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Visual MTHFR (Methylene Tetrahydrofolate Reductase) allele genotyping detection kit

A technology for detecting kits and alleles, which is applied in the fields of molecular biotechnology and genetic detection, can solve the problems of high price, and achieve the effect of low cost, low cost and high sensitivity closed-tube nucleic acid detection

Active Publication Date: 2017-03-08
广州市宝创生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the existing MTHFR allele typing detection kits on the market are basically based on the PCR amplification technology of fluorescent signal detection, which requires the use of real-time fluorescent PCR equipment, which is relatively expensive and is not conducive to medical units with poor facilities. use
At the same time, in clinical practice, so far there is no closed-tube method that can directly use ordinary PCR cycle instruments to achieve visual typing of MTHFR alleles

Method used

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  • Visual MTHFR (Methylene Tetrahydrofolate Reductase) allele genotyping detection kit
  • Visual MTHFR (Methylene Tetrahydrofolate Reductase) allele genotyping detection kit
  • Visual MTHFR (Methylene Tetrahydrofolate Reductase) allele genotyping detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Primer Probe

[0026] The corresponding primers and probes are specifically designed according to the specificity of the SNP sites of the MTHFR alleles, and the detection results are determined by a pair of specific primers and probes for the sites. The position and method of primer design are as follows: figure 1 shown.

[0027] The MTHFR allele typing detection kit includes 4 primers, 7 conventional probes, 2 nano-gold modified probes, a reaction solution, and an enzyme solution.

[0028] Primer, probe and nano gold probe sequence such as primer 1-primer 4, probe 1-probe 7, nano gold probe 1-nano gold probe 2, or the complementary sequence of the above sequence:

[0029] Primer 1: GCT GAC CTG AAG CAC TTG A; SEQ ID NO.1

[0030] Primer 2: GCG GAA GAA TGT GTC AGC C; SEQ ID NO.2

[0031] Primer 3: GGA GGA GCT GCT GAA GAT G; SEQ ID NO.3

[0032] Primer 4: CGG TTT GGT TCT CCC GAG A; SEQ ID NO.4

[0033] Probe 1: CGC GCC GAG GGC TCC CGC A; SEQ ID NO.5

[003...

Embodiment 2

[0046] Example 2 MTHFR allele SNP site typing detection sensitivity

[0047] In this embodiment, the MTHFR allele typing detection kit described in Example 1 was used to detect the templates of different concentrations of MTHFR allele SNP site typing, which was used to verify the visual detection method stated in the present invention. feasibility.

[0048] Reaction conditions:

[0049] The detection of each sample in the kit consists of 4 tubes of reaction, respectively containing primers and probes for the four genotypes, and the total volume of each tube reaction is 20 μL. MTHFR allele SNP site detection reaction system consists of: 10mM Tris buffer (pH 8.5), 1μM primer (primer 1 sequence: SEQ ID NO.1; primer 2 sequence: SEQ ID NO.2; primer 3 sequence: SEQ ID NO.2; ID NO.3; Primer 4 sequence: SEQ ID NO.4), 1 μ M probe (probe 1 sequence SEQ ID NO.5; Probe 2 sequence: SEQ ID NO.6; Probe 3 sequence SEQ ID NO.7; Needle 4 sequence: SEQ ID NO.8; Probe 5 sequence: SEQ ID NO.9; ...

Embodiment 3

[0051] Example 3 MTHFR allele SNP site typing detection matching genotype sample detection

[0052] In this example, the MTHFR allele typing detection kit described in Example 1 was used to detect the genomic DNA templates of different types of peripheral blood samples, which were used to verify the effectiveness of the visual detection method stated in the present invention in detecting actual samples. feasibility.

[0053] Reaction conditions:

[0054] The detection of each sample in the kit consists of 4 tubes of reaction, respectively containing primers and probes for the four genotypes, and the total volume of each tube reaction is 20 μL. MTHFR allele SNP site detection reaction system consists of: 10mM Tris buffer (pH 8.5), 1μM primer (primer 1 sequence: SEQ ID NO.1; primer 2 sequence: SEQ ID NO.2; primer 3 sequence: SEQ ID NO.2; ID NO.3; Primer 4 sequence: SEQ ID NO.4), 1 μ M probe (probe 1 sequence SEQ ID NO.5; Probe 2 sequence: SEQ ID NO.6; Probe 3 sequence SEQ ID N...

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Abstract

The invention relates to a visual MTHFR (Methylene Tetrahydrofolate Reductase) allele genotyping detection kit. The visual MTHFR allele genotyping detection kit comprises four primers, seven conventional probes and two probes connected with gold nanoparticles, wherein the primers are as shown from SEQ ID NO. 1 to SEQ ID NO. 4; the conventional probes are respectively as shown from SEQ ID NO.5 to SEQ ID NO.11, or are respectively as shown in reverse complementary sequences of sequences from SEQ ID NO.5 to SEQ ID NO. 11; base compositions of the probes connected with the gold nanoparticles are as shown in SEQ ID NO. 12 and SEQ ID NO. 13, or are as shown in reverse complementary sequences of SEQ ID NO. 12 and SEQ ID NO. 13. The visual MTHFR allele genotyping detection kit disclosed by the invention is capable of realizing visual, low-cost and high-sensitivity genotyping detection of MTHFR allele rs1801133 and rs1801131.

Description

technical field [0001] The invention belongs to the fields of molecular biotechnology and gene detection, in particular to a typing detection kit for MTHFR alleles (rs1801133 and rs1801131). Background technique [0002] Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in the methionine-folate metabolic system, maintaining the normal metabolism of folic acid. MTHFR allele loci rs1801133 and rs1801131 (c.677 and c.1298) mutations can lead to decreased activity of key folic acid metabolism enzymes encoded by them, causing folic acid metabolism disorders, resulting in decreased folic acid levels and hyperhomocysteinemia disease. For people with abnormal MTHFR gene, MTHFR enzyme activity is significantly reduced, resulting in folic acid metabolism disorders, leading to a significantly increased risk of neonatal neural tube defects, Down syndrome, cleft lip and palate and other diseases, such people need to supplement more folic acid to achieve the desired effect. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6858C12Q1/6883C12Q2600/106C12Q2600/156C12Q2521/301C12Q2563/137C12Q2563/155
Inventor 王建平潘腾飞
Owner 广州市宝创生物技术有限公司
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