Detection kit based on 19 mutations of human CTNNB1 gene

Abstract

The invention discloses a detection kit based on 19 mutations of a human CTNNB1 gene, and the kit provided by the invention constructs a fluorescent quantitative PCR (Polymerase Chain Reaction) and digital PCR detection system capable of simultaneously detecting and quantifying 19 mutations in a tube by providing a long LNA blocking probe capable of covering 19 mutation types contained in 8 mutation sites of a third exon of CTNNB1. The detection limit of a fluorescent PCR system is as low as 0.1%, some mutation sites even reach 0.01%, the detection limit of a digital PCR system is as low as 0.01%, and the kit can be suitable for accurate detection of various samples including tumor tissues, cerebrospinal fluid and blood. Therefore, compared with the existing detection technical scheme, the method has the characteristics and advantages of comprehensive target coverage, high sensitivity, high specificity and absolute quantification.

Description

Technical Field

[0001] The present invention relates to the field of in vitro diagnostic technology, and in particular to a detection kit based on 19 mutations of human CTNNB1 gene. Background Art

[0002] CTNNB1 is located on chromosome 3 and encodes β-catenin protein. β-catenin mediates cell-to-cell adhesion. As the central hub of the classical Wnt signaling pathway, it is a multifunctional protein for intracellular Wnt signaling. CTNNB1 gene mutations usually occur in the gene region of exon 3, concentrated in codons 32 to 37. Usually, each patient will only have one of the base mutations, and occasionally two mutations. This β-catenin amino acid change caused by a single base mutation prevents β-catenin from decomposing, causing it to accumulate in cells. Excessive β-catenin enters the cell nucleus and promotes unrestricted cell proliferation, leading to the occurrence and development of tumors.

[0003] Studies have shown that CTNNB1 gene mutations are associated with a variety of tumor diseases, including medulloblastoma, non-small cell lung cancer and liver cancer. Different CTNNB1 gene mutation states are extremely important for doctors to judge patients' tumor progression, prognosis, personalized medication, etc., so detecting CTNNB1 mutation status is of great significance for patients to develop personalized treatment plans.

[0004] For current molecular diagnostic technologies, it has always been a challenge to use easily accessible instruments to simultaneously achieve sensitive detection, especially accurate detection and quantification of low-frequency mutations. Traditional invasive tissue biopsy is currently the gold standard for tumor diagnosis, but it is limited by the high heterogeneity of tumors; non-invasive liquid biopsy can analyze the tumor genome with good consistency, but it is limited by the low content of ctDNA, high fragmentation, and coexistence with a large amount of wild-type DNA. The high heterogeneity of tumors and the interference of a large number of high-concentration wild-type background signals are the main factors affecting the sensitivity of low-abundance mutation detection. Therefore, it is necessary to develop a highly sensitive detection technology to meet the detection needs of low-quality clinical samples or trace samples, such as formalin-fixed paraffin-embedded tissue samples (FFPE samples) or puncture samples (ctDNA).

[0005] As the current mainstream gene mutation detection technology, the real-time fluorescence quantitative PCR (Quantitative Real-time PCR, QPCR) technology based on Taqman probes can detect mutation content as low as 1%, which can generally meet the clinical detection needs of tumor tissue samples, but it cannot accurately quantify the mutation frequency of genes and may also be affected by non-specific amplification. Digital PCR (Digital PCR, dPCR) realizes the absolute quantification of "single molecule template amplification" and can detect and quantify low-frequency mutations with VAF as low as 0.01%. It has obvious advantages in detecting precious samples and degraded samples, and is extremely suitable for liquid biopsy, but the dPCR throughput is limited and cannot detect a large number of targets in one reaction.

[0006] Locked Nucleic Acid (LNA) is a bicyclic nucleoside analog with a fixed N-type conformation, in which one ribonucleoside is connected between oxygen 2 and carbon 4 by a methylene unit, and like DNA, it also follows the Watson-Crick base pairing principle. The model constructed by Koshkin et al. shows that the LNA monomer is locked in the N-type conformation, so the single-stranded LNA oligonucleotide is pre-organized into an A-type helix to bind the complementary sequence, and an A-type helix is ​​generated when the LNA-DNA double helix is ​​formed. The stability of nucleic acid double helix is ​​affected by hydrogen bonds and base stacking interactions (such as hydrophobic and electrostatic interactions) in the base pair. Due to the short distance, the A-type helix may have a stronger base stacking interaction, and the enhanced base stacking interaction increases the Tm value. Therefore, the Tm of the LNA-DNA hybrid will be greatly increased compared with its corresponding DNA-DNA. When LNA is incorporated at a specific position of the oligonucleotide, there is a significant difference in the Tm value of LNA-DNA between perfect match and mismatch. Due to the above excellent characteristics, LNA technology is widely used in the field of molecular diagnostic technology, such as the detection of gene mutations. By using specific LNA-modified probes, the amplification efficiency of wild-type alleles is significantly reduced, so that mutant genes are enriched and sensitively detected. In 2010, Barbara Denys et al. developed an LNA-qPCR method that used LNA-modified wild-type blocking probes and dual-labeled hydrolysis probes to sensitively and specifically detect JAK2V617F mutations as low as 0.4%. In 2012, Daphne Ang et al. used competitive LNA blocking probes to partially inhibit the amplification of wild-type PIK3CA exon 9 or exon 20 sequences to detect PIK3CA hotspot mutations in small or mixed samples. Compared with standard Sanger sequencing, the sensitivity of this technology is greatly improved, ranging from 0.6% to 2.5%.

[0007] Although some patents have proposed methods for detecting CTNNB1 mutations, they all have the common problems of incomplete coverage of mutation sites, long time consumption, and high cost, and cannot quickly detect the 19 common mutation types of CTNNB1 in one tube. At the same time, competitive LNA blocking probes are rarely used for mutation detection of CTNNB1, and the length of current LNA blocking probes is usually relatively short (<16bp), and conventional blocking probes cannot cover all mutation sites in exon 3 of CTNNB1.

[0008] In view of this, the present invention is proposed. Summary of the invention

[0009] The purpose of the present invention is to provide a detection kit for 19 mutations of the human CTNNB1 gene. The LNA blocking probe of the kit can cover 19 mutation types contained in 8 mutation sites on exon 3 of CTNNB1, so as to achieve the effect of simultaneously detecting 19 mutations of the human CTNNB1 gene in one tube reaction, and has the characteristics of high specificity, high sensitivity and low cost.

[0010] Since most of the existing methods for detecting mutations in the human CTNNB1 gene only target a few hotspot mutations, they fail to fully cover the common mutation types of D32 to S37, or require multi-tube reactions to detect multiple mutations. In addition, due to the interference of a large number of wild-type sequences in tumor samples with detection, conventional PCR technology is difficult to achieve effective detection and quantification of low-frequency mutations in the context of a large number of wild-type templates, and the mutation frequency of genes cannot be calculated, resulting in sensitivity and specificity that cannot meet the detection requirements. Based on the above technical defects, the present invention designs a LNA-modified long blocking probe, and combines it with a TaqMan fluorescent probe to establish a fluorescent PCR and digital PCR detection technology system. The LNA probe can cover 19 types of mutations contained in 8 mutation sites on exon 3 of CTNNB1, and achieves efficient enrichment of mutant DNA molecules through specific blocking of wild-type DNA molecules, thereby greatly increasing the detection sensitivity of rare mutant DNA. The detection system of the present invention can simultaneously detect and quantify the above 19 mutations in one tube, increase the sensitivity and specificity of detection, and make up for the shortcomings of previous detection technologies. The present invention is implemented as follows:

[0011] In a first aspect, the present invention provides a nucleic acid composition for detecting 19 mutations of human CTNNB1 gene, comprising primers, TaqMan probes and LNA blocking probes.

[0012] The mutation sites detected are hotspot mutations of the human CTNNB1 gene, such as Figure 1shown, including: c.94G>T, p.D32Y; c.94G>C, p.D32H; c.94G>A, p.D32N; c.95A>C, p.D32A; c.95A>T, p.D32V; c.95A>G, p.D32G; c.97T>C, p.S33P; c.97T>G, p.S33A; c.98C>T, p.S33F; c.98C>G, p.S33C; c.98C>A, p.S33Y; c.100G>A, p.G34R; c.100G>C, p.G34R; c.101G>A, p.G34E; c.101 G>T, p.G34V; c.109T>C, p.S37P; c.110C>A, p.S37Y; c.110C>G, p.S37C; c.110C>T, p.S37F.

[0013] Among them, the primers include a forward primer and a reverse primer, the nucleotide sequence of the forward primer is shown in SEQ ID NO:1, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO:2; the nucleotide sequence of the TaqMan probe is shown in SEQ ID NO:3; and the nucleotide sequence of the LNA blocking probe is shown in SEQ ID NO:4.

[0014] In the present invention, the length of the LNA blocking probe is 23 bp. Compared with the conventional LNA blocking probe (<16 bp), the long LNA blocking probe can cover 19 mutations in exon 3 of CTNNB1. The long LNA modified blocking probe of the present invention is used to enrich the mutant template and block the amplification of the wild-type template. Different mutant templates are preferentially amplified 1000 to 65000 times more than the wild-type template, which increases the sensitivity and specificity of the detection, and can realize the detection of 19 types of mutations in one reaction tube at the same time.

[0015] The amplification principle of the primers, TaqMan probes and LNA blocking probes for detecting 19 mutations of the human CTNNB1 gene of the present invention is as follows: Figure 2 shown.

[0016] When designing the above LNA blocking probe, the bases at the mutation site are modified with LNA, the number of modified bases is 8, and an amino group (NH2C7) is added to the 3' end to prevent extension. The LNA blocking probe and the wild-type template sequence are completely complementary, and there is a mismatch with the mutant template. It is precisely because of the mismatch that the Tm value of the LNA blocking probe binding to the mutant template is reduced. The Tm value relationship under ideal conditions is: Tm LNA-WT >Tm F-MUT / WT >Tm LNA-MUT(WT is the wild-type template and MUT is the mutant template). This ensures that the LNA blocking probe preferentially binds to the wild-type template to block its amplification during the annealing process, and then the front primer (i.e., forward primer) preferentially binds to the mutant template before the LNA blocking probe to amplify it. There is a 4bp overlap between the 3' end of the front primer and the 5' end of the LNA blocking probe before the mutation site. On the one hand, the mutant template is enriched and the wild-type template is blocked by competitively binding to the template with the front primer. On the other hand, it is to prevent the LNA blocking probe from being hydrolyzed by DNA polymerase.

[0017] like Figure 2 As shown, the Taqman probe is located downstream of the LNA blocking probe, and the sequence complementary to it should not contain any possible mutations. When the template is a wild-type gene, the LNA blocking probe has a high affinity and a high Tm value, and will preferentially pair with the template before the front primer. The 3' end of the front primer cannot bind to the template and tilts up, and the DNA polymerase cannot bind and extend. The Taqman probe located downstream of the LNA blocking probe cannot be hydrolyzed to emit fluorescence, and the instrument cannot detect the fluorescence signal. On the contrary, when the template is a mutant sequence, the Tm value of the LNA blocking probe binding to the template is significantly reduced, which is lower than the Tm value of the primer binding to the template, and the thermal stability of the LNA blocking probe binding to the mismatched base is greatly reduced. Therefore, during the annealing process, the primer competitively binds to the template, the polymerase recognizes the 3'OH extension, and the downstream Taqman probe is hydrolyzed to release the fluorescent group, thereby collecting the fluorescent signal, and any gene mutation in the template chain corresponding to the LNA modification site can be detected.

[0018] In some embodiments, the nucleic acid composition also includes an internal reference primer and an internal reference probe; the internal reference primer includes an upstream internal reference primer and a downstream internal reference primer, the nucleotide sequence of the upstream internal reference primer is shown in SEQ ID NO:5, and the nucleotide sequence of the downstream internal reference primer is shown in SEQ ID NO:6; the nucleotide sequence of the internal reference probe is shown in SEQ ID NO:7.

[0019] The above-mentioned internal reference gene is selected as a sequence in the fifteenth exon of CTNNB1 that has not undergone mutation and has no SNP, and upstream and downstream primers and Taqman probes are designed for this sequence. In the present invention, the detection primers and Taqman probes of the internal reference gene are designed mainly for quality control of the detection system; at the same time, the internal reference gene can also be regarded as the sum of the amount of wild-type and mutant templates in the sample. In the QPCR system, a standard curve of VAF is established according to the linear relationship between the target Ct value and the gene mutation frequency VAF (Variant AllelFrequency), and the sample VAF is quantified. In the digital PCR system, the sample VAF is accurately quantified according to the ratio of the target software analysis value (indicating the number of template copies per microliter in the 16μL reaction system) to the internal reference software analysis value.

[0020] In some embodiments, the 5' end of the TaqMan probe and the internal reference probe are each labeled with a fluorescent reporter group, and the 3' end is each labeled with a fluorescent quencher group; wherein the fluorescent reporter group can be any one of FAM, VIC, ROX and TAMRA, and the fluorescent quencher group can be any one of BHQ1, BHQ2 and BHQ3. Those skilled in the art can perform conventional replacements for the fluorescent reporter group and the fluorescent quencher group according to actual needs.

[0021] In a second aspect, the present invention also provides the use of the above-mentioned nucleic acid composition in the preparation of a kit for detecting 19 mutations of the human CTNNB1 gene by fluorescent quantitative PCR and digital PCR.

[0022] In a third aspect, the present invention also provides a detection kit based on 19 mutations of the human CTNNB1 gene, which comprises the above-mentioned nucleic acid composition.

[0023] The above nucleic acid composition can be prepared into a fluorescent quantitative PCR detection system and a digital PCR detection system, and then can be used for fluorescent quantitative PCR detection of 19 mutations of the human CTNNB1 gene or digital PCR detection of 19 mutations of the human CTNNB1 gene, respectively.

[0024] In some embodiments, the above detection kit also includes an amplification reagent, including a buffer, dNTPs, TaqDNA polymerase, MgCl2 and other reaction reagents for amplification.

[0025] In some embodiments, the working concentration of the forward primer in the fluorescent quantitative PCR detection system and the digital PCR detection system is 0.15 μM-0.2 μM, and the working concentration of the LNA blocking probe is 0.25 μM-0.45 μM; the molar concentration ratio of the forward primer and the LNA blocking probe is: 3-4:5-9.

[0026] When performing fluorescent quantitative PCR detection, the detection system includes: forward primer, reverse primer, TaqMan probe, LNA blocking probe, forward internal reference primer, reverse internal reference primer, internal reference probe, amplification reagent, sample and ultrapure water.

[0027] Optionally, when the detection system of fluorescent quantitative PCR is 20 μL, the detection system includes: 3 μM forward primer, 4 μM reverse primer, 4 μM TaqMan probe, 5 μM LNA blocking probe, 3 μM forward internal reference primer, 4 μM reverse internal reference primer, 4 μM internal reference probe, 10 μL amplification reagent, 1 to 3 μL sample, and ultrapure water to 20 μL.

[0028] Optionally, the reaction procedure of fluorescent quantitative PCR is: pre-denaturation at 95°C for 30s; denaturation at 95°C for 5s, extension at 58-70°C for 30s, for a total of 40 cycles. The annealing temperature is preferably 66°C, but mutant templates and wild-type templates can be distinguished at 58-70°C, and the temperature range of 12°C greatly reduces the requirements for instrument precision for detection.

[0029] When digital PCR detection is performed, the detection system includes: forward primer, reverse primer, TaqMan probe, LNA blocking probe, forward internal reference primer, reverse internal reference primer, internal reference probe, amplification reagent, sample and ultrapure water.

[0030] Optionally, when the detection system of digital PCR is 16 μL, the detection system includes: 3 μM forward primer, 4 μM reverse primer, 4 μM TaqMan probe, 5 μM LNA blocking probe, 3 μM forward internal reference primer, 4 μM reverse internal reference primer, 4 μM internal reference probe, 8 μL 2× amplification reagent, 1 to 3 μL sample, and ultrapure water is added to 16 μL.

[0031] Optionally, the reaction program of digital PCR is: pre-denaturation at 95°C for 30s; denaturation at 95°C for 5s, extension at 63°C for 30s, for a total of 40 cycles.

[0032] The detection limit of the fluorescence quantitative system of the present invention is as low as 0.1%, and some mutation sites even reach 0.01%. VAF is quantified based on the linear relationship between Ct value and VAF, and then the mutation type is determined in combination with Sanger sequencing. The digital PCR system can detect mutations as low as 0.01% and accurately quantify VAF, achieving effective detection of ctDNA.

[0033] The detection samples of the kit provided by the present invention are not limited to blood, but can also be used for accurate detection of various samples such as formalin-fixed, paraffin-embedded tissue (FFPE), cerebrospinal fluid (CSF), pleural and peritoneal fluid (PF), etc.

[0034] The present invention has the following beneficial effects:

[0035] (1) Comprehensive target coverage and high throughput

[0036] The present invention uses a long LNA modified blocking probe to fully cover the target area, and can simultaneously detect 19 types of mutations in one tube, and detect the internal reference gene through multiple fluorescence channels, thereby achieving quality control and quantification of the reaction process. At the same time, Sanger sequencing is limited by low sensitivity, but the enrichment of mutant genes by LNA modified blocking probes can achieve high-sensitivity detection of mutant genes.

[0037] (2) High sensitivity and accurate quantification

[0038] The LNA blocking probe of the present invention is used to enrich the mutant template and block the amplification of the wild-type template. Compared with the wild-type template, the enrichment multiple of the mutant template is 1000 to 65000 times, thereby realizing high-sensitivity detection of mutant molecules. The detection limit of the QPCR system established by the present invention is as low as 0.01% to 0.1%, and the technology has strong scalability. By combining with Sanger sequencing, effective quantification of mutant molecules can be achieved; the detection limit of the established digital PCR system is as low as 0.01%, and the mutation frequency of the gene can be accurately quantified.

[0039] (3) Applicable to the detection of various types of samples

[0040] The detection kit and detection method of the present invention can be applied to the accurate detection of various samples including tumor tissue, cerebrospinal fluid, blood and pleural effusion. Liquid biopsy can break the limitations of traditional tissue biopsy, and the high sensitivity and accuracy of digital PCR play an important role in the early screening of tumors.

[0041] (4) Wider application scenarios and high clinical value

[0042] The fluorescent quantitative PCR detection system of the present invention can perform effective detection within the annealing temperature range of 58 to 70°C, greatly reducing the requirements for instrument precision; and after enrichment with LNA blocking probes, the sensitivity of Sanger sequencing is improved, and only 0.1 ng of sample is needed to detect paraffin section samples with a VAF as low as 1.86% to determine the mutation type, which is suitable for detection in hospitals. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0044] Figure 1 It is a common mutation site in the third exon of CTNNB1;

[0045] Figure 2 The technical principle of the present invention for detecting 19 types of mutations;

[0046] Figure 3 The melting curves of the forward primer binding to the complementary template and the melting curves of the LNA blocking probe binding to the wild-type template and seven common mismatch mutation templates in Example 2;

[0047] Figure 4 : amplification curves of wild-type and 19 mutant plasmids before and after adding LNA probes in Example 2. Figure 4 A is CT mismatch and wild-type template, Figure 4 B is GA mismatch and GG mismatch template, Figure 4 C is the AC mismatch and CC mismatch template, Figure 4 D is GT mismatch template;

[0048] Figure 5 A is the recognition ability of the LNA blocking probe to the mutant and wild-type templates at an annealing temperature of 58° C. to 70° C. in Example 2; Figure 5 B is the Ct value of qPCR results of different VAFs of 19 mutant templates; Figure 5 C indicates that the system can detect the amplification curve of the 110C>A mutant template as low as 0.01%; Figure 5 D represents the Sanger sequencing results of different VAF plasmid amplification products before and after the addition of LNA blocking probe to 97T>G;

[0049] Figure 6 A represents the linear relationship between Ct value and VAF; Figure 6B shows the dPCR results of the 94G>A plasmid template before and after the addition of the LNA blocking probe. It can be seen that the addition of the LNA blocking probe has almost no effect on the amplification of the template; Figure 6 C represents the dPCR results of negative samples before and after the addition of LNA blocking probe; Figure 6 D means that dPCR can detect mutations as low as 0.01%;

[0050] Figure 7 A represents the QPCR test results of CTNNB1 mutation-positive paraffin sections; Figure 7 B represents the QPCR test results of paraffin section samples with negative CTNNB1 mutation; Figure 7 C represents the dPCR test results of CTNNB1 mutation-positive paraffin sections. Figure 7 D represents the dPCR detection results of CTNNB1 mutation-negative paraffin section samples.

[0051] Figure 8 A represents the dPCR test results of free DNA in CTNNB1 mutation-positive plasma samples; Figure 8 B represents the dPCR test results of free DNA in CTNNB1 mutation-positive cerebrospinal fluid samples; Figure 8 C represents the sequencing graph of a positive sample with a VAF of 1.86% when 10 ng, 1 ng, and 0.1 ng of DNA template were added. It can be seen that a mutation can be detected with a sample input of 0.1 ng; Figure 8 D represents the expression level of the corresponding reference gene in 10ng, 1ng, and 0.1ng samples;

[0052] Fig. 9 Comparison of amplification effects of different forward primers in Experimental Example 1;

[0053] Fig.10 The comparison results of the detection effects of different LNA blocking probes in Experimental Example 2;

[0054] Fig.11 The comparison results of the detection effects of different Taqman probes in Experimental Example 3;

[0055] Fig.12 The comparison results of the detection effects of different internal reference primers and probes in Experimental Example 4;

[0056] Fig.13 This is a comparison of the detection effects of the combination of 0.15 μM forward primer and different concentrations of LNA blocking probe in Experimental Example 5;

[0057] Fig.14 This is a comparison of the detection effects of the combination of 0.2 μM forward primer and different concentrations of LNA blocking probe in Experimental Example 5;

[0058] Fig.15 This is a comparison of the detection effects of the combination of 0.25 μM forward primer and different concentrations of LNA blocking probe in Experimental Example 5. DETAILED DESCRIPTION

[0059] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0060] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0061] Example 1

[0062] This embodiment is a detection kit based on 19 mutations of the human CTNNB1 gene, including: forward primer, reverse primer, TaqMan probe, LNA blocking probe, forward internal reference primer, reverse internal reference primer, internal reference probe and standard, and also includes 2×PerfectStartⅡProbe qPCR SuperMix (Beijing Quanshijin Biotechnology Co., Ltd.) or 2×ProTaq HS Probe Premix (Hunan Aikerui Biotechnology Co., Ltd.).

[0063] Forward primer, reverse primer, TaqMan probe and LNA blocker probe follow Tm LNA-WT >Tm F-MUT / WT >Tm LNA-MUT The design was based on the principle of using COSMIC to find the conservative region of CTNNB1 without mutation and SNP, determine the internal reference gene sequence, and then design the forward internal reference primer, reverse internal reference primer and internal reference probe. The above-designed primers and probes were synthesized by a company in Shanghai. Primer, Probe, and LNA blocking probe were diluted to 10μM with TE buffer and stored at -20℃. The specific sequence information is shown in Table 1:

[0064] Table 1 Sequence information

[0065]

[0066] Note: "+" indicates that the following base is modified with LNA, and the fluorescence quenching group is BHQ1. BHQ: Blackhole quencher. FAM is 6-carboxyfluorescein; VIC is 5-VICphosphoramidite.

[0067] The sequence designed from the CTNNB1 reference genome in NCBI was mounted on the PUC57 vector to construct a positive plasmid standard, and the resistance gene was selected as the Amp gene. The extracted plasmid standard was diluted to 10 with TE buffer. 4 copies / μL, stored at -20℃. The plasmid was synthesized by a company in Shanghai.

[0068] Example 2

[0069] In this example, the kit of Example 1 was used to construct a QPCR detection system and verify its performance. The QPCR detection system is shown in Table 2:

[0070] Table 2 QPCR reaction system

[0071]

[0072] The reaction program was: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 5 s, and extension at 66°C for 30 s, for a total of 40 cycles.

[0073] The performance verification is as follows:

[0074] (1) Melting curves of the forward primer binding to the complementary template and the melting curves of the LNA blocking probe binding to the wild-type template and 19 mutant templates, such as Figure 3 As shown. Figure 3 It can be concluded that among the 7 types of mismatch binding contained in the 19 mutation sites, the Tm values ​​of the mutant templates of the 6 mismatch types, CT, AA, GA, GG, AC, and CC, combined with the forward primer and LNA blocking probe, meet the “Tm LNA-WT >Tm F-MUT / WT >Tm LNA-MUT "This design principle. The Tm value of the template with GT mismatch with the LNA blocking probe is higher, slightly higher than the Tm value of the forward primer binding to the template.

[0075] (2) Efficiency analysis of LNA blocking probes

[0076] After adding the LNA blocking probe, the wild-type template amplification can be perfectly blocked. Among the seven mismatch types, the mutant templates of the six mismatch types, CT, AA, GA, GG, AC, and CC, are not affected by the LNA blocking probe and amplify normally. Figure 4 As shown in the figure, the amplification effect of the template with GT mismatch with the LNA blocking probe is not ideal. The possible reason is that the base stacking interaction formed by the GT mismatch is enhanced, and the combination of the two is more stable, resulting in an increase in the Tm value, which is higher than the Tm value of the forward primer binding to the template. During the annealing process, some LNA blocking probes bind to the mutant template preferentially before the forward primer.

[0077] (3) Performance of LNA blocking probes at different annealing temperatures

[0078] When the annealing temperature of the reaction system is between 58 and 70°C, the LNA blocking probe can distinguish between mutant templates and wild-type templates, such as Figure 5 As shown, the 12°C temperature range greatly reduces the requirements for instrument precision, and the detection can be completed with only an ordinary PCR instrument capable of collecting fluorescence.

[0079] (4) Minimum detection limit

[0080] Take 10 4 The wild-type plasmid was used as the background, and 10 copies / μL 4 The mutant plasmids were diluted to a mixed plasmid with VAF of 100%, 10%, 1%, 0.1%, 0.01%, and 0% (100% WT) in sequence. Most mutations can be detected at 0.1% (e.g. Figure 5 B), where 110C>A can be detected at 0.01% (as shown in Figure 5 C). The amplification products of different VAF plasmids before and after the addition of LNA blocking probe to 97T>G were subjected to Sanger sequencing. It can be seen that after QPCR enrichment, Sanger sequencing can detect mutations as low as 0.1% (as shown in Figure 5 D).

[0081] (5) Quantitative performance of QPCR

[0082] Plot the Ct values ​​and log for the seven mismatch types 10 The linear relationship between VAF, such as Figure 6 As shown in A, it can be seen that the Ct value of 98C>T is related to log 10 There is a good linear relationship between VAF and R 2 The VAF value of the unknown sample is 0.98, so the VAF can be inferred based on the Ct value of the unknown sample, but further verification of the sample is required. For example, NGS detected a 98C>T sample with a mutation frequency VAF of 22.87%, and the Ct value after QPCR was 28.8. Figure 6 The VAF calculated by the formula in A is 23.4%, which is almost close to the result of NGS.

[0083] Example 3

[0084] This example uses the kit of Example 1 to construct a digital detection system and verify its performance. The digital detection system is shown in Table 3:

[0085] Table 3 Digital reaction system

[0086]

[0087] The detection was carried out on a pilot D3200 digital PCR instrument, and the reaction program was as follows: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 5 s, and extension at 63°C for 30 s, for a total of 40 cycles.

[0088] The performance verification is as follows:

[0089] (1) Specificity analysis of LNA blocking probes

[0090] Comparing the amplification of the mutant template 94G>A and the negative sample before and after the addition of the LNA blocking probe, it can be seen that the LNA blocking probe has excellent specificity, that is, it accurately distinguishes between wild-type and mutant templates, and perfectly blocks the amplification of the wild-type template, while the amplification of the mutant template is almost unaffected (e.g. Figure 6 B, as shown in 6C).

[0091] (2) Minimum detection limit

[0092] The preparation method of different VAF mixed plasmids is consistent with the above QPCR reaction system. The lowest mutation rate that can be detected is as low as 0.01% (such as Figure 6 D).

[0093] Example 4

[0094] This example uses the kit of Example 1 to perform clinical sample verification, as follows:

[0095] The clinical samples of this embodiment are FFPE samples, plasma, cerebrospinal fluid or pleural effusion of tumor patients obtained from the Xi'an Regional Medical Laboratory Center and Hospital. The slice nucleic acid was extracted using the Omega FFPE kit, and the free DNA was extracted using the Qiagen column extraction method. The extraction steps refer to the instructions of the corresponding kit. The concentration and purity of the extracted nucleic acid were determined using Qubit.

[0096] A total of 23 positive samples were tested, including 14 paraffin tissue section samples, 5 cerebrospinal fluid samples, 3 plasma samples, and 1 pleural and ascites sample; 17 negative samples that did not show CTNNB1 mutations by NGS were tested.

[0097] 1. In this example, QPCR and digital PCR were performed on clinical negative and positive samples, respectively, and the detection methods were the same as those in Examples 2 and 3.

[0098] QPCR test results Figure 7A and 7B. When the sample is paraffin section tissue genomic DNA, ΔCt (Ct target - Ct internal reference) is used as the judgment standard. According to the previous test results, the ΔCt value of the positive sample is <8, and the ΔCt value of the negative sample is >8, so the ΔCt value = 8 is used as the cutoff value, that is, the boundary value. If the ΔCt value is less than 8, the patient has a CTNNB1 mutation; if the ΔCt value is greater than 8, the patient has not had a CTNNB1 mutation.

[0099] The above determination method is adopted because the internal reference serves to indicate the amount of template added. The quality of clinical samples obtained varies, and the concentration of extracted nucleic acids varies. If the nucleic acid content of the added negative sample is particularly high, the LNA blocking probe in the system will not be sufficient to bind to the excess wild-type template, and some wild-type templates will still escape and amplify, but the Ct value of the internal reference will be smaller at this time; similarly, if the amount of nucleic acid added to the positive sample is relatively small, even if a mutation occurs, the target Ct will be too large, but the Ct value of the internal reference will also be too large at this time. Therefore, ΔCt (Ct target - Ct internal reference) is needed as the standard for judging positive and negative samples.

[0100] Digital PCR test results Figure 7 C, 7D, 8A and 8B.

[0101] When the sample is paraffin section tissue genomic DNA, if the patient does not have CTNNB1 mutation, the ddPCR software analysis value is less than 0.11; if the patient has CTNNB1 mutation, the ddPCR software analysis value is greater than 0.11, and positive droplets can be observed in the FAM channel. The sample test results are as follows Figure 7 C, as shown in 7D.

[0102] When the sample is free DNA, if the patient has no mutation, the ddPCR software analysis value is 0; if the patient has a mutation, the ddPCR software analysis value is greater than 0, and positive droplets can be observed in the FAM channel. The sample test results are as follows: Figure 8 As shown in A and 8B.

[0103] 2. Minimum amount of nucleic acid required to detect tissue samples with VAF < 2%

[0104] The VAF of a mutation-positive sample detected by NGS was 1.86%. Sample amounts of 10 ng, 1 ng, and 0.1 ng were respectively used. After enrichment, Sanger sequencing was performed. Only 0.1 ng was needed to detect a mutation with a VAF as low as 1.86% (e.g. Figure 8 C, as shown in 8D).

[0105] 3.Consistency analysis of QPCR, dPCR and NGS test results

[0106] The test results are shown in Table 4:

[0107] Table 4 Statistics of test samples

[0108]

[0109] Fourteen paraffin tissue section samples were tested on QPCR, and mutations were detected in all of them. Seventeen negative paraffin section samples were tested, and no mutations were detected in any of them, which was consistent with the NGS test results, with a sensitivity and specificity of 100%.

[0110] One paraffin section sample, five cerebrospinal fluid samples, three plasma samples, and one pleural and ascites fluid sample were tested using dPCR, and mutations were detected in all of them. One negative paraffin section sample was tested, and no mutation was detected, which was consistent with the NGS test results, with a sensitivity and specificity of 100%.

[0111] Experimental Example 1

[0112] This experiment compares the amplification effects of different forward primers under the same conditions. The sequence information of the forward primers is shown in Table 5:

[0113] Table 5 Sequence information of different forward primers

[0114]

[0115]

[0116] Test results such as Fig. 9 As shown, the forward primer shown in SEQ ID NO: 1 has a smaller Ct value and a better amplification effect than the other five primers.

[0117] Experimental Example 2

[0118] This experiment compares the amplification effects of different LNA blocking probes under the same conditions. The sequence information of the LNA blocking probe is shown in Table 6:

[0119] Table 6 Sequence information of different LNA blocking probes

[0120]

[0121] Test results such as Fig.10 As shown, for the mutant template, compared with the other five, the Ct value of the LNA blocking probe shown in SEQ ID NO: 4 is the smallest, and the Ct value is smaller after adding the LNA blocking probe than when not adding it; the blocking effect is better for the wild-type template,

[0122] Experimental Example 3

[0123] This experiment compares the amplification effects of different Taqman probes under the same conditions. The sequence information of the Taqman probe is shown in Table 7:

[0124] Table 7 Sequence information of different Taqman probes

[0125]

[0126] Test results such as Fig.11 As shown, the fluorescence increase of the Taqman probe shown in SEQ ID NO: 3 is high in comparison.

[0127] Experimental Example 4

[0128] This experiment compares the detection effects of different internal reference primers under the same conditions. The sequence information of the Taqman probe is shown in Table 8:

[0129] Table 8 Sequence information of different internal reference primers

[0130]

[0131] Test results such as Fig.12 As shown, the combinations of primers and probes for the internal reference genes shown in SEQ ID NOs: 19-24 failed to amplify successfully.

[0132] Experimental Example 5

[0133] This experiment compared the detection effects of different molar concentration ratios of forward primer and LNA blocking probe under the same conditions. The test results are as follows Figure 13-15 As shown, it can be seen that when the concentration of the primer is 0.5 μM, there will be non-specific amplification of the wild-type template. Therefore, when the concentration range of the forward primer is 0.15 μM-0.2 μM, the concentration range of the LNA blocking probe is 0.25 μM-0.45 μM, and the molar concentration ratio of the forward primer to the LNA blocking probe is 3-4:5-9, a better amplification effect can be achieved. This also shows that not all molar concentration ratios of the forward primer and the LNA blocking probe can achieve a good amplification effect.

[0134] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A nucleic acid composition for detecting 19 mutations in the human CTNNB1 gene, characterized in that: The nucleic acid composition includes a primer, a TaqMan probe and an LNA blocking probe; The mutation sites detected included: c.94G>T, c.94G>C, c.94G>A, c.95A>C, c.95A>T, c.95A>G, c.97T>C, c.97T>G, c.98C>T, c.98C>G, c.98C>A, c.100G>A, c.100G>C, c.101G>A, c.101G>T, c.109T>C, c.110C>A, c.110C>G, and c.110C>T; The primers include a forward primer and a reverse primer, the nucleotide sequence of the forward primer is shown in SEQ ID NO: 1, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO: 2; The nucleotide sequence of the TaqMan probe is shown in SEQ ID NO: 3; The nucleotide sequence of the LNA blocking probe is shown in SEQ ID NO:4; The 3' end of the forward primer and the 5' end of the LNA blocking probe overlap by 4 bases before the mutation site; 8 bases in the LNA blocking probe are LNA modified; the Taqman probe is located downstream of the LNA blocking probe, and the sequence complementary to it does not contain any mutation.

2. The nucleic acid composition according to claim 1, characterized in that The nucleic acid composition also includes an internal reference primer and an internal reference probe; wherein the internal reference gene is a sequence in the fifteenth exon of CTNNB1 that has not been mutated and has no SNP; Preferably, the internal reference primer includes a forward internal reference primer and a reverse internal reference primer, the nucleotide sequence of the forward internal reference primer is shown in SEQ ID NO:5, and the nucleotide sequence of the reverse internal reference primer is shown in SEQ ID NO:6; Preferably, the nucleotide sequence of the internal reference probe is as shown in SEQ ID NO:

7.

3. The nucleic acid composition according to claim 2, characterized in that The LNA blocking probe in the nucleic acid composition has a length of 23 bp, and its 3' end is connected to an amino group. The Tm values ​​of the forward primer and the LNA blocking probe are: Tm LNA-WT >Tm F-MUT / WT >Tm LNA-MUT , where WT is the wild-type template and MUT is the mutant template; The 5' ends of the TaqMan probe and the internal reference probe are each labeled with a fluorescent reporter group, and the 3' ends are each labeled with a fluorescent quencher group; the fluorescent reporter group is selected from any one of FAM, VIC, ROX and TAMRA, and the fluorescent quencher group is selected from any one of BHQ1, BHQ2 and BHQ3.

4. Use of the nucleic acid composition according to any one of claims 1 to 3 in preparing a kit for detecting 19 mutations of the human CTNNB1 gene by fluorescent quantitative PCR and digital PCR.

5. The use according to claim 4, characterized in that: The application includes simultaneous detection of c.94G>T, c.94G>C, c.94G>A, c.95A>C, c.95A>T, c.95A>G, c.97T>C, c.97T>G, c.98C>T, c.98C>G, c.98C>A, c.100G>A, c.100G>C, c.101G>A, c.101G>T, c.109T>C, c.110C>A, c.110C>G and c.110C>T mutation sites of the human CTNNB1 gene.

6. A detection kit based on 19 mutations of human CTNNB1 gene, characterized in that: The detection kit comprises the nucleic acid composition according to any one of claims 1 to 3.

7. The detection kit according to claim 6, characterized in that The working concentration of the forward primer in the detection kit is 0.15 μM-0.2 μM, and the working concentration of the LNA blocking probe is 0.25 μM-0.45 μM; the molar concentration ratio of the forward primer to the LNA blocking probe is: 3-4:5-9.

8. The detection kit according to claim 7, characterized in that The detection was performed based on fluorescent quantitative PCR, and the reaction procedure of the fluorescent quantitative PCR was: pre-denaturation at 95°C for 30s; denaturation at 95°C for 5s, extension at 58-70°C for 30s, for a total of 40 cycles; preferably, the extension temperature was 66°C; The detection was performed based on digital PCR, and the reaction procedure of the digital PCR was: pre-denaturation at 95° C. for 30 s; denaturation at 95° C. for 5 s, and extension at 63° C. for 30 s, for a total of 40 cycles.

9. The detection kit according to claim 7, characterized in that The detection kit also includes an amplification reagent, which contains a buffer, dNTPs, TaqDNA polymerase and MgCl2.

10. The detection kit according to any one of claims 6 to 9, characterized in that: The test samples of the detection kit include: tumor tissue, plasma, cerebrospinal fluid and pleural and abdominal fluid.

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