Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-05-01
2004-08-10
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S471000, C536S023100
Reexamination Certificate
active
06773882
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to compositions and methods for detecting one or more species of yeast in the genus Candida. More specifically, the invention relates to hybridization probes and accessory polynucleotides having specificity for ribosomal nucleic acids from a defined collection of Candida species.
BACKGROUND OF THE INVENTION
Candida spp. are frequently present as members of the normal flora of the mouth, throat, large intestine, vagina, and skin and are often contaminates in exudates or other specimens taken from these areas. In patients whose immune defenses have been compromised by disease or by the secondary effects of drugs used to treat their diseases, microorganisms that are part of the normal flora may invade deeper tissues and produce life-threatening infections.
Candida albicans
(
C. albicans
), the principal pathogenic species, causes mild to severe or chronic superficial infections of the skin, nails and mucous membranes in individuals with normal immune defenses, as well as serious systemic infections in debilitated patients.
Candida parapsilosis, C. tropicalis
, and
C. guilliermondii
have become important causes of endocarditis, pyelonephritis, arthritis, and disseminated candidaisis in patients with indwelling intravenous catheters, patients undergoing cardiovascular surgery, and drug addicts. (see
Manual of Clinical Microbiology
, 4th Edition, (1985) Lennette et al. (eds.), American Association for Microbiology, Washington, D.C., p. 535)
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), thymine (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 substantially cross react with nucleic acids from other fungal species or infectious agents under appropriate stringency conditions.
The present invention provides polynucleotide probes that can be used to detect
Candida albicans, Candida tropicalis, Candida dubliniensis, Candida viswanathii
and
Candida parapsilosis
in a highly specific manner.
SUMMARY OF THE INVENTION
A first aspect of the invention relates to an oligonucleotide having a sequence that is up to 100 nucleotides in length, and that includes at least 26 contiguous nucleotides contained within the sequence of SEQ ID NO:7 or the complement thereof. Preferably, the sequence of the oligonucleotide includes any one of SEQ ID NO:1 or the complement thereof, SEQ ID NO:2 or the complement thereof, SEQ ID NO:3 or the complement thereof, SEQ ID NO:4 or the complement thereof, SEQ ID NO:5 or the complement thereof, and SEQ ID NO:6 or the complement thereof. Even more preferably, the length of the oligonucleotide is up to 60 nucleotides. According to one embodiment of the invention, the oligonucleotide is made of DNA. According to a different embodiment, the oligonucleotide includes at least one nucleotide analog. For example, the nucleotide analog may have a methoxy group at the 2′ position of a ribose moiety. According to yet another embodiment, the sequence of the oligonucleotide consists of any one of SEQ ID NO:1 or the complement thereof, SEQ ID NO:2 or the complement thereof, SEQ ID NO:3 or the complement thereof, SEQ ID NO:4 or the complement thereof, SEQ ID NO:5 or the complement thereof, and SEQ ID NO:6 or the complement thereof. When this is the case, the oligonucleotide may further include a detectable label. Alternatively, the sequence of the oligonucleotide may be given by either SEQ ID NO:1 or SEQ ID NO:5. In a preferred embodiment, the oligonucleotide further includes a detectable label, which may be a chemiluminescent label. A particular example of a chemiluminescent label would be an acridinium ester.
A second aspect of the invention relates to a composition that is useful for detecting the nucleic acids of a yeast that is any of
C. albicans, C. tropicalis, C. dubliniensis, C. viswanathii
and
C. parapsilosis
. The invented composition includes an oligonucleotide probe having a length of up to 100 nucleotide bases and a sequence that includes the sequence of SEQ ID NO:1 or the complement thereof. According to one embodiment, the length of the oligonucleotide probe is up to 60 nucleotides. Preferably, the oligonucleotide probe includes a detectable label. According to another embodiment, the oligonucleotide probe is made of DNA. According to still another embodiment, the sequence of the oligonucleotide probe is given by SEQ ID NO:1. When this is the case, the oligonucleotide probe may further include a detectable label, such as a chemiluminescent label or a radiolabel. According to a preferred embodiment, when the sequence of the oligonucleotide is given by SEQ ID NO:1, and when the oligonucleotide includes a detectable label, the detectable label may be a chemiluminescent label or a radiolabel. According to a highly preferred embodiment, the detectable label is a chemiluminescent label, and the chemiluminescent label is an acridinium ester. Alternatively, when the sequence of the oligonucleotide is given by SEQ ID NO:1, and when the oligonucleotide includes a detectable label, the composition may further include at least one helper oligonucleotide.
Gordon Patricia C.
Hogan James J.
Gen-Probe Incorporated
Gilly Michael J.
Katcheves Konstantina
Ketter James
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