Relative quantification method and kit for detecting copy number of human DMD gene by multiple real-time fluorescence PCR

A gene copy number, real-time fluorescence technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of mutation hot spots, disappearance, inoperability, etc.

Inactive Publication Date: 2021-03-02
北京华瑞康源生物科技发展有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0014] Although the multiplex fluorescent quantitative PCR technology has the above-mentioned advantages, the 427m transcript of the DMD gene has 79 exons. If the fluorescent quantitative detection is performed on all 79 exons, it

Method used

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  • Relative quantification method and kit for detecting copy number of human DMD gene by multiple real-time fluorescence PCR
  • Relative quantification method and kit for detecting copy number of human DMD gene by multiple real-time fluorescence PCR
  • Relative quantification method and kit for detecting copy number of human DMD gene by multiple real-time fluorescence PCR

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0101] The multiple real-time fluorescent PCR method of the present embodiment detects the test kit (100 reactions / box) of human DMD gene copy number and comprises components as shown in table 2:

[0102] Table 2. Kit components

[0103]

[0104] Exon reaction solution one is the relative quantitative detection primer probe for the 4th, 17th, 45th exon copy number of DMD gene:

[0105] D4F:5'-AAGGCACTGCGGGTTTTG-3',

[0106] D4R: 5'-GTCACAGCATCCAGACCTTGTC-3',

[0107] D4P:5'-FAM-AGAACAATAATGTAAGTAGTACCC-3'-MGB-NFQ,

[0108] D17F: 5'-TGACCTCTGTTTCAATACTTCTCCACA-3',

[0109] D17R: 5'-GTCACCGTAGTTACTGTTTCCATTACA-3',

[0110] D17P:5'-ABY-ACCACCACTCAGCCATCACTAACACAGACA-3'-QSY,

[0111] D45 F: 5'-TCTTCCCCAGTTGCATTCAAT-3',

[0112] D45 R: 5'-CAGGAACTCCAGGATGGCATT-3',

[0113] D45P:5'-VIC-TTCTGACAACAGTTTGCCGCTGCC-3'-QSY,

[0114] CFTR1 F:5'-TTGTGCCTGTTGCAGCTTCT-3',

[0115] CFTR1 R: 5'-TGGAGTTACAGAAAGGCCTCATG-3',

[0116] CFTR1P:5'-Cy5-CGAATGGCACCACCTTCTCGGTGT-3'-QSY,

[...

Embodiment 2

[0155] Adopt the kit described in embodiment 1 to utilize human peripheral blood free DNA or gDNA to carry out quantitative molecular detection to human Duchenne / Becke muscular dystrophy pathogenic gene DMD copy number variation:

[0156] (1) Nucleic acid extraction:

[0157] 1 child with clinical diagnosis of DMD gave birth to 2 female carriers of children with clinical diagnosis of DMD, and also included whole blood samples from 3 normal females and 2 normal males. Use Tianlong Automatic Nucleic Acid Extractor (NP968-3S) and matching Tianlong Whole Blood Genomic DNA Extraction Kit to extract whole blood samples collected in EDTA anticoagulant tubes, and use a micro-ultraviolet spectrophotometer to determine the nucleic acid purity after extraction and concentration, its OD 260 / 280 Between 1.6-2.0; dilute the genomic DNA concentration with sterilized double distilled water to 20ng / μL for later use.

[0158] (2) Dilution of reference substance:

[0159] Dissolve the positiv...

Embodiment 3

[0195] Reagent Specificity Validation: Cross-reactivity to Common Clinical Pathogens.

[0196] (1) Experimental sample

[0197] Take 4 specific samples to verify the specificity of the reagent, hepatitis B virus (10 7 copies / ml), hepatitis C virus (10 5 copies / ml), human cytomegalovirus (10 4 copies / ml), Enterovirus 71 (10 4 copies / ml).

[0198] (2) Experimental process

[0199]Use the first reaction solution to the third reaction solution to test the above four specific samples respectively, analyze the test results, and verify the specificity of the reagents.

[0200] (3) Experimental results

[0201] The 4 specific samples tested by the 3 kinds of reaction solutions were all Undetermined, indicating that the specificity was good and there was no cross-reaction. The specific results are shown in Table 7.

[0202] Table 7. Results of cross-reaction between the three reaction solutions and common clinical pathogens (UD=Undetermined)

[0203]

[0204]

[0205] In ...

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Abstract

The invention discloses a relative quantification method and kit for detecting the copy number of a human DMD gene through multiple real-time fluorescence PCR. The kit comprises an exon reaction solution I, an exon reaction solution II, an exon reaction solution III, a main reaction mixed solution, a positive reference substance, a negative reference substance and a blank reference substance. According to the relative quantification method and kit for detecting the copy number of the human DMD gene through the multiple real-time fluorescence PCR, the 4th, 17th and 45th exons, 8th, 50th and 52nd exons, 47th, 48th and 51st exons of the DMD gene are respectively amplified through three independent PCR reactions, and each of three reaction solutions comprises four fluorescence channels, so that the quantitative detection of the copy number of the 4th, 8th, 17th, 45th, 47th, 48th, 50th, 51st and 52nd exons of the DMD gene is realized.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a relative quantitative method and kit for detecting the copy number of human DMD gene by multiple real-time fluorescent PCR method. Background technique [0002] Duchenne / Beckman muscμLar dystrophy (DMD / BMD) is an X-linked recessive genetic disease characterized by progressive muscle weakness and muscle atrophy caused by DMD gene mutations leading to dystrophin protein deficiency Hereditary Muscle Disease. Its clinical features include: muscle cramps, myalgia, quadriceps myopathy, asymptomatic hypercreatine kinaseemia, X-linked dilated cardiomyopathy, etc. [0003] The clinical manifestations of female DMD gene defect carriers are quite different. The severe cases can be typical DMD manifestations, and the mild cases can be manifested as mild proximal muscle weakness, gastrocnemius pseudohypertrophy, or basically normal muscle function. The incidence of DMD in mal...

Claims

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Application Information

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IPC IPC(8): C12Q1/6851C12Q1/6883
CPCC12Q1/6851C12Q1/6883C12Q2600/156C12Q2600/16C12Q2600/166C12Q2531/113C12Q2537/143C12Q2561/101C12Q2545/113
Inventor 于超计王倩玉赵立明
Owner 北京华瑞康源生物科技发展有限公司
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