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Fluorescent quantitative PCR kit for detecting copy number of virulence gene of human spinal muscular atrophy

A technology for spinal muscular atrophy and pathogenic genes, which is applied in the determination/examination of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc. It can solve problems such as deviation of quantitative results, avoid interference, and prevent false negatives and false positives. the effect of the presence of

Inactive Publication Date: 2018-05-18
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

AI Technical Summary

Problems solved by technology

Large-sample studies have shown that in the carrier population, the SMN2 gene copy number can be as many as 4 or more. For carriers with only 1 copy of SMN1, when SMN1 and SMN2 are amplified with common primers, it will be inevitable. The influence of multi-copy SMN2 gene dominant amplification on single-copy SMN1 gene (because the two PCR primers are the same at this time) will also lead to deviations in relative quantitative results

Method used

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  • Fluorescent quantitative PCR kit for detecting copy number of virulence gene of human spinal muscular atrophy
  • Fluorescent quantitative PCR kit for detecting copy number of virulence gene of human spinal muscular atrophy
  • Fluorescent quantitative PCR kit for detecting copy number of virulence gene of human spinal muscular atrophy

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Experimental program
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Effect test

Embodiment 1

[0053] Embodiment one: the composition of kit

[0054] 1. Common primers for specific amplification of exon 7 of SMN1 and SMN2 genes:

[0055] Upstream primer SMN-EX7-F;

[0056] Downstream primer SMN-EX7-R.

[0057] 2. Common primers for specifically amplifying exon 8 of SMN1 and SMN2 genes:

[0058] Upstream primer SMN-EX8-F;

[0059] Downstream primer SMN-EX8-R.

[0060] 3. PCR amplification primers of internal reference gene CFTR:

[0061] Upstream primer CFTR-F,

[0062] Downstream primer CFTR-R.

[0063] 4. A probe for detecting exon 7 of SMN1 gene: SMN1-EX7-P.

[0064] 5. A probe for detecting exon 8 of SMN1 gene: SMN1-EX8-P.

[0065] 6. A probe for detecting exon 7 of SMN2 gene: SMN2-EX7-P.

[0066] 7. A probe for detecting exon 8 of SMN2 gene: SMN2-EX8-P.

[0067] 8. A probe for detecting the internal reference gene CFTR: CFTR-P.

[0068] 9. PCR reaction reagents include:

[0069] PCR reaction buffer;

[0070] dNTPs;

[0071] magnesium chloride;

[0072...

Embodiment 2

[0079] Example 2: Genomic DNA Preparation

[0080]1ml of human peripheral venous blood was collected according to medical routine, and EDTA was added for anticoagulation. Use QIAGEN’s Human Peripheral Blood Genome Extraction Kit to extract genomic DNA, with an elution volume of 100 μL, and quantify with a UV spectrometer. The ratio of OD260nm / OD280nm should be between 1.8 and 2.0, and use deionized water to dilute the genomic DNA. Concentration to 10-20ng / μL.

Embodiment 3

[0081] Embodiment three: Gradient dilution of reference substance

[0082] Preparation of SMN1 gene control substance: Three concentration gradients of 1:2:4 were established for the SMN1 gene 1 copy quality control substance respectively, and the SMN1 gene 0 copy quality control substance was not serially diluted, and deionized water was used instead of DNA as a blank control. 5 groups of controls.

[0083] SMN2 gene control substance preparation: Three concentration gradients of 1:2:4 were established for the SMN2 gene 1 copy quality control substance respectively, and the SMN2 gene 0 copy quality control substance was not diluted in a gradient, and deionized water was used instead of DNA as a blank control. 5 groups of controls.

[0084] The method for establishing three concentration gradients of SMN1 gene 1 copy quality control product 1:2:4 is as follows: take 100 μL of SMN1 gene 1 copy control substance and add it to a 1.5 mL centrifuge tube, then add 100 μL of deioniz...

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Abstract

The invention discloses a fluorescent quantitative PCR kit for detecting the copy number of a virulence gene of human spinal muscular atrophy. The fluorescent quantitative PCR kit comprises amplimersand fluorescent probes, wherein the amplimers consist of a pair of shared primers for specific amplification of the seventh exon of SMN1 and SMN2 genes, a pair of shared primers for specific amplification of the eighth exon of the SMN1 and SMN2 genes, and a pair of specific primers for specific amplification of a reference gene, i.e., a CFTR gene; and the fluorescent probes consist of a fluorescent probe for specific detection of the seventh exon of the SMN1 gene, a fluorescent probe for specific detection of the eighth exon of the SMN1 gene, a fluorescent probe for specific detection of the seventh exon of the SMN2 gene, a fluorescent probe for specific detection of the eighth exon of the SMN2 gene and a fluorescent probe for specific detection of the reference gene CFTR. The fluorescentquantitative PCR kit for detecting the copy number of the virulence gene of human spinal muscular atrophy is directed at problems and insufficiencies in quantitative detection of the copy numbers of human motoneuron genes and is rapid and simple in detection and reliable in detection results.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a fluorescent quantitative PCR kit for detecting the copy number of human spinal muscular atrophy pathogenic gene. Background technique [0002] Spinal muscular atrophy (SMA) is a group of relatively common autosomal recessive genetic diseases, which is a neuromuscular disease caused by the degeneration of motor neurons in the anterior horn cells of the spinal cord. Clinical manifestations are progressive, symmetrical muscle weakness, muscle atrophy, and paralysis. The incidence rate of newborns is 1 / 6000-1 / 10000, and the incidence rate of disease-causing gene carriers in the normal population is 1 / 40-1 / 80. SMA is a fatal disease, a serious neuromuscular disease, and there is currently no effective clinical treatment. According to the age of onset and clinical manifestations of SMA patients, the International Society of Spinal Muscular Atrophy is divided into 4 types: ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6883C12Q2600/158C12Q2563/107C12Q2545/114C12Q2561/101C12Q2537/16
Inventor 魏星孙喆张可欣贾玮
Owner 北京华瑞康源生物科技发展有限公司
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