Kit for detecting high-frequency mutation of MMACHC gene by using melting curve

A kit and gene technology, applied in recombinant DNA technology, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problems of high quality requirements of the genome to be tested, cumbersome process operation, high detection sensitivity, and meet the needs of genetic testing. The effect of demand, simple operation, and short detection time period

Pending Publication Date: 2022-08-09
SHANDONG FIRST MEDICAL UNIV & SHANDONG ACADEMY OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The high-throughput sequencing method has a large sequencing throughput and high coverage, but is expensive and cumbersome to operate, which limits the sequencing speed
As the gold standard for sequencing inspection, sanger sequencing method has high accuracy, but it has high requirements on the quality of the genome to be tested and the process is cumbersome and time-consuming
High-resolution melting curve analysis (HRM) method combines saturated dyes with PCR amplification products to monitor changes in product melting curves to analyze gene mutations. It has high detection sensitivity and can detect mutant genes with a content as low as 1% in the sample, and is easy to operate Fast, low cost, and accurate results, but this method is mostly used to detect single gene mutations, and there are very few products for multiple gene mutation detection

Method used

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  • Kit for detecting high-frequency mutation of MMACHC gene by using melting curve
  • Kit for detecting high-frequency mutation of MMACHC gene by using melting curve
  • Kit for detecting high-frequency mutation of MMACHC gene by using melting curve

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 Design of primers

[0055] The preparation of primer-probe composition for detecting high-frequency mutation of MMACHC gene includes the following steps:

[0056] Sequence acquisition: The source of the gene sequence used is NCBI (National Center for Biotechnology Information)

[0057] Table 1 Hot spot mutation site information of MMACHC gene

[0058] Gene base change amino acid change rs number MMACHC c.80A>G p.Gln27Arg rs546099787 MMACHC c.217C>T p.Arg73Ter rs796051995 MMACHC c.482G>A p.Arg161Gln rs121918243 MMACHC c.567dupT p.Ile190fs rs1463495909 MMACHC c.609G>A p.Trp203Ter rs587776889 MMACHC c.658_660del p.Lys220del rs398124296

[0059] Design method: Primer probes were designed by software such as PrimerExpress 3.0 according to the hot spot mutation sequence of MMACHC gene published in NCBI database. Select 100-150bp to amplify the target fragment, and design specific PCR p...

Embodiment 2

[0063] Example 2: Kit performance verification

[0064] A kit for detecting MMACHC gene high frequency mutation using melting curve was used to detect hot spot mutations c.80A>G, c.217C>T, c.482G>A, c.567dupT of MMACHC gene in neonatal dried blood spot samples The mutation status of c.609G>A and c.658_660delAAG, the specific primer pair set for detecting the high frequency mutation of MMACHC gene in the kit was prepared in Example 1.

[0065] 2.1 Sample DNA acquisition

[0066] Take 1 neonatal dried blood spot sample with known normal type (named as sample 1); 6 neonatal dried blood spot samples from patients with known mutation type methylmalonic acidemia (from Jinan Maternal and Child Health Hospital) , named as sample 2, sample 3, sample 4, sample 5, sample 6, and sample 7, of which sample 2 is known to have the c.80A>G mutation, sample 3 has the c.217C>T mutation, and sample 4 has the c. 482G>A mutation, sample 5 is c.567dupT mutation, sample 6 is c.609G>A mutation, samp...

Embodiment 3

[0090] Example 3: Kit stability test

[0091] Randomly divided into 3 groups according to the number of years of laboratory study, one group: within 1 year of laboratory study; the second group: laboratory study for 1-3 years; the third group: laboratory study for 3 years above. One person from each group was randomly selected for the stability test of the kit. Three people (lab members, named member A, member B, member C) performed experiments on the same batch of samples using a kit for the detection of high-frequency mutations in the MMACHC gene using melting curves. Detection of hot spot mutations c.80A>G, c.217C>T, c.482G>A, c.567dupT, c.609G>A and c.658_660delAAG of MMACHC gene in fresh blood samples The set of specific primer pairs for detecting high-frequency mutation of MMACHC gene was prepared in Example 1.

[0092] Blood samples were collected from 6 subjects of physical examination items in Jinan Maternal and Child Health Hospital, and venous blood was collected...

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Abstract

The invention discloses a kit for detecting high-frequency mutation of an MMACHC gene by using a melting curve. The kit can detect the high-frequency mutation site c.80Agt of the MMACHC gene; g, c, 217Cgt; the Ggt is c.567dupT, c.482Ggt, c.567dupT, the A, the c, the 658660delAAG and the c, the 609Ggt are selected from a group consisting of a group A, a group According to the present invention, the mutation conditions of the 6 sites A and A are detected, such that the detection cost is low, the operation is simple, the accuracy is high, the good specificity is provided, and the clinical screening application can be met.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a kit for detecting high-frequency mutation of MMACHC gene using melting curve. Background technique [0002] Methylmalonic acidemia (MMA), also known as methylmalonic aciduria, is a common organic acid metabolism disease. The abnormal accumulation of toxic substances such as methylmalonic acid and methylcitric acid causes multiple organ damage, especially the nervous system damage, which is often life-threatening in severe cases. The disease is an autosomal recessive inheritance, with an incidence of about 1:48,000 to 1:250,000 worldwide, 1 / 28,004 in China, and 1 / 3,920 in Shandong Province. The clinical manifestations of the disease vary greatly among individuals, and it can manifest as multi-system involvement. It can occur from fetal period to adulthood, and it is easy to be missed and misdiagnosed. Genetic testing is a reliable basis for clinical classification, and early molecu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/6858C12N15/11
CPCC12Q1/6883C12Q1/6858C12Q2600/156C12Q2531/113C12Q2527/107
Inventor 赵燕杨海宁崔亚洲韩金祥邹卉
Owner SHANDONG FIRST MEDICAL UNIV & SHANDONG ACADEMY OF MEDICAL SCI
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