KR-102963790-B1 - Method and apparatus for concentrating and detecting microorganisms in biological samples

Abstract

The present invention provides a method and apparatus for enriching and detecting microorganisms in a biological sample, wherein the biological sample is filtered by a polymeric modified substrate having a specific ability to capture or separate nucleated cells, and microorganisms can pass through the polymeric modified substrate or flow through the filtrate, thereby enriching microorganisms (including bacteria, mycoplasma, fungi, viruses, spores, etc.) in the biological sample during the process of removing nucleated cells, and thereby reducing interference in the detection of pathogenicity by nucleated cells (leukocytes, etc.).

Inventors

• 융 창
• 멍추 우
• 정펑 종
• 얀원 천
• 하우 헝

Assignees

• 마이크론브레인 메디컬 씨오., 엘티디.

Dates

Publication Date

20260511

Application Date

20210315

Priority Date

20200327

Claims (20)

1. A method for enriching and detecting microorganisms in a biological sample, comprising: a) collecting a biological sample; b) filtering the sample through a polymer modification substrate, wherein human-derived nucleated cells in the sample are captured or separated by the polymer modification substrate and microorganisms in the sample pass through the polymer modification substrate or flow into the filtrate; and c) detecting microorganisms present in the filtrate; wherein the nucleated cells comprise one or more of erythroblasts, leukocytes, and cancer cells; and the polymer is prepared by the polymerization of one or more monomers having the structure of the following chemical formula (1). R1 is independently selected from the group consisting of hydrogen, methyl, ethyl, hydroxyl, C1-12 alkyl, and phenyl; R2 is independently selected from the group consisting of hydrogen, methyl, ethyl, C1-6 alkyl, amino, and phenyl; and n is an integer from 1 to 5.
2. In paragraph 1, the human-derived nucleated cell is a leukocyte, method.
3. In paragraph 1, the microorganism is a bacterium, method.
4. In paragraph 1, the microorganism is a fungus, method.
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12. In claim 1, the monomer of formula (1) is N-(2-hydroxyethyl)acrylamide, HEAA.
13. A method according to claim 1, wherein the polymer further comprises an additional monomer which is butyl methacrylate, and the monomer of formula (1) copolymerizes with the additional monomer to form a copolymer.
14. In claim 1, the polymer has the structure of formula (2), method: n is an integer from 10 to 50.
15. In claim 1, the polymer has the structure of formula (4), method: t is an integer from 50 to 90, n is an integer from 10 to 50, and R 2 is am.
16. In claim 1, the polymer is a segmented polymer.
17. A method according to claim 1, wherein the polymer is disposed on a substrate by coating, spraying, or impregnation.
18. In paragraph 17, the method described is polypropylene, polyethylene terephthalate, cellulose, and polybutylene terephthalate.
19. A method according to claim 18, wherein the surface element of the modified substrate comprises carbon, oxygen, and nitrogen; the total molar percentage of carbon, oxygen, and nitrogen is defined as 100%, the molar percentage of carbon is about 76.22% to 79.84%, the molar percentage of oxygen is about 18.1% to 21.04%, and the molar percentage of nitrogen is about 2.05% to 2.75%.
20. In paragraph 19, a method used for testing the pathogenicity of biological samples.

Description

Method and apparatus for enriching and detecting microorganisms in biological samples The present invention relates to the field of microorganism detection technology; in particular, to a method and apparatus for enriching and detecting microorganisms in a biological sample by reducing interference from human-derived nucleated cells in the biological sample. When using molecular detection methods to detect pathogenic microorganisms (including bacteria, mycoplasmas, fungi, viruses, spores, etc.) in biological samples, the number of human cells and the genome size of the human cells in the sample are much larger than those of the pathogenic microorganisms, and the amount of human DNA is generally tens of thousands or millions of times greater than that of the pathogenic microorganisms' DNA. Therefore, background interference of human DNA has always been a major challenge in the molecular detection process of pathogenic microorganisms. Currently, there are differential lysis methods such as the QIAamp DNA Microbiome Kit and recently published modified methods (Nanopore metagenomic enables rapid clinical diagnosis of bacterial lower respiratory infection. Charalampous et al., Nature Biotechnology, 2019); as well as methylation modification methods to remove human DNAs, e.g., NEBNext® Microbiome DNA Enrichment Kit (New England Biolabs)). However, these methods have distinct disadvantages, such as complex operations and inconsistent effects. FIG. 1: Schematic diagram of the method of the present invention for filtering a biological sample through a polymer-modified substrate. FIG. 2: Filtration device of the present invention for enriching and detecting microorganisms in a biological sample. Fig. 3: Structural formulas of the monomer and polymer of the present invention, as well as theoretically predicted values of the chemical shift of the nuclear magnetic resonance (NMR) spectrum signal. Fig. 4: Measured map of the NMR spectra of the monomer and polymer of the present invention. Fig. 5: Coating density results of the substrate of the present invention, namely PP, PET, cellulose, and PBT modified with BrH and BrD. Symbol Explanation: 1. Upper housing; 2. Filter; 3. Lower housing. Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings generally understood by those skilled in the art. Additionally, unless otherwise required by the context, singular terms include the plural and plural terms include the singular. Generally, the nomenclature and techniques used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, protein and nucleic acid chemistry, and hybridization described herein are those well known and commonly used in the art. Known methods and techniques are generally performed according to conventional methods widely known in the art and, unless otherwise indicated, as described in the various general and more specific references discussed throughout this specification. Purification and DNA sequencing techniques are generally achieved in the art or are performed according to the manufacturer's specifications as described herein. The nomenclature used in connection with the laboratory procedures and techniques described herein is a nomenclature well known and commonly used in the art. In the present invention, biological samples can be filtered through a Sterile Acrodisc® White Blood Cell Syringe Filter (PALL) or a polymer-modified substrate. Both the filter and the modified substrate possess highly specific capabilities for capturing or separating human-derived nucleated cells. Additionally, microorganisms within the biological sample can pass through the filter or the polymer-modified substrate and enter the filtrate. During the reduction of human-derived nucleated cells, high levels of microorganisms (bacteria, mycoplasmas, fungi, viruses, spores, etc.) are enriched in the sample, thereby reducing interference from human-derived nucleated cells. Biological samples effectively enriched with microorganisms can undergo DNA purification. A sequencing library is constructed using DNA-purified samples at appropriate concentrations according to the Oxford Nanopore rapid library construction process and sequenced using an Oxford Nanopore GridION sequencer. The sequencing results demonstrate that the Sterile Acrodisc® White Blood Cell Syringe Filter (PALL) and the modified substrate can specifically remove human-derived nucleated cells and enrich microorganisms. The Sterile Acrodisc® White Blood Cell Syringe Filter used in the present invention is provided by PALL (catalog numbers AP-4951 and AP-4952). The filter is a proven filtration device designed to separate white blood cells from whole blood samples while allowing red blood cells (RBCs) and platelets to pass through the membrane. The method for microbial enrichment and detection in a biological sample according to the present invention f