Use method for de-host extraction test kit of a metagenome sample

Abstract

The invention provides a use method for a de-host extraction test kit of a metagenome sample, and relates to the field of biological detection. The method comprises the following steps: pathogen enrichment: a step of adding a sample into a first centrifuge tube with a volume of 1.5 mL, carrying out centrifuging for 5-10 min at a room temperature, and removing a supernatant; host cell lysis: a stepof adding 200-1000 [mu]L of a lysis buffer solution, namely Buffer G1 into the first centrifuge tube, and carrying out incubating at 20-40 DEG C for 10-30 min; host DNA removal: a step of centrifuging the first centrifuge tube at a room temperature for 2-5 min so as to remove a supernatant, adding 200-500 [mu]L of the lysis buffer solution, namely the Buffer G1 and 1-3 [mu]L of totipotent nuclease into the first centrifuge tube again, carrying out incubating at 30-40 DEG C for 10-30 min, carrying out centrifuging so as to remove the supernatant, adding 500-1000 [mu]L of the Buffer G1, and washing a precipitate twice; wall breaking of microbial thalli: a step of adding 100-300 [mu]L of lysozyme to resuspend the precipitate, transferring a resuspension to a lysis tube filled with glass beads, placing the lysis tube at 30-40 DEG C, carrying out incubating for 10-30 min, and carrying out vortex uniform mixing treatment for 5-10 min; and microbial lysis and nucleic acid extraction. The usemethod provided by the invention is wide in application range, good in compatibility, good in extraction effect, high in efficiency and low in cost.

Description

technical field [0001] The invention belongs to the field of biological detection, and in particular relates to a method for using a metagenomic sample dehostization extraction kit. Background technique [0002] Compared with traditional culture identification methods, metagenomic sequencing identification has the advantages of short identification period and low requirements on the technical level of operators and identification personnel, and is increasingly used in microbial identification, especially for unknown pathogenic microorganisms. Identification. The current mainstream metagenomic sequencing methods are mainly high-throughput sequencing methods, including next-generation sequencing and third-generation sequencing. However, in most clinical samples, there is often a large amount of host DNA background, and pathogen DNA only accounts for a small part. In whole metagenomic shotgun sequencing studies of different sample types by the Human Microbiome Project, it was...

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Problems solved by technology

[0003] Existing methods for removing host nucleic acids include (1) removing host cells based on size differences between host cells and microorganisms or other physical differences (such as sedimentation velocity), usually with low removal efficiency, unable to remove free host DNA, and also Loss of nucleic acid of fungi, parasites and some intracellular microorganisms; (2) by destroying the free host DNA of the host cell membrane, and then degrading the host DNA by the salt-dependent HL-SAN enzyme, centrifuging to collect bacteria-based microbial cells, and then targeting the bacteria DNA extraction, usually under the condition of relatively high salt ion concentration, many bacteria may also be lost, and the amount of microbial nucleic acid obtained after this process is often very low; (3) Based on the fact that human cells are more fragile than microbial shells, use Hypotonic solution, or mild detergent treatment, only destroys the host cells, frees the host DNA into the solution, and then removes the host DNA by centrifugation to remove the supernatant, such as a sputum microbial metagenomic dehostization extraction And method for building a library (CN108048450 A, Guangzhou Saizhe Biological Technology Co., Ltd.)

Since the host DNA is a macromolecular polymer with high viscosity, some free host DNA may adhere to the surface of the bacteria, so the physical method of simple buffer washing and centrifugation cannot make the free DNA come out. The host DNA is removed completely, resulting in residual host DNA

[0004] In addition, in the process of microbial nucleic acid extraction, due to the wide variety of microorganisms and different shapes, the structure and composition of the cell walls of different microorganisms are not the same, resulting in differences in susceptibility to different cell wall lysis methods. Therefore, any cell wall lysis method preparation Most of the samples are likely to be unrepresentative, which may lead to biases in sample representativeness and detection of relative components of the microbiome

However, the instantaneous high temperature wall-breaking method will cause relatively large damage to the bacterial DNA, resulting in serious loss of microbial nucleic acid and greatly reducing the extraction amount.

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