Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1996-12-23
2000-02-15
Horlick, Kenneth R.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 912, 536 2432, 536 2433, C12Q 168, C12P 1934, C07H 2104
Patent
active
060251328
DESCRIPTION:
BRIEF SUMMARY
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-O 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 simultaneous
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Jannes Geert
Rossau Rudi
Van Heuverswyn Hugo
Horlick Kenneth R.
Innogenetics N.V.
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