WO2002022866A2 - Procede et kit d'essai pour analyser une matiere contenant de l'acide nucleique - Google Patents

Procede et kit d'essai pour analyser une matiere contenant de l'acide nucleique Download PDF

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Publication number
WO2002022866A2
WO2002022866A2 PCT/EP2001/010363 EP0110363W WO0222866A2 WO 2002022866 A2 WO2002022866 A2 WO 2002022866A2 EP 0110363 W EP0110363 W EP 0110363W WO 0222866 A2 WO0222866 A2 WO 0222866A2
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WO
WIPO (PCT)
Prior art keywords
filter material
nucleic acid
sample
filter
reagents
Prior art date
Application number
PCT/EP2001/010363
Other languages
German (de)
English (en)
Other versions
WO2002022866A3 (fr
Inventor
Gudrun Vogeser
Original Assignee
Pika Weihenstephan Gmbh
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 Pika Weihenstephan Gmbh filed Critical Pika Weihenstephan Gmbh
Priority to AU2002212215A priority Critical patent/AU2002212215A1/en
Publication of WO2002022866A2 publication Critical patent/WO2002022866A2/fr
Publication of WO2002022866A3 publication Critical patent/WO2002022866A3/fr

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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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1017—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by filtration, e.g. using filters, frits, membranes

Definitions

  • the present invention relates to a method for the analysis of nucleic acid-containing material, which comprises depositing the nucleic acid-containing material on a filter material and dissolving the filter material in a solvent. Furthermore, the present invention also relates to a sample kit for carrying out the method according to the invention.
  • centrifugation or membrane filtration are used for sample preparation.
  • a disadvantage of centrifugation is that not all organisms can be detected with certainty, so that filtration is the preferred method for detecting a trace infection.
  • the problem with filtration is that after filtration, the separated organisms adhere very firmly to the filter material and, depending on the filter type, can be firmly enclosed in its pores. It is therefore practically impossible for subsequent tests of the organisms to quantitatively detach the collected organisms from the filter material.
  • nucleic acids are obtained quantitatively from samples of material containing nucleic acid.
  • the invention is based on the knowledge that these objects can be achieved if the nucleic acid-containing material accumulated on a filter material is digested on this filter material.
  • the present invention therefore provides a method for the analysis of nucleic acid-containing material which comprises depositing the nucleic acid-containing material on a filter material and dissolving the filter material in a solvent, wherein the nucleic acid-containing material on the filter material is digested.
  • the present invention provides a sample kit for carrying out the method according to the invention, which comprises a filter material, a buffer solution and an aid for improved phase separation.
  • the method according to the invention makes it possible to obtain nucleic acids such as DNA and RNA quantitatively from all types of material containing nucleic acid. This ensures that no nucleic acids are lost from the material containing nucleic acid and are consequently not recognized in the analysis.
  • this ensures that when analyzing traces of material containing nucleic acid, no nucleic acids are overlooked and their content in the sample can be reliably determined quantitatively.
  • nucleic acids are obtained in high purity by the method according to the invention.
  • the nucleic acids obtained by the method according to the invention can be used for further analyzes, such as DNA hybridizations, cloning, polymerase chain reaction (PCR) etc. Because of the high purity of the nucleic acids, this can also be done directly without further purification or preparation steps.
  • nucleic acid concentration is still too low for further analysis, it can be enriched with common concentration methods, such as alcohol precipitation or with the help of nucleic acid binding columns.
  • the sample kit according to the invention enables the method according to the invention to be carried out in a user-friendly manner.
  • the nucleic acid-containing material can be deposited on the filter material from any medium suitable for filtration.
  • the nucleic acid-containing material is filtered off from a fluid phase, such as a liquid phase.
  • any material that is suitable for separating the nucleic acid-containing material and that can also be dissolved in a solvent can be used as the filter material.
  • a polycarbonate or cellulose acetate filter is preferably used as the filter material in the process according to the invention.
  • Polycarbonate filters have that Advantage that they can be dissolved without residue in a suitable solvent, while cellulose acetate filters are very inexpensive.
  • Chloroform or phenol are preferably used as solvents for dissolving the filter material. Chloroform is particularly suitable for dissolving polycarbonate filters, phenol is particularly suitable for dissolving cellulose acetate filters. These solvents have the advantage that they do not attack the nucleic acids and can completely dissolve the filter materials in the case of the polycarbonate filter and largely in the case of the cellulose acetate filter.
  • the nucleic acid-containing material deposited on the filter material can be disrupted using all common methods, such as treatment with ultrasound or boiling the samples in a water bath for several minutes.
  • the filter material with the nucleic acid-containing material deposited thereon is exposed to the radiation generated in a microwave device, a heat treatment, a treatment with ultrasound or a mechanical method such as stirring or shaking together with solid particles.
  • a microwave device a heat treatment
  • a treatment with ultrasound a mechanical method such as stirring or shaking together with solid particles.
  • cell-containing material for example, this enables the cell walls to be unlocked quickly and quantitatively, thus releasing the nucleic acids.
  • the method according to the invention can be used for the analysis of nucleic acid-containing material of all kinds, such as, for example, material which comprises microorganisms such as yeasts and bacteria, fungi, viruses, algae or vegetable material.
  • these organisms After being deposited on the filter material, these organisms can be processed using other methods before being digested. On the one hand, this can be done by incubating them on solid or in liquid nutrient media in order to increase the number of organisms. This enables the detection limits of these organisms to be improved.
  • the organisms on the filter material can also be microscoped and / or dyed before digestion.
  • fluorescent dyes can be used as dyes.
  • the organisms By microscoping and / or staining the organisms, it can be determined, for example, whether the organisms accumulated on the filter material are alive or dead. After examining their viability, the organisms can be further differentiated using molecular biological methods. It is a particular advantage of the method according to the invention that the differentiation with molecular biological logical methods concerns exactly the same organisms that were previously microscoped and / or analyzed by staining.
  • the nucleic acids can e.g. extracted from the solution with the aid of a buffer.
  • a buffer phase which is immiscible with the solution is added to the solution.
  • a means for improving the phase separation is used for the two-phase system.
  • This is preferably a phase lock gel as it is driven by the Eppendorf company.
  • the nucleic acids in the buffer phase can be used directly in the PCR, for example.
  • the isolated nucleic acids can be enriched with common concentration methods, such as, for example, alcoholic precipitation or with the aid of nucleic acid-binding columns.
  • the method according to the invention and / or the sample kit according to the invention can be used for the analysis of fluid media of all kinds.
  • the method / sample kit is preferably used where fluid media such as liquids are to be analyzed, which are to be used over a longer period of time or several times.
  • the method according to the invention and / or the sample kit according to the invention for the analysis of well water or in waterworks, for example for testing for E. coli and coliform germs, or for the analysis of fresh water, as used for example for hospitals or old people's homes at regular intervals Example for testing for Legionella, are prescribed.
  • the method according to the invention and / or the sample kit according to the invention is used for testing for pathogens or other contaminants, for example for analyzing blood serum, dialysis fluids or foods, in particular beverages such as beer or soft drinks and juices.
  • the method / the sample kit can also be used for the analysis of technical liquids such as lubricating oils, hydraulic fluids, cleaning solutions, coolants, etc.
  • the sample kit for carrying out the method according to the invention comprises: a) filter material for filtering a certain number of samples with material containing nucleic acid,
  • one or more solvents for dissolving the filter material as well as further reagents for the detection of the nucleic acids isolated from the sample can be added to the kit.
  • This sample kit contains the material necessary for the routine analysis of samples that contain nucleic acid-containing material and that is to be examined, for example, for possible contamination with microorganisms by means of DNA analysis.
  • the sample kit according to the invention comprises:
  • c) means for improving the phase separation, in each case one vessel is provided with the amount per sample required for processing a sample,
  • control reagents for PCR i.e. Reagents and control DNA for the necessary positive reactions.
  • the agent for improving the phase separation comprises a phase lock gel as can be obtained from Eppendorf.
  • Example 1 A water sample is filtered off by membrane filtration over a polycarbonate membrane filter. The filter is then transferred to a 2 ml Eppendorf tube. The contents of this jar are then exposed to radiation from a 650 watt microwave for 3 minutes. Then 200 ⁇ l water and 800 ⁇ l chloroform / isoamyl alcohol mixture (volume fraction 24/1) are added to the sample. The sample is shaken until the filter floats freely in the sample. The sample is then shaken overhead by shaking at 30 rpm for 10 minutes until the filter has completely dissolved. 300 ⁇ l of Phase Lock Gel from Eppendorf are then placed in a further 2 ml Eppendorf tube. The entire content of the first Eppendorf tube is then added to the Phase Lock Gel.
  • Centrifugation is carried out at 14,000 rpm for 2 minutes to separate the phases. Then 150 ⁇ l of the upper phase are transferred to a fresh vessel. 10 ⁇ l of the sample prepared in this way are used for a PCR analysis with a batch volume of 50 ⁇ l.
  • Reagents for carrying out a PCR consisting of 2 oligonucleotides, dNTPs and PCR buffers, 48 times predosed in the amount required for the analysis of one sample (50 ⁇ l), and dried

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

Abstract

La présente invention concerne un procédé pour analyser une matière contenant de l'acide nucléique, consistant à déposer ladite matière sur une matière filtrante et à dissoudre cette matière filtrante dans un solvant. Ce procédé se caractérise en ce que la matière contenant de l'acide nucléique se désagrège sur la matière filtrante. L'invention concerne également un kit d'essai permettant de mettre en oeuvre le procédé selon l'invention. Ce procédé et ce kit d'essai peuvent être employés pour analyser toutes sortes d'échantillons contenant de l'acide nucléique.
PCT/EP2001/010363 2000-09-11 2001-09-07 Procede et kit d'essai pour analyser une matiere contenant de l'acide nucleique WO2002022866A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2002212215A AU2002212215A1 (en) 2000-09-11 2001-09-07 Method and sampling kit for analysing material containing nucleic acid

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE10044856.9 2000-09-11
DE10044856A DE10044856A1 (de) 2000-09-11 2000-09-11 Verfahren und Probenkit zur Analyse von nucleinsäurehaltigem Material

Publications (2)

Publication Number Publication Date
WO2002022866A2 true WO2002022866A2 (fr) 2002-03-21
WO2002022866A3 WO2002022866A3 (fr) 2003-06-05

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PCT/EP2001/010363 WO2002022866A2 (fr) 2000-09-11 2001-09-07 Procede et kit d'essai pour analyser une matiere contenant de l'acide nucleique

Country Status (3)

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AU (1) AU2002212215A1 (fr)
DE (1) DE10044856A1 (fr)
WO (1) WO2002022866A2 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1394544A1 (fr) * 2002-08-30 2004-03-03 Biowell Technology Inc. Mèthode de mélange d'acides nucléiques dans un milieu insoluble dans l'eau, et son utilisation
EP2402456A1 (fr) * 2010-06-30 2012-01-04 Imeth AG Procédé de détermination d'organismes dans l'eau
WO2014019634A1 (fr) * 2012-07-31 2014-02-06 Sartorius Stedim Biotech Gmbh Dispositif et procédé de traitement d'un milieu de filtration
CN113430193A (zh) * 2021-06-30 2021-09-24 上海农林职业技术学院 一种核酸采集试剂盒及其使用方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5187083A (en) * 1990-11-13 1993-02-16 Specialty Laboratories, Inc. Rapid purification of DNA
US5654179A (en) * 1990-11-14 1997-08-05 Hri Research, Inc. Nucleic acid preparation methods
WO1999045021A1 (fr) * 1998-03-03 1999-09-10 Affymetrix, Inc. Genes regules par le cycle cellulaire
WO2002012560A1 (fr) * 2000-08-04 2002-02-14 E. & J. Gallo Winery Procede pour amplifier des acides nucleiques de micro-organismes presents dans des jus de fruit

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5187083A (en) * 1990-11-13 1993-02-16 Specialty Laboratories, Inc. Rapid purification of DNA
US5654179A (en) * 1990-11-14 1997-08-05 Hri Research, Inc. Nucleic acid preparation methods
WO1999045021A1 (fr) * 1998-03-03 1999-09-10 Affymetrix, Inc. Genes regules par le cycle cellulaire
WO2002012560A1 (fr) * 2000-08-04 2002-02-14 E. & J. Gallo Winery Procede pour amplifier des acides nucleiques de micro-organismes presents dans des jus de fruit

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
STARBUCK M A B ET AL: "Ultra sensitive detection of Listeria monocytogenes in milk by the polymerase chain reaction (PCR)." LETTERS IN APPLIED MICROBIOLOGY, Bd. 15, Nr. 6, 1992, Seiten 248-252, XP009005698 ISSN: 0266-8254 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1394544A1 (fr) * 2002-08-30 2004-03-03 Biowell Technology Inc. Mèthode de mélange d'acides nucléiques dans un milieu insoluble dans l'eau, et son utilisation
EP2402456A1 (fr) * 2010-06-30 2012-01-04 Imeth AG Procédé de détermination d'organismes dans l'eau
WO2012001111A3 (fr) * 2010-06-30 2012-03-08 Imeth Ag Procédé pour détecter la présence d'organismes dans l'eau
WO2014019634A1 (fr) * 2012-07-31 2014-02-06 Sartorius Stedim Biotech Gmbh Dispositif et procédé de traitement d'un milieu de filtration
US9790462B2 (en) 2012-07-31 2017-10-17 Sartorius Stedim Biotech Gmbh Device and method for treating a filtration medium
CN113430193A (zh) * 2021-06-30 2021-09-24 上海农林职业技术学院 一种核酸采集试剂盒及其使用方法

Also Published As

Publication number Publication date
AU2002212215A1 (en) 2002-03-26
DE10044856A1 (de) 2002-04-04
WO2002022866A3 (fr) 2003-06-05

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