Simultaneous detection,identification and differentiation of eubacterial taxa using a hybridization assay
AssigneeInnogenetics N.V.
InventorsGeert Jannes, Rudi Rossau, Hugo Van Heuverswyn
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Abstract
The present invention relates to a method for detection and identification of at least one microorganism, or for the simultaneous detection of several microorganisms in a sample.
Claims53
Method for the detection and identification of at least one micro-organism, or for the simultaneous detection of several micro-organisms in a sample, comprising the steps of:
- (i) if need be releasing, isolating and/or concentrating the polynucleic acids from the micro-organism(s) to be detected in the sample;
- (ii) if need be amplifying the 16S-23S rRNA spacer region, or a part of it, from the micro-organism(s) to be detected, with at least one suitable primer pair;
- (iii) hybridizing the polynucleic acids of step (i) or (ii) with a set of probes comprising at least two probes under the same hybridization and wash conditions, with said probes being selected from the sequences of table 1a or equivalents thereof, and/or from taxon-specific probes derived from any of the spacer sequences as represented in FIGS. 1-103, with said taxon-specific probe being selected such that it is capable of hybridizing under the same hybridization and wash conditions as at least one of the probes of table 1a;
- (iv) detecting the hybrids formed in step (iii);
- (v) identification of the micro-organism(s) present in the sample from the differential hybridization signals obtained in step (iv).
Method according to claim i to detect and identify one or more Listeria strains in a sample, wherein step (iii) comprises hybridizing to at least one of the following probes:
- 97 (SEQ ID NO 39) LIS-ICG 1:
- CAAGTAACCGAGAATCATCTGAAAGTGAATC (SEQ ID NO 40) LMO-ICG 1:
- AAACAACCTTTACTTCGTAGAAGTAAATTGGTTAAG (SEQ ID NO 41) LMO-ICG 2:
- TGAGAGGTTAGTACTTCTCAGTATGTTTGTTC (SEQ ID NO 42) LMO-ICG 3:
- AGGCACTATGCTTGAAGCATCGC (SEQ ID NO 43) LIV-ICG 1:
- GTTAGCATAAATAGGTAACTATTTATGACACAAGTAAC LSE-ICG 1:
- AGTTAGCATAAGTAGTGTAACTATTTATGACACAAG (SEQ ID NO 212) LISP-ICG 1:
- CGTTTTCATAAGCGATCGCACGTT and most preferabiy to at least one of the following probes:
- (SEQ ID NO 39) LIS-ICG 1:
- CAAGTAACCGAGAATCATCTGAAAGTGAATC (SEQ ID NO 42) LMO-ICG 3:
- AGGCACTATGCTTGAAGCATCGC (SEQ ID NO 212) LISP-ICG 1:
- CGTTTTCATAAGCGATCGCACGTT or to equivalents of said probes,
- and/or to any probe derived from SEQ ID NO 116, 118, 119, 120, 121, 213, 214 or 215 provided said probe hybridizes specifically to Listeria species.
Composition comprising at least one of the probes or primers as defined in claims 1 to 45 and 51 to 53.
Probe as defined in any of claims 1 to 42 and 51.
Primer as defined in any of claims 43 to 45 and 52 to 53.
Reverse hybridization method comprising any of the probes as defined in claims 1 to 42 and 51 wherein said probes are immobilized on a known location on a solid support, more preferably on a membrane strip.
Kit for the detection and identification of at least one micro-organism, or the simultaneous detection and identification of several micro-organisms in a sample, comprising the following components:
- (i) when appropiate, at least one suitable primer pair to allow amplification of the 16S-23S rRNA spacer region, or a part of it;
- (ii) at least one of the probes as defined in claims 1 to 42 and 51;
- (iii) a buffer, or components necessary to produce the buffer, enabling a hybridization reaction between said probes and the polynucleic acids present in the sample, or the amplified products thereof;
- (iv) a solution, or components necessary for producing the solution, enabling washing of the hybrids formed under the appropiate wash conditions;
- (v) when appropiate, a means for detecting the hybrids resulting from the preceding hybridization.
Dependencies are inferred from the claim text.