WO2021190340A1 - 一种用于富集和检测生物样品中微生物的方法和装置 - Google Patents

一种用于富集和检测生物样品中微生物的方法和装置 Download PDF

Info

Publication number
WO2021190340A1
WO2021190340A1 PCT/CN2021/080829 CN2021080829W WO2021190340A1 WO 2021190340 A1 WO2021190340 A1 WO 2021190340A1 CN 2021080829 W CN2021080829 W CN 2021080829W WO 2021190340 A1 WO2021190340 A1 WO 2021190340A1
Authority
WO
WIPO (PCT)
Prior art keywords
biological sample
filtrate
microorganisms
substrate
high molecular
Prior art date
Application number
PCT/CN2021/080829
Other languages
English (en)
French (fr)
Inventor
张雍
吴梦楚
钟政峯
陈彦文
洪灏
Original Assignee
康博医创股份有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 康博医创股份有限公司 filed Critical 康博医创股份有限公司
Priority to AU2021240657A priority Critical patent/AU2021240657B2/en
Priority to CA3171415A priority patent/CA3171415A1/en
Priority to KR1020227035799A priority patent/KR20230002416A/ko
Priority to JP2022558095A priority patent/JP7407486B2/ja
Publication of WO2021190340A1 publication Critical patent/WO2021190340A1/zh

Links

Images

Classifications

    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61MDEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/34Filtering material out of the blood by passing it through a membrane, i.e. hemofiltration or diafiltration
    • A61M1/3496Plasmapheresis; Leucopheresis; Lymphopheresis
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/24Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01DSEPARATION
    • B01D39/00Filtering material for liquid or gaseous fluids
    • B01D39/14Other self-supporting filtering material ; Other filtering material
    • B01D39/16Other self-supporting filtering material ; Other filtering material of organic material, e.g. synthetic fibres
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869Methods for sequencing
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701Specific hybridization probes
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911Bacteria
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01DSEPARATION
    • B01D2239/00Aspects relating to filtering material for liquid or gaseous fluids
    • B01D2239/04Additives and treatments of the filtering material
    • B01D2239/0407Additives and treatments of the filtering material comprising particulate additives, e.g. adsorbents
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01DSEPARATION
    • B01D2239/00Aspects relating to filtering material for liquid or gaseous fluids
    • B01D2239/04Additives and treatments of the filtering material
    • B01D2239/0471Surface coating material
    • B01D2239/0478Surface coating material on a layer of the filter

Definitions

  • the invention belongs to the technical field of microbial detection, and specifically relates to a method and device for reducing the interference of nucleated cells in a biological sample, enriching and detecting microbes.
  • the purpose of the present invention is to provide a method and device for reducing the interference of nucleated cells in biological samples, enriching and detecting microorganisms in biological samples.
  • the present invention provides a method for enriching and detecting microorganisms in a biological sample, which includes the following steps: a) obtaining a biological sample; b) passing the biological sample through Leukocyte needle filter or high molecular polymer modified substrate is filtered, the nucleated cells in the biological sample are captured or separated, and the microorganisms can pass through The white blood cell needle filter or the base material modified by high molecular polymer enters the filtrate; c) Perform microbiological testing on the filtrate; among them, the nucleated cells include one or more of erythrocytes, leukocytes and cancer cells; high
  • the molecular polymer is prepared by polymerization of one or more monomers, and one of the monomers has the structure of formula (1):
  • R 1 is independently selected from hydrogen, methyl, ethyl, hydroxyl, C 1 to C 12 alkyl and phenyl
  • R 2 is independently selected from hydrogen, methyl, ethyl, C 1 to C 6 alkyl, amino and benzene Base
  • n is an integer from 1 to 5.
  • the nucleated cells in the biological sample are leukocytes.
  • the microorganisms in the biological sample are bacteria.
  • the microorganisms in the biological sample are fungi.
  • the retention rate of microorganisms in the resulting filtrate is more than 65%.
  • the retention rate of microorganisms in the resulting filtrate is more than 80%.
  • the red blood cells in the biological sample can pass or flow through the substrate modified by the high molecular polymer into the filtrate, and the retention rate of the red blood cells in the filtrate is more than 80%.
  • the platelets in the biological sample can pass through or flow through the high-molecular polymer-modified substrate into the filtrate, and the retention rate of the platelets in the filtrate is more than 80%.
  • the plasma fibrinogen in the biological sample can pass through or flow through the high-molecular polymer-modified substrate into the filtrate, and the retention rate of plasma fibrinogen in the filtrate is more than 80%.
  • the detection rate of microorganisms in the resulting filtrate is more than 2 times the detection rate of unfiltered biological samples.
  • the detection rate of microorganisms in the resulting filtrate is more than 40 times that of unfiltered biological samples.
  • the monomer of the above formula (1) may be N-hydroxyethyl acrylamide or N-(2-hydroxyethyl) acrylamide.
  • another monomer is further included.
  • the other monomer may be butyl methacrylate, and the monomer of the formula (1) is copolymerized with the other monomer to form a copolymer.
  • the above-mentioned high molecular polymer has the structure of formula (2):
  • n is an integer of 10-50.
  • the above-mentioned high molecular polymer has the structure of formula (4):
  • t is an integer from 50 to 90
  • n is an integer from 10 to 50
  • the polymer of the present invention is a segmented polymer
  • the high molecular polymer of the present invention is disposed on the substrate by coating, spraying or dipping.
  • the substrate can be polypropylene, polyethylene terephthalate, cellulose, polybutylene terephthalate.
  • the surface elements of the substrate modified by the polymer include carbon, oxygen and nitrogen, and the total mole percentage of carbon, oxygen and nitrogen is defined as 100%, the mole percentage of carbon is about 76.22% to about 79.84%, and the mole of oxygen The percentage is from about 18.1% to about 21.04%, and the mole percentage of nitrogen is from about 2.05% to about 2.75%.
  • the obtained filtrate is subjected to DNA purification treatment, and can be analyzed by PCR, qPCR, digital PCR, NGS, MassSpec or Nanopore sequencing.
  • the obtained filtrate is subjected to DNA purification treatment, a sequencing library is constructed according to the Oxford Nanopore rapid library construction method, and the Oxford Nanopore GridION sequencer is used for sequencing analysis.
  • the biological sample is selected from blood, serum, saliva, urine, stool, throat or nasal swabs, spinal fluid, cells, lymph, peritoneal fluid, vitreous fluid, tears, semen, vaginal secretions, lungs Effusion, serous fluid, bronchoalveolar lavage fluid, cell culture or tissue sample.
  • the method for enriching and detecting microorganisms in biological samples of the present invention can be used for pathogen detection of biological samples.
  • the present invention also provides a device for the method of enriching and detecting microorganisms in a biological sample.
  • the device includes an upper shell, a filter material and a lower shell, and the filter material is located on the upper shell and the lower shell. Between the shells, the filter material is prepared from the base material modified by the above-mentioned high molecular polymer.
  • the upper shell is provided with an inlet
  • the lower shell is provided with an outlet. The biological sample enters from the inlet of the upper shell, is filtered by the filter material, and flows out through the outlet of the lower shell.
  • the method and device for enriching and detecting microorganisms in biological samples enable biological samples to pass through Leukocyte syringe filters or high molecular polymer modified substrates are filtered.
  • the above-mentioned commercial filters and modified substrates have highly specific nucleated cell capture or separation capabilities, and microorganisms in biological samples can pass Leukocyte needle filter or high molecular polymer modified substrate enters the filtrate; thus, in the process of nucleated cell removal, the microorganisms (including bacteria, mycoplasma, fungi, viruses, spores, etc.) in biological samples are enriched ), thereby reducing the interference of pathogen detection caused by nucleated cells.
  • capture refers to the nucleated cells in the sample that come in contact with the surface of the material and are attracted by the hydrophobic, hydrogen bonds, or electrostatic molecular forces between the material and the cells, causing all kinds of cells to be directly attached to the material. On the surface, smaller plasma proteins and platelets may be adsorbed first before larger cells attach. These processes are defined as the “capture” of nucleated cells.
  • separation means that the nucleated cells can be separated from the sample after a sample containing human cells is passed through the material for separating nucleated cells. It also means that the content of nucleated cells in the sample can be reduced, or even the amount of nucleated cells in the sample can be greatly reduced. The nucleated cell content of the separated filtrate is less than the original sample containing human cells.
  • nucleated cells reduction of nucleated cells
  • reduction of nucleated cells does not mean that all or substantially all of the nucleated cells are completely removed.
  • the term is used to broadly indicate that the number of nucleated cells is reduced during the separation or filtration process.
  • Figure 1 A schematic diagram of the biological sample of the present invention being filtered through a modified substrate.
  • Figure 2 The filter device of the present invention for the enrichment and detection of microorganisms in biological samples.
  • Figure 3 The structural formulas of the monomers and high-molecular polymers of the present invention and the theoretical prediction values of chemical shifts of their nuclear magnetic resonance spectrum signals.
  • Figure 4 The measured nuclear magnetic resonance spectrum of the monomer and polymer of the present invention.
  • Figure 5 The results of coating density of PP, PET, Cellulose, and PBT substrates modified by B-r-H and B-r-D of the present invention.
  • the biological sample of the present invention can be passed Leukocyte needle filter or high molecular polymer modified substrate for filtration, the commercial filter and modified substrate have highly specific nucleated cell capture or separation capabilities, and the microorganisms in it can pass Leukocyte needle filter or high molecular polymer modified substrate enters the filtrate; thus, in the process of nucleated cell removal, the microorganisms (including bacteria, mycoplasma, fungi, viruses, spores, etc.) in biological samples are enriched ), thereby reducing the interference of pathogen detection caused by nucleated cells, and the obtained biological samples that are effectively enriched in microorganisms can be subjected to DNA purification processing, and the purified DNA with an appropriate concentration is constructed according to the method of Oxford Nanopore rapid library construction.
  • the library was sequenced and analyzed using Oxford Nanopore GridION sequencer. The sequencing results showed The white blood cell needle filter and modified substrate can specifically remove nucleated cells and achieve the effect of enriching microorganisms.
  • White blood cell syringe filter (Sterile White Blood Cell Syringe Filter, Catalog Nos. AP-4951 & AP-4952) is a validated filter device sold by PALL, which can separate white blood cells from whole blood samples while allowing red blood cells (RBC) and platelets to pass through the filter membrane.
  • RBC red blood cells
  • the method for enriching and detecting microorganisms in a biological sample of the present invention includes: firstly, obtaining a biological sample such as blood; secondly, filtering the biological sample through a substrate modified by a polymer, and the nucleated cells in the biological sample are captured or After separation, the content of human-derived nucleated cells in the obtained filtrate is greatly reduced, in order to reduce the filtrate of human-derived nucleated cells, and microorganisms can be enriched in the filtrate through the modified substrate.
  • the polymer of the present invention is prepared by polymerization of one or more monomers.
  • the monomer may be a monomer containing an amide group and a hydroxyl group, and the monomer containing an amide group and a hydroxyl group has the formula (1) Structure:
  • R 1 is independently selected from hydrogen, methyl, ethyl, hydroxyl, C1 to C12 alkyl and phenyl
  • R2 is independently selected from hydrogen, methyl, ethyl, C1 to C6 alkyl, amino And phenyl
  • n is an integer of 1 to 5.
  • R1 in formula (1) is hydrogen
  • R2 is hydrogen
  • n is 1.
  • the monomer containing an amide group and a hydroxyl group is N-hydroxyethyl acrylamide ((N-Hydroxyethyl acrylamide), or N-(2-hydroxyethyl) acrylamide (N-(2 -Hyroxyethyl)acrylamide).
  • the high molecular polymer is copolymerized by the monomer of formula (1) and at least another monomer to form a copolymer, and the other monomer is butyl methacrylate (BMA).
  • the high-molecular polymer is a segmented polymer.
  • the high molecular polymer prepared from the monomer of formula (1) may have the structure of formula (2):
  • n is an integer of 10-50.
  • the high molecular polymer prepared from the monomer of formula (1) may also have the structure of formula (4):
  • t is an integer from 50 to 90
  • n is an integer from 10 to 50.
  • the preparation of high molecular polymer is carried out by known technology.
  • One monomer is used as the base material biting end, and another monomer is used as the functional end.
  • the mixture is mixed in proportion, and the initiator ACVA is added, and the solvent is ethanol.
  • the polymerization reaction is carried out in an environment of 70° C. to prepare the desired high molecular polymer.
  • the product is separated out and dried using deionized water as the precipitation agent.
  • the high molecular polymer of the present invention can be applied to the substrate by coating, spraying or dipping to achieve the purpose of substrate modification. Specifically, take an appropriate amount of high molecular polymer, use ethanol as the solvent to prepare a high molecular solution, select a suitable substrate, cut to a suitable size, soak in the high molecular solution for about 1 minute, and then wash off the surface with deionized water Residual solution and dry. According to this, a surface-modified substrate coated with a high molecular polymer can be obtained.
  • the material of the substrate can be polypropylene (PP), polyethylene terephthalate (PET), cellulose, polybutylene terephthalate (PBT), etc. .
  • the surface elements of the modified base material include carbon, oxygen, and nitrogen, and the total mole percentage of carbon, oxygen, and nitrogen is defined as 100%, the mole percentage of carbon is about 76.22% to about 79.84%, and the mole percentage of oxygen is about 18.1% to about 21.04%, and the mole percentage of nitrogen is about 2.05% to about 2.75%.
  • the biological sample of the present invention is filtered through the above-mentioned polymer modified substrate.
  • Figure 1 is a schematic diagram of the biological sample of the present invention through the modified substrate filtration treatment.
  • the modified substrate can be , Attach or adhere to nucleated cells (including leukocytes) to separate them from biological samples such as whole blood. What is important is that there is almost no adsorption of plasma proteins during the filtration process, and almost no platelet adhesion, which improves The retention rate of platelets is improved, and the remaining substances in biological samples such as blood will pass (flow through) the modified substrate to achieve the effect of purification, such as red blood cells, platelets, bacteria, viruses, spores, etc.
  • the test results show that the removal rate of white blood cells in the filtered sample is greater than 70%, and even can reach more than 90%, and the retention rate of red blood cells in the filtered sample is greater than 80%, and even can reach more than 90%.
  • the platelet retention in the filtered sample The rate is greater than 80%, and can even reach more than 85%, and the retention rate of plasma fibrinogen in the filtered sample is greater than 80%, and even can reach more than 90%.
  • the detection rate of microorganisms in the filtered sample is at least 2 times that of the sample before filtration, and can even reach a higher multiple, such as 40 times.
  • the present invention also provides a filtering device used in conjunction with the high-molecular polymer modified substrate.
  • the device includes an upper casing 1, a filter material 2, a lower casing 3, and the filter material 2 is located on the upper casing.
  • the upper housing 1 is provided with a sample inlet
  • the lower housing 3 is provided with a sample outlet.
  • the sample enters from the inlet of the upper housing 1 and is filtered by the filter material 2. Then it flows out through the outlet of the lower shell 3, and the filter material is prepared from a base material modified by a high molecular polymer.
  • the method for enriching and detecting microorganisms of the present invention can be applied to the filter device: first, a biological sample such as blood is obtained; secondly, the biological sample enters from the inlet of the upper shell 1, filtered by the filter material 2, and then passed through the lower shell 3 When the outlet flows out, the nucleated cells in the biological sample are captured or separated, and the content of human-derived nucleated cells in the resulting filtrate is greatly reduced, in order to reduce the filtrate of human-derived nucleated cells, and microorganisms can pass or flow through the filter material 2. And then get enriched in the filtrate, and remove the interference from human nucleated cells.
  • the microorganisms are effectively enriched in the filtrate for DNA purification, and the purified DNA with the appropriate concentration is used to construct a sequencing library according to the Oxford Nanopore rapid library construction method, and the Oxford Nanopore GridION sequencer is used for sequencing analysis.
  • the sequencing results indicate the modified substrate It can specifically remove the interference of human-derived nucleated cells and achieve the effect of enriching microorganisms, which greatly increases the proportion of microorganisms.
  • the DNA measurement in the present invention can be performed by methods known in the art, including but not limited to PCR, qPCR, digital PCR, NGS, MassSpec or Nanopore sequencing technology.
  • Nanopore sequencing uses Oxford Nanopore sequencing technology. Compared with the second-generation NGS technology that requires amplification and signal amplification, this single-molecule third-generation sequencing technology has the advantages of simple, fast sample processing and long sequencing length (>10kbp) . And these advantages are very suitable for the rapid identification of clinically unknown microbial pathogens, so this method can be applied to the detection of sepsis, pathogen testing and other fields.
  • test material of this embodiment is N-Hydroxyethyl acrylamide (HEAA), which has both a hydroxyl functional group and an amide functional group, and has the following chemical structure:
  • HEAA N-Hydroxyethyl acrylamide
  • the test material of the comparative example is the positively charged N,N-dimethylaminoethyl methacrylate (DMAEMA), which has the following chemical structure:
  • BMA butyl methacrylate
  • the material of the substrate is polypropylene (PP), polyethylene terephthalate (PET), cellulose, and polybutylene terephthalate (PBT).
  • PP polypropylene
  • PET polyethylene terephthalate
  • PBT polybutylene terephthalate
  • the chemical structures of PP, PET, Cellulose, and PBT are as follows:
  • BMA as the base material occlusal end
  • HEAA or DMAEMA as the functional action end
  • the solvent is ethanol.
  • the polymerization reaction was carried out at 70° C. for 24 hours to prepare BMA-r-HEAA and BMA-r-DMAEMA high-molecular polymers.
  • the product is separated out and dried using deionized water as the precipitation agent.
  • BMA-r-HEAA and BMA-r-DMAEMA high molecular polymers respectively, and use ethanol as solvent to prepare high molecular solutions.
  • BMA-r-HEAA and BMA-r-DMAEMA high molecular polymers respectively, and use ethanol as solvent to prepare high molecular solutions.
  • BMA-r-HEAA and BMA-r-DMAEMA high molecular polymers respectively, and use ethanol as solvent to prepare high molecular solutions.
  • BMA-r-HEAA and BMA-r-DMAEMA high molecular polymers respectively, and use ethanol as solvent to prepare high molecular solutions.
  • the weight of PP, PET, Cellulose, and PBT substrates is weighed with a microbalance. After the surface is modified, the substrate is dried and the weight of the surface-modified substrate is weighed by a microbalance. By calculating the weight difference of the substrate before and after the experiment, the weight of the high molecular polymer modified on the surface of the substrate can be obtained. Finally, the weight of the polymer per unit area can be obtained by conversion, which is the surface coating density.
  • Method 1 Take 10 mL of whole blood and filter it through the modified substrate in the filter. Then use a blood cell counter to test the blood before and after filtration to calculate the white blood cell removal rate and platelet retention rate.
  • Method 2 Add 1 mL of E. coli bacterial solution (6 ⁇ 10 9 cells/mL) to 9 mL of blood. After adding, shake for 5 minutes in the blood collection tube shaker, take out the blood sample and filter. Mixtures of other bacteria (such as Staphylococcus aureus) or fungi (such as Aspergillus brasiliensis) are also prepared in the same way.
  • the blood obtained before and after the filtration in the aforementioned 3.7 Method 1 and Method 2 was centrifuged in a centrifuge, and the supernatant (ie plasma) was extracted, and diluted with PBS for 10 times. After the dilution, 0.25 mL was taken out and transferred to a 24-well plate (24well -tissue culture polystyrene plate, that is, a 24-well TCPS plate), add 0.25 mL of the first antibody (Monoclonal Anti-human Fibrinogen, Clone) specific to fibrinogen to the sample, production company: Sigma Aldrich Co ., model: F4639), placed in an oven at 37°C for 30 minutes, and added 0.25mL of secondary antibody (anti-mouse IgG, rabbit IgG whole, HRP conjugated, production company: Wako Co., model: 014-1761), its It is specific to the primary antibody and will only bond with the primary antibody.
  • the first antibody Monitoringoclonal Anti-human Fibrinogen,
  • Gram-positive and negative (Gram-) bacteria such as Escherichia coli (E.coli), Shewanella algae (SA), Staphylococcus (Staphylococcus) aureus), Staphylococcus pseudintermedius (Staphylococcus pseudintermedius, SP), Klebsiella pneumoniae (CRKP), Imtechella halotolerans (I.Halo) and Allobacillus halotolerans (A.Halo), etc., and fungi, such as black yeast Mold (Aspergillus brasiliensis).
  • E.coli Escherichia coli
  • SA Shewanella algae
  • Staphylococcus Staphylococcus aureus
  • Staphylococcus pseudintermedius Staphylococcus pseudintermedius
  • SP Klebsiella pneumoniae
  • CKP Klebsiella pneumoniae
  • Group 1 Shewanella algae (Gram-), Staphylococcus pseudotermedius (Gram-), Klebsiella pneumoniae (Gram-).
  • Group 2 E.coli (Gram-), Staphylococcus aureus (Gram+), Aspergillus brasiliensis (fungi).
  • Group 3 Imtechella halotolerans (Gram-), Allobacillus halotolerans (Gram+).
  • the polymer compounds such as BMA-r-HEAA and BMA-r-DMAEMA are abbreviated as B-r-H and B-r-D.
  • Figure 3 shows the structural formulas of various monomers and polymers and their theoretical predictions of chemical shifts.
  • Figure 4 shows the measured NMR spectrum of each monomer and polymer. The specific experimental conditions and actual test values are listed in Table 1. After NMR analysis, the characteristic peak of HEAA is mainly reflected at a, and the characteristic peak of DMAEMA is mainly reflected at b. From the NMR spectrum and data, it can be known that the polymer used in this experiment has been successfully synthesized.
  • the coating density of PP, PET, Cellulose, and PBT substrates modified by BrH and BrD is the results of the coating density of PP, PET, Cellulose, and PBT substrates modified by BrH and BrD.
  • the coating density of each polymer on the surface of the substrate can be calculated to be between about 0.1 and about 0.3 mg/cm 2 .
  • the modified substrate is used to filter blood and blood with added bacteria.
  • the contents of various blood cells in the blood samples before and after filtration can be known. Based on this, the leukocyte removal rate and the retention rate of each cell can be calculated.
  • Plasma Fibrinogen uses ELISA enzyme immune link The reaction is detected, and E. coli and other bacteria are detected by an absorption spectrometer.
  • RBC Red blood cell
  • WBC White blood cell
  • PLT Platelet
  • E.coil Escherichia coli
  • Fibrinogen plasma fibrinogen.
  • Table 2 it can be seen that the substrate modified by HEAA can capture at least 94% of white blood cells, retain more than 93% of red blood cells and 87% of platelets, and retain more than 93% of plasma fibrinogen. This means that when whole blood flows through the modified substrate, most of the white blood cells will adhere to the substrate, and most of the red blood cells, platelets and plasma fibrinogen can be retained in the remaining filtrate.
  • the B-r-H modified substrate of the present invention has extremely high specificity for capturing leukocytes, does not cause the capture or attachment of red blood cells and platelets, and is a good material for depleting leukocytes from whole blood.
  • red blood cell concentrate or platelet concentrate can also be used to pass the above-mentioned modified substrate to achieve the effect of specifically removing leukocytes.
  • Table 3 shows the filtration results after adding E.coil bacteria to the blood.
  • the results show that the substrate modified by HEAA can capture at least 95% of white blood cells, retain more than 92% of red blood cells and 87% of platelets, and E. The retention rate of coli is more than 82%. This means that when whole blood flows through the modified substrate, most of the white blood cells will adhere to the substrate, and the remaining filtrate can retain other cells and bacteria in the blood.
  • the BrH modified substrate of the present invention has extremely high specificity for capturing leukocytes (not Causes the capture or attachment of red blood cells and platelets), which can retain most of the red blood cells, platelets and plasma proteinogen, and most of the bacteria can pass through the modified substrate without being attached, so the filtrate that specifically removes white blood cells can be Effectively enhance the sensitivity of pathogen detection and improve the accuracy of pathogen detection.
  • the bacterial filtration test mentioned in the aforementioned test method uses the combination of group 1 microorganisms to filter other types of bacteria using the BrH modified substrate that has high specificity for capturing leukocytes and can retain other cells and E. coli. test.
  • Table 4 shows the same amount of bacterial solution, including Shewanella algae (SA), Staphylococcus pseudontermedius (SP), Klebsiella pneumoniae (CRKP), which have been modified by BrH. After the material is filtered, the amount of DNA recovered can reach more than 92% of the unfiltered DNA, indicating that most of the common bacteria can capture the highly specific modified substrate through this white blood cell capture.
  • SA Shewanella algae
  • SP Staphylococcus pseudontermedius
  • CKP Klebsiella pneumoniae
  • E. coli Staphylococcus aureus
  • Black Aspergillus Aspergillus brasiliensis
  • the purified DNA uses primers of specific bacteria (see Table 5), and uses the SYBR-green Semi-quantitative Real-Time PCR semi-quantitative method for semi-quantitative comparison on the Light Cycler 96 (Roche) PCR machine.
  • the semi-quantitative analysis results are shown in Table 6.
  • W/O filter represents the unfiltered sample
  • W/filter represents the filtered sample.
  • Staphylococcus aureus Gram+
  • Black aspergillus Aspergillus brasiliensis, fungus
  • the results show that after the BrH modified substrate is filtered, most of the bacteria and fungi in the blood can capture the highly specific modified substrate through this white blood cell.
  • ZymoBIOMICS TM Spike-in Control I High Microbial Load
  • ZYMO RESEARCH Catalog Nos. D6320 & D6320-10
  • the product contains two strains in equal amounts: Imtechella halotolerans and Allobacillus halotolerans.
  • a total of 1 ⁇ 10 4 and 1 ⁇ 10 6 cells/mL of bacteria were added to the whole blood of normal people, and 5 mL of the sample was centrifuged at 2,000 g for 15 minutes, and the upper plasma was taken out, with or without After filtering the white blood cell syringe filter and the BrH modified substrate, centrifuge at 13,500 rpm for 10 minutes, remove the supernatant, and use Nucleic Acid Extraction kit Blood Bacterial DNA Auto Plate (TAN Bead, CatM6BGA45) is used to purify the bottom product. After the purified DNA uses Oxford Nanopore Technologies (ONT) SQK-RBK004 Rapid Barcoding Kit to establish a sequencing library, use ONT FLOMIN106 (revD) The flow cell is sequenced.
  • ONT FLOMIN106 ONT FLOMIN106
  • W/O represents the unfiltered sample
  • W/f Devin and W/f PALL represent the BrH modified substrate
  • the sample filtered by the white blood cell syringe filter ⁇ A and ⁇ I refer to whether the number of bacteria is relatively greater than the number of human cells when the value of human cells is used as the basic value. When the value of ⁇ A and ⁇ I is lower, the relative total amount of bacteria is larger.
  • ⁇ A and ⁇ I refers to whether the number of bacteria is relatively greater than the number of human cells when the value of human cells is used as the basic value. When the value of ⁇ A and ⁇ I is lower, the relative total amount of bacteria is larger.
  • the results show that through After filtering with a white blood cell syringe filter or BrH modified substrate, it can indeed enrich microorganisms in biological samples. Among them, 1 ⁇ 10 4 cells/mL is the most effective sample. Generally speaking, the concentration of pathogenic microorganisms in the human body is also quite low. , So the result is in
  • W/O fiter-1 and W/O fiter-2 represent unfiltered samples
  • W/fiter-1 and W/fiter-2 represent filtered samples, modified by BrH
  • the proportion of K.Pneumonia in the samples filtered by the quality substrate increased from 0.2% and 0.3% to 10.6% and 13.4% compared with the samples without filtration treatment; E.coli increased from 0.05% and 0.03% to 2.8% and 5.2%.
  • the results show that the BrH modified base material can indeed enrich the microorganisms in blood samples, and increase the detection rate of microorganisms by more than 40 times.
  • the cells were recovered and suspended in 5mL PBS, 10 ⁇ L of cells were taken for counting, and then the cells were diluted to 1 ⁇ 10 6 cells/mL with PBS, and the final volume of the sample was adjusted to be greater than 3mL. Take 20 ⁇ L of cell samples and count them with LUNA-II Automated Cell Counter (Logos Biosystems), repeat three times. Take 1mL sample and filter through BrH modified substrate, and finally take 20 ⁇ L of filtrate for counting, repeat three times.
  • the B-r-H modified substrate has a nucleated cell capture rate of at least 99%, which means that when a biological sample flows into the modified substrate, most human-derived nucleated cells will adhere to it. It can be seen from the results in Table 10 that after filtering through the B-r-H modified substrate, the human nucleated cells in the sample can be specifically removed.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Pathology (AREA)
  • Virology (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Botany (AREA)
  • Mycology (AREA)
  • Urology & Nephrology (AREA)
  • Public Health (AREA)
  • Vascular Medicine (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Anesthesiology (AREA)
  • Medicinal Chemistry (AREA)
  • Toxicology (AREA)
  • General Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Food Science & Technology (AREA)

Abstract

本发明提供一种用于富集和检测生物样品中微生物的方法及装置,使得生物样品可通过高分子聚合物改质的基材进行过滤,改质的基材具有高度专一性的有核细胞捕捉或分离能力,并且其中的微生物可通过或流过高分子聚合物改质的基材进入到滤液中,从而在有核细胞减除过程中富集生物样品中的微生物(包括细菌、霉浆菌、真菌、病毒与孢子等),进而降低因有核细胞(如白细胞)所造成的病源检验干扰。

Description

一种用于富集和检测生物样品中微生物的方法和装置 技术领域
本发明属于微生物检测技术领域,具体涉及一种减除生物样品中有核细胞的干扰,富集和检测微生物的方法和装置。
背景技术
利用分子检测的方法在生物样本中侦测致病微生物(包括细菌、霉浆菌、真菌、病毒与孢子等)时,由于生物样品中的人源细胞数量和基因体大小都远远大于致病微生物的细胞数量和基因体大小,人源DNA通常高达致病微生物DNA的几万倍甚至是几百万倍之多,故而在致病微生物的分子侦测中,人源DNA的背景干扰一直是一个挑战。目前有通过differential lysis的方法,例如QIAamp DNA Microbiome Kit和最近发表的改良方法(Nanopore metagenomics enables rapid clinical diagnosis of bacterial lower respiratory infection.Charalampous et al.,Nature Biotechnology,2019),也有人类DNA有甲基化修饰来去除人源DNA的方法,例如
Figure PCTCN2021080829-appb-000001
Microbiome DNA Enrichment Kit(New England Biolabs)。但是这些方法都有操作复杂,效果不一致的缺点。
发明内容
本发明的目的在于提供一种减除生物样品中的有核细胞的干扰,富集和检测生物样品中的微生物的方法和装置。
为了实现上述目的,本发明提供一种富集和检测生物样品中微生物的方法,包括以下步骤:a)取得生物样品;b)将生物样品通过
Figure PCTCN2021080829-appb-000002
白细胞针头过滤器或高分子聚合物改质的基材进行过滤,生物样品中的有核细胞被捕捉或分离,并且其中的微生物可通过
Figure PCTCN2021080829-appb-000003
白细胞针头过滤器或高分子聚合物改质的基材进入到滤液中;c)对该滤液进行微生物检测;其中,有核细胞包括红血球母细胞、白细胞和癌细胞的一种或多种;高分子聚合物通过一种或多种单体进行聚合反应制备而得,且其中一种单体具有式(1)结构:
Figure PCTCN2021080829-appb-000004
R 1独立选自氢、甲基、乙基、羟基、C 1至C 12烷基及苯基,R 2独立选自氢、甲基、乙基、C 1至C 6烷基、氨基及苯基,n为1至5的整数。
优选地,生物样品中的有核细胞为白细胞。
优选地,生物样品中的微生物为细菌。
优选地,生物样品中的微生物为真菌。
在一些实施方案中,所得滤液中微生物的保留率为65%以上。
在一些实施方案中,所得滤液中微生物的保留率为80%以上。
在一些实施方案中,生物样品中的红细胞可通过或流过高分子聚合物改质的基材进入到滤液中,滤液中红细胞的保留率为80%以上。
在一些实施方案中,生物样品中的血小板可通过或流过高分子聚合物改质的基材进入到滤液中,滤液中血小板的保留率为80%以上。
在一些实施方案中,生物样品中的血浆纤维蛋白原可通过或流过高分子聚合物改质的基材进入到滤液中,滤液中血浆纤维蛋白原的保留率为80%以上。
在一些实施方案中,所得滤液中微生物的检出率是未经过过滤的生物样品的检出率的2倍以上。
在一些实施方案中,所得滤液中微生物的检出率是未经过过滤的生物样品的检出率的40倍以上。
在一些实施方案中,上述式(1)结构的单体可以为N-羟乙基丙烯酰胺或N-(2-羟乙基)丙烯酰胺。
在一些实施方案中,还包括另一单体,该另一单体可以是甲基丙烯酸丁酯,式(1)结构的单体与该另一单体共聚合形成共聚物。
在一些实施方案中,上述的高分子聚合物具有式(2)的结构:
Figure PCTCN2021080829-appb-000005
式(2)中,n为10至50的整数。
在一些实施方案中,上述高分子聚合物具有式(4)的结构:
Figure PCTCN2021080829-appb-000006
式(4)中,t为50至90的整数,n为10至50的整数,
R2为
Figure PCTCN2021080829-appb-000007
在一些实施方案中,本发明的高分子聚合物为链段高分子
在一些实施方案中,本发明的高分子聚合物以涂布、喷洒或浸渍的方式设置于基材上。该基材可以为聚丙烯、聚对苯二甲酸乙二酯、纤维素、聚对苯二甲酸丁二酯。高分子聚合物改质的基材的表面元素包含碳、氧及氮,且碳、氧及氮的总摩尔百分比定义为100%,碳的摩尔百分比为约76.22%至约79.84%,氧的摩尔百分比为约18.1%至约21.04%,以及氮的摩尔百分比为约2.05%至约2.75%。
在一些实施方案中,所得滤液经过DNA纯化处理,可以通过PCR,qPCR,digital PCR,NGS,MassSpec或Nanopore sequencing进行分析。
优选地,所得滤液经过DNA纯化处理,依照Oxford Nanopore快速建库的方法构建测序文库,使用Oxford Nanopore GridION测序仪进行测序分析。
在一些实施方案中,生物样品选自血液、血清、唾液、尿液、粪便、喉咙或鼻拭子、脊髓液、细胞、淋巴液、腹膜液、玻璃体液、泪液、精液、阴道分泌物、肺积液、浆膜液、支气管肺泡灌洗液、细胞培养物或组织样品。
在一些实施方案中,本发明富集和检测生物样品中微生物的方法可用于生物样品的病源检验。
在一些实施方案中,本发明还提供一种用于富集和检测生物样品中微生物的方法的装置,该装置包括:上壳体,过滤材料和下壳体,过滤材料位于上壳体和 下壳体之间,过滤材料由上述高分子聚合物改质的基材制备而成。上壳体上设有进口,所述下壳体上设有出口,生物样品从上壳体的进口进入,经过所述过滤材料的过滤,经下壳体出口流出。
本发明提供的富集和检测生物样品中微生物的方法及装置,使得生物样品可通过
Figure PCTCN2021080829-appb-000008
白细胞针头过滤器或高分子聚合物改质的基材进行过滤,上述商业化过滤器和改质的基材具有高度专一性的有核细胞捕捉或分离能力,并且生物样品中的微生物可通过
Figure PCTCN2021080829-appb-000009
白细胞针头过滤器或高分子聚合物改质的基材进入到滤液中;从而在有核细胞减除过程中,富集生物样品中的微生物(包括细菌、霉浆菌、真菌、病毒与孢子等),进而降低因有核细胞所造成的病源检验干扰。
术语“捕捉”是指样品中的有核细胞,接触到材料表面上,受到材料与细胞之间的疏水、氢键、或静电分子作用力的吸引,造成各式细胞可能直接吸贴附于材料表面上,或先吸附体积较小的血浆蛋白质与血小板,才导致较大型的细胞贴附,这些过程被定义成有核细胞「捕捉」。
术语“分离”是指将含有人体细胞的样品通过分离有核细胞的材料后,可将有核细胞由样品中分离出来,亦指可减少样品中的有核细胞含量,甚至可大量减少样品中的有核细胞含量,而使得分离后的滤液的有核细胞浓度小于原来含有人体细胞的样品。
术语“减除有核细胞”并非是指所有的或实质上所有的有核细胞被完全去除,该用语是用以广义地指出有核细胞的数目在分离或过滤的过程中减少。
附图说明
图1:本发明生物样品通过改质的基材过滤处理的示意图。
图2:本发明用于富集和检测生物样品中微生物方法的过滤装置。
图3:本发明单体、高分子聚合物的结构式及其核磁共振图谱信号的化学位移理论预测值。
图4:本发明单体、高分子聚合物的核磁共振图谱实测图。
图5:本发明的B-r-H与B-r-D改质的PP、PET、Cellulose、PBT基材的披覆密度结果图。
符号说明:
1,上壳体;2,过滤材料;3,下壳体。
具体实施方式
本发明的生物样品可通过
Figure PCTCN2021080829-appb-000010
白细胞针头过滤器或高分子聚合物改质的基材进行过滤,该商业化过滤器和改质的基材具有高度专一性的有核细胞捕捉或分离能力,并且其中的微生物可通过
Figure PCTCN2021080829-appb-000011
白细胞针头过滤器或高分子聚合物改质的基材进入到滤液中;从而在有核细胞减除过程中,富集生物样品中的微生物(包括细菌、霉浆菌、真菌、病毒与孢子等),进而降低因有核细胞所造成的病源检验干扰,并可将得到的有效富集微生物的生物样品进行DNA纯化处理,取适当浓度的纯化后的DNA依照Oxford Nanopore快速建库的方法构建测序文库,使用Oxford Nanopore GridION测序仪进行测序分析,测序结果表明
Figure PCTCN2021080829-appb-000012
白细胞针头过滤器和改质的基材可专一性去除有核细胞并达到富集微生物的效果。
本发明所使用的
Figure PCTCN2021080829-appb-000013
白细胞针头过滤器(Sterile
Figure PCTCN2021080829-appb-000014
White Blood Cell Syringe Filter,Catalog Nos.AP-4951&AP-4952)是由PALL销售的一种经过验证的过滤装置,其能够将白细胞与全血样品分离,同时允许红细胞(RBC)和血小板通过滤膜。
本发明富集和检测生物样品中微生物的方法包括:首先,取得生物样品如血液;其次,使得生物样品通过高分子聚合物改质的基材进行过滤,生物样品中的有核细胞被捕捉或分离,所得滤液中人源有核细胞含量大大降低,为减除人源有核细胞的滤液,并且微生物可通过改质的基材进而在滤液中得到富集。
本发明的高分子聚合物通过一种或多种单体进行聚合反应制备而得,该单体可以是含酰胺基及羟基的单体,该含酰胺基及羟基的单体具有式(1)的结构:
Figure PCTCN2021080829-appb-000015
在式(1)中,R 1独立选自氢、甲基、乙基、羟基、C1至C12烷基及苯基,R2独立选自氢、甲基、乙基、C1至C6烷基、氨基及苯基,n为1至5的整数。
根据本发明的一些实施方式,其中式(1)中的R1为氢,R2为氢,以及n为1。
根据本发明的一些实施方式,该含酰胺基及羟基的单体为N-羟乙基丙烯酰 胺((N-Hydroxyethyl acrylamide),或N-(2-羟乙基)丙烯酰胺(N-(2-Hyroxyethyl)acrylamide)。
根据本发明的一些实施方式,该高分子聚合物由式(1)的单体及至少另一单体共聚合形成共聚物,另一单体是甲基丙烯酸丁酯(Butyl methacrylate,BMA)。
根据本发明的一些实施方式,该高分子聚合物为链段高分子。
根据本发明的一些实施方式,通过式(1)单体制备而得的高分子聚合物可具有式(2)的结构:
Figure PCTCN2021080829-appb-000016
式(2)中,n为10至50的整数。
根据本发明的一些实施方式,通过式(1)单体制备而得的高分子聚合物还可具有式(4)的结构:
Figure PCTCN2021080829-appb-000017
在式(4)中,t为50至90的整数,n为10至50的整数。
R2为
Figure PCTCN2021080829-appb-000018
高分子聚合物的制备以已知技术进行,以一种单体作为基材咬合端,以另一种单体作为功能作用端,依比例进行混合,并加入起始剂ACVA,溶剂为乙醇,接着,在70℃的环境下进行聚合反应,从而制备出所需高分子聚合物。待反应完成后,以去离子水作为析出剂将产物析出并干燥。
本发明的高分子聚合物可以涂布(coating)、喷洒或浸渍的方式设置于基材上, 达到基材改质的目的。具体而言,取适量高分子聚合物,以乙醇为溶剂,配制成高分子溶液,选取合适的基材,裁剪为合适大小,浸泡于高分子溶液中约1分钟,再以去离子水洗去表面残余溶液并干燥。据此,可获得经高分子聚合物涂布(coating)的表面改质的基材。基材的材料可以为聚丙烯(polypropylene,PP)、聚对苯二甲酸乙二酯(polyethylene terephthalate,PET)、纤维素(cellulose)、聚对苯二甲酸丁二酯(polybutylene terephthalate,PBT)等。改质的基材材料的表面元素包含碳、氧及氮,且碳、氧及氮的总摩尔百分比定义为100%,碳的摩尔百分比为约76.22%至约79.84%,氧的摩尔百分比为约18.1%至约21.04%,以及氮的摩尔百分比为约2.05%至约2.75%。
本发明的生物样品通过上述高分子聚合物改质的基材进行过滤,请参照图1,是本发明生物样品通过改质的基材过滤处理的示意图,改质的基材可通过捕捉、吸附、贴附或粘附有核细胞(包括白细胞)而使其从生物样品如全血中分离出来,重要的是,过滤过程中几乎不会吸附血浆蛋白质,亦几乎不会造成血小板贴附,提高了血小板的保留率,并且会使生物样品如血液中其余的物质通过(流过)改质的基材,达到纯化之效果,例如红细胞、血小板、细菌、病毒、孢子等。检测所得结果表明,过滤后样品中白细胞的去除率大于70%,甚至可以达到90%以上,过滤后样品中红细胞的保留率大于80%,甚至可以达到90%以上,过滤后样品中血小板的保留率大于80%,甚至可以达到85%以上,过滤后样品中血浆纤维蛋白原的保留率大于80%,甚至可以达到90%以上。而过滤后样品的微生物检出率至少是过滤前样品微生物检出率的2倍以上,甚至可以达到更高的倍数,比如40倍。
本发明还提供一种该高分子聚合物改质的基材配合使用的过滤装置,如图2所示,该装置包括上壳体1,过滤材料2,下壳体3,过滤材料2位于上壳体1和下壳体3之间,该上壳体1上设有样品进口,该下壳体3上设有样品出口,样品从上壳体1的进口进入,经过过滤材料2的过滤,之后经下壳体3出口流出,该过滤材料由高分子聚合物改质的基材制备而成。
本发明富集和检测微生物的方法可应用于该过滤装置:首先,取得生物样品如血液;其次,使得生物样品从上壳体1的进口进入,经过过滤材料2的过滤,之后经下壳体3出口流出,生物样品中的有核细胞被捕捉或分离,所得滤液中人源有核细胞含量大大降低,为减除人源有核细胞的滤液,并且微生物可通过或流 过过滤材料2,进而在滤液中得到富集,并去除来自人体的有核细胞的干扰。微生物得到有效富集的滤液进行DNA纯化处理,取适当浓度的纯化后的DNA依照Oxford Nanopore快速建库的方法构建测序文库,使用Oxford Nanopore GridION测序仪进行测序分析,测序结果表明改质的基材可专一性去除人源有核细胞干扰并达到富集微生物的效果,使得微生物比例得到大幅的增加。
本发明中DNA的测定可以利用本领域公知的方法进行,包括但不限于PCR、qPCR、digital PCR、NGS、MassSpec或Nanopore sequencing技术。
Nanopore sequencing采用的Oxford Nanopore定序技术,这种单分子三代定序技术和需要扩增放大讯号的NGS二代技术相比,具有样品处理简单,快速,定序长度超长的优势(>10kbp)。而这些优点都非常适合应用于临床上未知微生物致病源的快速鉴定,因此此方法可应用于检验败血症、病源检验等领域。
下面结合附图和具体实施例,进一步阐明本发明,应理解这些实施例仅用于说明本发明而不用于限制本发明的范围,在阅读了本发明之后,本领域技术人员对本发明的各种等价形式的修改均落于本申请所附权利要求所限定的范围。
实验方法,材料及测试结果
细胞株
下列实施例中使用的所有细胞系均来自美国典型培养物保藏中心(ATCC)。TF1
Figure PCTCN2021080829-appb-000019
和Jurkat克隆E6-1
Figure PCTCN2021080829-appb-000020
在含有10%FBS的RPMI培养基中培养;PC-3
Figure PCTCN2021080829-appb-000021
在含有10%FBS的F12K培养基中培养;SK-BR-3
Figure PCTCN2021080829-appb-000022
在含有10%FBS的McCoy's 5A培养基中培养;K-562
Figure PCTCN2021080829-appb-000023
在含有10%FBS的IMDM培养基培养。于37℃,5%CO 2的培养箱中培养。
1.基材改质的实验材料
本实施例的试验材料为同时具有羟基官能基和酰胺官能基N-羟乙基丙烯酰胺(N-Hydroxyethyl acrylamide,HEAA),具有化学结构如下:
Figure PCTCN2021080829-appb-000024
比较例的试验材料为带正电荷的聚甲基丙烯酸N,N-二甲基氨基乙酯 (N,N-dimethylaminoethyl methacrylate,DMAEMA),具有化学结构如下:
Figure PCTCN2021080829-appb-000025
另外,甲基丙烯酸丁酯(Butyl methacrylate,BMA)用于本发明聚合物中的咬合端,使得聚合物能以物理方式吸附于基材表面。BMA具有化学结构如下:
Figure PCTCN2021080829-appb-000026
4,4'-偶氮双(4-氰戊酸)(4,4'-Azobis(4-cyanovaleric acid),ACVA)做为起始剂,具有化学结构如下:
Figure PCTCN2021080829-appb-000027
基材的材料为聚丙烯(polypropylene,PP)、聚对苯二甲酸乙二酯(polyethylene terephthalate,PET)、纤维素(cellulose)、聚对苯二甲酸丁二酯(polybutylene terephthalate,PBT)。PP、PET、Cellulose、PBT分别具有化学结构如下:
PP
Figure PCTCN2021080829-appb-000028
PET
Figure PCTCN2021080829-appb-000029
Cellulose
Figure PCTCN2021080829-appb-000030
PBT
Figure PCTCN2021080829-appb-000031
2.用于基材改质的高分子聚合物制备
以BMA作为基材咬合端,以HEAA或DMAEMA作为功能作用端,依比例(咬合端约70%,作用端约30%)混合,并加入起始剂ACVA,而溶剂为乙醇。接着,在70℃的环境下进行聚合反应24小时,分别制备出BMA-r-HEAA、BMA-r-DMAEMA高分子聚合物。待反应完成后,以去离子水作为析出剂将产物析出并干燥。
3.基材表面改质及测试
选用PP、PET、Cellulose、PBT作为欲改质的基材。
3.1物理吸附作用力改质PP基材
分别取BMA-r-HEAA、BMA-r-DMAEMA高分子聚合物,以乙醇为溶剂,配制成高分子溶液。取PP基材,裁剪为合适大小,浸泡于高分子溶液中1分钟,再以去离子水洗去表面残余溶液并干燥。据此,可分别获得经BMA-r-HEAA、BMA-r-DMAEMA涂布(coating)的表面改质的PP基材。
3.2物理吸附作用力改质PET基材
分别取BMA-r-HEAA、BMA-r-DMAEMA高分子聚合物,以乙醇为溶剂,配制成高分子溶液。取PET基材,裁剪为合适大小,浸泡于高分子溶液中1分钟,再以去离子水洗去表面残余溶液并干燥。据此,可分别获得经BMA-r-HEAA、BMA-r-DMAEMA涂布(coating)的表面改质的PET基材。
3.3物理吸附作用力改质Cellulose基材
分别取BMA-r-HEAA、BMA-r-DMAEMA高分子聚合物,以乙醇为溶剂,配制成高分子溶液。取Cellulose基材,裁剪为合适大小,浸泡于高分子溶液中1分钟,再以去离子水洗去表面残余溶液并干燥。据此,可分别获得经BMA-r-HEAA、BMA-r-DMAEMA涂布(coating)的表面改质的Cellulose基材。
3.4物理吸附作用力改质PBT基材
分别取BMA-r-HEAA、BMA-r-DMAEMA高分子聚合物,以乙醇为溶剂,配制成高分子溶液。取PBT基材,裁剪为合适大小,浸泡于高分子溶液中1分 钟,再以去离子水洗去表面残余溶液并干燥。据此,可分别获得经BMA-r-HEAA、BMA-r-DMAEMA涂布(coating)的表面改质的PBT基材。
3.5核磁共振(NMR)的鉴定
[根据细则26改正25.03.2021] 
将上述高分子聚合物各秤取10mg,分别溶于1mL的甲醇(d-MeOH)中,配置为浓度10mg/mL的溶液并装入NMR试管,由图谱的特征峰,分析并计算高分子的化学结构与单体比例。
3.6表面改质密度量测
进行改质前,先将PP、PET、Cellulose、PBT基材以微量天平秤量其重量。当表面完成改质后,将基材干燥后并经由微量天平秤量该表面改质的基材重量。经由计算实验前后的基材的重量差,可得到改质于基材表面的高分子聚合物重量。最后经由换算可得每单位面积的高分子聚合物重量,即为表面披覆密度。
3.7血液过滤测试
将改质后的PP、PET、Cellulose、PBT基材,裁剪成直径2.6cm的圆形并堆栈20层,置于过滤装置中锁紧(类似于图2中所示装置)。本部分实验分为两种测试方法:
方法一:取10mL全血通过过滤器中改质后的基材进行过滤。然后使用血球计数仪对过滤前、过滤后的血品进行检验,计算出白细胞移除率以及血小板保留率。
方法二:于9mL血液中添加1mL大肠杆菌(E.coli)菌液(6×10 9cells/mL),添加后于采血管震荡器摇晃5分钟,取出血液样品进行过滤。其他细菌(如金黄色葡萄球菌(Staphylococcus aureus))或真菌(如黑麹菌(Aspergillus brasiliensis))的混合液也比照同样方法准备。
3.8酶结合免疫吸附(ELISA)
将前述3.7方法一和方法二过滤前后取得的血液,分别使用离心机进行离心,抽取上清液(即血浆),并使用PBS进行10倍稀释,稀释后取出0.25mL移入24孔盘中(24well-tissue culture polystyrene plate,即24孔的TCPS盘),于样品中加入0.25mL对纤维蛋白原(Fibrinogen)具有专一性的第一抗体(Monoclonal Anti-human Fibrinogen,Clone,生产公司:Sigma Aldrich Co.,型号:F4639),放置于37℃烘箱30分钟,加入0.25mL的第二抗体(anti-mouse IgG,rabbit IgG whole, HRP conjugated,生产公司:Wako Co.,型号:014-1761),其对第一抗体具有专一性,只会与第一抗体产生键结,放入37℃烘箱反应30分钟后,加入0.25mL显色剂3,3',5,5'-四甲基联苯胺(3,3',5,5'-Tetramethylbenzidine,TMB),等待6分钟使其显色后,再于每个样品中加入0.25mL 1M的硫酸以终止反应。由每个样品中(包括TCPS空孔位)吸取200微升溶液置于96孔盘中,藉由Bio-tek型号PowerWare XS的微量盘式分析仪(microplate reader)在UV波长450nm下检视读值,取得过滤前后UV读值后,经回推得知过滤后蛋白质回收比率。
3.9细菌过滤测试
使用各种不同的细微生物,包括革兰氏阳性(Gram+)和阴性(Gram-)菌,例如大肠杆菌(E.coli)、海藻希瓦菌(Shewanella algae,SA)、金黄色葡萄球菌(Staphylococcus aureus)、假中間葡萄球菌(Staphylococcus pseudintermedius,SP)、肺炎克雷伯氏菌(Klebsiella pneumoniae,CRKP)、Imtechella halotolerans(I.Halo)和Allobacillus halotolerans(A.Halo)等,以及真菌,例如黑麴黴(Aspergillus brasiliensis)。将等量的菌液分别经过过滤或不经过过滤,分别使用
Figure PCTCN2021080829-appb-000032
白细胞针头过滤器或改质后的PP、PET、Cellulose、PBT基材进行细菌过滤能力的测试。
组别1:Shewanella algae(Gram-)、Staphylococcus pseudintermedius(Gram-)、Klebsiella pneumoniae(Gram-)。
组别2:E.coli(Gram-)、Staphylococcus aureus(Gram+)、Aspergillus brasiliensis(真菌)。
组别3:Imtechella halotolerans(Gram-)、Allobacillus halotolerans(Gram+)。
3.10 DNA纯化、浓度测量、Semi-quantitative Real-Time PCR半定量PCR和宏基因定序
将前述3.7方法二中加入细菌的血液分为两份,一份进行过滤,一份不经过滤作为对照组,分别使用离心机进行离心并抽取上清夜(即血浆),以及3.9中细菌过滤测试的菌液,均使用DNeasy Blood and Tissue Kit进行DNA纯化,纯化后的DNA使用nanodrop spectrophotometer测量浓度,进行过滤前后的浓度对比,以及对应的细菌或真菌的Semi-quantitative Real-Time PCR的半定量对比。并取适当浓度的DNA,依照Oxford Nanopore快速建库的方法构建测序文库,使用 Oxford Nanopore GridION测序仪进行测序分析,从测序结果的分析证明改质后的基材去除宿主白细胞/人源DNA并达到富集微生物DNA的效果。
4.基材改质的检测结果
4.1核磁共振(NMR)的分析结果
如下表一所示,为方便后续说明,在此将高分子化合物如BMA-r-HEAA、BMA-r-DMAEMA各缩写为B-r-H与B-r-D。图3为各单体、高分子聚合物的结构式及其化学位移理论预测值。图4为各单体及高分子聚合物的NMR图谱实测图,具体实验条件及实际检验数值列于表一。核磁共振分析后,HEAA的特征峰主要体现于a处,DMAEMA的特征峰主要体现于b处。由核磁共振的图谱及数据可得知本试验所用的高分子聚合物已成功进行合成。
表一
Figure PCTCN2021080829-appb-000033
4.2 PP、PET、Cellulose、PBT基材披覆密度量测结果
如图5所示,为B-r-H与B-r-D改质的PP、PET、Cellulose、PBT基材的披覆密度结果图。藉由对改质前后的基材重量进行秤量,并量测基材的表面积,可算得基材表面的各高分子披覆密度,介于约0.1至约0.3mg/cm 2
4.3血液过滤测试结果
如前述试验方法中所提的血液过滤测试,利用改质后的基材对血液及添加细 菌之血液进行过滤。经由血球计数仪检测后可得知过滤前与过滤后的血样中各种血液细胞含量,依此计算出白细胞移除率与各细胞保留率,血浆纤维蛋白原(Fibrinogen)使用ELISA酵素免疫链结反应进行检测,E.coli等细菌使用吸收光谱仪进行检测。
RBC(Red blood cell)是红细胞,WBC(White blood cell)是白细胞,PLT(Platelet)是指血小板,E.coil(Escherichia coli)为大肠杆菌,Fibrinogen为血浆纤维蛋白原。根据表二可知经HEAA改质后的基材对白细胞的捕捉率至少达到94%,保留93%以上紅細胞与87%以上的血小板,且血浆纤维蛋白原可保留93%以上。意即,全血流经改质后的基材时,大部分白细胞会贴附于基材上,剩下的滤液中能保留大部分的红细胞、血小板与血浆纤维蛋白原。据此,本发明的B-r-H改质基材对于白细胞捕捉有极高的专一性,不会造成红细胞与血小板的捕捉或贴附,是作为全血减除白细胞的良好材料。当然,在其他实施方式中,亦可以使用例如红细胞浓缩液或血小板浓缩液通过上述改质基材,均可达到专一性去除白细胞的效果。
相对于使用B-r-D以及DMAEMA等聚合物进行改质的习知捕捉、分离或过滤白细胞的方式而言,不仅是白细胞捕捉率提高,更重要的是能保留大部分的红细胞、血小板与血浆蛋白原,是故明显可知,血液通过B-r-H是通过不同于过去的作用机制来达成功效。
表三显示血液中添加E.coil细菌后过滤结果,由结果显示经HEAA改质后的基材对白细胞的捕捉率至少达到95%,保留92%以上红细胞与87%以上的血小板,且E.coli保留率达82%以上。意即,全血流经改质后的基材时,大部分白细胞会贴附于基材上,剩下的滤液中能保留血液中其他细胞与细菌。据此,相对于使用B-r-D以及DMAEMA等聚合物进行改质的习知捕捉、分离或过滤白细胞的方式而言,本发明的B-r-H改质基材对于白细胞捕捉有极高的专一性(不会造成红细胞与血小板的捕捉或贴附),能保留大部分的红细胞、血小板与血浆蛋白原,并且大部分细菌可不被贴附从而通过改质后的基材,因此专一性去除白细胞之滤液可有效提升病源之检验灵敏度,提高病源检验之准确性。
表二
Figure PCTCN2021080829-appb-000034
表三
Figure PCTCN2021080829-appb-000035
4.4细菌过滤DNA浓度测试结果
如前述试验方法中所提的细菌过滤测试,使用组别1的微生物组合,利用对白细胞捕捉专一性高并可保留其他细胞和E.coli的B-r-H改质基材对其他种类的细菌进行过滤测试。表四显示等量的菌液,包括海藻希瓦菌(Shewanella algae,SA),金黄色葡萄球菌(Staphylococcus pseudintermedius,SP),肺炎克雷伯氏菌(Klebsiella pneumoniae,CRKP),经过B-r-H改质基材过滤后,DNA回收量可以达到不经过过滤的92%以上,说明常见的细菌大部分都可以通过这种白细胞捕捉专一性高的改质基材。
表四
Figure PCTCN2021080829-appb-000036
4.5血液添加模拟样本Semi-quantitative Real-Time PCR半定量对比结果
正常人全血中加入组别2的微生物组合,具体组成为10 6CFU/mL或10 4CFU/mL的混合微生物(大肠杆菌(E.coli):金黄色葡萄球菌(Staphylococcus aureus):黑麹菌(Aspergillus brasiliensis)=1:1:1)。5mL全血以2,000g离心15分钟后,取出上部的血浆,经过或不经过B-r-H改质基材过滤后,再用13,500rpm高速离心10分钟,去掉上清液,对底部产物进行DNA纯化。纯化后的DNA使用特定菌的primers(如表五),使用SYBR-green Semi-quantitative Real-Time PCR半定量方法在Light Cycler 96(Roche)PCR儀上進行半定量比較。半定量分析结果如表六所示,W/O filter代表未经过过滤的样品,W/filter代表经过过滤的样品,经过B-r-H改质基材过滤的血液样本中,大肠杆菌(E.coli,Gram-)、金黄色葡萄球菌(Staphylococcus aureus,Gram+)、黑麹菌(Aspergillus brasiliensis,真菌)的相应浓度和未经过滤的血液样本里的浓度沒有显着差异。结果说明经过B-r-H改质基材过滤后,血液中的细菌和真菌大部分都可以通过这种白细胞捕捉专一性高的改质基材。
表五
Figure PCTCN2021080829-appb-000037
表六
Figure PCTCN2021080829-appb-000038
4.6血液添加模拟样本宏基因测序结果
4.6.1阳性对照测序结果:
使用ZymoBIOMICS TM Spike-in Control I(High Microbial Load)(ZYMO RESEARCH,Catalog Nos.D6320&D6320-10)作为阳性对照,该产品含有等量的两个菌株:Imtechella halotolerans和Allobacillus halotolerans。正常人全血中分别加入总菌量1×10 4和1×10 6cells/mL的菌,取5mL样本以2,000g离心15分钟后,取出上部的血浆,分别经过或不经过
Figure PCTCN2021080829-appb-000039
白细胞针头过滤器和B-r-H改质基材过滤后,再用13,500rpm高速离心10分钟,去掉上清液,使用
Figure PCTCN2021080829-appb-000040
Nucleic Acid Extraction kit Blood Bacterial DNA Auto Plate(TAN Bead,CatM6BGA45)对底部产物进行DNA纯化,纯化后的DNA使用Oxford Nanopore Technologies(ONT)SQK-RBK004Rapid Barcoding Kit建立定序文库后,使用ONT FLOMIN106(revD)flow cell进行定序。定序分析结果如表七所示,W/O代表未经过滤的样品,W/f Devin和W/f PALL分别代表经过B-r-H改质基材和
Figure PCTCN2021080829-appb-000041
白细胞针头过滤器过滤的样品;ΔA和ΔI是指以人体细胞的数值为基本值时,细菌的数量是否相对大于人体细胞量,当ΔA和ΔI值越低,代表細菌的相对總量越多。结果表明,通过
Figure PCTCN2021080829-appb-000042
白细胞针头过滤器或B-r-H修饰的底物过滤后,确实可以富集生物样本中的微生物,其中1×10 4cells/mL的样品效果最顯著,一般来说人體中的致病微生物濃度也相當低,因此该結果符合我們的预期。
表七
Figure PCTCN2021080829-appb-000043
4.6.2病原微生物测序结果:
正常人全血中加入2.5×10 3CFU E.coli/mL和1ng/mL Klebsiella  pneumonia gDNA。取5mL样本以2,000g离心15分钟后,取出上部的血浆,经过或不经过B-r-H改质基材过滤后,再用13,500rpm高速离心10分钟,去掉上清液,对底部产物进行DNA纯化。纯化后的DNA使用Oxford Nanopore Technologies(ONT)SQK-RBK004 Rapid Barcoding Kit建立定序文库后,使用ONT FLOMIN106(revD)flow cell进行定序。定序分析结果如表八所示,W/O fiter-1和W/O fiter-2代表未经过过滤的样品,W/fiter-1和W/fiter-2代表经过过滤的样品,经过B-r-H改质基材过滤的样本中K.Pneumonia的占比和不经过过滤处理的样本相比,分别从0.2%和0.3%增加到10.6%和13.4%;E.coli则从0.05%和0.03%增加到2.8%和5.2%。结果说明经过B-r-H改质基材过滤后,的确可以起到富集血液样品中微生物的作用,使微生物的检出率提高40倍以上。
表八
Figure PCTCN2021080829-appb-000044
4.7有核细胞过滤测试结果
五个人源有核细胞株(见表九)用于测试B-r-H改质基材捕捉分离有核细胞的能力。将细胞回收并悬浮于5mL PBS中,取10μL细胞进行计数,接着用PBS将细胞稀释至1×10 6cells/mL,并调整样品的最终体积大于3mL。取20μL细胞样品以LUNA-II Automated Cell Counter(Logos Biosystems)计数,重复三次。取1mL样本经过B-r-H改质基材过滤,最后取20μL滤液进行计数,重复三次。
结果显示,B-r-H改质基材具有至少99%的有核细胞捕获率,意味着当生物样品流改质基材时,大多数人源有核细胞将粘附在上面。由表十的结果可得知,通过B-r-H改质基材过滤后,能够特异性除去样品中的人源有核细胞。
表九
Figure PCTCN2021080829-appb-000045
Figure PCTCN2021080829-appb-000046
表十
Figure PCTCN2021080829-appb-000047

Claims (27)

  1. 一种富集和检测生物样品中微生物的方法,其特征在于,所述方法包括以下步骤:a)取得生物样品;b)所述生物样品通过
    Figure PCTCN2021080829-appb-100001
    白细胞针头过滤器或高分子聚合物改质的基材进行过滤,所述生物样品中的有核细胞被过滤器或高分子聚合物改质的基材捕捉或分离,并且所述生物样品中的微生物可通过所述
    Figure PCTCN2021080829-appb-100002
    白细胞针头过滤器或高分子聚合物改质的基材进入到滤液中;c)对所述滤液进行微生物检测;其中,所述有核细胞包括红血球母细胞、白细胞和癌细胞的一种或多种;所述高分子聚合物通过一种或多种单体进行聚合反应制备而得,且其中一种单体具有式(1)结构:
    Figure PCTCN2021080829-appb-100003
    R 1独立选自氢、甲基、乙基、羟基、C 1至C 12烷基及苯基,R 2独立选自氢、甲基、乙基、C 1至C 6烷基、氨基及苯基,n为1至5的整数。
  2. 根据权利要求1所述的方法,其特征在于,所述生物样品中的微生物为细菌。
  3. 根据权利要求1所述的方法,其特征在于,所述生物样品中的微生物为真菌。
  4. 根据权利要求1所述的方法,其特征在于,所述生物样品中的有核细胞为白细胞。
  5. 根据权利要求1至4中任一所述的方法,其特征在于,所述滤液中微生物的保留率为65%以上。
  6. 根据权利要求5所述的方法,其特征在于,所述滤液中微生物的保留率为80%以上。
  7. 根据权利要求5所述的方法,其特征在于,所述生物样品中的红细胞可通过或流过所述高分子聚合物改质的基材进入到滤液中,所述红细胞的保留率为80%以上。
  8. 根据权利要求5所述的方法,其特征在于,所述生物样品中的血小板可通过或流过所述高分子聚合物改质的基材进入到滤液中,所述血小板的保留率为 80%以上。
  9. 根据权利要求5所述的方法,其特征在于,所述生物样品中的血浆纤维蛋白原可通过或流过所述高分子聚合物改质的基材进入到滤液中,所述血浆纤维蛋白原的保留率为80%以上。
  10. 根据权利要求1至4中任一所述的方法,其特征在于,所述滤液中微生物的检出率是未经过过滤的所述生物样品的检出率的2倍以上。
  11. 根据权利要求1至3中任一所述的方法,其特征在于,所述滤液中微生物的检出率是未经过过滤的所述生物样品的检出率的40倍以上。
  12. 根据权利要求1至4中任一所述的方法,其特征在于,所述式(1)结构的单体为N-羟乙基丙烯酰胺或N-(2-羟乙基)丙烯酰胺。
  13. 根据权利要求1至4中任一所述的方法,其特征在于,还包括另一单体,所述另一单体是甲基丙烯酸丁酯,式(1)结构的单体与所述另一单体共聚合形成共聚物。
  14. 根据权利要求1所述的方法,其特征在于,所述高分子聚合物具有式(2)的结构:
    Figure PCTCN2021080829-appb-100004
    式(2)中,n为10至50的整数。
  15. 根据权利要求1所述的方法,其特征在于,所述高分子聚合物具有式(4)的结构:
    Figure PCTCN2021080829-appb-100005
    式(4)中,t为50至90的整数,n为10至50的整数,
    R2为
    Figure PCTCN2021080829-appb-100006
  16. 根据权利要求1至4中任一所述的方法,其特征在于,所述高分子聚合物为链段高分子。
  17. 根据权利要求1至4中任一所述的方法,其特征在于,所述高分子聚合物以涂布、喷洒或浸渍的方式设置于基材上。
  18. 根据权利要求17所述的方法,其特征在于,所述基材为聚丙烯、聚对苯二甲酸乙二酯、纤维素、聚对苯二甲酸丁二酯。
  19. 根据权利要求18所述的方法,其特征在于,所述改质的基材的表面元素包含碳、氧及氮,且碳、氧及氮的总摩尔百分比定义为100%,碳的摩尔百分比为约76.22%至约79.84%,氧的摩尔百分比为约18.1%至约21.04%,以及氮的摩尔百分比为约2.05%至约2.75%。
  20. 根据权利要求19所述的方法,其特征在于,所述方法用于生物样品的病源检验。
  21. 根据权利要求20所述的方法,其特征在于,所述生物样品选自血液、血清、唾液、尿液、粪便、喉咙或鼻拭子、脊髓液、细胞、淋巴液、腹膜液、玻璃体液、泪液、精液、阴道分泌物、肺积液、浆膜液、支气管肺泡灌洗液、细胞培养物或组织样品。
  22. 根据权利要求19所述的方法,其特征在于,所述滤液经过DNA纯化处理,可以通过PCR、qPCR、digital PCR、NGS、MassSpec或Nanopore sequencing进行分析。
  23. 根据权利要求22所述的方法,其特征在于,所述滤液经过DNA纯化处理,依照Oxford Nanopore快速建库的方法构建测序文库,使用Oxford Nanopore GridION测序仪进行测序分析。
  24. 根据权利要求21所述的方法,其特征在于,所述方法用于生物样品的病源检验。
  25. 一种用于权利要求1至4中任一所述的方法的装置,其特征在于,所述装置包括:上壳体,过滤材料和下壳体,所述过滤材料位于上壳体和下壳体之间,所述过滤材料由权利要求1的高分子聚合物改质的基材制备而成。
  26. 根据权利要求25所述的装置,其特征在于,所述上壳体上设有进口,所述下壳体上设有出口,生物样品从上壳体的进口进入,经过所述过滤材料的过滤,经下壳体出口流出。
  27. 根据权利要求26所述的装置,其特征在于,所述装置可用于生物样品的病源检验的用途。
PCT/CN2021/080829 2020-03-27 2021-03-15 一种用于富集和检测生物样品中微生物的方法和装置 WO2021190340A1 (zh)

Priority Applications (4)

Application Number Priority Date Filing Date Title
AU2021240657A AU2021240657B2 (en) 2020-03-27 2021-03-15 Method and device for enriching and detecting microorganisms in biological samples
CA3171415A CA3171415A1 (en) 2020-03-27 2021-03-15 Method and device for enriching and detecting microorganisms in biological samples
KR1020227035799A KR20230002416A (ko) 2020-03-27 2021-03-15 생물학적 샘플에서 미생물을 농축 및 검출하기 위한 방법 및 장치
JP2022558095A JP7407486B2 (ja) 2020-03-27 2021-03-15 生物学的サンプル中の微生物を富化および検出するための方法およびデバイス

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010230413.4 2020-03-27
CN202010230413 2020-03-27

Publications (1)

Publication Number Publication Date
WO2021190340A1 true WO2021190340A1 (zh) 2021-09-30

Family

ID=74884907

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/080829 WO2021190340A1 (zh) 2020-03-27 2021-03-15 一种用于富集和检测生物样品中微生物的方法和装置

Country Status (9)

Country Link
US (1) US20210308351A1 (zh)
EP (1) EP3885444B1 (zh)
JP (1) JP7407486B2 (zh)
KR (1) KR20230002416A (zh)
CN (1) CN113444767A (zh)
AU (1) AU2021240657B2 (zh)
CA (1) CA3171415A1 (zh)
TW (1) TWI790567B (zh)
WO (1) WO2021190340A1 (zh)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1555418A (zh) * 2001-09-13 2004-12-15 ��Ĭϵͳ��˾ 从血液制品和/或其衍生物中浓缩和检测致病微生物的装置和方法
CN201189412Y (zh) * 2008-04-30 2009-02-04 王新平 用于收集血液中感染源类物质的滤器
WO2012016929A1 (en) * 2010-08-02 2012-02-09 Bruker Daltonik Gmbh Mass spectrometric diagnosis of sepsis without blood culture
CN107384865A (zh) * 2014-07-30 2017-11-24 日立化成株式会社 血中稀少细胞捕获方法

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FI91977C (fi) * 1991-01-24 1994-09-12 Orion Yhtymae Oy Menetelmä mikro-organismien määrittämiseksi ja erottamiseksi
KR101083454B1 (ko) * 2001-12-07 2011-11-16 사이토리 테라퓨틱스, 인크. 처리된 리포애스퍼레이트 세포로 환자를 치료하기 위한 시스템 및 방법
US8481265B2 (en) * 2007-08-02 2013-07-09 Universite Laval Concentration and enrichment of microbial cells and microbial nucleic acids from bodily fluids
JP2009118780A (ja) 2007-11-15 2009-06-04 Ihi Corp 微生物検出方法およびろ過装置
US20120270248A1 (en) * 2011-04-21 2012-10-25 Pall Corporation Detection of bacteria in biological fluids
JP2017038567A (ja) * 2015-08-20 2017-02-23 セイコーエプソン株式会社 単球分離用フィルター、単球分離方法、単球分離器具およびゲル材料
WO2018086556A1 (zh) * 2016-11-09 2018-05-17 普瑞博生技股份有限公司 捕捉或分离白血球的聚合物、装置、其制造方法及其应用
CN110339732A (zh) * 2019-06-12 2019-10-18 南京工业大学 一种高亲水接枝改性pvdf膜及其制备方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1555418A (zh) * 2001-09-13 2004-12-15 ��Ĭϵͳ��˾ 从血液制品和/或其衍生物中浓缩和检测致病微生物的装置和方法
CN201189412Y (zh) * 2008-04-30 2009-02-04 王新平 用于收集血液中感染源类物质的滤器
WO2012016929A1 (en) * 2010-08-02 2012-02-09 Bruker Daltonik Gmbh Mass spectrometric diagnosis of sepsis without blood culture
CN107384865A (zh) * 2014-07-30 2017-11-24 日立化成株式会社 血中稀少细胞捕获方法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BI, XIAOLIN , AN WAN , XINMING ORGAN, XU YAN,SUN GUANG ,NIE JINGXIN ,MA HUA: "Comparison of Blood Quality and Clinical Outcomes before and after Leukocyte Filter Filtration", CHINESE JOURNAL OF BLOOD TRANSFUSION, vol. 13, no. 2, 30 June 2000 (2000-06-30), pages 99 - 100, XP055852686, ISSN: 1004-549x, DOI: 10.13303/j.cjbt.issn.1004-549x.2000.02.020 *
D.M. KIM,M.E. BRECHER,L.A. BLAND,T.J. ESTES,S.K. MCALLISTER,S.M. AGUERO,R.A. CARMEN,E.J. NELSON: "Prestorage Removal of Yersinia Enterocolitica from Red Cells with White Cell-Reduction Filters", TRANSFUSION, vol. 32, no. 7, 30 September 1992 (1992-09-30), pages 658 - 662, XP055852680, ISSN: 1537-2995, DOI: 10.1046/j.1537-2995.1992.32792391041.x *
LIU FUPING , LIU RENQIANG ,YE ZHUJIANG , LIU JINGCHUN , ZOU WENTAO , WANG DEWEN , CHEN JINFENG , HE ZIYI: "Measures of Preventing Contamination of Blood Platelets with Bacteria and Clinical Application", CHINA TROPICAL MEDICINE, vol. 7, no. 7, 18 July 2007 (2007-07-18), pages 1102 - 1103+1114, XP055852672 *

Also Published As

Publication number Publication date
EP3885444A1 (en) 2021-09-29
JP2023518525A (ja) 2023-05-01
EP3885444B1 (en) 2022-11-09
KR20230002416A (ko) 2023-01-05
AU2021240657B2 (en) 2024-01-25
TWI790567B (zh) 2023-01-21
CN113444767A (zh) 2021-09-28
TW202136525A (zh) 2021-10-01
US20210308351A1 (en) 2021-10-07
CA3171415A1 (en) 2021-09-16
AU2021240657A1 (en) 2022-10-20
JP7407486B2 (ja) 2024-01-04

Similar Documents

Publication Publication Date Title
US6746841B1 (en) FTA- coated media for use as a molecular diagnostic tool
JP2019515662A (ja) 細菌の同定、および抗生物質感受性プロファイリング装置
EP1326877A4 (en) METHOD FOR SEPARATING NUCLEIC ACIDS, QUANTITATIVE DNA EXTRACTION AND DETECTION FOR EVALUATING LEUKOCYTES IN BLOOD PRODUCTS
US8574890B2 (en) Nucleic acid extraction from complex matrices
CN104810126A (zh) 具有超薄封闭二氧化硅层的磁性粒子及其制造和使用方法
EP2734280A1 (en) Method and kit isolating microorganisms from culture
CN105980846B (zh) 包括聚(酸)膜分离基质的旋转柱及其制造和使用的方法
EP1177420B1 (en) Fta-coated media for use as a molecular diagnostic tool
WO2021190340A1 (zh) 一种用于富集和检测生物样品中微生物的方法和装置
EP3989840B1 (en) Sampling device for biological specimen
CN109280664A (zh) 一种适用于eb病毒dna提取的方法及提取试剂盒
US20120070879A1 (en) Reduction of antibiotic activity or concentration in biological samples using molecularly imprinted polymers
CN115505590A (zh) 一种用于血液样本的菌体核酸快速提取试剂盒及其应用
JP2002125695A (ja) 微生物および生体由来物質の検出方法
EP1475387B1 (en) Method for preparing assay samples
Vahid et al. Designing a molecularly imprinted polymer-based nanomembrane for the selective removal of Staphylococcus aureus from aqueous media
CN114908190A (zh) Hiv dna检测方法
KR20190122120A (ko) 동형2기능성 이미도에스터를 이용한 병원체 농축 방법
JP2010207180A (ja) ノロウイルスrnaの精製用反応容器および精製方法
CN103388035B (zh) 一种羊关节炎-脑炎病毒前病毒dna实时荧光pcr试剂盒
CN115354028B (zh) 一种中脑多巴胺细胞群、其制造方法以及用途
Wilcox Comparison of formalin and Bouin's reagent for fixation of coagulase negative staphylococcal biofilm.
TR202008937A2 (tr) Elastik Polimer ile Mikroorganizma Konsantrasyon Yöntemi
CN113403285A (zh) 一种基于pamam介导的生物素/链霉素级联放大免疫磁富集牡蛎中诺如病毒的方法
WO2019190046A1 (ko) 규조류 및 쿠커비투릴을 이용한 병원체 농축 방법

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21775424

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 3171415

Country of ref document: CA

ENP Entry into the national phase

Ref document number: 2022558095

Country of ref document: JP

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 2021240657

Country of ref document: AU

Date of ref document: 20210315

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21775424

Country of ref document: EP

Kind code of ref document: A1