Amplifying system and reagent kit for detecting DMD (Duchenne muscular dystrophy) gene exon copy number variation

Abstract

The invention relates to an amplifying system for detecting human DMD (Duchenne muscular dystrophy) gene exon copy number variation. The amplifying system comprises an amplifying composition corresponding to the detection of 10 groups of exon copy number variation, wherein the amplifying composition comprises oligonucleotide sequences as shown from SEQID No.1 to SEQID No.36; the 10 exon purpose fragments including human DMD gene exons 4,8,17,44,45,47,48,50,51,52 are amplified through a real-time multiple fluorescent quantitation PCR reaction; and according to the detection result, whether thehuman DMD gene exon is subjected to deletion mutation or repeat mutation is judged. The invention also discloses a reagent kit for detecting human DMD (Duchenne muscular dystrophy) gene exon copy number variation. The amplifying system disclosed by the invention has the beneficial effects that through the detection result, the distinguishing of DMD carriers from DMD patients and normal people canbe realized, the result is reliable, the repeatability is good, the operation is easy, the equipment requirement is low, and the reagent cost is low; and the amplifying system can cover about 90% of copy number variation in the DMD carriers or the DMD patients, so that good balance between the detection cover degree and the detection exon number is achieved.

Description

technical field

[0001] The invention relates to the field of gene molecular detection, in particular to an amplification system and a kit for detecting the variation of the exon copy number of the DMD gene. Background technique

[0002] Duchenne / Beckman muscular dystrophy (DMD / BMD) is a group of genetic diseases caused by DMD gene mutation leading to dystrophin protein deficiency. Clinical phenotypes include: asymptomatic hypercreatine kinaseemia, muscle spasm / myalgia, quadriceps myopathy, X-linked dilated cardiomyopathy (X-linked dilated cardiomyopathy, XLDCM), etc. The incidence of DMD in male infants is about 1 / 3500-1 / 4000, and the onset is more than 5-6 years old. It manifests as slow walking, easy to fall, and abnormal walking posture. Died of cardiopulmonary failure. The incidence of BMD in males is about 1 / 8000-1 / 10000. The clinical symptoms appear later than DMD, and the ability to walk can be maintained until the age of 16. The disease progresses relatively slowly...

Examples

Embodiment 1

[0091] 1. Nucleic acid extraction:

[0092] 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 OD260 / OD280 is between 1.6-2.0; dilute the genomic DNA concentration with sterilized double distilled water to 20ng / μL for later use.

[0093] 2. Dilution of reference substance:

[0094] Take 10 μL of the first control and the second control respectively and add them to 2 eppendorffs, then add 10 μL of sterilized double distilled water, and mix well to obtain 20 μL each of the two 2-fold diluted control substances; 10 μL was taken out...

Embodiment 2

[0115] Reagent Specificity Validation: Cross-reactivity to Common Clinical Pathogens.

[0116] 1. Experimental sample:

[0117] 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).

[0118] 2. Experimental process:

[0119] Use the first group of amplification composition reaction solutions to the tenth group of amplification composition reaction solutions to detect the above four specific samples respectively, analyze the detection results, and verify the specificity of the reagents.

[0120] 3. Experimental results:

[0121] The 4 specific samples tested by the ten kinds of reaction solutions were all Undetermined, indicating good specificity and no cross-reaction. The specific results are shown in Table 7 below.

[0122] Table 7: Results of cross-reaction between ten kinds of reaction solutions and common clinic...

technical field

[0001] The invention relates to the field of gene molecular detection, in particular to an amplification system and a kit for detecting the variation of the exon copy number of the DMD gene. Background technique

[0002] Duchenne / Beckman muscular dystrophy (DMD / BMD) is a group of genetic diseases caused by DMD gene mutation leading to dystrophin protein deficiency. Clinical phenotypes include: asymptomatic hypercreatine kinaseemia, muscle spasm / myalgia, quadriceps myopathy, X-linked dilated cardiomyopathy (X-linked dilated cardiomyopathy, XLDCM), etc. The incidence of DMD in male infants is about 1 / 3500-1 / 4000, and the onset is more than 5-6 years old. It manifests as slow walking, easy to fall, and abnormal walking posture. Died of cardiopulmonary failure. The incidence of BMD in males is about 1 / 8000-1 / 10000. The clinical symptoms appear later than DMD, and the ability to walk can be maintained until the age of 16. The disease progresses relatively slowly...

Examples

Embodiment 1

[0091] 1. Nucleic acid extraction:

[0092] 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 OD260 / OD280 is between 1.6-2.0; dilute the genomic DNA concentration with sterilized double distilled water to 20ng / μL for later use.

[0093] 2. Dilution of reference substance:

[0094] Take 10 μL of the first control and the second control respectively and add them to 2 eppendorffs, then add 10 μL of sterilized double distilled water, and mix well to obtain 20 μL each of the two 2-fold diluted control substances; 10 μL was taken out...

Embodiment 2

[0115] Reagent Specificity Validation: Cross-reactivity to Common Clinical Pathogens.

[0116] 1. Experimental sample:

[0117] 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).

[0118] 2. Experimental process:

[0119] Use the first group of amplification composition reaction solutions to the tenth group of amplification composition reaction solutions to detect the above four specific samples respectively, analyze the detection results, and verify the specificity of the reagents.

[0120] 3. Experimental results:

[0121] The 4 specific samples tested by the ten kinds of reaction solutions were all Undetermined, indicating good specificity and no cross-reaction. The specific results are shown in Table 7 below.

[0122] Table 7: Results of cross-reaction between ten kinds of reaction solutions and common clinic...

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