Kit and detection method for detecting MTHFR (methylenetetrahydrofolate reductase) gene polymorphism

Abstract

The invention discloses a kit for detecting MTHFR (Methylene Tetrahydrofolate Reductase) gene polymorphism and a detection method, relates to gene detection, and aims to solve the problem that an existing detection method lacks convenience, timeliness and economy or cannot meet SNP (Single Nucleotide Polymorphism) genetic typing detection requirements. Comprising a component 1, a component 2, a component 3 and a component 4, wherein the component 1 is a primer and a probe for detecting the gene polymorphism of the C677T site of the MTHFR gene; and the component 2 is a primer and a probe for detecting an internal reference beta-globin. The kit for detecting MTHFR gene polymorphism disclosed by the invention not only has the technical effects of rapidness and convenience, but also is accurate and reliable in detection result.

Description

Technical Field

[0001] The present invention relates to gene detection, and more specifically, to a kit and a method for detecting MTHFR gene polymorphism. Background Art

[0002] The MTHFR (5,10-methylenetetrahydrofolate reductase) gene is an important gene in the human body, located at position 1p36.3 on chromosome 1. The enzyme it encodes plays a key role in folate metabolism and DNA methylation. The C677T (rs1801133) site is the most clinically studied MTHFR single nucleotide polymorphism site. Its polymorphism can affect the activity of the MTHFR enzyme, thereby affecting folate metabolism and homocysteine ​​levels.

[0003] Previous studies have shown that the MTHFR-C677T mutation is widely present worldwide, with different carrier rates in different races and regions. The C677T mutation is more common in European and Asian populations. In the Han Chinese population, the genotype distribution of the MTHFR-C677T locus is as follows: the normal type (CC) is the majority, followed by the heterozygous mutant type (CT), and the homozygous mutant type (TT) is relatively rare, but the specific proportion may vary depending on the research sample and regional differences.

[0004] In recent years, molecular biology technology has developed rapidly, and PCR amplification technology has been widely used. This technology has high sensitivity, good specificity, and good clinical application prospects, especially in the detection of gene polymorphisms. Commonly used detection methods for MTHFR gene polymorphisms are: PCR-gene chip method, PCR-fluorescent probe method, PCR-melting curve method and PCR-electrophoresis analysis method. In my country, many MTHFR gene polymorphism detection reagents have been approved for marketing, and some products are still under review. The methodology of the currently approved products is mainly PCR-fluorescent probe method.

[0005] With the rapid development of molecular biology technology, there are currently a variety of methods for SNP detection; such as PCR-gene chip method, PCR-fluorescent probe method, PCR-melting curve method and PCR-electrophoresis analysis method. Among these methods, PCR-fluorescent probe method is the most commonly used detection method on the market, but most of them require DNA extraction and then genotyping after PCR amplification, which prolongs the detection time and increases the detection timeliness and cost accordingly; there is currently no fast, convenient and economical full-typing gene detection kit for SNP typing detection; and most of the test kits use human peripheral blood DNA, which makes it difficult to collect samples; in view of the above-mentioned shortcomings of the existing technology, in view of the current situation that the existing detection methods lack convenience, timeliness, economy or cannot meet the requirements of SNP genotyping detection, there is an urgent need for a detection method that can be used for grassroots testing or even home testing, which is fast, accurate, convenient, economical and comprehensive. Summary of the invention

[0006] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a kit for detecting MTHFR gene polymorphism, which not only has a fast and convenient technical effect, but also has accurate and reliable detection results.

[0007] The above technical purpose of the present invention is achieved through the following technical scheme: a kit for detecting MTHFR gene polymorphism, comprising: Component 1: primers and probes for detecting the gene polymorphism of the C677T site of the MTHFR gene; Component 2: primers and probes for detecting the internal reference β-globin; Characterized in that: the component 1 comprises a C677T upstream primer having a nucleotide sequence as shown in SEQ ID NO.1, and a C677T downstream primer having a nucleotide sequence as shown in SEQ ID NO.2; The 5' end of the C677T probe 1 sequence is labeled with a FAM fluorescent group, and the 3' end is labeled with an MGB quenching fluorescent group. The nucleotide sequence of the C677T probe 1 is shown in SEQ ID NO. 3; The 5' end of the C677T probe 2 sequence is labeled with a VIC fluorescent group, and the 3' end is labeled with an MGB quenching fluorescent group. The nucleotide sequence of the C677T probe 2 is shown in SEQ ID NO.4; Component 2 includes a β-globin upstream primer having a nucleotide sequence as shown in SEQ ID NO.5, and a β-globin downstream primer having a nucleotide sequence as shown in SEQ ID NO.6; The 5' end of the β-globin probe sequence is labeled with a ROX fluorescent group, and the 3' end is labeled with an MGB quenching fluorescent group. The nucleotide sequence of the β-globin probe is shown in SEQ ID NO.7.

[0008] By adopting the above technical scheme, the primer and probe combination used in the kit can adopt the swab direct amplification combined with fluorescent PCR method to complete the detection of three genotypes of CC genotype, CT genotype and TT genotype of the MTHFR-C677T site, which not only has a fast and convenient technical effect, but also has accurate test results; at the same time, the kit has designed a detection method of primer and double probe combination, which contains detection primers and probes for internal reference β-globin, so as to effectively control the quality of sample collection, sample processing and PCR detection, and further improve the reliability of the test results.

[0009] The present invention is further configured to include an amplification enzyme, which is used to amplify the nucleic acid in the oral swab and participate in the fluorescent PCR reaction procedure.

[0010] The present invention is further configured as follows: the final concentration of the C677T upstream primer and the C677T downstream primer for amplification is 0.4 μmol / L; the final concentration of the C677T probe 1 and the C677T probe 2 for detection is 0.1 μmol / L.

[0011] The present invention is further configured as follows: the final concentration of the β-globin upstream primer and the β-globin downstream primer for amplification is 0.2 μmol / L, and the final concentration of the β-globin probe for detection is 0.05 μmol / L.

[0012] The present invention is further configured as follows: the liquid reagent in the reagent kit is freeze-dried in situ through a freeze-drying procedure to form a reagent freeze-dried powder.

[0013] The present invention is further configured to include: a positive control substance A1 tube and a blank control substance B1 tube; The positive control substance is a natural cell line heterozygous for the C677T site of the MTHFR gene; the blank control substance is enzyme-free water; The liquid reagent in the positive control substance A1 tube is freeze-dried in situ through a freeze-drying procedure to prepare the corresponding quality control substance positive control powder; The liquid in the blank control substance B1 tube is freeze-dried in situ through a freeze-drying procedure to prepare the corresponding quality control substance blank control powder.

[0014] In view of the deficiencies in the prior art, the purpose of the present invention is to provide a method for using a kit for detecting MTHFR gene polymorphism, which effectively meets the application needs of grassroots testing.

[0015] The above technical objectives of the present invention are achieved through the following technical solutions: A method for using a kit for detecting MTHFR gene polymorphism, characterized in that it comprises the following steps: S1. Obtain an oral swab sample to be tested, place it in a sample processing solution, allow the oral mucosal cells to release nucleic acids, and prepare a swab solution as an amplification template; S2. The prepared component 1, component 2 and DNA polymerase Mix are thoroughly mixed, and the liquid reagent is freeze-dried in situ through a freeze-drying procedure to prepare multiple portions of freeze-dried reagent powder; S4. Take 25 μL of the amplification template prepared in S1 and directly put it into a portion of the reagent freeze-dried powder prepared in S2 for dissolution, and transfer the solution to a reaction tube for amplification, and judge the result by the Ct value.

[0016] By adopting the above technical solution, the reagents are freeze-dried under the premise of being fast, convenient, accurate and reliable, which effectively reduces the difficulty of storage and transportation of reagents and effectively meets the application needs of grassroots testing.

[0017] The present invention is further configured as follows: the amplification program is pre-denaturation at 95° C. for 2 minutes, 1 cycle; denaturation at 95° C. for 10 seconds, annealing at 60° C. for 20 seconds, alternating for 40 cycles.

[0018] In view of the deficiencies in the prior art, the purpose of the present invention is to provide a method for using a kit for detecting MTHFR gene polymorphism, which realizes the detection of the quality of the reagent itself and whether the reagent is contaminated, thereby ensuring the actual detection efficacy of the reagent.

[0019] The above technical objectives of the present invention are achieved through the following technical solutions: A quality control method for a kit for detecting MTHFR gene polymorphism, characterized in that it comprises the following steps: S2. The prepared component 1, component 2 and DNA polymerase Mix are thoroughly mixed, and the liquid reagent is freeze-dried in situ through a freeze-drying procedure to prepare multiple portions of freeze-dried reagent powder; S3. Dissolve the positive control powder of the quality control product with 25 μL of enzyme-free water, then take a portion of the lyophilized powder of the reagent configured in S2 to dissolve it, transfer it to the corresponding reaction tube, mark it as tube B1, and amplify it; dissolve the blank control powder of the quality control product with 25 μL of enzyme-free water, then take a portion of the lyophilized powder of the reagent configured in S2 to dissolve it, transfer it to the corresponding reaction tube, mark it as tube B2, and amplify it; judge the quality control results by the Ct value.

[0020] By adopting the above technical solution, the quality of the reagent itself can be tested, and whether the reagent is contaminated can be tested, thereby ensuring the actual detection effectiveness of the reagent.

[0021] The present invention is further configured as follows: the quality control result judgment: the B1 tube configured in S3 is marked as a positive control tube, and if a typical S-shaped curve is shown in the three channels of FAM, VIC and ROX and Ct (FAM) ≤ 37, Ct (VIC) ≤ 37, and Ct (ROX) ≤ 37, then the reagent is considered qualified; the B2 tube is marked as a blank control tube, and if there is no Ct value in the four channels, then the reagent is considered to be uncontaminated and the reagent result is credible.

[0022] In summary, the present invention has the following beneficial effects: 1. Accuracy: The results of the comparison of 60 samples completed by the swab direct amplification combined with fluorescent PCR method used in the present invention and 60 samples completed by Sanger sequencing are completely consistent; 2. Time saving: The present invention takes only 40 minutes from sample collection to result issuance, while the fluorescence PCR method takes about 3 hours and the Sanger sequencing takes about 9 hours. In comparison, the present invention can save a lot of time; 3. Reduce costs: The present invention omits the step of DNA extraction, which saves time and reduces the corresponding costs. At the same time, the present invention is simple to operate, the results are fast and accurate, and a lot of labor costs are saved. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of PCR amplification curve of sample 1; Figure 2 is a schematic diagram of the PCR amplification curve of sample 2; Figure 3 is a schematic diagram of the PCR amplification curve of sample 3; Figure 4 Schematic diagram of PCR amplification curve of positive control; Figure 5 Schematic diagram of the PCR amplification curve of the blank control. DETAILED DESCRIPTION

[0024] The present invention is described in further detail below.

[0025] Embodiment 1: A primer-probe combination for detecting MTHFR gene polymorphism, comprising: Component 1: primers and probes for detecting the gene polymorphism of the C677T site of the MTHFR gene; Component 2: primers and probes for detecting the internal reference β-globin; Component 1 includes a C677T upstream primer having a nucleotide sequence as shown in SEQ ID NO.1, and a C677T downstream primer having a nucleotide sequence as shown in SEQ ID NO.2; The 5' end of the C677T probe 1 sequence is labeled with a FAM fluorescent group, and the 3' end is labeled with an MGB quenching fluorescent group. The nucleotide sequence of the C677T probe 1 is shown in SEQ ID NO. 3; The 5' end of the C677T probe 2 sequence is labeled with a VIC fluorescent group, and the 3' end is labeled with an MGB quenching fluorescent group. The nucleotide sequence of the C677T probe 2 is shown in SEQ ID NO.4; Component 2 includes a β-globin upstream primer having a nucleotide sequence as shown in SEQ ID NO.5, and a β-globin downstream primer having a nucleotide sequence as shown in SEQ ID NO.6; The 5' end of the β-globin probe sequence is labeled with a ROX fluorescent group, and the 3' end is labeled with an MGB quenching fluorescent group. The nucleotide sequence of the β-globin probe is shown in SEQ ID NO.7.

[0026] Example 2: A kit for detecting MTHFR gene polymorphism, comprising component 1, component 2 and an amplification enzyme in Example 1, wherein the amplification enzyme is preferably a DNA polymerase Mix, and the amplification enzyme is used to amplify nucleic acids in oral swabs and participate in a fluorescent PCR reaction procedure.

[0027] The final concentration of the amplification C677T upstream primer and the C677T downstream primer was 0.4 μmol / L; the final concentration of the detection C677T probe 1 and the C677T probe 2 was 0.1 μmol / L.

[0028] The final concentration of the β-globin upstream primer and the β-globin downstream primer was 0.2 μmol / L, and the final concentration of the β-globin detection probe was 0.05 μmol / L.

[0029] Table 1: Reagent composition table

[0030] Example 3: A kit for detecting MTHFR gene polymorphism. Based on Example 2, this example further includes a positive control tube A1 and a blank control tube B1; wherein the positive control is a natural cell line heterozygous for the C677T site of the MTHFR gene; and the blank control is enzyme-free water.

[0031] In this embodiment, a positive control substance A1 tube and a blank control substance B1 tube are additionally introduced, so that the reagent itself can be verified on the basis of detecting the MTHFR gene polymorphism, including the quality of the reagent and whether contamination occurs.

[0032] Example 4. A kit for detecting MTHFR gene polymorphism. In this example, based on Example 3, the liquid part of the kit is freeze-dried. Specifically, the liquid reagents involved in Table 1 in Example 2 are freeze-dried in situ through a freeze-drying procedure to prepare reagent freeze-dried powders, and the liquids in the positive control substance A1 tube and the blank control substance B1 tube are freeze-dried in situ through a freeze-drying procedure to prepare corresponding quality control substance positive control powders and quality control substance blank control powders.

[0033] Table 2: Reagent freeze-drying schedule

[0034] Table 3: Lyophilization schedule for positive and blank controls

[0035] The kit provided in this embodiment prepares the liquid reagent into a lyophilized powder, so that the product is in the form of three groups of independently packaged powders when it leaves the factory, wherein Group 1 is 3 portions of reagent lyophilized powder, Group 2 is 1 portion of quality control positive control powder, and Group 3 is 1 portion of quality control blank control powder. Compared with traditional liquid materials, not only is the packaging simpler, but it also solves the problem of insufficient reagent storage conditions in grassroots applications, and is more suitable for grassroots detection applications.

[0036] Example 5. This example is a method for using the kit disclosed in Example 4 to detect sample 1 (known to be CC genotype), sample 2 (known to be CT genotype), and sample 3 (known to be TT genotype).

[0037] A method for using a kit for detecting MTHFR gene polymorphism, characterized in that it comprises the following steps: S1. Obtain an oral swab sample to be tested, place it in a sample processing solution, allow the oral mucosal cells to release nucleic acids, and prepare a swab solution as an amplification template; S3. Dissolve the positive control powder of the quality control product with 25 μL of enzyme-free water, then dissolve a portion of the lyophilized powder of the reagent, and transfer it to the corresponding reaction tube (marked as tube B1) for amplification; dissolve the blank control powder of the quality control product with 25 μL of enzyme-free water, then dissolve a portion of the lyophilized powder of the reagent, and transfer it to the corresponding reaction tube (marked as tube B2) for amplification; judge the quality control results by the Ct value; S4. Take 25 μL of the amplification template prepared in S1 and directly put it into a portion of the reagent freeze-dried powder for dissolution, and transfer the solution to a reaction tube for amplification; Table 4: Reaction conditions used for amplification in steps S3 and S4

[0038] S5. Determine the test result by Ct value; Table 5: Ct value result judgment table

[0039] like Figure 1 The FAM channel Ct value of sample 1 is ≤37, the VIC channel NoCt, and the ROX channel Ct value is ≤37, which is consistent with the qPCR result of genotype 1 in the Ct value result judgment table, and the identification result is CC genotype; Table 6: Figure 1 Curve CT value calculation table

[0040] like Figure 2 The FAM channel, VIC channel, and ROX channel of sample 2 all have Ct values ​​≤ 37, which is consistent with the qPCR result of genotype 2 in the Ct value result judgment table, and the identification result is CT genotype; Table 7: Figure 2 Curve CT value calculation table

[0041] like Figure 3 The FAM channel NoCt, VIC channel Ct value ≤37, and ROX channel Ct value ≤37 of sample 3 are shown, which are consistent with the qPCR results of genotype three in the Ct value result judgment table, and the identification result is TT genotype; Table 8: Figure 3 Curve CT value calculation table

[0042] ( Figure 1 , Figure 2 , Figure 3 In the figure, the horizontal axis is the cycle number, the vertical axis is the fluorescence intensity, the blue curve is the FAM channel, the green curve is the VIC channel, the orange curve is the ROX channel, and the red curve is the unused CY5 channel).

[0043] S6. Quality control result judgment: If the B1 tube (positive control tube) configured in S3 shows a typical S-shaped curve in the three channels of FAM, VIC and ROX and Ct (FAM) ≤37, Ct (VIC) ≤37, Ct (ROX) ≤37, the reagent is considered qualified; if the B2 tube (blank control tube) has no Ct value in the four channels, the reagent is considered to be uncontaminated and the reagent result is credible.

[0044] like Figure 4 The FAM channel, VIC channel ≤ 37 and ROX channel Ct values ​​of the positive control shown are ≤ 37, and the identification results are consistent with the actual results, proving that the reagents are qualified; Table 9: Figure 4 Curve CT value calculation table

[0045] like Figure 5 The FAM channel, VIC channel ≤ 37 and ROX channel of the blank control shown are all NoCt, and the identification results are consistent with the actual results, proving that the reagents are not contaminated; Table 10: Figure 5 Curve CT value calculation table

[0046] ( Figure 4 , Figure 5 In the figure, the horizontal axis is the number of cycles, the vertical axis is the fluorescence intensity, the blue curve is the FAM channel, the green curve is the VIC channel, the orange curve is the ROX channel, and the red curve is the unused CY5 channel) Table 11: Quality control result judgment table

[0047] The specific embodiments are merely explanations of the present invention and are not limitations of the present invention. After reading this specification, those skilled in the art may make modifications to the embodiments without any creative contribution as needed. However, such modifications are protected by the patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A kit for detecting MTHFR gene polymorphism, comprising: Component 1: primers and probes for detecting the MTHFR gene C677T polymorphism; Component 2: primers and probe for detecting the internal reference β-globin; Characterized in that: the component 1 comprises a C677T upstream primer having a nucleotide sequence as shown in SEQ ID NO.1, and a C677T downstream primer having a nucleotide sequence as shown in SEQ ID NO.2; The 5' end of the C677T probe 1 sequence is labeled with a FAM fluorescent group and the 3' end is labeled with an MGB quenching fluorescent group. The nucleotide sequence of the C677T probe 1 is shown in SEQ ID NO. 3; The 5' end of the C677T probe 2 sequence is labeled with a VIC fluorescent group, and the 3' end is labeled with an MGB quenching fluorescent group. The nucleotide sequence of the C677T probe 2 is shown in SEQ ID NO. 4; Component 2 includes a β-globin upstream primer having a nucleotide sequence as shown in SEQ ID NO.5, and a β-globin downstream primer having a nucleotide sequence as shown in SEQ ID NO.6; The 5' end of the β-globin probe sequence is labeled with a ROX fluorescent group, and the 3' end is labeled with an MGB quenching fluorescent group. The nucleotide sequence of the β-globin probe is shown in SEQ ID NO.

7.

2. A kit for detecting MTHFR gene polymorphism according to claim 1, characterized in that: The invention also includes an amplification enzyme, which is used for amplifying nucleic acids in oral swabs and participating in a fluorescent PCR reaction procedure.

3. A kit for detecting MTHFR gene polymorphism according to claim 2, characterized in that: The final concentration of the C677T upstream primer and the C677T downstream primer for amplification was 0.4 μmol / L; the final concentration of the C677T probe 1 and the C677T probe 2 for detection was 0.1 μmol / L.

4. A kit for detecting MTHFR gene polymorphism according to claim 2, characterized in that: The final concentration of the β-globin upstream primer and the β-globin downstream primer for amplification was 0.2 μmol / L, and the final concentration of the β-globin probe for detection was 0.05 μmol / L.

5. The kit for detecting MTHFR gene polymorphism according to claim 1, characterized in that: The liquid reagents in the test kit are freeze-dried in situ through a freeze-drying process to produce freeze-dried powder.

6. A kit for detecting MTHFR gene polymorphism according to claim 2, characterized in that: Also included are positive control substance A1 tube and blank control substance B1 tube; The positive control substance is a natural cell line heterozygous for the C677T site of the MTHFR gene; the blank control substance is enzyme-free water; The liquid reagent in the positive control substance A1 tube is freeze-dried in situ through a freeze-drying procedure to prepare the corresponding quality control substance positive control powder; The liquid in the blank control substance B1 tube is freeze-dried in situ through a freeze-drying procedure to prepare the corresponding quality control substance blank control powder.

7. A method for using the kit for detecting MTHFR gene polymorphism according to any one of claims 2 to 4, characterized in that: The steps include: S1. Obtain an oral swab sample to be tested and place it in a sample processing solution to allow the oral mucosal cells to release nucleic acids, thereby preparing a swab solution as an amplification template; S2. The prepared components 1, 2, and amplification enzyme are thoroughly mixed, and the liquid reagent is freeze-dried in situ to prepare multiple lyophilized powders of the reagent; S4. Take 25 μL of the amplification template prepared in S1 and directly add it to a portion of the lyophilized powder of the reagent prepared in S2 for dissolution. Transfer the solution to a reaction tube for amplification and judge the result by the Ct value.

8. The method for using the kit for detecting MTHFR gene polymorphism according to claim 7, characterized in that: The amplification program was as follows: pre-denaturation at 95° C. for 2 minutes, one cycle; denaturation at 95° C. for 10 seconds, annealing at 60° C. for 20 seconds, alternating for 40 cycles.

9. A quality control method for a kit for detecting MTHFR gene polymorphism according to claim 6, characterized in that: The following steps are involved: S2. Thoroughly mix the prepared components 1, 2, and DNA polymerase Mix, and freeze-dry the liquid reagents in situ to prepare multiple freeze-dried powders. S3. Dissolve the positive control powder of the quality control product with 25 μL of enzyme-free water, then dissolve it with a portion of the lyophilized powder of the reagent configured in S2, transfer it to the corresponding reaction tube, mark it as tube B1, and amplify it; dissolve the blank control powder of the quality control product with 25 μL of enzyme-free water, then dissolve it with a portion of the lyophilized powder of the reagent configured in S2, transfer it to the corresponding reaction tube, mark it as tube B2, and amplify it; judge the quality control results by the Ct value.

10. The quality control method for a kit for detecting MTHFR gene polymorphism according to claim 9, characterized in that: The quality control result judgment is as follows: the B1 tube configured in S3 is marked as the positive control tube. If the three channels of FAM, VIC and ROX show a typical S-shaped curve and Ct (FAM) ≤ 37, Ct (VIC) ≤ 37, and Ct (ROX) ≤ 37, the reagent is considered qualified; the B2 tube is marked as the blank control tube. If there is no Ct value in the four channels, the reagent is considered to be uncontaminated and the reagent results are credible.

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