Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-04-14
2004-03-16
Souaya, Jehanne (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C536S023100, C536S024300, C536S024320
Reexamination Certificate
active
06706472
ABSTRACT:
In the foodstuffs and pharmaceutical industries the contamination of products by pathogenic microorganisms by way of the raw materials used or during the production or packaging processes poses a major problem. Salmonellae are among the most serious pathogens transmitted to humans through foodstuffs. Since the detection and identification of Salmonellae by conventional microbiological detection processes is very time-consuming—at least five days are required for the increase in quantity and subsequent serotyping required by legal regulations (LMBG, FDA)—there is a great need for alternative rapid methods.
In recent years, a number of new methods have been developed for routine use to detect microorganisms. These include immunological processes based on the use of polyvalent or monoclonal antibodies and processes in which nucleic acid probes are used for detection by means of hybridisation to organism-specific nucleic acids. Further methods that have been described are those processes based on a specific nucleic acid amplification, with or without a subsequent confirmation reaction by nucleic acid hybridisation. Suitable processes for the amplification of nucleic acids are, for example, polymerase chain reaction [PCR] [U.S. Pat. Nos. 4,683,195; 4,683,202; and 4,965,188], ligase chain reaction [WO Publication 89/09835], “self-sustained sequence replication” [EP 329 822], the “transcription based amplification system” [EP 310 229] and the Q&bgr; RNA-replicase system [U.S. Pat. No. 4,957,858].
The mentioned nucleic-acid-based processes are so sensitive that, unlike conventional microbiological processes, a lengthy increase in quantity of the microorganism to be detected from the sample to be investigated is unnecessary. An investigation of the presence or absence of, for example, Salmonellae is therefore generally concluded within one working day when using the mentioned nucleic-acid-based processes.
Some nucleic acid sequences for detecting Salmonellae by polymerase chain reaction are known. A disadvantage is, however, that when using those nucleic acid sequences as primers in the polymerase chain reaction false positive results [WO 95/33854] or false negative results [WO 92/01056; WO 95/00664; WO 92/01056; WO 93/04202] occur. In other cases, only an insufficient number of strains of all 7 Salmonellae subspecies have been studied [WO 92/08805; WO 94/25597; DE 4337295], so that thus far it is unclear whether the nucleic acid sequences in question are suitable for detecting all Salmonella strains.
An advantage of, for example, the primers and probes described in International Patent Application WO 95/00664 is that they allow the highly selective detection of bacteria of the Salmonella genus without the occurrence of false positive results. A disadvantage when using the oligonucleotides according to WO 95/00664 in amplification processes such as polymerase chain reaction is, however, the fact that none of the described primer pairs enable detection of all the representatives of the 7 Salmonella subspecies. For example, when using the primers ST11/ST15, a number of representatives of subspecies IIIa (subsp.
arizonae
) are not detected, and when using the primers ST11/ST14 a number of representatives of subspecies I (subsp.
enterica
Serovar. Blockley) and of subspecies IIIa (subsp.
arizonae
) are not detected.
An aim of the invention described herein was to optimise the detection processes described in WO 95/00664 by finding nucleic acid sequences the use of which as primers and/or probes ensures as complete detection as possible of all the representatives of the Salmonella genus.
According to an embodiment, the problem underlying the invention is solved by a set of nucleic acid molecules by means of which, in a process for the detection of representatives of
Salmonella enterica
subsp.
enterica, salamae, arizonae, diarizonae, houtenae, bongori
and
indica
, all the representatives of those subspecies can be detected, the set being obtainable by
(a) obtaining or deriving a first nucleic acid molecule (nucleic acid molecule 1) in a manner known per se using a nucleic acid isolate of a representative of one of the mentioned
Salmonella enterica
subspecies, which first nucleic acid molecule is specifically suitable as primer or probe for the detection of that representative or of further or all representatives of that one
Salmonella enterica
subspecies and possibly also of representatives of further
Salmonella enterica
subspecies,
(b) obtaining or deriving a second nucleic acid molecule (nucleic acid molecule 2) in a manner known per se using a nucleic acid isolate of a different representative of one of the mentioned
Salmonella enterica
subspecies, which second nucleic acid molecule is specifically suitable as primer or probe for the detection of that representative or of further or all representatives of that different
Salmonella enterica
subspecies and possibly also of representatives of others of the mentioned Salmonella enterica subspecies, and
(c) unless it is already possible to detect all the representatives of the mentioned
Salmonella enterica
subspecies using the nucleic acid molecules obtainable according to (a) and (b), continuing to obtain or derive nucleic acid molecules according to (a) and/or (b) until all the representatives of the mentioned
Salmonella enterica
subspecies can be detected using the obtained or derived set of nucleic acid molecules.
A derived nucleic acid molecule may be a nucleic acid molecule that can be hybridised with the obtained nucleic acid molecule and that preferably has the same number of bases, possible hybridisation conditions being:
temperature≧25° C. and 1M NaCl concentration.
A derived nucleic acid molecule may be, for example, a nucleic acid molecule the sequence of which has been determined by computer design and that has subsequently been manufactured and obtained by chemical synthesis.
The solution to the problem underlying the invention can also be described as the provision of one or more nucleic acid molecule(s) Y (Z, . . . ), that(those) nucleic acid molecule(s) being characterised in that the use of that(those) nucleic acid molecule(s)—in addition to the use of a nucleic acid molecule (X)—in a process for the detection of bacteria of the Salmonella genus enables the detection also of Salmonella strains or Salmonella isolates that cannot be detected or can be detected only with relatively low sensitivity using the nucleic acid molecule (X).
The set of nucleic acid molecules according to the invention can be characterised in that the nucleic acid isolates comprise or are phylogenetically conserved base sequences or regions of those base sequences. For the term “phylogenetically conserved base sequence”, see, for example, WO 95/00664 or Herder's Lexikon der Biochemie und Molekularbiologie, supplemented 1995, page 132, spectrum, production, etc.
The set of nucleic acid molecules according to the invention can be characterised in that the individual nucleic acid molecules or some of the nucleic acid molecules hybridise to
(i) different phylogenetically conserved base sequences, or
(ii) one and the same phylogenetically conserved base sequence at non-overlapping sequence regions, or
(iii) one and the same phylogenetically conserved base sequence at overlapping sequence regions.
The set of nucleic acid molecules according to the invention or a set of nucleic acid molecules according to the invention by means of which, in a process for the detection of representatives of
Salmonella
enterica subsp.
enterica, salamae, arizonae, diarizonae, houtenae, bongori
and
indica
, all the representatives of those subspecies can be detected, can be characterised in that the set for an individual nucleic acid molecule, for a number of its individual nucleic acid molecules or for each of its individual nucleic acid molecules in each case comprises at least one further nucleic acid molecule that, in a region of at least 10 successive
Berghof Cornelia
Gasch Alexander
Scheu Pia
Wilborn Freimut
Biotecon Diagnostics GmbH
Frommer & Lawrence & Haug LLP
Santucci Ronald R.
Souaya Jehanne
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