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Stargardt disease mutation detection kit

A technology of mutation detection and kit, which is applied in the field of genes, can solve the problems of retinal pigment epithelium and photoreceptor degeneration, the inability to obtain a clear diagnosis of patients, and the loss of central vision, and achieve rich diagnostic sites, high accuracy, and strong specificity Effect

Inactive Publication Date: 2022-05-13
SHANDONG PROVINCIAL HOSPITAL AFFILIATED TO SHANDONG FIRST MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Mutations in the ABCA4 gene lead to dysfunction of the ABCA4 transporter, accumulation of toxic tretinoin, and degeneration of the retinal pigment epithelium and photoreceptors, resulting in irreversible loss of central vision
[0004] The ABCA4 gene was first discovered by Allikmets et al. in 1997. So far, more than 2,000 ABCA4 mutations have been reported in the literature (www.lovd.nl / ABCA4), but there are still many patients who cannot be diagnosed clearly. Related disease-causing loci still need further research, and the discovery of any new mutation plays an important role in the diagnosis and prevention of diseases

Method used

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  • Stargardt disease mutation detection kit
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  • Stargardt disease mutation detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 Mutation site determination

[0056] First, a family with Stargardt disease in my country ( figure 1 Shown) conducted research, through whole exome sequencing, a new mutation site on the ABCA4 gene was discovered for the first time, specifically the mutation site from G to A of c.215, and family members and 200 1 normal control for verification and co-segregation analysis ( figure 2 shown), which was finally determined to be a new mutation site. The corresponding inventor has disclosed the DNA fragment where the mutation site is located, its nucleotide sequence is shown in Seq ID No: 1, its encoded amino acid sequence is shown in Seq ID No: 2, the unmutated DNA fragment, its nucleotide sequence The sequence is shown in Seq ID No:3, and the encoded amino acid sequence is shown in Seq ID No:4. According to the comparison, the mutated DNA fragment contains the c.215G>A mutation, and the corresponding amino acid sequence contains p.G72E mutation.

Embodiment 2

[0057] The composition of embodiment 2 detection kits

[0058] The inventors provide a set of detection primers:

[0059] The upstream primer is 5'-CACTACACCGGGAGCTTGAGA-3', the nucleotide sequence of which is shown in Seq ID No:5,

[0060] The downstream primer is 5'-TGACCAGAAGGCAGTGGACACAT-3', the nucleotide sequence of which is shown in Seq ID No:6, which can be used for the amplification of the above-mentioned DNA fragments;

[0061] Furthermore, the inventors provided a kit for detecting the above-mentioned mutations. The main components of the kit are as follows: 10 μM amplification primer, 10 μM specific probe, 10 μM padlock probe, 10 μM rolling circle primer, Taq DNA ligase, Bst DNA polymerase and reaction buffer; the more specific composition is:

[0062] Amplification primers, the upstream primer is 5'-CACTACACCGGGAGCTTGAGA-3', its nucleotide sequence is shown in Seq ID No:5, and the downstream primer is 5'-TGACCAGAAGGCAGTGGACACAT-3', its nucleotide sequence is sho...

Embodiment 3

[0078] The use of embodiment 3 kits

[0079] The method for detecting the c.215G>A mutation of the ABCA4 gene using the kit obtained in Example 2, the specific steps are as follows:

[0080] Step 1, sample preprocessing

[0081] (1) Genomic DNA was extracted using a conventional DNA extraction kit (the present embodiment uses the brand Axygen of Corning Company, the article number is AP-MD-FBL-GDNA-20), and the specific steps are as follows:

[0082] 1) Add 2ml of the blood sample to be tested into 5ml Buffer AP1, and mix vigorously;

[0083] 2) Add 1ml Buffer AP2, mix immediately, and centrifuge at 4630g for 15min;

[0084] 3) Transfer the supernatant to the medium preparation tube, turn on the negative pressure, keep the negative pressure, add 7ml Buffer W1, 8ml Buffer W2, 4ml Buffer W2;

[0085] 4) Remove the medium volume tube head and centrifuge at 12000g for 2min;

[0086] 5) Add 0.4ml Elution buffer, let stand at room temperature for 5 minutes, centrifuge at 12000g ...

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Abstract

The invention relates to the technical field of biological genes, in particular to a Stargardt disease mutation detection kit.The inventor firstly discovers a new mutation site from G to A of c.215 on an ABCA4 gene, then the corresponding kit is researched and developed, and the kit comprises an amplification primer, a specific probe, a lock type probe, a rolling ring primer, Taq DNA ligase, Bst DNA polymerase, a reaction buffer solution and the like; according to the kit, firstly, a human DNA fragment is used as a template, under the action of Taq DNA ligase, a lock-type probe is connected into a ring-shaped product, then isothermal rolling circle amplification is carried out under the action of a rolling circle primer, and whether a new mutation site from G to A of c.215 on an ABCA4 gene exists in human DNA or not is detected through agarose gel electrophoresis. According to the novel mutation found firstly, an ABCA4 gene mutation spectrum is added, meanwhile, the provided kit is high in specificity, high in sensitivity, free of temperature circulation and easy to operate, and guidance is provided for clinical detection of the Stargardt disease.

Description

technical field [0001] The invention relates to the field of gene technology, in particular to a Stargardt disease mutation detection kit. Background technique [0002] Stargardt disease (STGD1 OMIM#248200), also known as Stargardt macular dystrophy, juvenile macular degeneration, or macular fundus, is one of the most common causes of inherited retinal disease, accounting for 12% of blindness associated with inherited retinal disease. Stargardt's disease, first described by Karl Stargardt in 1909, is characterized clinically by adolescent macular dystrophy with decreased central vision and bilateral atrophic changes in the macula, including macular coppery changes, bull's-eye maculopathy, progressive macular atrophy and retinal atrophy A yellowish-white spot at the posterior pole; histologically, the disease is closely associated with photoreceptor cell death and retinal degeneration caused by massive lipofuscin deposition in the retinal pigment epithelium. In 1998, Blachar...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/6844C12N15/11
CPCC12Q1/6883C12Q1/6844C12Q2600/156C12Q2531/125C12Q2525/307C12Q2521/501C12Q2521/101C12Q2565/125
Inventor 白晓卉徐璐璐王榕榕刘静文
Owner SHANDONG PROVINCIAL HOSPITAL AFFILIATED TO SHANDONG FIRST MEDICAL UNIVERSITY
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