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WO2017096110A1 - Procédé et dispositif pour optimiser le processus d'identification de pathogènes - Google Patents

Procédé et dispositif pour optimiser le processus d'identification de pathogènes Download PDF

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Publication number
WO2017096110A1
WO2017096110A1 PCT/US2016/064533 US2016064533W WO2017096110A1 WO 2017096110 A1 WO2017096110 A1 WO 2017096110A1 US 2016064533 W US2016064533 W US 2016064533W WO 2017096110 A1 WO2017096110 A1 WO 2017096110A1
Authority
WO
WIPO (PCT)
Prior art keywords
pathogen
nucleic acids
cartridge
pathogens
nucleic acid
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/US2016/064533
Other languages
English (en)
Inventor
Praveen Bhat GURPUR
Nivedita MITRA
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Siemens Healthcare Diagnostics Inc
Original Assignee
Siemens Healthcare Diagnostics Inc
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 Siemens Healthcare Diagnostics Inc filed Critical Siemens Healthcare Diagnostics Inc
Publication of WO2017096110A1 publication Critical patent/WO2017096110A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING 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
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502715—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502753—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING 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
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms

Definitions

  • the present disclosure generally relates to processing of nucleic acids, and more particularly to method and device for optimizing the process of identification of pathogens.
  • pathogen deoxyribonucleic acid is isolated from the pathogen in patient's blood, the pathogen DNA is subjected to polymerase chain reaction (PGR) that is designed to determine the type of pathogen such as bacteria, fungus, virus, etc.
  • PGR polymerase chain reaction
  • the PGR may also be designed to determine the species and/or sub type of the pathogen such as Gram positive/Gram negative bacteria, Candida, Aspergillus, etc. and the presence of any drug resistance in the pathogen.
  • the current methods of diagnosing pathogens have certain limitations due to the need to gather multiple layers of information. For example, a large number of primers need to be used for multiplexing and/or a large amount of time is necessary to gather and analyze data. This may lead to a delay in administration of the appropriate antibiotic and high cost of the procedure.
  • Embodiments provide a method and a device for optimizing the process of identification of pathogens in a given sample so as to reduce delays in administration of appropriate antibiotics.
  • a method of optimizing the process of identification of pathogens includes obtaining nucleic acids specific to the pathogen, in another aspect, the method includes processing the obtained nucleic acids to identify gene sequence of at least a part of the nucleic acid. In another aspect, the method includes identifying the pathogen based on the gene sequence of the nucleic acid. [0007] Therein, obtaining nucleic acids specific to the pathogen includes isolating the pathogens from the given sample; extracting nucleic acids from the isolated pathogens; and amplifying the extracted nucleic acids using polymerase chain reaction; and generating amplified nucleic acids specific to the pathogen.
  • processing the obtained nucleic acids includes performing a nested polymerase chain reaction using the amplified nucleic acids specific to the pathogen.
  • processing the obtained nucleic acids includes determining the gene sequence of the amplified nucleic acids specific to the pathogen.
  • the device includes a first cartridge adapted to produce amplified pathogen nucleic acid. In another aspect, the device includes a second cartridge adapted to identify the gene sequence of the amplified pathogen nucleic acid.
  • Figure 1 illustrates a flowchart of an exemplary method of optimizing the process of identification of pathogens in a sample, according to an embodiment.
  • Figure 2A illustrates a device for optimizing the process of identification of pathogens in a sample, according to an embodiment.
  • Figure 2B illustrates a schematic diagram of an exemplary embodiment of the interaction between the first cartridge and the second cartridge.
  • FIG. 1 provides an illustration of a flowchart of an exemplary method 10 for optimizing the process of identification of a pathogen in a sample.
  • one or more pathogens are isolated from the given sample.
  • the sample may be, for example, whole blood, saliva, urine, or any other body fluid.
  • the one or more pathogens may be isolated from the given sample by filtration, a positive enrichment method, a negative enrichment method or a combination of one or more methods of isolation of pathogens that may be well-known to a person ordinarily skilled in the art.
  • pathogen nucleic acid for example, deoxyribonucleic acid (DNA) is extracted from the one or more isolated pathogens.
  • Pathogen nucleic acid may be extracted from the pathogens using physical methods and/or chemical methods that are well-known in the art. Alternatively, the method 10 may also be performed with pathogen nucleic acid that is already extracted from one or more pathogens.
  • the extracted pathogen nucleic acids are amplified using polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • Polymerase chain reaction is a molecular biology technique for amplifying or making several copies of a given sequence of deoxyribonucleic acid (DNA). Amplification of pathogen nucleic acids may be done in multiple rounds of polymerase chain reaction.
  • the primers used in the first round of amplification may be chosen so as to make the identification of the species or type of pathogen easy.
  • the primers used in the first round of amplification may be specific to Gram positive or Gram negative bacteria, parasite, virus, fungi, etc.
  • the amplified nucleic acids may also be further processed, for example, using gel electrophoresis. [0017] At act 14, the amplified pathogen nucleic acid is further processed to identify the pathogen more specifically.
  • the amplified pathogen nucleic acid derived from the first round of polymerase chain reaction may further be amplified by performing a nested polymerase chain reaction.
  • Nested polymerase chain reaction makes use of two pairs of polymerase chain reaction primers for a single locus of nucleic acid.
  • the first pair of primers amplifies the nucleic acid sequence as in a usual polymerase chain reaction.
  • the second pair of primers binds within the product of the first polymerase chain reaction and produces a second product that is shorter than the first one. Therefore, nested polymerase chain reaction provides for very specific results, thereby providing for identification of pathogen sub-species.
  • the first round of polymerase chain reaction may also be followed by a round of nucleic acid sequencing.
  • the nucleic acid sequencing may be performed by using, for example, Next-generation sequencing (NGS) technology.
  • NGS Next-generation sequencing
  • the Next-generation sequencing technology identifies the sequence of the nucleic acid using DNA polymerase enzyme that incorporates deoxyribonucleotide triphosphates (dNTPs) into a nucleic acid template strand.
  • the dNTPs are labelled and may be identified by, for example, fiuorophore excitation.
  • the Next-generation sequencing technology processes the sequencing of millions of fragments of nucleic acids at the same time in a parallel manner. Gene sequencing increases the range of pathogens that may be identified.
  • FIG. 2 A illustrates a schematic diagram of an exemplary embodiment of a device 20 used to optimize the process of identification of pathogens in a sample.
  • the device includes a first cartridge 21 that has one or more chemical components specific for amplification of nucleic acids.
  • the first cartridge 21 may further include one or more chambers within which the chemical components are housed.
  • the chemical components may include Taq polymerase enzyme, primers, nucleotides, and one or more buffer solutions.
  • the primers used in the first cartridge 21 may be specific to different types of pathogens.
  • the first cartridge 21 has an inlet 22 through which the nucleic acids extracted from the pathogens are introduced into the cartridge 21.
  • the first cartridge 21 is designed to be suitable to be used for a polymerase chain reaction.
  • the first cartridge 21 may further include an outlet 23 from which the pathogen nucleic acids may be removed, after amplification, for further processing such as gel electrophoresis.
  • the inlet 22 and the outlet 23 of the first cartridge 21 are sealed, for example, with a rubber seal, such that the contents of the cartridge 21 do not leak to the outer environment and also such that air or any other fluid does not enter the cartridge 21 unless the seal is broken.
  • the device 20 further includes a second cartridge 24 that may include chemical components for second round of polymerase chain reaction.
  • the chemical components may include Taq polymerase enzyme, primers, nucleotides, and one or more buffer solutions.
  • the second cartridge may be of several different types based on the specificity of the primers included in the cartridge.
  • the second cartridge 24 is chosen based on the results of the first round of amplification in the first cartridge 21. For example, if after the first round of amplification using the first cartridge 21 , the pathogen nucleic acid is determined to be that of fungi, then the second cartridge 24 used would be specific to fungi, i.e., the primers included in the second cartridge 24 would be fungi specific. The primers included in the second cartridges 24 are such that they determine the sub-species of the pathogen. The primers included in the second cartridge 24 specifically bind to certain regions in the nucleic acid. Alternatively, the chemical components in the second cartridge 24 may also pertain to genetic sequencing. The amplified nucleic acids from the first cartridge 21 may be further processed for genetic sequencing.
  • the chemical components that may be included in the second cartridge include deoxyribonucleic acid polymerase, labelled deoxyribonucleotide triphosphates (dNTPs), and one or more buffers.
  • the second cartridge 24 has an inlet 25 through which the amplified nucleic acids from the first cartridge 21 are transferred.
  • the second cartridge 24 may also have an outlet 26 through which the processed nucleic acids may be extracted out.
  • the inlet 25 and the outlet 26 of the second cartridge 24 are sealed, for example, with a rubber seal, such that the contents of the cartridge 24 do not leak to the outer environment and also such that air or any other fluid does not enter the cartridge 24 unless the seal is broken.
  • FIG. 2B illustrates a schematic diagram of an exemplary embodiment of the interaction between the cartridges 21, 24 in the device 20.
  • the first cartridge 21 may be positioned such that the outlet 23 of the first cartridge 21 is in line with the inlet 25 of the second cartridge 24.
  • the first cartridge 21 may be moved or rotated mechanically or automatically such the outlet 23 of the first cartridge 21 is aligned with the inlet 25 of the second cartridge 24.
  • the first cartridge 21 may be brought in contact with the second cartridge 24 such that the contents of the first cartridge 21 may be transferred to the second cartridge 24.
  • the contents of the first cartridge 21 may be transferred to the second cartridge 24 using positive pressure, such as a pump, or using negative pressure, such as vacuum.
  • the processed nucleic acids in the second cartridge 24 may be removed from the outlet 26 for further identification of the pathogen.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne un procédé et un dispositif pour optimiser le processus d'identification de pathogènes. Le procédé comprend l'obtention d'acides nucléiques spécifiques au pathogène, l'obtention d'acides nucléiques spécifiques au pathogène comprenant l'amplification au moyen d'une réaction de polymérase en chaîne d'acides nucléiques extraits à partir du pathogène ; et la génération d'acides nucléiques amplifiés spécifiques au pathogène. Le procédé comprend en outre le traitement des acides nucléiques obtenus pour identifier la séquence génique au moins une partie de l'acide nucléique. Le procédé comprend en outre l'identification du pathogène sur la base de la séquence génique de l'acide nucléique. L'invention concerne en outre des dispositifs qui comprennent des cartouches combinables pour amplifier pour déterminer la séquence de pathogènes.
PCT/US2016/064533 2015-12-04 2016-12-02 Procédé et dispositif pour optimiser le processus d'identification de pathogènes Ceased WO2017096110A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US201562263021P 2015-12-04 2015-12-04
US62/263,021 2015-12-04
US201562265173P 2015-12-09 2015-12-09
US62/265,173 2015-12-09

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WO2017096110A1 true WO2017096110A1 (fr) 2017-06-08

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Cited By (1)

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US20220177981A1 (en) * 2020-04-16 2022-06-09 Safetraces, Inc. Airborne Pathogen Simulants and Mobility Testing

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WO2007082480A1 (fr) * 2006-01-19 2007-07-26 Capitalbio Corporation Analyseur microfluidique utilisant une cartouche
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WO2005003375A2 (fr) * 2003-01-29 2005-01-13 454 Corporation Procede d'amplification et de sequençage d'acides nucleiques
WO2007082480A1 (fr) * 2006-01-19 2007-07-26 Capitalbio Corporation Analyseur microfluidique utilisant une cartouche
WO2015031691A1 (fr) * 2013-08-28 2015-03-05 Cellular Research, Inc. Analyse massivement parallèle de cellules uniques

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20220177981A1 (en) * 2020-04-16 2022-06-09 Safetraces, Inc. Airborne Pathogen Simulants and Mobility Testing
US12258638B2 (en) 2020-04-16 2025-03-25 Safetraces, Inc. Airborne pathogen simulants and mobility testing
US12404560B2 (en) * 2020-04-16 2025-09-02 Safetraces, Inc. Airborne pathogen simulants and mobility testing

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