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MMACHC (methylmalonic academia-combined homocysteine) and MTHFR (methylenetetrahydrofolate reductase) gene mutation detection kit

A gene and mutation site technology, applied in the MTHFR gene mutation detection kit, abnormal folic acid metabolism detection, MMAHCC field, can solve the problems of low throughput, high cost, unsatisfactory detection rate, etc., and achieve the effect of simplifying the interpretation process

Inactive Publication Date: 2018-09-07
北京金准基因科技有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method has low throughput, poor efficiency, high cost, and unsatisfactory detection rate

Method used

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  • MMACHC (methylmalonic academia-combined homocysteine) and MTHFR (methylenetetrahydrofolate reductase) gene mutation detection kit
  • MMACHC (methylmalonic academia-combined homocysteine) and MTHFR (methylenetetrahydrofolate reductase) gene mutation detection kit
  • MMACHC (methylmalonic academia-combined homocysteine) and MTHFR (methylenetetrahydrofolate reductase) gene mutation detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Preparation of sample DNA

[0038] A conventional peripheral blood nucleic acid extraction kit (magnetic bead method) was used to extract genomic DNA from peripheral blood samples of 23 patients with clinical symptoms consistent with CblC type of methylmalonic acidemia associated with homocysteinemia. For DNA quality control, it is required that the DNA concentration is not lower than 20ng / μL, the total amount of DNA is not lower than 1μg, and the DNA purity A260 / A280 is between 1.75-1.90. Store the extracted samples in a refrigerator at 4°C for no more than 7 days, and store them in a refrigerator below -18°C for long-term storage. If the extraction effect does not meet the above conditions, re-extraction is required.

Embodiment 2

[0039] Example 2 Determination of Mutation Sites in Samples

[0040] The DNA library construction and hybridization capture of the genomic DNA of the above 23 patients were carried out using the detection kit for the preparation of MMAHC and MTHFR gene mutations described in the present invention to obtain the current fragments for subsequent next-generation sequencing and data analysis.

[0041] The specific operation process is as follows:

[0042] 1 Library preparation

[0043] 1.1 Genomic DNA fragmentation and end repair

[0044] Take a certain amount of the genomic DNA extracted above, and add 10 μL of the interruption repair reaction mixture to the genomic DNA. Place on a PCR machine and incubate at 37°C for 20min, at 65°C for 15min, and cool to 4°C to maintain.

[0045] 1.2 Connector connection

[0046] Add 50 μL of the ligation reaction mixture to the end-repaired DNA in the previous step, place it on a PCR machine and incubate at 23°C for 1 hour, then cool to 4°C ...

Embodiment 3

[0092] Test sample verification:

[0093]The primers described in the patent of the present invention were used to verify the next-generation sequencing results of the above 23 patients with clinical symptoms consistent with methylmalonic acidemia associated with homocysteinemia CblC type.

[0094] The extraction method of sample DNA is the same as in Example 1.

[0095] Use the primer sequences shown in SEQ ID NO: 6-13 to amplify the extracted DNA, and use Sanger sequencing for the amplification results, and judge the amplification results according to the result judging method in the table of Example 2.

[0096] The overall detection situation:

[0097] 23 patients with suspected methylmalonic acidemia and homocysteinemia CblC type, 2 cases of c.80A>G heterozygous mutation on MMAHC gene; 4 cases of c.482G>A heterozygous mutation; c. 2 cases of 567dupT heterozygous mutation, 4 cases of c.609G>A homozygous mutation, 11 cases of c.609G>A heterozygous mutation; 6 cases of 656_...

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Abstract

The invention provides a kit for detecting a mutation site associated with methylmalonic academia-combined homocysteine CbIC (cobalamin C) type disease and a mutation site associated with medication guidance, and further provides a probe set for detecting the mutation sites and sequence information of a primer set for amplifying the probe set, mutation situations of related gene loci of a patientcan be identified by use of the probe set or the primer, so that acurate screening and diagnosis of a methylmalonic academia-combined homocysteine CbIC (cobalamin C) type patient can be realized, andtargeted guidance for folic acid medication for treatment can be provided.

Description

technical field [0001] The invention belongs to the field of gene detection, and specifically relates to a detection kit for MMACHC and MTHFR gene mutations, which is mainly used in the detection of methylmalonic acidemia associated with homocysteinemia CblC type, and simultaneously applied in the detection of abnormal folic acid metabolism. Background technique [0002] Methylmalonic acidemia (methylmalonic academia, MMA) is the most common disease among congenital organic acid metabolism disorders, mainly due to the defect of methylmalonyl-CoA mutase (mutasepoenzyme, mut) or its coenzyme cobalt Amine (Cobalamin, Cbl, VitB12) metabolic defects. Mutase deficiency includes complete defect mut0 type, partial defect mut-type. Cobalamin metabolism defects include 8 subtypes of cblA, cblB, cblC, cblD, cblE, cblF, cblG and cblH, among which cblC, cblD and cblF can lead to coenzyme deoxyadenosylcobalamin and methylcobalamin synthesis disorders , causing abnormal accumulation of m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/6883C12Q2600/106C12Q2600/156
Inventor 不公告发明人
Owner 北京金准基因科技有限责任公司
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