De-host extraction kit for phlegm macrogenome

Abstract

The invention provides a de-host extraction method for a sputum pathogenic microorganism metagenome. The method comprises steps of sputum pretreatment, differential lysis to remove the host and nucleic acid extraction. The invention combines DTT and protease to quickly homogenize sputum, and then uses surfactant to differentially lyse cells, reduces interference of human genes, realizes enrichmentof microbial genome, and effectively improves detection rate of metagenomic pathogens in sputum samples.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to a kit for extracting sputum metagenomic dehostization. Background technique [0002] In the environment of increasingly serious air pollution, respiratory diseases, such as tuberculosis, severe pneumonia, cystic fibrosis, chronic obstructive pulmonary disease, etc., have become major public health problems that seriously threaten human health. Common respiratory pathogenic microorganisms include Streptococcus pneumoniae, Mycoplasma pneumoniae, Haemophilus influenzae, Klebsiella pneumoniae, Mycobacterium tuberculosis, Aspergillus, respiratory syncytial virus, adenovirus, etc. Traditional pathogenic microorganism detection methods, such as microscopy, isolation and culture, serological and immunological detection, have disadvantages such as low sensitivity / specificity, time-consuming, and susceptibility to the influence of antibacterial drugs. With the mutation of pathogenic m...

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Problems solved by technology

[0004] 1. NaOH alkali lysis method: refers to the liquefaction of sputum in a certain concentration of sodium hydroxide solution, which takes a short time and has obvious effects, but the strong alkali environment is easy to cause cell rupture and nucleic acid loss

[0005] 2. DTT reduction method: use sulfhydryl (-SH) to break mucin, the main viscous component in sputum, which has a mild effect on cells and can preserve cell integrity, but it is often unable to restore the protein embedded in the structure. And the disulfide bond, so the phenomenon of incomplete digestion often occurs, and it takes a long time

However, in the operation of this method, problems such as insufficient amount of extracted microbial samples will be caused.

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