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Preparing method of diabetes early warning and/or diagnosis kit based on hsa-miR-320a

A hsa-mir-320a, early warning technology, applied in biochemical equipment and methods, microbial determination/inspection, medical preparations containing active ingredients, etc., can solve problems such as lack of products or drugs for diabetes

Active Publication Date: 2019-10-01
纽诺生物医药(武汉)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the relationship between miR-320a and diabetes has not been found in this field, and the existing technology lacks effective products or drugs in the molecular diagnosis and molecular treatment of diabetes

Method used

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  • Preparing method of diabetes early warning and/or diagnosis kit based on hsa-miR-320a
  • Preparing method of diabetes early warning and/or diagnosis kit based on hsa-miR-320a
  • Preparing method of diabetes early warning and/or diagnosis kit based on hsa-miR-320a

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preparation example Construction

[0081] This group of embodiments provides a preparation method of a medicine for preventing and treating diabetes. In all the examples of this group, the preparation method has the following common features: the preparation method includes: degrading and / or down-regulating the substance expressing the hsa-miR-320a; antisense to hsa-miR-320a Complementary sequence fragment anti-hsa-miR-320a; recombinant plasmid expressing antisense complementary to hsa-miR-320a sequence fragment anti-hsa-miR-320a; and / or, expressing antisense complementary to hsa-miR-320a Sequence fragment of anti-hsa-miR-320a recombinant adeno-associated virus plasmid as active ingredient of antidiabetic drug.

[0082] In a further embodiment, the preparation method further includes: inserting a primer pair capable of expressing a sequence fragment anti-hsa-miR-320a complementary to hsa-miR-320a into an expression vector, thereby preparing a hsa-miR-320a antisense complementary sequence fragment anti-hsa-miR-...

experiment example 1

[0084] Experimental example 1. Detection of miRNA in peripheral blood of diabetic patients

[0085] 1. Collect peripheral blood from 200 diabetic patients and 200 normal controls. After sampling, centrifuge at 3,500 rpm for 6 minutes at room temperature, take the upper layer of plasma, and store it in a -80°C refrigerator. Add 1ml TRIZOL LS (Invitrogen Company) to every 0.25ml peripheral blood plasma, extract RNA, and RNasey Mini Kit (Qiagen) processes the sample. use ND-1000 detects RNA quality.

[0086] 2. Real-time PCR detection of the expression of hsa-miR-320a was performed using the miRNA detection kit of Guangzhou Ruibo Company:

[0087] reverse transcription of miRNAs:

[0088] Reverse transcription primer (RT Primer Mix) configuration:

[0089] miRNA RT Primer 1μl

[0090] U6RT Primer 1μl

[0091] RNase free H 2 O 78μl

[0092] Reverse transcription reaction system:

[0093] RNA template 2μg

[0094] RT Primer Mix 4μl

[0095] RNase free H 2 O up to 19μl...

experiment example 2

[0112] Experimental example 2. Preparation of a kit for detecting diabetes risk assessment

[0113] Kit components: The detection kit includes a set of specific reverse transcription primers and real-time PCR primers for amplifying miR-320a; specific reverse transcription primers and real-time PCR primers for amplifying control RNA (U6) A set of PCR primers and related reagents, the composition and content are as follows (100 times), stored at -20 degrees:

[0114]

[0115] The above reagents are provided by various source companies and have been commercialized. Refer to Example 1 for specific detection methods and relevant reaction parameters.

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Abstract

The invention discloses a preparing method of a diabetes early warning and / or diagnosis kit based on hsa-miR-320a, and belongs to the field of molecular medicines and diagnosis, prevention and treatment of metabolic diseases. A biological marker for diagnosis and / or early warning of diabetes comprises a sequence segment with hsa-miR-320a, and the hsa-miR-320a is shown as SEQ ID No.1. The kit for diabetes diagnosis and / or early warning comprises an agent for quantitatively detecting the biological marker. By detecting the biological marker and / or using the kit, the diabetes can be efficiently and accurately diagnosed, and corresponding diabetes early warning and risk evaluating are conducted.

Description

technical field [0001] The invention belongs to the field of diagnosis, prevention and treatment of molecular medicine and metabolic diseases, and specifically relates to a preparation method of an early warning and / or diagnosis kit of diabetes based on hsa-miR-320a. Background technique [0002] microRNA (miRNA, miR) is a newly discovered endogenous small molecule non-coding single-stranded RNA with a length of about 22 nucleotides derived from endogenous hairpin transcripts. The untranslated region (3'untranslated region, 3'UTR) specifically binds to promote the degradation of the target mRNA, or inhibits the translation of the target mRNA, and reduces the level of the encoded protein, thereby achieving post-transcriptional expression regulation of the gene. miRNA is closely related to many diseases in the body, including neurodegenerative diseases, heart disease, kidney disease, and tumors. Recent studies have found that miRNA plays an important role in the regulation of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883A61K31/7088A61P3/10
CPCA61K31/7088A61P3/10C12Q1/6883C12Q2600/136C12Q2600/178
Inventor 汪道文陈琛李华萍王峰殷中伟侯慧英
Owner 纽诺生物医药(武汉)有限公司
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