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Kit used for detecting ophthalmic diseases, and detection method thereof

A technology for ophthalmic diseases and kits, applied in disease diagnosis, biological testing, biochemical equipment and methods, etc., can solve the problems of complex and time-consuming detection of human leptin protein, and achieve easy storage, low cost, and easy preparation Effect

Inactive Publication Date: 2016-10-12
李倩
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the traditional detection of human leptin protein expression has the disadvantages of complicated operation and time-consuming

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1, Screening and Preparation of Nucleic Aptamers

[0019] Design a random nucleic acid library comprising approximately 20 nucleotides at both ends and 39 nucleotides in the middle as follows:

[0020] 5'-TAGACTATCATGTGACTT(N39)GAGTGCTCGATGCTACTAG-3'; N39 represents 39 random nucleotides.

[0021] The single-stranded DNA library is amplified into double-stranded DNA, and the product is subjected to 2% agarose gel electrophoresis and then gel-cut to recover and purify; the recovered double-stranded DNA is used as a template to transcribe a single-stranded RNA random library in vitro, and the transcript is purified by PAGE . 75 μg of the RNA library was reverse-screened through a nitrocellulose membrane to remove membrane-bound RNA molecules, and then incubated with 2ugleptin protein (the target protein expressed by the method disclosed in CN100366738C) at 37°C for 30 min, and the reaction solution was filtered through a nitrocellulose membrane , wash the filter...

Embodiment 2

[0052] Example 2 The performance measurement of protein binding suitable gametes

[0053] Take 2.0 μg of RNA aptamers and digest them with calf intestinal alkaline phosphatase (CIP) at 37°C for 1 hour, purify and recover the dephosphorylated RNA; label [γ-32P]ATP with T4 polynucleotide kinase to dephosphorylate end of the RNA molecule. 10nmol of radioactively labeled RNA aptamers were incubated with different concentrations (1-200nM) of leptin at 37°C for 30min, and the reaction solution of each group was filtered through a nitrocellulose membrane, the filter membrane was washed, dried, and the filtration rate was measured by a liquid scintillation counter. The amount of radioactivity remaining on the film was measured twice in parallel on the same sample. The dissociation constants of each aptamer and leptin were calculated. The result is as follows:

[0054] name

Embodiment 3

[0055] Adapter specificity analysis and stability analysis described in embodiment 3

[0056] Human serum albumin, immune serum globulin, Vibrio cholerae VgrG3C protein, Escherichia coli outer membrane protein A, Lp-PLA2 protein, leptin, and 14 aptamers were used for specific detection. Gametes do not bind to these proteins, but only bind to leptin with high specificity.

[0057] 0.2ug of the aptamer was taken and placed in normal temperature serum and aqueous solution respectively for two weeks. Through RT-PCR detection, it was found that its structure was stable and not degraded after two weeks of storage.

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PUM

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Abstract

The invention discloses a kit including an aptamer specifically bound with a human leptin protein. The aptamer has good affinity ability to the human leptin. The aptamer being able to capture the human leptin in a solution is used to make the corresponding kit used for screening the ophthalmic diseases. The kit has the advantages of high sensitivity, low cost, easiness in production, and easiness in preservation.

Description

technical field [0001] The invention relates to a kit for detecting ophthalmic diseases and a detection method thereof. Background technique [0002] Graves' disease (Graves'disease) is a common disease, and the female incidence rate is 1 / 1000 person / year. In addition to hyperthyroidism, 25-50% of individuals with Graves' disease progress to clinically involving the eye, ie, thyroid eye disease. Graves' Ophthalmopathy (GO or TAO) is a typical form of thyroid eye disease. While some GO patients suffer only mild ocular discomfort, 3-5% suffer from severe pain and inflammation with diplopia and even vision loss. At present, the cause of this disease is unknown, and the clinical treatment is mainly based on hormones, immunosuppressants, growth hormone analogs, antithyroid drugs, intraorbital radiation, orbital decompression, etc., but the effect is not satisfactory due to various reasons. So far, no drug can effectively treat TAO and achieve long-term remission. Therefore, i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68C12N15/115
CPCC12N15/115C12N2310/16G01N33/68G01N33/6893G01N2333/47G01N2333/575G01N2800/046G01N2800/16
Inventor 李倩
Owner 李倩