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
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
  • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria

Definitions

• the present invention relates to a method for the specific detection and/or identification of Enterococcus species, in particular Enterococcus faecalis and/or Enterococcus faecium , using new nucleic acid sequences derived from the ITS (Internal Transcribed Spacer) region.
• ITS Internal Transcribed Spacer
• the present invention relates also to said new nucleic acid sequences derived from the ITS region, between the 16S and 23S ribosomal ribonucleic acid (rRNA) or rRNA genes, to be used for the specific detection and/or identification of Enterococcus species, in particular of Enterococcus faecalis and/or Enterococcus faecium , in a biological sample.
• rRNA ribosomal ribonucleic acid
• nucleic acid primers to be used for the amplification of said spacer region of Enterococcus species in a sample.
• the genus Enterococcus includes currently 27 described species. From the human clinical point of view, E. faecalis and/or E. faecium are the most important species: E. faecalis , and E. faecium together make up 95% of all nosocomial enterococcal infections distributed respectively as 80 to 90% and 5 to 10%. Occasionally E. casseliflavus and E. gallinarum are isolated.
• Enterococci are increasingly recognized as common causes of infection that become difficult to treat because of both inherent and acquired antibiotic resistance.
• Effective control of E. faecalis and/or E. faecium within the hospital and community requires more aggressive measures that include earlier diagnosis of colonized patients, in other words, that include a step of screening.
• the present invention thus provides an isolated nucleic acid molecule consisting of SEQ ID NO 1 or 2, the RNA form of said SEQ ID NO 1 or 2 wherein T is replaced by U, the complementary form of said SEQ ID NO 1 or 2, or any homologue, and the use of said nucleic acid molecule as a target for the detection and/or identification of Enterococcus species.
• An aspect of the present invention relates to new polynucleotides for use as probes and/or primers, which have as target a particular region of the 16S-23S rRNA spacer region of Enterococcus species, and which allow the detection and/or identification of Enterococcus species, in particular of Enterococcus faecalis and/or Enterococcus faecium.
• the present invention thus provides an isolated nucleic acid molecule that specifically hybridizes to SEQ ID NO 1 or 2, or to the RNA form of said SEQ ID NO 1 or 2 wherein T is replaced by U, or to the complementary form of said SEQ ID NO 1 or 2, or to any homologous sequences thereof, or to a fragment of at least 20 contiguous nucleotides thereof, for the detection and/or identification of Enterococcus species, in particular of Enterococcus faecalis and/or Enterococcus faecium.
• Another aspect of the present invention relates to sets of probes for the detection and/or identification of Enterococcus species, in particular of Enterococcus faecalis and/or Enterococcus faecium in a sample.
• Another aspect of the present invention concerns primers allowing specific amplification of the 16S-23S rRNA spacer region of Enterococcus species, in particular of E. faecalis and/or E. faecium.
• Another object of the present invention is a composition containing any of the new sequences of the invention, or any of the new sets of probes and/or primers of the invention; or a combination thereof.
• Another object of the present invention is a kit, in which said probes and/or primers are used, for the detection and/or identification Enterococcus species, in particular of Enterococcus faecalis and/or Enterococcus faecium.
• Another object of the present invention is a rapid and reliable hybridization method for detection and/or identification of Enterococcus species, in particular of Enterococcus faecalis and/or Enterococcus faecium.
• Another object of the present invention is a hybridization method based on real time PCR for detection and/or identification of Enterococcus species, in particular of Enterococcus faecalis and/or Enterococcus faecium.
• spacer and “ITS” (Internal Transcribed Spacer) are abbreviated terms both referring to the region between the 16S and 23S rRNA or between the 16S and 23S rRNA genes.
• probe refers to single stranded oligonucleotides or polynucleotides which have a sequence which is sufficiently complementary to hybridize to the target sequence to be detected.
• the probes of the invention are 70%, 80%, 90%, or more than 95% homologous to the exact complement of the target sequence to be detected.
• target sequences are either genomic DNA or precursor RNA, or amplified versions thereof.
• the probes of the invention can be formed by cloning of recombinant plasmids containing inserts including the corresponding nucleotide sequences, if need be by cleaving the latter out from the cloned plasmids upon using the adequate nucleases and recovering them, e.g. by fractionation according to molecular weight.
• the probes according to the present invention can also be synthesized chemically, for instance by the conventional phospho-triester method.
• nucleic acid sequences can form a perfect base-paired double helix with each other.
• polynucleic acid corresponds to either double-stranded or single-stranded cDNA or genomic DNA or RNA, containing at least 5, 10, 20, 30, 40 or 50 contiguous nucleotides.
• a polynucleic acid which is smaller than 100 nucleotides in length is referred to as an “oligonucleotide”.
• modified nucleotides such as inosine or nucleotides containing modified groups which do not essentially alter their hybridization characteristics.
• Single stranded polynucleic acid sequences are always represented in the current invention from the 5′ end to the 3′ end.
• closest neighbor means the taxon which is known or expected to be most closely related in terms of DNA homology and which has to be differentiated from the organism of interest.
• taxon-specific hybridization or “taxon-specific probe” means that the probe only hybridizes to the DNA or RNA from the taxon for which it was designed and not to DNA or RNA from other taxa.
• taxon can refer to a complete genus or a sub-group within a genus, a species or even subtype within a species (subspecies, serovars, sequevars, biovars . . . ).
• the oligonucleotides or polynucleotides selected as being “preferential” show a sensitivity and specificity of more than 80%, preferably more than 90% and most preferably more than 95%.
• solid support can refer to any substrate to which a polynucleotide probe can be coupled, provided that it retains its hybridization characteristics and provided that the background level of hybridization remains low.
• the solid substrate will be a microtiter plate, a membrane (e.g. nylon or nitrocellulose) or a microsphere (bead).
• a membrane e.g. nylon or nitrocellulose
• a microsphere bead
• modifications may encompass homopolymer tailing, coupling with different reactive groups such as aliphatic groups, NH 2 groups, SH groups, carboxylic groups, or coupling with biotin, haptens or proteins.
• labeled refers to the use of labeled nucleic acids. Labeling may be carried out by the use of labeled nucleotides incorporated during the polymerase step of the amplification such as illustrated by Saiki et al. (1988) or Bej et al. (1990) or by the use of labeled primers, or by any other method known to the person skilled in the art.
• the nature of the label may be isotopic ( 32 P, 35 S, etc.) or non-isotopic (biotin, digoxigenin, fluorescent dye, biotin, enzyme, etc.).
• signal refers to a series of electromagnetic waves (for example fluorescence), or changes in electrical current which carry information.
• the signal can be directly visible, or can be made visible and/or interpretable by different means or devices.
• sample may be any biological material. This biological material may be taken either directly from the infected human being, or animal, or after culturing or enrichment, or from food, from the environment, etc.
• Biological material may be for example expectoration of any kind, broncheolavages, blood, skin tissue, biopsies, lymphocyte blood culture material, colonies, etc. Said samples may be prepared or extracted according to any of the techniques known in the art.
• the Enterococcus species that are clinically relevant in the meaning of the present invention are E. faecalis, E. faecium, E. avium, E. cecorum, E. columbae, E. durans, E. flavescens, E. hirae, E. malodoratus, E. mundtii, E. pseudoavium, E. raffinosus, E. casseliflavus, E. gallinarum (Table 4).
• the ITS is already known for some Enterococcus species.
• E. faecalis strains show two different type of spacer, and E. faecium strains show four different types.
• the present invention provides a particular region of the ITS, identified and delimited for its great advantage of offering a unique target sequence for the detection and/or identification of all Enterococcus species, and in particular of all Enterococcus species clinically relevant, and more particularly of E. faecalis and/or E. faecium.
• the target sequence of the invention are found in all type of spacer of every Enterococcus species, in particular of every Enterococcus species that are clinically relevant.
• This particular region of the ITS also referred to as the “target region” or “target sequence” can be defined as a nucleic acid molecule consisting of SEQ ID NO 1 or SEQ ID NO 2, or as a nucleic acid molecule that is homologous to SEQ ID NO 1 or 2, their RNA form wherein T is replaced by U, or their complementary form.
• target sequence covers all the homologous sequences found in the ITS of any Enterococcus species, said homologous sequences are also referred to herein after as “homologues”. The degree of homology is then higher than 75%, generally higher than 80%, and even higher than 90%.
• homologues are then homologous sequences to SEQ ID NO 1 or 2 or to any fragment thereof, localized in the ITS region of any Enterococcus species, SEQ ID NO 1 and 2 being derived respectively from E. faecalis and E. faecium stras.
• New polynucleotides for use as probes and/or primers designed from the target sequence of the invention for the detection and/or identification of Enterococcus species are also an object of the invention.
• an object of the invention relates to new polynucleotides for use as probes and/or primers, which hybridize with the target sequence of the invention for the detection and/or identification of Enterococcus species.
• an object of the invention is an isolated nucleic acid molecule that specifically hybridizes to SEQ ID NO 1 or 2, or to the RNA form of said SEQ ID NO 1 or 2 wherein T is replaced by U, or to the complementary form of said SEQ ID NO 1 or 2, or to a fragment of at least 20 contiguous nucleotides thereof, or to any of their homologues, for the detection and/or identification of Enterococcus species, in particular of E. faecalis and/or E. faecium
• Preferred polynucleotide probes are between about 5 to about 50 bases in length, more preferably from about 10 to about 25 nucleotides and are sufficiently homologous to the target sequence.
• Polynucleotides of SEQ IDs NO 3 to 84 and any of their homologues may be used as probes.
• Preferred probes are polynucleotides of SEQ IDs NO 22 to 26, 28 to 43, 45 to 65 and 67 to 84, and homologues, in particular polynucleotides of SEQ IDs NO 28 to 36, 45 to 58, 67 to 84.
• Preferred primers of the invention are single stranded DNA polynucleotides capable of acting as a point of initiation for synthesis of the target sequence of the invention.
• the length and the sequence of a primer of the invention must be such that they allow to prime the synthesis of the extension products.
• a primer of the invention is about 5 to about 50 nucleotides long, preferably about 15 to about 25. Its specific length and sequence is to be chosen depending on the conditions used such as temperature and ionic strength.
• Preferred primers of the invention amplify the target sequence.
• preferred primers of the invention amplify SEQ ID NO 1 or SEQ ID NO 2 and/or homologues.
• Preferred primers of the invention are polynucleotides of SEQ IDs NO 3 to 10 and 12 to 20, and homologues.
• the amplification method used can be either polymerase chain reaction (PCR; Saiki et al., 1988), ligase chain reaction (LCR; Landgren et al., 1988; Wu & Wallace, 1989; Barany, 1991), nucleic acid sequence-based amplification (NASBA; Guatelli et al., 1990; Compton, 1991), transcription-based amplification system (TAS; Kwoh et al., 1989), strand displacement amplification (SDA; Duck, 1990; Walker et al., 1992) or amplification by means of Q ⁇ replicase Qizardi et al., 1988; Lomeli et al., 1989) or any other suitable method to amplify nucleic acid molecules known in the art.
• PCR polymerase chain reaction
• LCR Landgren et al., 1988; Wu & Wallace, 1989
• NASBA nucleic acid sequence-based amplification
• TAS transcription-based amplification system
• SDA strand displacement
• the preferred polynucleotides of the invention for use as primers or as probes are listed in Table 1.
• Polynucleotides of the invention may differ in sequence from any of the polynucleotides specified in Table 1, or from any of their homologues, either by addition to or removal from any of their respective extremities of one or several nucleotides, or by changing one or more nucleotides within said sequences, or a combination of both, provided that the equivalents then obtained still hybridize with the target sequence as the corresponding unmodified polynucleotides.
• Said equivalent polynucleotides share at least 75% homology, preferably more than 80%, most preferably more than 85% homology with the corresponding unmodified polynucleotides.
• the polynucleotides primers and/or probes of the invention may also comprise nucleotide analogues such as phosphorothioates (Matsukra et al., 1987), alkylphosphorothioates (Miller et al., 1979) or peptide nucleic acids (Nielsen et al., 1991; Nielsen et al., 1993) or may contain intercalating agents (Asseline et al., 1984), etc.
• nucleotide analogues such as phosphorothioates (Matsukra et al., 1987), alkylphosphorothioates (Miller et al., 1979) or peptide nucleic acids (Nielsen et al., 1991; Nielsen et al., 1993) or may contain intercalating agents (Asseline et al., 1984), etc.
• the modified primers or probes require adaptations with respect to the conditions under which they are used in order to obtain the required specificity and sensitivity. However the results of hybridization should remain essentially the same as those obtained with the unmodified polynucleotides.
• the probes and primers of the invention are used in methods, also objects of the present invention, for the detection and/or identification of Enterococcus species, in particular of E. faecalis and/or E. faecium.
• Detection and/or identification of the target sequence can be performed by using a electrophoresis method, a hybridization method or a sequencing method.
• a method of the invention for the detection of one or more Enterococcus species in a sample comprises the following steps:
• the nucleic acids present in the sample are made available for amplification and/or hybridization.
• the nucleic acids are amplified with one or another target amplification system, as specified below.
• amplification is needed to enhance the subsequent hybridization signal.
• amplification might not be necessary.
• the nucleic acids present in the sample or the resulting amplified product are contacted with probes, and hybridization is allowed to proceed.
• hybrids are detected using a convenient and compatible detection system. From the hybridization signals or patterns observed the presence or absence of one or several Enterococcus species can be deduced.
• the amplification system used may be more or less universal, depending on the specific application needed.
• step (iii) hybridizing the polynucleic acids of step (i) or (ii) with at least one polynucleotide probe that hybridizes to the target sequence, wherein the target sequence consists of SEQ ID NO 1 or 2 or homologues thereof, or to their RNA form wherein T is replaced by U, or to their complementary form, or a to a fragment of at least 20 contiguous nucleotides thereof,
• the probes of the inventions hybridize under conditions of high stringency.
• the stringency of the assay conditions determines the amount of complementarity needed between two nucleic acid strands forming a hybrid. Stringency is chosen to maximize the difference in stability between the hybrid formed with the target and the non-target nucleic acid.
• the hybridization conditions are chosen in such a way that the signal of hybridization obtained when a polynucleotide of the invention hybridizes specifically to a target sequence, is different from the signal obtained when said polynucleotide hybridizes to a target sequence in a non-specific manner.
• the different signals may be visualized for example when its intensity is two, five, ten or more times stronger with a specific hybridization to the target, as compared to non-specific hybridization to the target sequence, LiPA system for example.
• the different signals may also be visualized when different peaks are drawn in a melting curve analysis, for instance when using a real time PCR method.
• the fragment mentioned in the amplification or the hybridization step of any method of the invention may comprise 20 to 50, 20 to 80 or 20 to 100 contiguous nucleotides of SEQ ID NO 1 or 2 or of any homologues.
• a very convenient and advantageous technique for the detection of target sequences that are possibly present in the sample is the real time PCR method.
• a single-stranded hybridization probe is labeled with two components.
• the first component the so-called fluorescer
• the second component the so-called quencher
• the hybridization probe binds to the target DNA and is degraded by the 5′-3′ exonuclease activity of the polymerase, for example Taq Polymerase, during the elongation phase.
• the excited fluorescent component and the quencher are spatially separated from one another and thus a fluorescence emission of the first component can be measured (EP B 0 543 942 and U.S. Pat. No. 5,210,015).
• the probes are also labeled with a first component and with a quencher, the labels preferably being located at different ends of an at least partially self-complementary probe.
• both components are in spatial vicinity in solution.
• After hybridization to the target nucleic acids both components are separated from one another such that after excitation with light of a suitable wavelength the fluorescence emission of the first component can be measured (U.S. Pat. No. 5,118,801).
• the Fluorescence Resonance Energy Transfer (FRET) hybridization probe test format is especially useful for all kinds of homogenous hybridization assays (Matthews, J. A. and Kricka, L. J., Anal Biochem 169 (1988) 1-25). It is characterized by two single-stranded hybridization probes which are used simultaneously and are complementary to adjacent sites of the same strand of an (amplified) target nucleic acid. Both probes are labeled with different fluorescent components. When excited with light of a suitable wavelength, a first component transfers the absorbed energy to the second component according to the principle of fluorescence resonance energy transfer such that a fluorescence emission of the second component can be measured only when both hybridization probes bind to adjacent positions of the target molecule to be detected.
• FRET Fluorescence Resonance Energy Transfer
• the hybridization probes When annealed to the target sequence, the hybridization probes must be located very close to each other, in a head to tail arrangement.
• the gap between the labeled 3′ end of the first probe and the labeled 5′ end or the second probe is as small as possible, and notably consists of about 0 to 25 bases, and preferably of about 1 to about 5 bases. This allows for a close vicinity of the FRET donor compound and the FRET acceptor compound, which is typically 10-100 ⁇ ngstrom.
• the FRET-hybridization probe format has been proven to be highly sensitive, exact and reliable (WO 97/46707; WO 97/46712; WO 97/46714). Yet, the design of appropriate FRET hybridization probe sequences may sometimes be limited by the special characteristics of the target nucleic acid sequence to be detected.
• FRET hybridization probes can also be used for melting curve analysis (WO 97/46707; WO 97/46712; WO 97/46714).
• the target nucleic acid is amplified first in a typical PCR reaction with suitable amplification primers.
• the hybridization probes may already be present during the amplification reaction or be added subsequently.
• the temperature of the sample is consecutively increased. Fluorescence is detected as long as the hybridization probe is bound to the target DNA.
• the hybridization probe is released from their target, and the fluorescent signal is decreasing immediately down to the background level. This decrease is monitored with an appropriate fluorescence versus temperature-time plot such that the negative of a first derivative function can be calculated.
• the temperature value corresponding to the obtained maximum of such a function is then taken as the determined melting temperature of said pair of FRET hybridization probes.
• Point mutations or polymorphisms within the target nucleic acid result in a less then 100% complementarity between the target nucleic acid and the FRET probes, thus resulting in a decreased melting temperature. This enables for a common detection of a pool of sequence variants by means of FRET-Hybprobe hybridization, whereas subsequently, different members of said pool may become discriminated by means of performing melting curve analysis.
• Molecular Beacons may alternatively be used for melting curve analysis.
• melting temperature of the generated double stranded PCR product has to be determined. Yet, this method has only limited applications since few differences cannot be monitored efficiently, because minor sequence variations only result in subtle melting temperature differences.
• hybridization probes may be used in such a way that the melting temperature of the probe/target nucleic acid hybrid is being determined.
• the melting peak data are characteristic of a particular [probe:target] sequence because mismatches between probe and target affect the kinetics of melting, producing different melting peaks for each species of interest
• the LightCyclerTM platform offers many advantages and in particular a gain of time and the possible use of several different sequence-specific fluorescent probe detection systems such as hybridization probes (HybProbes), TaqManTM probes, Molecular Beacons and biprobes (SYBR Green I).
• HybProbes hybridization probes
• TaqManTM probes TaqManTM probes
• Molecular Beacons Molecular Beacons
• biprobes SYBR Green I
• the HybProbe system consisting of two adjacent polynucleotide probes derived from the target region of the invention, in a head-to-tail orientation, spaced by a few nucleotides, generally 0 to 25, preferably about 1 to about 5.
• One of the probes is labeled at its 3′ end by a donor dye, the other is labeled with an acceptor molecule at its 5′ end, and is phosphate blocked at the 3′ end (to prevent its acting as a primer).
• the donor dye is generally fluorescein, and the acceptor molecule generally LC Red640 or 705.
• the detection of the target sequence of the invention may be achieved also by an internal labeled PCR strand and a detection probe located on the opposite strand.
• the signal is dependent on the spatial approximation of the dyes, and is dependent on the amount of the target.
• the emitted light of the donor is transmitted to the acceptor fluorophore by Fluorescence Resonance Energy Transfer (FRET), and the emitted fluorescence (640 or 705 nm) can be detected.
• FRET Fluorescence Resonance Energy Transfer
• the intensity of the emitted fluorescence increases in parallel with the target DNA, product of the amplification.
• the LightCycler probes offer the advantage over the TaqManTM probes of not requiring hydrolysis and, therefore, no additional extension of the PCR times (annealing-elongation ⁇ 12 s). It is therefore possible to take advantage of the high-speed thermal cycling of the LightCycler, and complete the PCR program in only 45 minutes.
• Another object of the invention relates to sets of at least 2 polynucleotide probes, also referred to as HybProbes, both HybProbes hybridizing to the same target sequence, adjacent to each other, with no more than 25 nucleotides between said 2 HybProbes, preferably with no more than 10 nucleotides, in particular with no more than 5 nucleotides.
• HybProbes When there are two HybProbes, one is labeled with an acceptor fluorophore and the other with a donor fluorophore of a fluorescence energy transfer pair such that upon hybridization of the two HybProbes with the target sequence, the donor and acceptor fluorophores are within 0 to 25 nucleotides of one another, and preferably within 0 to 5 nucleotides of one another.
• the donor and acceptor fluorophores are within 0 to 25 nucleotides of one another, and preferably within 0 to 5 nucleotides of one another.
• a set of at least two polynucleotide probes may be used, said probes hybridizing to SEQ ID NO 1 or SEQ ID NO 2, or to the RNA form of said SEQ ID NO 1 or 2 wherein T is replaced by U, or to the complementary form of said SEQ ID NO 1 or 2, or to homologues, wherein there are no more than 25 nucleotides, preferably no more than 5 nucleotides, between said probes.
• a set of probes of the invention may also consist of 3, 4, 5, 6, 7, 8, 9, 10, or more, probes, but it preferably consists of 2 to 5 probes, and more preferably of 3 probes.
• Sets of 2 polynucleotides, one for use as primer, the other for use as probe, may also be used, both said primer and probe hybridizing to the target sequence consisting of SEQ ID NO 1 or 2, of the RNA form of said SEQ ID NO 1 or 2 wherein T is replaced by U, of the complementary form of said SEQ ID NO 1 or 2, or of any homologues, wherein there are no more than 25 nucleotides, preferably no more than 5 nucleotides, between said primer and said probe.
• the sets of at least 2 polynucleotides of the invention are used in methods for the detection and/or identification of Enterococcus species, in particular of E. faecalis and/or E. faecium.
• a method of the present invention for detection and/or identification of Enterococcus species in a sample comprises the steps of:
• step (iv) detecting the hybrids formed in step (iii);
• step (v) inferring the presence of Enterococcus species, or identifying the Enterococcus species in the sample from the differential hybridization signals obtained in step (iv).
• a primer pair used in the amplification step is any combination of a forward primer consisting of SEQ ID NO 3 to 11 or their homologues, and a reverse primer consisting of SEQ ID 12 to 21 or their homologues.
• a set of 2 or 3 HybProbes used in the hybridization step is any combination of 2 or 3 HybProbes chosen among polynucleotides of SEQ IDs NO 22 to 26, 28 to 43, 45 to 65 or 67 to 84 or their homologues, preferably among polynucleotides of SEQ IDs NO 28 to 36, 45 to 56 or 67 to 84 or their homologues, provided that the gap between two of said HybProbes when hybridized to the target sequence is less than 25 nucleotides, preferably less than S nucleotides.
• HybProbes system resides in the fact that it allows the detection of sequence variation, including mutations, polymorphisms and other variant nucleic acid species, based on the following molecular concept: one of the HybProbe is a tightly binding “anchor probe” whereas the adjacent “sensor probe” spans the region of sequence variation. During melting of the final PCR product, the sequence alteration is detected as a change in the melting temperature (Tm) of the sensor probe.
• Tm melting temperature
• the fluorescence may be measured during the amplification step, generating then amplification curves, or after the amplification step, for a melting curve analysis generating melting curves.
• the signal obtained may be visualized in the form of amplification curves or in the form of melting curves, from which it is possible to infer the presence of Enterococcus species, and/or to infer which one(s) of the Enterococci are present.
• a method for detection and/or identification of Enterococcus species in a sample comprises also the steps of
• step (v) inferring the presence of Enterococcus species, and/or identifying the Enterococcus species in the sample from the signals obtained in step (iv).
• a method of the invention using the HybProbes system may be adapted for the detection and identification of Enterococcus faecalis and/or Enterococcus faecium , allowing the distinction of E. faecalis and/or E. faecium from other species.
• suitable primers are primer pairs that specifically amplify the target sequence which consists of SEQ ID NO 1 or 2, or of the RNA form of said SEQ ID NO 1 or 2 wherein T is replaced by U, or of the complementary form of said SEQ ID NO 1 or 2.
• the HybProbes should hybridize specifically to SEQ ID NO 1 or 2, or to the RNA form wherein T is replaced by U, or to the complementary form.
• E. faecalis and/or E. faecium strains can be unequivocally distinguished from all other organisms examined by melting curve analysis.
• Preferred primer pairs used in this particular example are any combinations of forward primers chosen among SEQ ID NO 3 to 11 or their homologues and reverse primers chosen among SEQ ID NO 12 to 21 or their homologues.
• HybProbes listed in Table 3 or their homologues are the preferred sets of HybProbes of the invention.
• a more preferred set of 3 Hybprobes consists of SEQ ID NO 36 or homologues and SEQ ID NO 56 or homologues and SEQ ID NO 73 or homologues.
• the set of HybProbes consisting of SEQ ID NO 36, 56 and 73 is able to detect E. faecalis and/or E. faecium with a high sensitivity.
• Each polynucleotide listed in Table 1, corresponding to SEQ ID NO 1 to SEQ ID NO 84 and any of their homologues, may be used in any methods of the present invention as a primer and/or as a probe, alone or in combination.
• a second embodiment based also on a hybridization method is the Line Probe Assay technique.
• the Line Probe Assay (LiPA) is a reverse hybridization format (Saiki et al., 1989) using membrane strips onto which several polynucleotide probes (including negative or positive control polynucleotides) can be conveniently applied as parallel lines.
• the LiPA technique provides a rapid and user-friendly hybridization test. Results can be read within 4 h. after the start of the amplification. After amplification during which usually a non-isotopic label is incorporated in the amplified product, and alkaline denaturation, the amplified product is contacted with the probes on the membrane and the hybridization is carried out for about 1 to 1,5 h. Consequently, the hybrids formed are detected by an enzymatic procedure resulting in a visual purple-brown precipitate.
• the LiPA format is completely compatible with commercially available scanning devices, thus rendering automatic interpretation of the results possible. All those advantages make the LiPA format liable for use in a routine setting.
• the LiPA format is an advantageous tool for detection and/or identification of pathogens at the species level but also at higher or lower taxonomical levels.
• probe-configurations on LiPA strips can be selected in such a manner that they can detect the complete genus of Enterococcus or can identify species within the genus (e.g. Enterococcus faecalis and/or Enterococcus faecium , etc) or can in some cases even detect subtypes (subspecies, serovars, sequevars, biovars, etc. whatever is clinically relevant) within a species.
• the ability to simultaneously generate hybridization results with a large number of probes is another benefit of the LiPA technology.
• the amount of information which can be obtained by a particular combination of probes greatly outnumbers the data obtained by using single probe assays. Therefore the selection of probes on the membrane strip is of utmost importance since an optimized set of probes will generate the maximum of information possible.
• These probes can be applied to membrane strips at different locations and the result is interpreted as positive if at least one of these probes is positive.
• these probes can be applied as a mixture at the same location, hereby reducing the number of lines on a strip. This reduction may be convenient in order to make the strip more concise or to be able to extend the total number of probes on one strip.
• probes with nucleotide sequences A and B are both required to detect all strains of taxon X.
• a probe can be synthesized having the nucleotide sequence AB. This probe will have the combined characteristics of probes A and B.
• the LiPA system can be considered as an efficient format for a hybridization method wherein several organisms need to be detected simultaneously in a sample.
• any other hybridization assay whereby different probes are used under the same hybridization and wash conditions can be used for the above-mentioned detection and/or selection methods.
• many different supports are available.
• the nucleic acids present in the sample are made available for amplification and/or hybridization.
• the nucleic acids are amplified with one or another target amplification system, as specified below. Usually, amplification is needed to enhance the subsequent hybridization signal.
• the nucleic acids present in the sample or the resulting amplified product are contacted with LiPA strips onto which one or more probes (DNA-, RNA-, degenerate or modified probes), allowing the detection of the organisms of interest, are immobilized, and hybridization is allowed to proceed.
• probes DNA-, RNA-, degenerate or modified probes
• the hybrids are detected using a convenient and compatible detection system. From the hybridization signals or patterns observed the presence or absence of one or several organisms screened for in that particular biological sample can be deduced.
• the amplification system used may be more or less universal, depending on the specific application needed.
• a method of the invention for detection and/or identification of Enterococcus species in a sample comprises the steps of:
• step (iv) detecting the hybrids formed in step (iii);
• step (v) identification of the micro-organism(s) present in the sample from the differential hybridization signals obtained in step (iv).
• the part of the ITS mentioned in the step of amplification is a polynucleotide comprising the target sequence, or the target sequence itself, the target sequence consisting of SEQ ID NO 1 or 2, or of the RNA form of said SEQ ID NO 1 or 2 wherein T is replaced by U, or of the complementary form of said SEQ ID NO 1 or 2, or of any homologues, or of a fragment of at least 20 contiguous nucleotides thereof.
• the present invention provides for a method as described above wherein at least 2 micro-organisms are detected simultaneously.
• a set of probes as described in step (iii) comprises at least two, three, four, five, six, seven, eight, nine or more probes of the invention, or equivalents thereof.
• set of probes as described in step (iii) comprises at least two probes.
• Preferred probes are polynucleotides of SEQ ID NO 1 to 84 and their homologues.
• the present invention also provides for a method as described above, wherein the probes as specified in step (iii) are combined with at least one other probe, preferentially also from the 16S-23S rRNA spacer region, enabling the simultaneous detection of different pathogenic bacteria liable to be present in the same sample.
• Preferred probes are designed for attaining optimal performance under the same hybridization conditions so that they can be used in sets for simultaneous hybridization; this highly increases the usability of these probes and results in a significant gain in time and labor.
• a kit containing any of the polynucleotides of the present invention is also an object of the invention.
• a kit of the invention comprise the following components:
• a preferred kit comprises
• the method used in the examples is a method for the detection of Enterococci, in particular E. faecalis and/or E. faecium , using the HybProbe system consisting of two Fluorescein-labeled probes of SEQ IDs NO 36 and 56, acting as sensor, and one LC-Red-labeled probe of SEQ ID NO 73 as anchor, in combination with a Enterococcus -genus primer pair of SEQ IDs NO 5 and 18.
• the gDNA was retyped by t-RNA PCR (Vaneechoutte, M. et al., 1998, Int. J. Syst. Bacteriol. (48) 127-139) and/or the culture was retyped by ApiSTREP (Biomérieux).
• the instrumentation is the LightCyclerTM (version 1.2) provided with the adequate software (LC-software version 3.5) enabling a Real-Time fluorescence PCR detection.
• Genomic DNA was prepared as described in the pack insert of the MPLC DNA Isolation Kit III. As recommended for Organon Teknika blood culture bottles, prior to PCR the eluate was centrifuged 10 sec at 14000 rpm to spin down the extracted carbon particles.
• the amplification and melting conditions are described herein after.
• the LC software version 3.5 was used.
• the quantification settings were F2/back F1 (samples).
• the crossing point (Ct) calculation was based on the second derivative maximum.
• the calculation method for the melting peak was polynomial.
• the peak area was used to calculate the Tm.
• Amplification and melting curve program Temp. Hold Slope Acquisition (° C.) time (° C./sec.) mode Denaturation 95 10 min 20 None Cycles 95 10 sec 20 None 45 ⁇ ⁇ open oversize brace ⁇ 50 15 sec 20 SINGLE 72 10 sec 20 None Melting 95 60 sec 20 None 40 60 sec 20 None 80 0 sec 0.1 CONTIN- UOUS Cooling 30 0 sec 20 None
• the discrepant samples were analyzed using biochemical identification techniques (Api 20Strep, Biomerieux) and tRNA PCR analysis. For all 7 samples it was shown that the initial identification was not correct and for 6 samples the results coincided with the in results of the assay.
• E. faecalis and E. faecium are detected (Ct & melting curves) and differentiated from other organisms including other Enterococci.

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