Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-08-17
2004-11-02
Horlick, Kenneth R. (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S024320
Reexamination Certificate
active
06811978
ABSTRACT:
The present invention relates to nucleic acid probes derived from the spacer region between the 16S and 23S ribosomal ribonucleic acid (rRNA) genes, to be used for the specific detection of eubacterial organisms in, a biological sample by a hybridization procedure, as well as to nucleic acid primers to be used for the amplification of said spacer region of eubacterial organisms in a biological sample. The present invention also relates to new spacer region sequences from which said probes or primers may be derived.
Since the advent of the polymerase chain reaction and some other nucleic acid amplification techniques the impact of DNA-probe technology in the diagnosis of micro-organisms in biological samples of all sorts is increasing. Being often more specific and potentially more sensitive—if an adequate amplification and or detection system is used the DNA probe approach may eventually replace the conventional identification techniques.
The reliability of nucleic acid based tests essentially depends on the sensitivity and specificaty of the probes and/or primers used. Thus the corner stone of this type of assay is the identification of nucleic acid sequences which are unique to the group of organisms of interest.
Most of the nucleic acid based tests either described in literature and/or commercially available aim at the detection of just one particular organism in a biological sample, Since most biological samples usually may contain a great variety of clinically relevant micro-organisms, a multitude of separate assays have to be performed to detect all relevant organisms possibly present. This approach would be very expensive, laborious and time-consuming. Consequently, the number of tests actually performed in most routine diagnostic labs on a particular sample is restricted to the detection of just a few of the most relevant organisms. Therefore it would be extremely convenient to have access to a system which enables the fast, easy and simultaneous detection of a multitude of different organisms. The more organisms that can be screened for in the same assay, the more cost-effective the procedure would be.
As put forward in earlier published documents, the spacer region situated between the 16S rRNA and the 23S rRNA gene, also referred to as the internal transcribed spacer (ITS), is an advantageous target region for probe development for detection of pathogens of bacterial origin (International application WO 91/16454; Rossau et al., 1992; EP-A-0 395 292).
One of its most appreciated advantages, is that sequences unique to a great variety of bacterial taxa can be found in a very limited area of the bacterial genome. This characteristic allows for an advantageous design of “probe-panels” enabling the simultaneous detection of a set of organisms possibly present in a particular type of a biological sample. Moreover, being flanked by quasi-universally conserved nucleotide sequences—more particularly located in the 3′-part of the 16S rRNA gene and the 5′-part of the 23S rRNA gene respectively—almost all spacers can be simultaneously amplified with a limited set of amplification primers. Alternatively, specific primer sets can be derived from the spacer sequences themselves, thereby allowing species- or group-specific amplifications.
The 16S-23S rRNA spacer region is a relatively short (about 200 to 1000 base pairs) stretch of DNA present in one or multiple copies in the genome of almost all eubacterial organisms. If multiple copies are present in the genome of one bacterium these copies can either be identical (as is most probably the case in some
Neisseria
species) or may differ from each other (as is the case for
E. coli
). This difference can be limited to a few nucleotides but also deletions and insertions of considerable length may be present.
Uptil now, spacer probes are only described and made publicly available for a limited number of organisms many of which were disclosed in international application WO 91/16454. As described above, it would be very advantageous to be able to detect simultaneously a panel of pathogens: e.g. a panel of pathogens possibly present in the same type of biological sample, or a panel of pathogens possibly causing the same type of disease symptoms, which are difficult to differentiate clinically and/or biochemically, or a panel of organisms belonging to the same taxon. In order to make the different panels as complete as possible, additional probes or sets of probes located in the spacer region and enabling the identification of at least the following bacterial groups or species are required:
Mycobacterium
species
Listeria
species
Chlamydia
species
Acinetobacter
species
Mycoplasma
species
Streptococcus
species
Staphylococcus
species
Salmonella
species
Brucella
species
Yersinia
species
Pseudomonas
species
These additional spacer probes need to be meticulously designed such that they can be used simultaneously with at least one other probe, under the same hybridization and wash conditions, allowing the detection of a particular panel of organisms.
It is thus the aim of the present invention to select probes or sets of probes, which have as target the 16S-23S rRNA spacer region, and which allow the detection and identification of at least one, and preferably more than one, of the above mentioned micro-organisms. The probes or probe sets are selected in such a way that they can be used in combination with at least one other probe, preferably also originating from the 16S-23S rRNA spacer region, under the same hybridization and wash conditions, to allow possibly the simultaneous detection of several micro-organisms in a sample.
It is also an aim of the present invention to provide for a selection method for use in the selection of said spacer probes or probe sets.
It is also an aim of the present invention to provide a rapid and reliable hybridization method for detection and identification of at least one micro-organism in a sample, or for the simultaneous detection and identification of several micro-organisms in a sample.
It is more particularly an aim of the present invention to provide a hybridization method allowing simultaneous detection and identification of a set of micro-organisms, liable to be present in a particular type of sample.
It is more particularly an aim of the present invention to provide probes or sets of probes for the possible simultaneous detection of micro-organisms in a sample originating from respiratory tract.
It is another particular aim of the present invention to provide probes or sets of probes for the possible simultaneous detection of micro-organisms in a sample originating from cerebrospinal fluid.
It is still another particular aim of the present invention to provide probes or sets of probes for the possible simultaneous detection of micro-organisms in a sample originating from urogenital tract.
It is still another particular aim of the present invention to provide probes or sets of probes for the possible simultaneous detection of micro-organisms in a sample taken from the gastro-intestinal tract of a patient.
It is still another particular aim of the present invention to provide probes or sets of probes for the possible simultaneous detection of micro-organisms in a sample originating from food or environmental samples.
It is moreover an aim of the present invention to provide a method for detection and identification of a particular taxon in a sample, or a set of particular taxa, said taxon being either a complete genus, or a subgroup within a genus, a species or even subtypes within a species (subspecies, serovars, sequevars, biovars . . . ).
It is more particularly an aim of the present invention to provide probes or sets of probes for the detection of
Mycobacterium
species and subspecies, more particularly for the detection of
M. tuberculosis
complex strains.
Mycobacterium
strains from the MAIS-complex,
M. avium
and
M. paratuberculosis, M. intracellulare
and
M. intracellulare
-like strains,
M. scrofulaceum, M. kansasii, M. chelonae, M. gordonae, M. ulcerans, M. gena
Jannes Geert
Rossau Rudi
Van Heuverswyn Hugo
Horlick Kenneth R.
Innogenetics N.V.
Nixon & Vanderhye P.C
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