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A reagent for detecting different subtypes of breast cancer cells based on fluorescence resonance energy transfer

A technology of fluorescence resonance energy and breast cancer cells, which is applied in fluorescence/phosphorescence, measuring devices, and material analysis through optical means, and can solve the problems of high cost of instrument and chip production, low specificity and repeatability, and limited application range , to achieve the effects of improving detection accuracy and sensitivity, improving detection accuracy, and good biocompatibility

Active Publication Date: 2022-01-28
NANJING NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The current traditional detection methods Northern Blot, microarray technology, and real-time quantitative polymerase chain reaction can only detect miRNAs in vitro, but cannot detect miRNAs in situ in living cells
Northern Blot blotting is a standard method for early detection of miRNAs. Although this method allows partial mismatch of probes and the membrane after hybridization can be reused, a large amount of toxic reagents are required in the experimental process, and the processing process is cumbersome and complicated. Large sample volume leads to low sensitivity, and insufficient specificity leads to high false positive results; although microarray technology can analyze multiple components at the same time, this method will cause cross-hybridization and low repeatability due to insufficient specificity , the high cost of making instruments and chips limits its application; real-time quantitative polymerase chain reaction technology is a kind of method with wide practicability. The reaction process does not generate heat, has good accuracy and high sensitivity, but the experimental process requires distribution. Manipulation and precise cycle temperature control, and complex primer design limits its range of applications

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  • A reagent for detecting different subtypes of breast cancer cells based on fluorescence resonance energy transfer
  • A reagent for detecting different subtypes of breast cancer cells based on fluorescence resonance energy transfer
  • A reagent for detecting different subtypes of breast cancer cells based on fluorescence resonance energy transfer

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Embodiment 1

[0058] A reagent for detecting different subtypes of breast cancer cells based on fluorescence resonance energy transfer

[0059] Solution A and Solution B, solution A contains Tris solutions of three hairpin molecular beacon DNA chains HP1, HP2, and HP3, the 3' end of HP1 is labeled with Cy3 fluorescent donor group, and the 3' end of HP2 is labeled with Alexa488 fluorescent donor group The 3' end of HP3 is labeled with the Alexa405 fluorescent donor group; solution B contains the Tris solution of three hairpin molecular beacon DNA chains HP4, HP5, and HP6, and the 5' end of HP4 is labeled with the Cy5 fluorescent acceptor group group, the 5' end of HP5 is labeled with Cy3 fluorescent acceptor group, and the 5' end of HP6 is labeled with Alexa488 fluorescent acceptor group.

[0060] HP1 is composed of a 15-nucleotide (nucleotide, nt) loop and a 12-base-pair (base-pair, bp) stem part, and the stem part has 1 pair of base mismatches, and HP2 is a 5nt loop The HP3 is composed of...

Embodiment 2

[0065] Take 100 μL of solution A and 400 μL of solution B in Example 1 with the concentration of each molecular beacon being 10 μmol / L, and dilute to 1 mL with 20 mmol / L Tris solution. At this time, the concentration of each molecular beacon in solution A was 1 μmol / L before working, and the concentration of each molecular beacon in solution B was 4 μmol / L before working. miR-let-7a was dissolved in 410 μL 20 mmol / L Tris solution as 10 μmol / L long-term storage concentration; then 50 μL 10 μmol / L miR-let-7a solution was dissolved in 950 μL 20 mmol / L Tris solution as 0.5 μmol / L working previous concentration.

[0066] Take 100 μL of 1 μmol / L A solution after the above dilution, 100 μL of 4 μmol / L B solution, and 20 μL of 0.5 μmol / L miR-let-7a solution, and add them together to a quartz dish with a total volume of 1 mL containing 20 mmol / L Tris solution (780 μL). The final working concentration of each molecular beacon in solution A was 0.1 μmol / L, the final working concentratio...

Embodiment 3

[0069]Take 100 μL of the diluted 1 μmol / L A solution in Example 2, 100 μL of the 4 μmol / L B solution, and 20 μL of the 0.5 μmol / L miR-let-7a solution, and add them together to a quartz dish with a total volume of 1 mL containing the 20 mmol / L Tris solution. The final working concentration of each molecular beacon in solution A was 0.1 μmol / L, the final working concentration of each molecular beacon in solution B was 0.4 μmol / L, and the final working concentration of miR-let-7a was 0.01 μmol / L.

[0070] After excitation at the optimal excitation wavelength of 488nm for Alexa488, the fluorescence signal at the Cy3 emission wavelength of 562nm was enhanced, indicating that HP2-HP5 formed a multi-level hairpin-shaped DNA structural unit and could be used for the detection of the target molecule miR-141.

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Abstract

The invention discloses a reagent for detecting different subtypes of breast cancer cells based on fluorescence resonance energy transfer. The 3' end is labeled with the Cy3 fluorescence donor group, the 3' end of HP2 is labeled with the Alexa488 fluorescence donor group, and the 3' end of HP3 is labeled with the Alexa405 fluorescence donor group; solution B contains three hairpin molecular beacons HP4 , HP5, HP6 Tris solution, the 5' end of HP4 is labeled with Cy5 fluorescent acceptor group, the 5' end of HP5 is labeled with Cy3 fluorescent acceptor group, and the 5' end of HP6 is labeled with Alexa488 fluorescent acceptor group. The reagent of the present invention realizes the detection and analysis of the miRNAs target sequence by observing the enhancement of the fluorescence signal of the receptor fluorophore, and then realizes the simultaneous and accurate detection of different subtypes of breast cancer cells.

Description

technical field [0001] The invention belongs to the technical field of biological detection, in particular to a reagent for detecting different subtypes of breast cancer cells based on fluorescence resonance energy transfer. Background technique [0002] Breast cancer has become one of the killers of women's health that cannot be ignored. Due to its strong heterogeneity, Perou first proposed the concept of molecular classification of breast cancer in 2000, and after many scholars and researchers gradually improved it into three categories: Normal-like subtype, basal-like subtype, luminal subtype, human epidermal growth factor receptor 2 overexpression type (Her2++). This is a method to determine the molecular type of breast cancer by detecting the expression of cell surface receptors (mainly estrogen receptor ER, progesterone receptor PgR, human epidermal growth factor receptor 2Her2), although this method has been It is used for the clinical diagnosis of breast cancer clas...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/64
CPCG01N21/6486
Inventor 蔡称心乔玲蔡哲炜胡耀娟吴传丽粱文慧孙宇杰吴萍
Owner NANJING NORMAL UNIVERSITY