Method and device for quantitatively detecting metagenome pathogens based on internal reference

Abstract

The invention relates to a method and a device for quantitatively detecting metagenome pathogens based on internal reference. The method comprises the following steps of: adding an internal referencesequence with a set content into a sample to be detected containing a pathogen nucleic acid sequence and a human-derived nucleic acid sequence; extracting nucleic acid from the to-be-detected sample added with the internal reference sequence, and performing sequencing library construction and computer sequencing by using the nucleic acid to obtain sequencing data; counting the total sequencing sequence number, the internal reference specific detection sequence number and the pathogen specific detection sequence number; calculating the concentration of the human-derived nucleic acid in the sample to be detected; and calculating the concentration of the to-be-detected pathogenic nucleic acid according to the concentration of the human-derived nucleic acid. According to the invention, a theoretical calculation model based on a metagenomics pathogen detection technology is established, and the model can carry out auxiliary analysis on abnormal conditions occurring in pathogen metagenome detection.

Description

technical field [0001] The invention relates to the technical field of pathogen detection, in particular to a method and device for quantitative detection of metagenomic pathogens based on internal references. Background technique [0002] With the development of molecular biology technology and the reduction of the cost of next-generation sequencing, the detection technology of pathogenic microorganisms based on metagenomic method is more and more clinically applied, and its rapid, comprehensive and accurate characteristics are the pathogenic microorganisms of clinically infected patients. Diagnostics are of great help. [0003] However, the detection based on metagenomics method also has certain limitations, because it is affected by many factors, including the content of human nucleic acid, pathogen concentration, pathogen genome size and sequencing depth, etc. Negative phenomenon. In order to explain this false negative phenomenon, it is often necessary to check the ab...

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Problems solved by technology

[0003] However, the detection based on metagenomics method also has certain limitations, because it is affected by many factors, including the content of human nucleic acid, pathogen concentration, pathogen genome size and sequencing depth, etc. Negative phenomenon

In order to explain this false negative phenomenon, it is often necessary to check the above influencing factors one by one, which is time-consuming and labor-intensive, which has caused great limitations in the promotion of this technology in clinical practice.

[0004] At present, there are related literatures (Miller S, Naccache S N, Samayoa E, et al. Laboratory validation of a clinical metagenomic sequencing assay for pathogen detection in cerebrospinal fluid [J]. Genome research, 2019, 29(5): 831-842.) report , in the metagenomic pathogen detection process, adding phage internal reference quality control products for quality control can mainly achieve two aspects of quality control. On the one hand, it can check the integrity of the reagents used in the entire detection process, equipment functions and inhibitors. At the same time, the detection of internal standard quality control products can also be used to prompt the nucleic acid content in the sample. The article believes that in the process of metagenomic detection of cerebrospinal fluid samples, when the detection value of internal reference is lower than 100 sequences , indicating that the sample contained more human nucleic acid, but did not give a clear range of human nucleic acid content. At the same time, this method failed to analyze the content of pathogenic nucleic acid in the sample, and did not make full use of the metagenomic sequencing. The role of internal quality control products

[0005] In addition, there are also literature (Blauwkamp T A, Thair S, Rosen M J, et al.Analytical and clinical validation of a microbial cell-free DNA sequencing test for infectious disease[J].Nature microbiology,2019,4(4):663.) report, In the plasma metagenomic sequencing, a degenerate base sequence is added as an internal reference quality control product, and the content of the pathogen molecule to be tested is evaluated according to the test results of the internal reference quality control product, but the estimated pathogen content is not consistent with the general clinical concentration calculation method. And the conversion relationship is not clear, so it cannot provide guidance in the clinical use process

[0006] The existing internal reference quality control can only perform quality control on the detection process, and cannot provide additional auxiliary analysis, including the calculation of the human nucleic acid content in the sample and the calculation of the pathogen content through the internal reference, which cannot fully demonstrate the multiple quality of the internal reference quality control. effect

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