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Synthetic detection kit for hybridization in situ and detection method

A comprehensive detection and in situ hybridization technology, applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of human and environmental harm, and achieve the effect of convenient operation, strong specificity and high sensitivity

Inactive Publication Date: 2011-10-05
李学莹
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although isotope markers have the advantages of high sensitivity and clear background, but because radioactive isotopes can cause harm to people and the environment, they have recently tended to be replaced by non-isotopes.

Method used

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  • Synthetic detection kit for hybridization in situ and detection method
  • Synthetic detection kit for hybridization in situ and detection method
  • Synthetic detection kit for hybridization in situ and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Prepare the in situ hybridization kit of this embodiment according to conventional methods, the kit includes hybridization probes, markers, and instructions designed with AFPmRNA and RhoGDI2 genes as detection target genes, wherein:

[0049] Digoxigenin was selected as the probe label in this embodiment.

[0050] Kit hybridization solution composition:

[0051] Digestive solution 100μL / tube 1 tube / box Colorless transparent liquid

[0052] Protective solution 100μL / tube 1 tube / box Colorless transparent liquid

[0053] Pre-hybridization solution 1300μL / tube 2 tubes / box Colorless transparent liquid

[0054] Sense hybridization solution 10μL / tube 1 tube / box Colorless transparent liquid

[0055] Antisense hybridization solution 10μL / tube 1 tube / box Colorless transparent liquid

[0056] Blocking solution 1000μL / tube 1 tube / box Colorless transparent liquid

[0057] Alkaline phosphatase antibody 1μL / tube 1 tube / box Colorless transparent liquid

[0058] Chromogen A 175μL / t...

Embodiment 2

[0073] The implementation process of applying nucleic acid in situ hybridization detection method to the expression of AFP mRNA and RhoGDI2 gene in the early stage of cancer:

[0074] 1).Take two specimens to be tested;

[0075] 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;

[0076] 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;

[0077] 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...

Embodiment 3

[0093] 20 liver cancer patients and 10 normal controls. Take 3-5 milliliters of peripheral blood (leukocyte separation) from all persons to be tested for in situ hybridization. The results showed that the AFP gene was overexpressed in all cancer patients, and the cells were stained; the AFP gene was not expressed in the normal control group, and the cells were not stained. The expression of RhoGDI2 gene decreased in 30 cases of clinically diagnosed metastases in the interim period, and the expression of RhoGDI2 gene was not expressed in 10 cases of patients, and the cells were not stained. For specific results, see figure 2 , image 3 .

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Abstract

The invention relates to an in situ hybridization comprehensive detection kit, which comprises two groups of hybrid probes and labels. The invention also discloses a method for performing in situ hybridization detection on AFP mRNA genes and RhoGDI2 genes related to liver cancer genesis and metastasis by utilization of the kit. The method comprises the following steps: (1) RNA to be detected in asubstrate contacts the hybrid probes to form a hybrid complex under the condition that the hybrid probes and target sequences can form a stable hybrid complex; and (2) the hybrid complex is detected.The kit can detect the expression amount of the AFP mRNA genes and the RhoGDI2 genes in the genetic level, diagnose earlier than AFP detection, can achieve the real aim of early diagnosis, and can effectively improve the cure rate of liver cancer; and simultaneously the detection method is simple and convenient, and is convenient to apply.

Description

technical field [0001] The invention relates to the fields of biological detection and disease diagnosis, and more specifically relates to the detection technology of genes related to the occurrence and metastasis of liver cancer. Background technique [0002] The incidence of liver cancer is relatively high in my country and Asia, and its mortality rate ranks fourth in the world and second in my country. The 5-year survival rate, no matter in the East or the West, generally does not exceed 5%. One of the main reasons is related to the metastasis of liver cancer. How to overcome the metastasis and recurrence of liver cancer after treatment is still a very difficult problem. Task. The mechanism of liver cancer metastasis, like other cancers, is a dynamic process involving multiple genes. If early diagnosis and detection of metastasis and recurrence after treatment are achieved, this is the hope of prolonging survival and curing. Because the liver is hidden in the deep part ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 裘建英张云福
Owner 李学莹
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