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Prader-Willi Syndrome detection kit and use method thereof

A detection kit and technology for Wiley syndrome, applied in the field of diagnosis and research of chubby Wiley syndrome, can solve the problems of difficult detection of microdeletions, large sample input, complicated operation, etc.

Pending Publication Date: 2020-04-21
CENT FOR EXCELLENCE IN MOLECULAR CELL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above two methods have disadvantages such as large amount of sample input, complex operation, and difficulty in detecting microdeletions.

Method used

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  • Prader-Willi Syndrome detection kit and use method thereof
  • Prader-Willi Syndrome detection kit and use method thereof
  • Prader-Willi Syndrome detection kit and use method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0093] Example 1 Rapid extraction of nucleic acid from clinical samples (such as blood, saliva, urine)

[0094] In order to realize rapid extraction of nucleic acid, the inventors added an RNase inactivator to the sample, and then lysed the cells at 95° to release the nucleic acid. The specific steps are as follows, add RNase inactivator (TCEP / EDTA with a final concentration of 100mM / 1mM) to the sample (such as blood, saliva, urine), pipette and mix with a sterile pipette tip (Thermo QSP), 95° Heat for 10 minutes, centrifuge at 14000 rpm for 10 minutes, and take the supernatant. RNase activity in the supernatant was detected with RNaseAlert Lab Test Kit v2 (Thermo), and the real-time fluorescence change was monitored with a multifunctional microplate detector (BioTek SynergyNEO) (such as figure 1 shown), the RNase inactivator can effectively inhibit the RNase activity in the sample (blood), thereby inhibiting its degradation of RNA, effectively protecting the RNA to be detect...

Embodiment 2

[0095] Example 2 Screening for efficient and specific primers for sno-lncRNAs molecular markers for recombinase polymerase amplification

[0096] 2.1 Design and synthesis of specific recombinase polymerase amplification primers

[0097] The sno-lncRNAs molecular marker combination of chubby-Willi syndrome includes 5 long noncoding RNAs, namely sno-lncRNA1, sno-lncRNA2, sno-lncRNA3, sno-lncRNA4, and sno-lncRNA5. Sequences are in the NCBI GeneExpression Omnibus database ( https: / / www.ncbi.nlm.nih.gov / geo / ; accession number GSE38541). In order to realize trace detection, the present invention utilizes recombinase polymerase to amplify sno-lncRNAs molecular markers. The recombinase polymerase amplification primers of the chubby-Willi syndrome sno-lncRNAs molecular marker combination are multiple pairs of specific primers, and the primers are designed through the NICB Primer-BLAST online primer design tool ( https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ), and the prime...

Embodiment 3

[0113] Example 3 Screening gRNA (CRISPR guide RNA) for efficient and specific targeting of sno-lncRNAs molecular markers

[0114] The nucleotide sequence of the gRNA is as follows:

[0115] Table 2

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[0127] Screen gRNAs that are highly efficient and specifically target molecular markers of sno-lncRNAs:

[0128] Under the "guidance" of gRNA, Cas13a binds to the target RNA to exert its cutting activity, and the "activated" Cas13a will not only cut the target RNA, but also non-specifically cut other RNAs. Therefore, gRNAs with high efficiency were screened by the ability of Cas13 to cleave fluorescent RNA substrates. The fluorescent RNA substrate RNAse Alert v2 was purchased from a company (Integrated DNA Technologies). Cas13 cutting RNA reaction system is as follows:

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[0131] 37 ° reaction, 3h, real-time monitoring substr...

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Abstract

The invention belongs to the field of biotechnology detection, and particularly provides a Prader-Willi Syndrome detection kit and a use method thereof. According to the present invention, snO-lncRNA1, snO-lncRNA2, snO-lncRNA3, snO-lncRNA4 and snO-lncRNA5 are extremely highly expressed in human embryonic stem cells, and abnormally stable, so that the detection kit prepared by taking snO-lncRNA1, snO-lncRNA2, snO-lncRNA3, snO-lncRNA4 and snO-lncRNA5 as detection genes can detect samples such as common blood, saliva, urine and the like so as to provide guarantee for realizing non-invasive examination of Prader-Willi Syndrome; the use method is simple to operate and has low requirements on instruments and equipment; the input amount of the required sample is small, and the sample source is highly inclusive; the kit has the characteristics of high sensitivity, high specificity and the like; the detection result is visual, simple and easy to understand; and by using the detection method, Prader-Willi Syndrome can be detected in a short time.

Description

technical field [0001] The invention belongs to the field of diagnosing and researching chubby-Willi syndrome, and in particular relates to a detection kit for chubby-Willi syndrome and a use method thereof. Background technique [0002] Prader-Willi syndrome (Prader-Willi syndrome, referred to as "PWS") is a complex clinical syndrome with multiple system disorders, with an incidence rate of 1 / 12,000 to 1 / 15,000 newborns. On May 11, 2018, the National The National Health Commission, the Ministry of Science and Technology, the Ministry of Industry and Information Technology, the State Drug Administration, and the State Administration of Traditional Chinese Medicine jointly formulated and issued the "First List of Rare Diseases", including Pang-Willi Syndrome and others121 Diseases are included in it. The main clinical features of Pang-Willi syndrome include hypotonia in newborns or infants, slow development, prominent facial features (narrow face, narrow forehead, long head,...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/6844
CPCC12Q1/6883C12Q1/6844C12Q2600/158C12Q2600/178C12Q2521/507C12Q2521/301C12Q2563/107
Inventor 陈玲玲吴煌
Owner CENT FOR EXCELLENCE IN MOLECULAR CELL SCI CHINESE ACAD OF SCI
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