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

A detection kit and in situ hybridization technology, applied in biochemical equipment and methods, microbiological determination/inspection, etc., can solve the problems of reducing the sensitivity of in situ hybridization, affecting clinical differential diagnosis, weak hybridization signal intensity, etc., and achieve signal and clear background, faster hybridization speed, and higher hybridization temperature

Active Publication Date: 2022-08-05
XIAMEN TALENT BIOMEDICAL TECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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

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preparation example Construction

[0028] A method for preparing the above-mentioned hybridization solution for in situ hybridization, comprising the following steps:

[0029] (1) 1.25 g of sodium fluoride, 2.5 mL of 1 M disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, 5 mL of 20-fold concentration SSC solution, 0.1 mL of 0.5 M EDTA solution, 1 mL of 50-fold concentration The concentrated Denhardts solution is mixed with 2.5 g 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 with a final concentration of 0.1 mg / mL, and then add ultrapure water to make the volume 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 hybridization solution of the in situ hybridization of the prese...

Embodiment 1

[0041] Embodiment 1: 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) Take 5mL of 20x SSC solution and add it to a 50mL centrifuge tube;

[0046] (4) Take 0.1mL of 0.5M EDTA solution and add it to a 50mL centrifuge tube;

[0047] (5) Take 1mL of 50x Denhardts solution and add it to a 50mL centrifuge tube;

[0048] (6) Weigh 2.5g of dextran sulfate into a 50mL centrifuge tube and mix thoroughly, 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 50mL.

[0051] Finally, the hybridization solution was used to dilute the Kappa and Lambda probes to a final concentration of 1 ng / uL.

[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) Take 5mL of 20x SSC solution and add it to a 50mL centrifuge tube;

[0074] (4) Take 0.1mL of 0.5M EDTA solution and add it to a 50mL centrifuge tube;

[0075] (5) Take 1mL of 50x Denhardts solution and add it to a 50mL centrifuge tube;

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

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

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

[0079] Finally, use hybridization solution to prepare HER2 probe, including final concentration of 10ng / uL HER2 green fluorescent probe, fina...

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Abstract

The present invention relates to a hybridization solution for in-situ hybridization, a preparation method thereof and an in-situ hybridization detection kit. The hybridization solution for in situ hybridization includes the following components at final concentrations: formamide with a volume percent concentration of not more than 2%, fluoride with a weight-volume percent concentration of 0.1-10%, 2-4 times the concentration of fluoride SSC, 1-10 mM EDTA, 1-5 times the concentration of Denhardts solution, 5-10% w / v dextran sulfate, 0.1-1 mg / mL salmon sperm DNA, and 50-100 mM with a pH of 7.0 Sodium Hydrogen Phosphate-Sodium Phosphate Buffer or Tris-HCl Buffer. The hybridization solution for in situ hybridization of the present invention can improve the hybridization efficiency and shorten the hybridization time under the condition of ensuring the hybridization signal.

Description

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

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6841
CPCC12Q1/6841C12Q2527/125C12Q2527/101
Inventor 郭金灿
Owner XIAMEN TALENT BIOMEDICAL TECH CO LTD
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