Point mutation gene specificity loop-mediated isothermal amplification detection method
By introducing mismatched base primers into the LAMP detection method, a dual-specificity recognition mechanism was constructed, which solved the false positive problem in LAMP detection and achieved efficient and sensitive point mutation gene detection, applicable to various types of clinical samples.
Patent Information
- Application Number
- CN202511610431.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-05
- Publication Date
- 2025-12-19
AI Technical Summary
Existing LAMP detection methods suffer from problems such as non-specific dyes, primer dimers, or mismatched melting temperatures in the specific detection of point mutation genes, leading to false positive results. Furthermore, they require sophisticated equipment, making it difficult to achieve efficient and sensitive detection in resource-constrained scenarios.
Using wild-type and mutant primer sets, a dual-specific recognition mechanism of "complementary pairing of point mutation gene sites and mismatch of adjacent bases" was constructed by introducing mismatched bases into the second base at the 5' end of the forward and backward primers, thereby blocking non-specific adsorption and amplification.
It improves the specificity of detection without sacrificing sensitivity, achieves stable detection of low-abundance target point mutation genes, is suitable for the detection of various types of clinical samples, does not require complex temperature-changing equipment, and reduces detection costs.
Smart Images
Figure CN121160862A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the technical field of point mutation gene-specific detection, and in particular relates to a loop-mediated isothermal amplification detection method specific to point mutation genes. Background Technology
[0002] Cancer originates from the accumulation of mutations in key driver genes, and their mutation profiles can reveal the evolutionary process of tumors. Systematic discovery and effective detection of these mutations are fundamental to precision oncology, driving targeted therapies and biomarker development. However, mutation detection, especially of low-frequency variants, remains a significant challenge due to high-frequency wild-type background noise. While isothermal amplification techniques (such as RPA, RCA, and LAMP) simplify the detection process by eliminating thermal cycling, there is an urgent need to develop more efficient, sensitive, and resource-constrained point mutation-specific detection technologies.
[0003] Traditional LAMP detection methods have inherent limitations in achieving specific detection of point mutation genes due to issues such as non-specific dyes, primer dimers, or mismatches between melting temperature (Tm) and reaction temperature. Currently, three main strategies exist to enhance the specificity of point mutation detection without introducing additional reaction principles or enzyme tools: fluorescence probe-based detection, primer design optimization, and melting curve analysis. Fluorescence probe-based LAMP methods for specific detection of point mutation genes have high probe preparation costs; melting curve analysis methods require sophisticated equipment and high-resolution temperature control to detect differences in individual bases; and specific primer design is challenging, with primer non-specificity easily leading to false positives.
[0004] Liu et al. successfully identified clinically relevant single nucleotide variants (SNVs) of the MTHFR (C677T) and ALDH2 (Glu504Lys) genes by positioning the 5' ends of the forward inward primer (FIP) and backward inward primer (BIP) at the mutation site, demonstrating the effectiveness of the specific primer design strategy. Biosensors and Bioelectronics (2018, 115, 70–76), but this strategy requires strict optimization of reaction conditions, including: FIP / BIP concentration (0.8 μM) and amplification reaction temperature (60 °C). However, at a lower temperature of 60 °C, non-specific primer adsorption is aggravated, causing false positive results in normal LAMP reactions. Summary of the Invention
[0005] To address the technical problems existing in the background art, the present invention provides a loop-mediated isothermal amplification detection method specific to point mutation genes.
[0006] This invention employs the following technical solution: a method for detecting point mutation genes specifically using loop-mediated isothermal amplification, comprising the following steps: The wild type primer group and the mutant primer group are synthesized for a target point mutation gene site, and each of the wild type primer group and the mutant primer group comprises a forward inner primer FIP and a backward inner primer BIP; wherein the first base at the 5' end of the forward inner primer FIP is complementary to the base at the target point mutation gene site of the corresponding template to be detected, the first base at the 5' end of the backward inner primer BIP is the same as the base at the target mutation site of the corresponding template to be detected, and a base that is not matched with the base of the target detection gene is introduced at the second base at the 5' end of the forward inner primer FIP and the backward inner primer BIP; The wild type primer group is used for LAMP amplification reaction with the target point mutation gene as the template to be detected, and an amplification reaction result is obtained. The relative quantitative concentration of the target point mutation gene in the template to be detected relative to the wild type gene is determined according to the amplification reaction result.
[0007] In further embodiments, the wild type primer group and the mutant primer group further comprise a forward outer primer F3 and a backward outer primer B3.
[0008] In further embodiments, the template to be detected comprises a wild type template and a mutant template.
[0009] In further embodiments, when the template to be detected matches the wild type primer group, a stem-loop structure with 3' end and 5' end matching is formed in subsequent explosive amplification, and when the template to be detected matches the mutant primer group, it is difficult to form a stem-loop structure with 3' end and 5' end matching, so that the template to be detected is determined as the wild type template, wherein the target mutation site is the wild type.
[0010] In further embodiments, when the template to be detected matches the wild type primer group, it is difficult to form a stem-loop structure with 3' end and 5' end matching, and when the template to be detected matches the mutant primer group, a stem-loop structure with 3' end and 5' end matching is formed in subsequent explosive amplification, so that the template to be detected is determined as the mutant template, wherein the target mutation site is the mutant type.
[0011] In further embodiments, the second base at the 5' end of the forward inner primer FIP-WT of the wild type primer group is not complementary to the base at the corresponding position of the wild type template, forming a base mismatch. The second base at the 5' end of the backward inner primer BIP-WT of the wild type primer group is not the same as the base at the corresponding position of the wild type template, forming a base mismatch.
[0012] In further embodiments, the second base at the 5' end of the forward inner primer FIP-MUT of the mutant primer group is not complementary to the base at the corresponding position of the mutant template, forming a base mismatch. The second base at the 5' end of the backward inner primer BIP-MUT of the mutant primer set is not identical to the base at the corresponding position of the mutant template, forming a base mismatch.
[0013] In a further embodiment, the first base at the 5' end of the forward inner primer FIP-WT of the wild-type primer set is complementary to the wild-type template at the target mutation site, and the first base at the 5' end of the backward inner primer BIP-WT is identical to the wild-type template at the target mutation site, and the second base at the 5' end of FIP-WT and BIP-WT forms a specific recognition structure of the primer pair for the wild-type template.
[0014] In a further embodiment, the first base at the 5' end of the forward inner primer FIP-MUT of the mutant primer set is complementary to the mutant template at the target mutation site, and the first base at the 5' end of the backward inner primer BIP-MUT is identical to the mutant template at the target mutation site, and the second base at the 5' end of FIP-MUT and BIP-MUT forms a specific recognition structure of the primer pair for the mutant template.
[0015] In a further embodiment, the application is applied to the typing detection of different point mutation sites related to diseases in clinical samples, including blood, tissue, saliva or cell samples.
[0016] The present application has the following advantages: The present application introduces mismatched bases that are not complementary (or not identical) to the target detection gene at the second base at the 5' end of the forward inner primer (FIP) and the backward inner primer (BIP) of the wild-type primer set and the mutant primer set, thereby constructing a dual-specific recognition mechanism of "point mutation gene site complementary pairing and adjacent base mismatching", which blocks non-specific adsorption and amplification.
[0017] The present application improves specificity without sacrificing detection sensitivity, and realizes stable detection of low-abundance target point mutation genes by optimizing the binding efficiency of primers and templates and the LAMP amplification kinetics.
[0018] The present application can be directly applied to the typing detection of point mutation genes in common types of clinical samples (blood, tissue, saliva, and cell samples), without the need for complex temperature-changing equipment, and is suitable for the diversity needs of clinical samples. At the same time, the primer design logic is universal, and the primer sequence can be flexibly adjusted for different point mutation genes related to diseases (such as mental illness, cancer, etc.), thereby realizing specific detection of multiple targets, providing a unified technical framework for molecular diagnosis in multiple disease fields, and reducing the research and development and promotion costs of multiple disease detection platforms. BRIEF DESCRIPTION OF DRAWINGS
[0019] Figure 1is a schematic diagram of primer specificity and nucleic acid amplification principle of Example 1.
[0020] Figure 2 is a real-time fluorescent quantitative loop-mediated isothermal amplification curve chart of Example 1.
[0021] Figure 3 is a real-time fluorescent quantitative loop-mediated isothermal amplification curve chart of Comparative Example 1.
[0022] Figure 4 is an amplification curve chart of concentration-dependent fluorescent quantitative detection of Example 2. DETAILED DESCRIPTION
[0023] The technical solutions of the present application are described in detail through specific examples in combination with the accompanying drawings and experimental data, which aims to further verify the effectiveness of the present application in the specific detection of point mutation genes. All experimental operations follow the conventional molecular biology experimental specifications, and the reagents not specifically mentioned are commercially available molecular biology grade.
[0024] Example 1 In this embodiment, the key point mutation gene sites of the cancer-related PIK3CA gene (Exon9 region WT, E542K, E545K, E545D; Exon20 region WT, H1047R, H1047L) are detected as the detection target.
[0025] First, prepare the template to be detected. The PIK3CA gene mutation genomic DNA standard substance is used as the target detection target. Three groups of mixed templates containing the target point mutation gene are prepared by gradient dilution with enzyme-free water, and the specific concentrations are as follows: Template 1: 1.09x10³ copies / μL PIK3CA Exon9 wild type (WT) template + 3.18x10³ copies / μL PIK3CA E542K mutant (MUT) template; Template 2: 1.26x10³ copies / μL PIK3CA Exon9 wild type (WT) template + 1.29x10³ copies / μL PIK3CA E545K mutant template; Template 3: 0.781x10³ copies / μL PIK3CA Exon20 wild type (WT) template + 3.7x10³ copies / μL PIK3CA H1047R mutant template.
[0026] It should be noted that the experimental sample concentrations of template 1, template 2 and template 3 given in this embodiment are to ensure that the clinical common mutation abundance scene is covered, and to facilitate intuitive comparison of specificity differences.
[0027] The wild type primer group and the mutant primer group are synthesized for the target point mutation gene site, and the wild type primer group and the mutant primer group each include a forward inner primer FIP, a backward inner primer BIP, a forward outer primer F3 and a backward outer primer B3. The first base at the 5' end of the forward inner primer FIP is complementary to the base at the target mutation site of the corresponding template to be detected, the first base at the 5' end of the backward inner primer BIP is the same as the base at the target mutation site of the corresponding template to be detected, and a base that is not matched with the target detection gene base is introduced at the second base at the 5' end of the forward inner primer FIP and the backward inner primer BIP.
[0028] In combination Figure 1 The judgment principle is that when the LAMP starts to amplify, if the template to be detected matches the wild type primer group, a stem-loop structure with 3' end and 5' end matching is formed for subsequent explosive amplification; if the template to be detected matches the mutant primer group, it is difficult to form a stem-loop structure with 3' end and 5' end matching, so it is judged that the template to be detected is a wild type template, and the target mutation site is a wild type.
[0029] Correspondingly, when the LAMP starts to amplify, if the template to be detected matches the wild type primer group, it is difficult to form a stem-loop structure with 3' end and 5' end matching; if the template to be detected matches the mutant primer group, a stem-loop structure with 3' end and 5' end matching is formed for subsequent explosive amplification, so it is judged that the template to be detected is a mutant template, and the target mutation site is a mutant type.
[0030] Specifically, for the wild type point mutation gene site (Exon9 WT): the first base at the 5' end of the forward inner primer FIP is complementary to the wild type site, and the first base at the 5' end of the backward inner primer BIP is the same as the wild type site (for example, the first base at the 5' end of FIP-WT of Exon9 WT is thymine T, which is complementary to adenine A at the corresponding position of the wild type template; the first base at the 5' end of BIP-WT of BIP-WT is adenine A, which is the same as adenine A at the corresponding position of the wild type template); a mismatch is introduced at the second base at the 5' end of the forward inner primer FIP and the backward inner primer BIP (for example, the second base at the 5' end of FIP-WT of Exon9 WT is adenine A, which is not complementary to cytosine C at the corresponding position of the wild type template; the second base at the 5' end of BIP-WT is cytosine C, which is not the same as adenine A at the corresponding position of the wild type template).
[0031] For mutant point mutation gene sites (E542K, E545K, H1047R, H1047L): introduce mismatches according to the same logic (for example, the first base at the 5' end of the FIP-WT of E542K is cytosine C, which is complementary to the guanine G at the mutant site of the mutant template; the first base at the 5' end of the BIP-WT of E542K is guanine G, which is the same as the guanine G at the mutant site of the mutant template; the second base at the 5' end of the FIP-E542K of E542K is adenine A, which is not complementary to the cytosine C at the corresponding position of the E542K mutant template; the second base at the 5' end of the BIP-E542K is cytosine C, which is not the same as the adenine A at the corresponding position of the E542K mutant template).
[0032] It should be noted that the sequences of the forward external primer F3 and the reverse external primer B3 in this embodiment remain unchanged in subsequent experimental groups and are not adjusted.
[0033] After the synthesis of the two groups of primers, they were purified by HAP, dissolved in enzyme-free water and diluted to 100 μM stock solution, and stored at -20°C for standby.
[0034] The LAMP amplification system was prepared, and 25 μL of the system contained: Bst DNA polymerase 8U, dNTPs mixture 0.8 mM, amplification buffer containing 1.0 M betaine (20 mM Tris-HCl, 10 mM KCl, 10 mM (NH4)2SO4, 8 mM MgSO4), 300 μM SYBGreen indicator, primer mixture (FIP / BIP each 1.6 μM, F3 / B3 each 0.2 μM), template DNA 2 μL, and the rest was supplemented with enzyme-free water.
[0035] The prepared reaction system was transferred to a 96-well fluorescent quantitative PCR plate, the plate was sealed, and then placed in a constant temperature metal bath (or a fluorescent quantitative PCR instrument), and the reaction temperature was set to 65°C and the reaction time was 90 minutes.
[0036] Reference Figure 2 The template 1 (WT+E542K) detection: only the Exon9 WT primer group (ct value of 30), the E542K primer group (ct value of 45), and the Exon20 WT primer group (ct value of 20) showed positive amplification, and the E545K primer group, the E545D primer group, the H1047R primer group, and the H1047L primer group had no amplification within 90 minutes.
[0037] Template 2 (WT+E545K) detection: Exon9 WT primer group (ct value is 42), E545K primer group (ct value is 31), Exon20 WT primer group (ct value is 24) appear positive amplification, and the rest of the primer groups are negative.
[0038] Template 3 (WT+H1047R) detection: Exon20 WT primer group (ct value is 45), Exon20 WT primer group (ct value is 24), H1047R primer group (ct value is 24) positive amplification, and the rest of the primer groups are negative.
[0039] The blank control and the repeated experiment results are consistent, and there is no abnormal amplification signal, and the repeatability deviation is less than 3%.
[0040] Comparative Example 1 A control group is created, and the first base at the 5' end of the forward inner primer FIP and the backward inner primer BIP is designed to match the target mutation site base without additional mismatch, that is, no mismatch is introduced at the second base. Other preparations and steps are the same as those in Example 1 and are not described herein.
[0041] Combination Figure 3 Template 1 (WT+E542K) detection: In addition to Exon9 WT primer group, E542K primer group, Exon20 WT primer group appearing positive amplification (ct value is less than 30), E545K primer group, H1047R primer group also appear non-specific amplification (ct value is less than 30); Template 2 (WT+E545K) detection: Exon9 WT primer group, E545K primer group, Exon20 WT primer group normal amplification, E542K primer group, H1047R primer group appear non-specific amplification; Template 3 (WT+H1047R) detection: Exon20 WT primer group, H1047R primer group, Exon20 WT primer group normal amplification, E542K primer group, E545K primer group appear non-specific amplification; The blank control has no amplification signal, proving that the non-specific amplification is caused by the non-specific combination of the primer and the template, rather than contamination.
[0042] Comparing Comparative Example 1 and Example 1, the design of introducing a mismatch base at the second base at the 5' end of the forward inner primer FIP and the backward inner primer BIP in Example 1 is beneficial to reduce the non-specific combination of the 3' and 5' ends of the stem-loop structure, significantly inhibit non-specific amplification, realize no non-specific amplification within 80 minutes, and fully verify the promotion effect of the primer design strategy of the application on detection specificity, solving the core problem of false positive caused by non-specific combination of the primer in traditional LAMP detection of point mutation genes.
[0043] Example 2 This embodiment focuses on the wild-type primer set and mutant primer set with "introduced mismatched bases". Targeting five key mutation sites of the PIK3CA gene – Exon9 wild-type (WT), Exon20 wild-type (WT), E542K mutant, E545K mutant, and H1047R mutant – the limit of detection (LOD) of the detection method for each target site was determined by gradient dilution of template concentration, thus verifying its applicability in clinical low-abundance mutation detection scenarios.
[0044] Serial dilutions were performed with enzyme-free water to construct concentration gradients covering the clinically low abundance range: 10¹ copies / μL, 10² copies / μL, 10³ copies / μL, 10 4 copies / μL, 10 5 The experiment was repeated 3 times for each concentration, with a "template-free blank control" (using 2 μL of enzyme-free water instead of template) to eliminate contamination interference.
[0045] The same specific primer set and LAMP amplification system as in Example 1, which "introduces mismatched bases," were used. Figure 4 The fluorescence curve data for each target site and the limits of detection (LOD) results for the five PIK3CA gene point mutation sites are as follows: Exon9 WT site: When the template concentration was 273 copies / μL, clear amplification curves were observed in all three replicate experiments (ct value of 70); when the template concentration was lower than 273 copies / μL (e.g., 100 copies / μL), only one replicate showed a weak fluorescence signal, which did not meet the positive judgment criteria. Therefore, the detection limit of Exon9 WT site is 273 copies / μL. Exon20 WT site: When the template concentration was 781 copies / μL, all three replicates met the positive criteria (ct value of 39); when the template concentration was reduced to 391 copies / μL, only one weak positive signal was observed. Therefore, the detection limit of Exon20 WT site is 781 copies / μL. E542K mutation site: When the template concentration was 794 copies / μL, stable amplification curves (ct value of 79) were observed in all three replicates; when the template concentration was 398 copies / μL, the positive standard could not be met, therefore the detection limit of the E542K mutation site is 794 copies / μL; The E545K mutation site: when the template concentration is 322 copies / μL, 3 repetitions all meet the positive judgment (ct value is 80); when the template concentration is 161 copies / μL, no positive signal can be obtained, and therefore the detection limit of the E545K mutation site is 322 copies / μL; The H1047R mutation site: when the template concentration is 463 copies / μL, 3 repetitions all have obvious amplification curves (ct value is 58); when the template concentration is 231 copies / μL, only 1 repetition reaches the positive standard, and therefore the detection limit of the H1047R mutation site is 463 copies / μL.
[0046] The blank control has no fluorescence signal amplification in all experiments, proving that the experimental results have no exogenous pollution interference, and the data is reliable. The above detection limit results show that the detection method of the present application can effectively detect the low-abundance PIK3CA gene mutations commonly seen in clinic, and meet the detection needs of precise medical scenes such as early diagnosis of cancer, monitoring of targeted therapy efficacy, etc.
Claims
1. A method for detecting a point mutation gene-specific by loop-mediated isothermal amplification, characterized in that, The method comprises the following steps: Synthesizing a wild type primer group and a mutant primer group for a target point mutant gene; The wild type primer group and the mutant primer group each comprise a forward inner primer FIP and a backward inner primer BIP; wherein the first base at the 5' end of the forward inner primer FIP is complementary to the base at the target point mutant gene site of the corresponding template to be detected, the first base at the 5' end of the backward inner primer BIP is the same as the base at the target mutation site of the corresponding template to be detected, and a base that is not matched with the base of the target detection gene is introduced at the second base at the 5' end of the forward inner primer FIP and the backward inner primer BIP; Using the wild type primer group to perform LAMP amplification reaction on the template containing the target point mutant gene site as the template to be detected, and obtaining an amplification reaction result; According to the amplification reaction result, judging the relative quantitative concentration of the target point mutant gene in the template to be detected relative to the wild type gene.
2. The method according to claim 1, wherein the point mutation is selected from the group consisting of a single nucleotide substitution, a single nucleotide insertion, and a single nucleotide deletion. The wild type primer group and the mutant primer group further comprise a forward outer primer F3 and a backward outer primer B3.
3. The method according to claim 1, wherein the point mutation is selected from the group consisting of a single nucleotide substitution, a single nucleotide insertion, and a single nucleotide deletion. The template to be detected comprises a wild type template and a mutant template.
4. The method according to any one of claims 1 or 3, wherein the method is a method for detecting a point mutation-specific LAMP. When the template to be detected matches the wild type primer group during the initial amplification of LAMP, a stem-loop structure with 3' end and 5' end matching required for subsequent explosive amplification is formed; when the template to be detected matches the mutant primer group, it is difficult to form the stem-loop structure with 3' end and 5' end matching, and thus it is judged that the template is the wild type template.
5. The method according to any one of claims 1 or 3, wherein the method is a point mutation gene-specific LAMP method, characterized in that, When the template to be detected matches the wild type primer group during the initial amplification of LAMP, it is difficult to form the stem-loop structure with 3' end and 5' end matching; when the template to be detected matches the mutant primer group, the stem-loop structure with 3' end and 5' end matching for subsequent explosive amplification is formed, and thus it is judged that the template is the mutant template.
6. The method according to claim 1, wherein the point mutation is selected from the group consisting of a single nucleotide substitution, a single nucleotide insertion, and a single nucleotide deletion. The second base at the 5' end of the forward inner primer FIP-WT of the wild type primer group is not complementary to the base at the corresponding position of the wild type template, forming a base mismatch; The second base at the 5' end of the backward inner primer BIP-WT of the wild type primer group is not the same as the base at the corresponding position of the wild type template, forming a base mismatch.
7. The method according to claim 1, wherein the point mutation is selected from the group consisting of a single nucleotide substitution, a single nucleotide insertion, and a single nucleotide deletion. The second base at the 5' end of the forward inner primer FIP-MUT of the mutant primer group is not complementary to the base at the corresponding position of the mutant template, forming a base mismatch; The second base at the 5' end of the backward inner primer BIP-MUT of the mutant primer group is not the same as the base at the corresponding position of the mutant template, forming a base mismatch.
8. The method according to any one of claims 1 to 7, wherein the method is a method for detecting a point mutation-specific LAMP. The first base at the 5' end of the forward inner primer FIP-WT of the wild type primer group is complementary to the base at the target mutation site of the wild type template, and the second base at the 5' end thereof is not complementary to the corresponding position of the wild type template, which together constitute specific recognition of the wild type template by the primer; The first base at the 5' end of the backward inner primer BIP-WT of the wild type primer group is the same as the base at the target mutation site of the wild type template, and the second base at the 5' end thereof is not the same as the corresponding position of the wild type template, which together constitute specific recognition of the wild type template by the primer.
9. The method according to any one of claims 1 to 7, wherein the method is a method for detecting a point mutation-specific LAMP. The first base at the 5' end of the forward inner primer FIP-MUT of the mutant primer set is complementary to the base at the target mutation site of the mutant template, and the second base at the 5' end thereof is not complementary to the corresponding position of the mutant template, which together constitute specific recognition of the primer to the mutant template; The first base at the 5' end of the backward inner primer BIP-MUT of the mutant primer set is the same as the base at the target mutation site of the mutant template, and the second base at the 5' end thereof is not the same as the corresponding position of the mutant template, which together constitute specific recognition of the primer to the mutant template.
10. A method for detecting a point mutation gene-specific by loop-mediated isothermal amplification, characterized by, The application is applied to the typing detection of different point mutation sites related to diseases in clinical samples, including blood, tissue, saliva or cell samples.