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Legionella DNA (deoxyribonucleic acid) extraction kit and method for extracting legionella DNA

A Legionella and kit technology, applied in the direction of DNA preparation, recombinant DNA technology, etc., to achieve the effect of simple configuration, simple and fast operation method, and avoid cross-contamination

Inactive Publication Date: 2014-01-15
SHANGHAI KINGMED DIAGNOSTICS INST
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Problems solved by technology

At present, common bacterial genomic DNA extraction methods include alkaline lysis method, Chelex-100 method, lysozyme method, boiling method and SDS lysis method, etc. Chelex-100 method and boiling method are simple and effective rapid DNA extraction methods. It is widely used in the extraction of bacterial DNA, but the conventional Chelex-100 method and boiling method have certain limitations when dealing with clinical specimens

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Embodiment Construction

[0016] The present invention extracts Legionella genome DNA from clinical specimens and environmental water samples as specific examples to further illustrate the present invention.

[0017] 1. Preparation of Legionella DNA Extraction Kit

[0018] Element concentration best value Chelex100 solution 5～10% 5% Tris-HCL Hydrochloride (Tris-HCL) 8~12mM 10mM Disodium ethylenediaminetetraacetic acid (EDTA-Na 2 ) 0.1~0.3mM 0.1mM Sodium azide (NaN3) 0.1～0.3% 0.1% Proteinase K (Protease-k) 100~200μg / ml 200μg / ml Water H 2 o ----

[0019] Table 1 The main components of Legionella DNA extract: (mM in the table is millimole per liter)

[0020] 2. Configuration of 200ml Legionella DNA Extraction Kit:

[0021] First weigh 0.24g of trishydroxymethylammonium methane, add sterile water to 100ml, mix and dissolve; add 10μl of concentrated hydrochloric acid, adjust the pH value to 8.3; add 7.5mg of disodium edetate, mix and diss...

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Abstract

The invention relates to a method for extracting legionella DNA (deoxyribonucleic acid) from a clinic sample and an environmental water sample, and an extraction kit. The method comprises the following steps: denaturating protein in the samples by virtue of sodium azide and a Chelex-100 solution, after proteinase K is adequately digested, chelating non-nucleic acid organic matters by virtue of Chelex-100 solution, then centrifuging to remove Chelex particles, and separating other impurities from the DNA, thus dissolving the DNA of a supernatant in trihydroxymethyl aminomethane hydrochloride and ethylenediamine tetraacetic acid solution, and purifying and recovering the DNA. According to the method disclosed by the invention, a plurality of samples can be extracted simultaneously in a short time, the normal working time is less than 60 minutes, and DNA samples suitable for subsequent molecular biological analysis are obtained. The DNA samples obtained by the kit established by the method disclosed by the invention are high in purity; moreover, due to a single-tube operation, the pollution caused by transformation can be effectively avoided; the kit is low in cost and wide in use range.

Description

technical field [0001] The invention belongs to the technical field of genomic DNA separation and purification, in particular to a genomic DNA kit and a method for rapidly extracting Legionella DNA from clinical specimens and environmental water samples. Background technique [0002] Legionella, a facultative intracellular parasite, is an important pathogen that causes legionnaires' disease in humans. In recent years, Legionnaires' disease has broken out frequently around the world, and my country has listed it as one of the 14 emerging infectious diseases. According to foreign reports, 10% of nosocomial pneumonia infections of unknown cause are caused by Legionella. Although currently known Legionella has 52 species, more than 70 serotypes. Therefore, the identification of Legionella is of great value for the diagnosis of Legionnaires' disease. [0003] DNA extraction is the basic technology of molecular biology research, and the purity and structural integrity of the ex...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 朱庆义胡朝晖赵利伟
Owner SHANGHAI KINGMED DIAGNOSTICS INST
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