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Method for detecting and identifying pathogens of children infectious diseases based on metagenomic sequencing

A technology for infectious diseases and metagenomics, which is applied in the field of pathogen detection and identification of infectious diseases in children, and can solve problems such as difficulty in obtaining, few applications, and small sample size

Inactive Publication Date: 2020-07-10
CHILDRENS HOSPITAL OF FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, mNGS is mainly used in the detection of infectious diseases in adults, and the application in children is less
Due to the difficulty in obtaining children's samples and the small sample size (for example, cerebrospinal fluid is usually less than 1ml, blood 1-2ml), it poses challenges for nucleic acid extraction and mNGS library construction
In addition, since mNGS is the sequencing of the genomes of all microbial populations, the background contamination of laboratories or reagents and the presence of colonized flora in different parts of the human body make the detection results of mNGS usually more complicated

Method used

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  • Method for detecting and identifying pathogens of children infectious diseases based on metagenomic sequencing
  • Method for detecting and identifying pathogens of children infectious diseases based on metagenomic sequencing
  • Method for detecting and identifying pathogens of children infectious diseases based on metagenomic sequencing

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Embodiment 1

[0064] This embodiment provides a method for detecting and identifying pathogens of infectious diseases in children based on metagenomic sequencing. The specific processing method includes the following steps:

[0065] (1) Process the collected cerebrospinal fluid: take 1.5ml of the sample to be tested and centrifuge at 20000G for 5min, discard the supernatant, and obtain the processed sample;

[0066] (2) Use the Qiagen UCP Pathogen Small Kit to extract the DNA of the treated sample; then use the Qubit fluorescence quantitative instrument to measure the concentration of the obtained DNA, and use agarose gel electrophoresis for quality control. It is required that the sample DNA can be clearly The band of genomic DNA was observed, and the sample DNA was obtained;

[0067] (3) The steps of using the KAPA DNA library preparation kit to construct the sample sequencing library include DNA fragmentation, end repair, adapter ligation, screening of DNA with a fragment size of 250-450...

Embodiment 2

[0073] This embodiment provides a method for detecting and identifying pathogens of infectious diseases in children based on metagenomic sequencing. The specific processing method includes the following steps:

[0074] (1) Process the collected throat swab samples: first, shake and squeeze, first add 2 mL of sterile PBS to soak the throat swab for 10 minutes, and shake for 30 seconds every 2 minutes, squeeze the throat swab cotton swab to fully obtain the eluent , and then draw 2mL of eluent as the processed sample;

[0075] (2) Use the Qiagen UCP Pathogen Mini Kit to extract the DNA of the treated sample, measure the concentration of the obtained DNA with a Qubit fluorescence quantitator, and use electrophoresis for quality control to obtain the sample DNA;

[0076] (3) The steps of using the KAPA DNA library preparation kit to construct the sequencing library of the sample DNA include DNA fragmentation, end repair, adapter connection, screening of DNA with a fragment size of...

Embodiment 3

[0082] This embodiment provides a method for detecting and identifying pathogens of infectious diseases in children based on metagenomic sequencing. The specific processing method includes the following steps:

[0083] (1) Process the collected whole blood samples: First, separate the plasma, take 2ml of whole blood and centrifuge at 1600G, 4°C for 10min, separate the plasma without touching the buffy coat, then centrifuge at 16000G, 4°C for 10min, and absorb the supernatant as processed samples;

[0084] (2) Extract the DNA of the processed sample: extract the processed sample DNA from whole blood, use the MagMAX free DNA separation kit, then use the Qubit fluorescence quantitator to measure the concentration of the obtained DNA, and use electrophoresis to determine the quality Control, obtain sample DNA;

[0085] (3) Use the KAPA DNA library preparation kit to construct the sample sequencing library: the steps of constructing the sequencing library from the sample DNA obtai...

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Abstract

The invention relates to a method for detecting and identifying pathogens of children infectious diseases based on metagenomic sequencing. The method comprises the following specific steps: extractingtrace sample DNA after infection of a child, and carrying out concentration determination and quality control on the DNA; establishing a sequencing library of the sample DNA, and then carrying out high-throughput sequencing and screening; and comparing the treated sample sequence with a reference database to obtain a sequence with a comparison ratio of 70% or above and no multiple comparison, andthen carrying out screening of pathogenic microorganisms to complete the detection and identification method. According to the method disclosed by the invention, a DNA extraction method and a database establishment method are optimized aiming at trace samples of children, so that the success rate of database establishment of the trace samples and the effective data volume of sequencing are improved. A pathogenic microorganism screening and identifying strategy is established, laboratory and reagent pollution is effectively eliminated, and identification of pathogenic microorganisms can be effectively completed.

Description

technical field [0001] The invention belongs to the technical field of microbial detection, in particular to a method for detecting and identifying pathogens of infectious diseases in children based on metagenomic sequencing. Background technique [0002] Infectious disease is one of the most common diseases in children, and it is the focus of clinical attention in pediatrics. The clinical manifestations of infection in children are atypical and mostly non-specific, making early identification difficult through clinical manifestations. On the basis of quickly and accurately identifying the pathogen of infection and choosing a reasonable treatment plan, the occurrence and development of the disease can be effectively controlled. Therefore, early diagnosis and early treatment of infectious diseases in children are of great significance to reduce child mortality. [0003] Traditional pathogen detection methods include smear microscopy, immunological detection, microbial cultu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6869G16B30/10
CPCC12Q1/689C12Q1/6869G16B30/10
Inventor 甘明宇吴冰冰周文浩卢宇蓝王立波王传清
Owner CHILDRENS HOSPITAL OF FUDAN UNIV
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