Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-02-04
2003-04-22
Jones, W. Gary (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S024320, C536S024300, C536S023100
Reexamination Certificate
active
06551776
ABSTRACT:
The invention relates to oligonucleotides for the detection and visualization of bacteria belonging to the genomic species
Escherichia coli
in a sample. More particularly, it relates to an oligonucleotide capable of hybridizing specifically with the ribosomal RNA (rRNA) or to the corresponding gene (rDNA) of the genomic species
Escherichia coli
(including Shigellae with the exception of
S. boydii
serotype 13)/
Escherichia fergusonii.
It likewise relates to a procedure for detection of the genomic species in question employing this oligonucleotide as well as the use of said oligonucleotide in a gene amplification procedure.
In this document, the term “
Escherichia coli
” (
E. coli
) denotes the genomic species (genomospecies) containing the strain-type
Escherichia coli
ATCC 11775 (=CIP 58-8). A genomic species is a collection of strains whose deoxyribonucleic acid (DNA) has a homology of more than 70% with the DNA of the strain-type of the species considered with a thermal instability of the hybridized DNA of lower than 5°0 C. (Grimont, 1988; Wayne et al., 1987). Following these criteria, the genomic species
E. coli
includes, apart from the strains usually identified as
E. coli
, the strains traditionally classed as Shigella (
S. dysenteriae, S. flexneri, S. boydii, S. sonnei
) with the exception of the serotype 13 of
S. boydii
(Brenner et al., 1973). In strictly applying these criteria, it is possible to argue that
Escherichia fergusonii
belongs to the genomic species
E. coli
(Farmer et al., 1985).
E. coli
is usually a commensal bacterium of the colon of man and of warm-blooded animals. For this reason, its presence in a sample of water, of food, or from the environment, is interpreted as an indication of fecal contamination (indicative bacterium). Thus, an alimentary product must not contain more than a certain number of living cells of
E. coli
(being able to form a colony on a solid culture medium) in a defined mass of product (these numbers vary according to the product). For example, drinking water must not contain any living cell of
E. coli
in 100 ml (De Zuane, 1997). The counting of the
E. coli
is essential in order to estimate the hygienic quality of a food.
Strains of the genomic species
E. coli
can be pathogenic. Among these strains is found any which is commonly called Shigella, the agent of bacillary human dysentery. The strains commonly called
E. coli
can cause different infections in man or in animals according to the provision with pathogenic genes (urinary infections, choleriform or hemorrhagic diarrhea, dysentery syndrome, hemolytic and uremic syndrome, septicemia, neonatal meningitis, various purulent infections).
The identification of a strain of the genomic species
E. coli
(taxonomic identification) is important in order to question or demonstrate the fecal contamination of water or food. It is likewise important in the case where the bacterium is isolated in a normally sterile or almost sterile biological medium (urine, blood, cerebrospinal fluid, collection of fluid in a tissue or in a closed space of the body). In the open spaces of the body (digestive tract) or the feces, the presence of
E. coli
is commonplace and the identification of pathogenic factors of
E. coli
is of paramount importance in the taxonomic identification.
The taxonomic identification of
E. coli
is conventionally based on the isolation and the culture of the bacterium on a solid gelatinous medium and the application of some biochemical tests. The appearance of colonies on a gelatinous medium requires at least 18 hours. In the case of samples from the environment, culture for some days is often necessary in order that all the colonies which ought to develop appear. The application of biochemical tests starting from an isolated colony again requires 18 to 48 hours. By way of example, the counting of
E. coli
in water necessitates the filtration of a volume of water through a sterile membrane, the placing of the membrane on a semi-selective and/or indicator medium, incubation (48 hours) allowing colonies of a characteristic (but not absolutely specific) color to develop, which are then counted. As each isolated colony is supposed to be derived from a bacterial cell, the counting of the
E. coli
by volume units can be carried out. It is wise to check that the isolated colonies indeed correspond to the species
E. coli
and this requires at least 18 hours more.
Recently, techniques based on the detection of specific nucleotide sequences of the genomic species
E. coli
have been described. Thus, the detection by gene amplification (PCR type) of the gene encoding beta-glucuronidase allows the presence of
E. coli
in a sample to be identified. This method is especially used qualitatively and the interpretation of the gene amplification is frequently hampered by the possibility of contamination due to the dispersion on the apparatus and experimental tools of some nucleic acid fragments.
In situ hybridization is an interesting alternative in gene amplification. An oligonucleotide probe which is labeled (generally by a fluorescent substance) penetrates into the previously treated bacterial cells in order to facilitate this step. According to whether the ribosomal nucleic acids have or do not have a complementary (target) sequence to the probe, the probe will fix to its target and will not be removed by washing. The bacteria having retained the probe in this way become labeled (for example fluorescent) and visible by microscopic examination.
The ribosomal ribonucleic acids (rRNA) form the preferred target in hybridization in situ because of the number of copies per cell (10,000 to 30,000), which is higher than the number of copies of messenger RNA after induction (100 to 200) or of a given gene (one to several). These ribosomal RNAs (rRNAs) are identified according to their sedimentation constant (for the bacteria: 5S, 16S and 23S), present in the small subunit (16S rRNA) or the large subunit (23S and 5S RNA) of the ribosome.
The largest rRNAs are the 16S (approximately 1500 nucleotides) and the 23S (approximately 3000 nucleotides). A complementary nucleic probe of a part of an rRNA would be able to hybridize with this rRNA but also with the complementary strand of the gene (rDNA) which has encoded this rRNA. Various applications of this methodology have been published (Amann et al., 1990; DeLong et al., 1989; Giovannoni et al., 1988; Trebesius et al., 1994).
These rRNAs in fact appeared as the most appropriate molecules to serve as a molecular chronometer in the evolution of bacteria (Brenner et al., 1969; Doi & Iragashi, 1965; Moore and McCarthy, 1967; Pace & Campbell, 1971; Takahashi et al., 1967). The primary structure (sequence) of the rRNAs contains highly conserved regions and others which are hypervariable (Sogin et al., 1972; Woese et al., 1975). The perfection of a DNA-rRNA hybridization method (Gillespie & Spiegelman, 1965) has been followed by a very large number of publications applying this approach to the taxonomy and the phylogeny of bacteria and to the identification of badly classified bacteria (Johnson et al., 1970; Palleroni et al., 1973; De Smedt & De Ley, 1977).
Generally speaking, in a hybridization experiment bringing into play given sequences, the result depends greatly on the temperature and on the molarity of sodium ions of the reaction medium. For a reaction medium of given composition, an optimal hybridization temperature is defined. If the temperature is increased, the reassociated strands will finish by separating. The temperature necessary for this separation depends on the length of the perfectly hybridized (apparently perfect) part of the sequence and on its nucleotide composition. A temperature only allowing hybridization of the longest sequences is called restrictive (in opposition to optimal). Mispairings during hybridization make the thermal stability of the hybridized molecules fall.
The specificity of the hybridization in situ will therefore depend on the quality of the probe capable of recognizing and of hybri
Collin Monique
Grimont Patrick
Regnault Béatrice
Einsmann Juliet
Institut Pasteur
Jones W. Gary
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