Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-02-02
2004-04-27
Sitton, Jehanne Souaya (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C536S023100, C536S024300, C536S024320
Reexamination Certificate
active
06727061
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to a method for distinguishing among bacteria within the same taxonomic group based on the reactivity of specific 16S subsequences found within the ribosomal operons of the organisms, using probes during hybridization under conditions of increasing severity (stringency). Hybridization is the process whereby two strands of nucleic acid can interact and, if sufficiently matched in sequence, form a double-stranded structure. By the term probe is meant a marked, single-stranded nucleic acid sequence that is complementary to the nucleic acid sequences to be detected (target sequences). The use of this method of operon analysis for distinguishing the genera Escherichia from Shigella and for distinguishing among species of Shigella is demonstrated together with nucleic acid probes needed for conducting the analyses.
BACKGROUND OF THE INVENTION AND DESCRIPTION OF THE INVENTION
The terms “
Escherichia coli,” “Shigella boydii,” “Shigella dysenteriae,” “Shigella flexneri
,” and “
Shigella sonnei
” refer to the bacteria classified as such in Bergey's Manual of Determinative Bacteriology, 8th Edition, R. D. Buchanan and N. E. Gibbons, Eds., William & Wilkins, 1974, pp. 290-339. Unless specified otherwise the term Shigella will refer collectively to the four species mentioned above.
Detection of Shigella is important for medical diagnosis, public health surveillance, food safety, and other applications. Cases of Shigella, which must be identified by species, are required to be reported to the Centers for Disease Control and Prevention, which tracks the incidence and prevalence of Shigella in every state in the United States and in the District of Columbia. Current methods of detection are neither simple, straightforward, nor absolute (J. J. Farmer, III and M. T. Kelly, Enterobacteriaceae, in A. Balows, Ed, Fifth Edition, Manual of Clinical Microbiology, Washington, D.C., American Society for Microbiology, 1991.)
Suspected colonies usually are grown on both MacConkey agar and xylose-lysine-deoxycholate agar. Colonies of Shigella missed on one medium may show up on the other. Many laboratories also use Hektoen enteric agar. Enrichment of less than optimum cultures may require GN broth. Selenite broth may be useful for isolating
S. sonnei
. Suspected colonies of Shigella require confirmation by culture on other types of growth media such as triple sugar iron or Kliger iron agar slants. Colonies that show an alkaline/acid reaction with no H
2
S or gas then must be screened further by serological analysis with antisera in order to identify each of the species of Shigella.
Even with these procedures, differentiating strains of Shigella from
Escherichia coli
has proved to be one of the most difficult problems for a clinical microbiology laboratory. Recommended guidelines are complicated by exceptions related to one or more Shigella species. The difficulties inherent in distinguishing these organisms often forces investigators to depend merely on the fact that two Shigella species (
S. boydii
and
S. flexneri
) are not as prevalent in the United States, although a significant number of cases do occur, as are the other two species in order to help solidify their diagnoses. Still, the guidelines conclude with the realization that no definitive rules on the identification of Shigella isolates can be made and complete biochemical and serological typing must be done in each instance.
It is yet another aspect of the invention to avoid the disadvantages associated with the traditional culturing and serological identification techniques and to employ nucleic acid probes to distinguish
Escherichia coli
from Shigella and to identify each of its four associated species.
Efforts to circumvent the difficult, expensive, and time-consuming procedures with a simple yet rapid molecular procedure for differentiating the genus Shigella from the genus Escherichia and for separately identifying each of the four individual species of Shigella has also proved difficult. This has been attributed, in particular, to the very close relatedness of
E. coli
and all four Shigella species by DNA-DNA hybridization (J. J. Farmer, III and M. T. Kelly, Enterobacteriaceae, in A. Balows, Ed, Fifth Edition, Manual of Clinical Microbiology, Washington, D.C., American Society for Microbiology, 1991.)
Kyriaki Parados and Janice McCarty (U.S. Pat. No. 5,648,481) have identified a set of nucleic acid probes for detection of the genus Shigella and/or
E. coli
(EIBC) based on specific chromosomal sequences and fragments of Shigella. These probes can neither distinguish between
E. coli
and Shigella nor can they separately distinguish between one species of Shigella and another. In addition, the relatively large probes (approximately 40 nucleotides each) require hybridization overnight followed by exposure for 15 hours to x-ray film to produce autoradiographs.
Alessio Fasano, Myron M. Levine, James P. Nataro, and Fernando Noriega (U.S. Pat. No. 5,589,380) targeted the enterotoxins of
Shigella flexneri
2a and produced antibodies to the same, which might be useful primarily for the identification of
S. flexneri
. Kyriaki Parodos, Hsien-Yeh Hsu, Daid Sobell, Janice M. McCarty, and David J. Lane (U.S. Pat. No. 5,084,565) devised probes capable of hybridizing to rRNA of both
E. coli
and Shigella species but unable to discriminate among them. Phillippe Sansonetti, Catherine Boileau and Hélène D'Hauteville (U.S. Pat. No. 4,992,364) targeted the 140 MDalton virulence plasmid of
S. flexneri
. Their probes are relatively large, ranging in size from about 11.5 kbases to 27 kbases, and identify only combined strains of Shigella and
E. coli
carrying the virulence plasmid. Long-term hybridization (overnight) is followed by 6 hours of exposure to produce autoradiographs.
A subsequence of ribosomal RNA (rRNA) or its gene presents a potential target for separate identification of
E. coli
and each of the four species of Shigella through hybridization with appropriate DNA or RNA probes. Portions of rRNA have been found not to be conserved among diverse bacterial species, making them potential hybridization targets for distinguishing between one taxonomic group and another. David E. Kohne (U.S. Pat. No. 5,601,984) discusses such a method for detecting and quantitating organisms. But Kohne does not provide the teaching necessary to make Shigella species-specific probes.
Furthermore, Kohne does not teach how to distinguish among very closely related organisms using probes where a subsequence of a rRNA subunit or rRNA subunit gene is not specific to the taxonomic group (qualitative difference) but rather occurs as multiple but slightly differentiated copies in different proportions among multiple operons for the RNA genes (quantitative difference). An operon is defined as a group of contiguous genes that are coordinately regulated by controlling elements. Nor does Kohne teach the use of probes of sequence specific neither to genus nor species or other taxonomic grouping.
The
E. coli
chromosome is circular and contains seven operons for rRNA (FIG.
1
). A typical rRNA (rrn) operon contains two promoters and genes for 16S, 23S, and 5S rRNA and a single 4S tRNA gene (FIG.
2
). When analyzed, the 16S genes of the different
E. coli
rrn operons have been found to have regions where the sequences have been altered through mutations (Table 1). In some operons the mutations are the same in one particular region and in other operons they are different. Other organisms such as Shigella may either have a different number of operons, different types of operons, a different proportion of a particular mutation in one or more of its operons, one or more mutations in its operons distinct from
E. coli
or all of these possibilities.
FIG. 1
is an illustration of the ribosomal RNA operon on the
E. coli
chromosome. Each line marks the relative positions of one of the seven rrn operons found on the
E. coli
chromosome.
FIG. 2
is an illustration of a ribosomal RNA operon.
TABLE 1
Subsequence Variation i
Cabtec, Inc.
Millen White Zelano & Branigan P.C.
Sitton Jehanne Souaya
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