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Rapid detection kit for lung cancer marker GK5 in exosome

A technology for detecting kits and exosomes, applied in the field of kits, can solve the problems of low specificity and sensitivity, and achieve the effect of ultra-high sensitivity

Active Publication Date: 2018-11-23
HANGZHOU DIXIANG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the specificity and sensitivity of these methods are low. If the specific nucleic acid derived from tumor cells can be detected in the body fluid samples of patients, it will play a positive role in the early diagnosis of lung cancer.

Method used

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  • Rapid detection kit for lung cancer marker GK5 in exosome
  • Rapid detection kit for lung cancer marker GK5 in exosome
  • Rapid detection kit for lung cancer marker GK5 in exosome

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1: Specific Molecular Beacon Design

[0020] Designing specific molecular beacons to detect target genes is crucial for the detection of specific nucleic acids in exosome capture well plates. To this end, combined with the characteristics of the targeted gene, the applicant designed a molecular beacon with a special stem-loop structure. The 5' end stem and loop are completely complementary to the target gene, the 3' end stem is partially complementary to the 5' end stem, and the 5' end stem is partially complementary to the 5' end stem. The 3' and 3' ends are respectively modified with a fluorescent group and a quencher group, and some bases on the ring are modified with a locked nucleic acid. The specific sequence of the specific molecular beacon is shown in Table 1.

[0021] The specific molecular beacon designed in the present invention maximizes the specificity of binding the molecular beacon to the target gene, and simultaneously reduces the background fluo...

Embodiment 2

[0029] Embodiment 2: detection test

[0030] 1. Exosome isolation

[0031] 1. Take 200ul serum samples, centrifuge at room temperature 12000×g for 30min, remove cells and debris;

[0032] 2. Transfer the supernatant to a new EP tube, add 100ul exosome precipitation reagent;

[0033] 3. Mix well and incubate at 4°C for 30 minutes;

[0034] 4. Centrifuge at 10,000×g for 10 minutes at room temperature;

[0035] 5. Discard the supernatant, take 100ul 1×PBS to resuspend the exosome-rich precipitate, and let stand at 4°C for later use.

[0036] 2. Column purification of exosomes

[0037] 1. Balance column: add 100ul balance solution, centrifuge at 9000×g for 1min;

[0038] 2. Sample loading: put 100ul resuspension on the column, centrifuge at 9000×g for 1min;

[0039] 3. Elution: add 50ul eluent, centrifuge at 9000×g for 3min.

[0040] 3. Detection of exosome capture well plate

[0041] 1. Take out the orifice plate (each well of the orifice plate can be coated with 1-3 kind...

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Abstract

The invention provides a rapid detection kit for a lung cancer marker GK5 in exosome and belongs to the technical field of kits. The kit contains an exosome in-situ capturing pore plate, wherein eachpore in the exosome in-situ capturing pore plate contains a fluorescein-labeled molecular beacon; the molecular beacon is a specific DNA (Deoxyribonucleic Acid) probe of targeting tumor marker GK5 mRNA (micro Ribonucleic Acid). The kit provided by the invention applies an exosome in-situ capturing pore plate technology and is used for detecting the tumor marker GK5 mRNA in a body fluid sample of alung cancer patient; the kit can be used for carrying out early diagnosis on medical clinical lung tumors. The technology is a third-generation body fluid biopsy technology based on exosome detection, has the advantages of non-invasive or minimally invasive effect, ultrahigh sensitivity, rapidness, specificity and the like and is applicable to molecular diagnosis and epidemiological survey of themedical clinical lung tumors.

Description

technical field [0001] The invention belongs to the technical field of kits, and in particular relates to a rapid detection kit for the lung cancer marker GK5 in exosomes. Background technique [0002] Liquid biopsy can obtain disease information through non-invasive or minimally invasive sampling methods, and is a representative diagnostic technology of "precision medicine". Secretion (exosome). In essence, liquid biopsy is an extension of molecular diagnosis in terms of sample sources, an expansion of the molecular diagnosis market capacity, and an entry point for better diagnostic services for patients. Due to its rich content, specific source of contents, and stability, exosomes have gradually become a new favorite in the field of liquid biopsy and an important development direction for molecular diagnostic applications. [0003] Exosomes (exosomes) are tiny membrane vesicles that can be secreted by most cells, with a diameter of about 30-150nm. They have a lipid bilay...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/88
CPCG01N30/02G01N30/88G01N2030/8827
Inventor 白茹潘晓春曾恒山
Owner HANGZHOU DIXIANG CO LTD
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