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Probe combination for diagnosing fibula amyotrophy, kit and application

A technology of Charcot-Marie-Tooth disease and a kit, which is applied in the direction of recombinant DNA technology, microbial measurement/inspection, DNA/RNA fragments, etc., can solve problems such as missed diagnosis, improve accuracy, improve accurate diagnosis rate, reduce time and The effect of financial burden

Pending Publication Date: 2022-04-05
THE THIRD XIANGYA HOSPITAL OF CENT SOUTH UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Although whole exome sequencing has been widely used clinically, the molecular diagnosis of CMT still faces many challenges: (1) Mutations in the 3′UTR region of the GJB1 gene can also lead to CMT1X phenotype
(2) Large repeat mutations of PMP22 can co-exist with other point mutations, resulting in a common pathogenic effect (Double trouble effect). Single detection of PMP22 gene copy number variation or point mutations of CMT pathogenic genes may lead to missed diagnosis

Method used

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  • Probe combination for diagnosing fibula amyotrophy, kit and application
  • Probe combination for diagnosing fibula amyotrophy, kit and application
  • Probe combination for diagnosing fibula amyotrophy, kit and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1 probe design

[0050] This embodiment uses the following methods to design specific probes for CMT-related gene detection:

[0051] 1) The probe covers the exon region and 50bp at both ends of the exon;

[0052] 2) For regulatory regions other than exons, use 100 bp as a sliding window. When there are 2 or more known pathogenic mutations in one window, keep this interval and design related probes;

[0053] 3) The probe design uses the shingled style, and each base in the reference interval is covered by at least 3 probes;

[0054] 4) For the target gene with CNV, add a 200bp CNV-plus probe interval at every interval of 1-3k. This probe interval needs to include the SNP positions with a mutation frequency of 20%-80% in the East Asian population in the gnomad population database point and there is no similar sequence on the genome in this interval (using 200bp as the sliding window, the whole genome comparison is performed, and there is no sequence with a s...

Embodiment 2

[0076] Example 2 Target Region Capture Sequencing

[0077] In this example, the capture probe set provided in Example 1 is used, and the sequence capture technology based on liquid phase hybridization is used to enrich the target sequence. The technical route of liquid phase hybridization is: (1) prepare a hybridization probe library, (2) use the probe to enrich the target gene, (3) sequence the enriched DNA sequence with a high-throughput sequencer.

[0078] The use of liquid phase hybridization technology can effectively enrich the target gene, reduce the requirement of sample size, and better control the cost of sequencing. The library construction method comprises the following steps:

[0079] 1. DNA extraction and fragmentation

[0080] DNA was extracted from 4 peripheral blood samples using the QIAamp DNA Blood Mini Kit.

[0081] Use the Bioruptor Pico DNA interrupter, after the temperature of the cold cycler drops to 4°C, set the parameters ON for 30s, OFF for 30s as...

Embodiment 3

[0161] Example 3 Sample Detection

[0162]20 samples were selected for detection by the method shown in Example 2, and WES and MLPA were used to conduct parallel comparison experiments on the CNV of the PMP22 gene of the samples. The kit used for WES is: SureSelect human whole exon V7 exome; the kit used for MLPA is: MRC-Holland P405 detection kit.

[0163] The results are shown in Table 11, which shows that the detection result of the method in Example 2 has a coincidence rate of 100% with MLPA, which is higher than the result based solely on the Reads depth analysis.

[0164] Table 11 Sample test results

[0165]

[0166]

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Abstract

The invention discloses a probe combination for diagnosing fibula amyotrophy, a kit and application, and belongs to the technical field of gene detection. Wherein the probe combination comprises at least one probe for targeting at least one gene of PMP22, GJB1, MFN2, MPZ, GDAP1, MORC2, SORD, NEFH, HSPB1, NEFL, PMP2, GLA, PRPS1 and TTR. The invention further discloses a kit containing the probe combination and application of the kit in diagnosis of bone amyotrophy. According to the probe combination or the kit, the accuracy of target gene variation detection can be remarkably improved, accurate auxiliary diagnosis of the fibula amyotrophy is further facilitated, and the probe combination or the kit has huge clinical application value.

Description

technical field [0001] The invention belongs to the technical field of gene detection, and in particular relates to a probe combination, a kit and an application for diagnosing Charcot-Marie-Tooth disease. Background technique [0002] Charcot-marie-tooth disease (CMT) is the most common group of hereditary peripheral neuropathies with high clinical and genetic heterogeneity, and its overall incidence is about 1 / 2500. According to the median motor nerve conduction velocity (MNCV), CMT can be further subdivided into demyelinating (CMT1, MNCV≤38m / s), axonal (CMT2, MNCV>38m / s) and intermediate There are three types (ICMT, 25m / s<MNCV<45m / s). So far, more than 100 genes have been identified to be associated with CMT, among which: PMP22 is the most common gene in CMT type 1, followed by MPZ; MFN2 is the most common gene in CMT type 2 and GJB1 is the most common gene in CMT intermediate type. According to literature reports, PMP22, GJB1, MPZ and MFN2 are the four most co...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/6869C12N15/11
Inventor 张如旭李小波黄顺祥林志强刘蕾谢雍之马卧龙吴志强鲁铤刘宇萍金瑜剑詹兴王阳马赛勇范嘉庚
Owner THE THIRD XIANGYA HOSPITAL OF CENT SOUTH UNIV
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