EP1994175A1 - Pre-traitement d'une solution destinée à être utilisée dans des réactions d'amplification de l'adn - Google Patents

Pre-traitement d'une solution destinée à être utilisée dans des réactions d'amplification de l'adn

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Publication number
EP1994175A1
EP1994175A1 EP07747673A EP07747673A EP1994175A1 EP 1994175 A1 EP1994175 A1 EP 1994175A1 EP 07747673 A EP07747673 A EP 07747673A EP 07747673 A EP07747673 A EP 07747673A EP 1994175 A1 EP1994175 A1 EP 1994175A1
Authority
EP
European Patent Office
Prior art keywords
dna
solution
pcr
binding agent
ema
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.)
Withdrawn
Application number
EP07747673A
Other languages
German (de)
English (en)
Other versions
EP1994175A4 (fr
Inventor
Hugh William Morgan
Andreas Rueckert
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.)
ZyGEM Corp Ltd
Original Assignee
WaikatoLink Ltd
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 WaikatoLink Ltd filed Critical WaikatoLink Ltd
Publication of EP1994175A1 publication Critical patent/EP1994175A1/fr
Publication of EP1994175A4 publication Critical patent/EP1994175A4/fr
Withdrawn legal-status Critical Current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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction

Definitions

  • This invention relates to a solution pre-treatment.
  • this invention relates to the pre-treatment of protein solutions.
  • the protein solutions are for use in deoxyribonucleic acid (DNA) amplification reactions, and the pre-treatment ensures that any contaminating DNA present in the protein solution cannot take part in the DNA amplification reaction.
  • DNA deoxyribonucleic acid
  • Amplification of nucleic acids especially DNA has become a widely used tool in DNA analysis work. This is especially so in cases where only minute amounts of DNA are present.
  • PCR polymerase chain reaction
  • PCR is a method of amplifying a DNA base sequence using a heat-stable DNA polymerase and two approximately 20-base primers, one complementary to the (+)-strand at one end of the sequence to be amplified and the other complementary to the (- )-strand at the other end of the sequence to be amplified.
  • PCR also can be used to detect the existence of a defined sequence in a DNA sample, if relevant primers can be developed.
  • contamination of DNA polymerase and other protein PCR reagents with bacterial (or other) DNA is a major problem for PCR methodology. This problem has previously been addressed by several authors (Bottger, 1990; Rand and Houck, 1990; Schmitd et al., 1991 ; Hughes et al., 1994; Maiwald et al., 1994; HiIaIi et al., 1997).
  • Eubacterial DNA is ubiquitous in the environment and contaminating DNA can also be introduced into the PCR mix at practically any stage of processing by laboratory personnel (Kitchin et al., 1990; Millar et al., 2002).
  • DNA polymerases have been repeatedly reported to be one of the most likely sources of exogenous bacterial DNA contamination (Rand and Houck, 1990; Bottger, 1990; Schmidt et al., 1991 ; HiIaIi et. al., 1997; Newsome et al., 2004). Schmidt et al., (1991) regard micro-organisms used for the production of DNA polymerases and enzyme purification equipment, such as chromatography columns, as the most likely source for introducing exogenous DNA into DNA polymerases.
  • restriction enzymes can also be problematic as the enzymes themselves may be a source of exogenous eubacterial DNA (Jinno et al, 1990). There has been some work in the area of capturing DNA after amplification as discussed in NZ 514707. However, this composition and method requires the use of binding enhancers and a label which is used for subsequent analysis and detection of amplicons.
  • a solution for use in DNA amplification reactions wherein the solution is produced via the method substantially as described above.
  • a method of DNA amplification including the steps of:
  • the binding agent binding to any DNA in the base solution so that it is unreactive and is not co-amplified during the DNA amplification reaction.
  • the base solution may be a protein solution, and shall be referred to as such herein.
  • base solution should be taken as meaning any protein or other solution which has been prepared or is commercially available for use in DNA amplification reactions, but which may still include contaminating DNA.
  • the protein solution may be a solution of DNA polymerase, used for the extension of nucleic acid strands in PCR reactions.
  • DNA polymerase used for the extension of nucleic acid strands in PCR reactions.
  • this should not be seen as limiting as the present invention may also be used for other protein solutions for DNA amplification or detection, for example restriction enzymes.
  • the protein solution may be a solution of Taq polymerase, a heat-stable DNA polymerase isolated from the bacterium Therrnus aquaticus.
  • the base solution may be a PCR master mix, containing all the required components to carry out a PCR reaction except the template DNA to be amplified.
  • the base solution may be an RNA preparation.
  • RNA analysis often includes the steps of: extracting RNA from a cell, making DNA copies (cDNA) of the RNA (eg mRNA, rRNA) molecules using a reverse transcriptase and then amplifying the cDNA by PCR, or other amplification techniques.
  • RNA extract is not contaminated with cellular DNA.
  • the base solution may be an RNA extract prior to cDNA production.
  • Protein solutions obtained from any source are likely to contain contaminating DNA.
  • the inventors have investigated three of the most commonly used commercially available Taq polymerase preparations. All three contained roughly the same level of contaminating DNA. Commercially available preparations of Taq polymerase must be screened to be below an "acceptable" level of DNA contamination, however, this does not mean they are contamination free.
  • Universal primers are those that, for example will bind to DNA from any Eubacteria species.
  • the present invention is targeted at use for solutions which are used in DNA amplification reactions.
  • PCR RNA sequence complementary to the (+)-strand at one end of the sequence to be amplified and the other complementary to the (-)-strand at the other end. Repetitive cycles of primer annealing, strand elongation and dissociation produce rapid and highly specific amplification of the desired sequence.
  • the present invention provides a quick and easy method of binding contaminating DNA and preventing co-amplification of same.
  • the current method of preventing any contaminating DNA from participating in DNA amplification reactions could either be undertaken by a commercial supplier, thereby being able to provide guaranteed DNA free polymerase or master mixes, alternatively the current method could be undertaken by individual research institutions or researchers as and when required.
  • DNA should be taken as having its generic meaning, including any compound made up of purine or pyrimidine base nucleotides, each with a deoxyribose sugar, and phosphate group.
  • the binding agent will prevent contaminating DNA from taking part in amplification reactions requiring the least possible number of opening, adding or removals from the reaction tube.
  • the binding agent may be one which has two different forms, one being an inactive form wherein it will not react or bind to DNA, and one being an active form wherein it will bind to DNA. It must be possible for the binding agent to be able to go between the inactive and active form (and vice versa) easily and quickly.
  • binding agent It is important that the activation and deactivation of the binding agent is easily undertaken and controllable. This ensures that binding of the agent to any contaminating DNA will only take place prior to the addition of template (target) DNA and initiation of the DNA amplification reaction.
  • the binding agent will only be in an active form in the presence of a stimulus.
  • the binding agent does not break down or destroy the contaminating DNA, this is also advantageous as such fragments or portions may themselves interfere in the amplification reaction.
  • Another advantage is that there is no possibility of any other solution constituents, such as enzymes being affected by the binding agent.
  • the binding agent may be activated by an external stimulus such as UV or ordinary light.
  • an external stimulus such as UV or ordinary light.
  • the stimulus will depend on the binding agent selected, and stimuli which may result in the binding agent being transferred from an inactive to an active form (or vice versa).
  • intercalating agent should be taken to include any agent that is capable of intercalating with DNA, that is to be reversibly included between two adjacent base pairs.
  • Some well studied DNA intercalators include ethidium, proflavin and thalidomide.
  • the binding agent may be ethidium monoazide (EMA).
  • EMA ethidium monoazide
  • the method is based on the complexation of the contaminating DNA with the intercalating agent EMA.
  • EMA is a photoreactive analogue of ethidium bromide, which in the dark, interacts with DNA in a similar manner as its parent compound (Bolton and Kearns, 1978; Garland et al, 1980).
  • the amount of binding agent added may be just sufficient to bind contaminating DNA.
  • the EMA concentration in the order of 3 ⁇ g ml-1 may be used.
  • An EMA concentration of 3 ⁇ g ml-1 may be sufficient to completely eliminate contaminating DNA from the master mix preparations, without affecting the amplification of template DNA concentrations as low as 10 fg
  • EMA would be added to a minimal titre, just sufficient to bind all exogenous DNA, while minimizing the formation of free nitrene in solution associated with the formation of hydroxylamine.
  • EMA is an analogue of ethidium bromide in which the amino group in the 8-position has been replaced with an azido group.
  • EMA intercalates with double stranded DNA in the dark in the same manner as its parent compound but can additionally covalently bind DNA after photo-activation with visible light (Bolton and Kearns, 1978; Garland et. al., 1980). Accordingly, DNA that has chemically reacted with the azide, is unable to be amplified by PCR.
  • the azido-group in the 8-position of EMA is photo-chemically activated using long wavelength light greater than substantially 400 nm (Bolton and Kearns, 1978) to covalently crosslink with nucleic acid at the site of binding via a nitrene radical (Hardwick et al., 1984; Cantrell et al., 1978). Residual unbound nitrene in the solution is converted to hydroxylamine (Graves et al., 1981 ), which reacts with aldehyde and/or ketone components of the solution - preferentially with cytosine - to form oximes (Freese et al., 1961 ). The covalently cross-linked DNA is unable to participate in PCR amplification and therefore its interference is removed (Nogva et al., 2003).
  • PCR master mix should be taken as meaning a mixture of some, or all of the required components for a PCR reaction, but excluding the template DNA to be amplified.
  • PCR master mix may contain: 1X Taq Master Mix: 10 mM Tris-HCI 50 mM KCI 1.5 mM MgCI 2 0.2 mM dNTPs 0.5 mM DTT 5 % glycerol 0.8 % NP-40 0.05 m M Tween-20 25 U/ml Taq DNA Polymerase
  • DTT DTT
  • glycerol glycerol
  • Tween Tween
  • concentration used can vary, for example primers and MgCI.
  • composition of the master mix is not so important as most of the contaminating DNA is introduced with the Taq polymerase and this is nearly always used at between 0.5-2.0 units per reaction (with 1 unit per reaction considered a good average).
  • template DNA should be taken as meaning target DNA which is amplified during a PCR reaction.
  • Advantages of the current invention include the following: • It provides a quick and easy method for preventing contaminating DNA in protein or other solutions from being co-amplified in DNA amplification reactions such as PCR,
  • the binding agent does not lead to the break down of contaminating DNA thereby preventing fragments of same interfering in DNA amplification reactions
  • the binding agent, with bound contaminating DNA does not have to be removed from the solution, thus preventing further possible contamination by limiting the number of times the container/reaction tube needs to be opened.
  • the binding agent does not require preparation, other than adjustment to the correct concentration before the method is undertaken,
  • the binding agent, or binding agent bound to contaminating DNA does not inhibit Tag polymerase, • Only a low level of the binding agent is required to be added to the base solution or reaction mixture.
  • Figure 1 illustrates PCR amplification product with decreasing template DNA concentration in the presence of 0.1 ⁇ g ml "1 EMA, and pixel density profile of product concentration
  • Figure 2 illustrates pixel density profile for 1 ⁇ g ml "1 ;
  • Figure 3 illustrates pixel density profile for 3 ⁇ g ml "1 ;
  • Figure 4 illustrates pixel density profile for 5 ⁇ g ml "1 ;
  • Figure 5 illustrates pixel density profile for 10_ ⁇ g ml "1 ;
  • Figure 6 illustrates pixel density profile using 10 ⁇ g ml "1 EMA and 200 pg bovine DNA
  • Figure 7 illustrates pixel density profile for 3 ⁇ g ml "1 EMA on JumpStart TM Taq DNA polymerase
  • Figure 8 illustrates pixel density profile for 3 ⁇ g ml "1 EMA on Platinum ® Taq DNA Polymerase. BEST MODES FOR CARRYING OUT THE INVENTION
  • the amount of EMA required to decontaminate a PCR master mix preparation depends on the amount of contaminating DNA present.
  • This software was used to calculate the pixel value statistics of a selected area in the agarose gel images, from which the line density profile plots were established.
  • This computer controlled measurement of the relative quantities of the amplicons had advantages over direct visual observation, particularly for the negative controls. This method minimized ambiguous results due to misinterpretations or inconsistencies in ethidium bromide staining procedures and gel to gel variations.
  • the amount of contaminating DNA in commercially available PCR reagents will vary and every batch of DNA polymerase or PCR master mix will require an optimization of the EMA titer to ensure maximal removal of contaminating DNA while maintaining PCR sensitivity.
  • EMA would be added to a minimal titer - just sufficient to bind all exogenous DNA, while minimizing the formation of free nitrene in solution associated with the formation of hydroxylamine.
  • Hydroxylamine has been shown to be mutagenic, being able to bind and chemically modify DNA over the concentration range of 250 to 500 mM, with incubation times greater than 30 minutes (Aslanzadeh, 1992; Freese et al., 1961 ).
  • hydroxylamine induces DNA damage in the presence of Cu(II), but not in the presence of Mn(III), Mn(II), Fe(III) or Fe(II) (Yamamoto et al., 1993).
  • the EMA optimization step could be redundant when the template DNA concentration is above 100 pg, i.e. where a small loss of template DNA due to unbound EMA can be sustained. In these situations an EMA concentration of 4 to 5 ⁇ g ml "1 could be recommended.
  • the current method is able to completely remove any contaminating DNA associated with commercially available DNA polymerases and/or PCR reagents from being co-amplified during PCR after 41 cycles of amplification (i.e. with no added template DNA).
  • the decontamination protocol presented is rapid and simple to employ, being accomplished within 10 minutes on PCR amplifications.
  • the current methodology is well suited as a routine molecular diagnostic tool for the analysis of subtle/delicate PCR samples with extremely low template concentrations where no background amplification is desired, i.e. the analysis of clinical and forensic specimens.
  • DNA was extracted from A. flavithermus and B. subtilis by a modification of the procedure described by Sambrook and Russel (2001). Following growth, 10 ml of cell suspension was harvested and re-suspended in 1 ml of 50 mM Tris HCI (pH 8.0), 100 mM NaCI and 20 mM EDTA.
  • Deoxyribonucleic acid from calf thymus was purchased from Sigma (D4764) as lyophilized powder and re-suspended in 1X TE. An aliquot of 25 ⁇ g of the bovine DNA was partially digested in a total sample volume of 100 ⁇ l with 1 Kunitz units of DNase I (Sigma, DN25) and 10 ⁇ l of 10X DNase buffer (10 mmol I "1 EDTA, 75 mmol I "1 MgCI2 and 200 mmol I "1 Tris-HCI, pH 7.5) at 37 0 C for 10 minutes.
  • the size of the PCR amplicon was expected to be approximately 300 bp depending on the template DNA present in the sample.
  • the D-alanine racemase gene from B. subtilis BS was amplified and sequenced using primers derived from the D-alanine racemase gene B. subtilis strain 168 (NCBI accession number M 16207).
  • the forward primer and reverse primer were 5'-AGC ACA AAA CCT TTT TAC AGA GAT AC-3' and 5'-TTA ATT GCT TAT ATT TAC CTG CAA TAA AG-3', respectively.
  • the amplification reactions contained 4 mM of either MgCI 2 or MgSO 4 , 0.2 mM dNTPs, 1x Taq PCR reaction buffer, 1.25 units of appropriate Taq polymerase, 100 nM of forward and reverse primers and EMA in the range of 0.1 and 10 ⁇ g ml "1 .
  • the amplification reaction contained DNA from A. flavithermus in the decimal dilution range from 10 ng to 10 fg.
  • Some PCR reactions also contained calf thymus DNA (after treatment with DNase I) of between 200 and 20 pg ⁇ l "1 .
  • DNA sequencing was carried out for each sample in the forward and reverse priming direction by the Waikato DNA Sequencing Facility on a MegaBACE capillary DNA Analysis System.
  • the master mix was aliquoted into PCR tubes and AmpliTaq® polymerase added in two-fold serial dilutions ranging from 1.25 to 0.0097 U. PCR reactions were run on each of the dilutions after adding 100 pg of A. flavithermus template DNA.
  • a control master mix was similarly prepared, but without addition of EMA.
  • the PCR master mix was incubated in darkness for 5 minutes and illuminated for 3 minutes. Subsequent to light exposure either 200 or 20 pg ⁇ l "1 of partially DNase I digested bovine DNA was added to the master mix, and the samples incubated in darkness for a further 5 minutes on ice in order to allow the restricted bovine DNA to react with any unbound EMA. The master mixes were then distributed and an aliquot of the decimal serially diluted template DNA added to the PCR tubes and amplification reaction initiated as described under 2.4. 1.8. Agarose gel-electrophoresis, imaging and evaluation
  • the amplification products were electrophoresed through a 1.5% (w/v) agarose gel (SeaKem, LE Agarose) at 55 Volts (Power Pack Model 250; Life Technologies, GIBCO BRL) using a HORIZON 11 -14 electrophoresis box (Life Technologies, GIBCO BRL).
  • the optimal amount of EMA for the removal of exogenous DNA from the PCR master mix containing AmpliTaq® polymerase was determined over the concentration range of 0.1 , 1 , 3, 5 and 10 ⁇ g ml "1 .
  • Template DNA concentrations covering the range from 10 ng to 10 fg of A. flavithermus DNA were used to assess the sensitivity of the master mix to EMA incorporation. The results from these experiments are shown in Figures 1 to 5.
  • EMA concentrations below 1 ⁇ g ml "1 were unable to completely remove all the exogenous DNA present in the PCR master mix and both reactions produced an appropriate amplicon.
  • the amount of PCR product generated - measured relative to the gray value in the pixel density profile - varied significantly between both samples, with the negative control sample treated with 1 ⁇ g ml "1 EMA ( Figure 2, lane 9) producing a noticeably smaller quantity of PCR product than that treated with 0.1 ug ml "1 EMA ( Figure 1 , lane 9).
  • the optimal amount of EMA for the elimination of exogenous DNA in this master mix was 3 ⁇ g ml "1 ( Figure 3), while at the same time showing no interference of amplification of template DNA at concentrations as low as 10 fg. 2.2. Effect of EMA concentrations greater than 5 ⁇ g mf on PCR efficiency
  • PCR products were subsequently sequenced, and their sequences compared to the known D-alanine racemase (1170 bp) of B. subtilis 168 by ClustalW alignment (as shown in Table 1).
  • the results show that EMA at concentrations of 3 and 5 ⁇ g ml-1 , or any secondary products on photolysis, had " no adverse effect on amplification and sequencing.
  • the untreated negative control samples from lanes 10 in Figure 7 and 8 demonstrate the amplification of specific PCR amplicons, which substantiates that both DNA polymerases were the source of amplifiable DNA.
  • the density profiles indicate that quantitative difference in contaminating DNA were evident with the JumpStart Taq-polymerase introducing approximately 2 fg and the Platinum 7ag-Polymerase approximately 40 fg of amplifiable DNA per reaction.
  • Taq polymerase contains bacterial DNA of unknown origin. Molecular and Cellular Probes 4, 445-450.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biophysics (AREA)
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Abstract

L'invention porte sur un procédé de préparation d'une solution destinée à être utilisée dans des réactions d'amplification de l'ADN. Ce procédé consiste à mélanger un agent de liaison avec une solution de base; à soumettre le mélange (d'un agent de liaison et de la solution de base) à un stimulus qui active l'agent de liaison, et après activation, à lier l'agent de liaison à n'importe quel ADN dans la solution de base, de sorte qu'il ne soit pas réactif et co-amplifié pendant la réaction d'amplification de l'ADN.
EP07747673A 2006-03-10 2007-03-08 Pre-traitement d'une solution destinée à être utilisée dans des réactions d'amplification de l'adn Withdrawn EP1994175A4 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
NZ54589406A NZ545894A (en) 2006-03-10 2006-03-10 Pre-treatment of DNA amplification solutions
PCT/NZ2007/000045 WO2007105965A1 (fr) 2006-03-10 2007-03-08 Pre-traitement d'une solution destinée à être utilisée dans des réactions d'amplification de l'adn

Publications (2)

Publication Number Publication Date
EP1994175A1 true EP1994175A1 (fr) 2008-11-26
EP1994175A4 EP1994175A4 (fr) 2009-12-23

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EP07747673A Withdrawn EP1994175A4 (fr) 2006-03-10 2007-03-08 Pre-traitement d'une solution destinée à être utilisée dans des réactions d'amplification de l'adn

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EP (1) EP1994175A4 (fr)
JP (1) JP2009529346A (fr)
CN (1) CN101432443A (fr)
AU (1) AU2007225509A1 (fr)
NZ (1) NZ545894A (fr)
WO (1) WO2007105965A1 (fr)

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US8507662B2 (en) 2001-01-19 2013-08-13 General Electric Company Methods and kits for reducing non-specific nucleic acid amplification
US8361712B2 (en) 2007-12-17 2013-01-29 General Electric Company Contamination-free reagents for nucleic acid amplification
JP5295787B2 (ja) 2006-02-28 2013-09-18 モンタナ ステート ユニバーシティ 核酸に基づく分子的技法を用いて無傷細胞と膜損傷細胞とを識別するためのフェナントリジウム誘導体の使用

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US5532145A (en) * 1989-10-26 1996-07-02 Steritech, Inc. Methods for treatment of enzyme preparations
US6187566B1 (en) * 1999-03-09 2001-02-13 Applied Gene Technologies, Inc. Method of labeling a nucleic acid amplicon with simultaneous contamination prevention
EP1627926A1 (fr) * 2004-08-19 2006-02-22 Roche Diagnostics GmbH Une méthode pour reduire le nombre de résultats faux-positifs

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NOCKER ANDREAS ET AL: "Selective removal of DNA from dead cells of mixed bacterial communities by use of ethidium monoazide" APPLIED AND ENVIRONMENTAL MICROBIOLOGY, AMERICAN SOCIETY FOR MICROBIOLOGY, US, vol. 72, no. 3, 1 March 2006 (2006-03-01), pages 1997-2004, XP002541184 ISSN: 0099-2240 *
NOGVA HEGE KARIN ET AL: "Ethidium monoazide for DNA-based differentiation of viable and dead bacteria by 5'-nuclease PCR" BIOTECHNIQUES, INFORMA LIFE SCIENCES PUBLISHING, WESTBOROUGH, MA, US, vol. 34, no. 4, 1 April 2003 (2003-04-01), pages 804-813, XP008109791 ISSN: 0736-6205 *
RUDI K ET AL: "Detection of viable and dead Listeria monocytogenes on gouda-like cheeses by real-time PCR Comments : Real-time diagnostics to detect the presence of viable, dead or viable but not culturable cells" LETTERS IN APPLIED MICROBIOLOGY, OXFORD, GB, vol. 40, no. 4, 1 January 2005 (2005-01-01), pages 301-306, XP002482493 ISSN: 1472-765X *
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, AMERICAN SOCIETY FOR MICROBIOLOGY, US, vol. 71, no. 2, 1 January 2005 (2005-01-01), pages 1018-1024, XP003000006 ISSN: 0099-2240 *
RUECKERT A ET AL: "Rapid differentiation and enumeration of the total, viable vegetative cell and spore content of thermophilic bacilli in milk powders with reference to Anoxybacillus flavithermus" JOURNAL OF APPLIED MICROBIOLOGY, vol. 99, no. 5, 2005, pages 1246-1255, XP008109747 ISSN: 1364-5072 *
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Also Published As

Publication number Publication date
WO2007105965A1 (fr) 2007-09-20
AU2007225509A1 (en) 2007-09-20
EP1994175A4 (fr) 2009-12-23
NZ545894A (en) 2007-12-21
JP2009529346A (ja) 2009-08-20
CN101432443A (zh) 2009-05-13

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