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A primer, probe and kit for detecting human mthfr and mtrr gene mutations

A technology for detecting people and kits, which is applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc. , can not fully cover the problem of gene mutation sites, etc., to achieve the effect of intuitive typing, improving analysis performance, and accurate typing

Active Publication Date: 2021-09-07
深圳友一生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The existing detection kits for human MTHFR and MTRR gene mutations basically use the fluorescent PCR method based on the TaqMan hydrolysis probe, although compared with PCR-RELF, direct sequencing and other methods, it has the advantages of rapid detection, easy operation and low cross-contamination , but limited by the detection channel of the fluorescent PCR instrument, it can only detect a single site mutation (MTHFR C677T), cannot fully cover the relevant gene mutation sites, and cannot comprehensively evaluate the individual's folic acid utilization capacity
In addition, due to the difference of a single base, non-specific amplification curves (with non-specific Ct) are prone to appear regardless of conventional PCR or ARMS PCR. The added PCR template concentration will increase the workload of detection operation and result interpretation

Method used

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  • A primer, probe and kit for detecting human mthfr and mtrr gene mutations
  • A primer, probe and kit for detecting human mthfr and mtrr gene mutations
  • A primer, probe and kit for detecting human mthfr and mtrr gene mutations

Examples

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Effect test

Embodiment 1

[0067] Example 1 Production and assembly of a kit for detecting human MTHFR and MTRR gene mutations:

[0068] Primers and probes are:

[0069] MTHFR C677T Primer 1 (SEQ ID NO: 1);

[0070] MTHFR C677T Primer 2 (SEQ ID NO:2);

[0071] MTHFR C677T probe 1 (the nucleotide sequence is SEQ ID NO: 7), ROX-TGTCTGCGGGAGCCGATTTCATCATC-BHQ2;

[0072] MTHFR A1298C Primer 3 (SEQ ID NO:3);

[0073] MTHFR A1298C Primer 4 (SEQ ID NO:4);

[0074] MTHFR A1298C probe 2 (the nucleotide sequence is SEQ ID NO: 8), FAM-CCAGTGAAGAAAGTGTCTTT-MGB;

[0075] MTRR A66G Primer 5 (SEQ ID NO:5);

[0076] MTRR A66G Primer 6 (SEQ ID NO:6);

[0077] MTRR A66G probe 3 (nucleotide sequence is SEQ ID NO: 9), VIC-CAGAAGAAATGTGTGAGCAAG-MGB.

[0078] Kit includes:

[0079] Reaction solution (see Table 2 for detailed composition), enzyme (see Table 3 for detailed composition), positive quality control product (see Table 4 for detailed composition) and blank quality control product (see Table 4 for detailed co...

Embodiment 2

[0090] Embodiment 2 The kit provided by the present invention is used for the detection of clinical samples

[0091] 30 cases of clinical samples were tested using the 48-person kit in Example 1 of the present invention. The composition of the kit is shown in Table 5.

[0092] table 5

[0093] kit components Component Specifications Number of components The reaction solution 1109μL / tube 1 tube enzyme 53μL / tube 1 tube positive control 15μL / tube 1 tube blank quality control 1.5mL / tube 1 tube

[0094] 1. Nucleic acid extraction

[0095] Take 200 μL whole blood samples and 200 μL blank quality control products, use the magnetic bead method blood genome extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., DP329) in the automatic nucleic acid extraction instrument (Tiangen Biochemical Technology (Beijing) Co., Ltd., TGuideS32) for nucleic acid extraction, the extraction is carried out according to the instruction...

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Abstract

The invention discloses a primer, a probe and a kit for detecting human MTHFR and MTRR gene mutations, belonging to the technical field of gene detection. The invention provides primers and probes SEQ ID NO: 1 to SEQ ID NO: 9 for detecting human MTHFR C677T and A1298C gene mutations and MTRR A66G gene mutations; detection of human by asymmetric PCR amplification and TaqMan probe melting curve analysis technology MTHFR C677T and A1298C gene mutations and MTRR A66G gene mutations are used to judge the individual's folic acid utilization ability. The test results combined with other clinical test results and clinical symptoms can assist in the diagnosis of clinical individualized folic acid supplementation.

Description

technical field [0001] The invention belongs to the technical field of gene detection, in particular to a primer, a probe and a kit for detecting human MTHFR and MTRR gene mutations. Background technique [0002] Folic acid, also known as vitamin B9, is a water-soluble vitamin composed of pteridine, p-aminobenzoic acid and L-glutamic acid. Folic acid is an important methyl donor in the human body, involved in DNA synthesis, replication and repair, and methylation regulation of genome and protein. Folic acid deficiency in the body is closely related to neonatal birth defects such as neural tube closure defects, and also increases the risk of cardiovascular diseases, neurodegenerative diseases, diabetes, kidney diseases, osteoporosis and other diseases. Folic acid deficiency in the body is the result of the interaction between environment and genes. The human body cannot synthesize folic acid, and external intake is the only way for the human body to obtain folic acid. There...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/6883C12Q2600/106C12Q2600/156
Inventor 任维蔡泽加刘伟邓琳赵碧霞
Owner 深圳友一生物科技有限公司
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