Hybridization solution for in-situ hybridization, preparation method of solution and detection kit

A technology of in situ hybridization and hybridization liquid, which is applied in the direction of biochemical equipment and methods, microbiological determination/inspection, etc. It can solve the problems of reducing the sensitivity of in situ hybridization, affecting clinical differential diagnosis, and weak hybridization signal intensity, so as to speed up hybridization Speed, clear signal and background ratio, effect of increasing hybridization temperature

Active Publication Date: 2019-09-20
XIAMEN TALENT BIOMEDICAL TECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The probe hybridization time of most in situ hybridization products on the market is 4 to 16 hours, but the shorter the hybridization time, the less sufficient the hybridization, the weaker the hybridization signal intensity and the high non-specific background. In this case, Rapid hybridization for 4 hours may reduce the sensitivity of in situ hybridization and affect clinical differential diagnosis

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  • Hybridization solution for in-situ hybridization, preparation method of solution and detection kit
  • Hybridization solution for in-situ hybridization, preparation method of solution and detection kit
  • Hybridization solution for in-situ hybridization, preparation method of solution and detection kit

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preparation example Construction

[0028] A kind of preparation method of above-mentioned hybridization solution for in situ hybridization, comprises the following steps:

[0029] (1) Mix 1.25g of sodium fluoride, 2.5mL of 1M disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, 5mL of 20 times concentration of SSC solution, 0.1mL of 0.5M EDTA solution, 1mL of 50 times The concentration of Denhardts solution is mixed with 2.5g of dextran sulfate, and the pH of the disodium hydrogen phosphate-sodium dihydrogen phosphate buffer is 7.0;

[0030] (2) centrifuging the mixed solution obtained in step (1);

[0031] (3) Add salmon sperm DNA at a final concentration of 0.1 mg / mL, and then add ultrapure water to dilute to 50 mL.

[0032] The main mechanism of action of the hybridization solution for in situ hybridization of the present invention is now described:

[0033] Compared with the conventional hybridization solution, the in situ hybridization solution of the present invention adds the component of fl...

Embodiment 1

[0041] Embodiment one: Kappa and Lambda in situ hybridization detection kit

[0042] 1. Preparation of hybridization solution (50mL):

[0043] (1) Weigh 1.25g of sodium fluoride into a 50mL centrifuge tube;

[0044] (2) Take 2.5mL of 1M disodium hydrogen phosphate-sodium dihydrogen phosphate buffer pH7.0 solution and add it to a 50mL centrifuge tube;

[0045] (3) Add 5mL of 20x SSC solution into a 50mL centrifuge tube;

[0046] (4) Add 0.1mL of 0.5M EDTA solution into a 50mL centrifuge tube;

[0047] (5) Add 1mL of 50x Denhardts solution into a 50mL centrifuge tube;

[0048] (6) Weigh 2.5g of dextran sulfate and add it into a 50mL centrifuge tube to fully shake and mix, and centrifuge at 1000rpm for 5 minutes;

[0049] (7) adding a final concentration of 0.1 mg / mL salmon sperm DNA;

[0050] (8) Add ultrapure water to make the volume 50mL.

[0051] Finally, Kappa and Lambda probes were diluted to a final concentration of 1 ng / uL with the hybridization solution.

[0052] ...

Embodiment 2

[0069] Example 2: HER2 fluorescence in situ hybridization

[0070] 1. Preparation of hybridization solution (50mL):

[0071] (1) Weigh 1.25g of sodium fluoride into a 50mL centrifuge tube;

[0072] (2) Take 2.5mL of 1M disodium hydrogen phosphate-sodium dihydrogen phosphate buffer pH7.0 solution and add it to a 50mL centrifuge tube;

[0073] (3) Add 5mL of 20x SSC solution into a 50mL centrifuge tube;

[0074] (4) Add 0.1mL of 0.5M EDTA solution into a 50mL centrifuge tube;

[0075] (5) Add 1mL of 50x Denhardts solution into a 50mL centrifuge tube;

[0076] (6) Weigh 5g of dextran sulfate and add it to a 50mL centrifuge tube, shake and mix well, and centrifuge at 1000rpm for 5 minutes;

[0077] (7) adding a final concentration of 0.2 mg / mL salmon sperm DNA;

[0078] (8) Add ultrapure water to make the volume 50mL.

[0079] Finally, HER2 probes were prepared with the hybridization solution, including a final concentration of 10 ng / uL HER2 green fluorescent probe, a final ...

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Abstract

The invention relates to a hybridization solution for in-situ hybridization, a preparation method of the solution and an in-situ hybridization detection kit. The hybridization solution for in-situ hybridization comprises the following final concentration components: formamide with the volume percentage concentration not exceeding 2%, fluoride with the weight percentage concentration of 0.1-10%, SSC with the weight percentage concentration of 2-4 times, 1-10mM of EDTA, Denhardts solution with the concentration of 1-5 times, dextran sulfate with the weight percentage concentration of 5-10%, salmon sperm DNA with 0.1-1mg / mL, and 50-100mM of disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution or Tris-HCl buffer solution with the pH of 7.0. The hybridization solution for in-situ hybridization can improve hybridization efficiency and shorten hybridization time under the condition of ensuring hybridization signals.

Description

technical field [0001] The invention relates to in situ hybridization technology, in particular to a hybridization solution for in situ hybridization, its preparation method and in situ hybridization detection kit. Background technique [0002] In 1969, Gall and Pardue first used isotope-labeled probes to determine nucleic acid sequences in intact cells, and in situ hybridization (ISH) developed rapidly. The emergence of immunohistochemical staining methods has promoted the development of non-isotope-labeled nucleic acid probes, making it possible to detect DNA and RNA in conventional formalin-fixed and paraffin-embedded tissues. In formalin-fixed tissues, formaldehyde penetrates between nucleic acids and proteins, which can lead to DNA-DNA and DNA-protein cross-links, preserving antigens in situ. In situ hybridization is a technique that combines molecular hybridization with histology. By using labeled probes such as biotin and digoxin, under appropriate conditions, hydrog...

Claims

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Application Information

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IPC IPC(8): C12Q1/6841
CPCC12Q1/6841C12Q2527/125C12Q2527/101
Inventor 郭金灿
Owner XIAMEN TALENT BIOMEDICAL TECH CO LTD
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