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WO1991008305A1 - Fragments d'adn et sondes et amorces d'adn a base de ceux-ci - Google Patents

Fragments d'adn et sondes et amorces d'adn a base de ceux-ci Download PDF

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Publication number
WO1991008305A1
WO1991008305A1 PCT/NL1990/000178 NL9000178W WO9108305A1 WO 1991008305 A1 WO1991008305 A1 WO 1991008305A1 NL 9000178 W NL9000178 W NL 9000178W WO 9108305 A1 WO9108305 A1 WO 9108305A1
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Prior art keywords
dna
methicillin
resistant
staphylococcus
hybridization
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PCT/NL1990/000178
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English (en)
Inventor
Adriaan Camille Fluit
Jan Verhoef
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U-Gene Research B.V.
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Publication date
Priority claimed from NL8902926A external-priority patent/NL8902926A/nl
Application filed by U-Gene Research B.V. filed Critical U-Gene Research B.V.
Publication of WO1991008305A1 publication Critical patent/WO1991008305A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a DNA fragment derived from a methicillin-resistant Staphylococcus strain as well as to a DNA probe based on such a DNA fragment and adapted for the detection of Staphylococcus bacteria that are resistant to methicillin and related penicillins insensitive to ⁇ -lactamase and to a DNA-primer based on such a DNA fragment and adapted for DNA amplication by means of a polymerase chain reaction.
  • Staphylococcus aureus is the most important bacterium pathogenic to man. The appearance of methicillin-resistant strains of this bacterium is a serious problem in antibacterial chemotherapy (1,2) .
  • Such strains are resistant to methicillin and also to closely related penicillins insensitive to ⁇ -lactamase, such as cloxacillin, flucloxacillin, etc. In the following description these strains will be designated, for brevity's sake, as methicillin-resistant strains.
  • methicillin resistance is connected with the presence of a unique penicillin-binding protein (PBP2a or PBP2 * ) , which is apparently not present in sensitive Staphylococcus strains.
  • PBP2a or PBP2 * penicillin-binding protein
  • the genetic determinant for methicillin resistance (mec) is located in the chromosomal DNA (3) .
  • Various publications are devoted to the involvement of mec and possibly a second factor in the methicillin resistance of Staphylococcus strains (4-10) .
  • mec gene of a methicillin-resistant ⁇ aureus isolate was inactivated by insertion of a transposon have shown that the mec gene is necessary for resistance (7) .
  • DNA probes are small segments of single-stranded nucleic acid labelled with, e.g., an enzyme or a radioisotope which very specifically bind to complementary nucleic acid sequences (hybridization) .
  • the detection of bound label indicates the presence of the pathogen to be demonstrated in a clinical sample.
  • probes have been developed for detecting, e.g., bacteria, viruses, protozoa, and even fungi.
  • a number of probe kits are already commercially available.
  • a problem in the development of a probe is that it must be sensitive and very specific. The aim is to develop a smallest possible probe being sufficiently specific.
  • a probe for the detection of resistance to a certain antibiotic is known from, e.g., French patent application 2,602,794.
  • This application relates to a DNA fragment containing at least part of the tetM gene coding for a tetracycline-resistance protein and the nucleotide sequence of which is given.
  • Such a fragment can be used for the preparation of a DNA probe for the detection of tetracycline resistance in a cell culture.
  • the only probe found useful is a rather large DNA fragment of 850 bp.
  • the DNA probe used therein is a 3.9 kb Hindlll restriction fragment of chromosomal DNA of a methicillin-resistant Staphylococcus aureus strain, which fragment hybridized with the 3.5 kb B ⁇ lll restriction fragment previously described by Beck et al (5) .
  • the 3.9 kb DNA probe hybridized with chromosomal DNA of all the selected methicillin-resistant Staphylococcus haemplyticvs and Staphylococcus epidermidis strains, whereas no hybridization occurred with the negative hybridization controls, namely a methicillin-sensitive Staphylococcus haemolyticus isolate and a methicillin- sensitive Staphylococcus ep dermidis isolate.
  • the great length (3.9 kb) and unknown base sequence of this probe impede development of sensitive and specific tests.
  • the invention satisfies these needs by means of a DNA fragment derived from a methicillin-resistant Staphylococcus strain and consisting essentially of or being complementary to the nucleotide sequence shown in the sequence list under SEQ ID NO:l or a related nucleotide sequence, such as one of the nucleotide sequences shown in the sequence list under SEQ ID NO's:2-8, or part of such a nucleotide sequence having a length of at least 14 nucleotides.
  • the DNA fragments according to the invention are derived from a methicillin-resistant Staphylococcus strain.
  • the invention is not limited to DNA fragments derived from a certain species of Staphylococcus bacteria.
  • the DNA fragments may be derived from a methicillin- resistant strain of StaphylPCQCc ⁇ s aureus.
  • Stflphylppppc ⁇ S SflprpphytJCUS Staphylococcus haemolyticus.
  • the DNA fragments with the nucleotide sequences according to SEQ ID NO's:1-8 are all derived from methicillin-resistant Staphylococcus aureus strains. Yet they can also be used for the detection of methicillin-resistant strains of other species of Staphylococcus bacteria, which is due to the great homology between the relative sequences of the different Staphylococcus species .
  • nucleotide sequence shown in the sequence list under SEQ ID N0:1 is derived from the methicillin-resistant i _ aureus strain 05723.
  • a nucleotide sequence reletaed thereto is the corresponding sequence in other methicillin-resistant Staphylococcus strains, both methicillin-resistant strains of the species S. aureus and methicillin-resistant strains of other Staphylococcus species (such as the above-mentioned species) .
  • SEQ ID NO's:2-8 are derived from the methicillin-resistant S_,_ aureus strains 00646, A216, 02599, 214, 215C, 06231 and 1335, respectively.
  • S_,_ aureus strains 00646, A216, 02599, 214, 215C, 06231 and 1335 are derived from the methicillin-resistant S_,_ aureus strains 00646, A216, 02599, 214, 215C, 06231 and 1335, respectively.
  • S_,_ aureus strains 00646, A216, 02599, 214, 215C, 06231 and 1335 prove to be about 68 nucleotides shorter than those of the other examples shown.
  • the DNA sequences found which (like the sequences complementary thereo) may all function as a suitable probe for the detection of methicillin resistance in Staphylococcus aureus. have been compared with all the sequences occurring in the EMBL (European Molecular Biology Laboratory) data bank. Homology has been found with three sequences:
  • pp5mer 13
  • pp5mer 13
  • pp5cada 14
  • Tn4003 15
  • the sequences shown in the sequence list under SEQ ID NO's:1-8 can be subdivided into two groups, the first consisting of DNA fragments having a length of about 333 bp and the second consisting of DNA fragments lacking a sequence of about 68 bp (nucleotides 155-222) .
  • the fragments of about 333 bp three parts can be distinguished: nucleotides 1- 154 (Part I), nucleotides 155-222 (Part II.
  • Part III nucleotides 223-333 .
  • the observed homology with pp ⁇ mer extends over all the three parts; that with pp5cada and Tn4003 only over Part III.
  • Part III proves to form part of insertion sequence IS257, also designated by IS431, occurring in transposon Tn4003.
  • DNA probes and primers based on DNA fragments as defined above.
  • DNA probes designate labelled DNA fragments adapted for the detection of complementary sequences, but in a broader sense they can also designate non-labelled DNA fragments capable of specifically binding to complementary sequences.
  • Suitable labelling methods are known to those skilled in the art. By way of example, mention is made of labelling with radioisotopes, enzymes, fluorescent substances, pigments, the biotin-avidin system, etc.
  • Both labelled and unlabelled DNA probes according to the invention can be used for the detection of Staphylococcus bacteria resistant to methicillin and related penicillins insensitive to ⁇ -lactamase by means of hybridization analysis.
  • a classical hybridization analysis makes use of a labelled probe which, if necessary after a denaturation treatment, is contacted with the denatured target DNA bound to a solid carrier (such as nitrocellulose) .
  • the hybridization is followed by detection of the label, e.g., of a radioisotope by autoradiography by means of an X-ray film, of an enzyme label by means of a substrate of the enzyme, etc.
  • An alternative hybridization analysis uses both a labelled and an unlabelled probe which are complementary to different parts of the target DNA, the latter probe serving to bind the target DNA to a solid carrier and the former probe serving to provide the bound target DNA with a detectable label.
  • SEQ ID NO's:15 and 16 are examples of sequences that could function as the unlabelled "collecting" probe and the labelled "marking" probe, or conversely.
  • Polymer particles e.g., polystyrene globules
  • magnetic particles could function as a solid carrier, in which connection different methods can be used to bind the unlabelled probe to the carrier (e.g., by means of the avidin-biotin system, etc.) .
  • the different steps including a prehybridization with hybridization buffer to inhibit aspecific bindings and washing treatments after the hybridization step to remove unbound probe
  • the employed reagents and reaction conditions are fully known to those skilled in the art and need no further explanation.
  • the invention therefore also provides a diagnostic test kit for detection in a sample of Staphylococcus bacteria that are resistant to methicillin and related penicillins insensitive to ⁇ -lactamase by means of hybridization analysis, comprising at least a (labelled) DNA probe according to the invention as well as one or more further means required for hybridization analysis, such as a denaturation liquid, a hybridization liquid, a washing liquid, a solid carrier, a hybridization vessel and label detecting means .
  • the invention also provides a process for the detection in a sample of Staphylococcus bacteria that are resistant to methicillin and related penicillins insensitive to ⁇ -lactamase by means of hybridization analysis, which comprises isolating the DNA from the bacteria in the sample, contacting the isolated DNA under hybridization conditions with at least a (labelled) DNA probe according to the invention, washing non- hybridized probe and analyzing any hybridization by label detection.
  • the DNA probe according to the invention is preferably based on Parts I, II and III together, or on Parts I and III together. Part III alone forms a very suitable probe. Less suitable are probes on the basis of Parts I and II alone or Parts I and II together. Examples of relatively short probes are SEQ ID NO's:15 and 16.
  • DNA primers are DNA fragments that are complementary to part of a "target" DNA and, after hybridization thereto, may serve as starting-point for DNA polymerase which, in the presence of the necessary building materials, synthesizes the chain complementary to the target DNA.
  • the presence in a sample of DNA of methicillin-resistant Staphylococcus strains can be determined by means of the PCR method in a very sensitive and specific manner.
  • the different steps, reagents and reaction conditions are known to those skilled in the art and need no detailed explanation. This also applies to the methods by which the result of the PCR can be analyzed, e.g., separation according to length of the resulting DNA fragments and visualization thereof by means of ethidium bromide, or hybridization analysis of the resulting DNA fragments.
  • primers will be relatively short, provided they are still sufficiently specific for the contemplated target DNA.
  • DNA fragments having a length of 14 nucleotides are often sufficient, but slightly longer fragments, say from 15 to 40, preferably from 18 to 32 nucleotides, are preferred.
  • Such relative short DNA fragments may also be used for DNA probes, as elucidated by means of SEQ ID NO's:15 and 16 (but also larger fragments, particularly the complete sequences SEQ ID NO's:1-8 as given in the sequence list, are suitable as probes) .
  • primer sets are the DNA fragments shown in the sequence list under SEQ ID NO's: 9 and 10, or SEQ ID NO's:11 and 12.
  • one of the primers may be based on a sequence in Part I of the DNA fragments according to the invention, whereas the other primer is based on a sequence occurring in Part III. It is preferred, however, that both primers of a primer set are based on a sequence occurring in Part III.
  • Very suitable is a primer pair, of which the first primer is SEQ ID NO:13 and the second primer is SEQ ID NO:14.
  • the invention therefore also provides a diagnostic test kit for the detection in a sample of Staphylococcus bacteria that are resistant to methicillin and related penicillins insensitive to ⁇ -lactamase by means of PCR analysis, comprising a DNA primer set according to the invention, as well as one or more further means required for PCR analysis, such as a polymerase, a polymerization liquid, an oil overlay, a reaction vessel and means for detecting the amplified DNA.
  • the invention also provides a process for the detection in a sample of Staphylococcus bacteria that are resistant to methicillin and related penicillins insensitive to ⁇ -lactamase by means of PCR analysis, which comprises isolating, if required, the DNA from the bacteria in the sample, subjecting the DNA that may or may not have been isolated to several PCR cycles using a DNA primer set according to the invention, and analyzing the amplified DNA.
  • a suitable set of protein A-specific primers is shown in the sequence list with SEQ ID NO's:17 and 18. Also the primers SEQ ID NO's:18 and 19 form a suitable primer pair.
  • the invention further provides a plasmid, consisting essentially of a bacterial cloning vector including an insertion consisting of a DNA fragment according to the invention, and bacteria containing at least one such plasmid.
  • a DNA fragment with the nucleotide sequence according to SEQ ID NO:l is contained in plasmid pAA29-l in E. coli DH5CCF' , which strain was deposited with Centraal Bureau voor
  • a DNA fragment according to the invention may be obtained by the following process : a) partial digestion of purified DNA of a methicillin- resistant Staphylococcus strain with, e.g., Sau 3AI, b) hybridization of the resulting fragments with labelled DNA fragments of a methicillin-sensitive Staphylococcus strain, c) collecting the free DNA fragments, d) repetition of steps b) and c) , e) ligation of the resulting fragments in a plasmid and transformation of a suitable bacterial strain with this plasmid, f) culturing the transformants and isolation of the DNA from the transformants, g) hybridization of the isolated DNA with labelled DNA of a small number of methicillin-resistant and methicillin- sensitive Staphylococcus strains, h) selection of the transformants only showing hybridization with DNA of the methicillin-resistant strain or strains, i) digestion with, e.g., Pstl and EcoRI of the DNA of the
  • Staphylococcus strains used in steps a, b, d, g and k may in principle be nearly any Staphylococcus strain or isolate, provided they satisfy the relative criterion of methicillin resistance or methicillin sensitivity required in the different steps.
  • Step b) preferably consists in hybridization in solution at elevated temperature of the resulting fragments with sheared biotinylated DNA fragments of a methicillin-sensitive Staphylococcus strain.
  • Step e) preferably consists in ligation of the resulting fragments in the unique BamHI restriction site of pUC18 and transformation of E. coli DH50CF' with this plasmid.
  • Step f) preferably consists in culturing the transformants, lysis and DNA fixation on filters.
  • Step g) preferably consists in Southern hybridization of the isolated DNA with labelled DNA of not more than three methicillin-resistant Staphylococcus strains and not more than three methicillin-sensitive Staphylococcus strains.
  • Example I describes more extensively how to obtain the probe according to the invention. In combination with this process, another process will be described which began with digestion with i Lndlll but did not lead to a desired probe.
  • the specificity of the probe according to the invention appears from the hybridization tests carried out with it, which tests show a perfect correlation with methicillin resistance.
  • the MIC's for methicillin and gentamicin were determined using the agar dilution method.
  • the MIC's for methicillin were determined by inoculation of 100 ⁇ i of a 10 8 cfu/ml bacterial suspension on Mueller-Hinton agar followed by incubation at 30°C. Isolates with MIC > 4 were considered resistant.
  • the MIC's for gentamicin were determined by inoculation of 100 ⁇ i of a suspension of 10 6 cfu/ml on Isosensitest followed by incubation at 37°C. Isolates with MIC > 4 were considered resistant.
  • Staphylococcus aureus DNA was purified with the method as described by Lindberg et al (16) , except that after lysis of the bacteria the suspension was extracted with phenol/chloroform as described by Maniatis et al (17) . After the first extraction the mixture was treated with RNase and once more extracted with phenol/chloroform. Plasmid DNA was purified as described by Maniatis et al, followed by centrifugation with CsCl density gradient (17) . c) Digestions with restriction endonucleases were carried out according to the prescriptions of the manufacturer (GIBCO
  • the Staphylococcus aureus DNA used as probe was labelled with ⁇ - 32 P-dCTP (specific activity 800 Ci/mmol; Amersham, Great
  • Example J A method described by Poper et al (19) was used to enrich DNA fragments specific for methicillin resistance.
  • purified DNA from the methicillin-resistant isolate 05723 was partially digested with Sau 3AI, resulting in fragments smaller than 4 kb.
  • hybridization in solution was carried out using the Sau 3AI restriction fragments and sheared biotinylated DNA fragments of the methicillin-sensitive strain Ps-47-8325. 5 ⁇ g Sau 3AI fragments and 6 ⁇ g biotinylated DNA were denatured by heating, after which they were allowed to hybridize in solution at 62°C. After 4 hours at 62°C a suspension of avidin-agarose
  • a PCR was carried out in the following reaction medium: 10 mM Tris HC1 (pH 8.3), 50 mM KC1, 1.5 mM MgCl2, 0.001% (w/s) gelatin, 100 ⁇ M of each of the 4 dNTP's, 50 pmol of each primer and 1.25 U Ampli-Taq polymerase (Cetus/Permin Elmer) . The final volume was 50 or 100 ⁇ l. The incubation mixture was overlaid with a drop of mineral oil to inhibit evaporation. Two primer combinations were tested: (a) primers SEQ ID NO's: 9 and 10, (b) primers SEQ ID NO's:11 and 12. The PCR conditions were also varied.
  • the products formed were analyzed by means of agarose gel electrophoresis.
  • 1% (w/s) agarose was dissolved in 50 mM Tris, 50 mM boric acid, 1 mM EDTA (pH 8.3) .
  • the gel was poured into a commercially available gel receptacle designed for the purpose.
  • To 10 ⁇ l PCR incubation mixture were added 1 ⁇ l 40% sucrose, 0.1% bromphenol blue. After the bromphenol blue had entered the gel over a few cm under the influence of an electric field applied, the result was analyzed under UV light. Since DNA fragments of a known length were also applied to the gel, it could be established whether there had been formed a product of the correct length.
  • DNA sequence for the probe range of _2_,_ aureus strain 06231 length 265 nucleotides
  • DNA sequence of PCR primer length: 20 nucleotides
  • DNA sequence of PCR primer length: 20 nucleotides
  • DNA sequence of PCR primer length: 20 nucleotides
  • DNA sequence of PCR primer length: 20 nucleotides
  • DNA sequence of PCR primer length: 20 nucleotides
  • DNA sequence of PCR primer length: 20 nucleotides
  • DNA sequence of probe. length 20 nucleotides
  • DNA sequence of probe. length 20 nucleotides
  • DNA sequence of protein A-specific PCR primer length 20 nucleotides
  • DNA sequence of protein A-specific PCR primer length 20 nucleotides
  • DNA sequence of protein A-specific PCR primer length 20 nucleotides

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Abstract

On décrit un fragment d'ADN dérivé d'une souche de Staphylococcus résistante à la méthicilline, ainsi qu'une sonde d'ADN à base d'un tel fragment d'ADN et servant à déceler les bactéries de Staphylococcus qui sont résistantes à la méthicilline et aux pénicillines associées, lesquelles sont insensibles à la pénicillinase. On décrit également une amorce d'ADN à base d'un tel fragment d'ADN et servant à l'amplification d'ADN au moyen d'une réaction en chaîne de polymérase. Le fragment d'ADN est essentiellement constitué de, ou complémentaire à, la séquence représentée dans la liste des séquences sous SEQ ID NO:1 ou une séquence associée, telle qu'une des séquences représentées dans la liste des séquences sous SEQ ID NO's:2-8, ou une partie d'une telle séquence présentant une longueur d'au moins 14 nucléotides. On décrit également des ensembles et des procédés diagnostiques à base desdits fragments d'ADN.
PCT/NL1990/000178 1989-11-27 1990-11-27 Fragments d'adn et sondes et amorces d'adn a base de ceux-ci WO1991008305A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
NL8902926A NL8902926A (nl) 1989-11-27 1989-11-27 Werkwijze voor de bereiding van een dna-probe voor de detectie van methicilline-resistente staphylococcus-bacterien, daarmee verkrijgbare dna-probe en diagnostische test-kit die de dna-probe bevat.
NL8902926 1989-11-27
NL9002157 1990-10-04
NL9002157A NL9002157A (nl) 1989-11-27 1990-10-04 Dna fragmenten en daarop gebaseerde dna-probes en primers.

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WO1991008305A1 true WO1991008305A1 (fr) 1991-06-13

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AU (1) AU6950791A (fr)
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Cited By (18)

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EP0527628A1 (fr) * 1991-08-13 1993-02-17 Eli Lilly And Company Méthode rapide de détection de staphylocoques résistants à la méthycilline
FR2687168A1 (fr) * 1992-02-10 1993-08-13 Bio Merieux Fragment de l'adn genomique de streptococcus pneumoniae, sonde d'hybridation, amorce d'amplification, reactif et procede de detection de streptococcus pneumoniae.
EP0624650A2 (fr) * 1993-04-30 1994-11-17 Eli Lilly And Company Gène FemB de staphylococcus épidermis, protéine FemB, et vecteurs et micro-organismes contenant le gène FemB
EP0625575A2 (fr) * 1993-04-30 1994-11-23 Eli Lilly And Company Gène Fem A de staphylococcus épidermis, protéine Fem A, et vecteurs et micro-organismes contenant le gène Fem A
EP0652291A1 (fr) * 1992-07-07 1995-05-10 Fuso Pharmaceutical Industries Ltd. Sonde pour diagnostiquer des maladies infectieuses
US5620847A (en) * 1990-10-05 1997-04-15 Hoffman-La Roche Inc. Methods and reagents for detection of bacteria in cerebrospinal fluid
US5656432A (en) * 1992-02-10 1997-08-12 Bio Merieux Genomic DNA fragment of Streptococcus pneumoniae, hybridization probe, amplification primer, reagent and method for the detection of Streptococcus pneumoniae
DE19731292A1 (de) * 1997-07-21 1999-01-28 Biotecon Ges Fuer Biotechnologische Entwicklung & Consulting Mbh Nucleinsäuremolekül, Kit und Verwendung
US5994066A (en) * 1995-09-11 1999-11-30 Infectio Diagnostic, Inc. Species-specific and universal DNA probes and amplification primers to rapidly detect and identify common bacterial pathogens and associated antibiotic resistance genes from clinical specimens for routine diagnosis in microbiology laboratories
US6001564A (en) * 1994-09-12 1999-12-14 Infectio Diagnostic, Inc. Species specific and universal DNA probes and amplification primers to rapidly detect and identify common bacterial pathogens and associated antibiotic resistance genes from clinical specimens for routine diagnosis in microbiology laboratories
WO1999058713A3 (fr) * 1998-05-12 2000-08-10 Bioinside Gmbh Procede de detection de micro-organismes presents dans des produits
US6893846B2 (en) * 1998-09-28 2005-05-17 Creighton University Primers for use in detecting beta-lactamases
WO2006028601A2 (fr) * 2004-07-26 2006-03-16 Nanosphere, Inc. Technique permettant de distinguer un staphylocoque dore resistant a la methicilline d'un staphylocoque dore sensible a la methicilline dans une culture mixte
US7045291B2 (en) 2002-05-17 2006-05-16 Creighton University Multiplex PCR for the detection of AmpC beta-lactamase genes
US7943346B2 (en) 1994-09-12 2011-05-17 Geneohm Sciences Canada Inc. Probes and primers for detection of bacterial pathogens and antibiotic resistance genes
US8034588B2 (en) 1997-11-04 2011-10-11 Geneohm Sciences Canada Inc. Species-specific, genus-specific and universal DNA probes and amplification primers to rapidly detect and identify common bacterial and fungal pathogens and associated antibiotic resistance genes from clinical specimens for diagnosis in microbiology laboratories
US8114601B2 (en) 1999-09-28 2012-02-14 Geneohm Sciences Canada Inc. Highly conserved genes and their use to generate probes and primers for detection of microorganisms
US8426137B2 (en) 1996-11-04 2013-04-23 Genohm Sciences Canada, Inc. Methods and probes for detecting a vancomycin resistance gene

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Journal of General Microbiology, vol. 133, 1987, SGM, P.R. Matthews et al.: "The cloning of chromosomal DNA associated with methicillin and other resistances in staphylococcus aureus", pages 1919-1929 *
Proc. Natl. Acad. Sci. USA, vol. 84, August 1987, R.A. Laddaga et al.: "Nucleotide sequence and expression of the mercurial-resistance operon from Staphylococcus aureus plasmid pI258", pages 5106-5110 *

Cited By (47)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5620847A (en) * 1990-10-05 1997-04-15 Hoffman-La Roche Inc. Methods and reagents for detection of bacteria in cerebrospinal fluid
EP0527628A1 (fr) * 1991-08-13 1993-02-17 Eli Lilly And Company Méthode rapide de détection de staphylocoques résistants à la méthycilline
FR2687168A1 (fr) * 1992-02-10 1993-08-13 Bio Merieux Fragment de l'adn genomique de streptococcus pneumoniae, sonde d'hybridation, amorce d'amplification, reactif et procede de detection de streptococcus pneumoniae.
EP0577523A1 (fr) * 1992-02-10 1994-01-05 Bio Merieux Fragment de l'ADN génomique de Streptococcus pneumoniae, sonde d'hybridation, amorce d'amplification, réactif et procédé de détection de Streptococcus pneumoniae
US5776691A (en) * 1992-02-10 1998-07-07 Bio Merieux Genomic DNA fragment of Streptococcus pneumontiae, hybridization probe, amplification primer, reagent and method for the detection of Streptococcus pneumoniae
US5770362A (en) * 1992-02-10 1998-06-23 Bio Merieux Genomic DNA fragment of Streptococcus pneumoniae, hybridization probe, amplification primer, reagent and method for the detection of Streptococcus pneumoniae
US5656432A (en) * 1992-02-10 1997-08-12 Bio Merieux Genomic DNA fragment of Streptococcus pneumoniae, hybridization probe, amplification primer, reagent and method for the detection of Streptococcus pneumoniae
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EP1167543A3 (fr) * 1992-07-07 2004-01-07 Fuso Pharmaceutical Industries Ltd. Sonde pour diagnostiquer des maladies infectieuses liées à Staphylococcus epidermidis
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AU6950791A (en) 1991-06-26

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