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Exosome nucleic acid detection technology based on magnetism-enriched electrochemical luminescence

A detection technology, exosome technology, applied in the field of exosome separation and nucleic acid detection, can solve the problems of limited exosome content in biological fluids, difficulty in reaching the detection range of the target nucleic acid sequence concentration, detection interference, etc.

Inactive Publication Date: 2018-11-13
THE FIFTH AFFILIATED HOSPITAL SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, electrochemiluminescence technology still cannot be directly applied to the detection of exosomal nucleic acids in body fluids. The specific reasons are as follows: (1) The composition of biological fluids is relatively complex, and free nucleic acids may interfere with the detection; (2) Clinically available The biological fluid is limited and the content of exosomes is low, and the concentration of the target nucleic acid sequence after lysis is difficult to reach the detection range

Method used

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  • Exosome nucleic acid detection technology based on magnetism-enriched electrochemical luminescence
  • Exosome nucleic acid detection technology based on magnetism-enriched electrochemical luminescence
  • Exosome nucleic acid detection technology based on magnetism-enriched electrochemical luminescence

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0194] Example 1 Sample pretreatment and separation of exosome standard

[0195] (1) Sample collection

[0196] 1) Collection of animal or human cell culture fluid

[0197] The cell culture medium is DMEM, and the added volume fraction is 10% calf serum (FCS). Cells were trypsinized, transferred to a cell culture dish, and incubated at 37°C, 5% CO 2 The cells were cultured in a saturated water vapor carbon dioxide incubator for 24 hours to make the cells confluent to 50%-60%, and then replaced with serum-free DMEM culture medium for 24-48 hours. Recover the serum-free DMEM cell culture medium, store it at 4°C for a short period of time, and store it at -80°C for a long time.

[0198] 2) Animal or human blood sample collection

[0199] Draw blood from animal or human vein, add anticoagulant, centrifuge to remove cellular components in the blood, and obtain plasma; or take serum after blood coagulation, and the obtained blood sample can be stored at -80°C or used immediately...

Embodiment 2

[0208] Synthesis of Example 2 Immunomagnetic Beads

[0209] (1) Activation of streptavidin magnetic beads, the process is as follows:

[0210] 1) Electronic balance weighing: EDTA (0.292g) (CAS: 60-00-4), NaCl (29.22g) (CAS: 7647-14-5), TRIS (2.423g) (CAS: 77-86-1 ) in a 1L beaker.

[0211] 2) Add water to make the volume to 800ml, stir and mix evenly under a magnetic stirrer.

[0212] 3) Transfer the obtained solution to a 1L volumetric flask and dilute to 1L with water.

[0213] 4) Then adjust the pH value to 7.5 with 1mol / L HCl, and calibrate with precision pH test paper.

[0214] 5) The obtained solution was filtered with filter paper, and the obtained reaction solution I was divided into equipment for use.

[0215] 6) Mix the mother solution of streptavidin magnetic beads, take 500ug and put it in a 1.5ml EP tube, remove the protective reagent by magnetic separation, add 1ml PBS to the EP tube and mix well.

[0216] 7) Place the EP tube in the magnetic separation rac...

Embodiment 3

[0223] Example 3 The combination of immunomagnetic beads and exosomes (capture and enrichment of exosomes), the process is as follows:

[0224] 1) Mix the sample with the immunomagnetic beads obtained in Example 2. The mixing ratio of the sample and the immunomagnetic beads is: 1ml of serum: 50ug of immunomagnetic beads or 1L of urine: 30ug of immunomagnetic beads.

[0225] 2) Incubation: place on a shaker at room temperature for 10-30 minutes, and the time is appropriately adjusted according to the room temperature and the concentration of exosomes in the sample.

[0226] 3) Place the incubated sample on the magnetic separation rack and let it stand for 30 seconds to remove the liquid retaining magnetic beads.

[0227] 4) Wash the magnetic beads obtained in 3) 2-3 times with PBS to remove sample residues, and the process of pipetting should be gentle to avoid exosomes falling off.

[0228] 5) Magnetic separation to obtain purified magnetic bead-exosome complexes. The result...

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Abstract

The invention discloses an exosome nucleic acid detection technology based on magnetism-enriched electrochemical luminescence. The exosome nucleic acid detection technology comprises the steps of sample pretreatment and separation of an exosome standard substance, activation of streptavidin magnetic beads and a biotin antibody, binding of immunomagnetic magnetic beads and an exosome, preparation of exosome nucleic acid sample preparation and target nucleotide sequence detection through electrochemical luminescence. The exosome nucleic acid detection technology has the advantages that exosomesin biological samples such as urine, serum, saliva and a cell culture fluid can be separated and analyzed; antibodies are modified to the surfaces of the magnetic beads through specific binding of biotin and streptavidin, systems are stable, and nonspecific interference is avoided; the systems are controllable, different subtypes of exosomes are separated and purified by changing antibody types, and the technology can be used for nucleic acid expression profile studies and exosome somatotype studies; the final concentration is controllable, required concentration is obtained through re-dilution or re-releasing after magnetic enrichment, and follow-up detection is facilitated; the separation speed is high, the instrument requirement is low, and multiple samples can be treated simultaneously.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to an exosome isolation and nucleic acid detection technology. Background technique [0002] Exosomes contain a variety of nucleic acid components (including dsDNA, ssDNA, mRNA, microRNA, circRNA, etc.) that can be used as effective biomarkers for disease diagnosis. However, the content of exosomes in body fluids is low, and traditional methods cannot be directly detected. The classic ultracentrifugation method is time-consuming and laborious, relying on large precision equipment such as ultra-high-speed centrifuges, and it is difficult to remove protein aggregates and other structures of the same size. Although a rapid exosome isolation kit based on the PEG coagulation method has been launched on the market, PEG can aggregate viral particles and macromolecular proteins at the same time, and its application in the isolation of exosomes has been questioned. The shortag...

Claims

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Application Information

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IPC IPC(8): G01N33/543
CPCG01N33/54326
Inventor 黄曦廖玉辉樊志金肖铿
Owner THE FIFTH AFFILIATED HOSPITAL SUN YAT SEN UNIV
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