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Capture probe set, method and kit for detecting pathogenic microorganisms and application

A technology of pathogenic microorganisms and capture probes, applied in biochemical equipment and methods, microbial measurement/testing, combinatorial chemistry, etc., can solve the problems that mNGS hybrid capture detection products have not been established, and the effect is not significant

Pending Publication Date: 2021-05-18
GZ VISION GENE TECH CO LTD +4
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For various human host removal schemes through DNA libraries, each has its own shortcomings: for example, it can target the presence or absence of methylation sites in human nucleic acids and microbial nucleic acids, and use specific methylation antibodies to remove methylated sites. Generally, the enrichment efficiency of human nucleic acid is only 3-5 times, and the effect is not significant
Therefore, there are no products on the market that use this technology for broad-spectrum pathogen detection. They are often aimed at the detection of specific types of diseases or individual pathogens, and have not yet established a mNGS that is suitable for different sample types, disease types, and pathogen types. High sensitivity hybrid capture detection products

Method used

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  • Capture probe set, method and kit for detecting pathogenic microorganisms and application
  • Capture probe set, method and kit for detecting pathogenic microorganisms and application
  • Capture probe set, method and kit for detecting pathogenic microorganisms and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] A kit for detecting pathogenic microorganisms, used for rapid hybrid capture of mNGS, designed by the following method.

[0070] 1. Design and synthesis of capture probe sets.

[0071]Review the previous clinical samples of our company, screen the pathogenic microorganisms with high clinical frequency, covering bacteria, fungi, DNA viruses, RNA viruses, parasites, and common drug resistance genes, select the coding region of pathogenic microorganisms as the target region, and based on known For the genome sequence, analyze and screen the specific regions of each species, and design and generate probe groups in the form of tile arrays. The length of the probes is about 120bp, and the probes with biotin-labeled nucleotides are synthesized.

[0072] The design of the probe requires high specificity and is not affected by homologous sequences of other closely related species. And their sequences are not affected by each other. These sequences are located in the coding reg...

Embodiment 2

[0107] A kind of pathogenic microorganism detection method, adopts the test kit described in embodiment 1, comprises the following steps:

[0108] 1. Pre-capture library preparation.

[0109] The nucleic acid of the sample to be tested is extracted, and the library is constructed so that the final library fragment is 300-600 bp, and the concentration of the library is quantitatively detected after purification.

[0110] 2. Capture by liquid phase hybridization.

[0111] 2.1 Preparation of library mixture:

[0112] Take 8 to 16 adapter libraries, and quantitatively take 125ng of each library. After mixing, add concentrated reagents according to the table below, and add the corresponding adapter blocking solution according to the library construction process of Illumina or BGI, and select according to the subsequent sequencing platform.

[0113] Table 7. Library Mixture Composition

[0114] reaction system Amount (μL) / library library to be hybridized x J...

Embodiment 3

[0175] A comparative experiment of the detection limits of conventional metagenomic detection and the probe hybridization capture method of the present invention.

[0176] 1. Method.

[0177] 1.1 Prepare simulated mixed samples.

[0178] The simulated mixed sample contains human HELA cells, Acinetobacter baumannii (Baumann), Klebsiella pneumoniae (Klebsiella pneumoniae, lung grams), Streptococcus agalactiae (Streptococcus agalactiae, GBS) DNA, pyogenic Streptococcus pyogenes, pyogenes, Cryptococcus neoformans, Candida albicans, human herpesvirus type Ⅰ (HSV1), human herpesvirus type 4 (EBV).

[0179] Each mock-mixed sample contained 2 × 10 HELA cells 5 cells / ml, the prepared pathogenic microorganism mixture was diluted 3× with sterile PBS, and put into HELA cells in equal amounts, and finally the concentration of pathogenic microorganisms was obtained as shown in the table below.

[0180] Table 10. Pathogenic microorganisms and concentrations in simulated mixed samples

[...

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Abstract

The invention relates to a capture probe set, method and kit for detecting pathogenic microorganisms and application, and belongs to the technical field of gene detection. The capture probe set specifically detects 62 bacteria, 41 drug-resistant genes, 16 fungi, 14 parasites, 15 DNA viruses and 60 RNA viruses, can cover 95% or above of pathogenic microorganisms with the high detection rate in clinical samples, can greatly improve the detection sensitivity and specificity by combining the current situation that part of pathogenic microorganisms are difficult to detect, and particularly improves the detection sensitivity of pathogenic microorganisms which are significant to clinic.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a capture probe set, method, kit and application for detecting pathogenic microorganisms. Background technique [0002] Microbial pathogen detection has always been an important problem in infection diagnosis. For a long time, microbial culture has remained the gold standard for pathogen detection. Clinical applications also include antigen and antibody detection, PCR detection, staining, smear, microscopy, etc. However, the long time and low positive rate are the disadvantages of traditional detection methods, and they are also the main reasons for the difficulties in clinical diagnosis. [0003] In recent years, the development of meta-genomic next-generation sequencing (mNGS) has provided new ideas for pathogen detection. Metagenome, also known as the environmental genome of pathogenic microorganisms or metagenome of pathogenic microorganisms, refers to the sum of th...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/689C12Q1/6893C12Q1/6888C12Q1/6895C12Q1/6813C12Q1/04C12Q1/10C12N15/11C40B40/06
CPCC12Q1/701C12Q1/703C12Q1/705C12Q1/706C12Q1/689C12Q1/6893C12Q1/6888C12Q1/6895C12Q1/6813C40B40/06C12Q2531/113C12Q2535/122C12Q2563/143C12Q2563/149Y02A50/30
Inventor 许腾曾伟奇吴婉婷杨洁秦子颖刘足李永军王小锐苏杭
Owner GZ VISION GENE TECH CO LTD
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