Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-05-01
2002-12-17
Myers, Carla J. (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023740, C536S024320
Reexamination Certificate
active
06495327
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to compositions and methods for detecting the pathogenic yeast species,
Candida albicans
and
Candida dubliniensis.
More specifically, the invention relates to hybridization probes and accessory polynucleotides having specificity for the ribosomal nucleic acids of these species.
BACKGROUND OF THE INVENTION
The yeast
Candida albicans
(
C. albicans
) is one of the most common fungal pathogens that infect humans. Although it is part of the normal flora of the mucous membranes in the respiratory, gastrointestinal and female genital tracts, this opportunistic pathogen may gain dominance in these locations and result in disease conditions. Indeed, while
C. albicans
infections of otherwise healthy individuals are rarely fatal, infections can lead to life-threatening conditions in persons having comprised immune systems such as, for example, AIDS patients, cancer patients undergoing chemotherapy, and organ transplant patients receiving immunosuppressive drugs. Disseminated (systemic) candidiasis is most prevalent in immunosuppressed individuals and is usually established following passage of the organism across the mucosal epithelium into the bloodstream. Once in the bloodstream, this organism can cause thrombophlebitis, endocarditis, or infection of the eyes. Further,
C. albicans
can invade virtually any organ or tissue when introduced intravenously, e.g., via tubing, needles or narcotics abuse.
Candida is a heterogeneous genus of yeast. At least six Candida species have been implicated as human pathogens, although the majority of Candida infections are caused by
C. albicans
and
C. tropicalis.
Since proper diagnosis and early treatment of an infection by
C. albicans
provides a clear advantage with respect to medical treatment, it is highly desirable to have available methods of detecting a wide range of different strains of this organism.
The discovery of the novel opportunistic pathogen
Candida dubliniensis
has added to the range of organisms responsible for disease in humans (see Sullivan et al.,
Microbiology
141:1507 (1995)). Isolates of this yeast species have been primarily recovered from the oral cavities of HIV-infected patients, both with and without clinical symptoms of oral candidiasis. However,
C. dubliniensis
has also been recovered from the oral cavities of asymptomatic and symptomatic immunocompetent individuals, although to a much lesser extent. There are also a few reports of the isolation of
C. dubliniensis
from sputum, blood, fecal and vaginal specimens. In a study reported by Odds et al., in
J. Clin. Microbiol.
36:2869 (1998), about 2% of yeasts originally identified as
C. albicans
were reidentified as
C. dubliniensis
on the basis of DNA fingerprinting.
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 cross react with nucleic acids from any other bacterial species or infectious agents under appropriate stringency conditions.
The present invention provides polynucleotide probes that can be used to detect a broad range of
Candida albicans
strains and
Candida dubliniensis
in a highly specific manner.
SUMMARY OF THE INVENTION
A first aspect of the invention relates to an oligonucleotide that has a length of up to 100 nucleotides and a sequence that includes at least 17 contiguous nucleotides from SEQ ID NO:6 or the complement thereof. The sequence of the oligonucleotide may include 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, or SEQ ID NO:5 or the complement thereof. In each of these cases, rather than having a length of up to 100 nucleotides, the oligonucleotide may have a length of up to only 60 nucleotides. In certain embodiments, the oligonucleotide is made of DNA. In certain other embodiments of the invention the sequence of the oligonucleotide is given precisely by 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, and SEQ ID NO:5 or the complement thereof. When this is the case, the oligonucleotide optionally may include a detectable label. In a highly preferred embodiment, the sequence of the oligonucleotide is given by either of SEQ ID NO:1 or SEQ ID NO:4. In an even more highly preferred embodiment, the oligonucleotide which has the sequence of either SEQ ID NO:1 or SEQ ID NO:4 further includes a detectable label. Examples of detectable labels include chemiluminescent labels, such as acridinium esters.
A second aspect of the invention relates to a composition that can be used for detecting the nucleic acids of a yeast that is either
C. albicans
or
C. dubliniensis.
This composition includes an oligonucleotide probe having a length of up to 100 nucleotide bases and a sequence that includes at least 17 contiguous nucleotides from SEQ ID NO:6 or the complement thereof. In certain instances, rather than having a length of up t
Bee Gary G.
Hogan James J.
Milliman Curt L.
Gen-Probe Incorporated
Gilly Michael J.
Johannsen Diana
Myers Carla J.
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