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Quantitative fungus detection method based on metagenome sequencing

A metagenomic and genomic technology, applied in the field of quantitative detection of fungi based on metagenomic sequencing, can solve the problems of high proportion of human-sourced reads, large interference of genetic background, and influence on the accuracy of results, so as to reduce the proportion and improve the accuracy of sequencing results interference, low cost, and easy operation

Pending Publication Date: 2021-03-16
THE SECOND PEOPLES HOSPITAL OF SHENZHEN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] mNGS technology has a very high clinical application prospect, but it also faces severe problems, mainly manifested in the high proportion of human reads in the sequencing results, the large interference of gene background, and the GC preference. The so-called GC preference refers to the preferential amplification of the genome. Fragments with low GC content in the medium make the amplification efficiency of different fragments different, which may lead to missing or underdetection of important pathogenic bacteria and affect the accuracy of the results
At the same time, the high proportion of human reads in the sequencing read length affects the sequencing efficiency and increases the sequencing cost

Method used

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  • Quantitative fungus detection method based on metagenome sequencing
  • Quantitative fungus detection method based on metagenome sequencing
  • Quantitative fungus detection method based on metagenome sequencing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 Sample Pretreatment

[0056] (1) Remove the host genome

[0057] Take 1mL blood sample, centrifuge at 8000g for 3min, collect the precipitate, discard the supernatant, add 200μL PBS to resuspend the precipitate, and vortex to mix;

[0058] Add 200μL 5% saponin and incubate at room temperature for 10min;

[0059] Add 350μL water to incubate for 30s, centrifuge at 6000g for 5min, discard the supernatant;

[0060] Add 100 μL PBS to resuspend the pellet, add 100 μL HL-SAN buffer (5.5M NaCl and 100 mM MgCl 2 );

[0061] Add 5 μL DNaseI and incubate at 37°C for 40 minutes at 1000 rpm;

[0062] Centrifuge at 6000g for 3min, discard the supernatant, and wash twice with 500μL and 1mL PBS respectively;

[0063] Centrifuge at 10,000 g for 1 min, discard 800 μL supernatant, and obtain a sample from which the host genome has been removed;

[0064] (2) Ultrasonic wall breaking treatment

[0065] The sample from which the host genome was removed was ultrasonically bro...

Embodiment 2

[0067] Embodiment 2 sample pretreatment

[0068] (1) Remove the host genome

[0069] Take 1mL of stool sample, centrifuge at 8000g for 3min, collect the precipitate, discard the supernatant, add 200μL PBS to resuspend the precipitate, and vortex to mix;

[0070] Add 200 μL 2% saponin and incubate at room temperature for 15 minutes;

[0071] Add 350μL water to incubate for 30s, centrifuge at 6000g for 5min, discard the supernatant;

[0072] Add 100 μL PBS to resuspend the pellet, add 100 μL HL-SAN buffer (5.5M NaCl and 100 mM MgCl 2 );

[0073] Add 6 μL DNaseI and incubate at 40°C for 30 minutes at 1000 rpm;

[0074] Centrifuge at 6000g for 3min, discard the supernatant, and wash twice with 500μL and 1mL PBS respectively;

[0075] Centrifuge at 10,000 g for 1 min, discard 800 μL supernatant, and obtain a sample from which the host genome has been removed;

[0076] (2) Microwave wall breaking treatment

[0077] The sample from which the host genome was removed was subject...

Embodiment 3

[0079] Example 3 Sample Pretreatment

[0080] (1) Remove the host genome

[0081] Take 1mL soil sample, centrifuge at 8000g for 3min, collect the precipitate, discard the supernatant, add 200μL PBS to resuspend the precipitate, and vortex to mix;

[0082] Add 200 μL 6% saponin and incubate at room temperature for 10 minutes;

[0083] Add 350μL water to incubate for 30s, centrifuge at 6000g for 5min, discard the supernatant;

[0084] Add 100 μL PBS to resuspend the pellet, add 100 μL HL-SAN buffer (5.5M NaCl and 100 mM MgCl 2 );

[0085] Add 2 μL DNaseI and incubate at 35°C for 60 minutes at 1000 rpm;

[0086] Centrifuge at 6000g for 3min, discard the supernatant, and wash twice with 500μL and 1mL PBS respectively;

[0087] Centrifuge at 10,000 g for 1 min, discard 800 μL supernatant, and obtain a sample from which the host genome has been removed;

[0088] (2) Ultrasonic wall breaking treatment

[0089] The sample from which the host genome was removed was subjected to ...

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Abstract

The invention provides a quantitative fungus detection method based on metagenome sequencing. The method comprises the following steps of: (1) sequentially adding saponin and endonuclease into a sample, and removing a host genome; (2) performing wall breaking treatment on the sample without the host genome, and extracting nucleic acid to obtain a fungus genome; (3) performing library constructionand high-throughput sequencing on the fungus genome to obtain original sequencing data; and (4) comparing the original sequencing data with a fungus database, and performing fungus identification andabundance detection. According to the method, host DNA in the sample is removed by adopting the saponin and the endonuclease before high-throughput sequencing, so that the proportion of a human genomeand the interference to the accuracy of a sequencing result are remarkably reduced; and the wall breaking treatment operation is matched, so that the proportion of the fungus genome is increased, thesequencing cost is reduced, and the method has important significance in the field of fungus detection.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for quantitative detection of fungi based on metagenomic sequencing. Background technique [0002] In recent years, high-throughput sequencing technology (also known as infection metagenomic sequencing, meta next generation sequencing, mNGS for short) has been increasingly used in the detection of pathogenic bacteria. The genetic information of all microorganisms has incomparable advantages compared with other methods for providing typing information and abundance information of pathogenic bacteria. [0003] mNGS technology has a very high clinical application prospect, but it also faces severe problems, mainly manifested in the high proportion of human reads in the sequencing results, the large interference of gene background, and the GC preference. The so-called GC preference refers to the preferential amplification of the genome. Fragments with low GC content in the medium...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/6806C12Q1/06
CPCC12Q1/6895C12Q1/6806C12Q2523/301C12Q2521/301C12Q2525/173C12Q2525/191C12Q2531/113C12Q2535/122
Inventor 刘文兰徐迹黄建林
Owner THE SECOND PEOPLES HOSPITAL OF SHENZHEN
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