LAMP (loop-mediated isothermal amplification) primer probe composition for detecting folic acid MTHFR (methylenetetrahydrofolate reductase) gene polymorphism, kit and application of LAMP primer probe composition

Abstract

The invention belongs to the technical field of molecular biology and in-vitro diagnosis, and particularly discloses an LAMP (loop-mediated isothermal amplification) primer probe composition for detecting folic acid MTHFR (methylenetetrahydrofolate reductase) gene polymorphism, a kit and application of the LAMP primer probe composition. A probe-mediated LAMP melting curve analysis system is constructed, efficient nucleic acid amplification is realized under a constant-temperature condition by utilizing LAMP reaction, and rapid and accurate typing of C677T mutation sites is realized by combining high-specificity discrimination characteristics of a double-labeled probe melting curve. Compared with a traditional method, the method has the advantages of being short in reaction time, high in sensitivity, high in specificity, free of complex instruments and follow-up operation and the like, and is suitable for clinical rapid typing detection and basic molecular diagnosis application.

Description

Technical Field

[0001] This application relates to the fields of molecular biology and in vitro diagnostic technology, and in particular to a LAMP primer-probe composition, kit, and application for detecting folic acid MTHFR gene polymorphism. Background Technology

[0002] Folic acid, one of the most important water-soluble vitamins, plays a vital role in the human body by participating in the synthesis and metabolism of biomolecules such as DNA synthesis, methylation reactions, and amino acid metabolism. Folic acid deficiency can lead to neural tube defects during pregnancy, megaloblastic anemia, cardiovascular disease, and other chronic functional disorders. Normally, people can obtain sufficient folic acid through a normal daily diet. However, individuals carrying gene mutations related to folic acid metabolism require supplementation to prevent folic acid-related diseases. Therefore, genotyping of folic acid metabolism-related genes is of great significance for the identification, prevention, and treatment of folic acid deficiency.

[0003] Methylenetetrahydrofolate reductase (MTHFR) is one of two key enzymes involved in folate metabolism. The MTHFR gene exhibits polymorphism, with the C677T mutation being the most studied. Physiologically, MTHFR is responsible for reducing 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, providing a methyl donor for the remethylation of homocysteine ​​to methionine. When the C677T mutation results in the substitution of valine for alanine at position 222, the enzyme's thermostability and activity decrease significantly, especially in homozygous TT individuals, where activity is only about 30% of that in normal individuals, leading to impaired folate metabolism and homocysteine ​​accumulation. Therefore, MTHFR C677T site detection has significant clinical value in genetic risk assessment, disease prevention, preconception screening, and precision nutritional intervention.

[0004] Currently, methods for genotyping folate metabolism-related genes mainly include first-generation Sanger sequencing, gene chips, and PCR. Despite significant advancements in these technologies, drawbacks such as high cost, time-consuming and labor-intensive processes, and false positives remain. Sequencing is the most accurate method for mutation detection, but it is cumbersome and time-consuming. PCR, as a nucleic acid exponential amplification technique, is widely used in clinical molecular testing. PCR is often combined with other techniques (such as PCR sequencing, PCR-RFLP, and PCR-fluorescent probes) to achieve mutation detection. Among these techniques, PCR-fluorescent probes are the most commonly used SNP analysis method in clinical practice. However, this method still has some limitations: PCR-fluorescent probes rely on strict annealing temperatures; excessively high annealing temperatures can lead to probe dissociation from the target, while excessively low annealing temperatures can result in non-specific hybridization of the probe.

[0005] Isothermal amplification (LAMP) is characterized by its rapid, efficient, and constant-temperature amplification capabilities, making it easy to perform on-site detection. Many researchers consider LAMP a viable alternative to PCR. Among these, loop-mediated isothermal amplification (LAMP) shows significant potential as a replacement for PCR. However, the polymerases used in LAMP lack 5' to 3' exonuclease activity, preventing the use of TaqMan probes for hydrolysis as in PCR. Furthermore, real-time LAMP methods primarily rely on fluorescent dyes or binding to the Cas system to generate signals, which carries risks such as poor specificity and aerosol contamination. Summary of the Invention

[0006] In view of the shortcomings of the prior art described above, the purpose of this application is to provide a LAMP primer-probe composition, kit and application for detecting folic acid MTHFR gene polymorphism. The LAMP isothermal amplification technology combined with the melting curve mediated by the dual-labeled probe is used to achieve C677T genotyping under closed tube reaction conditions, which solves the problems of long detection cycle, high cost, complicated operation and high false positive rate of the existing SNP detection technology.

[0007] To achieve the above and other related objectives, this application provides a LAMP primer-probe composition for detecting folic acid MTHFR gene polymorphism, the LAMP primer-probe composition comprising an outer primer pair, an inner primer pair, and a fluorescent probe; The outer primer pair includes a forward outer primer (F3) with the nucleotide sequence shown in SEQ ID NO.1: CCCTCACCTGGATGGGAA, and a reverse outer primer (B3) with the nucleotide sequence shown in SEQ ID NO.2: TCTTCATCCCTCGCCTTGA; The inner primer pair includes a forward inner primer (FIP) with the nucleotide sequence shown in SEQ ID NO.3: AGGCTGACACATTCTTCCGCTTT-CATGTCGGTGCATGCCT, and a reverse inner primer (BIP) with the nucleotide sequence shown in SEQ ID NO.4: TGCTTCAGGTCAGCCTCAAAGC-TACCCCAAAGGCCACCC. The fluorescent probe comprises nucleotides with the sequence shown in SEQ ID NO.5: ATGAAATCGACTCCCGCAG.

[0008] Furthermore, the 5' end of the fluorescent probe is labeled with a fluorescent group, which is selected from any one of ROX, FAM, HEX, CY3, and CY5, and the 3' end of the fluorescent probe is labeled with a quenching group, which is selected from the BHQ series.

[0009] Furthermore, the fluorescent probe is: ROX-ATGAAATCGACTCCCGCAG-BHQ2.

[0010] This application also provides a LAMP kit for detecting folic acid MTHFR gene polymorphism, the LAMP kit comprising the LAMP primer-probe composition as described above.

[0011] Furthermore, the LAMP kit also includes at least one of the following components: reaction solution, DNA polymerase, fluorescent dye, water (preferably ultrapure water), and negative control.

[0012] Furthermore, the DNA polymerase is selected from at least one of Bst 2.0 polymerase and Bst 3.0 polymerase.

[0013] Furthermore, the LAMP kit also includes 2×LAMP Master Mix.

[0014] Furthermore, in the LAMP kit, the molar ratio of the outer primer pair, the inner primer pair, and the fluorescent probe is 1:(4-8):1.

[0015] This application also provides the use of the LAMP primer-probe composition as described above or the LAMP kit as described above in the preparation of reagents for detecting folic acid MTHFR gene polymorphism.

[0016] Furthermore, the polymorphism of the folic acid MTHFR gene was analyzed and detected using the loop-mediated isothermal amplification melting curve method. The loop-mediated isothermal amplification melting curve method includes a loop-mediated isothermal amplification reaction and melting curve analysis. The temperature of the loop-mediated isothermal amplification reaction is 55~65℃, and the amplification time is equal to or greater than 30 minutes. The melting curve analysis is performed at 50~70℃.

[0017] Furthermore, fluorescence signals were monitored in real time during the loop-mediated isothermal amplification reaction; after the loop-mediated isothermal amplification reaction was completed, melting curve analysis was performed, and the MTHFR gene was genotyped based on the melting curve. When the melting curve shows only one characteristic peak and the Tm peak is at 55±0.3℃, the target MTHFR gene is of type CC. When the melting curve shows only one characteristic peak and the Tm peak is at 61±0.3℃, the target MTHFR gene is of the TT type. When the melting curve shows two characteristic peaks simultaneously, and the two Tm peaks are at 55±0.3℃ and 61±0.3℃ respectively, the target MTHFR gene is CT heterozygous.

[0018] The beneficial effects of this application are: This application utilizes the LAMP reaction to achieve efficient nucleic acid amplification under isothermal conditions. Combined with the high specificity of the melting curve of a dual-labeled probe, it enables rapid and accurate typing of the C677T mutation site under closed-tube reaction conditions. The method involves LAMP amplification under isothermal conditions. By analyzing the melting curve within the range of 50–70 °C, different genotypes can be distinguished based on the characteristic melting peaks: CC genotype at approximately 55 ± 0.3 °C, TT genotype at approximately 61 ± 0.3 °C, and CT heterozygous genotype exhibiting both characteristic peaks simultaneously. Compared with the traditional PCR-fluorescent probe method, this invention has advantages such as short reaction time, high sensitivity, strong specificity, and no need for complex instruments and subsequent operations, making it suitable for rapid clinical typing and molecular diagnostic applications at the grassroots level. Attached Figure Description

[0019] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this application and, together with the description, serve to explain the principles of this application. It is obvious that the drawings described below are merely some embodiments of this application, and those skilled in the art can obtain other drawings based on these drawings without any inventive effort.

[0020] In the attached diagram: Figure 1 This is a schematic diagram of the binding and melting curves of the LAMP isothermal amplification product and the probe provided in this application.

[0021] Figure 2 The following are sequencing images of typical TT, CT, and CC types in Example 1 of this application (A), amplification curves of typical TT, CT, and CC types (B), raw melting curves of typical TT, CT, and CC types (C), melting curve integrals of typical TT, CT, and CC types (D), and the difference between the left and right peaks (E).

[0022] Figure 3 The following are the experimental results of clinical specimen verification in Example 1 of this application: A. LAMP melting curve results of clinical specimens, B. PCR amplification results of clinical specimens, C. PCR amplification heatmap of clinical specimens. Detailed Implementation

[0023] The following specific examples illustrate the implementation of this application. Those skilled in the art can easily understand other advantages and effects of this application from the content disclosed in this specification. This application can also be implemented or applied through other different specific embodiments. Various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of this application. In the absence of conflict, the following embodiments and features in the embodiments can be combined with each other.

[0024] In this application, unless otherwise stated, the term "multiple" means two or more.

[0025] The character " / " indicates that the objects before and after it are in an "or" relationship. For example, A / B means: A or B.

[0026] The term "and / or" describes an association between objects, indicating that three relationships can exist. For example, A and / or B means: A or B, or A and B.

[0027] Unlike traditional LAMP probe design, which relies on fixed loop regions or strand substitution mechanisms, this application is the first to discover that the strand dissociation-rehybridization dynamics of the LAMP amplification product itself can provide active sites for the probe, thereby achieving stable probe binding and melting analysis. This mechanism overcomes the limitation of traditional probe design, which must be located in the loop region, significantly improving the flexibility and applicability of probe design. Furthermore, the inventors of this application have also found that probe-based melting curve analysis (MCA) is more suitable for SNP analysis: the probe length is approximately 20 nt, and a single base change can significantly affect the thermodynamic stability of the hybrid double strand, leading to a significant change in the Tm value. Therefore, probe-based MCA is more sensitive than dye-based MCA. Probe-based MCA has advantages such as high sensitivity, low cost, and no need for post-PCR manipulation, making it suitable for SNP detection.

[0028] like Figure 1 As shown, in terms of technical implementation, this application constructs a probe-mediated LAMP melting curve analysis system. By utilizing the transient chain separation phenomenon generated by LAMP products during temperature changes, the fluorescent probe can reversibly bind to the amplification product, forming melting peaks with characteristic differences during the melting process, thereby achieving the differentiation of different SNP alleles. Based on this, this application proposes a genotyping method for the C677T site of folate metabolism-related genes based on LAMP isothermal amplification combined with double-labeled probe-mediated melting curve analysis. This is a probe melting curve analysis method based on the respiratory dynamic behavior of LAMP amplification product DNA. It not only achieves accurate identification of SNPs but also effectively solves the problems of long detection cycles, high costs, complex operations, and high false positive rates in existing SNP detection technologies. Compared with the traditional PCR-fluorescent probe method, this method has the advantages of short reaction time, high sensitivity, strong specificity, and no need for complex instruments and subsequent operations, making it suitable for rapid clinical genotyping and molecular diagnostic applications at the grassroots level.

[0029] Specifically, one embodiment of this application provides a LAMP primer-probe composition for detecting folic acid MTHFR gene polymorphism, the LAMP primer-probe composition comprising an outer primer pair, an inner primer pair, and a fluorescent probe; The outer primer pair includes: Forward outer primer (F3): CCCTCACCTGGATGGGAA (SEQ ID NO.1). Reverse outer primer (B3): TCTTCATCCCTCGCCTTGA (SEQ ID NO.2); The inner primer pair includes: Forward inner primer (FIP): AGGCTGACACATTCTCCGCTTT-CATGTCGGTGCATGCCT (SEQ ID NO. 3); Reverse inner primer (BIP): TGCTTCAGGTCAGCCTCAAAGC-TACCCCAAAGGCCACCC (SEQ ID NO. 4); The fluorescent probe comprises a nucleotide with the sequence shown in SEQ ID NO.5: ATGAAATCGACTCCCGCAG. The 5' end of the fluorescent probe is labeled with fluorescent groups such as ROX, FAM, HEX, CY3, CY5, etc., and the 3' end is labeled with a quenching group such as the BHQ series, but is not limited thereto. In an exemplary embodiment of this application, the fluorescent probe is: ROX-ATGAAATCGACTCCCGCAG-BHQ2.

[0030] Another embodiment of this application provides a LAMP kit for detecting folic acid MTHFR gene polymorphism, the LAMP kit comprising the LAMP primer-probe composition as described above.

[0031] In some embodiments of this application, the LAMP kit further includes at least one of the following components: reaction solution, DNA polymerase, fluorescent dye, water (preferably ultrapure water), and a negative control. The DNA polymerase includes, but is not limited to, Bst 2.0 polymerase, Bst 3.0 polymerase, etc., and the water is, for example, ultrapure water. Exemplarily, the LAMP kit also includes 2×LAMP Master Mix, which is commercially available.

[0032] In some embodiments of this application, the molar ratio of the outer primer pair, inner primer pair, and fluorescent probe in the LAMP kit is 1:4 to 8:1. Furthermore, the concentration and amount of each component in the LAMP kit are selected and designed according to actual needs.

[0033] Another embodiment of this application provides the application of the LAMP primer-probe composition or the LAMP kit described above in the preparation of reagents for detecting folic acid MTHFR gene polymorphism. An exemplary application is as follows: The LAMP melting curve method is used to analyze and detect folic acid MTHFR gene polymorphism. The LAMP melting curve method includes a LAMP reaction and melting curve analysis; the LAMP reaction temperature is 55-65°C, and the amplification time is equal to or greater than 30 minutes, for example, 30-40 minutes; the melting curve analysis is performed at 50-70°C. During the LAMP reaction, the fluorescence signal is monitored in real time; after the LAMP reaction is completed, melting curve analysis is performed, and the MTHFR gene is genotyped based on the melting curve. When the melting curve shows only one characteristic peak and the Tm peak is at 55±0.3℃, the target MTHFR gene is of type CC. When the melting curve shows only one characteristic peak and the Tm peak is at 61±0.3℃, the target MTHFR gene is of the TT type. When the melting curve shows two characteristic peaks simultaneously, and the two Tm peaks are at 55±0.3℃ and 61±0.3℃ respectively, the target MTHFR gene is CT heterozygous.

[0034] The following specific examples illustrate the present invention in detail. It should also be understood that the following examples are only for specific illustrative purposes and should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above description of the present invention are within the scope of protection of the present invention. The specific process parameters, etc., in the following examples are merely examples within a suitable range; that is, those skilled in the art can make appropriate selections within the appropriate range based on the description herein, and are not intended to be limited to the specific values ​​in the examples below.

[0035] Example 1 like Figure 1 As shown, this embodiment employs LAMP isothermal amplification technology combined with dual-labeled probe-mediated melting curve analysis to achieve C677T genotyping. The LAMP reaction is performed at 60 °C, and the melting curve analysis is conducted at 50–70 °C. The specific implementation scheme is as follows.

[0036] (1) Reaction system: (2) Reaction sequence: (3) Reaction procedure: react at 60 ℃ for 30 min, and perform melting curve analysis at 50~70 ℃.

[0037] (4) Results analysis: like Figure 1As shown, the melting curve peak at around 56 ℃ indicates the CC type. The melting curve peak at around 61 ℃ indicates the TT type. The melting curve peaks at 55 ℃ and 61 ℃ indicate the CT hybrid type.

[0038] To verify the feasibility and accuracy of the established LAMP melting curve detection system in MTHFR C677T SNP genotyping, Sanger sequencing was used as the gold standard to confirm the genotype of typical samples. TC showed a typical bimodal signal at the mutation site, demonstrating that the sample genotyping was accurate and reliable. Figure 2 A). Real-time fluorescence monitoring results showed that all three genotypes entered the exponential amplification phase and reached the plateau phase after approximately 20 minutes, indicating that the system had good amplification efficiency. Because the probes were designed to be completely complementary to the T allele, the endpoint fluorescence intensity showed a genotype-dependent gradient of TT > TC > CC, while the NTC control showed no fluorescence signal, indicating that the system had good specificity. Figure 2 B). Further analysis of the melting curves after amplification showed that different genotypes had distinct melting peaks: TT showed a high Tm peak at approximately 61 °C, CC showed a low Tm peak at approximately 55 °C, while TC exhibited both characteristic peaks simultaneously. Figure 2 (C and 2D). The melting temperature difference of approximately 6 °C between the two alleles allows this method to achieve reliable genotyping even under conditions with relatively low instrument precision requirements. Figure 2 E). In summary, the LAMP melting curve method constructed in this embodiment can achieve accurate, stable, and promising SNP genotyping detection of MTHFR C677T.

[0039] Next, this embodiment uses the constructed method to analyze 30 clinical blood samples. Figure 3 A) The experimental results are in good agreement with the TaqMan probe method. Figure 3 B and C) verified the reliability and application potential of this method in SNP typing of clinical samples.

[0040] The above embodiments are merely illustrative of the principles and effects of this application and are not intended to limit this application. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of this application. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in this application should still be covered by the claims of this application.

Claims

1. A LAMP primer-probe composition for detecting folic acid MTHFR gene polymorphism, characterized in that: The LAMP primer-probe composition includes an outer primer pair, an inner primer pair, and a fluorescent probe; The outer primer pair includes the forward outer primer F3 with the nucleotide sequence shown in SEQ ID NO.1: CCCTCACCTGGATGGGAA, and the reverse outer primer B3 with the nucleotide sequence shown in SEQ ID NO.2: TCTTCATCCCTCGCCTTGA; The inner primer pair includes a forward inner primer FIP with a nucleotide sequence as shown in SEQ ID NO.3: AGGCTGACACATTCTTCCGCTTT-CATGTCGGTGCATGCCT, and a reverse inner primer BIP with a nucleotide sequence as shown in SEQ ID NO.4: TGCTTCAGGTCAGCCTCAAAGC-TACCCCAAAGGCCACCC. The fluorescent probe comprises nucleotides with the sequence shown in SEQ ID NO.5: ATGAAATCGACTCCCGCAG.

2. The LAMP primer-probe composition according to claim 1, characterized in that: The fluorescent probe has a fluorescent group labeled at its 5' end, which is selected from any one of ROX, FAM, HEX, CY3, and CY5. The fluorescent probe also has a quenching group labeled at its 3' end, which is selected from the BHQ series.

3. The LAMP primer-probe composition according to claim 2, characterized in that: The fluorescent probe is: ROX-ATGAAATCGACTCCCGCAG-BHQ2.

4. A LAMP kit for detecting folic acid MTHFR gene polymorphism, characterized in that: The LAMP kit includes the LAMP primer and probe composition as described in any one of claims 1 to 3.

5. The LAMP kit according to claim 4, characterized in that: The LAMP kit also includes at least one of the following components: reaction solution, DNA polymerase, fluorescent dye, water (preferably ultrapure water), and negative control.

6. The LAMP kit according to claim 5, characterized in that: The DNA polymerase is selected from at least one of Bst 2.0 polymerase and Bst 3.0 polymerase; And / or, the LAMP kit also includes 2×LAMP Master Mix.

7. The LAMP kit according to any one of claims 4 to 6, characterized in that: In the LAMP kit, the molar ratio of the outer primer pair, inner primer pair, and fluorescent probe is 1:4 to 8:

1.

8. The use of the LAMP primer-probe composition according to any one of claims 1 to 3 or the LAMP kit according to any one of claims 4 to 7 in the preparation of reagents for detecting folic acid MTHFR gene polymorphism.

9. The application according to claim 8, characterized in that: The polymorphism of the folic acid MTHFR gene was detected by using the loop-mediated isothermal amplification melting curve method, which includes the loop-mediated isothermal amplification reaction and melting curve analysis. The temperature of the loop-mediated isothermal amplification reaction is 55~65℃, and the amplification time is equal to or greater than 30 minutes. The melting curve analysis was performed at 50–70 °C.

10. The application according to claim 1, characterized in that: During the loop-mediated isothermal amplification reaction, fluorescence signals were monitored in real time. After the loop-mediated isothermal amplification reaction was completed, melting curve analysis was performed, and the MTHFR gene was genotyped based on the melting curve. When the melting curve shows only one characteristic peak and the Tm peak is at 55±0.3℃, the target MTHFR gene is of type CC. When the melting curve shows only one characteristic peak and the Tm peak is at 61±0.3℃, the target MTHFR gene is of the TT type. When the melting curve shows two characteristic peaks simultaneously, and the two Tm peaks are at 55±0.3℃ and 61±0.3℃ respectively, the target MTHFR gene is CT heterozygous.

Images