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A method for dehosting extraction and library construction of sputum microbial metagenomics
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A technology of metagenomics and extraction methods, which is applied in the field of host-free extraction and library construction of sputum microbial metagenomics. Cost, good prospects for promotion and application, and good compatibility
Active Publication Date: 2021-10-29
湖南赛哲智造科技有限公司
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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
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Embodiment 1
[0050] A sputum microorganism metagenome dehosting extraction method, specifically comprising the following steps:
[0051] 1. Sample pretreatment
[0052] (1) Sample preparation: The sample materials were about 60 cases of sputum and airway lavage fluid from a group of severe pneumonia patients provided by the Southern Hospital of Southern Medical University. About 2 mL of sputum sample was loaded into a 5 cm high cylindrical sputum collection tube, collected at the bottom, and stored at -80°C.
[0053] (2) Instant dissolving of the sample: place the bottom of the sputum collection tube in a 37°C water bath and shake continuously to make the sample change from a solid state to a dissolved liquid state within 2 minutes. If the sample is in a frozen state, it needs to be instant-dissolved. If it is a 4°C or fresh sample, this step can be omitted. The instant solution allows the sample to be thawed in a short time without destroying the sample itself and its cellular integrity...
Embodiment 2
[0105] A sputum microbial metagenomics method for building a library, specifically comprising the following steps:
[0106] 1. Sample genomic DNA extraction
[0107] Extract according to the method of Example 1.
[0108] 2. Genomic DNA processing
[0109] (1) Add 10µL of reaction buffer and 5µL of gDNA (total QB accurately quantified 1ng) and mix, then add 5µL of transposase mixture (ATM), and mix well;
[0110] (2) The mixture was reacted on a PCR machine at 55°C for 6 minutes, the genome was fragmented to a length of 230bp, and then 5µL of termination buffer (NT) was added to terminate the enzymatic reaction.
[0111] 3. Library amplification
[0112] (1) adding a combination of index primers to the treated genomic DNA;
[0113] (2) Add 15µL PCR amplification enzyme master mix, and mix and centrifuge;
[0114] (3) The mixture was reacted on a PCR instrument, and the PCR amplification conditions were: 72°C for 3 min; 95°C for 30s; 95°C for 10s, 55°C for 30s, 72°C for 30s...
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Abstract
The invention belongs to the technical field of microbial molecular biology detection, and specifically discloses a sputum microbial metagenomic dehostization extraction method, including the first sputum liquefaction, the second sputum liquefaction, microbial wall breaking, DNA extraction and nucleic acid extraction. Quality testing and other steps. The invention also discloses a method for constructing a sputum microbial macrogene library, which includes the steps of sample genome DNA extraction, genome DNA processing, library amplification, library initial purification, two-step fragment selection, on-machine detection and the like. The invention adopts a gentle digestion method combined with trypsin and SDS to release the host genome into the solution, obtain microorganisms with intact cell wall structure, and realize dehostized microorganism nucleic acid extraction. The extraction method and database construction method provided by the present invention have good compatibility, can be widely used in mainstream sequencing platforms, have the advantages of flexibility, simplicity, high efficiency, quickness, and cost saving, and have good prospects for popularization and application.
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 metagenomes. 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 gut microbes and human health, and significant progress has been made. Respiratory microbiology research is mainly 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. Common bacteria include Staphylococcus aureus, Streptococcus pneumoniae, Hemolytic Streptococcus, Enterobacteriaceae, Mycobacterium tuberculosis, Hepatitis B virus, Oxygen Streptococcus, Miller Streptococcus, Bacteroidetes, Moraxella catarrhalis, Pseudomonas aeruginosa, respiratory fungi...
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