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WO2011068465A1 - Procede et appareil de recuperation de cellules et leur analyse - Google Patents

Procede et appareil de recuperation de cellules et leur analyse Download PDF

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Publication number
WO2011068465A1
WO2011068465A1 PCT/SG2009/000462 SG2009000462W WO2011068465A1 WO 2011068465 A1 WO2011068465 A1 WO 2011068465A1 SG 2009000462 W SG2009000462 W SG 2009000462W WO 2011068465 A1 WO2011068465 A1 WO 2011068465A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
filter
nucleic acid
membrane
acid molecules
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/SG2009/000462
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English (en)
Inventor
Haiqing Gong
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.)
Individual
Original Assignee
Individual
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 Individual filed Critical Individual
Priority to PCT/SG2009/000462 priority Critical patent/WO2011068465A1/fr
Priority to SG2012040408A priority patent/SG181161A1/en
Publication of WO2011068465A1 publication Critical patent/WO2011068465A1/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
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/4077—Concentrating samples by other techniques involving separation of suspended solids
    • G01N2001/4088—Concentrating samples by other techniques involving separation of suspended solids filtration

Definitions

  • the present invention relates to the field of separating and/or recovering and analysing cells.
  • the present invention relates to the field of recovering and analysing viable cells from fluid or gas.
  • the gas sample e.g. air sample
  • the membrane filter For capturing cells from gas (e.g. airborne cells), the gas sample (e.g. air sample) similarly enters the inlet, passes through the membrane filter and out through the outlet.
  • the same process as described above may be used to recover the trapped cells.
  • back-flushing Another method to recover the trapped cells, termed “back-flushing”, involves allowing an eluent to enter through the outlet and to flow through the membrane to flush out the trapped cells before exiting through the fluid inlet, followed by collecting the eluent comprising cells for further analysis.
  • a method of separating and/or recovering and analysing cells comprising:
  • separating cells from a fluid or gas with a filter apparatus comprising a filter
  • the method may be used for separating and/or recovering and analysing cells of any organism.
  • the method may be used for bacteria and/or protozoa cells and in particular, cells of pathogenic organisms.
  • Figure 1 shows an example of a filter apparatus for the exemplary method.
  • Figure 2 illustrates an example of the exemplary method with the filter apparatus of Figure 1.
  • the top view of the membrane filter after each step of the method is also illustrated in Figures 2(A) to (E).
  • Figure 2(F) also shows an illustration depicting the eluent comprising unbound DNA derived from viable and/or membrane-intact cells and covalently bound DNA from dead or membrane-compromised cells.
  • Figure 3 shows the (A) cross-sectional view and (B) top view of an example of a fluidic apparatus for the exemplary method.
  • the present exemplary embodiments relate to a method of separating and/or recovering and analysing cells, for example, from fluid or gas.
  • the fluid or gas may be filtered through a filter apparatus comprising a filter which separates the cells from the fluid or gas, trapping them on the filter. After filtration, the trapped cells on the filter may be lysed to release nucleic acid molecules.
  • Any suitable method for lysing the cells may be used. For example, various methods may be used for lysing the cells, including but not limited to using heat, chemicals, ultrasound and freeze-thawing.
  • the nucleic acid molecules may then be eluted from the filter apparatus for further analysis, detection and/or identification.
  • the method may also further include steps for differentiating viable cells.
  • a phenanthridium compound capable of preferentially penetrating dead or membrane-compromised cells over viable and/or substantially intact cells to intercalate with at least one nucleic acid molecule
  • the cells may be contacted with the phenanthridium compound before or after filtration.
  • the cells may be mixed with the phenanthridium compound and the mixture is then filtered.
  • the cells may be filtered first and the phenanthridium compound may then be loaded through the filter, contacting the cells on the filter while flowing through the filter.
  • the covalently bound nucleic acid molecules of the dead or membrane-compromised cells are unsuitable or unable to take part in further reactions, for example, the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • the filter may also be washed to substantially remove any excess, residual and/or unbound phenanthridium compound. Either exposing the filter to a light source may be performed before washing the filter or alternatively, washing the filter may be performed before exposing the filter to a light source.
  • any suitable phenanthridium compound may be used.
  • the compound propidium monoazide may be used. Propidium monoazide is able to preferentially penetrate dead or membrane-compromised cells over viable and/or substantially intact cells to intercalate with DNA molecules and on exposure to a blue light source, covalently binds with the DNA molecules.
  • the filter apparatus may comprise a filter cartridge comprising a membrane filter 3 as shown in Figure 1.
  • the filter cartridge comprises a lid 1 with an inlet 2, a membrane filter 3 on a filter support 4 and an outlet 5.
  • the method of the invention including the optional step differentiation of viable cells will now be described, with reference to Figure 2.
  • a fluid sample comprising Escherichia coli for example, was allowed to enter the inlet, pass through the membrane filter 3 and out through the outlet 5, as indicated by the arrows.
  • the fluid sample may comprise both viable and dead E. coli, for example, by mixing a sample of E. coli heated at 72 °C for 10 minutes with a sample of live E. coli before using for the filtration.
  • the membrane filter 3 traps particles including both viable and/or membrane intact E. coli 6 and dead or membrane-compromised E. coli 7 and other particles 8 (e.g. dirt) from the fluid.
  • a washing fluid e.g. 4 ml of water
  • the propidium monoazide molecules represented by the black dots 8 which had penetrated the dead or membrane-compromised cells were not washed away.
  • the lid of the filter cartridge may be removed and the membrane filter 3 may then be exposed to a light source 0, for example, a blue light from a light emitting diode (LED).
  • a light source for example, a blue light from a light emitting diode (LED).
  • the propidium monoazide covalently binds to the DNA 11 of the penetrated cells.
  • the lid of the filter cartridge may be made of a substantially optically transparent material (or substantially optically transparent to the blue light used) and in this case, the lid need not be removed when exposing the membrane filter 3 to the light source.
  • the lid 1 may then be replaced on the filter apparatus. 200 ⁇ of water 12 was then added to immerse the membrane fully within the filter apparatus and the cartridge may be immersed in ⁇ 95 °C hot liquid bath 13 to lyse the cells on the membrane filter 3. Heat causes the cell wall to break and the nucleic acid molecules are released into the water within the filter apparatus.
  • a hot gas stream also of ⁇ 95 °C may be allowed to enter via the inlet 2 to pass through the filter apparatus to lyse the cells on the membrane filter.
  • the entire filter apparatus may also be incubated at ⁇ 95 °C, for example in an oven or a hot gas stream of ⁇ 95 °C may also be directed to the exterior of the filter apparatus to lyse the cells.
  • a heater may be included as part of the filter apparatus for heating the membrane filter.
  • a hot liquid of ⁇ 95 °C may also be introduced via the inlet to submerge the filter as indicated by the arrow and lyse the cells on the membrane filter 3.
  • Both DNA from viable and/or membrane-intact cells 14 and covalently bound DNA from dead or membrane-compromised cells 11 are released with cell lysis. (See Figure 2(E)).
  • Cell debris 15 may also be present.
  • Other methods of cell lysis may be utilised, including but not limited to chemical lysis, freeze-thawing and ultrasonication.
  • the nucleic acid molecules may be eluted from the membrane filter 3 for example by adding 800 ⁇ of TE buffer as the elution buffer via the inlet of the filter apparatus as indicated by the arrow. Any suitable eluent including but not limited to water or other solution or buffer may also be used.
  • the eluted nucleic acid molecules may then be collected for further analysis, detection and/or identification. During elution, most of the cell debris 15 from lysis and other particles 8 remained on the membrane filter 3 while the nucleic acid molecules were eluted. Further analysis, detection and/or identification may be with PCR. For example, 1 ⁇ of the eluent may be used for PCR. During PCR, only DNA obtained from viable or membrane-intact cells 14 may be amplified. The covalently bound DNA from the dead or membrane- compromised cells 1 should not be amplified.
  • the filter apparatus may be in the form of a fluidic chip as illustrated in Figure 3.
  • the filter apparatus comprises three layers 16, 17 and 18 and a filter 3 bonded together to form a planar apparatus with an inlet 2, a channel 19 and an outlet 5.
  • Layer 16 may be substantially optically transparent to light (or substantially optically transparent to the blue light used).
  • the fluidic device is exposed to light from a LED (e.g. blue light), which may be positioned above layer 16.
  • a heater and/or an ultrasonic device may be positioned below layer 18 for lysing the cells.
  • the fluid enters via an inlet 2, passes through the membrane 3 which separates the particles including cells, passes through a channel 19 within the fluidic chip and out again via the outlet 5.
  • the subsequent steps including the optional step of differentiating viable cells with a phenanthrdium compound, as well as lysing the cells and eluting the nucleic acid molecules may also be carried out with the fluidic chip.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Immunology (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)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

L'invention concerne un procédé et un appareil permettant de récupérer des cellules et de les analyser. Plus précisément, l'invention concerne le domaine de la récupération et de l'analyse de cellules viables dans un fluide ou un gaz. Les cellules peuvent être récupérées par filtration et des molécules d'acide nucléique sont éluées du filtre. Avant l'élution, des cellules peuvent être traitées avec un composé phénanthridium pouvant pénétrer des cellules mortes ou à membrane fragilisée. Plus précisément, l'invention concerne l'utilisation de monioazide de propidium (PMA), qui empêche l'amplification par PCR d'acides nucléiques dérivés de cellules mortes ou à membrane fragilisée, ce qui permet la différentiation entre des cellules viables et des cellules mortes.
PCT/SG2009/000462 2009-12-02 2009-12-02 Procede et appareil de recuperation de cellules et leur analyse Ceased WO2011068465A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/SG2009/000462 WO2011068465A1 (fr) 2009-12-02 2009-12-02 Procede et appareil de recuperation de cellules et leur analyse
SG2012040408A SG181161A1 (en) 2009-12-02 2009-12-02 A method and apparatus for recovering cells and their analysis

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/SG2009/000462 WO2011068465A1 (fr) 2009-12-02 2009-12-02 Procede et appareil de recuperation de cellules et leur analyse

Publications (1)

Publication Number Publication Date
WO2011068465A1 true WO2011068465A1 (fr) 2011-06-09

Family

ID=44115162

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/SG2009/000462 Ceased WO2011068465A1 (fr) 2009-12-02 2009-12-02 Procede et appareil de recuperation de cellules et leur analyse

Country Status (2)

Country Link
SG (1) SG181161A1 (fr)
WO (1) WO2011068465A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014015925A1 (fr) 2012-07-26 2014-01-30 Sartorius Stedim Biotech Gmbh Procédé de différenciation entre cellules vivantes et mortes
WO2014151177A1 (fr) * 2013-03-15 2014-09-25 3M Innovative Properties Company Concentrateur d'échantillon et méthode d'utilisation
JPWO2017154349A1 (ja) * 2016-03-10 2018-08-30 パナソニックＩｐマネジメント株式会社 核酸抽出装置、核酸抽出ユニット及び核酸抽出方法
US11261476B2 (en) 2016-10-19 2022-03-01 Q-Linea Ab Method for recovering microbial cells

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996041810A1 (fr) * 1995-06-08 1996-12-27 Progen Industries Limited Procede et appareil d'extraction d'adn
US20080213870A1 (en) * 2007-03-01 2008-09-04 Sean Wuxiong Cao Methods for obtaining modified DNA from a biological specimen
US20090081772A1 (en) * 2007-09-21 2009-03-26 Metagenex Method and device for collecting cellular material from cells isolated on a filter
WO2009055810A1 (fr) * 2007-10-25 2009-04-30 U.S. Environmental Agency Discriminateur de microorganismes et procédé associé

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996041810A1 (fr) * 1995-06-08 1996-12-27 Progen Industries Limited Procede et appareil d'extraction d'adn
US20080213870A1 (en) * 2007-03-01 2008-09-04 Sean Wuxiong Cao Methods for obtaining modified DNA from a biological specimen
US20090081772A1 (en) * 2007-09-21 2009-03-26 Metagenex Method and device for collecting cellular material from cells isolated on a filter
WO2009055810A1 (fr) * 2007-10-25 2009-04-30 U.S. Environmental Agency Discriminateur de microorganismes et procédé associé

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
NOCKER, A. ET AL.: "Use of Propidium Monoazide for Live/Dead Distinction in Microbial Ecology.", APPLIED AND ENVIRONMENTAL MICROBIOLOGY., vol. 73, no. 16, 2007, pages 5111 - 5117, XP055018729, DOI: doi:10.1128/AEM.02987-06 *
RUDI, K. ET AL.: "Use of Ethidium Monoazide and PCR in Combination for Quantification of Viable and Dead Cells in Complex Samples.", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 71, no. 2, 2005, pages 1018 - 1024, XP003000006, DOI: doi:10.1128/AEM.71.2.1018-1024.2005 *
VESPER, S. ET AL.: "Quantifying fungal viability in air and water samples using quantitative PCR after treatment with propidium monoazide (PMA).", JOURNAL OF MICROBIOLOGICAL METHODS., vol. 72, no. 2, 2008, pages 180 - 184, XP022420007 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014015925A1 (fr) 2012-07-26 2014-01-30 Sartorius Stedim Biotech Gmbh Procédé de différenciation entre cellules vivantes et mortes
WO2014151177A1 (fr) * 2013-03-15 2014-09-25 3M Innovative Properties Company Concentrateur d'échantillon et méthode d'utilisation
US9677981B2 (en) 2013-03-15 2017-06-13 3M Innovative Properties Company Sample concentrator and method of use
JPWO2017154349A1 (ja) * 2016-03-10 2018-08-30 パナソニックＩｐマネジメント株式会社 核酸抽出装置、核酸抽出ユニット及び核酸抽出方法
US11261476B2 (en) 2016-10-19 2022-03-01 Q-Linea Ab Method for recovering microbial cells

Also Published As

Publication number Publication date
SG181161A1 (en) 2012-07-30

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