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Preserving solution for sputum and preparation method

A technology for preserving liquids and solutions, applied in microorganism-based methods, preservation of human or animal bodies, biochemical equipment and methods, etc., can solve the problems of high cost, inability to produce proteinase K, downstream industries relying on imports, etc. low cost effect

Pending Publication Date: 2020-11-17
长沙健路医学检验所有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But at present, my country is unable to produce pharmaceutical-grade proteinase K, and downstream industries rely heavily on imports
Moreover, proteinase K has a complex production process and high cost, which leads to the high price of commercially available proteinase K, and proteinase K needs to be stored below -20°C, the ideal incubation temperature is 58°C, and the ideal incubation time is 2 hours

Method used

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  • Preserving solution for sputum and preparation method
  • Preserving solution for sputum and preparation method
  • Preserving solution for sputum and preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] A kind of preparation method of sputum preservation solution 1000ml is as follows:

[0015] S1: Prepare 100ml of 1.5% phosphorus trichloride solution;

[0016] S2: Weigh each component according to the following components and contents: 2.5g dithiothreitol, 17.5g trishydroxymethylaminomethane, 0.85g sodium chloride, 0.35g disodium hydrogen phosphate, 0.05g potassium dihydrogen phosphate, 0.65g guanidine isothiocyanate, add step S1 and make phosphorus trichloride solution and 500ml nucleic acid-free water dissolve and stir;

[0017] S3: Adjust the pH of the solution obtained in step S2 to 5.5-8.5 to prepare a preservation solution.

[0018] Among them, Dithiothreitol (DTT for short) is a small molecule organic reducing agent with a chemical formula of C4H10O2S2. It is a linear molecule in the reduced state, and becomes a six-membered ring structure containing disulfide bonds after being oxidized. DTT is used as a reducing agent and deprotecting agent for thiolated DNA ...

Embodiment 2

[0027] A kind of preservation liquid of sputum, the preparation method of every 1000ml preservation liquid is as follows:

[0028] S1: Prepare 100ml of 0.90% phosphorus trichloride solution;

[0029] S2: Weigh each component according to the following components and contents: 0.85g dithiothreitol, 12.5g trishydroxymethylaminomethane, 4.5g sodium chloride, 0.35g disodium hydrogen phosphate, 0.15g potassium dihydrogen phosphate, 1.0 g of guanidine hydrochloride was added to step S1 to prepare phosphorus trichloride solution (0.9%) and 500 ml of nucleic acid-free water to dissolve and stir evenly;

[0030] S3: adjusting the pH of the solution obtained in step S2 to 5.5-8.5 to obtain the preservation solution.

[0031] The main difference between this example and the examples is that the virus is inactivated by replacing guanidine isothiocyanate with guanidine hydrochloride. Guanidine hydrochloride (CH6ClN3), used as a strong denaturant in the experiment of extracting total RNA ...

Embodiment 3

[0037] A kind of preservation liquid of sputum, the preparation method of every 1000ml preservation liquid is as follows:

[0038] S1: Prepare 100ml of 2.0% phosphorus trichloride solution;

[0039] S2: Weigh each component according to the following components and contents: 1.35g dithiothreitol, 8.0g trishydroxymethylaminomethane, 7.8g sodium chloride, 1.25g disodium hydrogen phosphate, 0.13g potassium dihydrogen phosphate , 2.13g guanidine isothiocyanate, add step S1 to make phosphorus trichloride solution and 500ml nucleic acid-free water dissolve and stir evenly, constant volume;

[0040] S3: adjusting the pH of the solution obtained in step S2 to 5.5-8.5 to obtain the preservation solution.

[0041] Use the sputum preserved in the preservation solution for the detection of the new crown nucleic acid detection reagent: take the sputum of 8 healthy people in the above preservation solution, mix well, and test it separately with the new crown nucleic acid detection reagent....

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Abstract

The invention relates to the technical field of biology, in particular to a preserving solution for sputum and a preparation method. The preserving solution of each 1000ML comprises the following components: 0.5-3.5g of dithiothreitol, 5.0-30g of tris(hydroxymethyl)methyl aminomethane, 0.1-10.5g of sodium chloride, 0.01-2.0g of phosphorus trichloride, 0.01-2.5g of disodium hydrogen phosphate, 0.01-2.5g of potassium dihydrogen phosphate, 0.2-3.5g of guanidinium isothiocyanate and the balance nucleic acid-free water, and the pH value is 5.5-8.5. The preserving solution prepared by the inventioncan be stored and applied at room temperature, has a digesting / liquefying function, can effectively digest / liquefy specimens (sputum), does not need to digest the specimens before detection, and meanwhile, has an inactivation effect on specimen (sputum) viruses, so that the biosafety risk of specimen preservation, transportation, processing and detection is lowered; after being collected, sputum specimens collected by using the preserving solution can be stored for 72 hours at 2-8 DEG C, and can be stored for 10 days or more at ultra-low temperature (-70 DEG C); and moreover, the components ofthe preserving solution are easy to obtain, and the preparation cost is low.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a preservation solution for sputum and a preparation method thereof. Background technique [0002] Nucleic acid detection, especially the new crown nucleic acid detection, sputum specimen preservation solution, digestive agent or liquefaction agent, the main ingredient is proteinase K. Proteinase K can degrade the histone tightly bound to nucleic acid, separate DNA from protein, and purify DNA better; proteinase K can degrade RNA hydrolase activity (Rnase), inhibit RNase from hydrolyzing template RNA, and can be used in RNA extraction and detection play an integral role in the process. But at present, my country is unable to produce pharmaceutical-grade proteinase K, and downstream industries rely heavily on imports. Moreover, proteinase K has a complex production process and high cost, which leads to the high price of commercially available proteinase K, and proteinase K needs to ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806C12Q1/70A01N1/02C12R1/93
CPCC12Q1/6806C12Q1/701A01N1/021A01N1/0215C12Q2527/125
Inventor 董存岩蒋峻峰李俊
Owner 长沙健路医学检验所有限公司
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