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Host removing method and kit for metagenomes of pathogen microorganisms

A metagenomic and microbial technology, applied in the field of pathogenic microbial metagenomic dehosting methods and kits, can solve the problems of pathogen reduction, human cell enrichment, etc., and achieve the effect of maintaining integrity and providing detection sensitivity

Pending Publication Date: 2021-02-05
SHAOXING INGENIGEN BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, for sputum samples, because they contain a large amount of mucus, cells and proteins, and pathogenic microorganisms are wrapped in sputum, the process of removing the host needs to liquefy the sputum first. Although the process can enrich pathogens such as bacteria and fungi, it will also lead to the enrichment of human cells and the reduction of pathogens such as viruses, chlamydia, and parasites. Therefore, the method of dehosting especially for sputum is not necessarily suitable for dehosting for viruses method

Method used

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  • Host removing method and kit for metagenomes of pathogen microorganisms
  • Host removing method and kit for metagenomes of pathogen microorganisms
  • Host removing method and kit for metagenomes of pathogen microorganisms

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] The denaturant composition of design experiment group is: the saponin of 5%, Triton X-100 1%, Digitonin2%, CHAPS 1%;

[0042] The denaturant composition of the control group is: Triton X-100 1%, saponin 1%;

[0043] The rest of the experimental conditions were controlled the same.

[0044] experiment procedure:

[0045] Sample preparation:

[0046] Take 1.5mL samples of alveolar lavage fluid from 3 patients with clinical infectious diseases, and place them in centrifuge tubes. The patients with clinical infectious diseases were selected from the First Affiliated Hospital of Zhejiang University.

[0047] experiment procedure:

[0048] Take the samples from patients with the same clinical infectious disease and divide them into three equal parts, each containing 1.5mL of samples, and use them as the experimental samples of the experimental group and the control group respectively. A total of 3 experiments were conducted on the same clinical infectious disease patients;...

Embodiment 2

[0055] The denaturant composition of design experiment group 1 is: Triton X-100 0.5%, saponin5%;

[0056] The denaturant components of experimental group 2 are: Triton X-100 0.5%, saponin 1%;

[0057] The denaturant components of experimental group 3 are: Triton X-100 1%, saponin 2%;

[0058] The denaturant composition of experimental group 4 is: Triton X-100 1%, saponin 3%;

[0059] The denaturant composition of experimental group 5 is: Triton X-100 1%, saponin10%;

[0060] The rest of the experimental conditions were controlled the same.

[0061] Sample preparation:

[0062] Take 1.5mL samples of alveolar lavage fluid from 3 patients with clinical infectious diseases, and place them in centrifuge tubes. The patients with clinical infectious diseases were selected from the First Affiliated Hospital of Zhejiang University.

[0063] experiment procedure:

[0064] Samples from patients with the same clinical infectious disease were divided into five equal parts, each contai...

Embodiment 3

[0072] Control group:

[0073] The denaturant composition of design experiment group is: the saponin of 5%, Triton X-100 1%, Digitonin2%, CHAPS 1%;

[0074] The control group does not apply to the human source kit;

[0075] Sample preparation:

[0076] Take 1.5mL samples of alveolar lavage fluid from multiple patients with clinical infectious diseases and place them in centrifuge tubes. The patients with clinical infectious diseases are selected from the First Affiliated Hospital of Zhejiang University.

[0077] Experiments in the experimental group:

[0078] Take the samples from patients with the same clinical infectious disease and divide them into two equal parts, each containing 1.5mL of samples, and use them as the experimental samples of the experimental group and the control group respectively, and conduct 3 experiments on the same clinical infectious disease patients;

[0079] Experiments in the control group: Directly use Xunminkang Second Generation Sequencing Li...

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Abstract

The invention provides a host removing method and kit for metagenomes of pathogen microorganisms, and is particularly suitable for detection of the metagenomes of the pathogen microorganisms includingbronchoalveolar lavage fluid, cerebrospinal fluid, sputum, ascites and the like; by utilizing the characteristics that human cells are inconsistent with bacteria, fungi, viruses and the like in cellstructure and the concentration and the genome size of the human cells are remarkably different from those of the fungi, the bacteria, the viruses and the like, a denaturing agent and an enzyme are selected to respectively dissolve the cells and digest nucleic acid of the human cells, so that the host removal effect is achieved, wherein the denaturing agent at least includes one or more of saponin, Tween 20, Triton X-100, digitonin and CHAPS; and the host removal operation of the metagenomes of the pathogen microorganisms can be completed within 5-20 minutes, and the method can improve the detection sensitivity of the metagenomes of the pathogen microorganisms.

Description

technical field [0001] The invention relates to the field of biological detection, in particular to a method and a kit for removing hosts of pathogenic microorganism metagenomics. Background technique [0002] High-throughput sequencing technology (also known as next-generation sequencing technology (NGS)) is more and more widely used in the traceability, detection, typing and drug resistance assessment of infectious diseases, and it is moving towards a fast and economical direction. develop. In 2008 and 2011, Palacios et al. and Xu et al. used metagenomics and metatranscriptomes to discover two new pathogens of clinical infection cases, namely arenavirus and new bunyavirus, which opened up metagenomics. A precedent for the application of sequencing (mNGS) technology in clinical infection. Since mNGS does not rely on the sequence amplification of specific gene primers, but instead sequences all the DNA or RNA of the sample to be tested, and compares the sequencing data wit...

Claims

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Application Information

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IPC IPC(8): C12Q1/6806
CPCC12Q1/6806
Inventor 胡彬王烨凤褚嘉豪蔡媛媛
Owner SHAOXING INGENIGEN BIOTECH CO LTD
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