Detection kit and method for Duchenne/Becker muscular dystrophy based on multiplex PCR (Polymerase Chain Reaction) trapping technique

A detection method and multiple technologies, applied in the direction of recombinant DNA technology, microbial measurement/inspection, biochemical equipment and methods, etc., can solve the problems of long detection cycle, low detection accuracy and low detection throughput, and achieve accuracy Good, low DNA sample quality requirements, high detection sensitivity

Active Publication Date: 2018-06-29
CAPITALBIO GENOMICS
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the complexity of the DMD gene and the diversity of mutation types, traditional DMD gene mutation detection methods such as Southern hybridization (Southern blotting), denaturing high performance liquid chromatography (DHPLC), multiple junction-dependent Probe amplification (Multiplex ligation-dependent probe amplification, MLPA), microarray-comparative genomic hybridizati

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  • Detection kit and method for Duchenne/Becker muscular dystrophy based on multiplex PCR (Polymerase Chain Reaction) trapping technique
  • Detection kit and method for Duchenne/Becker muscular dystrophy based on multiplex PCR (Polymerase Chain Reaction) trapping technique
  • Detection kit and method for Duchenne/Becker muscular dystrophy based on multiplex PCR (Polymerase Chain Reaction) trapping technique

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1, DMD gene multiplex PCR capture primer set

[0048] The inventor designed multiple PCR capture primers for the target DNA sequences of the exons and introns of the DMD gene, specifically including: according to the reference sequence of the DMD gene in the UCSC database, 23 high-frequency known introns were retrieved from relevant literature and databases at the same time. Containing pathogenic mutations, the inventor selected 79 exons and 50bp intron sequences on both sides of the DMD gene and 23 sequences near the known intron pathogenic mutations, designed multiple PCR capture primers, and screened through a large number of experiments , optimization, verification, and finally selected 145 pairs of 290 primers (primer sequences are shown in SEQ ID NO: 1 ~ SEQ ID NO: 290, see Table 1 for details), which can maintain the amplification efficiency within a certain number of amplification cycles High consistency, in addition, the inventor also added 3 pairs o...

Embodiment 2

[0057] Embodiment 2, DMD gene mutation detection method

[0058] This embodiment provides a DMD gene mutation detection method based on the proton sequencing platform and multiple PCR capture technology. This embodiment takes 20 cases of DMD patients / carriers provided by the Neurology Laboratory of the First Affiliated Hospital of Sun Yat-sen University as an example for DMD For gene mutation detection, the above samples have been molecularly diagnosed by conventional methods and the pathogenic mutation information has been clarified.

[0059] 1. Synthesis of DMD gene PCR capture primers

[0060] According to the library construction requirements of the proton sequencing platform, the universal sequence was connected to the 5' end of the primers shown in SEQ ID NO: 1 to SEQ ID NO: 296 provided in Example 1. The primer pooling coefficients are shown in Table 1. The primers were provided by Thermo Fisher Corporation of the United States. synthesis.

[0061] Wherein, the nucleo...

Embodiment 3

[0087] Embodiment 3, DMD gene mutation information detection of low-quality, low-concentration DNA samples

[0088] Based on DMD gene multiplex PCR capture primer set of the present invention, because its specificity is good, sensitivity is high and highly consistent amplification efficiency, find through a large amount of experimental studies, for low quality (OD260 / 230<1.0) and low concentration (Qbit Template DNA with a concentration of <10ng / uL) can also be accurately detected. Taking 5 cases of low-quality and low-concentration DNA samples as examples, the test method is the same as that in Example 2.

[0089] Table 4 provides the basic situation and detection results of 5 samples. It can be seen that the DMD gene mutation detection method based on the DMD gene multiplex PCR capture primer set of the present invention has low detection limit, high accuracy, and low requirements for DNA sample quality.

[0090] Table 4. Low-quality DNA detection results

[0091]

[009...

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Abstract

The invention discloses a multiplex PCR (Polymerase Chain Reaction) trapping primer group for a DMD (Duchenne Muscular Dystrophy) gene, a mutation detection kit for the DMD gene and a mutation detection method for the DMD gene. The inventors provide the multiplex PCR trapping primer aiming at the sequences of 79 exons and bilateral 50bp introns and 23 known intron pathogenic mutations of the DMD gene; moreover, the mutation detection method of the DMD gene is provided based on the primer group; the mutation detection method is high in detection sensitivity, good in accuracy, highly consistentin amplification efficiency and low in requirement on quality of a DNA (Deoxyribonucleic Acid) sample, and is used for solving the problem that the complete detection on the mutation information, particularly on loss of heterozygosity/repetition, of the DMD gene is difficultly carried out in one time by adopting a multiplex PCR trapping technique in the prior art.

Description

technical field [0001] The invention belongs to the field of gene sequencing, and in particular relates to a detection kit and a method for Duchenne / Bell muscular dystrophy based on multiplex PCR target sequence capture technology. Background technique [0002] Duchenne muscular dystrophy is a fatal X-linked recessive genetic disease, which can be divided into pseudohypertrophic muscular dystrophy (Duchenne muscular dystrophy, DMD) according to the degree of progress BMD (Becker muscular dystrophy, BMD), both are allelic clinically heterogeneous diseases. Among them, the main clinical features of DMD are progressive atrophy and weakness of the proximal skeletal muscles of the extremities, pseudohypertrophy of the gastrocnemius muscle of the calf, involving both the respiratory muscles and the myocardium, and some patients are accompanied by mental retardation. Loss of walking ability and death in the 20s occurs in up to 1 in 3500 male live births. Compared with DMD, the ag...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6883C12Q2600/156C12Q2537/143
Inventor 糜庆丰周幸芝吕来灰吴春求朱鹏远黄铨飞王杨陈雨刘丽菲
Owner CAPITALBIO GENOMICS
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