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Kit for detecting MMACHC gene mutation

A kit and gene technology, applied in the field of kits for detecting MMACHC gene mutation, can solve the problems of limited promotion and application, high detection cost and high false negative rate, and achieve the effects of simple operation, improved specificity and high accuracy.

Inactive Publication Date: 2019-02-19
QILU CHILDRENS HOSPITAL OF SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there are mainly sequencing methods and ARMS-PCR methods for the detection of gene mutations, but both methods have certain defects. Among them, the sequencing method has a lower sensitivity of about 20%, and the operation is complicated, the detection time is longer, and false negatives higher rate
The ARMS-PCR method can achieve a sensitivity of 1%, but the detection cost is too high, which limits its clinical promotion and application

Method used

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  • Kit for detecting MMACHC gene mutation
  • Kit for detecting MMACHC gene mutation
  • Kit for detecting MMACHC gene mutation

Examples

Experimental program
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Effect test

Embodiment 1

[0033] Example 1: A kit for detecting the c.492_493 insA mutation of the MMAHC gene

[0034] The composition of the kit:

[0035] (1) MMAHC gene-specific amplification primers (concentrations of both upstream primers and downstream primers are 10 μM, individually packaged or mixed packages):

[0036] Upstream primer: 5'-CAGGCTAGCTGCATGATTGA-3' (SEQ ID NO.1);

[0037] Downstream primer: 5'-GCTACTCGAAGGCAATTTC-3' (SEQ ID NO.2).

[0038] (2) PCR reaction solution: Each 10μL PCR reaction solution contains: DNA polymerase 0.5U, dNTPs 1.0mM, MgCl 2 3mM.

[0039] (3) BmtI endonuclease.

[0040] (4) Digestion solution: l0×Buffer.

[0041] (5) Positive reference product and negative reference product: The positive reference product is a DNA fragment containing the c.492_493 insA mutation of the MMAHC gene that can be digested; the negative reference product is a DNA fragment that does not contain the c.492_493 insA mutation of the MMAHC gene. Digested DNA fragments.

[0042] In ...

Embodiment 2

[0043]Example 2: Detection of MMAHC gene c.492_493 insA mutation

[0044] The kit of Example 1 is used to detect the c.492_493 insA mutation of the MMAHC gene, and the specific steps are as follows:

[0045] 1. Extraction of sample DNA:

[0046] Genomic DNA of the sample to be tested is extracted using a DNA extraction kit, and the extracted sample DNA is used as a template.

[0047] 2. PCR amplification:

[0048] Utilize the MMAHC gene-specific amplification primers (shown in SEQ ID NO.1-SEQ ID NO.2) in the kit to carry out PCR amplification according to the following system and procedure:

[0049] PCR reaction system (20μL):

[0050]

[0051]

[0052] PCR reaction program:

[0053] Pre-denaturation at 95°C for 10 minutes; followed by 30 cycles of 94°C for 15s, 62°C for 20s, and 70°C for 25s; store at 4°C.

[0054] 1% agarose gel was used to check whether the size of the PCR product was correct, and as a result, the target PCR product was obtained.

[0055] 3. Enz...

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Abstract

The invention relates to a kit for detecting MMACHC gene mutation. The kit comprises MMACHC gene specificity amplification primers and a BmtI incision enzyme. The sequences of the MMACHC gene specificity amplification primers are shown in SEQ ID NO.1 and SEQ ID NO.2. When the MMACHC gene c.492_493 insA mutation is detected through the kit, compared with other detection methods, the kit is low in cost, easy to operate, visual in result interpretation, high in accuracy, free of special requirements for equipment or operators and suitable for screening the MMACHC gene c.492_493 insA mutation in ordinary hospitals and molecular biology laboratories.

Description

technical field [0001] The invention relates to the technical field of gene mutation detection, in particular to a kit for detecting MMAHC gene mutation. 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, It causes abnormal accumulation of metabolites such as methylmalonic acid and homocysteine ​​in the body, so it is called methylmalonic acidemia with homocysteinemia (referred to as combined MMA). Clinical manifestations inc...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/6858
CPCC12Q1/6858C12Q1/6883C12Q2600/156C12Q2531/113C12Q2521/301C12Q2565/125
Inventor 张开慧王兴翠宋丽刘毅张春艳高敏律玉强
Owner QILU CHILDRENS HOSPITAL OF SHANDONG UNIV
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