Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-05-03
2001-12-04
Myers, Carla J. (Department: 1655)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023700, C435S006120
Reexamination Certificate
active
06326486
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to nucleic acid detection systems. More specifically, the invention relates to polynucleotide probes having binding specificity for rRNA or rDNA of bacteria in the family Enterobacteriaceae.
BACKGROUND OF THE INVENTION
Enterobacteriaceae are Gram-negative, oxidase negative, rod-shaped bacteria that cause disease in a wide variety of animals. This family of bacteria, which includes approximately 29 genera, 107 named species and at least 50 unnamed species, is responsible for major economic losses in the veterinary and agricultural areas. Based on rRNA sequencing analysis, the Enterobacteriaceae have been placed in the Proteobacteria “subphyla” or “subdivision” of gamma, more particularly subgroup gamma-3. Significantly, bacteria in the family Enterobacteriaceae cause up to 50% of the nosocomial infections in the United States.
It is well established that two single strands of deoxyribonucleic acid (“DNA”) or ribonucleic acid (“RNA”) can associate or “hybridize” with one another to form a double-stranded structure having two strands held together by hydrogen bonds between complementary base pairs. The individual strands of nucleic acid are formed from nucleotides that comprise the bases: adenine (A), cytosine (C), thymidine (T), guanine (G), uracil (U) and inosine (I). In the double helical structure of nucleic acids, the base adenine hydrogen bonds with the base thymine or uracil, the base guanine hydrogen bonds with the base cytosine and the base inosine hydrogen bonds with adenine, cytosine or uracil. At any point along the chain, therefore, one may find the classical “Watson-Crick” base pairs A:T or A:U, T:A or U:A, and G:C or C:G. However, one may also find A:G, G:U and other “wobble” or mismatched base pairs in addition to the traditional (“canonical”) base pairs.
A double-stranded nucleic acid hybrid will result if a first single-stranded polynucleotide is contacted under hybridization-promoting conditions with a second single-stranded polynucleotide having a sufficient number of contiguous bases complementary to the sequence of the first polynucleotide. DNA/DNA, RNA/DNA or RNA/RNA hybrids may be formed under appropriate conditions.
Generally, a probe is a single-stranded polynucleotide having some degree of complementarity with the nucleic acid sequence that is to be detected (“target sequence”). Probes commonly are labeled with a detectable moiety such as a radioisotope, an antigen or a chemiluminescent moiety.
Descriptions of nucleic acid hybridization as a procedure for detecting particular nucleic acid sequences are given by Kohne in U.S. Pat. No. 4,851,330, and by Hogan et al., in U.S. Pat. Nos. 5,541,308 and 5,681,698. These references also describe methods for determining the presence of RNA-containing organisms in a sample which might contain such organisms. These procedures require probes that are sufficiently complementary to the ribosomal RNA (rRNA) of one or more non-viral organisms or groups of non-viral organisms. According to the method, nucleic acids from a sample to be tested and an appropriate probe are first mixed and then incubated under specified hybridization conditions. Conventionally, but not necessarily, the probe will be labeled with a detectable label. The resulting hybridization reaction is then assayed to detect and quantitate the amount of labeled probe that has formed duplex structures in order to detect the presence of rRNA contained in the test sample.
With the exception of viruses, all prokaryotic organisms contain rRNA genes encoding homologs of the procaryotic 5S, 16S and 23S rRNA molecules. In eucaryotes, these rRNA molecules are the 5S rRNA, 5.8S rRNA, 18S rRNA and 28S rRNA which are substantially similar to the prokaryotic molecules. Probes for detecting specifically targeted rRNA subsequences in particular organisms or groups of organisms in a sample have been described previously. These highly specific probe sequences advantageously do not cross react with nucleic acids from any other bacterial species or infectious agent under appropriate stringency conditions.
The present invention provides polynucleotide probes that can be used to detect members of the family Enterobacteriaceae in a highly specific manner.
SUMMARY OF THE INVENTION
One aspect of the present invention relates to an oligonucleotide probe that specifically hybridizes a nucleic acid target region characteristic of bacteria in the family Enterobacteriaceae under a high stringency hybridization condition to form a detectable probe:target duplex. The invented oligonucleotide probe has a length of up to 100 nucleotides and includes at least 17 contiguous nucleotides contained within the sequence of SEQ ID NO:10 or the complement thereof. The high stringency hybridization conditions may be provided by either: (a) 0.48 M sodium phosphate buffer, 0.1% sodium dodecyl sulfate, and 1 mM each of EDTA and EGTA, or (b) 0.6 M LiCl, 1% lithium lauryl sulfate, 60 mM lithium succinate and 10 mM each of EDTA and EGTA. In certain embodiments the oligonucleotide probe is made of DNA. In other embodiments, the oligonucleotide probe includes at least one nucleotide analog. For example, the oligonucleotide probe may include at least one nucleotide analog having a methoxy group at the 2′ position of a ribose moiety. In still other embodiments of the invention, the oligonucleotide probe has the sequence of any one of SEQ ID NO:1 or the complement thereof, SEQ ID NO:2 or the complement thereof, and SEQ ID NO:3 or the complement thereof. When the oligonucleotide has the sequence of either SEQ ID NO:2 or SEQ ID NO:3, the oligonucleotide is particularly useful as a helper oligonucleotide. Any of the disclosed oligonucleotides can include a detectable label which may be either a chemiluminescent label or a radiolabel. In a highly preferred embodiment, the oligonucleotide probe has the sequence given by SEQ ID NO:1. When this is the case, the oligonucleotide probe may further include a detectable label. This detectable label may be a chemiluminescent label, such as an acridinium ester.
Another aspect of the invention relates to a probe composition that can be used for detecting nucleic acids of bacteria in the family Enterobacteriaceae. This composition includes an oligonucleotide probe that hybridizes under a high stringency condition to an Enterobacteriaceae target region corresponding to
E. coli
rRNA nucleotide positions 1222-1303 to form a detectable probe:target duplex. The oligonucleotide probe has a length of up to 100 nucleotide bases and includes at least 17 contiguous nucleotides contained within the sequence of SEQ ID NO:10 or the complement thereof. Under the high stringency hybridization conditions the oligonucleotide probe specifically hybridizes nucleic acids that are present in
Citrobacter diversus, Citrobacter freundii, Enterobacter aerogenes, Enterobacter agglomerans, Enterobacter cloacae, Enterobacter fragilis, Enterobacter gergoviae, Escherichia coli, Escherichia fergusonii, Escherichia hermanii, Hafnia alvei, Klebsiella oxytoca, Klebsiella ozaenae, Klebsiella pneumoniae, Klebsiella rhinoscleromatis, Proteus mirabilis, Proteus penneri, Proteus vulgaris, Providencia alcalifaciens, Providencia rettgeri, Providencia stuartii, Salmonella enteritidis, Salmonella paratyphi, Salmonella typhi, Salmonella typhimurium, Serratia liquefaciens, Serratia marcescens, Shigella dysenteriae, Shigella sonnei, Yersinia enterocolitica, Yersinia intermedia
and
Yersinia pseudotuberculosis.
In certain embodiments the oligonucleotide probe is made of DNA. Examples of useful high stringency hybridization conditions include: (a) 0.48 M sodium phosphate buffer, 0.1% sodium dodecyl sulfate, and 1 mM each of EDTA and EGTA, and (b) 0.6 M LiCl, 1% lithium lauryl sulfate, 60 mM lithium succinate and 10 mM each of EDTA and EGTA. In a highly preferred embodiment, the oligonucleotide probe has the sequence of SEQ ID NO:1 or the complement thereof. Rather than having a length of 100 nucleotides, the invented oligonucleotide probe can have a length
Gordon Patricia
Hogan James J.
Gen-Probe Incorporated
Gilly Michael J.
Johannson Diana
Myers Carla J.
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