MRNA level in-situ hybridization detection kit of KLF4 gene in earlier stage of pathologic evolution of human cardiovascular and cerebtovascular disease, detection method and application

A detection kit and detection method technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problem of non-decreasing mortality, and achieve the effects of strong specificity, convenient operation and high sensitivity

Inactive Publication Date: 2014-11-12
嘉兴瑞康生物科技有限公司
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

[0010] In view of the current clinical diagnosis of cardiovascular and cerebrovascular diseases (biochemical indicators are the diagnosis of most cardiovascular and cerebrovascular diseases) is a late diagnosis, treatment is also a late treatment, leading to a treatment model that does not reduce mortality

Method used

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  • MRNA level in-situ hybridization detection kit of KLF4 gene in earlier stage of pathologic evolution of human cardiovascular and cerebtovascular disease, detection method and application
  • MRNA level in-situ hybridization detection kit of KLF4 gene in earlier stage of pathologic evolution of human cardiovascular and cerebtovascular disease, detection method and application
  • MRNA level in-situ hybridization detection kit of KLF4 gene in earlier stage of pathologic evolution of human cardiovascular and cerebtovascular disease, detection method and application

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Experimental program
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Effect test

Embodiment 1

[0048] The in situ hybridization kit of this example was prepared according to conventional methods, and the kit included a hybridization probe designed with KLF4, a marker, and instructions, wherein: the probe marker of this example was selected from digoxigenin.

[0049] Kit hybridization solution composition:

[0050]

[0051] alkaline phosphatase

[0052]

[0053] positive control standard

[0054] 6 pieces / box

[0055] Concentration of prepared reagents

[0056] 1). Dilute 10× buffer I with triple distilled water 1:10 into 1× buffer I;

[0057] 2). Dilute 20× buffer II with triple distilled water 1:10 to form 2× buffer II;

[0058] Dilute 1:100 into 0.2×buffer II; dilute 1:200 into 0.1×buffer II;

[0059] 3). Dilute 10× buffer III with triple distilled water 1:10 to 1× buffer III;

[0060] 4). Dilute 10× buffer IV with triple distilled water at 1:10 to form × buffer IV (take 10 mL each of 1#, 2#, and 3#, and add water to 100 mL).

Embodiment 2

[0062] The implementation process of applying the nucleic acid in situ hybridization detection method to the KLF4 expression level of each group of blood samples:

[0063] 1).Take two specimens to be tested;

[0064] 2). Add 50ml of digestive solution (100μL of digestive solution plus 1× buffer I99.9ml, which is the concentration used) in a glass tank, preheat in a water bath at 37°C for 10 minutes, put 16 slides in, and treat at 37°C for 12 minutes, then Wash with 1× buffer I for 5 min;

[0065] 3). Wash with 0.2% protection solution (protection solution 1ml plus 1× buffer I, 99ml is the concentration used) for 10 minutes, three-distilled water for 5 minutes (the above process is carried out in a glass tank), take out the slide and let it naturally dry;

[0066] 4). Put the slides into the humidity box, add 25 μL / slice of pre-hybridization solution (add to the place where there are cells), cover with a cover glass, cover the humidity box tightly, and put it in a constant te...

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Abstract

The invention discloses an in-situ hybridization detection kit which comprises a hybridization probe and a marker. The invention also discloses a method for in-situ hybridization detection of vascular endothelial cell's genetic factor (KLF4) gene's mRNA which is closely related with pathologic evolution of cardiovascular and cerebtovascular vascular endothelial injury in the earlier stage by the use of the kit. The method comprises the following steps: (1) under the condition that a hybridization probe and a target sequence can form a stable hybrid complex, RNA to be tested in a substrate is contacted with the hybridization probe so as to form a hybrid complex; and (2) the hybrid complex is detected. According to the detection kit and the detection method, gene expression quantity can be detected at the RNA level, the index of the detection method is ealier than present clinical biochemical detection index (protein detection index), and genuine RNA level screening of the earlier stage of cardiovascular and cerebtovascular disease can be realized. Meanwhile, the detection method provided by the invention is simple and convenient; cost is low; and the method is convenient for popularization and application in district-level hospitals.

Description

technical field [0001] The present invention relates to the field of biological detection, more specifically, relates to the relevant detection technology related to the change of mRNA expression (pathological evolution process) in human and cerebrovascular pathological evolution. Background technique [0002] According to the information provided by authoritative organizations at home and abroad, there are more than 3 million new cases of cardiovascular disease in my country every year, nearly 2.5 million deaths, and more than 9 million patients. Globally, there are more than 12 million new cardiovascular disease patients every year, and the death toll is close to more than 1,000. There are more than 100 million patients. According to the forecast of the world authoritative organization, the number of patients will double by 2030. This is a group of terrible figures. The cost of diagnosis and treatment of cardiovascular diseases is getting higher and higher. The annual treat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6883C12Q1/6804C12Q2600/158
Inventor 裘建英张云福张玉丽裘霖
Owner 嘉兴瑞康生物科技有限公司
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