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Host removal extraction and database building method for sputum microorganism metagenome

A technology of metagenomics and extraction methods, which is applied in the field of host-free extraction and library construction of sputum microbial metagenomics, which can solve the problems of low initial quantity, inability to meet multiple library types and multiple sequencing platforms at the same time, and achieve compatibility Good performance, beneficial to the effect of quantitative stability

Active Publication Date: 2018-05-18
湖南赛哲智造科技有限公司
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  • Abstract
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AI Technical Summary

Problems solved by technology

At present, the existing transposase library construction kits cannot meet the requirements of multiple library types and multiple sequencing platforms at the same time, and meet the requirements of high efficiency and accurate quantification
[0005] At present, in the research of respiratory metagenomic sequencing, there are no nucleic acid extraction methods and kits specifically for sputum microorganisms to remove the host genome, as well as a complete low-input, rapid library construction method

Method used

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  • Host removal extraction and database building method for sputum microorganism metagenome
  • Host removal extraction and database building method for sputum microorganism metagenome
  • Host removal extraction and database building method for sputum microorganism metagenome

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Embodiment 1

[0050] A method for extracting host-free microbial metagenomic groups from sputum, specifically comprising the following steps:

[0051] 1. Sample pretreatment

[0052] (1) Sample preparation: The sample materials are about 60 cases of sputum and airway lavage fluid from a group of patients with severe pneumonia provided by Nanfang Hospital of Southern Medical University. About 2 mL of sputum sample was loaded into a 5 cm high cylindrical sputum collection tube, concentrated at the bottom, and stored at -80 °C.

[0053] (2) Sample instant dissolution: place the bottom of the sputum collection tube in a 37°C water bath and shake continuously to make the sample change from solid to dissolved liquid within 2 minutes. If the sample is in a frozen state, instant dissolution is required, and if it is at 4°C or a fresh sample, this step can be omitted. Instant thawing allows the sample to be thawed in a short time without destroying the sample itself and its cellular integrity.

...

Embodiment 2

[0105] A method for constructing a sputum microbial metagenomic library, specifically comprising the following steps:

[0106] 1. Sample genomic DNA extraction

[0107] Extract with reference to the method of embodiment 1.

[0108] 2. Genomic DNA processing

[0109] (1) Add 10 µL of reaction buffer and 5 µL of gDNA (accurately quantified 1 ng of total QB) to mix, then add 5 µL of transposase mixture (ATM), and mix well;

[0110] (2) The mixture was reacted on a PCR machine for 6 minutes at 55°C to fragment the genome into a length of 230 bp, and then 5 µL of termination reaction buffer (NT) was added to terminate the enzyme reaction.

[0111] 3. Library amplification

[0112] (1) Add a combination of index primers to the processed genomic DNA;

[0113] (2) Add 15µL PCR Enzyme Master Mix, mix and centrifuge;

[0114] (3) React the mixture on a PCR instrument. The PCR amplification conditions are: 72°C for 3 minutes; 95°C for 30s; 95°C for 10s, 55°C for 30s, 72°C for 30s, 1...

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Abstract

The invention belongs to the technical field of microorganism molecular biological detection, and specifically discloses a host removal extraction method for a sputum microorganism metagenome. The method comprises primary sputum liquefaction, secondary sputum liquefaction, microorganism wall breaking, DNA extraction and nucleic acid quality detection. The invention further discloses a database building method for a sputum microorganism metagenome, wherein the method comprises genome DNA extraction, genome DNA treatment, library amplification, library pre-purification, two-step fragment selection and computer detection. A mild digestion method combining trypsin with SDS is adopted to release a host genome to a solution, thereby obtaining microorganism with complete cell wall structure maintained and extracting nucleic acid from microorganisms with host removed. The provided extraction method and database building method are good in compatibility, and can be widely used to a mainstream sequencing platform. The methods are flexible and simple, efficient and fast, and cost-saving, and have bright popularization application prospects.

Description

technical field [0001] The invention relates to the technical field of microbial molecular biology detection, in particular to a method for dehosting extraction and library building of sputum microbial metagenomics. Background technique [0002] In recent years, research in the field of microbial metagenomics has become more and more popular, especially in the field of human intestinal microbes and human health. However, the progress of respiratory microbes has been slow due to the difficulties in processing respiratory samples and building libraries. Research on respiratory microbiology mainly focuses on respiratory diseases, such as severe pneumonia, tuberculosis, cystic fibrosis, influenza and other infectious diseases. Common sample types for these diseases are saliva, sputum, airway lavage fluid, etc. Oxystreptococcus, Streptococcus muller, Bacteroides, Moraxella catarrhalis, Pseudomonas aeruginosa, respiratory fungi, respiratory viruses, etc. Most of these pathogenic ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12N1/06C40B50/06
CPCC12N1/06C12N15/1003C12N15/1013C40B50/06C12Q2521/537
Inventor 伍泳彰陈杰
Owner 湖南赛哲智造科技有限公司
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