WO2001016363A2 - Rapid, highly specific detection method for pseudomonas aeruginosa using multi-probe detection - Google Patents
Rapid, highly specific detection method for pseudomonas aeruginosa using multi-probe detection Download PDFInfo
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- WO2001016363A2 WO2001016363A2 PCT/EP2000/008418 EP0008418W WO0116363A2 WO 2001016363 A2 WO2001016363 A2 WO 2001016363A2 EP 0008418 W EP0008418 W EP 0008418W WO 0116363 A2 WO0116363 A2 WO 0116363A2
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- probes
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- 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
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
Definitions
- the invention relates to agents for the detection of Pseudomonas ae ruginosa, methods for the specific detection of Ps. aeruginosa and the use of the agent disclosed according to the invention.
- Pseudomonas aeruginosa are widespread germs in the environment (e.g. in surface waters). They are gram negative, need oxygen for growth (aerobic) and are oxidase positive. As a rule, they grow at 41 ° C in the presence of cetyltrimethylammonium bromide (cetrimide) in the nutrient medium, form the pigments pyocyanin and pyoverdin (fluoresce weakly blue-green under UV excitation), form nitrate and nitrite and are sensitive towards polymyxin. They are able to utilize a wide range of nutrients and can multiply even with a very limited supply of usable carbon sources. Only a small part of all germs of the Pseudomonas aeruginosa bacterial species is pathogenic. This ubiquitous type of bacteria, however, has considerable secondary pathogenic significance as an opportunistic germ in connection with hospitalism.
- Pseudomonas aeruginosa can cause wound infections, sepsis, endocarditis, inflammation of the genitourinary tract, but in particular infections of the respiratory tract, including pneumonia.
- Immun-suppressed patients in particular are often affected. Patients with neutropenia or cystic fibrosis usually have very poor prognoses when they are infected with Pseudomonas aeruginosa. A quick and specific early detection of this germ is therefore of great importance in medicine, especially in medical diagnostics.
- this germ is also a valuable indicator of contamination with surface water and unhygienic production. Therefore, according to legal regulations in Germany and Europe, e.g. the regulation on mineral, spring and table water, bottled products containing mineral, spring or table water are checked for the presence of this germ.
- a means for the detection of Ps. aeruginosa which is characterized in that it contains at least two probes with e at least ten successive nucleotides of the following two sequences:
- PAE1 5 - tca ctc cgt ggt aac cgt ccc cct tgc ggt tag act agc tac ttc tg -3 x
- PAE2 5 - tct cct tag agt gcc cac ccg agg tgc tgg taa cta ag - 3
- the detection method according to the invention is based on the detection of defined nuclear acid sections of Ps. aeruginosa, with highly specific sequences of the 16S ⁇ bosomal ribonucleic acid (16S rRNA) from Pseudomonas aeruginosa and strains derived therefrom being recognized selectively.
- the agent provided according to the invention can be used for the rapid identification of Ps. Aeruginosa, since neither selective cultivation of Ps. aeruginosa to pure cultures still the formation of a defined phanotype are necessary. It differentiates Ps reliably and reproducibly. aeruginosa from other closely related Pseudomonas species, for example Pseudomonas alcaligenes, Pseudomonas mendocma and Pseudomonas fluorescens.
- the agent according to the invention is characterized in that it comprises a combination of two probes which are characterized by the nucleotide sequences PAE1 and PAE2. Only the combined use of both probes leads to a reliably discriminatory detection of Ps. aeruginosa and its strains from other Pseudomonas species and their strains.
- the probes disclosed here specifically bind to sequences of the 16S rRNA on Ps. aeruginosa.
- a germ that reacts positively with both probes is called Ps. aeruginosa identified.
- a single identification probe can identify Ps. Do not guarantee aeruginosa. Only the combination according to the invention tion from both probes allows the specific identification of Ps. aeruginosa.
- Standard detection in the beverage industry Incubation of the sample on cetrimides (US Pat. No. 4072573, Ald ⁇ dge et al., 1978) - agar at 41 ° C. for 48 h. Illuminate the agar plates with ultraviolet light. Identification of bluish green fluorescent colonies as Pseudomonas aeruginosa.
- the developed probe is directed against the 23S rRNA. It detects Ps. aeruginosa, but also other fluorescent pseudomonas, e.g. Ps. trim, Ps. mendocma, Ps fluorescens, Ps putida and many more. A distinction between Ps. aeruginosa and other Group I pseudomonas is not possible. Furthermore, this probe cannot be used for the detection of Pseudomonas aeruginosa cells using FISH, but only for the detection of m nuclear acid extracts.
- the probe designated PAE3 in FIGS. 1 and 2 was found as an additional probe. This is one of the 23S rRNA derived probe with the following sequence: 5 -GACCAGCCAGAGCTTACG-3. However, as Figure 2 shows, this PAE3 probe does not succeed in detecting all Pseudomonas aeruginosa isolates from mineral waters. Isolates NE54 and NE55 are not detected by the probe.
- the agent proposed according to the invention for the detection of Ps. aeruginosa comprises at least two probes with the sequences PAE1 and PAE2 mentioned in claim 1 and derivatives thereof.
- the detection method according to the invention at least 10 consecutive nucleotides from PAE1 and PAE2 are used in combination.
- “combination * ” means both the sequential use of PAEl and PAE2 and the use of PAEl and PAE2 in one step.
- the probes comprise more than 10 consecutive nucleotides, preferably 12 to 26 nucleotides, further preferably 14 to 22 nucleotides, more preferably 16 to 20 nucleotides and particularly preferably 18 nucleotides. It is obvious to the person skilled in the art that probes with lengths other than those specifically described here can also be used, provided they are in the range from 12 to 26 successive nucleotides of the sequences PAE1 and PAE2. According to the invention, probes with the following sequences are particularly preferably used in combination: gt aac cgt cccct tgc g
- modifications * are to be understood as meaning derivatives of the sequences in which one, two or three nucleotides on one or both edges of the probe have been replaced by other nucleotides. The prerequisite here is that a specific binding to the 16 rRNA on Ps. aeruginosa remains, so that the object of the invention is achieved.
- “derivatives *” are also to be understood to mean those probes which are derived from the probes PAE1 and PAE2 and in which one or two nucleotides have been deleted or replaced by another nucleotide in the interior of the probe, but of course only such ones Variations are to be considered in which the specific binding to Ps. aeruginosa nucleotide sequences are retained.
- the person skilled in the art can easily determine which modifications of the sequences for the highly specific detection of Ps. aeruginosa are suitable and which are not.
- the invention also encompasses the sequences and reverse-complementary sequences which are reverse to PAE1 and PAE2 and their derivatives.
- the probes provided in the present invention are used in methods for the specific detection of Ps. aeruginosa used. Methods in which DNA probes are specific Detection of a microorganism are known per se. The person skilled in the art can use these methods known per se with the present probes.
- the DNA or the RNA is based on the presence of Ps. aeruginosa sample to be examined with at least the two probes PAEl and PAE2 or derivatives of these probes, as described in more detail above, brought into contact.
- the hybridization of the probes with the RNA or the DNA of the sample is then detected in order to determine the presence of Ps. to show aeruginosa specific DNA and / or RNA sequences.
- the probes can be provided with a label, for example a radioactive label, digoxigenin, peroxidase-biotin label, or a fluorescent label in order to carry out a specific hybridization with Ps. to detect aeruginosa specific DNA or RNA.
- a label for example a radioactive label, digoxigenin, peroxidase-biotin label, or a fluorescent label in order to carry out a specific hybridization with Ps. to detect aeruginosa specific DNA or RNA.
- probes are e.g. PNAs in which the bases are bound to PNA and not to a sugar-phosphate backbone.
- the probes are bound to a matrix (reverse hybridizations) and hybridization is carried out with fluorescence-labeled DNA or a PCR amplificate.
- matrices are microchips and microtiter plates.
- the invention also encompasses derivatives of the probes or the individual nucleotides of the probes that are not described separately here, for example chemical changes. improvements to the probes that facilitate the detection procedure. Such derivatives are known to the person skilled in the art and can be applied to the probes provided according to the invention.
- the present invention serves for the highly specific detection of Pseudomonas aeruginosa with the aid of a combination of at least two probes. It is known that a species like Ps. aeruginosa is naturally inconsistent, i.e. split up into individual trunks. In terms of taxonomy, a "strain" is usually understood to mean a bacterial clone defined by a strain number and its descendants. There is a so-called type strain that represents the species and thus the properties of the species. Since all strains of a species, such as Ps. aeruginosa, closely related to each other, the detection methods and probes provided according to the invention can of course also be applied to all strains of Ps. aeruginosa.
- the DNA or RNA of the sample to be examined is either isolated from the sample organisms or the organisms are disrupted in a suitable manner so that direct contact of the probes with the DNA and / or the RNA of the sample organisms is made possible without extensive and time-consuming cleaning procedures beforehand must be carried out.
- the probes are hybridized with the DNA and / or the RNA of the sample organisms under stringent conditions, preferably highly stringent conditions. These conditions are explained in more detail below.
- reaction buffer and wash buffer are composed of the following functional components: a) Buffer system for adjusting and stabilizing the pH between 7 and 8 (eg Tris / HCl) b) Water as ⁇ solvent 'c) Possibly chelating agents, which at low concentrations of monovalent cations have the influence of divalent cations
- Nonionic, aprotic detergents eg formamide
- Salt functional units are cations that neutralize the negative charges of the nuclear acid phosphate groups and thereby facilitate the duplex formation of two single-stranded nucleic acids (eg Na + m NaCl).
- Components e) and f) influence the binding strengths of duplex nuclear acid. Increasing the monovalent cations in the reaction or wash solution stabilizes the duplex molecules formed, while with an increasing content of e.g. Formamide the duplex formulations are weakened.
- a suitable probe concentration must be used.
- the hybridization must take place at a suitable temperature ⁇ the higher the temperature, the weaker the binding of the hybrids.
- Strict hybridization and washing conditions are the reaction conditions (the correct choice of the four factors) under which only duplex molecules between the probe and the desired target molecules (perfect hybrids) are formed or only the desired target organism is detected.
- Strict reaction conditions mean, for example, a hybridization temperature of approximately 5-10 ° C. below the respective primer melting point.
- the stability of the DNA / DNA or RNA / DNA hybrids must be ensured even at low salt concentrations corresponding to 0.1 x SSC / 0.5% SDS. In this way undesirable cross-reactions with other species can be prevented.
- the respective temperature conditions can differ depending on the chosen test conditions and depending on the DNA sample to be examined and must then be adapted accordingly.
- the hybridization product can be detected, for example, by autoradiography in the case of radioactively labeled primer molecules or by fluorimetry when using fluorescence-labeled oligonucleotides.
- the above probes are complementary to areas of rRNA. Therefore these probes can bind to the rRNA. However, you can also bind to the anti-Smn DNA strand of the rRNA gene (which is the same sequence as the rRNA). In order to amplify the rRNA gene region between the two probe regions by means of PCR, a primer must bind to the anti-Smn strand, the second primer to the sense strand.
- PAE2 The area complementary to PAE2 (5'-ctt agt tac cag cac ctc ggg tgg gca Ctc taa gga ga -3 ⁇ ) offers itself as a forward primer '(PAE vorwa ⁇ : t ⁇ ), as a' backward primer '(PAE ruckwar r s ) Area PAEl (5'- tca ctc cgt ggt aac cgt ccc ct tgc ggt tag act agc tac ttc tg -3 ').
- the probes according to the invention thus enable highly specific and rapid detection of representatives of the bacterial species Ps. Aeruginosa. For example, by marking of probes with different fluorescent dyes and their use at FISH in fixed samples Ps. aeruginosa can be identified at the single cell level when a cell fluoresces in two colors. In this way Ps. aeruginosa can also be specifically detected in mixed cultures. Growing on selective nutrient media and obtaining pure cultures is no longer necessary. Since it is based on the optical perception or measurement of two different fluorescent dyes, the identification can also be carried out by laypersons.
- the PCR method has the advantage that very small amounts of DNA can be detected. Depending on the material to be detected, the temperature conditions and the cycle numbers of the PCR have to be modified. The optimal reaction conditions can be determined by hand tests in a manner known per se.
- An example of a PCR is as follows:
- the characteristic, species-specific DNA marker fragments formed in the course of the PCR amplification by the extension of the primer sequences can be detected, for example, by gel electrophoresis or fluorimetry using fluorescence-labeled oligonucleotides. Of course, other detection methods known to the person skilled in the art can also be used.
- a particular advantage of the probe combination provided according to the invention is its use in situ detection methods. Often there are problems with hybridization, and the proteins present in the cell can jeopardize the success of m situ hybridization by masking important nucleotides. The possibility of using the present probe combination for hybridizations, in particular for in situ hybridizations, and not only PCR methods was therefore also not predictable.
- Hybridization methods and PCR methods are known per se to the person skilled in the art, and reference is only made here, for example, to the publication by C. Mulhardt, The Experimentator: Molecular Biology, G. Fischer, Stuttgart, Jena, Luebeck, Ulm, 1999. Reference is hereby made in full to this publication.
- Fig. 1 Identification of Pseudomonas aeruginosa reference strains and kimic isolates with rRNA-directed probes.
- Fig. 2 25 isolates from bottled non-carbonated natural mineral water.
- Example 1
- Fluorescence microscopic detection of fixed Ps. aeruginosa m water samples (bottled mineral water, spring or table water) or river enrichments.
- 250 ml of mineral water or bacterial enrichment from 250 ml of liquid medium is filtered through bacteria-proof, white membrane filters (e.g. polycarbonate filter, Mill pore GTTP02500, Eschborn) with standard filtration systems.
- hybridization solution 62.5 ng PAE1-CY3 and 250 ng PAE2 fluorescence with hybridization buffer (0.9M NaCl, 0.01 M Tris / HCl (pH 7.2 - 8.0), 0.01% SDS , 60% formamide
- the filter is then m 50 ml wash solution (14 mM NaCl, To remove unbound probe, incubate 0.01 M Tris / HCl, 0.01% SDS, 5mM EDTA (pH8.0) for 15 minutes at 48 ° C. After drying, the filter is placed on a carrier surface (eg glass object carrier) and with an anti-thread g - Dripped medium (eg Citifluor AF1, Citifluor Ltd., London) and covered with a cover glass To detect the signals, the filter is placed in the epifluorescence microscope under excitation with blue and green light (equipped with fluorescence filters for fluorescem and rhodam) at 400- bis 1000x Magnification examined. Pseudomonas aeruginosa cells fluoresce green when excited blue and red when excited green.
- a carrier surface eg glass object carrier
- an anti-thread g - Dripped medium eg Citifluor AF1, Citifluor Ltd., London
- Example 2 Identification of Pseudomonas aeruginosa m liquid enrichments from clinical samples m selective or non-selective nutrient medium on slides.
- 1 ml of slightly cloudy liquid enrichment is mixed with 1 ml of absolute ethanol and incubated at 4 ° C for at least 15 minutes. Centrifuge at 5000g for 3 minutes. Discard the supernatant. Resuspend the pellet in 500 ⁇ l phosphate-buffered NaCl solution (PBS buffer), add 500 ⁇ l 100% ethanol. Mix well. Distribute 10 ⁇ l of the enrichment thus prepared in a reaction field of the hybridization slide (e.g. Paul Marienfeld KG, Bad Mergentheim, Germany; Art. No. 1215130 - slide with 6 reaction fields) and allow it to dry 10 mm at 46 ° C. 6 different samples can be placed on one of the slides mentioned above and examined at the same time.
- a reaction field of the hybridization slide e.g. Paul Marienfeld KG, Bad Mergentheim, Germany; Art. No. 1215130 - slide with 6 reaction fields
- hybridization solution 12.5 ng PAE1-CY3 and 50 ng PAE2 fluorescence in in hybridization buffer (0.9M NaCl, 0.01M Tris / HCl (pH 7.2 - 8.0 ), 0.01% SDS, 60% formamide
- Wells of a sterile microtiter plate are filled with 50 ⁇ l PBS buffer each. Samples of the bacterial colonies are now suspended from the agar plate in the wells using sterile toothpicks. Add 50 ⁇ l of 100% ethanol each to the wells that contain suspended colonial samples. Mix. Distribute 10 ⁇ l of the samples fixed in this way per reaction field of the hybridization slide (e.g. Paul Marienfeld KG, Bad Mergentheim, Germany; Art. No. 1215130 - slide with 6 reaction fields) and allow it to dry 10 mm at 46 ° C. 6 different fixed colonies can be placed on one of the slides mentioned above and examined at the same time.
- reaction field of the hybridization slide e.g. Paul Marienfeld KG, Bad Mergentheim, Germany; Art. No. 1215130 - slide with 6 reaction fields
- 10 ⁇ l hybridization solution 12.5 ng PAE1-CY3 and 50 ng PAE2-Fluorescem m Hyb ⁇ dleitersbuffer (0.9M NaCl, 0.01 M Tris / HCl (pH 7.2 - 8.0;, 0.01% SDS, 60-formamide)
- the hybridization solution is then rinsed from the microscope slide with a wash solution preheated to 48 ° C. (14 mM NaCl, 0.01 M Tris / HCl, 0.01% SDS, 5 mM EDTA (pH 8.0)) and this 50 ml wash solution for 15 minutes at 48 ° C. mcubated to remove unbound probe. Rinse the slides carefully with distilled water and let them dry. Drip slides with anti-dmg medium (eg Citifluor AF1, Citifluor Ltd., London) and cover with cover glass.
- a wash solution preheated to 48 ° C. (14 mM NaCl, 0.01 M Tris / HCl, 0.01% SDS, 5 mM EDTA (pH 8.0)
- this 50 ml wash solution for 15 minutes at 48 ° C. mcubated to remove unbound probe. Rinse the slides carefully with distilled water and let them dry. Drip slides with anti-dmg medium (eg Citifluor
- the individual reaction fields are examined in the epifluorescence microscope under excitation with blue and green light (equipped with fluorescence filters for Fluoresce and Rhodamm) at a magnification of 400 to 1000 times.
- Pseudomonas aeruginosa cells fluoresce green when excited blue and red when excited green.
- Mucus sample is placed in 100-1000 ul PBS buffer (pH 7.2-7.4) and mixed with 100-1000 ul absolute ethanol and incubated at 4 ° C for at least 15 minutes.
- Spread the fixed mucus sample over the reaction field of the hybridization slide eg Paul Marienfeld KG, Bad Mergentheim, Germany; Art. No. 1215130 - slide with 6 reaction fields
- let it dry 10 mm at 46 ° C. 6 different samples can be placed on one of the slides mentioned above brought and examined at the same time.
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Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
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AU68415/00A AU6841500A (en) | 1999-08-31 | 2000-08-29 | Rapid, highly specific detection method for pseudomonas aeruginosa using multi-probe detection |
EP00956495A EP1208237A2 (en) | 1999-08-31 | 2000-08-29 | Rapid, highly specific detection method for pseudomonas aeruginosa using multi-probe detection |
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DE19941359.2 | 1999-08-31 | ||
DE19941359A DE19941359C2 (en) | 1999-08-31 | 1999-08-31 | Fast, highly specific detection of Pseudomonas aeruginosa by multiple probe detection |
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WO2001016363A2 true WO2001016363A2 (en) | 2001-03-08 |
WO2001016363A3 WO2001016363A3 (en) | 2001-11-01 |
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PCT/EP2000/008418 WO2001016363A2 (en) | 1999-08-31 | 2000-08-29 | Rapid, highly specific detection method for pseudomonas aeruginosa using multi-probe detection |
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EP (1) | EP1208237A2 (en) |
AU (1) | AU6841500A (en) |
DE (1) | DE19941359C2 (en) |
WO (1) | WO2001016363A2 (en) |
YU (1) | YU24002A (en) |
Cited By (1)
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EP1801239A2 (en) * | 2005-12-20 | 2007-06-27 | Millipore Corporation | Composition for increasing microorganism wall permeability and method for detecting said microorganisms on a membrane |
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JP2004313181A (en) * | 2003-04-02 | 2004-11-11 | Canon Inc | Probe for detecting infection-inducing microbe, probe set, carrier and method for testing gene |
US7449337B2 (en) | 2007-01-24 | 2008-11-11 | Idexx Laboratories, Inc. | Lytic reagent and method for leukocytes differential in whole blood |
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WO1999022023A2 (en) * | 1997-10-29 | 1999-05-06 | Mira Diagnostica Gmbh | Method for identifying micro-organisms |
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1999
- 1999-08-31 DE DE19941359A patent/DE19941359C2/en not_active Expired - Fee Related
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2000
- 2000-08-29 WO PCT/EP2000/008418 patent/WO2001016363A2/en not_active Application Discontinuation
- 2000-08-29 AU AU68415/00A patent/AU6841500A/en not_active Abandoned
- 2000-08-29 EP EP00956495A patent/EP1208237A2/en not_active Withdrawn
- 2000-08-29 YU YU24002A patent/YU24002A/en unknown
Patent Citations (1)
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WO1999022023A2 (en) * | 1997-10-29 | 1999-05-06 | Mira Diagnostica Gmbh | Method for identifying micro-organisms |
Non-Patent Citations (2)
Title |
---|
KINGSFORD ET AL. : "Detection of Pseudomonas aeruginosa from ovine fleece washings by PCR amplification of 16S ribosomal RNA" VETERINARIAN MICROBIOLOGY, Bd. 47, Nr. 1-2, November 1995 (1995-11), Seiten 61-70, XP000990889 * |
O'CALLAGHAN E M ET AL: "DEVELOPMENT OF A PCR PROBE TEST FOR IDENTIFYING PSEUDOMONAS AERUGINOSA AND PSEUDOMONAS (BURKHOLDERIA) CEPACIA" JOURNAL OF CLINICAL PATHOLOGY,GB,LONDON, Bd. 47, Nr. 3, 1. M{rz 1994 (1994-03-01), Seiten 222-226, XP000603979 ISSN: 0021-9746 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1801239A2 (en) * | 2005-12-20 | 2007-06-27 | Millipore Corporation | Composition for increasing microorganism wall permeability and method for detecting said microorganisms on a membrane |
EP1801239A3 (en) * | 2005-12-20 | 2007-08-15 | Millipore Corporation | Composition for increasing microorganism wall permeability and method for detecting said microorganisms on a membrane |
US8148081B2 (en) | 2005-12-20 | 2012-04-03 | Emd Millipore Corporation | Method for detecting microorganisms on a membrane |
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Publication number | Publication date |
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YU24002A (en) | 2005-06-10 |
DE19941359C2 (en) | 2002-12-05 |
EP1208237A2 (en) | 2002-05-29 |
WO2001016363A3 (en) | 2001-11-01 |
AU6841500A (en) | 2001-03-26 |
DE19941359A1 (en) | 2001-03-01 |
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