An extracting method of an alveolar lavage fluid metagenome

A technology of alveolar lavage fluid and extraction method, which is applied in the field of alveolar lavage fluid macrogenome extraction, can solve the problems of affecting diagnostic accuracy, easy pollution, and difficulty in ensuring the quality of sputum, and achieves improved DNA elution efficiency, bacteria Cracking is sufficient and the effect of reducing viscosity

Active Publication Date: 2016-12-21
厦门基源医疗科技有限公司
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Problems solved by technology

At present, sputum culture is commonly used for pathogenic diagnosis, but because of its easy contamination, the quality of liquid sputum is difficult to guarantee, which seriously affects the accuracy of diagnosis

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  • An extracting method of an alveolar lavage fluid metagenome
  • An extracting method of an alveolar lavage fluid metagenome

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Embodiment

[0019] A method for extracting the metagenome of alveolar lavage fluid. First, the bronchoalveolar lavage fluid of patients with chronic obstructive diseases is clinically obtained, and then the microbial metagenome in the alveolar lavage fluid is extracted. Specific steps include:

[0020] (1) Sample liquefaction and cell disruption: After getting the fresh lavage fluid sample, add 10 times the volume of 1 mol / L NaOH solution, and let it stand at room temperature for 10 min; after the liquefaction is complete, centrifuge at 14,000 r / min for 5 min, discard the supernatant and keep the precipitate; add 1 ml of deionized water to resuspend the precipitate, and pipette the solution into a new 2 ml centrifuge tube; centrifuge at 12000-r / min for 5 min, keep the precipitate for use; add Incubate 150 μl of lysis buffer in a water bath at 56°C for 10 minutes in advance and 20 μl of lysozyme with a concentration of 10 mg / ml and 3 mmol / L DTT to resuspend by blowing with a pipette tip; a...

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Abstract

A safe, simple, convenient, low-pollution and good-repeatability extracting method of alveolar lavage fluid metagenome DNAs is provided. The method includes steps of liquefying a sample, crushing cells, isolating target DNAs, removing impurity proteins and salt ions, performing DNA adsorption and performing DNA elution. The method can increase the yield and purity of the metagenome DNAs and can meet PCR, DNA library construction, gene sequencing, and other molecular biological experiments.

Description

technical field [0001] The invention provides a method for extracting the metagenome of alveolar lavage fluid, which belongs to the field of biotechnology. Background technique [0002] Lower respiratory tract infection is a relatively common clinical infection, with many types of pathogenic bacteria and strong drug resistance. At present, sputum culture is commonly used for pathogenic diagnosis, but because of its easy contamination, the quality of liquid sputum is difficult to guarantee, which seriously affects the accuracy of diagnosis. Compared with liquid sputum, alveolar lavage fluid has more opportunities to obtain pathogenic bacteria because it is directly collected from the lesion, and can greatly reduce the contamination of samples by oropharyngeal colonization microbiota and improve the isolation rate of pathogenic bacteria. The diagnosis of respiratory infection has higher specificity and sensitivity. The use of alveolar lavage fluid for pathogen diagnosis can ...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/101
Inventor 李珊谢文龙刘青青王松林陈荣山肖辛野李奇渊姚迅
Owner 厦门基源医疗科技有限公司
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