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WO1994021780A1 - Appareil de determination quantitative d'analytes particulaires - Google Patents

Appareil de determination quantitative d'analytes particulaires Download PDF

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Publication number
WO1994021780A1
WO1994021780A1 PCT/CA1994/000101 CA9400101W WO9421780A1 WO 1994021780 A1 WO1994021780 A1 WO 1994021780A1 CA 9400101 W CA9400101 W CA 9400101W WO 9421780 A1 WO9421780 A1 WO 9421780A1
Authority
WO
WIPO (PCT)
Prior art keywords
filter element
filter
liquid sample
pump
particulate analyte
Prior art date
Application number
PCT/CA1994/000101
Other languages
English (en)
Inventor
Roger G. L. Wheatcroft
William B. Berndt
Original Assignee
Her Majesty The Queen In Right Of Canada, As Represented By The Minister Of Agriculture
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 Her Majesty The Queen In Right Of Canada, As Represented By The Minister Of Agriculture filed Critical Her Majesty The Queen In Right Of Canada, As Represented By The Minister Of Agriculture
Priority to AU61049/94A priority Critical patent/AU6104994A/en
Publication of WO1994021780A1 publication Critical patent/WO1994021780A1/fr

Links

Classifications

    • 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
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
    • C12M41/36—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of biomass, e.g. colony counters or by turbidity measurements
    • 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
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/06—Quantitative determination

Definitions

  • the invention relates to an apparatus for the quantitative determination of particulate analytes, including microorganisms.
  • Species and strains of microorganisms in such samples can be detected and identified by probing for specific DNA sequences, as described in U.S. Patent No. 4358535 to Falkow et al. This method, however, is not usually quantitative.
  • PCP polymerase chain reaction
  • This process involves repeated cycles of incubation of the sample with an appropriate reagent, which includes a DNA polymerase enzyme and DNA synthesis oligonucleotides as primers.
  • the initial step in each cycle thermally denatures the DNA. This is followed by annealing and new DNA synthesis steps at lower temperatures.
  • Samples for analysis by selective amplification by PCR are usually contained in micro-test tubes during the necessary manipulations.
  • test tubes can be subjected to the necessary time/temperature cycles in apparatus such as the Perkin- Elmer/Cetus DNA Thermal Cycler which comprises a heating block with apertures to hold the tubes and appropriate controls to maintain the block and the tubes at the desired temperatures for the desired time periods.
  • apparatus such as the Perkin- Elmer/Cetus DNA Thermal Cycler which comprises a heating block with apertures to hold the tubes and appropriate controls to maintain the block and the tubes at the desired temperatures for the desired time periods.
  • the results obtainable by this method are, at most, semi-quantitative.
  • the reaction mixture resulting from PCR amplification may be analysed by agarose gel electrophoresis, the intensity of the resulting bands providing only a very rough quantitation of the original target DNA sequence.
  • the invention provides an apparatus for detecting and enumerating a particulate analyte present in a liquid sample comprising a filter element, a holder to support said filter element, an inlet duct to deliver said liquid sample to said filter element, an outlet duct to receive filtrate from said filter element, a heat transfer means to transfer heat to and from said filter element, a controller operable upon said heat transfer means to maintain said filter element at preselected temperatures for preselected time periods, a flow controller operable in a first position to connect said inlet and outlet ducts for recirculation of fluid through said filter element and in a second position to prevent recirculation of fluid through said filter element and a pump for recirculating fluid through said filter element.
  • an tit apparatus for detecting and enumerating a particulate analyte present in a liquid sample comprising at least one filter for retaining the particulate analyte, a holder for supporting said filter, temperature control means for adjusting the filter to a selected temperature, a first conduit for delivering liquids to said filter and a second conduit for removing filtrates from said filter, flow control means whereby said first and second conduits may be connected directly or indirectly to form a closed loop for recirculation of fluid through the filter, a controller for controlling operation of said temperature control means and a pump for recirculation of fluid through the filter.
  • a method for detecting and enumerating a particulate analyte present in a liquid sample comprising delivering said liquid sample through an inlet duct to a filter element, removing filtrate flowing through the filter element through an outlet duct while retaining the particulate analyte on the filter element, delivering a suitable reagent to said filter element while maintaining the filter element at a suitable temperature to fix a selected component of said particulate analyte to said filter element, and delivering to the filter element suitable detection reagents to render the selected component detectable, whereupon, on delivery of said reagents, said inlet duct is connected directly or indirectly to said outlet duct to permit recirculation of said reagents through said filter element, and said filter element is subjected for a selected number of repetitions to a selected temperature cycle by operation of a heat transfer means operable to transfer heat to and from said filter element, said reagents being recirculated through said filter element
  • Figure I shows a schematic representation of on embodiment of the invention.
  • Figure 2 shows greater detail of a portion of the apparatus of Figure 1.
  • Figure 3 shows a schematic representation of fluid flow through the apparatus of the invention in closed configuration.
  • Figure 4 shows a schematic representation of fluid flow through the apparatus of the invention when in open configuration.
  • the present invention provides an apparatus which facilitates the quantitative determination of a particulate analyte in a sample.
  • the particulate analyte is trapped as discrete particles on the filter of the apparatus and fixed to the filter by a suitable method.
  • the particulate analyte is then exposed to various reagents and reaction conditions selected to render the particulate analyte of interest detectable and countable.
  • one or mora of the reaction steps may be carried out by means of the apparatus of the invention, with an additional step or steps being carried out on the filter after its removal from the apparatus.
  • a sample dilution is employed which will result in the trapping of the particulate analyte on the filter as discrete particles sufficiently spaced from each other to be counted.
  • An appropriate dilution can be determined empirically.
  • any particulate analyte which can be trapped on and fixed to the filter and rendered detectable and countable thereon by exposure to suitable reagents may be quantitated using the apparatus of the invention.
  • the apparatus is particularly useful for the determination of microorganisms, which term is used to include any unicellular organisms such as bacteria, fungi, protozoa and single call preparations of eukaryotic cells.
  • Microorganism-containing samples may include water samples, soil samples, biological samples such as body fluids or tissue samples and food or food-processing samples.
  • a suitable aqueous suspension is prepared and filtered through the apparatus of invention, as will be understood by those skilled n the art.
  • FIGS 1 to 4 illustrate one embodiment of the apparatus of the invention.
  • a filter 1 of appropriate porosity is supported in a holder 2.
  • Reagent or sample solutions are introduced into the apparatus through inlet port 3 and reservoir 4 to inlet duct 5 which delivers solution to filter 1.
  • a closed system can be formed by adjustment of flow controller 10 to direct liquid flow through return line 11 back to reservoir 4, so that once the system is charged with a desired reagent, this may be pumped continuously through filter 1 and around the closed loop 9 for a desired time interval by means of pump 8.
  • the filter holder 2 is supported in filter housing 12.
  • filter holder 2 and housing 12 may be of integral construction.
  • Heat gun 13 is positioned adjacent to filter 1 and can be operated to direct a stream of hot or cool air over filter 1 so as to maintain filter 1 at preselected temperatures.
  • a commercially available hand held-type heat gun has been found to be satisfactory.
  • heat transfer means may be employed to transfer heat to and from the filter, including use of heating and cooling coils, for example located in filter holder 2, and use of a liquid as a heat transfer medium.
  • Operation of heat gun 13 is controlled by controller 14.
  • the temperature of the filter 1 is monitored by an integral thermocouple 15 placed directly in the liquid flow adjacent the filter with read-out on digital thermometer 16.
  • Controller 14 is programmed to provide thermostatic control of the temperature of filter 1, the heat gun being activated by the controller to transfer heat to and from filter 1 so as to maintain filter 1 at a preselected temperature for a preselected period of time.
  • the controller 14 is also programmed to control operation of the pump 8. As it may be desirable for certain applications to switch off the pump when the filter is in a particular temperature range, as will be described, the pump controller is preferable thermostatically controlled.
  • Bubbling may be controlled by returning the recirculated solution from return duct 11 into the top of reservoir 4 as seen in Figures 3 and 4.
  • Other bubbling control systems will be known to those skilled in the art.
  • the filter 1 must be of a porosity to retain the organisms of interest.
  • the material of the filter will be selected depending on the method to be used for detection, of the particles. For example, if high temperatures are involved, the filter must be able to withstand these temperatures
  • the filter holder 2, filter housing 12 and tubing of the circulation loop 9 must also be of materials compatible with the reagents and temperatures of the reactions to be employed. For reactions involving high temperatures, a nylon filter holder and housing and teflon tubing have been found to be satisfactory. A filter holder with minimal void volume and thin walls is preferred to facilitate rapid temperature change of both holder 2 and filter 1.
  • Filter holder 2 was machined from a solid block of nylon and had an outer diameter of 40 mm, wall thickness of 2 mm and a void volume of approximately 3.0 mL.
  • two or more filters supported in filter holders may be arranged in parallel, being supplied with reagents from a common reservoir by branches of inlet line 5 and being drained by outlet lines which converge into a common outlet line 6.
  • Such parallel filter holders and their filters may, for example, be used for samples of different dilutions or from different sites or sources.
  • two or more filters of differing porosity may be arranged in stacked filter holders to retain analytes of differing particle sizes.
  • the void volume of the apparatus will be larger, and longer cycle times may be required to attain a predetermined temperature, as will be understood by those skilled in the art.
  • a liquid sample containing the microorganisms to be enumerated is introduced into the apparatus through the inlet port and pumped through the filter with the system in open configuration as in Figure 4, so that the microorganisms are deposited on the filter and the filtrate is pumped to waste.
  • the microorganisms are fixed to the filter by heating the filter, by means of the heat gun, to an appropriate temperature for an appropriate time, followed by pumping of a suitable lysing reagent through the filter.
  • the DNA of the lysed microorganisms becomes fixed to the filter, along with other components of the cell lysate, at the sites on the filter where the microorganisms were deposited.
  • the DNA is then directly subjected to amplification by PCR at these sites.
  • a solution containing the necessary reagents and diagnostic primers appropriate to the organisms of interest is introduced into the apparatus through the inlet port and reagent reservoir and the system is converted to a closed configuration by operation of flow controller 10.
  • the reagent mixture is circulated as required, as will be described, and the filter is adjusted to and held at the temperature required for each stage of the PCR process by operation of the heat transfer means.
  • Operation of the heat gun or other heat transfer means is controlled by the controller 14, which is programmed to provide thermostatic control of the filter temperature, so as to maintain the filter at a suitable temperature for a suitable period of time for each step of the polymerase chain reaction.
  • thermostatic control of the filter temperature so as to maintain the filter at a suitable temperature for a suitable period of time for each step of the polymerase chain reaction.
  • Such temperatures and times are known to those skilled in the art.
  • the cycle of temperature steps is repeated a sufficient number of times to achieve the desired degree of DNA amplification, as will be understood by those skilled in the art.
  • the inventors have found, using the apparatus of the invention, that about 25 to about 50 cycles of DNA synthesis are satisfactory for enumeration of microorganisms.
  • the controller may also be programmed to control operation of the pump. As it is desirable to stop the pump when the filter is at high temperatures, as described below, the pump controller may preferably be thermostatically controlled.
  • filters of appropriate size have been cut from Biotrans nylon membrane, Catalogue Number 810205, 0.45 ⁇ m pore size.
  • Other filter materials are also suitable, including teflon or nylon filters such as Millipore, Type FH-Fluorpore and Whatman, Type 7404, respectively.
  • a filter of fibrous texture is preferred for optimal retention of single-stranded DNA target template and of newly synthesised double-stranded DNA product.
  • the apparatus is emptied of reagent mixture and the filter is raised to 920 for 15 in. to denature tie DNA and fix it to the filter.
  • the DNA may then be rendered visible by application of appropriate reagents to the filter, either by pumping these through the filter while it is contained in the apparatus of the invention or the filter may be removed and the reagents applied to the filter by spraying or dipping.
  • DNA may be rendered visible by staining the filter with a DNA stain such as Hoechst 33258, DAPI or ethidiu bromide and the resulting stained spots on the filter are counted either directly or under UV light.
  • the amplified DNA spots on the filter may also be directly visualised and counted under UV light.
  • the selected primers may be diagnostic of a particular species of organism and strains of this species may be distinguished and counted on the filter by means of strain specific probes as will be understood by those skilled in the art.
  • the probe carries a suitable label, such as a radioactive label, which permits detection and enumeration of the strain of interest. Where the probe is radioactive, the filter is placed in contact with x-ray film and spots on the developed film are counted to provide enumeration of the organism of interest.
  • probe labels will be known to those skilled in the art and include chromogenic compounds which can be reacted with a suitable reagent to give a detectable coloured compound.
  • two or more cell types or strains may be enumerated on the same filter by using a primer mixture containing a primer pair diagnostic for each cell type or strain in the amplification process, followed by probing with a probe selective for each of the cell types or strains of interest.
  • These selective probes may optionally carry labels giving rise to different coloured compounds, so that spots of different colours are produced on the filter, each colour corresponding to a cell type or strain of interest.
  • the apparatus of the invention can be used for detection and enumeration of particulate analytes by methods other than PCR amplification.
  • ligase chain reactions may be carried out in the apparatus, or analytes may be rendered detectable by enzyme immunoassay techniques.
  • the following example further illustrates the use of the apparatus of the invention.
  • Rhizobium meliloti strain 825 is a soil inoculant strain used to enhance symbiotic nitrogen fixation of alfalfa. It contains a diagnostic DNA sequence containing a novel transposition copy of the DNA insertion sequence ISRml. This DNA locus has been sequenced and PCR primers selected and prepared for its amplication, as described in U.S. Patent Application, Serial No. 07/613,061 (Wheatcroft and Wyndham) the contents of which are incorporated herein by reference) . After introduction of R. meliloti 825 into soil, a soil sample was obtained for enumeration of the organism of interest.
  • 0.1 g soil was shaken up with 10 ml suspension buffer (50 mM Tris/HCl, pH 8.0, containing 25% W/v sucrose) and after centrifugation of the suspension at 1000 rpm for 5 min to pellet soil particles, a series of dilutions (xlO"*, xlO" 5 , xlO "6 ) of the supernatant were made in water.
  • the appropriate dilution for optimal enumeration is determined by filtering and processing a sample of each dilution in the series and selecting a dilution which gives good distribution of the microorganism as discrete countable spots.
  • the heat gun was switched off and 0.5 ml lysing reagent (10 M EDTA in suspension buffer with 1.5 mg/ml lysozyme added immediately before use) , was introduced through inlet port and drawn through filter to waste by pumping.
  • the filter was heated to 92°C for 15 min without pumping and then was washed with 2 ml. water which was pumped to waste.
  • reaction mixture contained 50 mM/KCl; 10 mM Tris/HCl, pH 9.0; 0.1% v/v Triton X-100; dATP, dCTP, dTTP and dGTP, each 0.2 mM; and (added immediately before use) 0.05 ml of each primer at 0.01 mM and 5 units of Taq DNA polymerase.
  • Flow controller 10 was adjusted to the closed loop position so that the reaction mixture could be recirculated through the filter and round closed loop 9 by means of the pump.
  • the filter was raised to 94°C and held at that temperature for 2 min, followed by 2.5 min. at 50°C and 3 min. at 74°C.
  • Filter temperature was raised or lowered as required by blowing warn or cool air over the filter and filter holder by means of the heat gun.
  • the circulation pump was stopped during steps carried out at about 90°C or higher to reduce loss of enzyme.
  • the filter was taken through the described temperature/time cycle for 30 repetitions. After the amplification period was complete, the reaction mixture was voided and the filter raised to 92°C for 15 min. to denature the newly synthesised DNA. The filter was removed from the apparatus and treated by probing with a radio labelled probe homologous to the diagnostic sequence of strain 825, followed by autoradiography and counting of spots where the cells of this specific strain had been lysed. As indicated above, it was found preferable for optimal conservation of enzyme to stop the circulation pump during high temperature stages of the amplification process when using Taq DNA polymerase. As will be understood by those skilled in the art, a less heat stable DNA polymerase such as Klenow DNA polymerase may also be employed for amplification. In this case, the filter is raised to 94°C for 2 min. followed by 5 min. at 42°C. When using this enzyme, the circulation pump is operated only when the filter temperature is below about 45°C for optimal preservation of enzyme.

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

Abstract

L'invention se rapporte à un appareil servant à détecter et à dénombrer un analyte particulaire présent dans un échantillon liquide, et comprenant un filtre (1); un élément de support (2) destiné au filtre (1); un conduit d'entrée (3) transportant un échantillon liquide vers le filtre (1); un conduit de sortie (6) destiné à recevoir un filtrat du filtre (1); un moyen de transfert de chaleur (13) transférant la chaleur vers le filtre (1) et à partir de celui-ci; un contrôleur (14) commandant le moyen de transfert de chaleur afin de maintenir le filtre (1) à des températures présélectionnées pendant des laps de temps présélectionnés; un contrôleur d'écoulement (10) pouvant être actionné en une première position afin de raccorder les conduits d'entrée et de sortie (3, 6) pour permettre le recyclage d'un fluide à travers le filtre (1), et en une seconde position empêchant le recyclage de fluide à travers le filtre (1); ainsi qu'une pompe (8) servant à faire repasser le fluide à travers le filtre (1). L'invention se rapporte également à un procédé de détection et de dénombrement d'un analyte particulaire dans un échantillon liquide, au moyen de l'appareil de l'invention.
PCT/CA1994/000101 1993-03-18 1994-02-25 Appareil de determination quantitative d'analytes particulaires WO1994021780A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU61049/94A AU6104994A (en) 1993-03-18 1994-02-25 Apparatus for the quantitative determination of particulate analytes

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US3322893A 1993-03-18 1993-03-18
US033,228 1993-03-18

Publications (1)

Publication Number Publication Date
WO1994021780A1 true WO1994021780A1 (fr) 1994-09-29

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Application Number Title Priority Date Filing Date
PCT/CA1994/000101 WO1994021780A1 (fr) 1993-03-18 1994-02-25 Appareil de determination quantitative d'analytes particulaires

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AU (1) AU6104994A (fr)
WO (1) WO1994021780A1 (fr)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995025815A1 (fr) * 1994-03-24 1995-09-28 Gamera Bioscience Corporation Fondometre a adn et procedes d'utilisation
WO2000033962A1 (fr) * 1998-12-07 2000-06-15 Her Majesty The Queen In Right Of Canada As Represented By The Minister Of Agriculture And Agri-Food Appareil de thermocyclage rotatif
WO2003085109A2 (fr) * 2002-04-09 2003-10-16 Genesystems Procede et automate d'extraction d'acides nucleiques a partir d'un melange complexe
WO2006117676A2 (fr) 2005-04-29 2006-11-09 Millipore Corporation Procede de detection et de caracterisation de micro-organismes sur un filtre
EP2298931A1 (fr) * 1996-06-04 2011-03-23 University of Utah Research Foundation Système et procédé d'exécution et de suivi de processus biologiques
EP1593747B2 (fr) † 2004-05-04 2013-05-15 EMD Millipore Corporation Procédé d'énumeration et d'identification de micro-organismes
WO2015162059A1 (fr) * 2014-04-25 2015-10-29 Robert Bosch Gmbh Procédé et dispositif de purification de molécules biologiques
EP3191576A4 (fr) * 2014-09-12 2018-04-18 Anthrogenesis Corporation Procédés de quantification de particules dans une culture cellulaire

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4912059A (en) * 1988-10-21 1990-03-27 The Johns Hopkins University Phase sensitive differential polarimetry technique and apparatus
WO1991006670A1 (fr) * 1989-11-07 1991-05-16 Infometrix, Incorporated Procede electrochimiques de quantification de populations de bacteries

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4912059A (en) * 1988-10-21 1990-03-27 The Johns Hopkins University Phase sensitive differential polarimetry technique and apparatus
WO1991006670A1 (fr) * 1989-11-07 1991-05-16 Infometrix, Incorporated Procede electrochimiques de quantification de populations de bacteries

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995025815A1 (fr) * 1994-03-24 1995-09-28 Gamera Bioscience Corporation Fondometre a adn et procedes d'utilisation
EP2298931A1 (fr) * 1996-06-04 2011-03-23 University of Utah Research Foundation Système et procédé d'exécution et de suivi de processus biologiques
WO2000033962A1 (fr) * 1998-12-07 2000-06-15 Her Majesty The Queen In Right Of Canada As Represented By The Minister Of Agriculture And Agri-Food Appareil de thermocyclage rotatif
US6448066B1 (en) 1998-12-07 2002-09-10 Her Majesty The Queen In Right Of Canada, As Represented By The Minister Of Agriculture And Agri-Food Rotary thermocycling apparatus
WO2003085109A2 (fr) * 2002-04-09 2003-10-16 Genesystems Procede et automate d'extraction d'acides nucleiques a partir d'un melange complexe
WO2003085109A3 (fr) * 2002-04-09 2004-04-01 Genesystems Procede et automate d'extraction d'acides nucleiques a partir d'un melange complexe
EP1593747B2 (fr) † 2004-05-04 2013-05-15 EMD Millipore Corporation Procédé d'énumeration et d'identification de micro-organismes
WO2006117676A2 (fr) 2005-04-29 2006-11-09 Millipore Corporation Procede de detection et de caracterisation de micro-organismes sur un filtre
EP1877573B1 (fr) * 2005-04-29 2013-06-05 EMD Millipore Corporation Procede de detection et de caracterisation de micro-organismes sur un filtre
WO2015162059A1 (fr) * 2014-04-25 2015-10-29 Robert Bosch Gmbh Procédé et dispositif de purification de molécules biologiques
CN106232799A (zh) * 2014-04-25 2016-12-14 罗伯特·博世有限公司 用于净化生物分子的方法和装置
JP2017515500A (ja) * 2014-04-25 2017-06-15 ローベルト ボツシユ ゲゼルシヤフト ミツト ベシユレンクテル ハフツングＲｏｂｅｒｔ Ｂｏｓｃｈ Ｇｍｂｈ 生体分子を精製するための方法および装置
EP3191576A4 (fr) * 2014-09-12 2018-04-18 Anthrogenesis Corporation Procédés de quantification de particules dans une culture cellulaire

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Publication number Publication date
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