A primer set, probe set and kit for detecting high-frequency gene pathogenic variants

A technology of primer set and probe set, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/testing, etc., to achieve the effects of simple result interpretation, reduced birth rate, and low false positive rate

Active Publication Date: 2022-05-13
北京华诺奥美医学检验实验室有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no fluorescent quantitative PCR primers, probes and kits specifically targeting this variant site

Method used

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  • A primer set, probe set and kit for detecting high-frequency gene pathogenic variants
  • A primer set, probe set and kit for detecting high-frequency gene pathogenic variants
  • A primer set, probe set and kit for detecting high-frequency gene pathogenic variants

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Experimental program
Comparison scheme
Effect test

Embodiment

[0041] This kit is a specific primer / probe set for detecting pathogenic variants of high-frequency gene loci (GALC:NM_000153:exon16:c.1901T>C:p.L634S), and is suitable for efficient DNA polymerization of high-GC sequences Enzyme, specific ratio of dNTPs, buffer system suitable for high GC sequences, ROX reference fluorescent dye, blank control and positive control.

[0042] Detection site and its primer set nucleotide sequence list 1:

[0043]

[0044]

[0045] The above primers and probes are designed independently, and the detection sites can be effectively distinguished by adjusting the specific design parameters. Each of the above primer sequences is a nucleotide sequence with detection significance for the high-frequency GALC gene locus.

[0046] The temperature setting of the primer sequence determines the extension temperature during the PCR reaction. Too high or too low will have adverse effects on the PCR reaction.

[0047] The primer pair of the first forwar...

experiment example

[0060] 1. When using wild-type DNA as a template:

[0061]

[0062] The samples in the above table (111001139725, 111001139717, 111001139700, 111001152842, 111001139720, 111001155336) are wild-type human genomic DNA, the threshold line of FAM and VIC signal is set to 0.1, and the threshold line below 0.1 is background signal, which has no reference significance.

[0063] From the above table and as figure 1 The schematic diagram of the log logarithmic curve experimental results shown shows that the average Ct value of the VIC signal is 23.42 with a standard deviation of 0.08; the average Ct value of the FAM signal is 41.60 with a standard deviation of 0.86; and the Ct values ​​of the FAM signals of the three samples is undetermined.

[0064] The experimental results of Linear linear amplification curve are as follows: figure 2 shown. figure 2 The results show that the amplification curve of the FAM channel is hardly observed in the linear curve. And the difference of ...

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Abstract

The invention belongs to the technical field of biological detection, and discloses a primer set, a probe set and a kit for detecting high-frequency gene pathogenic variation. The kit can conveniently and quickly assist doctors to provide scientific data when diagnosing patients or provide data support for carrier screening, which helps doctors diagnose patients as soon as possible, or predict fertility risks in advance during the pregnancy preparation stage. It is a method for detecting genetic material DNA / RNA, which has high accuracy, low false positive rate, low cost, simple interpretation of results, and effectively reduces the birth rate of children with Krabbe disease. The reaction system maximizes the difference in amplification efficiency between alleles, and is suitable for Ct value interpretation and endpoint method interpretation; it takes less time and has high accuracy. It can be used to quickly and accurately detect the c.1901T>C pathogenic variant on the GALC gene NM_000153 transcript in human whole blood samples, and is used for the auxiliary diagnosis of Krabbe disease.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a primer set, a probe set and a kit for detecting high-frequency gene pathogenic variation. Background technique [0002] Krabbe disease (Krabbe disease, also known as spherical cell leukodystrophy) is a serious neurological disease. It is also classified as a type of leukodystrophy disease. This disease is usually due to demyelination of the nervous system. Myelin is the protective covering around nerve cells that ensures the efficient transmission of nerve signals. Krabbe disease is also characterized by the presence of globular cells in the brain, which are large and often have more than one nucleus. [0003] The most common form of Krabbe disease is the infantile form, which usually begins before the age of 1. Initial signs and symptoms often include irritability, muscle weakness, difficulty eating, fever without any signs of infection, rigid post...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6883C12Q1/6851C12N15/11
CPCC12Q1/6883C12Q1/6851C12Q2600/156C12Q2531/113C12Q2561/101C12Q2545/113
Inventor 张可欣王慧魏星
Owner 北京华诺奥美医学检验实验室有限公司
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