Nucleic acid detection method for separating luminous marker based on magnetic beads and nucleic acid hydrolysis

A technology of luminescent markers and detection methods, which is applied in the field of nucleic acid detection based on magnetic beads and nucleic acid hydrolysis to separate luminescent markers, and can solve problems such as limited detection sensitivity and weakened luminescent signal intensity

Inactive Publication Date: 2015-04-08
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] Technical problem: The main technical problem to be solved by the present invention is to overcome the limited detection sensitivity of the existing nucleic acid detection method

Method used

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  • Nucleic acid detection method for separating luminous marker based on magnetic beads and nucleic acid hydrolysis
  • Nucleic acid detection method for separating luminous marker based on magnetic beads and nucleic acid hydrolysis
  • Nucleic acid detection method for separating luminous marker based on magnetic beads and nucleic acid hydrolysis

Examples

Experimental program
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Effect test

Embodiment 1

[0040] Embodiment 1 detects with hepatitis B virus (Hepatitis B virus, HBV) DNA as the object

[0041] 1. Carry out functional modification on the surface of magnetic beads: (1) Amination modification of magnetic beads: 1mL Fe 3 o 4 SiO 2 Magnetic beads (30 mg / mL) were magnetically separated, 6 mL of ethanol / water mixture was added and dispersed ultrasonically, then 12 μL of aminopropyltriethoxysilane (APTES) was added to the above mixture, and stirred at room temperature for 7 h. Use an external magnetic field to separate the APTES-modified magnetic particles, wash them with ethanol and dimethylformamide (N,N-dimethylformamide, DMF) several times, and disperse them in DMF at 5 mg / mL for later use; (2) magnetic Bead carboxylation modification: 1 mL of aminated magnetic beads (5 mg / mL) was added dropwise to an equal volume of succinic anhydride solution (succinic anhydride, SA, 1 mM) dissolved in DMF, and shaken at 37 ° C for 12 h. Wash several times with deionized water and...

Embodiment 2

[0048] Example 2: Detection of serum miR-21 as an object

[0049] 1. Carry out functional modification on the surface of magnetic beads: the same method as described in Example 1 for functional modification on the surface of magnetic beads.

[0050] 2. Design a functional capture probe and a reporter probe that are complementary to the miR-21 sequence. The 3' end of the capture probe is modified with amino groups, and the 5' end of the reporter probe is modified with biotin. The probe sequences are as follows: 5'-CTGATAAGCTA-(T)15-NH2-3', 5'-Biotin-(T)15-TCAACATCAG-3'.

[0051] 3. Modification of capture probes on the surface of magnetic beads: Dilute carboxylated magnetic beads (5mg / mL) with an equal volume of 2-N-morpholinoethanesulfonic acid hydrate solution (2-(N-morpholino)ethanesulfonic acid hydrate, MES , 25mM, pH 6) Wash several times, discard the supernatant by magnetic separation, add the probe (1μM) diluted in MES solution to the washed carboxylated magnetic beads,...

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Abstract

The invention discloses a nucleic acid detection method for separating a luminous marker based on magnetic beads and nucleic acid hydrolysis. The method comprises the following steps: designing a functional probe for detecting a target nucleic acid fragment; modifying the functional probe on the surface of functional magnetic beads; capturing the target nucleic acid fragment and a luminous marker to the surface of the magnetic beads by virtue of hybridization; performing magnetic separation; cleaning, thereby obtaining the magnetic beads on which the luminous marker is obtained; adding a nucleic acid hydrolysis reagent, separating the luminous marker from the surface of the magnetic beads by virtue of nucleic acid hydrolysis, thereby obtaining a luminous marker solution which does not contain the magnetic beads; and performing luminous detection on the solution, thereby acquiring the target nucleic acid information. According to the method disclosed by the invention, the luminous signal loss caused by a shielding effect of the magnetic beads on the luminous signal is completely overcome, 0.1pM of nucleic acid fragments can be detected at least, and the detection sensitivity is high.

Description

technical field [0001] The invention relates to the fields of biochemical detection and molecular diagnosis, in particular to a nucleic acid detection method based on magnetic beads and nucleic acid hydrolysis to separate luminescent markers. The invention can be applied to the field involving the detection of ultrasensitive nucleic acid molecules. Background technique [0002] In recent years, due to frequent vicious public health incidents, such as the outbreak of Escherichia coli (E.coli) O157:H7 food poisoning in Japan in 1996, it caused several deaths and more than 10,000 infections, causing huge social panic; 2003 The raging SARS virus in my country in 2005; the harm of Streptococcus suis in 2005; the recent avian influenza virus and the H1N1 influenza virus that spread around the world in 2009 have all caused serious threats to people's health and lives. In addition, pathogenic microbial contamination is also the most important factor affecting food hygiene and safety...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/70C12R1/93
CPCC12Q1/6823C12Q2563/143C12Q2563/103
Inventor 何农跃杨淏文李智洋苏恩本
Owner SOUTHEAST UNIV
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