Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1997-08-07
2001-04-24
Campbell, Eggerton A. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S022100
Reexamination Certificate
active
06221582
ABSTRACT:
The present invention relates to the use of the GTPase gene family as a target for nucleic acid based assays for the detection and differentiation of prokaryotic organisms.
The present invention relates to polynucleic acids derived from gene sequences encoding prokaryotic GTPase (=GTP-binding) proteins, as well as their use in the detection and identification of prokaryotic organisms; primers and probes derived from said polynucleic acid sequences, for specific amplification and detection of prokaryotic DNA in a biological sample; as well as methods and kits allowing the detection and identification of at least one micro-organism, and preferentially several micro-organisms simultaneously in a sample.
More specifically, the invention relates to new polynucleic acid sequences encoding GTPase proteins from Campylobacter species, primers and probes derived from them, and methods and kits comprising these reagents for the detection and differentiation of species belonging to the genus Campylobacter.
GTP-binding proteins (also called GTPases because of the GTP hydrolysis that they catalize) constitute a large family of proteins that all have a similar GTP-binding globular domain. When its bound GTP is hydrolysed to GDP, this domain undergoes a conformational change that inactivates the protein. It is well known that GTP-binding proteins in pro- and eukaryotic organisms show conserved structures and contain common amino acid sequence motifs at their GTP-binding sites (for a review see: Bourne et al., 1991). Although GTP-binding sites show a conserved motif at the amino acid level, sequences are only partially conserved at the nucleic acid level. Prokaryotic GTP-binding proteins have been amply described in literature, and often they are responsable for vital molecular functions in the cell, e.g. they function as elongation and initiation factors in protein synthesis (e.g. for
E. coli
; Zengel et al., 1984; March and Inouye, 1985; Laursen et al., 1981; Sacerdot et al., 1984) and they may have a role in protein translocation across membranes (e.g. for
E. coli;
Bernstein et al., 1989; Römisch et al., 1989; Gill et al. 1986). Some prokaryotic GTP-binding proteins have a still unknown function, like the era-protein from
E. coli
(Ahnn et al. 1986) or the spoOB-associated protein from
Bacillus subtilis
(Trach and Hoch, 1989).
It is an aim of the present invention to use the GTPase gene family as a target for nucleic acid based assays for the detection and differentiation of prokaryotic organisms.
It is an aim of the present invention to provide polynucleic acids derived from the GTPase gene family to be used for the detection and identification of one or several micro-organisms simultaneously in a sample. The use of the polynucleic acids of the invention can be embodied by the use as an oligonucleotide primer, capable of amplifying the prokaryotic polynucleic acids present in the sample, or by the use as an oligonucleotide probe, capable of hybridizing specifically to the polynucleic acids of the different micro-organisms present in the sample.
It is therefore an aim of the present invention to select primer sequences derived from the polynucleic acid sequences encoding prokaryotic GTPase proteins, said primer sequences allowing amplification of part of a GTPase gene of one micro-organism, or of several micro-organisms simultaneously.
It is a more specific aim of the present invention to select primer sequences from the polynucleic acid sequences encoding the GTP-binding sites, said primer sequences allowing the simultaneous amplification of part of a GTPase gene of several micro-organisms.
Another aim of the present invention is to select probe sequences from the polynucleic acid sequences encoding prokaryotic GTPase proteins, said probe sequences allowing specific detection and identification of one or several micro-organisms.
It is a more specific aim of the present invention to select probe sequences from the polynucleic acid sequences encoding the GTP-sites enclosed region, said probe sequences allowing the specific detection and differentiation of several micro-organisms.
It is also an aim of the present invention to provide a rapid and reliable method for the detection and identification of one or several micro-organisms simultaneously in a sample, using the above-mentioned polynucleic acids and/or oligonucleotides derived from them as principle reagents.
It is moreover an aim of the present invention to provide a kit enabling the detection and identification of one or several micro-organisms simultaneously in a sample, comprising the above-mentioned polynucleic acids and/or oligonucleotides derived from them as principle reagents.
In particular, it is an aim of the present invention to provide new polynucleic acid sequences encoding novel GTP-binding proteins, or part of them, from Campylobacter species.
It is more specifically an aim of the present invention to use said new GTPase genes as a target for the detection and differentiation of Campylobacter species.
It is in particular an aim of the present invention to provide for oligonucleotides, primers and probes, derived from said new polynucleic acid sequences, to be used in the specific detection and differentiation of Campylobacter species.
It is also a specific aim of the present invention to provide for a method and a kit enabling the detection and differentiation of Campylobacter species in a sample, and using the above-mentioned new polynucleic acids and/or oligonucleotides derived from them, as principle reagents.
All of these aims are achieved by the polynucleic acids of this invention.
The current invention makes use of the semi-conserved nature of the GTPase gene family, a property which proved to be advantageous for use in nucleic acid based assays applied to bacterial detection, differentiation and identification. Said nucleic acid based assays consist of amplification of the target sequences and/or hybridization to the target sequences. It is shown in the current invention that both universal as well as specific oligonucleotides can be derived from the polynucleic acid sequences encoding GTPases. Both kinds of oligonucleotides have their proper place in bacterial diagnosis, depending on the application.
The term “GTPase gene family” encompasses genes encoding GTPase (GTP binding) proteins (E.C.3.6.1.) in different prokaryotic organisms. These GTPase proteins are structurally related, especially at their GTP-binding sites which show conserved sequence motifs, but they may have different functions in vivo. Although GTPase proteins show a structural relatedness on the protein level, the relatedness on the DNA level may be much less clear.
The term “universal” oligonucleotide (probe and/or primer) signifies that this oligonucleotide hybridizes to and/or allows amplification of part of the GTPase genes from different taxa.
The term “specific” oligonucleotide (probe and/or primer) means that this oligonucleotide hybridizes to and/or allows amplification of part of the GTPase genes from only one taxon.
The term “taxon” may refer to a complete genus, or a subgroup within a genus, a species, or even a subtype within a species.
Depending on the application, the term “universal” oligonucleotide may thus refer to an oligonucleotide allowing the amplification of and/or the hybridization to part of the GTP-ase genes of most of the organisms of one genus, while, in that same application, the term “specific” oligonucleotide may refer to oligonucleotides allowing the amplification of and/or the hybridization to part of the the GTPase gene(s) of only one species of that genus (species-specific oligonucleotides).
The current invention shows that “universal” oligonucleotides may be derived from the polynucleic acid sequences encoding the GTP-binding sites of GTPase proteins, while “specific” oligonucleotides may be derived from the polynucleic acids sequences encoding the GTP-sites enclosed region. It is shown in the current invention that the polynucleic acid sequences encoding the “GTP-sites enclosed regions” are suffic
Giesendorf Belinda
Quint Wilhelmus
Van Doorn Leendert-Jan
Campbell Eggerton A.
Howrey Simon Arnold & White , LLP
Innogenetics N.V.
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