[0169] The above results show that, compared with Comparative Examples 1-4, the R175H wild type primer with the nucleotide sequence shown in SEQ ID NO: 9 and the R175H mutant primer with the nucleotide sequence shown in SEQ ID NO: 11 in the kit provided by Example 1 are more suitable for detecting the target site R175H of TP53 gene, and can well distinguish the R175H wild type and mutant sites.

[0170] 2. R249S wild type primer and R249S mutant primer for target mutant site R249S in Example 1 and Comparative Examples 5-8

[0171] For the convenience of comparison, the nucleotide sequences of the R249S wild type primer and the R249S mutant primer for detecting the target mutant site R249S in the kit for detecting TP53 gene mutation provided by Example 1 and Comparative Examples 5-8 are integrated into Table 5.

[0172] Table 5 Nucleotide sequences of R249S wild type primer and R249S mutant primer for target mutation site R249S in the kit for detecting TP53 gene mutation provided by Example 1 and Comparative Examples 5-8

[0173]

[0174] The R249S wild type specimen and mutant specimen were detected by using the R249S wild type primer and R249S mutant primer in the kit for detecting TP53 gene mutation provided by Example 1 and Comparative Examples 5-8 respectively, and the Ct value detection results are shown in Table 6, and the amplification curve results are shown in Figure 2 Figure 2 A-B are the amplification curve detection results of the R249S wild type specimen and mutant specimen by using the R249S wild type primer in the kit provided by Example 1 respectively, Figure 2 C-D are the amplification curve detection results of the R249S wild type specimen and mutant specimen by using the R249S wild type primer in the kit provided by Comparative Example 5 respectively, Figure 2 E-F are the amplification curve detection results of the R249S wild type specimen and mutant specimen by using the R249S wild type primer in the kit provided by Comparative Example 6 respectively, Figure 2 G-H are the amplification curve detection results of the R249S wild type specimen and mutant specimen by using the R249S mutant primer in the kit provided by Example 1 respectively, Figure 2 I-J are the amplification curve detection results of the R249S wild type specimen and mutant specimen by using the R249S mutant primer in the kit provided by Comparative Example 7 respectively, Figure 2 K-L are the amplification curve detection results of the R249S wild type specimen and mutant specimen by using the R249S mutant primer in the kit provided by Comparative Example 8 respectively.

[0175] Table 6 Detection results of R249S wild type specimen and mutant specimen by R249S wild type primer and R249S mutant primer in the kit for detecting TP53 gene mutation provided by Example 1 and Comparative Examples 5-8

[0176]

[0177] From Figure 2 ​As shown in Table 6 and Table 7, when the R249S wild type primer with the nucleotide sequence shown in SEQ ID NO: 18 in the kit provided in Example 1 was used to detect the R249S wild type sample and the mutant sample, the efficiency of the R249S wild type primer with the nucleotide sequence shown in SEQ ID NO: 18 in amplifying the R249S wild type sample met the requirements and no detection was made when the R249S mutant sample was amplified, while when the R249S wild type primer in the kit provided in Comparative Examples 5-6 was used to detect the R249S wild type sample and the mutant sample, the efficiency of the R249S wild type primer with the nucleotide sequence shown in SEQ ID NO: 37 or 38 in amplifying the R249S wild type sample met the requirements but detection was made when the R249S mutant sample was amplified; when the R249S mutant primer in the kit provided in Example 1 was used to detect the R249S wild type sample and the mutant sample, the efficiency of the R249S mutant primer with the nucleotide sequence shown in SEQ ID NO: 20 in amplifying the R249S mutant sample met the requirements and no detection was made when the R249S wild type sample was amplified, while when the R249S mutant primer in the kit provided in Comparative Examples 7-8 was used to detect the R249S wild type sample and the mutant sample, the efficiency of the R249S mutant primer with the nucleotide sequence shown in SEQ ID NO: 39 or 40 in amplifying the R249S mutant sample met the requirements but detection was made when the R249S wild type sample was amplified.

[0178] The above results show that, compared with Comparative Examples 5-8, the R249S wild type primer with the nucleotide sequence shown in SEQ ID NO: 18 and the R249S mutant primer with the nucleotide sequence 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 well distinguish the R249 wild type and mutant sites.

[0179] 3. R282W wild type primer and R282W mutant primer for the target mutant site R282W in Example 1 and Comparative Examples 9-12

[0180] For the purpose of comparison, the nucleotide sequences of the R282W wild type primer and the R282W mutant primer for the target mutant site R282W in the kit for detecting the mutation of the TP53 gene provided in Example 1 and Comparative Examples 9-12 are integrated into Table 7.

[0181] Table 7 Nucleotide sequences of the R282W wild type primer and the R282W mutant primer for the target mutant site R282W in the kit for detecting the mutation of the TP53 gene provided in Example 1 and Comparative Examples 9-12

[0182]

[0183] The R282W wild type primer and the R282W mutant primer in the kit for detecting TP53 gene mutation provided by Example 1 and Comparative Examples 9-12 were used to detect the R282W wild type sample and the mutant sample, respectively, and the Ct value detection results are shown in Table 8, and the amplification curve results are shown in Figure 3 Figure 3 A-B are the amplification curve detection results of the R282W wild type sample and the mutant sample using the R282W wild type primer in the kit provided by Example 1, Figure 3 C-D are the amplification curve detection results of the R282W wild type sample and the mutant sample using the R282W wild type primer in the kit provided by Comparative Example 9, Figure 3 E-F are the amplification curve detection results of the R282W wild type sample and the mutant sample using the R282W wild type primer in the kit provided by Comparative Example 10, Figure 3 G-H are the amplification curve detection results of the R282W wild type sample and the mutant sample using the R282W mutant primer in the kit provided by Example 1, Figure 3 I-J are the amplification curve detection results of the R282W wild type sample and the mutant sample using the R282W mutant primer in the kit provided by Comparative Example 11, Figure 3 K-L are the amplification curve detection results of the R282W wild type sample and the mutant sample using the R282W mutant primer in the kit provided by Comparative Example 12.

[0184] Table 8 Detection results of the R282W wild type sample and the mutant sample using the R282W wild type primer and the R282W mutant primer in the kit for detecting TP53 gene mutation provided by Example 1 and Comparative Examples 9-12

[0185]

[0186] Figure 3 ​​As shown in Table 8 and Table 9, when the R282W wild type primer with the nucleotide sequence shown in SEQ ID NO: 1 in the kit provided in Example 1 was used to detect the R282W wild type sample and the mutant sample, the efficiency of the R282W wild type primer with the nucleotide sequence shown in SEQ ID NO: 1 in amplifying the R282W wild type sample met the requirements and no detection was made when the R282W wild type primer was used to amplify the R282W mutant sample; when the R282W wild type primer with the nucleotide sequence shown in SEQ ID NO: 41 or 42 in the kit provided in Comparative Example 9 or 10 was used to detect the R282W wild type sample and the mutant sample, the efficiency of the R282W wild type primer with the nucleotide sequence shown in SEQ ID NO: 41 or 42 in amplifying the R282W wild type sample met the requirements but detection was made when the R282W wild type primer was used to amplify the R282W mutant sample; when the R282W mutant primer with the nucleotide sequence shown in SEQ ID NO: 3 in the kit provided in Example 1 was used to detect the R282W wild type sample and the mutant sample, the efficiency of the R282W mutant primer with the nucleotide sequence shown in SEQ ID NO: 3 in amplifying the R282W mutant sample met the requirements and no detection was made when the R282W mutant primer was used to amplify the R282W wild type sample; when the R282W mutant primer with the nucleotide sequence shown in SEQ ID NO: 43 or 44 in the kit provided in Comparative Example 11 or 12 was used to detect the R282W wild type sample and the mutant sample, the efficiency of the R282W mutant primer with the nucleotide sequence shown in SEQ ID NO: 43 or 44 in amplifying the R282W mutant sample met the requirements but detection was made when the R282W mutant primer was used to amplify the R282W wild type sample.

[0187] The above results show that, compared with Comparative Examples 9 to 12, the R282W wild type primer with the nucleotide sequence shown in SEQ ID NO: 1 and the R282W mutant primer with the nucleotide sequence 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 well distinguish the R282W wild type and mutant sites.

[0188] 4. R248Q wild type primer and R248Q mutant primer for the target mutant site R248Q in Example 1 and Comparative Examples 13 to 16

[0189] For the convenience of comparison, the nucleotide sequences of the R248Q wild type primer and the R248Q mutant primer for the target mutant site R248Q in the kit for detecting the mutation of the TP53 gene provided in Example 1 and Comparative Examples 13 to 16 are integrated into Table 9.

[0190] Table 9 Nucleotide sequences of R248Q wild type primer and R248Q mutant primer for the target mutant site R248Q in the kit for detecting the mutation of the TP53 gene provided in Example 1 and Comparative Examples 13 to 16

[0191]

[0192] The R248Q wild type primer and the R248Q mutant primer in the kit for detecting TP53 gene mutation provided by Example 1 and Comparative Examples 13-16 were used to detect the R248Q wild type sample and the mutant sample, respectively, and the Ct value detection results are shown in Table 10, and the amplification curve results are shown in Figure 4 Figure 4 A-B are the amplification curve detection results of the R248Q wild type sample and the mutant sample using the R248Q wild type primer in the kit provided by Example 1, respectively, Figure 4 C-D are the amplification curve detection results of the R248Q wild type sample and the mutant sample using the R248Q wild type primer in the kit provided by Comparative Example 13, respectively, Figure 4 E-F are the amplification curve detection results of the R248Q wild type sample and the mutant sample using the R248Q wild type primer in the kit provided by Comparative Example 14, respectively, Figure 4 G-H are the amplification curve detection results of the R248Q wild type sample and the mutant sample using the R248Q mutant primer in the kit provided by Example 1, respectively, Figure 4 I-J are the amplification curve detection results of the R248Q wild type sample and the mutant sample using the R248Q mutant primer in the kit provided by Comparative Example 15, respectively, Figure 4 K-L are the amplification curve detection results of the R248Q wild type sample and the mutant sample using the R248Q mutant primer in the kit provided by Comparative Example 16, respectively.

[0193] Table 10 Detection results of the R248Q wild type sample and the mutant sample using the R248Q wild type primer and the R248Q mutant primer in the kit for detecting TP53 gene mutation provided by Example 1 and Comparative Examples 13-16

[0194]

[0195] Figure 4 ​​As shown in Table 10 and Table 11, when the R248Q wild type primer in the kit provided in Example 1 was used to detect the R248Q wild type sample and the mutant sample, the R248Q wild type primer with the nucleotide sequence shown in SEQ ID NO: 2 could meet the requirement for amplifying the R248Q wild type sample and had no detection for the R248Q mutant sample, while the R248Q wild type primer in the kit provided in Comparative Examples 13-14 was used to detect the R248Q wild type sample and the mutant sample, the R248Q wild type primer with the nucleotide sequence shown in SEQ ID NO: 45, 46 could meet the requirement for amplifying the R248Q wild type sample but had detection for the R248Q mutant sample; when the R248Q mutant primer in the kit provided in Example 1 was used to detect the R248Q wild type sample and the mutant sample, the R248Q mutant primer with the nucleotide sequence shown in SEQ ID NO: 4 could meet the requirement for amplifying the R248Q mutant sample and had no detection for the R248Q wild type sample, while the R248Q mutant primer in the kit provided in Comparative Examples 15-16 was used to detect the R248Q wild type sample and the mutant sample, the R248Q mutant primer with the nucleotide sequence shown in SEQ ID NO: 47, 48 could meet the requirement for amplifying the R248Q mutant sample but had detection for the R248Q wild type sample.

[0196] The above results show that, compared with Comparative Examples 13-16, the R248Q wild type primer with the nucleotide sequence shown in SEQ ID NO: 2 and the R248Q mutant primer with the nucleotide sequence shown in SEQ ID NO: 4 in the kit provided in Example 1 are more suitable for detecting the target site R248Q of TP53 gene, which can well distinguish the R248Q wild type and mutant sites.

[0197] 5. R273H wild type primer and R273H mutant primer for target mutant site R273H in Example 1 and Comparative Examples 17-20

[0198] For the convenience of comparison, the nucleotide sequences of the R273H wild type primer and the R273H mutant primer for detecting the target mutant site R273H in the kit for detecting the mutation of TP53 gene provided in Example 1 and Comparative Examples 17-20 are integrated into Table 11.

[0199] Table 11 Nucleotide sequences of R273H wild type primer and R273H mutant primer for target mutant site R273H in the kit for detecting the mutation of TP53 gene provided in Example 1 and Comparative Examples 17-20

[0200]

[0201] The R273H wild type primer and the R273H mutant primer in the kit for detecting TP53 gene mutation provided by Example 1 and Comparative Examples 17-20 were used to detect the R273H wild type sample and the mutant sample, respectively, and the Ct value detection results are shown in Table 12, and the amplification curve results are shown in Figure 5 Figure 5 A-B are the amplification curve detection results of the R273H wild type sample and the mutant sample using the R273H wild type primer in the kit provided by Example 1, Figure 5 C-D are the amplification curve detection results of the R273H wild type sample and the mutant sample using the R273H wild type primer in the kit provided by Comparative Example 17, Figure 5 E-F are the amplification curve detection results of the R273H wild type sample and the mutant sample using the R273H wild type primer in the kit provided by Comparative Example 18, Figure 5 G-H are the amplification curve detection results of the R273H wild type sample and the mutant sample using the R273H mutant primer in the kit provided by Example 1, Figure 5 I-J are the amplification curve detection results of the R273H wild type sample and the mutant sample using the R273H mutant primer in the kit provided by Comparative Example 19, Figure 5 K-L are the amplification curve detection results of the R273H wild type sample and the mutant sample using the R273H mutant primer in the kit provided by Comparative Example 20.

[0202] Table 12 Detection results of the R273H wild type sample and the mutant sample using the R273H wild type primer and the R273H mutant primer in the kit for detecting TP53 gene mutation provided by Example 1 and Comparative Examples 17-20

[0203]

[0204] From Figure 5 ​As shown in Table 12, when the R273H wild-type primer in the kit provided in Example 1 was used to detect the R273H wild-type sample and the mutant sample, the R273H wild-type primer with the nucleotide sequence shown in SEQ ID NO: 9 had a satisfactory amplification efficiency for the R273H wild-type sample and no detection for the R273H mutant sample, while the R273H wild-type primer in the kit provided in Comparative Examples 17-18 was used to detect the R273H wild-type sample and the mutant sample, the R273H wild-type primer with the nucleotide sequence shown in SEQ ID NO: 49, 50 had a satisfactory amplification efficiency for the R273H wild-type sample but had detection for the R248Q mutant sample; when the R273H mutant primer in the kit provided in Example 1 was used to detect the R273H wild-type sample and the mutant sample, the R273H mutant primer with the nucleotide sequence shown in SEQ ID NO: 11 had a satisfactory amplification efficiency for the R273H mutant sample and no detection for the R273H wild-type sample, while the R273H mutant primer in the kit provided in Comparative Examples 19-20 was used to detect the R273H wild-type sample and the mutant sample, the R273H mutant primer with the nucleotide sequence shown in SEQ ID NO: 51, 52 had a satisfactory amplification efficiency for the R273H mutant sample but had detection for the R273H wild-type sample.

[0205] The above results show that, compared with Comparative Examples 17-20, the R273H wild-type primer with the nucleotide sequence shown in SEQ ID NO: 9 and the R273H mutant primer with the nucleotide sequence 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 well distinguish the R273H wild-type and mutant sites.

[0206] 6. G245S wild-type primer and G245S mutant primer for the target mutant site G245S in Example 1 and Comparative Examples 21-24

[0207] For the purpose of comparison, the nucleotide sequences of the G245S wild-type primer and the G245S mutant primer for the target mutant site G245S in the kit for detecting the mutation of the TP53 gene provided in Example 1 and Comparative Examples 21-24 are integrated into Table 13.

[0208] Table 13 Nucleotide sequences of the G245S wild-type primer and the G245S mutant primer for the target mutant site G245S in the kit for detecting the mutation of the TP53 gene provided in Example 1 and Comparative Examples 21-24

[0209]

[0210] The G245S wild type specimen and mutant specimen were detected by the G245S wild type primer and G245S mutant primer in the kit for detecting TP53 gene mutation provided by Example 1 and Comparative Examples 21-24 respectively, and the Ct value detection results are shown in Table 14, and the amplification curve results are shown in Figure 6 Figure 6 A-B are the amplification curve detection results of the G245S wild type specimen and mutant specimen by the G245S wild type primer in the kit provided by Example 1, Figure 6 C-D are the amplification curve detection results of the G245S wild type specimen and mutant specimen by the G245S wild type primer in the kit provided by Comparative Example 21, Figure 6 E-F are the amplification curve detection results of the G245S wild type specimen and mutant specimen by the G245S wild type primer in the kit provided by Comparative Example 22, Figure 6 G-H are the amplification curve detection results of the G245S wild type specimen and mutant specimen by the G245S mutant primer in the kit provided by Example 1, Figure 6 I-J are the amplification curve detection results of the G245S wild type specimen and mutant specimen by the G245S mutant primer in the kit provided by Comparative Example 23, Figure 6 K-L are the amplification curve detection results of the G245S wild type specimen and mutant specimen by the G245S mutant primer in the kit provided by Comparative Example 24.

[0211] Table 14 Detection results of G245S wild type specimen and mutant specimen by G245S wild type primer and G245S mutant primer in the kit for detecting TP53 gene mutation provided by Example 1 and Comparative Examples 21-24

[0212]

[0213] From Figure 6 ​As shown in Table 14 and Table 15, when the G245S wild type primer with the nucleotide sequence as shown in SEQ ID NO: 10 in the kit provided in Example 1 was used to detect the G245S wild type sample and the mutant sample, the G245S wild type primer with the nucleotide sequence as shown in SEQ ID NO: 10 could meet the requirement for amplifying the G245S wild type sample and had no detection for the G245S mutant sample, while when the G245S wild type primer with the nucleotide sequence as shown in SEQ ID NO: 53, 54 in the kit provided in Comparative Example 21~22 was used to detect the G245S wild type sample and the mutant sample, the G245S wild type primer with the nucleotide sequence as shown in SEQ ID NO: 53, 54 could meet the requirement for amplifying the G245S wild type sample but had detection for the R248Q mutant sample; when the G245S mutant primer with the nucleotide sequence as shown in SEQ ID NO: 12 in the kit provided in Example 1 was used to detect the G245S wild type sample and the mutant sample, the G245S mutant primer with the nucleotide sequence as shown in SEQ ID NO: 12 could meet the requirement for amplifying the G245S mutant sample and had no detection for the G245S wild type sample, while when the G245S mutant primer with the nucleotide sequence as shown in SEQ ID NO: 55, 56 in the kit provided in Comparative Example 23~24 was used to detect the G245S wild type sample and the mutant sample, the G245S mutant primer with the nucleotide sequence as shown in SEQ ID NO: 55, 56 could meet the requirement for amplifying the G245S mutant sample but had detection for the G245S wild type sample.

[0214] The above results show that, compared with Comparative Examples 21~24, the G245S wild type primer with the nucleotide sequence as shown in SEQ ID NO: 10 and the G245S mutant primer with the 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 well distinguish the G245S wild type and mutant sites.

[0215] Test Example 2

[0216] The present test example aims to study the detection effect of the internal control probe in the kit for detecting the mutation of the TP53 gene provided in Example 1 and Example 2 and the internal control primer set in the kit for detecting the mutation of the TP53 gene provided in Example 3 on different clinical samples (i.e. clinical sample 1 and clinical sample 2).

[0217] In order to facilitate comparison, the nucleotide sequences of the internal control probe and the internal control primer set in the kit for detecting the mutation of the TP53 gene provided in Examples 1~3 are integrated into Table 15.

[0218] Table 15 Nucleotide sequences of the internal control probe and the internal control primer set in the kit for detecting the mutation of the TP53 gene provided in Examples 1~3

[0219]

[0220] The kit for detecting TP53 gene mutation provided by Examples 1-3 was used to detect clinical sample 1 and clinical sample 2 according to the detection method of Test Example 1, and the Ct value detection results are shown in Table 16, and the amplification curve results are shown in Figure 7 Figure 7 A-B are the amplification curve results of detecting clinical sample 1 and clinical sample 2, respectively, using the kit provided by Example 1, Figure 7 C-D are the amplification curve results of detecting clinical sample 1 and clinical sample 2, respectively, using the kit provided by Example 2, Figure 7 E-F are the amplification curve results of detecting clinical sample 1 and clinical sample 2, respectively, using the kit provided by Example 3.

[0221] Table 16 Detection results of different clinical samples by the kit for detecting TP53 gene mutation provided by Examples 1-3

[0222]

[0223] From Table 16 and Figure 7 It can be seen that when the kit provided by Example 1 is used to detect clinical sample 1 and clinical sample 2, the amplification efficiency of clinical sample 1 and 2 meets the requirements, and when the kits provided by Examples 2-3 are used to detect clinical sample 1 and clinical sample 2, the amplification efficiency of clinical sample 1 and 2 does not meet the requirements. The above results can show that compared with Examples 2-3, the kit provided by Example 1 is suitable for detecting TP53 gene mutation, and has higher amplification or detection efficiency.

[0224] Test Example 3

[0225] The present test example aims to detect the sensitivity (FAM signal, ROX signal, CY5 signal) of the kit for detecting TP53 gene mutation provided by Example 1 in detecting TP53 gene mutation according to the detection method of Test Example 1, and the housekeeping gene RPPH1 is used as an internal reference (its role in detection is to monitor whether the specimen is added to the PCR reaction system and whether the PCR reaction process of the to-be-tested hole position is normal), and the amplification curve results of the internal reference amplified by the internal control primer set are shown in Figure 8 The amplification curve results of wild-type specimens and mutant specimens for wild-type primers and mutant primers for two target mutation sites R282W and R248Q are shown in Figure 9 The amplification curve results of wild-type specimens and mutant specimens for wild-type primers and mutant primers for two target mutation sites R273H and G245S are shown in Figure 10 ​As shown, the amplification curve results of wild type samples and mutant samples for wild type primers and mutant primers targeting the two target mutation sites R175H and R249S are as follows Figure 11 As shown, the amplification curve results of wild type samples and mutant samples for wild type primers and mutant primers targeting the two target mutation sites R175H and R249S are as follows Figure 9 A-B are respectively the amplification curve detection results of R282W and R248Q wild type samples by the kit for detecting TP53 gene mutations provided in Example 1, Figure 9 C-D are respectively the amplification curve detection results of R282W and R248Q mutant samples by the kit for detecting TP53 gene mutations provided in Example 1, Figure 10 A-B are respectively the amplification curve detection results of R273H and G245S wild type samples by the kit for detecting TP53 gene mutations provided in Example 1, Figure 10 C-D are respectively the amplification curve detection results of R273H and G245S mutant samples by the kit for detecting TP53 gene mutations provided in Example 1, Figure 11 A-B are respectively the amplification curve detection results of R175H and R249S wild type samples by the kit for detecting TP53 gene mutations provided in Example 1, Figure 11 C-D are respectively the amplification curve detection results of R175H and R249S mutant samples by the kit for detecting TP53 gene mutations provided in Example 1.

[0226] As shown in Table 17, the results of the kit for detecting TP53 gene mutations provided in Example 1 and Sanger sequencing in detecting TP53 gene mutations in different clinical samples are as follows, wherein 20 healthy people (Sample1#-Sample20#) and 50 tumor patients (Ca1#-Ca50#) were detected. Figures 9-11 It can be seen that the minimum detection limit of the kit provided in Example 1 is 50 copies (FAM Ct value = 34.5 ± 0.3), there is no cross with wild type background, and it can be normally detected by adding only 1 ng of genomic DNA (i.e. the DNA to be detected) in the PCR system.

[0227] Test Example 4

[0228] The present test example aims to study the consistency of the results of the kit for detecting TP53 gene mutations provided in Example 1 and Sanger sequencing in detecting TP53 gene mutations in clinical samples by referring to the detection method of Test Example 1, and the results are shown in Table 17, wherein 70 clinical samples were detected, of which 20 were from healthy people (Sample1-20#) and 50 were from tumor patients (Ca1-50#).

[0229] Table 17 Comparison of the results of the kit for detecting TP53 gene mutations provided in Example 1 and Sanger sequencing in testing different clinical samples, wherein 20 healthy people (Sample1#-Sample20#) and 50 tumor patients (Ca1#-Ca50#) were detected.

[0230]

[0231] As shown in Table 17, when the kit for detecting TP53 gene mutation provided in Example 1 is used to detect clinical samples, the detection results have good consistency with the detection results of Sanger sequencing (first-generation sequencing), and the accuracy reaches 100%.

[0232] The above examples are only used to illustrate the technical solutions of the present application, but not to limit the protection scope of the present application. Although the present application has been described in detail with reference to the above examples, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or replaced equivalently, and these modifications or replacements are within the protection scope of the present application.

Claims

1. A kit for detecting a mutation in a TP53 gene, characterized in that: The kit comprises reagent A, reagent B, reagent C, reagent D, reagent E, reagent F, an internal control primer set and an internal control probe; The reagent A comprises 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 as SEQ ID NO: 1, the nucleotide sequence of the R248Q wild type primers is shown as SEQ ID NO: 2; The reagent B comprises 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 as SEQ ID NO: 3, the nucleotide sequence of the R248Q mutant primers is shown as SEQ ID NO: 4; In the reagent A and the reagent B, the nucleotide sequence of the R282W detection probe is shown as SEQ ID NO: 5, the nucleotide sequence of the R248Q detection probe is shown as SEQ ID NO: 6, the nucleotide sequence of the R282W universal primer is shown as SEQ ID NO: 7, and the nucleotide sequence of the R248Q universal primer is shown as SEQ ID NO: 8; The reagent C comprises 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 as SEQ ID NO: 9, and the nucleotide sequence of the G245S wild type primers is shown as SEQ ID NO: 10; The reagent D comprises 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 as SEQ ID NO: 11, and the nucleotide sequence of the G245S mutant primers is shown as SEQ ID NO: 12; In the reagent C and the reagent D, the nucleotide sequence of the R273H detection probe is shown as SEQ ID NO: 13, the nucleotide sequence of the G245S detection probe is shown as SEQ ID NO: 14, the nucleotide sequence of the R273H universal primer is shown as SEQ ID NO: 15, and the nucleotide sequence of the G245S universal primer is shown as SEQ ID NO: 16; The reagent E comprises R175H wild type primers, R249S wild type primers, R175H detection probes, R249S detection probes, R175H universal primers and R249S universal primers, the nucleotide sequence of the R175H wild type primers is shown as SEQ ID NO: 17, and the nucleotide sequence of the R249S wild type primers is shown as SEQ ID NO: 18; The reagent F comprises 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 as SEQ ID NO: 19, and the nucleotide sequence of the R249S mutant primers is shown as SEQ ID NO: 20; In the reagent E and the reagent F, the nucleotide sequence of the R175H detection probe is shown as SEQ ID NO: 21, the nucleotide sequence of the R249S detection probe is shown as SEQ ID NO: 22, the nucleotide sequence of the R175H universal primer is shown as SEQ ID NO: 23, and the nucleotide sequence of the R249S universal primer is shown as SEQ ID NO: 24; The internal control primer set comprises a first internal control primer and a second internal control primer, the nucleotide sequence of the first internal control primer is shown as SEQ ID NO: 25, and the nucleotide sequence of the second internal control primer is shown as SEQ ID NO: 26; The nucleotide sequence of the internal control probe is shown as SEQ ID NO:

27.

2. The kit for detecting TP53 gene mutation according to claim 1, wherein: 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 all contain a quencher and a fluorescent group, the quencher 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 quencher 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 mutation according to claim 1, wherein: The 3' end of the internal control probe is labeled with a quencher MGB, and the 5' end of the internal control probe is labeled with a fluorescent group CY5.

5. The kit for detecting TP53 gene mutation according to claim 1, wherein: The kit further comprises an external control plasmid set, the external control plasmid set comprises a first external control plasmid and a second external control plasmid, the nucleotide sequence of the first external control plasmid is shown as SEQ ID NO: 28, and the nucleotide sequence of the second external control plasmid is shown as SEQ ID NO:

29.

6. The kit for detecting a mutation of TP53 gene according to claim 1, wherein The kit further comprises a PCR master mix comprising the following components: hot start Taq DNA polymerase, 10-1000 μΜ dNTPs, 2-10 mM MgCl2, 0.01-0.5 wt% DMSO, 0.01-0.5 wt% formamide.

7. The kit for detecting a mutation in TP53 gene according to claim 6, wherein: The PCR master mix further comprises a PCR buffer comprising Tris, NaCl, NP-40, MgCl2.

8. The kit for detecting TP53 gene mutation according to claim 1, wherein The kit further comprises a nucleic acid releasing agent comprising the following components: 0.01-0.1 M HCl, 0.01-0.05 wt% SDS, 1-10 mM Surfactin, 0.05-3 wt% trehalose.

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