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A method for the co-characterization of exosomal membrane markers and RNA based on aptamer immuno-PCR

A nucleic acid aptamer and exosome technology, which is applied in biochemical equipment and methods, microbial measurement/inspection, animal cells, etc., can solve the problem of simultaneous detection of exosome protein and RNA, and save test samples , simplification of operation steps, and the effect of mature PCR technology

Active Publication Date: 2021-08-03
INST OF CHEM CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, no method has been reported for the simultaneous detection of exosomal proteins and RNA

Method used

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  • A method for the co-characterization of exosomal membrane markers and RNA based on aptamer immuno-PCR
  • A method for the co-characterization of exosomal membrane markers and RNA based on aptamer immuno-PCR
  • A method for the co-characterization of exosomal membrane markers and RNA based on aptamer immuno-PCR

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1: Establishment of an aptamer-based immuno-PCR detection method

[0053] Step 1: Synthesis of DSPE-S-S-MBs;

[0054] Step 1.1: Wash 1 mL of carboxy-modified magnetic beads 3 times with PBS, and finally resuspend in 1 mL of PBS;

[0055] Step 1.2: Add 15 mg of cystamine hydrochloride to step 1.1 and mix well, then add 10 mg of EDC ((1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and 10 mg of hydroxysuccinimide (N-Hydroxysuccinimide, NHS ), stirring and reacting at room temperature for 5h;

[0056] Step 1.3: Separate the mixed solution in step 1.2 under an external magnetic field, repeat washing 3 times, and finally resuspend in 1mL PBS;

[0057] Step 1.4: Add 10 mg NHS and DSPE co-modified polyethylene glycol (NHS-PEG-DSPE) to step 1.3, mix well, and react at room temperature for 16 hours;

[0058] Step 1.5: Separate the mixed solution in step 1.4 under an external magnetic field, repeat washing 5 times, and finally resuspend in 1mL PBS.

[005...

Embodiment 2

[0089] Example 2: Characterization of Exosome Membrane Marker CD63 Protein by Fluorescent Quantitative PCR for Nucleic Aptamers

[0090] Taking exosomal CD63 protein as an example, the nucleic acid aptamer shown in SEQ ID NO:1 was used to detect different concentrations (50-5×10 5 Each / μL) of exosomes, using the nucleic acid aptamer SEQ ID NO:1 of CD63 protein to perform fluorescent quantitative PCR detection, the amplification primers include SEQ ID NO:5-6, and the probe includes SEQ ID NO:7 (5' The end is labeled FAM, the 3' end is labeled MGB). The result is as image 3 shown. It can be seen that the number of cycles and Ct values ​​detected by fluorescent quantitative PCR of nucleic acid aptamers combined with CD63 protein are negatively correlated with the concentration of exosomes. Therefore, the detection of nucleic acid aptamers by fluorescent quantitative PCR can quantitatively detect exosomal membrane markers.

Embodiment 3

[0091] Example 3: Characterization of exosomal PD-L1 protein, exosomal RNA SLC25A6 and IDO1 in the same reaction system

[0092] The exosomal PD-L1 protein, exosomal RNA SLC25A6 and IDO1 in the plasma samples of 3 healthy people (H1-H3) and 5 lung cancer patients (P1-P5) were simultaneously characterized in the same reaction system.

[0093]In the reaction system, the nucleic acid aptamer SEQ ID NO:2 of the PD-L1 protein is detected by fluorescent quantitative PCR, the amplification primers include SEQ ID NO:8-9, and the probe includes SEQ ID NO:7 (the 5' end labeled FAM , 3' end labeled MGB). Real-time quantitative PCR detection for exosomal RNA SLC25A6 and IDO1, primers for exosomal RNA IDO1 include SEQ ID NO: 11-12, probes include SEQ ID NO: 13 (5' end labeled VIC, 3' end labeled BHQ1 ); the primers of exosomal RNA SLC25A6 include SEQ ID NO: 14-15, and the probe includes SEQ ID NO: 16 (the 5' end is marked with Cy5, and the 3' end is marked with BHQ3).

[0094] The result...

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Abstract

The present invention relates to a method for jointly characterizing markers and RNAs on exosome membranes based on nucleic acid aptamer immuno-PCR. This method mainly involves the specific recognition and binding of the markers on the exosome membrane through nucleic acid aptamers, and then the polymerase chain reaction (Polymerase Chain Reaction, PCR) is used to simultaneously amplify the nucleic acid aptamers and exosomes bound to the exosomes. Somatic RNA, enabling simultaneous characterization of markers and RNAs on exosome membranes. By using the method proposed in the present invention, simultaneous high-sensitivity detection of markers and RNAs on exosome membranes can be realized, samples are saved, operation is simple, and has high clinical application value.

Description

technical field [0001] The invention belongs to the field of biological detection, and in particular relates to a detection method for exosome molecules, in particular to a detection method for simultaneous detection of exosome membrane markers and RNA based on nucleic acid aptamer immuno-PCR. Background technique [0002] Exosomes are lipid vesicles secreted by cells with a diameter of 30-150nm, carrying information such as various proteins, RNA and DNA of the source cells. Among them, exosomes secreted by cancer cells play an important role in tumor growth, metastasis, immune escape and angiogenesis. Tumor cell exosomes have specific proteins and RNAs associated with cancer, and detection of these markers can provide more accurate disease diagnosis and treatment guidance. At the same time, increasing evidence shows that the diagnostic value of proteins on exosomes is significantly different from that present in plasma. Therefore, exosomes are considered as emerging marke...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/07C12Q1/6844
CPCC12N5/06C12Q1/6844
Inventor 方晓红董再再赵立波张振周卫徐丽
Owner INST OF CHEM CHINESE ACAD OF SCI
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