Metagenomics-based respiratory tract throat swab sample library building method and pathogen detection method

A technology of metagenomics and respiratory tract, which is applied in the field of database construction and pathogen detection of respiratory throat swab samples based on metagenomics, which can solve the problem that the nucleic acid content is difficult to meet the requirements of two library constructions, increase the detection time and cost, and reduce the nucleic acid content. and other problems, to achieve the effect of optimizing the cDNA synthesis process, saving detection time and cost, and obtaining accurate results.

Active Publication Date: 2021-04-13
中国人民解放军疾病预防控制中心
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Problems solved by technology

However, in many cases in actual work, it may not be possible to judge based on experience that the pathogens causing the infection are bacteria, viruses or other types of pathogens, and once the wrong library construction method is selected, the pathogens may be missed.
On the other hand,...

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  • Metagenomics-based respiratory tract throat swab sample library building method and pathogen detection method
  • Metagenomics-based respiratory tract throat swab sample library building method and pathogen detection method
  • Metagenomics-based respiratory tract throat swab sample library building method and pathogen detection method

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Embodiment

[0081] In the throat swabs of healthy people, a specific pathogen of different concentrations was artificially added, and the metagenomic pathogen detection based on the above method and the pathogen detection by fluorescent quantitative PCR method were carried out respectively. It has high consistency, and the number of pathogen sequences obtained by detection has a high correlation with the concentration of pathogens in the sample and the Ct value of fluorescent quantitative PCR.

[0082] On this basis, a variety of different types of pathogens were added to the throat swabs of healthy people at the same time. It is expected to be further promoted and applied to pathogenic metagenomic detection of throat swab samples.

[0083] The specific steps and results of the experiment are as follows:

[0084] Metagenome sequencing detection was performed by adding mock samples of known concentrations of pathogens to throat swabs. Pharyngeal swab samples from 7 healthy people were co...

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Abstract

The invention provides a metagenomics-based respiratory tract throat swab sample library building method and a pathogen detection method, and relates to the technical field of microbiological detection. The library building method comprises the following steps: (a) pretreatment of a to-be-detected sample solution: adding lysozyme for digestion; (b) extraction of total nucleic acid of the sample: not adding carrier RNA in an extraction process; (c) cDNA synthesis: carrying out reverse transcription by adopting a random primer; (d) purifying a product synthesized in step (c) by using magnetic beads; and (e) construction of a sample DNA library. Based on a DNA-RNA co-sequencing principle, the invention provides the metagenomics-based method suitable for throat swab sample library building and detection and pathogen detection of a throat swab sample metagenome can be realized through a single reaction.

Description

technical field [0001] The invention relates to the technical field, in particular to a metagenomics-based method for building a library of respiratory and throat swab samples and a method for detecting pathogens. Background technique [0002] There are various types of pathogens that cause respiratory infections, including viruses, bacteria, fungi, etc. Most of them use DNA as the carrier of genetic information, but some viruses (such as influenza virus, coronavirus, etc.) use RNA as the carrier of genetic information. Metagenome sequencing of respiratory clinical samples has now become an important method for the detection of respiratory infection pathogens. By directly detecting all microbial nucleic acids in the sample, it can not only detect multiple pathogens in the sample at the same time, but also discover some conventional methods. New or rare pathogens that are difficult to identify. Due to some differences in the sequencing methods for DNA or RNA, the current con...

Claims

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Application Information

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IPC IPC(8): C12Q1/6806C12N15/10C12Q1/6869C12Q1/689C12Q1/70C40B40/06C40B50/06
CPCC12Q1/6806C12Q1/6869C12Q1/689C12Q1/701C40B50/06C40B40/06C12N15/1013C12Q2535/122
Inventor 李鹏宋宏彬林彦锋王凯英李瑾慧杨朗李沛翰贾雷立
Owner 中国人民解放军疾病预防控制中心
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