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Product for simultaneously detecting polymorphism of MTHFR and MTRR genes, as well as method and application thereof

A gene polymorphism, gene technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the limitations of TaqManprobe method, poor discrimination, false positive test results, and cumbersome operation process and other problems, to achieve the effect of low cost, comprehensive detection and improved detection efficiency

Inactive Publication Date: 2018-12-07
生工生物工程(上海)股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The experimental operation process of Sanger sequencing method is cumbersome, the experimental cycle is long, the cost is high, and it is not suitable for the detection of a large number of samples
The chip hybridization method also has the disadvantages of cumbersome experimental operation process, long experimental cycle, and high cost.
The fluorescent quantitative PCR method usually uses the Taqman probe method. Due to its own limitations, the TaqMan probe method has poor discrimination, and the test results may have false positives or false negatives.

Method used

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  • Product for simultaneously detecting polymorphism of MTHFR and MTRR genes, as well as method and application thereof
  • Product for simultaneously detecting polymorphism of MTHFR and MTRR genes, as well as method and application thereof
  • Product for simultaneously detecting polymorphism of MTHFR and MTRR genes, as well as method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0091] The following is the specific implementation of the wild-type, heterozygous and mutant standard product gene detection of the three sites of rs1801133, rs1801131 and rs1801394 constructed by the present invention.

[0092] 1. Construction of wild-type, heterozygous and mutant standards for three SNP sites

[0093] Construct wild-type, heterozygous, and mutant standards for each locus as described in the Summary of the Invention. That is, first search the sequences of the three SNP sites rs1801133, rs1801131 and rs1801394 from the NCBI (National Center for Biotechnology Information) database, and then use the searched sequences as the target sequence to design primers, and the positions of the forward and reverse primers for each site are at a distance from the corresponding The SNP sites are in the range of 100-300bp.

[0094] Among them, the construction of the rs1801133 CC wild-type plasmid and the rs1801133 TT mutant plasmid first used human genomic DNA as a templat...

Embodiment 2

[0111] The following is the specific implementation of the detection of rs1801133, rs1801131 and rs1801394 in the TY-1 saliva sample by the present invention, and the standard product of each site is used as the positive quality control product.

[0112] 1. Construction of wild-type, heterozygous and mutant standards for three SNP sites

[0113] The specific construction is as described in Example 1.

[0114] 2. Genomic DNA extraction of YT-1 saliva samples

[0115] Genomic DNA extraction from YT-1 saliva samples was performed in strict accordance with the instructions of Sangon Biotech (Shanghai) Co., Ltd. Magnetic Bead Method Saliva and Urine Genomic DNA Extraction Kit (Product No.: B518766-0050). The obtained genomic DNA was measured by a micro-spectrophotometer and its quality met the requirements by agarose gel electrophoresis, and a portion was aspirated and diluted to 25 ng / μl for later use.

[0116] 3. Establishment of YT-1 saliva sample detection reaction system

...

Embodiment 3

[0133] In order to further verify the accuracy of the detection results of the present invention, the rs1801133, rs1801131 and rs1801394 genotypes of 50 cases of blood sample genomic DNA were respectively adopted by the present invention and the Sanger sequencing method, and the consistency of the results of the two detection methods of the present invention and the Sanger sequencing method was compared .

[0134] 1. Construction of wild-type, heterozygous and mutant standards for three SNP sites

[0135] The specific construction process is as described in Example 1.

[0136] 2. Establishment of Genomic DNA Detection Reaction System for 50 Blood Samples

[0137] The establishment of the rs1801133, rs1801131 and rs1801394 reaction systems of 50 cases of blood sample genomic DNA is the same as the genomic DNA reaction system of TY-1 saliva samples described in Example 2, and the reaction system of each site is also set with negative controls and corresponding sites Positive c...

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Abstract

The invention provides a product for simultaneously detecting the polymorphism of MTHFR and MTRR genes, as well as a method and application thereof, and relates to the technical field of molecular diagnosis. The primer composition for simultaneously detecting the polymorphism of MTHFR and MTRR genes comprises a primer group for detecting rs 1801133 site, rs1801131 site and rs1801394 site, can be used for simultaneously and comprehensively detecting the polymorphic site of different types of genes with comprehensive detection with strong specificity and high sensitivity, can be used for accurately distinguish three genotypes of each site respectively, and can be used for detecting the lowest concentration of 1ng / mu l. The detection result can be used in clinical directed administration. Furthermore, locked nucleic acid is introduced into a probe for modifying, the probe with enough short length and excellent parting effect can be screened, each parameter of the probe accords with the designing principle, the accuracy of the detection result can be effectively improved, and an optimized parting effect is achieved.

Description

technical field [0001] The invention relates to the technical field of molecular diagnosis, in particular to a product for simultaneous detection of MTHFR and MTRR gene polymorphisms and its method and application. Background technique [0002] Folic acid is a water-soluble vitamin, namely vitamin B9, which cannot be synthesized in the body and can only be taken from the outside. Folic acid plays an important role in the synthesis of protein and nucleic acid in the body and the metabolism of various amino acids, so folic acid plays an important role in maintaining individual health. Folic acid deficiency can cause fetal neural tube defects, Down syndrome, cleft lip and palate, and congenital heart disease; it can also cause pregnancy-induced hypertension, premature delivery, preeclampsia, habitual abortion, and megaloblastic anemia in pregnant women. Excessive supplementation of folic acid can affect the absorption of zinc, leading to abnormal fetal development and neonatal...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6858C12Q1/6883C12N15/11
CPCC12Q1/6858C12Q1/6883C12Q2600/156C12Q2600/166C12Q2531/113C12Q2563/107C12Q2545/113
Inventor 李威张云静
Owner 生工生物工程(上海)股份有限公司
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