Pathogen detection background microorganism judgment method and application

A microbiological and background technology, applied in the field of bioinformatics analysis, can solve the problems of batch false positives, fluctuations, high internal references, etc., and achieve the effect of wide range of functions and high application scalability

Active Publication Date: 2022-05-17
GZ VISION GENE TECH CO LTD +3
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This type of method can only obtain a list of potential background contamination, and use a one-size-fits-all approach to directly remove these microorganisms
However, common clinical pathogens, such as Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Stenotrophomonas maltophilia, Escherichia coli, Enterococcus faecalis, Serratia marcescens, etc., all exist at the same time Microorganisms in the background of nucleic acid extraction or reagents cannot determine whether the microorganisms in the sample are background contamination or originate from the sample itself.
[0007] The other method is to quantify by adding internal references to remove microorganisms whose relative abundance is below the threshold. This method has higher requirements for internal references and requires an internal reference set with accurate quantification and high complexity, and the operation is more complicated.
However, in actual operation, we found that different sample types require different pre-processing steps. When adding internal references will have different effects on the final quantitative results. Different sample types need to calculate the corresponding thresholds separately. In addition, this method is based on strict Assuming that the nucleic acid of background microorganisms is a stable constant, it is often found that the background contamination of different batches of experimental consumables will still fluctuate in practical operations, so it is easy to have batches of false positives or false negatives

Method used

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  • Pathogen detection background microorganism judgment method and application
  • Pathogen detection background microorganism judgment method and application
  • Pathogen detection background microorganism judgment method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0107] A method for determining pathogen detection background microorganisms, such as figure 1 shown, including the following steps:

[0108] 1. Determine the list of core background microorganisms.

[0109] 1.1 Sample analysis.

[0110] Take about 20,000 mNGS DNA samples sequenced in our company's Guangzhou laboratory. According to the time sequence of the samples, every 2,000 samples are used as a data set (each data set contains various types of samples at the same time, such as Alveolar lavage fluid, sputum, cerebrospinal fluid, throat swab, blood, tissue, etc.).

[0111] Firstly, obtain the microorganisms whose occurrence frequency is greater than 25% in the data set and appear in all the data sets, that is, the microorganisms that appear frequently and stably in the samples.

[0112] Then calculate the pearson correlation test and spearman correlation test between the nucleic acid extraction concentration or library concentration of these samples and the number of spe...

Embodiment 2

[0138] 639 clinical samples were collected as a verification set, and the method in Example 1 was used to analyze and judge whether the characteristic sequence in the sample was a background sequence.

[0139] Sequence according to the method of Example 1, obtain wherein Acinetobacter baumannii gene sequence data, analyze according to the above-mentioned method, build a model, draw the ROC curve, the result is as follows Figure 4 As shown, judging by the threshold value of 0.041, the AUC value was 0.992, the specificity was 0.942, and the sensitivity was 0.983.

[0140] The above results show that the method for determining background microorganisms in pathogen detection of the present invention can achieve a better discrimination effect on samples from different source batches, and has the advantages of high determination stability and strong practicability.

[0141] It can be understood that the judgment method in the above embodiment is applicable to a wide range of sample...

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Abstract

The invention relates to a pathogen detection background microorganism judgment method and its application, and belongs to the technical field of bioinformatics analysis. The judgment method includes the following steps: determine the list of core background microorganisms: take a number of biological samples, compare the gene sequence data of each microorganism to the characteristic sequence region of the corresponding microorganism, and perform a correlation test with the nucleic acid extraction concentration or library concentration to obtain the correlation The list of microorganisms that are negatively correlated can be used to obtain the list of core background microorganisms; determine the core background microorganism correction index CBI: the sum of the specific comparison sequence numbers of all microorganisms in the above core background microorganism list is the CBI; background sequence judgment: the sample to be tested The judgment value was obtained by dividing the number of specific alignment sequences of microorganisms in the medium and the CBI. Using this method, the background microorganism index can be used to correct the background-related microorganisms, and the presence or absence of the pathogenic microorganisms in the sample can be judged according to the corrected amount, so that the result can be judged more accurately.

Description

technical field [0001] The invention relates to the technical field of bioinformatics analysis, in particular to a method and application for determining background microorganisms in pathogen detection. Background technique [0002] Pathogenic metagenomics (mNGS) is a high-throughput sequencing technology that does not depend on culture, directly extracts nucleic acids from clinical specimens and detects pathogens. Compared with traditional clinical laboratory detection methods, pathogenic mNGS is based on the detection of sequences at the nucleic acid level, which can break through the limitations of different pathogen types, comprehensively cover thousands of pathogens without bias, and simultaneously identify bacteria, fungi, viruses and Various types of pathogenic microorganisms such as parasites, pathogenic mNGS has gradually become an important tool in the field of clinical microbial identification. [0003] However, the accuracy of mNGS is affected by contaminants—DN...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G16B40/00G16B20/30G16B30/10
CPCG16B40/00G16B20/30G16B30/10Y02A50/30
Inventor 许腾何福生李晓蕾谢淑媚王小锐李永军苏杭
Owner GZ VISION GENE TECH CO LTD
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