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A CRISPR-Cas system for diagnosing spinal muscular atrophy and its application

A technology for spinal muscular atrophy and diagnostic kits, which is applied in the determination/testing of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., and can solve the problem of expensive detection costs, unsuitable for large-scale promotion and use, and long detection cycles, etc. problem, to achieve the effect of low cost, high accuracy rate, high specificity and sensitivity

Active Publication Date: 2020-08-18
JINAN UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the methods used for the detection of SMA gene mutations mainly include restriction fragment length polymorphism, PCR, denaturing high performance liquid chromatography and Sanger sequencing, etc. The above methods have their own shortcomings, such as long detection cycle and expensive instruments and detection costs are high, and are not suitable for clinical large-scale promotion and use

Method used

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  • A CRISPR-Cas system for diagnosing spinal muscular atrophy and its application
  • A CRISPR-Cas system for diagnosing spinal muscular atrophy and its application
  • A CRISPR-Cas system for diagnosing spinal muscular atrophy and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] An embodiment of the spinal muscular atrophy diagnostic kit of the present invention, the kit includes the following components: FnCas12a, crRNA, dsDNA and reporter RNA strand; the crRNA sequence is shown in SEQ ID No.1; The dsDNA amplification primers include an upstream primer shown in SEQ ID No.2 and a downstream primer shown in SEQ ID No.3; the reporter RNA chain sequence is shown in SEQ ID No.4, and the reporter RNA chain The 5' end is modified with a fluorescent group FAM, and the 3' end is modified with a quenching group BHQ1.

[0028] SEQ ID No.1: 5'-GAAATTAATACGACTCACTATAGGGTAATTTCTACTAAGTGTAGATAGACAAAATCAAAAAGAAGG-3';

[0029] SEQ ID No.2: 5'-TATAAAGCTATCTATATA-3';

[0030] SEQ ID No. 3: 5'-AGGTGCTCACATTCCTTAAATTAA-3'.

[0031] SEQ ID No. 4: 5'FAM-TTATT-BHQ1 3'.

[0032] The crRNA preparation method comprises the following steps: (1) designing and synthesizing the crRNA whose sequence is shown in SEQ ID No.1; (2) transcribing the crRNA obtained in step (1) ...

Embodiment 2

[0038] In this example, the kit described in Example 1 was used to detect 20 suspected spinal muscular atrophy patient samples, and at the same time compared with the final clinical diagnosis results.

[0039] 1. Detection method

[0040] 1. Use the DNA extraction kit to extract the DNA of the patient's saliva sample, and the operation steps are strictly in accordance with the instructions;

[0041] 2. Prepare dsDNA corresponding to each sample according to the dsDNA preparation method described in Example 1;

[0042]3. Configure the following reaction system (100uL): FnCas12a 45nM, crRNA 67.5nM, dsDNA 100ng, reporter RNA strand 125nM.

[0043] 4. Incubate the FnCas protein and crRNA at 37°C for 10 minutes before using the machine, and detect the fluorescence every 1 minute on a microplate reader.

[0044] 2. Test results

[0045] The test results are shown in Table 1:

[0046] Table 1 Test results of 20 samples

[0047]

[0048]

[0049] From the above results, it ...

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Abstract

The invention discloses a CRISPR-Cas system for diagnosing spinal muscular atrophy. The system comprises Cas 12. Preferably, the CRISPR-Cas system includes Cas12, crRNA, dsDNA, and reporter RNA strands. At that same time, the invention also disclose application of the CRISPR-Cas system in that preparation of kit for diagnosis of spinal muscular atrophy. The CRISPR-CAS system can quickly detect themutation of SMN1 gene, the pathogenic gene of spinal muscular atrophy, and has the advantages of high specificity and sensitivity, intuitive analysis of results, accurate rate as high as 100%, etc. The CRISPR-Cas system is very suitable for large-scale clinical sample detection.

Description

technical field [0001] The invention belongs to the technical field of gene detection, and in particular relates to a CRISPR-Cas system for diagnosing spinal muscular atrophy and its application. Background technique [0002] Spinal muscular atrophy (SMA), also known as spinal muscular atrophy, is an autosomal recessive genetic disease characterized by pathological degeneration of the anterior horn cells of the spinal cord and brainstem motor nerve nuclei. Weakness and muscle atrophy, the patient eventually died of respiratory failure and lung infection, ranking second among fatal autosomal genetic diseases. The incidence rate of the disease in newborn babies is about 1 / 6000 to 1 / 10000, and the carrier rate of the normal population is about 1 / 40 to 1 / 50. According to the age of onset, prognosis and genetic pattern, it is divided into four types. There are 3 clinical types (types Ⅰ~Ⅰ) in children, and type Ⅳ is the adult type. [0003] Studies have shown that the main patho...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/6883C12Q2600/156
Inventor 罗良平唐佳李旭马巍
Owner JINAN UNIVERSITY
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