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Gene chip for detecting hypertension and kit thereof

A technology of gene chips and kits, applied in the field of prepared kits, can solve the problems of low efficiency, time-consuming and laborious detection, etc., and achieve the effect of high detection efficiency, good sensitivity, and wide application range

Active Publication Date: 2016-12-07
GENE CRAB BIOTECH CO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the prior art, the expression level of GPD2 is mainly detected by ELISA, which is time-consuming, laborious and inefficient.

Method used

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  • Gene chip for detecting hypertension and kit thereof
  • Gene chip for detecting hypertension and kit thereof
  • Gene chip for detecting hypertension and kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] The acquisition of embodiment 1 aptamer

[0028] Recombinant human glycerol triphosphate dehydrogenase-2 was purchased from ProSpec, product number ENZ-437.

[0029] Construct a random single-stranded DNA (ssDNA) library and primers: construct a ssDNA library with fixed sequences at both ends and a random sequence of 35 nucleotides in the middle, where N represents any one of A, T, C, and G: 5'- ACTTTAGTAGCCAGTGAGAC-N35-GGAGCAGTACAGTGATCAGT-3'.

[0030] The upstream primer 1 is 5'-ACTTTAGTAGCCAGTGAGAC-3', and the downstream primer 2 is 5'-ACTGATCACTGTACTGCTCC-3'. The 5' end of the downstream primer sequence needs to be labeled with biotin. Random single-stranded DNA library and primers can be synthesized by primer synthesis company.

[0031] (1) Use primer 1 and primer 2 to amplify the single-stranded DNA library: use asymmetric PCR, the concentration ratio of primer 1 / primer 2 is 100:1, and the amplification conditions are: 94°C pre-denaturation: 3min, then 94°C den...

Embodiment 3

[0058] Example 3 Aptamer-coated biochip

[0059] We coated the most affinity aptamer SEQ ID NO: 1-26 on the surface substrate, and then combined with SPR biosensor for detection, the specific method is as follows:

[0060] Biochip Surface Pretreatment Put the biochip with streptavidin-coated gold film electrode on the surface substrate into 1.0mol / L NaOH, soak and clean for 20min, take it out, wash it with deionized water for 3 times, and then put the biochip into Piranha It was treated in the solution for 15 minutes, and immediately put it into absolute ethanol to soak for 5 minutes after taking it out, and then dried it with nitrogen gas for later use.

[0061] Aptamer pretreatment: dissolve in PBS buffer, denature at 95°C for 3 minutes, quickly ice-bath for 2 minutes, and then fix with thiol method.

[0062] Thiol-based probe immobilization: the denatured biotinylated aptamer covers the pretreated chip surface matrix, so that the aptamer is firmly fixed on the biochip, and...

Embodiment 4

[0065] Adapter specificity analysis and stability analysis described in embodiment 4

[0066] NADH dehydrogenase, human type III alcohol dehydrogenase, BSA, human type 2 11β-hydroxysteroid dehydrogenase protein and 26 aptamers were used for specificity detection. Binds to these proteins, but retains binding activity only to human glycerol triphosphate dehydrogenase.

[0067] 0.5 ug of the aptamer was taken and placed in normal temperature serum and aqueous solution respectively for four weeks. Through RT-PCR detection, it was found that its structure was stable and not degraded after being placed for four weeks. It shows that the aptamer has strong stability.

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Abstract

The invention provides a gene chip for detecting hypertension and a kit of the gene chip. The gene chip is provided with an aptamer. The kit can rapidly detect human glycerol 3-phosphate dehydrogenase, and whether a sample is essential hypertension is identified by detecting the quantity of the glycerol 3-phosphate dehydrogenase. The kit detects in a mode of the chip, the detection with the chip has the effects of high detection efficiency, high sensitivity and wide scope of application, and the chip and the kit can be widely popularized and used.

Description

technical field [0001] The invention belongs to the field of biology, and specifically relates to a biochip specifically combined with human glycerol triphosphate dehydrogenase, and a correspondingly prepared kit. Background technique [0002] Hypertension (hypertension) is a common chronic disease characterized by sustained increase in arterial blood pressure, which refers to arterial diastolic blood pressure >90mmHg in a resting state. A systolic blood pressure of 120-139 mmHg and / or a diastolic blood pressure of 80-89 mmHg is called prehypertension or high normal. [0003] Hypertension is the most important risk factor for cardiovascular and cerebrovascular diseases. Its major complications such as stroke, myocardial infarction, heart failure and chronic kidney disease not only cause disability and high mortality rate, but also seriously consume medical and social resources. The state is heavily burdened. A large number of long-term clinical observations have shown t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B40/06C12Q1/68G01N33/573C12N15/115
CPCC12N15/115C12Q1/6837C40B40/06G01N33/573
Inventor 朱继平
Owner GENE CRAB BIOTECH CO
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