WO2000014274A1 - PROCEDE D'IDENTIFICATION ET DE SPECIATION DE BACTERIES DU COMPLEXE $i(BURKHOLDERIA CEPACIA) - Google Patents

PROCEDE D'IDENTIFICATION ET DE SPECIATION DE BACTERIES DU COMPLEXE $i(BURKHOLDERIA CEPACIA) Download PDF

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WO2000014274A1
WO2000014274A1 PCT/CA1999/000813 CA9900813W WO0014274A1 WO 2000014274 A1 WO2000014274 A1 WO 2000014274A1 CA 9900813 W CA9900813 W CA 9900813W WO 0014274 A1 WO0014274 A1 WO 0014274A1
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bacteria
cepacia complex
genomovar
burkholderia cepacia
seq
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PCT/CA1999/000813
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English (en)
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Eshwar Mahenthiralingam
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The University Of British Columbia
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Priority to GB0104647A priority Critical patent/GB2362212A/en
Priority to AU56120/99A priority patent/AU5612099A/en
Publication of WO2000014274A1 publication Critical patent/WO2000014274A1/fr

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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents

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  • This application relates to a new method for the identification and speciation of bacteria of the Burkholderia cepacia complex, and to vaccines developed specific for certain bacteria characterized using this method.
  • Burkholderia cepacia has recently been shown to consist of five different genomovars or new species, and as a collective the bacteria have been called the B. cep ⁇ ci ⁇ complex.
  • Two of these genomovars have been given new species names: Burkholderia multivorans (formerly genomovar II) and Burkholderia vietnamiensis (formerly genomovar V).
  • Burkholderia multivorans formerly genomovar II
  • Burkholderia vietnamiensis originally genomovar V.
  • These bacteria cause problematic infections in patients with cystic fibrosis (CF) and chronic granulomatous disease as well as often causing infection outbreaks among vulnerable hospitalized patients.
  • cystic fibrosis clinical outcome and epidemiology of infection may vary depending on the type of species patients are colonized with.
  • the species of a bacteria of the B. cepacia complex which is responsible for a given infection may be determined.
  • the challenge of provide effective therapy if the strain is determined to be an epidemic strain it is therefore still a further object of the present invention to provide an answer to this challenge by providing a vaccine which promotes a therapeutically beneficial immune response to epidemic strains of the B. cepacia.
  • identification and speciation of a target group of microorganisms based on nucleotide sequences, it is necessary to identify a gene or genes within the target group which on the one hand contain conserved regions which are common to all microorganisms in the group such that non-specific amplification can be performed, and on the other hand contain regions which are dissimilar in ways that are diagnostic of the species.
  • identification and speciation of bacteria of the Burkholderia cepacia complex in a sample can be accomplished by a method comprising the steps of
  • the nucleotide sequence information is obtained by evaluation of restriction fragment length polymorphism (RFLP).
  • RFLP restriction fragment length polymorphism
  • Other techniques for obtaining sequence information can also be used, including base-by-base determination of the sequence of the region of interest, sequence-specific oligonucleotide hybridization probes, and ligation techniques.
  • the invention also provides universal primers which can be used for amplification of all known members of the Burkholderia cepacia complex, and genomovar-specific primers which can be used for selective amplification of the recA gene from bacteria of one genomovar.
  • the present invention also provides a vaccine composition which is useful in providing a therapeutically beneficial immune response in warm-blooded animals, particularly mammals (including humans) infected with epidemic strains of bacteria of the B. cepacia complex belonging to genomovar III.
  • the vaccine comprises a protein or peptide antigen, or an expressible polynucleotide encoding a protein or peptide antigen derived from the flagellin of such bacteria.
  • Speciation can also be used as a basis for selection and/or isolation of industrially useful bacterial species.
  • a number of strains of B. cepacia can be used in as biocontrol strains in agricultural applications. It has been found using the speciation method of the invention that these strains frequently fall into one of two classes based on recA RFLPpattern, one of which is a class associated with epidemic strains and one of which is not.
  • recA RFLPpattern one of which is a class associated with epidemic strains and one of which is not.
  • one can use the test methodology of the invention to select among biocontrol strains to reduce the likelihood of public health hazards.
  • Figs. 1 and 2 show phylogenetic trees for B. cepacia based on the recA sequence evaluation
  • Fig. 3 shows the RFLP patterns obtained for representative strains of B. cepacia complex.
  • DNA sequence variation in conserved bacterial genes such as those encoding the ribosomal RNA (rRNA) molecules
  • rRNA ribosomal RNA
  • Speciation of B. cepacia complex based on the genes encoding the 16S rRNA gene has been examined, but there is insufficient DNA variation within the gene to enable it to distinguish among all the genomovars of B. cepacia.
  • the present invention it has now been determined that there is sufficient sequence variation for speciation of B. cepacia in the gene encoding the RecA protein (recA).
  • the present invention provides a rapid molecular diagnostic test which speciates bacteria of the B. cepacia complex based on the detection of sequence variation within the gene encoding the RecA protein.
  • CTCTTCTTCGTCCATCGCCTC SEQ ID No. 4 were subsequently used to amplify the recA gene from additional strains, and were found to be effective as amplification primers for the recA gene from all known members of the B. cepacia complex, including LMG 14191 ⁇ , the type strain for Burkholderia pyrrocinia.
  • the amplified genes from these other strains were sequenced (Seq. ID Nos. 5-19), and the sequences aligned with the two previously known recA gene sequences. While there are substantial similarities, no two strains were identical except the sequences for 70431 and ATCC 17616.
  • recA genes from additional strains were sequenced and have strain numbers and GenBank Accession numbers, respectively, as follows: HI-2308, AF143777; C5424, AF143781; C1394, AF143783; ATCC 25416, AF143786; C2822, AF143792; LMG 10929, AF143793; LMG 14191 ⁇ B.pyrrocinia), AF143794; Ral-3, AF143795; M54, AF143796; M36, AF143797; ATCC 29424, AF143798; ATCC 53617, AF143799; ATCC 49709, AF143800; ATCC 39277, AF143801; ATCC 53266, AF143802.
  • the primers BCR1 and BCR2 are located at the 5'-ends of the sense and anti-sense strands, such that substantially the entire gene is amplified. Persons skilled in the art will appreciate, however, that other primers could be developed based on the sequences provided, for example to amplify only a portion of the gene in which mutations of diagnostic significance are found. Such alternative primers would be targeted to conserved regions of the recA gene, and could be degenerate if necessary to obtain amplification of all species within the B. cepacia complex.
  • nucleotide sequence regions may be used to design alternative primers for alternative amplification of recA from all bacteria of the B. cepacia complex. For example, there is substantial nucleotide variation from position 100 to 600 in the recA gene. Thus, a universal primer pair which amplified only this region would permit development of smaller amplified fragments effective for speciation. This can be accomplished using the primers: Forward Primer (BCRU1 *)
  • BCRU1 ATCATGCGGATGGGCGACG SEQ ID No. 36 Reverse Primer (BCRU2)
  • CAGTTCTGTCGCTTGATCG SEQ ID No. 37 to produce a 488 base pair PCR product spanning a region of considerable sequence variation.
  • the DNA sequence variation in the recA gene used to speciate the B. cepacia can be detected using restriction enzyme digestion, separation of the digested fragments followed by pattern matching of the resulting profile. Prior to the digestion, the recA gene is amplified to increase the relative abundance of this gene as compared to other nucleic acid polymers in the sample.
  • the preferred amplification technique is PCR amplification, but other known amplification techniques may be used as well.
  • the restriction fragment patterns contain sufficient variation due to natural variation in the 16 recA sequences to enable the sequence to speciate among all 5 genomovars of B. cepacia.
  • the enzymes Haelll and Alul were found to be suitably discriminatory, with Haelll providing the highest degree of discrimination. Other enzymes which might be possess adequate discrimination can be determined either by experimental comparison of RFLP patterns, or by computer analysis of the sequences to identify restriction sites based on the known cleavage sites of a given enzyme.
  • nucleotide sequence information used in the method of the invention can be obtained by any other technique, including sequencing through at least the relevant regions of the recA gene; and the use of sequence- specific hybridization probes or ligation techniques adapted to identify sequence variations.
  • the 485 base pair fragment generated using primers BRCUl and BRCU2 is particularly suited for direct sequencing, since its length is amenable to complete processing in conventional commercial sequencing apparatus.
  • the primers BRCUl and/or BRCU2 may also be used as sequencing primers. Comparison of this 485 bp fragment of the B. cepacia recA gene using phylogenetic tree software and alignment software is sufficient to separate the species clusters of the B. cepacia complex in the same fashion as comparison of the entire gene sequence. It is, however, a more rapid approach than determination of the entire gene sequence, since only one PCR product is involved which is of an optimal size for sequencing. Genomovar-specific RFLP of this fragment can also be performed to provide speciation of B. cepacia complex.
  • a further alternative for obtaining nucleotide sequence information indicative of the genomovar to which a sample bacterium of the B. cepacia complex belongs is through the use of PCR primers which are genomovar-specific. Such primers are selected such that at least one primer in the pair hybridizes to a region of the recA gene which is not conserved, i.e., to a variable region, in such a manner that amplification only occurs if the sequence of the variable region is complementary to the primer. These specific PCR primers enable a single PCR test to be used for identification and direct detection of strains of each genomovar.
  • genomovar-specific primers The following are non-limiting examples of genomovar-specific primers.
  • the specificity of these primers is such that other genomovars, other than the targeted genomovar, do not produce amplification products under stringent PCR conditions.
  • B. multivorans specific recA primers Forward primer (BCRBMl):
  • Reverse primer 5' - TAC GCC ATC GGG CAT GCT - 3' SEQ ID No. 29
  • BCRG32 5' - TCG AGA CGC ACC GAC GAG - 3' SEQ ID No. 33
  • a second novel recA group, RG-D was identified which includes B. pyrrocinia LMG 14191 ⁇ and ATCC 32977, a strain of B. cepacia which produces the antibiotic xylocladin. This group is also shown in Fig. 2.
  • the invention also provides reagents and kits suitable for carrying out this method.
  • the reagents are generally polynucleotide primers or probes which bind to the recA gene of one or more strains of bacteria of the B. cepacia complex.
  • One subset of the reagents of the invention are non-specific primers, such as used in Example 4 below, which are complementary to conserved regions found identically in strains of bacteria of the B. cepacia complex for which the sequences are given.
  • a second subset of reagents in accordance with the invention are primers/probes which can be used to selectively amplify and/or detect one genomovar of bacteria of the B. cepacia complex.
  • the reagents of the invention may have a detectable or capturable label, for example a radioactive or fluorescent label or biotin, incorporated therein to facilitate evaluation of nucleotide sequence information.
  • Either of these types of primers/probes may be packaged in a kit with suitable reagents.
  • reagents may include discriminatory restriction enzymes, which are capable of producing distinctive fragment patterns to permit speciation of a bacteria- containing sample, or reagents suitable for PCR, nucleic acid sequencing and the like.
  • the present invention further provides a vaccine composition based upon the antigenic properties of the flagellin of epidemic strains of B. cepacia complex for use in treating infections caused by certain species of the B. cepacia complex.
  • flagellins as an antigen for vaccine purposes has been proposed in a variety of instances because of their location on the outside of bacterial cells.
  • flagellin gene fliC
  • Such variability is inconsistent with the normal requirements that a vaccine antigen be highly conserved, such that its will be generally effective against variants of the target species.
  • a total of 30 strains of bacteria of the B. cepacia complex were classified using the speciation method of the invention into groups based on the sequence of the recA gene, and were in addition characterized with respect to the BCESM and cblA markers for highly transmissible strains of B. cepacia. As reflected in Table 1, a substantial portion of the genomovar III strains which were positive for one or both of these markers produced a single RFLP pattern (Fig. 3, pattern G) after treatment with the restriction endonuclease Haelll.
  • B. cepacia flagellin gene which encodes the major subunit protein of the bacterial flagellum of B. cepacia
  • CF cystic fibrosis
  • flagellin gene of 5 the flagellin gene of 5.
  • multivorans strains of recA type F which appear less problematic in patients with CF and do not generally spread among patients, have flagellin genes which are highly variable in sequence.
  • a vaccine based on the flagellum may not protect against infection with all strains types as has been the case with the bacterium Pseudomonas aeruginosa in CF.
  • the flagellin gene is actually highly conserved in the most devastating epidemic B. cepacia strains infecting patients with CF is apparently unique to this subset of the species of the B. cepacia complex.
  • the vaccine can be prepared in a variety of ways. First, protein can be purified from bacterial strains representative of this group to obtain a purified antigen. Methods for purification of flagellin from bacteria are known in the art, and can be applied to recovery of purified flagellin from epidemic strains of B. cepacia. Purified antigen is then used as a vaccine, with or without an adjuvant. Vaccines of this type are generally administered by subcutaneous or intramuscular injection, although other routes of administration may also be suitable. Therapeutically effective levels and frequency of vaccine administration are determined by routine monitoring of antibody titers.
  • flagellin isolated directly from bacteria it will be appreciated that the same protein, or an immunologically effective portion thereof may also be prepared using, for example, recombinant technology.
  • cDNA encoding flagellin or an immunologically effective portion thereof may be cloned into a host organism and expressed to produce flagellin antigen.
  • Smaller antigenic peptides may also be made synthetically.
  • the term "derived from” refers to proteins or peptides which are either isolated directly from bacteria of the B. cepacia complex, or which have the same amino acid sequence but which are obtained synthetically or by expression in a host organism.
  • Vaccine compositions in accordance with the invention comprise the flagellin or flagellin-derived antigen, in a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier will be aqueous media, and may include buffers, emulsifiers, or adjuvants to enhance the immune response.
  • Antigenic proteins or peptides may also be provided in association with lipid-carriers, e.g., liposomes or other lipid particles, in the vaccine composition.
  • composition of the invention may also include other materials which are more indirectly derived from flagellin, and which provide immunoprotective therapeutic benefits.
  • human antibodies specific to B. cepacia flagellin or immunologically effective portion thereof are considered to be "derived- from" flagellin in accordance with the invention.
  • Such antibodies may be administered to individuals with B. cepacia, genomovar III infections, to help combat the progression of infection.
  • Such therapy is particularly suitable in end-stage B. cepacia infection when antibiotics and anti- inflammatory therapy have failed.
  • DNA vaccines may also be carried out using DNA vaccines of the type described generally in US Patent No. 5,580,859, which is incorporated herein by reference.
  • DNA vaccines comprise a sequence encoding the desired protein or peptide antigen, optionally in combination with a regulatory element to control expression of the antigen.
  • the DNA vaccine which may be naked or incorporated in a carrier such as a liposome, is administered by subcutaneous or intramuscular injection.
  • a "gene gun” may be used for administration.
  • EXAMPLE 1 Restriction fragment length polymorphisms (RFLP) of the 16S rRNA gene amplified from strains from the B. cepacia complex were determined.
  • RFLP Restriction fragment length polymorphisms
  • the amplified fragment of the 16S rRNA gene was digested with the enzyme Dde I and the resulting products separated by agarose gel electrophoresis. Species specific patterns where found for B. vietnamiensis (genomovar V) and B. multivorans (genomovar II) strains, however sequence variation in the 16S rRNA gene was insufficient to distinguish among strains from genomovars I, III and IV.
  • EXAMPLE 2 The novel DNA sequences of 16 recA genes, from 16 strains of the genomovar representative panel shown in Table 1 , have now been obtained by conventional DNA sequence analysis of portions of the recA gene amplified by PCR. DNA sequence analysis was performed in collaboration with Prof. Julian Davies at TerraGen Diversity Inc., Vancouver, BC, using an ABI 377 Nucleotide Sequencer. The 1040 bp recA gene was split into two -520 bp PCR products to facilitate complete sequence analysis. The primer pairs used were as follows: (i) 5' - portion of the recA gene: Forward primer BCR1 :
  • primers amplified a 527 bp product corresponding to the 5' half of the recA gene from all members of the B. cepacia complex tested. Nucleotide sequence from both strands of the amplified products was determined by direct nucleotide sequence analysis using primers BCR1 and BCR4 respectively, (ii) 3' - portion of the recA gene: Forward primer BCR 3:
  • Reverse primer BCR 2 5' - CTC TTC TTC GTC CAT CGC CTC - 3' SEQ ID No. 4
  • These primers amplified a 529 bp product corresponding to the 3' half of the recA gene from all members of the B. cepacia complex tested (see Table 1).
  • Nucleotide sequence from both strands of the amplified products was determined by direct nucleotide sequence analysis using primers BCR2 and BCR3 respectively.
  • cepacia strain from the same cable-pilus gene encoding lineage cited in PCT Patent Applications Nos. WO 97/01647 and WO97/07237. From such a sequence alignment, the sequence variation in the recA gene detected by the Hae III RFLP analysis is clearly visible. These sequences can also be used for the design of genomovar-specific amplification/sequencing primers, and genomovar-specific hybridization or ligation probes.
  • EXAMPLE 3 A B. cepacia complex phylogenetic tree based on the sequence alignment of the novel recA genes determined was formulated as shown in Fig. 1. The alteration of single bases in the nucleotide sequence of genes by natural mutation over time can be quantitated by computer programs to an evolutionary distant which separates strains and species. Phylogenetic analysis of the recA sequences which I have determined clearly demonstrates that sequence variation in this gene can separate all five genomovars of the B. cepacia complex, shown by the tree in the figure. Even more interesting is the novel finding of two distinct groups within the strains otherwise classified within genomovar III.
  • recA sequences derived from strains CEP511, C1394 and PC 184, respectively cluster as a separate group on a different branch of the tree from recA sequences derived form strains C6433, C4455m K56-2 and C5425. These two groups have been designated a RG-B and RG-A, respectively.
  • the three strains of the RG-B are all epidemic amongst patients with CF (Mahenthiralingam et al., J. Clin. Microbiol. 34: 2914- 2920 (1996) and encode the BCESM (Mahenthiralingam et al., J. Clin. Microbiol 35: 808- 816 (1996).
  • the separate classification of these three strains based on the recA suggest that they may constitute a new species/genomovar group within the B. cepacia complex.
  • CTCTTCTTCGTCCATCGCCTC SEQ ID No. 4 using a standard polymerase chain reaction mixture of 25 microlitres in volume (described in Mahenthiralingam et al., J. Clin. Microbiol. 35: 808-816 (1996)) containing 1.5 mM MgCl 2 and 10-20 ng of B. cepacia DNA.
  • Amplification was performed as follows: 30 cycles of 1 min. at 94°C, 1 min. at 56°C , and 2 min. at 72°C, follow by a final 6 min. cycle at 72°C. This resulted in the amplification of a 1 kb DNA band corresponding to the recA gene of the B. cepacia strain being tested.
  • CTCTTCTTCGTCCATCGCCTC SEQ ID No. 4 using a standard polymerase chain reaction mixture of 25 microlitres in volume (described in Mahenthiralingam et al, J. Clin. Microbiol. 35: 808-816 (1996)) containing 1.5 mM MgCl 2 and 10-20 ng of B. cepacia DNA.
  • Amplification was performed as follows: 30 cycles of 1 min. at 94°C, 1 min. at 56°C , and 2 min. at 72°C, follow by a final 6 min. cycle at 72°C. This resulted in the amplification of a 1 kb DNA band corresponding to the recA gene of the B. cepacia strain being tested (Fig. 2 A).
  • restriction enzyme were screened for their ability to reveal DNA sequence variation in this amplified gene which would be suitable for speciation of B. cepacia.
  • the enzymes Hae III and Alu I were found to be suitably discriminatory.
  • EXAMPLE 6 Bacterial strains identified in Example 5 as belonging to RFLP group G were epidemic strains of genomovar III which encode the BCESM DNA. When the flagellin gene of these strains was amplified using the PCR primers as described in Hales et al., a 1 kb product corresponding to the flagellin gene was obtained. Sequence variation in amplified flagellin gene was again detected using the restriction enzyme Hae III. The RFLP of the flagellin genes was highly conserved demonstrating that both the gene and the encoded protein are very conserved in sequence. The conservation in sequence of the flagellin of these epidemic strains suggests that the flagellum is an ideal candidate upon which to develop a vaccine able to protect against B.
  • EXAMPLE 8 Thirteen isolates having useful biological properties were evaluated to determine the recA group to which the isolates belong. The results of these and other tests are summarized in Table 2. Two strains within the ATCC collection with interesting catabolic properties (ATCC 29424 and ATCC 53617) were classified as B. vietnamensis on the basis of both 16S rRNA ARDRA and recA analysis. Of the remaining 11 commercially useful strains, all possessed ARDRA polymorphisms characteristic of B. cepacia genomovars I, III(RG-A and RG-B) and IV. Strain ATCC 49709, a seed- treatment biocontrol strain, was classified as genomovar I by recA RFLP and specific recA-VCR.
  • recA analysis may provide a simple method for screening biocontrol strains and other potentially useful members of the B. cepacia complex to identify strains that are less likely to raise health and public safety issues in their use. Furthermore, a number of the biocontrol strains were found to occupy a novel recA phylogenetic subgroup, RG-C.
  • the RG-C specific PCR primers, or other tests based on the sequence analysis performed on clinical isolates indicate that RG- C strains are not encountered in human infections, and appear specifically adapted to the plant rhizosphere, they may prove to be the best template upon which to continue to development of safe biocontrol strains.
  • Genomovar Source: 3 RAPD b
  • BCESM C cblA: Gene RFLP 16S
  • Footnotes a Source of isolate; CF, cystic fibrosis infection; CF-e, epidemic amongst CF patients (31):
  • Footnotes a Biocontrol strains demonstrated protection of crops against a various of phytopathogens; these included either fungal infection or nematode infections.

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Abstract

On identifie, dans un échantillon, des bactéries du complexe Burkholderia cepacia et on procède à leur spéciation, (a), en se procurant une information de séquence nucléotidique relative au gène recA au moyen des bactéries appartenant au complexe Burkholderia cepacia et présentes dans l'échantillon et (b), en comparant cette information de séquence nucléotidique à une bibliothèque d'informations de séquences nucléotidiques renfermant une information de séquence nucléotidique classique relativement à trois espèces au moins de bactéries du complexe Burkholderia cepacia. On se procure, de préférence, l'information de séquence nucléotidique par évaluation du polymorphisme de la longueur des fragments de restriction (RFLP). Il est également possible de faire appel à d'autres techniques pour obtenir cette information, notamment à la détermination base par base de la séquence de la région à étudier, ou d'employer des sondes d'hybridation oligonucléotidiques et des techniques de ligation. L'utilisation éventuelle d'amorces universelles aux fins de l'amplification de tous les éléments connus du complexe Burkholderia cepacia ainsi que celle d'amorces spécifiques d'un génomovar pour l'amplification sélective du gène recA provenant de bactéries d'un génomovar, fournissent d'autres modalités analytiques. On peut se servir de la spéciation du complexe Burkholderia cepacia comme base aux fins d'une administration d'un vaccin spécifique de la flagelline des bactéries, dans la mesure où il est démontré que cette flagelline est conservée par les membres du génomovar III, sous-groupe RG-B.
PCT/CA1999/000813 1998-09-03 1999-09-03 PROCEDE D'IDENTIFICATION ET DE SPECIATION DE BACTERIES DU COMPLEXE $i(BURKHOLDERIA CEPACIA) WO2000014274A1 (fr)

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GB0104647A GB2362212A (en) 1998-09-03 1999-09-03 Method for the identification and speciation of bacteria of the burkholderia cepacia complex
AU56120/99A AU5612099A (en) 1998-09-03 1999-09-03 Method for the identification and speciation of bacteria of the (burkholderia cepacia) complex

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WO2001023604A2 (fr) 1999-09-28 2001-04-05 Infectio Diagnostic (I.D.I.) Inc. Genes a fort pouvoir de conservation et leur utilisation pour produire des sondes d'acide nucleique specifiques a l'espece, specifiques au gene, specifiques a la famille, specifiques au groupe et universelles et des sondes d'amplification, en vue de detecter et d'identifier rapidement des micro-organismes algaires, archeoba
CN100432228C (zh) * 2003-11-21 2008-11-12 广东省微生物研究所 一种2－萘酸降解菌dna片段的核苷酸序列及其制备方法和应用
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
US8426137B2 (en) 1996-11-04 2013-04-23 Genohm Sciences Canada, Inc. Methods and probes for detecting a vancomycin resistance gene
CN116064866A (zh) * 2022-09-23 2023-05-05 中国食品药品检定研究院 用于检测洋葱伯克霍尔德菌群的试剂盒、引物和方法及应用
CN117265140A (zh) * 2023-05-05 2023-12-22 浙江省食品药品检验研究院 洋葱伯克霍尔德菌复合群pcr检测引物及其检测方法

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US8426137B2 (en) 1996-11-04 2013-04-23 Genohm Sciences Canada, Inc. Methods and probes for detecting a vancomycin resistance gene
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
US8067207B2 (en) 1997-11-04 2011-11-29 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
EP2322666A2 (fr) 1999-09-28 2011-05-18 Geneohm Sciences Canada, Inc. Gene a fort pouvoir de conservation et l'utilisation pour produire des sondes d'acide nucleique specifiques a l'espece, specifiques au gene, specifiques a la famille, specifiques au groupe et universelles et des sondes d'amplification, en vue de detecter et d'identifier rapidement des micro-organismes
EP2322668A2 (fr) 1999-09-28 2011-05-18 Geneohm Sciences Canada Inc. GENE A FORT POUVOIR DE CONSERVATION ET l'UTILISATION POUR PRODUIRE DES SONDES D'ACIDE NUCLEIQUE SPECIFIQUES A L'ESPECE, SPECIFIQUES AU GENE, SPECIFIQUES A LA FAMILLE, SPECIFIQUES AU GROUPE ET UNIVERSELLES ET DES SONDES D'AMPLIFICATION, EN VUE DE DETECTER ET D'IDENTIFIER RAPIDEMENT DES MICRO-ORGANISMES
EP2322667A2 (fr) 1999-09-28 2011-05-18 Geneohm Sciences Canada, Inc. Gene a fort pouvoir de conservation et l'utilisation pour produire des sondes d'acide nucleique specifiques a l'espece, specifiques au gene, specifiques a la famille, specifiques au groupe et universelles et des sondes d'amplification, en vue de detecter et d'identifier rapidement des micro-organismes
WO2001023604A2 (fr) 1999-09-28 2001-04-05 Infectio Diagnostic (I.D.I.) Inc. Genes a fort pouvoir de conservation et leur utilisation pour produire des sondes d'acide nucleique specifiques a l'espece, specifiques au gene, specifiques a la famille, specifiques au groupe et universelles et des sondes d'amplification, en vue de detecter et d'identifier rapidement des micro-organismes algaires, archeoba
US8182996B2 (en) 1999-09-28 2012-05-22 Geneohm Sciences Canada Inc. Compositions and methods for detecting Klebsiella pneumoniae
EP2660335A2 (fr) 1999-09-28 2013-11-06 Geneohm Sciences Canada, Inc. Gènes hautement conservées et leur utilisation pour générer des sondes d'acide nucléique universelles et spécifiques aux espèces, genres, familles, groupes et amorces d'amplification pour détecter rapidement et identifier des microorganismes algales, archéales, bactériennes, fongiques ou parasitaires à partir de spécimens cliniques pour diagnostic
US10047404B2 (en) 1999-09-28 2018-08-14 Geneohm Sciences Canada, Inc. Highly conserved tuf genes and their use to generate probes and primers for detection of coagulase-negative Staphylococcus
CN100432228C (zh) * 2003-11-21 2008-11-12 广东省微生物研究所 一种2－萘酸降解菌dna片段的核苷酸序列及其制备方法和应用
CN116064866A (zh) * 2022-09-23 2023-05-05 中国食品药品检定研究院 用于检测洋葱伯克霍尔德菌群的试剂盒、引物和方法及应用
CN116064866B (zh) * 2022-09-23 2023-09-29 中国食品药品检定研究院 用于检测洋葱伯克霍尔德菌群的试剂盒、引物和方法及应用
CN117265140A (zh) * 2023-05-05 2023-12-22 浙江省食品药品检验研究院 洋葱伯克霍尔德菌复合群pcr检测引物及其检测方法

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