Methods and compositions for detecting candida species

Abstract

To provide methods and compositions for detecting Candida species.SOLUTION: There are disclosed methods utilizing specific amplification of Candida sp. target nucleic acid for detecting the presence or absence of Candida sp. in a sample. Also there are disclosed corresponding oligomers, including amplification oligomers, capture probes and detection probes, and combinations thereof, as well as corresponding reaction mixtures and kits. In some embodiments of a method as above, for determining the presence or absence of Candida sp. in a sample as above, the method includes contacting the sample with both the first and second amplification oligomer combinations.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority under 35 U.S.C. § 119(e) to Provisional Application No. 62 / 274,610, filed January 4, 2016, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing Reference This application contains a Sequence Listing that has been submitted in ASCII format via EFS-Web and is incorporated herein by reference in its entirety. The ASCII copy, created on January 4, 2017, is entitled "DIA.0003.02(PCT)Seq Listing2_ST25" and is 26KB in size. [Background technology]

[0003] Vulvovaginal Candida (VVC) is sometimes called a "yeast infection." It is a common infection that occurs when there is an overgrowth of a yeast called Candida. Candida is always present in and on the body in small amounts. However, when an imbalance occurs, for example, when the normal acidity of the vagina changes or when hormone balance changes, Candida can grow. When this happens, symptoms of Candida can appear.

[0004] Women with VVC typically experience genital itching, a burning sensation, and sometimes a "cottage cheese-like" vaginal discharge. Men with genital Candida infection may experience a pruritic rash on the penis. The symptoms of VVC are similar to those of many other genital infections, so it is important to receive a proper diagnosis to ensure appropriate treatment.

[0005] There is currently no single standard for diagnosing VVC. Diagnosis must be based on a clinical evaluation of several signs, both from laboratory findings and the patient's condition. Diagnosis of yeast infection by physical examination alone can be difficult. Diagnosis is usually made by taking a sample of vaginal secretions and examining the sample under a microscope to determine whether an abnormal number of Candida organisms are present. Because Candida species are normal inhabitants of the body, fungal cultures may not always be useful.

[0006] Treatment of Candida infections is often postponed until the causative organism is identified because the antifungal drugs used are toxic to the host. However, if antifungal therapy is initiated promptly, the prognosis is greatly improved. Therefore, any treatment regimen focuses on the specificity and speed of diagnosing the infection. Therefore, there is a need for a rapid, sensitive, and specific test to help diagnose infections, such as Candidemia, caused by pathogenic yeasts.

[0007] It is therefore an object of the present invention to provide compositions, reaction mixtures, methods and kits for the specific, sensitive and rapid detection of Candida species in a sample. Summary of the Invention [Means for solving the problem]

[0008] In one aspect, the present invention provides a method for determining the presence or absence of Candida species in a sample. The method generally comprises: (1) contacting a sample suspected of containing Candida species with at least one of a first amplification oligomer combination and a second amplification oligomer combination; (a) a first amplification oligomer combination comprising first and second Candida-specific amplification oligomers for amplifying a first Candida species target nucleic acid region or a second Candida species target nucleic acid region, wherein the first target region corresponds to a region from about nucleotide 133 or 161 to about nucleotide 259 of SEQ ID NO: 129, and the second region corresponds to a region from about nucleotide 202 to about nucleotide 308 of SEQ ID NO: 130, and wherein the first and second Candida-specific amplification oligomers comprise first and second Candida-specific target hybridizing sequences, respectively; and (b) contacting a second amplification oligomer combination comprising first and second C. glabrata-specific amplification oligomers for amplifying a third Candida species target nucleic acid region, the third target region corresponding to a region from about nucleotide 355 to about nucleotide 554 of SEQ ID NO: 131, the first and second C. glabrata-specific amplification oligomers comprising first and second C. glabrata-specific target hybridizing sequences, respectively; (2) performing an in vitro nucleic acid amplification reaction, in which any Candida species target nucleic acid, if present in the sample, is used as a template to generate one or more amplification products corresponding to at least one of the first, second, and third target regions; (3) detecting the presence or absence of one or more amplification products, thereby determining the presence or absence of Candida species in the sample.

[0009] In some embodiments of the methods for determining the presence or absence of Candida species in a sample described above, the method comprises contacting the sample with both a first and a second combination of amplification oligomers. For example, in certain variations, the method is a multiplex method comprising contacting the sample with both a first and a second combination of amplification oligomers in the same reaction mixture.

[0010] In some embodiments of the above methods, the first Candida-specific target hybridizing sequence substantially corresponds to the nucleotide sequence of residues 28-46 of SEQ ID NO:9, and / or the second Candida-specific target hybridizing sequence is (i) a sequence comprising 15-24 contiguous nucleotides contained in SEQ ID NO:132 and at least the sequence of SEQ ID NO:133, (ii) a sequence comprising 20-23 contiguous nucleotides contained in SEQ ID NO:152 and at least the sequence of SEQ ID NO:151, or (iii) a sequence substantially corresponding to the nucleotide sequence of SEQ ID NO:34.

[0011] In some embodiments of the above method, the first C. glabrata-specific target hybridizing sequence is a sequence comprising 15 to 24 contiguous nucleotides contained in the sequence of SEQ ID NO: 134 and at least the sequence of SEQ ID NO: 135, and / or the second C. glabrata-specific target hybridizing sequence is a sequence comprising 16 to 21 contiguous nucleotides contained in the sequence of SEQ ID NO: 136 and at least the sequence of SEQ ID NO: 137.

[0012] In some embodiments of the above methods, the method further comprises contacting the sample with a third Candida-specific amplification oligomer, wherein the first and second Candida-specific amplification oligomers are for amplifying a first Candida species target nucleic acid region, and the second Candida-specific target hybridizing sequence is a sequence comprising 15 to 24 contiguous nucleotides contained in the sequence of SEQ ID NO: 134 and at least the sequence of SEQ ID NO: 135, and the first and third Candida-specific amplification oligomers are for amplifying a second Candida species target region, and the third Candida-specific amplification oligomer comprises a third Candida-specific target hybridizing sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 34. In some such embodiments, the third Candida-specific target hybridizing sequence comprises or consists of the nucleotide sequence of SEQ ID NO: 34.

[0013] In some embodiments of the above methods, the first Candida-specific target hybridizing sequence comprises the nucleotide sequence of residues 28-46 of SEQ ID NO:9, the second Candida-specific target hybridizing sequence comprises the nucleotide sequence of SEQ ID NO:26, residues 3-22 of SEQ ID NO:74, or the nucleotide sequence of SEQ ID NO:34, the first C. glabrata-specific target hybridizing sequence comprises the nucleotide sequence of residues 28-49 of SEQ ID NO:14, and / or the second C. glabrata-specific target hybridizing sequence comprises the nucleotide sequence of SEQ ID NO:12. In more specific variations, the first Candida-specific target hybridizing sequence consists of the nucleotide sequence of residues 28-46 of SEQ ID NO:9, the second Candida-specific target hybridizing sequence consists of the nucleotide sequence of SEQ ID NO:26, SEQ ID NO:74, or SEQ ID NO:34, the first C. glabrata-specific target hybridizing sequence consists of the nucleotide sequence of residues 28-49 of SEQ ID NO:14, and / or the second C. glabrata-specific target hybridizing sequence consists of the nucleotide sequence of SEQ ID NO:12.

[0014] In some embodiments of the methods for determining the presence or absence of Candida species in a sample described above, at least one of the first Candida-specific amplification oligomer and the first C. glabrata-specific amplification oligomer is a promoter primer or promoter provider that further comprises a promoter sequence located 5' to the respective target hybridizing sequence. A particularly suitable promoter sequence is a T7 promoter sequence, such as the T7 promoter sequence having the sequence set forth in residues 1-27 of SEQ ID NO:9. In some such embodiments, the first Candida-specific amplification oligomer has the nucleotide sequence of SEQ ID NO:9 and / or the first C. glabrata-specific amplification oligomer has the nucleotide sequence of SEQ ID NO:14.

[0015] Typically, methods for determining the presence or absence of Candida species further include purifying any Candida species target nucleic acid, if present, from other components in the sample prior to step (2). In certain embodiments, the purification step includes contacting the sample with at least one capture probe oligomer comprising a target-hybridizing sequence covalently attached to a sequence or moiety that binds to the immobilized probe. In some such variations, the sample is contacted with a first Candida-specific capture probe oligomer and a first C. glabrata-specific capture probe oligomer, wherein the first Candida-specific capture probe oligomer comprises a first Candida-specific capture probe target hybridizing sequence that specifically hybridizes to a target sequence within a first or second Candida species target nucleic acid, and the first C. glabrata-specific capture probe oligomer comprises a first C. glabrata capture probe target hybridizing sequence that specifically hybridizes to a target sequence within a third Candida species target nucleic acid, and wherein each of the first Candida-specific and C. glabrata-specific capture probe target hybridizing sequences is covalently linked to a sequence or moiety that binds to an immobilized probe. Particularly suitable target hybridizing sequences for the first Candida-specific capture probe include (i) 16 to 21 contiguous nucleotides contained within the sequence of SEQ ID NO: 138 and comprising at least the sequence of SEQ ID NO: 139, or (ii) 15 to 25 contiguous nucleotides contained within the sequence of SEQ ID NO: 140 and comprising at least the sequence of SEQ ID NO: 141. Particularly suitable target hybridizing sequences for the first C. glabrata-specific capture probe include 16 to 27 contiguous nucleotides contained within the sequence of SEQ ID NO: 142 and comprising at least the sequence of SEQ ID NO: 143 or SEQ ID NO: 144.

[0016] In some embodiments in which the sample is contacted with a first Candida-specific capture probe oligomer, as described above, the method further comprises contacting the sample with a second Candida-specific capture probe oligomer. In some such variations, the first Candida-specific capture probe target hybridizing sequence comprises 16-21 contiguous nucleotides contained in SEQ ID NO: 138 and at least the sequence of SEQ ID NO: 139, and the second Candida-specific capture probe oligomer comprises a second Candida-specific capture probe target hybridizing sequence that specifically hybridizes to a target sequence within the first or second Candida species target nucleic acid, the second Candida-specific capture probe target hybridizing sequence comprising 15-25 contiguous nucleotides contained in SEQ ID NO: 140 and at least the sequence of SEQ ID NO: 141. In more specific variations, the second Candida-specific capture probe target hybridizing sequence comprises or consists of the nucleotide sequence of residues 1-17 of SEQ ID NO:66, and in some such embodiments, the second Candida-specific capture probe oligomer has the nucleotide sequence of SEQ ID NO:66.

[0017] In some embodiments in which the sample is contacted with a first Candida-specific capture probe oligomer and a first C. glabrata-specific capture probe oligomer, as described above, the first Candida-specific capture probe target hybridizing sequence comprises or consists of the nucleotide sequence of residues 1-20 of SEQ ID NO:24 or residues 1-17 of SEQ ID NO:66, and / or the first C. glabrata-specific capture probe target hybridizing sequence comprises or consists of the nucleotide sequence of residues 1-26 of SEQ ID NO:48. In more particular variations, the first Candida-specific capture probe oligomer has the nucleotide sequence of SEQ ID NO:24 or SEQ ID NO:66, and / or the first C. glabrata-specific capture probe oligomer has the nucleotide sequence of SEQ ID NO:48.

[0018] In certain embodiments, the detecting step (3) comprises contacting one or more amplification products with a Candida-specific detection probe that specifically hybridizes to the first or second Candida species target region and a C. glabrata-specific detection probe that specifically hybridizes to the third Candida species target region, and detecting the presence or absence of any target-hybridizing Candida-specific and / or C. glabrata-specific detection probe. Particularly suitable Candida-specific detection probes include probes comprising a Candida-specific detection probe target-hybridizing sequence selected from (A1) 18-22 contiguous nucleotides contained in the sequence of SEQ ID NO: 145 and a sequence comprising at least the sequence of SEQ ID NO: 146, (B1) a DNA equivalent or RNA / DNA chimera of (A1), and (C1) the exact complement of (A1) or (B1). Particularly suitable C. glabrata-specific detection probes include probes comprising a C. glabrata-specific detection probe target hybridizing sequence selected from (A2) a sequence comprising 17 to 23 consecutive nucleotides contained in the sequence of SEQ ID NO: 147 and at least the sequence of SEQ ID NO: 148, (B2) a sequence substantially corresponding to the sequence of residues 1 to 17 of SEQ ID NO: 18, (C2) a sequence substantially corresponding to the sequence of residues 1 to 20 of SEQ ID NO: 21, (D2) a DNA equivalent or RNA / DNA chimera of any one of (A2) to (C2), and (E2) a perfect complement of any one of (A2) to (D2). In some embodiments, the Candida-specific detection probe target hybridizing sequence comprises or consists of residues 1-22 of SEQ ID NO:27, its DNA equivalent or RNA / DNA chimera, or the complete complement of any of the foregoing, and / or the C. glabrata-specific detection probe target hybridizing sequence comprises or consists of residues 1-17 of SEQ ID NO:60, residues 1-23 of SEQ ID NO:45, residues 1-17 of SEQ ID NO:18, residues 1-20 of SEQ ID NO:21, the DNA equivalent or RNA / DNA chimera of any of the foregoing, or the complete complement of any of the foregoing.In more specific variations, the Candida-specific detection probe has the sequence of SEQ ID NO: 27, its DNA equivalent or RNA / DNA chimera, or the complete complement of any of the foregoing, and / or the C. glabrata-specific detection probe has the sequence of SEQ ID NO: 60, SEQ ID NO: 45, SEQ ID NO: 18, SEQ ID NO: 21, its DNA equivalent or RNA / DNA chimera, or the complete complement of any of the foregoing.

[0019] In some embodiments of methods utilizing detection probes, the Candida-specific and C. glabrata-specific detection probes each comprise at least one label. In certain variations, the label is a chemiluminescent or fluorescent label. In certain embodiments utilizing labeled detection probes, the detection step (3) occurs during the amplification step (2), and in some such embodiments, the Candida-specific and C. glabrata-specific detection probes each comprise a fluorescent label and a quencher. Suitable detection probes comprising a fluorescent label and a quencher include molecular torches, molecular beacons, and TaqMan detection probes.

[0020] In some embodiments of methods utilizing detection probes, at least one of the Candida-specific and C. glabrata-specific detection probes further comprises a non-target hybridizing sequence. For example, in some variations, each of the Candida-specific and C. glabrata-specific detection probes is a molecular torch or molecular beacon.

[0021] In certain variations of the above-described methods for determining the presence or absence of Candida species, the amplification reaction of step (2) is an isothermal amplification reaction, such as, for example, a transcription-mediated amplification (TMA) reaction. In some such embodiments, the amplification reaction is a real-time amplification reaction.

[0022] In another aspect, the present invention provides oligomer combinations for determining the presence or absence of Candida species in a sample, the oligomer combinations generally comprising at least one of a first amplification oligomer combination and a second amplification oligomer combination; (a) a first amplification oligomer combination comprising first and second Candida-specific amplification oligomers for amplifying a first Candida species target nucleic acid region or a second Candida species target nucleic acid region, wherein the first target region corresponds to a region from about nucleotide 133 or 161 to about nucleotide 259 of SEQ ID NO: 129, and the second region corresponds to a region from about nucleotide 202 to about nucleotide 308 of SEQ ID NO: 130, and wherein the first and second Candida-specific amplification oligomers comprise first and second Candida-specific target hybridizing sequences, respectively; and (b) a second amplification oligomer combination comprising first and second C. glabrata-specific amplification oligomers for amplifying a third Candida species target nucleic acid region, the third target region corresponding to a region from about nucleotide 355 to about nucleotide 554 of SEQ ID NO: 131, and the first and second C. glabrata-specific amplification oligomers comprising first and second C. glabrata-specific target hybridizing sequences, respectively;

[0023] In some embodiments of the oligomer combinations for determining the presence or absence of Candida species in a sample described above, the oligomer combination includes a combination of both a first and a second amplification oligomer. For example, in certain variations, the oligomer combination includes a combination of both a first and a second amplification oligomer in the same reaction mixture.

[0024] In some embodiments of the above oligomer combinations, the first Candida-specific target hybridizing sequence substantially corresponds to the nucleotide sequence of residues 28-46 of SEQ ID NO:9, and / or the second Candida-specific target hybridizing sequence is (i) a sequence comprising 15-24 contiguous nucleotides contained in SEQ ID NO:132 and at least the sequence of SEQ ID NO:133, (ii) a sequence comprising 20-23 contiguous nucleotides contained in SEQ ID NO:152 and at least the sequence of SEQ ID NO:151, or (iii) a sequence substantially corresponding to the nucleotide sequence of SEQ ID NO:34.

[0025] In some embodiments of the above oligomer combinations, the first C. glabrata-specific target hybridizing sequence is a sequence comprising 15 to 24 contiguous nucleotides contained in the sequence of SEQ ID NO: 134 and at least the sequence of SEQ ID NO: 135, and / or the second C. glabrata-specific target hybridizing sequence is a sequence comprising 16 to 21 contiguous nucleotides contained in the sequence of SEQ ID NO: 136 and at least the sequence of SEQ ID NO: 137.

[0026] In some embodiments of the above-described oligomer combinations, the oligomer combination further comprises a third Candida-specific amplification oligomer, wherein the first and second Candida-specific amplification oligomers are for amplifying a first Candida species target nucleic acid region, and the second Candida-specific target hybridizing sequence is a sequence comprising 15 to 24 contiguous nucleotides contained in the sequence of SEQ ID NO: 134 and at least the sequence of SEQ ID NO: 135, and the first and third Candida-specific amplification oligomers are for amplifying a second Candida species target region, and the third Candida-specific amplification oligomer comprises a third Candida-specific target hybridizing sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 34. In some such embodiments, the third Candida-specific target hybridizing sequence comprises or consists of the nucleotide sequence of SEQ ID NO: 34.

[0027] In some embodiments of the above oligomer combinations, the first Candida-specific target hybridizing sequence comprises the nucleotide sequence of residues 28-46 of SEQ ID NO:9, the second Candida-specific target hybridizing sequence comprises the nucleotide sequence of SEQ ID NO:26, residues 3-22 of SEQ ID NO:74, or the nucleotide sequence of SEQ ID NO:34, the first C. glabrata-specific target hybridizing sequence comprises the nucleotide sequence of residues 28-49 of SEQ ID NO:14, and / or the second C. glabrata-specific target hybridizing sequence comprises the nucleotide sequence of SEQ ID NO:12. In more specific variations, the first Candida-specific target hybridizing sequence consists of the nucleotide sequence of residues 28-46 of SEQ ID NO:9, the second Candida-specific target hybridizing sequence consists of the nucleotide sequence of SEQ ID NO:26, SEQ ID NO:74, or SEQ ID NO:34, the first C. glabrata-specific target hybridizing sequence consists of the nucleotide sequence of residues 28-49 of SEQ ID NO:14, and / or the second C. glabrata-specific target hybridizing sequence consists of the nucleotide sequence of SEQ ID NO:12.

[0028] In some embodiments of the oligomer combinations for determining the presence or absence of Candida species in a sample described above, at least one of the first Candida-specific amplification oligomer and the first C. glabrata-specific amplification oligomer is a promoter primer or promoter provider that further comprises a promoter sequence located 5' to the respective target hybridizing sequence. A particularly suitable promoter sequence is a T7 promoter sequence, such as the T7 promoter sequence having the sequence set forth in residues 1-27 of SEQ ID NO:9. In some such embodiments, the first Candida-specific amplification oligomer has the nucleotide sequence of SEQ ID NO:9 and / or the first C. glabrata-specific amplification oligomer has the nucleotide sequence of SEQ ID NO:14.

[0029] In certain embodiments, the oligomer combination further comprises at least one capture probe oligomer comprising a target-hybridizing sequence covalently linked to a sequence or moiety that binds to the immobilized probe. In some such variations, the oligomer combination comprises a first Candida-specific capture probe oligomer and a first C. glabrata-specific capture probe oligomer, wherein the first Candida-specific capture probe oligomer comprises a first Candida-specific capture probe target-hybridizing sequence that specifically hybridizes to a target sequence within the first or second Candida species target nucleic acid, and the first C. glabrata-specific capture probe oligomer comprises a first C. glabrata capture probe target-hybridizing sequence that specifically hybridizes to a target sequence within a third Candida species target nucleic acid, and wherein each of the first Candida-specific and C. glabrata-specific capture probe target-hybridizing sequences is covalently linked to a sequence or moiety that binds to the immobilized probe. Particularly suitable target hybridizing sequences for the first Candida-specific capture probe include (i) 16 to 21 contiguous nucleotides contained in the sequence of SEQ ID NO: 138 and comprising at least the sequence of SEQ ID NO: 139, or (ii) 15 to 25 contiguous nucleotides contained in the sequence of SEQ ID NO: 140 and comprising at least the sequence of SEQ ID NO: 141. Particularly suitable target hybridizing sequences for the first C. glabrata-specific capture probe include 16 to 27 contiguous nucleotides contained in the sequence of SEQ ID NO: 142 and comprising at least the sequence of SEQ ID NO: 143 or SEQ ID NO: 144.

[0030] In some embodiments in which the oligomer combination comprises a first Candida-specific capture probe oligomer, as described above, the oligomer combination further comprises a second Candida-specific capture probe oligomer. In some such variations, the first Candida-specific capture probe target hybridizing sequence comprises 16-21 contiguous nucleotides contained within SEQ ID NO: 138 and at least the sequence of SEQ ID NO: 139, and the second Candida-specific capture probe oligomer comprises a second Candida-specific capture probe target hybridizing sequence that specifically hybridizes to a target sequence within the first or second Candida species target nucleic acid, the second Candida-specific capture probe target hybridizing sequence comprising 15-25 contiguous nucleotides contained within SEQ ID NO: 140 and at least the sequence of SEQ ID NO: 141. In more specific variations, the second Candida-specific capture probe target hybridizing sequence comprises or consists of the nucleotide sequence of residues 1-17 of SEQ ID NO:66, and in some such embodiments, the second Candida-specific capture probe oligomer has the nucleotide sequence of SEQ ID NO:66.

[0031] As described above, in some embodiments in which the oligomer combination comprises a first Candida-specific capture probe oligomer and a first C. glabrata-specific capture probe oligomer, the first Candida-specific capture probe target hybridizing sequence comprises or consists of the nucleotide sequence of residues 1-20 of SEQ ID NO:24 or residues 1-17 of SEQ ID NO:66, and / or the first C. glabrata-specific capture probe target hybridizing sequence comprises or consists of the nucleotide sequence of residues 1-26 of SEQ ID NO:48. In more particular variations, the first Candida-specific capture probe oligomer has the nucleotide sequence of SEQ ID NO:24 or SEQ ID NO:66, and / or the first C. glabrata-specific capture probe oligomer has the nucleotide sequence of SEQ ID NO:48.

[0032] In certain embodiments, the above-described oligomer combinations further comprise a Candida-specific detection probe that specifically hybridizes to a first or second Candida species target region, and a C. glabrata-specific detection probe that specifically hybridizes to a third Candida species target region. Particularly suitable Candida-specific detection probes include probes comprising a Candida-specific detection probe target hybridizing sequence selected from: (A1) a sequence comprising 18-22 contiguous nucleotides contained in the sequence of SEQ ID NO: 145 and at least the sequence of SEQ ID NO: 146; (B1) a DNA equivalent or RNA / DNA chimera of (A1); and (C1) the exact complement of (A1) or (B1). Particularly suitable C. glabrata-specific detection probes include probes comprising a C. glabrata-specific detection probe target hybridizing sequence selected from (A2) a sequence comprising 17 to 23 consecutive nucleotides contained in the sequence of SEQ ID NO: 147 and at least the sequence of SEQ ID NO: 148, (B2) a sequence substantially corresponding to the sequence of residues 1 to 17 of SEQ ID NO: 18, (C2) a sequence substantially corresponding to the sequence of residues 1 to 20 of SEQ ID NO: 21, (D2) a DNA equivalent or RNA / DNA chimera of any one of (A2) to (C2), and (E2) a perfect complement of any one of (A2) to (D2). In some embodiments, the Candida-specific detection probe target hybridizing sequence comprises or consists of residues 1-22 of SEQ ID NO:27, its DNA equivalent or RNA / DNA chimera, or the complete complement of any of the foregoing, and / or the C. glabrata-specific detection probe target hybridizing sequence comprises or consists of residues 1-17 of SEQ ID NO:60, residues 1-23 of SEQ ID NO:45, residues 1-17 of SEQ ID NO:18, residues 1-20 of SEQ ID NO:21, the DNA equivalent or RNA / DNA chimera of any of the foregoing, or the complete complement of any of the foregoing.In more specific variations, the Candida-specific detection probe has the sequence of SEQ ID NO: 27, its DNA equivalent or RNA / DNA chimera, or the complete complement of any of the foregoing, and / or the C. glabrata-specific detection probe has the sequence of SEQ ID NO: 60, SEQ ID NO: 45, SEQ ID NO: 18, SEQ ID NO: 21, its DNA equivalent or RNA / DNA chimera, or the complete complement of any of the foregoing.

[0033] In some embodiments of oligomer combinations further comprising detection probes, the Candida-specific and C. glabrata-specific detection probes each comprise at least one label. In particular variations, the labels are chemiluminescent or fluorescent labels. In some embodiments, the Candida-specific and C. glabrata-specific detection probes each comprise a fluorescent label and a quencher. Suitable detection probes comprising a fluorescent label and a quencher include molecular torches, molecular beacons, and TaqMan detection probes.

[0034] In some embodiments of oligomer combinations further comprising a detection probe, at least one of the Candida-specific and C. glabrata-specific detection probes further comprises a non-target hybridizing sequence. For example, in some variations, each of the Candida-specific and C. glabrata-specific detection probes is a molecular torch or molecular beacon.

[0035] In another aspect, the present invention provides a detection probe for detecting a Candida species target nucleic acid, wherein the detection probe is a Candida-specific detection probe or a C. glabrata-specific detection probe described above. In certain embodiments, for example, the following are provided: (Item 1) 1. A method for determining the presence or absence of Candida species in a sample, comprising: (1) contacting a sample suspected of containing Candida species with at least one of a first amplification oligomer combination and a second amplification oligomer combination; (a) the first amplification oligomer combination comprises first and second Candida-specific amplification oligomers for amplifying a first Candida species target nucleic acid region or a second Candida species target nucleic acid region, wherein the first target region corresponds to a region from about nucleotide 133 or 161 to about nucleotide 259 of SEQ ID NO: 129, and the second region corresponds to a region from about nucleotide 202 to about nucleotide 308 of SEQ ID NO: 130, and the first and second Candida-specific amplification oligomers comprise first and second Candida-specific target hybridizing sequences, respectively; and (b) contacting the second amplification oligomer combination with a C. glabrata-specific amplification oligomer for amplifying a third Candida species target nucleic acid region, the third target region corresponding to a region from about nucleotide 355 to about nucleotide 554 of SEQ ID NO: 131, the first and second C. glabrata-specific amplification oligomers comprising first and second C. glabrata-specific target hybridizing sequences, respectively; (2) performing an in vitro nucleic acid amplification reaction, in which any Candida species target nucleic acid, if present in the sample, is used as a template to generate one or more amplification products corresponding to at least one of the first, second, and third target regions; (3) detecting the presence or absence of the one or more amplification products, thereby determining the presence or absence of Candida species in the sample. (Item 2) 2. The method of claim 1, wherein the method comprises contacting the sample with a combination of both the first and second amplification oligomers. (Item 3) 3. The method of claim 2, wherein the method is a multiplex method comprising contacting the sample with both the first and second amplification oligomer combinations in the same reaction mixture. (Item 4) the first Candida-specific target hybridizing sequence substantially corresponds to the nucleotide sequence of residues 28-46 of SEQ ID NO:9; and / or 4. The method of any one of Items 1 to 3, wherein the second Candida-specific target-hybridizing sequence is (i) a sequence comprising 15 to 24 consecutive nucleotides contained in the sequence of SEQ ID NO: 132 and at least the sequence of SEQ ID NO: 133, (ii) a sequence comprising 20 to 23 consecutive nucleotides contained in the sequence of SEQ ID NO: 152 and at least the sequence of SEQ ID NO: 151, or (iii) a sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 34. (Item 5) the first C. glabrata-specific target hybridizing sequence is a sequence comprising 15 to 24 consecutive nucleotides contained in the sequence of SEQ ID NO: 134 and at least the sequence of SEQ ID NO: 135; and / or 5. The method according to any one of Items 1 to 4, wherein the second C. glabrata-specific target-hybridizing sequence is a sequence comprising 16 to 21 consecutive nucleotides contained in the sequence of SEQ ID NO: 136 and at least the sequence of SEQ ID NO: 137. (Item 6) contacting the sample with a third Candida-specific amplification oligomer; the first and second Candida-specific amplification oligomers are for amplifying the first Candida species target nucleic acid region, and the second Candida-specific target hybridizing sequence is a sequence comprising 15 to 24 consecutive nucleotides contained in the sequence of SEQ ID NO: 134 and at least the sequence of SEQ ID NO: 135; 6. The method of claim 4 or 5, wherein the first and third Candida-specific amplification oligomers are for amplifying the second Candida species target region, and the third Candida-specific amplification oligomer comprises a third Candida-specific target-hybridizing sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 34. (Item 7) the first Candida-specific target-hybridizing sequence comprises the nucleotide sequence of residues 28 to 46 of SEQ ID NO:9; the second Candida-specific target-hybridizing sequence comprises the nucleotide sequence of SEQ ID NO:26, residues 3-22 of SEQ ID NO:74, or SEQ ID NO:34; the first C. glabrata-specific target hybridizing sequence comprises the nucleotide sequence of residues 28-49 of SEQ ID NO: 14; and / or 6. The method of claim 4 or 5, wherein the second C. glabrata-specific target hybridizing sequence comprises the nucleotide sequence of SEQ ID NO: 12. (Item 8) contacting the sample with a third Candida-specific amplification oligomer; the first and second Candida-specific amplification oligomers are for amplifying the first Candida species target nucleic acid region, and the second Candida-specific target hybridizing sequence is a sequence comprising 15 to 24 consecutive nucleotides contained in the sequence of SEQ ID NO: 134 and at least the sequence of SEQ ID NO: 135; 8. The method of any one of items 4, 5, and 7, wherein the first and third Candida-specific amplification oligomers are for amplifying the second Candida species target region, and the third Candida-specific amplification oligomer comprises a third Candida-specific target-hybridizing sequence comprising the nucleotide sequence of SEQ ID NO: 34. (Item 9) the first Candida-specific target-hybridizing sequence consists of the nucleotide sequence of residues 28 to 46 of SEQ ID NO:9; the second Candida-specific target-hybridizing sequence consists of the nucleotide sequence of SEQ ID NO:26, SEQ ID NO:74, or SEQ ID NO:34; the first C. glabrata-specific target hybridizing sequence consists of the nucleotide sequence of residues 28-49 of SEQ ID NO: 14; and / or 8. The method of any of items 4, 5, and 7, wherein the second C. glabrata-specific target hybridizing sequence consists of the nucleotide sequence of SEQ ID NO: 12. (Item 10) contacting the sample with a third Candida-specific amplification oligomer; the first and second Candida-specific amplification oligomers are for amplifying the first Candida species target nucleic acid region, and the second Candida-specific target hybridizing sequence is a sequence comprising 15 to 24 consecutive nucleotides contained in the sequence of SEQ ID NO: 134 and at least the sequence of SEQ ID NO: 135; 10. The method of any of items 4, 5, 7, and 9, wherein the first and third Candida-specific amplification oligomers are for amplifying the second Candida species target region, and the third Candida-specific amplification oligomer comprises a third Candida-specific target-hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO: 34. (Item 11) 9. The method of claim 6 or 8, wherein the second Candida-specific target hybridizing sequence comprises the nucleotide sequence of SEQ ID NO: 26. (Item 12) 11. The method of any one of items 6, 8, and 10, wherein the second Candida-specific target-hybridizing sequence consists of the nucleotide sequence of SEQ ID NO: 26. (Item 13) 13. The method of any one of items 1 to 12, wherein at least one of the first Candida-specific amplification oligomer and the first C. glabrata-specific amplification oligomer is a promoter primer or a promoter provider that further comprises a promoter sequence located 5' to the respective target hybridizing sequence. (Item 14) Item 14. The method of item 13, wherein the promoter sequence is a T7 promoter sequence. (Item 15) Item 15. The method of item 14, wherein the promoter sequence has the nucleotide sequence of residues 1 to 27 of SEQ ID NO:9. (Item 16) the first Candida-specific amplification oligomer has the nucleotide sequence of SEQ ID NO: 9; and / or Item 14. The method of item 13, wherein the first C. glabrata-specific amplification oligomer has the nucleotide sequence of SEQ ID NO: 14. (Item 17) 17. The method of any one of items 1 to 16, further comprising purifying any Candida species target nucleic acid, if present, from other components in the sample prior to step (2). (Item 18) 18. The method of claim 17, wherein the purifying step comprises contacting the sample with at least one capture probe oligomer comprising a target-hybridizing sequence covalently linked to a sequence or moiety that binds to an immobilized probe. (Item 19) contacting the sample with a first Candida-specific capture probe oligomer and a first C. glabrata-specific capture probe oligomer; the first Candida-specific capture probe oligomer comprises a first Candida-specific capture probe target-hybridizing sequence that specifically hybridizes to a target sequence within the first or second Candida species target nucleic acid; the first C. glabrata-specific capture probe oligomer comprises a first C. glabrata capture probe target-hybridizing sequence that specifically hybridizes to a target sequence within the third Candida species target nucleic acid; 19. The method of claim 18, wherein each of the first Candida-specific and C. glabrata-specific capture probe target-hybridizing sequences is covalently linked to a sequence or moiety that binds to the immobilized probe. (Item 20) the first Candida-specific capture probe target hybridizing sequence is (i) a sequence comprising 16 to 21 consecutive nucleotides contained in the sequence of SEQ ID NO: 138 and at least the sequence of SEQ ID NO: 139, or (ii) a sequence comprising 15 to 25 consecutive nucleotides contained in the sequence of SEQ ID NO: 140 and at least the sequence of SEQ ID NO: 141; and / or 20. The method of item 19, wherein the first C. glabrata-specific capture probe target hybridizing sequence is a sequence comprising 16 to 27 consecutive nucleotides contained in the sequence of SEQ ID NO: 142 and at least the sequence of SEQ ID NO: 143 or SEQ ID NO: 144. (Item 21) contacting the sample with a second Candida-specific capture probe oligomer; the first Candida-specific capture probe target hybridizing sequence is a sequence comprising 16 to 21 consecutive nucleotides contained in the sequence of SEQ ID NO: 138 and at least the sequence of SEQ ID NO: 139; 21. The method of claim 20, wherein the second Candida-specific capture probe oligomer comprises a second Candida-specific capture probe target hybridizing sequence that specifically hybridizes to a target sequence within the first or second Candida species target nucleic acid, and the second Candida-specific capture probe target hybridizing sequence is a sequence comprising 15 to 25 contiguous nucleotides contained in the sequence of SEQ ID NO: 140 and at least the sequence of SEQ ID NO: 141. (Item 22) the first Candida-specific capture probe target hybridizing sequence comprises the nucleotide sequence of residues 1-20 of SEQ ID NO:24 or residues 1-17 of SEQ ID NO:66; and / or 21. The method of claim 20, wherein the first C. glabrata-specific capture probe target hybridizing sequence comprises the nucleotide sequence of residues 1 to 26 of SEQ ID NO: 48. (Item 23) contacting the sample with a second Candida-specific capture probe oligomer; the first Candida-specific capture probe target hybridizing sequence is a sequence comprising 16 to 21 consecutive nucleotides contained in the sequence of SEQ ID NO: 138 and at least the sequence of SEQ ID NO: 139; 23. The method of claim 20 or 22, wherein the second Candida-specific capture probe oligomer comprises a second Candida-specific capture probe target hybridizing sequence that specifically hybridizes to a target sequence within the first or second Candida species target nucleic acid comprising the nucleotide sequence of residues 1-17 of SEQ ID NO:66. (Item 24) the first Candida-specific capture probe target hybridizing sequence consists of the nucleotide sequence of residues 1-20 of SEQ ID NO:24 or residues 1-17 of SEQ ID NO:66; and / or 23. The method of claim 20 or 22, wherein the first C. glabrata-specific capture probe target hybridizing sequence consists of the nucleotide sequence of residues 1 to 26 of SEQ ID NO: 48. (Item 25) contacting the sample with a second Candida-specific capture probe oligomer; the first Candida-specific capture probe target hybridizing sequence is a sequence comprising 16 to 21 consecutive nucleotides contained in the sequence of SEQ ID NO: 138 and at least the sequence of SEQ ID NO: 139; 25. The method of any one of items 20, 22, and 24, wherein the second Candida-specific capture probe oligomer comprises a second Candida-specific capture probe target-hybridizing sequence consisting of the nucleotide sequence of residues 1-17 of SEQ ID NO: 66. (Item 26) 24. The method of claim 21 or 23, wherein the first Candida-specific capture probe target hybridizing sequence comprises the nucleotide sequence of residues 1 to 20 of SEQ ID NO:24. (Item 27) 26. The method of any one of items 21, 23, and 25, wherein the first Candida-specific capture probe target hybridizing sequence consists of the nucleotide sequence of residues 1 to 20 of SEQ ID NO:24. (Item 28) the first Candida-specific capture probe oligomer has the nucleotide sequence of SEQ ID NO:24 or SEQ ID NO:66; and / or 25. The method of any one of items 20, 22, and 24, wherein the first C. glabrata-specific capture probe oligomer has the nucleotide sequence of SEQ ID NO: 48. (Item 29) 26. The method of any one of items 21, 23, and 25, wherein the second Candida-specific capture probe oligomer has the nucleotide sequence of SEQ ID NO: 66. (Item 30) The detecting step (3) contacting one or more amplification products with a Candida-specific detection probe that specifically hybridizes to the first or second Candida species target region and a C. glabrata-specific detection probe that specifically hybridizes to the third Candida species target region; and detecting the presence or absence of any target-hybridized Candida-specific and / or C. glabrata-specific detection probes. (Item 31) The Candida-specific detection probe (A1) a sequence comprising 18 to 22 consecutive nucleotides contained in the sequence of SEQ ID NO: 145 and at least the sequence of SEQ ID NO: 146; (B1) a DNA equivalent or RNA / DNA chimera of (A1), and (C1) a Candida-specific detection probe target hybridizing sequence selected from the group consisting of the perfect complement of (A1) or (B1); and / or The C. glabrata specific detection probe is (A2) a sequence comprising 17 to 23 consecutive nucleotides contained in the sequence of SEQ ID NO: 147 and at least the sequence of SEQ ID NO: 148; (B2) a sequence substantially corresponding to the sequence of residues 1 to 17 of SEQ ID NO: 18; (C2) a sequence substantially corresponding to residues 1 to 20 of SEQ ID NO: 21; (D2) A DNA equivalent or RNA / DNA chimera of any of (A2) to (C2), and (E2) The method of item 30, comprising a C. glabrata-specific detection probe target hybridizing sequence selected from the group consisting of the complete complement of any one of (A2) to (D2). (Item 32) the Candida-specific detection probe target hybridizing sequence is the sequence of residues 1 to 22 of SEQ ID NO: 27; its DNA equivalent or RNA / DNA chimera, and and / or comprising a sequence selected from the group consisting of the full complement of any of the foregoing. The C. glabrata specific detection probe target hybridizing sequence is the sequence of residues 1 to 17 of SEQ ID NO: 60; the sequence of residues 1 to 23 of SEQ ID NO: 45; the sequence of residues 1 to 17 of SEQ ID NO: 18; the sequence of residues 1 to 20 of SEQ ID NO: 21; DNA equivalents or RNA / DNA chimeras of any of the foregoing, and 32. The method of claim 31, comprising a sequence selected from the group consisting of the full complement of any of the foregoing. (Item 33) the Candida-specific detection probe target hybridizing sequence is the sequence of residues 1 to 22 of SEQ ID NO: 27; its DNA equivalent or RNA / DNA chimera, and and / or consisting of a sequence selected from the group consisting of the full complement of any of the foregoing. the C. glabrata-specific detection probe target hybridizing sequence is the sequence of residues 1 to 17 of SEQ ID NO: 60; the sequence of residues 1 to 23 of SEQ ID NO: 45; the sequence of residues 1 to 17 of SEQ ID NO: 18; the sequence of residues 1 to 20 of SEQ ID NO: 21; DNA equivalents or RNA / DNA chimeras of any of the foregoing, and 33. The method of item 31 or 32, wherein the sequence is selected from the group consisting of the full complement of any of the foregoing. (Item 34) the Candida-specific detection probe has a sequence selected from the group consisting of SEQ ID NO: 27, a DNA or RNA / DNA chimera thereof, and the exact complement of any of the foregoing; and / or 34. The method of any one of Items 31 to 33, wherein the C. glabrata-specific detection probe target hybridizing sequence has a sequence selected from the group consisting of SEQ ID NO: 60, SEQ ID NO: 45, SEQ ID NO: 18, SEQ ID NO: 21, a DNA or RNA / DNA chimera of any of the foregoing, and a perfect complement of any of the foregoing. (Item 35) 34. The method according to any one of items 30 to 33, wherein each of the Candida-specific and C. glabrata-specific detection probes comprises a label. (Item 36) 36. The method of claim 35, wherein the label is a chemiluminescent label or a fluorescent label. (Item 37) 36. The method of claim 35, wherein the detection step (3) occurs during the amplification step (2). (Item 38) 38. The method of claim 37, wherein each of the Candida-specific and C. glabrata-specific detection probes comprises a fluorescent label and a quencher. (Item 39) 39. The method of claim 38, wherein each of the Candida-specific and C. glabrata-specific detection probes is a molecular torch, a molecular beacon, or a TaqMan detection probe. (Item 40) 34. The method of any one of items 30 to 33, wherein at least one of the Candida-specific and C. glabrata-specific detection probes further comprises a non-target hybridizing sequence. (Item 41) 41. The method of claim 40, wherein each of the Candida-specific and C. glabrata-specific detection probes is a molecular torch or a molecular beacon. (Item 42) 42. The method according to any one of items 1 to 41, wherein the amplification reaction in step (2) is an isothermal amplification reaction. (Item 43) 43. The method of claim 42, wherein the amplification reaction is a transcription-mediated amplification (TMA) reaction. (Item 44) 44. The method of claim 42 or 43, wherein the amplification reaction is a real-time amplification reaction. (Item 45) 1. A combination of oligomers for determining the presence or absence of Candida species in a sample, said combination of oligomers comprising: comprising at least one of a first amplification oligomer combination and a second amplification oligomer combination; (a) the first amplification oligomer combination comprises first and second Candida-specific amplification oligomers for amplifying a first Candida species target nucleic acid region or a second Candida species target nucleic acid region, wherein the first target region corresponds to a region from about nucleotide 133 or 161 to about nucleotide 259 of SEQ ID NO: 129, and the second region corresponds to a region from about nucleotide 202 to about nucleotide 308 of SEQ ID NO: 130, and the first and second Candida-specific amplification oligomers comprise first and second Candida-specific target hybridizing sequences, respectively; and (b) a combination of oligomers, wherein the second amplification oligomer combination comprises first and second C. glabrata-specific amplification oligomers for amplifying a third Candida species target nucleic acid region, the third target region corresponding to a region from about nucleotide 355 to about nucleotide 554 of SEQ ID NO: 131, and the first and second C. glabrata-specific amplification oligomers comprise first and second C. glabrata-specific target hybridizing sequences, respectively. (Item 46) 46. The oligomer combination of item 45, wherein the oligomer combination comprises a combination of both the first and second amplification oligomers. (Item 47) 47. The oligomer combination of item 46, wherein the oligomer combination comprises both the first and second amplification oligomer combinations in the same reaction mixture. (Item 48) the first Candida-specific target hybridizing sequence substantially corresponds to the nucleotide sequence of residues 28-46 of SEQ ID NO:9; and / or 48. The oligomer combination according to any one of Items 45 to 47, wherein the second Candida-specific target-hybridizing sequence is (i) a sequence comprising 15 to 24 consecutive nucleotides contained in the sequence of SEQ ID NO: 132 and at least the sequence of SEQ ID NO: 133, (ii) a sequence comprising 20 to 23 consecutive nucleotides contained in the sequence of SEQ ID NO: 152 and at least the sequence of SEQ ID NO: 151, or (iii) a sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 34. (Item 49) the first C. glabrata-specific target hybridizing sequence is a sequence comprising 15 to 24 consecutive nucleotides contained in the sequence of SEQ ID NO: 134 and at least the sequence of SEQ ID NO: 135; and / or 49. The oligomer combination according to any one of Items 47 to 48, wherein the second C. glabrata-specific target-hybridizing sequence is a sequence comprising 16 to 21 consecutive nucleotides contained in the sequence of SEQ ID NO: 136 and at least the sequence of SEQ ID NO: 137. (Item 50) further comprising a third Candida-specific amplification oligomer; the first and second Candida-specific amplification oligomers are for amplifying the first Candida species target nucleic acid region, and the second Candida-specific target hybridizing sequence is a sequence comprising 15 to 24 consecutive nucleotides contained in the sequence of SEQ ID NO: 134 and at least the sequence of SEQ ID NO: 135; 50. The oligomer combination of claim 48 or 49, wherein the first and third Candida-specific amplification oligomers are for amplifying the second Candida species target region, and the third Candida-specific amplification oligomer comprises a third Candida-specific target-hybridizing sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 34. (Item 51) the first Candida-specific target-hybridizing sequence comprises the nucleotide sequence of residues 28 to 46 of SEQ ID NO:9; the second Candida-specific target-hybridizing sequence comprises the nucleotide sequence of SEQ ID NO:26, residues 3-22 of SEQ ID NO:74, or SEQ ID NO:34; the first C. glabrata-specific target hybridizing sequence comprises the nucleotide sequence of residues 28-49 of SEQ ID NO: 14; and / or 50. The oligomer combination according to item 48 or 49, wherein the second C. glabrata-specific target hybridizing sequence comprises the nucleotide sequence of SEQ ID NO: 12. (Item 52) further comprising a third Candida-specific amplification oligomer; the first and second Candida-specific amplification oligomers are for amplifying the first Candida species target nucleic acid region, and the second Candida-specific target hybridizing sequence is a sequence comprising 15 to 24 consecutive nucleotides contained in the sequence of SEQ ID NO: 134 and at least the sequence of SEQ ID NO: 135; 52. The combination of oligomers of any one of items 48, 49, and 51, wherein the first and third Candida-specific amplification oligomers are for amplifying the second Candida species target region, and the third Candida-specific amplification oligomer comprises a third Candida-specific target-hybridizing sequence comprising the nucleotide sequence of SEQ ID NO: 34. (Item 53) the first Candida-specific target-hybridizing sequence consists of the nucleotide sequence of residues 28 to 46 of SEQ ID NO:9; the second Candida-specific target-hybridizing sequence consists of the nucleotide sequence of SEQ ID NO:26, SEQ ID NO:74, or SEQ ID NO:34; the first C. glabrata-specific target hybridizing sequence consists of the nucleotide sequence of residues 28-49 of SEQ ID NO: 14; and / or 52. The oligomer combination according to any one of items 48, 49 and 51, wherein the second C. glabrata-specific target-hybridizing sequence consists of the nucleotide sequence of SEQ ID NO: 12. (Item 54) further comprising a third Candida-specific amplification oligomer; the first and second Candida-specific amplification oligomers are for amplifying the first Candida species target nucleic acid region, and the second Candida-specific target hybridizing sequence is a sequence comprising 15 to 24 consecutive nucleotides contained in the sequence of SEQ ID NO: 134 and at least the sequence of SEQ ID NO: 135; 54. The combination of oligomers of any of items 48, 49, 51, and 53, wherein the first and third Candida-specific amplification oligomers are for amplifying the second Candida species target region, and the third Candida-specific amplification oligomer comprises a third Candida-specific target-hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO: 34. (Item 55) 53. The oligomer combination according to item 50 or 52, wherein the second Candida-specific target-hybridizing sequence comprises the nucleotide sequence of SEQ ID NO: 26. (Item 56) 55. The oligomer combination of any one of items 50, 52, and 54, wherein the second Candida-specific target-hybridizing sequence consists of the nucleotide sequence of SEQ ID NO: 26. (Item 57) 57. The oligomer combination of any one of items 45 to 56, wherein at least one of the first Candida-specific amplification oligomer and the first C. glabrata-specific amplification oligomer is a promoter primer or a promoter provider, further comprising a promoter sequence located 5' to the respective target hybridizing sequence. (Item 58) 58. The oligomeric combination according to item 57, wherein the promoter sequence is a T7 promoter sequence. (Item 59) 59. The oligomeric combination according to item 58, wherein the promoter sequence has the nucleotide sequence of residues 1 to 27 of SEQ ID NO:9. (Item 60) the first Candida-specific amplification oligomer has the nucleotide sequence of SEQ ID NO: 9; and / or 60. The combination of oligomers described in Item 59, wherein the first C. glabrata-specific amplification oligomer has the nucleotide sequence of SEQ ID NO: 14. (Item 61) 61. The oligomer combination of any one of items 45 to 60, further comprising at least one capture probe oligomer comprising a target-hybridizing sequence covalently linked to a sequence or moiety that binds to the immobilized probe. (Item 62) the combination of oligomers comprises a first Candida-specific capture probe oligomer and a first C. glabrata-specific capture probe oligomer; the first Candida-specific capture probe oligomer comprises a first Candida-specific capture probe target-hybridizing sequence that specifically hybridizes to a target sequence within the first or second Candida species target nucleic acid; the first C. glabrata-specific capture probe oligomer comprises a first C. glabrata capture probe target-hybridizing sequence that specifically hybridizes to a target sequence within the third Candida species target nucleic acid; 62. The oligomeric combination of claim 61, wherein each of the first Candida-specific and C. glabrata-specific capture probe target-hybridizing sequences is covalently linked to a sequence or moiety that binds to the immobilized probe. (Item 63) the first Candida-specific capture probe target hybridizing sequence is (i) a sequence comprising 16 to 21 consecutive nucleotides contained in the sequence of SEQ ID NO: 138 and at least the sequence of SEQ ID NO: 139, or (ii) a sequence comprising 15 to 25 consecutive nucleotides contained in the sequence of SEQ ID NO: 140 and at least the sequence of SEQ ID NO: 141; and / or 63. The oligomer combination according to Item 62, wherein the first C. glabrata-specific capture probe target-hybridizing sequence is a sequence comprising 16 to 27 contiguous nucleotides contained in the sequence of SEQ ID NO: 142 and at least the sequence of SEQ ID NO: 143 or SEQ ID NO: 144. (Item 64) further comprising a second Candida-specific capture probe oligomer; the first Candida-specific capture probe target hybridizing sequence is a sequence comprising 16 to 21 consecutive nucleotides contained in the sequence of SEQ ID NO: 138 and at least the sequence of SEQ ID NO: 139; 64. The oligomer combination of item 63, wherein the second Candida-specific capture probe oligomer comprises a second Candida-specific capture probe target hybridizing sequence that specifically hybridizes to a target sequence within the first or second Candida species target nucleic acid, and the second Candida-specific capture probe target hybridizing sequence is a sequence comprising 15 to 25 contiguous nucleotides contained in the sequence of SEQ ID NO: 140 and at least the sequence of SEQ ID NO: 141. (Item 65) the first Candida-specific capture probe target hybridizing sequence comprises the nucleotide sequence of residues 1-20 of SEQ ID NO:24 or residues 1-17 of SEQ ID NO:66; and / or 64. The oligomer combination according to item 63, wherein the first C. glabrata-specific capture probe target hybridizing sequence comprises the nucleotide sequence of residues 1 to 26 of SEQ ID NO: 48. (Item 66) further comprising a second Candida-specific capture probe oligomer; the first Candida-specific capture probe target hybridizing sequence is a sequence comprising 16 to 21 consecutive nucleotides contained in the sequence of SEQ ID NO: 138 and at least the sequence of SEQ ID NO: 139; 66. The oligomer combination of claim 63 or 65, wherein the second Candida-specific capture probe oligomer comprises a second Candida-specific capture probe target hybridizing sequence that specifically hybridizes to a target sequence within the first or second Candida species target nucleic acid comprising the nucleotide sequence of residues 1-17 of SEQ ID NO: 66. (Item 67) the first Candida-specific capture probe target hybridizing sequence consists of the nucleotide sequence of residues 1-20 of SEQ ID NO:24 or residues 1-17 of SEQ ID NO:66; and / or 66. The oligomer combination according to item 63 or 65, wherein the first C. glabrata-specific capture probe target hybridizing sequence consists of the nucleotide sequence of residues 1 to 26 of SEQ ID NO: 48. (Item 68) further comprising a second Candida-specific capture probe oligomer; the first Candida-specific capture probe target hybridizing sequence is a sequence comprising 16 to 21 consecutive nucleotides contained in the sequence of SEQ ID NO: 138 and at least the sequence of SEQ ID NO: 139; 68. The oligomer combination of any one of items 63, 65, and 67, wherein the second Candida-specific capture probe oligomer comprises a second Candida-specific capture probe target-hybridizing sequence consisting of the nucleotide sequence of residues 1 to 17 of SEQ ID NO: 66. (Item 69) 67. The oligomer combination according to item 64 or 66, wherein the first Candida-specific capture probe target hybridizing sequence comprises the nucleotide sequence of residues 1 to 20 of SEQ ID NO: 24. (Item 70) 69. The oligomeric combination of any one of items 64, 66, and 68, wherein the first Candida-specific capture probe target hybridizing sequence consists of the nucleotide sequence of residues 1 to 20 of SEQ ID NO: 24. (Item 71) the first Candida-specific capture probe oligomer has the nucleotide sequence of SEQ ID NO:24 or SEQ ID NO:66; and / or 68. The combination of oligomers according to any one of items 63, 65, and 67, wherein the first C. glabrata-specific capture probe oligomer has the nucleotide sequence of SEQ ID NO: 48. (Item 72) 69. The combination of oligomers of any one of items 64, 66, and 68, wherein the second Candida-specific capture probe oligomer has the nucleotide sequence of SEQ ID NO: 66. (Item 73) 73. The oligomer combination of any one of items 45 to 72, further comprising a Candida-specific detection probe that specifically hybridizes to the first or second Candida species target region and a C. glabrata-specific detection probe that specifically hybridizes to the third Candida species target region. (Item 74) The Candida-specific detection probe (A1) a sequence comprising 18 to 22 consecutive nucleotides contained in the sequence of SEQ ID NO: 145 and at least the sequence of SEQ ID NO: 146; (B1) a DNA equivalent or RNA / DNA chimera of (A1), and (C1) a Candida-specific detection probe target hybridizing sequence selected from the group consisting of the perfect complement of (A1) or (B1); and / or The C. glabrata specific detection probe is (A2) a sequence comprising 17 to 23 consecutive nucleotides contained in the sequence of SEQ ID NO: 147 and at least the sequence of SEQ ID NO: 148; (B2) a sequence substantially corresponding to the sequence of residues 1 to 17 of SEQ ID NO: 18; (C2) a sequence substantially corresponding to residues 1 to 20 of SEQ ID NO: 21; 74. The oligomer combination of Item 73, comprising a C. glabrata-specific detection probe target-hybridizing sequence selected from the group consisting of: (D2) the DNA equivalent or RNA / DNA chimera of any of (A2) to (C2), and (E2) the perfect complement of any one of (A2) to (D2). (Item 75) the Candida-specific detection probe target hybridizing sequence is the sequence of residues 1 to 22 of SEQ ID NO: 27; its DNA equivalent or RNA / DNA chimera, and and / or comprising a sequence selected from the group consisting of the full complement of any of the foregoing. The C. glabrata specific detection probe target hybridizing sequence is the sequence of residues 1 to 17 of SEQ ID NO: 60; the sequence of residues 1 to 23 of SEQ ID NO: 45; the sequence of residues 1 to 17 of SEQ ID NO: 18; the sequence of residues 1 to 20 of SEQ ID NO: 21; DNA equivalents or RNA / DNA chimeras of any of the foregoing, and 75. The oligomeric combination according to item 74, comprising a sequence selected from the group consisting of the full complement of any of the foregoing. (Item 76) the Candida-specific detection probe target hybridizing sequence is the sequence of residues 1 to 22 of SEQ ID NO: 27; its DNA equivalent or RNA / DNA chimera, and and / or consisting of a sequence selected from the group consisting of the full complement of any of the foregoing. the C. glabrata-specific detection probe target hybridizing sequence is the sequence of residues 1 to 17 of SEQ ID NO: 60; the sequence of residues 1 to 23 of SEQ ID NO: 45; the sequence of residues 1 to 17 of SEQ ID NO: 18; the sequence of residues 1 to 20 of SEQ ID NO: 21; DNA equivalents or RNA / DNA chimeras of any of the foregoing, and 76. The oligomeric combination according to item 74 or 75, consisting of a sequence selected from the group consisting of the full complement of any of the foregoing. (Item 77) the Candida-specific detection probe has a sequence selected from the group consisting of SEQ ID NO: 27, its DNA equivalent or RNA / DNA chimera, and the exact complement of any of the foregoing; and / or 77. The oligomeric combination of any one of items 74 to 76, wherein the C. glabrata-specific detection probe target hybridizing sequence has a sequence selected from the group consisting of SEQ ID NO: 60, SEQ ID NO: 45, SEQ ID NO: 18, SEQ ID NO: 21, a DNA equivalent or RNA / DNA chimera of any of the foregoing, and a perfect complement of any of the foregoing. (Item 78) 77. The oligomeric combination according to any one of items 73 to 76, wherein each of the Candida-specific and C. glabrata-specific detection probes comprises a label. (Item 79) 79. The oligomeric combination according to item 78, wherein the label is a chemiluminescent or fluorescent label. (Item 80) 79. The oligomeric combination according to item 78, wherein each of the Candida-specific and C. glabrata-specific detection probes comprises a fluorescent label and a quencher. (Item 81) 81. The oligomeric combination according to item 80, wherein each of the Candida-specific and C. glabrata-specific detection probes is a molecular torch, a molecular beacon, or a TaqMan detection probe. (Item 82) 77. The oligomer combination according to any one of items 73 to 76, wherein at least one of the Candida-specific and C. glabrata-specific detection probes further comprises a non-target hybridizing sequence. (Item 83) 83. The oligomeric combination according to item 82, wherein each of the Candida-specific and C. glabrata-specific detection probes is a molecular torch or a molecular beacon. (Item 84) 1. A detection probe for detecting a Candida species target nucleic acid, said detection probe comprising: A Candida-specific detection probe, (A1) a sequence comprising 18 to 22 consecutive nucleotides contained in the sequence of SEQ ID NO: 145 and at least the sequence of SEQ ID NO: 146; (B1) a DNA equivalent or RNA / DNA chimera of (A1), and (C1) a Candida-specific detection probe comprising a Candida-specific detection probe target hybridizing sequence selected from the group consisting of the perfect complement of (A1) or (B1); or A C. glabrata-specific detection probe, (A2) a sequence comprising 17 to 23 consecutive nucleotides contained in the sequence of SEQ ID NO: 147 and at least the sequence of SEQ ID NO: 148; (B2) a sequence substantially corresponding to the sequence of residues 1 to 17 of SEQ ID NO: 18; (C2) a sequence substantially corresponding to residues 1 to 20 of SEQ ID NO: 21; (D2) A DNA equivalent or RNA / DNA chimera of any of (A2) to (C2), and (E2) A detection probe that is a C. glabrata-specific detection probe, comprising a C. glabrata-specific detection probe target hybridizing sequence selected from the group consisting of the complete complement of any one of (A2) to (D2). (Item 85) the Candida-specific detection probe target hybridizing sequence is the sequence of residues 1 to 22 of SEQ ID NO: 27; its DNA equivalent or RNA / DNA chimera, and a sequence selected from the group consisting of the full complement of any of the foregoing; or The C. glabrata specific detection probe target hybridizing sequence is the sequence of residues 1 to 17 of SEQ ID NO: 60; the sequence of residues 1 to 23 of SEQ ID NO: 45; the sequence of residues 1 to 17 of SEQ ID NO: 18; the sequence of residues 1 to 20 of SEQ ID NO: 21; DNA equivalents or RNA / DNA chimeras of any of the foregoing, and 85. The detection probe of item 84, comprising a sequence selected from the group consisting of the perfect complement of any of the foregoing. (Item 86) the Candida-specific detection probe target hybridizing sequence is the sequence of residues 1 to 22 of SEQ ID NO: 27; its DNA equivalent or RNA / DNA chimera, and consisting of a sequence selected from the group consisting of the full complement of any of the foregoing; or the C. glabrata-specific detection probe target hybridizing sequence is the sequence of residues 1 to 17 of SEQ ID NO: 60; the sequence of residues 1 to 23 of SEQ ID NO: 45; the sequence of residues 1 to 17 of SEQ ID NO: 18; the sequence of residues 1 to 20 of SEQ ID NO: 21; DNA equivalents or RNA / DNA chimeras of any of the foregoing, and 86. The detection probe according to item 84 or 85, consisting of a sequence selected from the group consisting of the perfect complement of any of the foregoing. (Item 87) the Candida-specific detection probe has a sequence selected from the group consisting of SEQ ID NO: 27, its DNA equivalent or RNA / DNA chimera, and the exact complement of any of the foregoing; or 87. The detection probe of any one of Items 84 to 86, wherein the C. glabrata-specific detection probe target hybridizing sequence has a sequence selected from the group consisting of SEQ ID NO: 60, SEQ ID NO: 45, SEQ ID NO: 18, SEQ ID NO: 21, a DNA equivalent or RNA / DNA chimera of any of the foregoing, and a perfect complement of any of the foregoing. (Item 88) 87. The detection probe according to any one of items 84 to 86, wherein the detection probe comprises a label. (Item 89) 89. The detection probe according to item 88, wherein the label is a chemiluminescent label or a fluorescent label. (Item 90) 89. The detection probe of claim 88, wherein the detection probe comprises a fluorescent label and a quencher. (Item 91) 91. The detection probe of item 90, wherein the detection probe is a molecular torch, a molecular beacon, or a TaqMan detection probe. (Item 92) 87. The detection probe according to any one of Items 84 to 86, wherein the detection probe further comprises a non-target hybridizing sequence. (Item 93) 93. The detection probe according to item 92, wherein the detection probe is a molecular torch or a molecular beacon.

[0036] These and other aspects of the present invention will become evident upon reference to the following detailed description of the invention.

[0037] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods and compositions are described. As used herein, the following terms and phrases have the meanings ascribed to them unless specified otherwise.

[0038] The terms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise.

[0039] As used herein, "Candida species" refers to at least one or more of C. albicans, C. parapsilosis, C. dubliniensis, C. tropicalis, and C. glabrata. As used herein with respect to an oligomer, "Candida-specific" refers to specificity for at least one or more of C. albicans, C. parapsilosis, C. dubliniensis, and C. tropicalis target nucleic acids. As used herein with respect to an oligomer, "C. glabrata-specific" refers to specificity for at least a C. glabrata target nucleic acid.

[0040] A "sample" includes any specimen that may contain Candida species, or components thereof (such as nucleic acids or fragments of nucleic acids). Samples include "biological samples," including any tissue or material from living or dead humans that may contain Candida species or components thereof (e.g., target nucleic acids derived therefrom), including, for example, vaginal swab samples, cervical brush samples, respiratory tissue or exudates (such as those obtained from bronchoscopy, bronchoalveolar lavage (BAL), or lung biopsies, sputum, saliva, peripheral blood, plasma, serum, lymph nodes, gastrointestinal tissue, feces, urine, semen, or other bodily fluids or materials). Biological samples may be treated to physically or mechanically disrupt tissue or cellular structures, thus releasing intracellular components into solutions that may further contain enzymes, buffers, salts, detergents, etc., which are used to prepare the biological sample for analysis using standard methods. Samples can also include processed samples, such as those obtained by passing the sample over or through a filtration device, or after centrifugation, or by attaching to a medium, matrix, or support.

[0041] "Nucleic acid" refers to a polymeric compound containing two or more covalently linked nucleosides, or nucleoside analogs with nitrogen-containing heterocyclic bases, or base analogs, where the nucleosides are linked together by phosphodiester or other linkages to form a polynucleotide. Nucleic acids include RNA, DNA, or chimeric DNA-RNA polymers, or oligonucleotides, and their analogs. The nucleic acid "backbone" can be composed of various linkages, including one or more of sugar-phosphodiester linkages, peptide nucleic acid linkages ("peptide nucleic acids" or PNAs, see PCT Publication WO 95 / 32305), phosphorothioate linkages, methylphosphonate linkages, or combinations thereof. The sugar moiety of a nucleic acid can be either ribose or deoxyribose, or similar compounds with known substitutions, such as 2'-methoxy and 2'-halogenated substitutions (e.g., 2'-F). The nitrogenous base may be a conventional base (A, G, C, T, U), its analogs (e.g., inosine, 5-methylisocytosine, isoguanine, The Biochemistry of the Nucleic Acids 5-36, Adams et al., ed., 11th ed., 1992; Abraham et al., 2007, BioTechniques 43:617-24), including derivatives of purine or pyrimidine bases (e.g., N 4 -methyldeoxyguanosine, deaza- or aza-purines, deaza- or aza-pyrimidines, pyrimidine bases with substituents at the 5 or 6 positions, purine bases with modified or replaced substituents at the 2, 6, and / or 8 positions, e.g., 2-amino-6-methylaminopurine, O 6 -methylguanine, 4-thio-pyrimidine, 4-amino-pyrimidine, 4-dimethylhydrazine-pyrimidine, and O 4These include 3-alkyl-pyrimidines, and pyrazolo-compounds, such as unsubstituted or 3-substituted pyrazolo[3,4-d]pyrimidines (U.S. Pat. Nos. 5,378,825, 6,949,367, and PCT WO 93 / 13121). Nucleic acids can contain "abasic" residues in which the backbone does not contain a nitrogenous base for one or more residues (U.S. Pat. No. 5,585,481). Nucleic acids can contain only conventional sugars, bases, and linkages found in RNA and DNA, or can contain conventional components and substitutions (e.g., nucleic acids containing conventional bases linked by a 2' methoxy backbone, or mixtures of conventional bases with one or more base analogs). Nucleic acids may include "locked nucleic acids" (LNAs), in which one or more nucleotide monomers have a bicyclic furanose unit locked in an RNA-mimicking sugar conformation that enhances hybridization affinity to complementary sequences of single-stranded RNA (ssRNA), single-stranded DNA (ssDNA), or double-stranded DNA (dsDNA) (Vester et al., Biochemistry 43:13233-41, 2004). Nucleic acids may also contain modified bases to alter the function or behavior of the nucleic acid, for example, the addition of a 3'-terminal dideoxynucleotide to block the addition of additional nucleotides to the nucleic acid. While synthetic methods for producing nucleic acids in vitro are well known in the art, nucleic acids can also be purified from natural sources using conventional techniques.

[0042] As used herein, the term "polynucleotide" refers to a nucleic acid strand. Throughout this application, nucleic acids are designated from the 5' to the 3' end. Standard nucleic acids, such as DNA and RNA, are typically synthesized "5' to 3'," i.e., by the addition of nucleotides to the 3' end of a growing nucleic acid.

[0043] As used herein, a "nucleotide" is a nucleic acid subunit consisting of a phosphate group, a 5-carbon sugar, and a nitrogenous base. The 5-carbon sugar found in RNA is ribose. In DNA, the 5-carbon sugar is 2'-deoxyribose. The term also includes analogs of such subunits, such as a methoxy group at the 2' position of ribose (2'-O-Me).

[0044] As used herein, "target nucleic acid" refers to the nucleic acid that contains the target sequence to be detected.Target nucleic acid can be DNA or RNA as described herein, and can be either single-stranded or double-stranded.Target nucleic acid can also contain other sequences other than the target sequence.

[0045] By "isolated" it is meant that the sample containing the target nucleic acid is removed from its natural environment, although the term does not imply any degree of purification.

[0046] As used herein, the term "target sequence" refers to a specific nucleotide sequence of a target nucleic acid to be detected. "Target sequence" includes a complexing sequence to which an oligonucleotide (e.g., a probe oligonucleotide, a priming oligonucleotide, and / or a promoter oligonucleotide) is complexed during the detection process (e.g., an amplification-based detection assay such as TMA or PCR). If the target nucleic acid is originally single-stranded, the term "target sequence" also refers to a sequence complementary to the "target sequence" present in the target nucleic acid. If the target nucleic acid is originally double-stranded, the term "target sequence" refers to both the sense (+) and antisense (-) strands. In selecting a target sequence, those skilled in the art will understand that a "unique" sequence should be selected so as to distinguish between unrelated or closely related target nucleic acids.

[0047] The term "target hybridizing sequence" is used herein to refer to a portion of an oligomer configured to hybridize with a target nucleic acid sequence. Preferably, the target hybridizing sequence is configured to specifically hybridize with the target nucleic acid sequence. Target hybridizing sequences can be, but are not necessarily, 100% complementary to the portion of the target sequence to which they are configured to hybridize. Target hybridizing sequences can also contain inserted, deleted, and / or substituted nucleotide residues relative to the target sequence. For example, when the target nucleic acid is multiple strains within a species, such as is the case with oligomers configured to hybridize with various Lactobacillus strains, the target hybridizing sequence may be less than 100% complementary to the target sequence. It is understood that there are other reasons for configuring a target hybridizing sequence to have less than 100% complementarity to the target nucleic acid.

[0048] As used herein with respect to a region of a Candida species nucleic acid, the term "targeting a sequence" refers to the process by which an oligonucleotide hybridizes to a target sequence in a manner that allows for detection as described herein. In one embodiment, the oligonucleotide is complementary to the targeted Candida species nucleic acid sequence and contains no mismatches. In another embodiment, the oligonucleotide is complementary to the targeted Candida species nucleic acid sequence but contains 1, 2, 3, 4, or 5 mismatches. Preferably, an oligonucleotide that hybridizes to a target nucleic acid sequence contains at least 10 to 50 or so nucleotides that are complementary to the target sequence. At least 10 to 50 or so is an inclusive range that includes 10, 50, and each integer therebetween. Preferably, the oligomer specifically hybridizes to the target sequence.

[0049] The term "configured to" refers to the actual arrangement of the polynucleotide sequence composition of a referenced oligonucleotide target hybridizing sequence. For example, an oligonucleotide configured to specifically hybridize to a target sequence has a polynucleotide sequence that specifically hybridizes to the referenced sequence under stringent hybridization conditions.

[0050] As used herein, the term "configured to specifically hybridize to" means that the target hybridizing region of an oligonucleotide is designed to have a polynucleotide sequence capable of targeting the sequence of the referenced Candida species target region. Such oligonucleotides are not limited to targeting only that sequence, but rather are useful as compositions in kits or methods for targeting Candida species target nucleic acids. The oligonucleotides are designed to function as components of an assay for the detection of Candida species from a sample and, therefore, are designed to target Candida species in the presence of other nucleic acids commonly found in test samples. "Specifically hybridizing to" does not mean exclusively hybridizing, as is understood in the art, since some small levels of hybridization with non-target nucleic acids may occur. Rather, "specifically hybridizing to" means that the oligonucleotide is configured to function in an assay that primarily hybridizes to the target, such that accurate detection of the target nucleic acid in a sample can be determined. The term "configured to" refers to the actual arrangement of the polynucleotide sequence configuration of the oligonucleotide target hybridizing sequence.

[0051] As used herein with respect to nucleic acids targeting Candida species, the term "fragment" refers to a piece of contiguous nucleic acid. In certain embodiments, the fragment comprises contiguous nucleotides from the non-coding RNA ribozyme that is the RNA component of Candida species RNAse P, or contiguous nucleotides of the Candida gene RPR1 that encodes the RNA ribozyme component of RNAse P, wherein the number of contiguous nucleotides in the fragment is less than that of the entire RNA ribozyme encoded by the RPR1 gene.

[0052] As used herein, the term "region" refers to a portion of a nucleic acid that is smaller than the entire nucleic acid. For example, if the reference nucleic acid is an oligonucleotide promoter primer, the term "region" can be used to refer to the smaller promoter portion of the entire oligonucleotide. Similarly, and by way of example only, if the nucleic acid is an RNA ribozyme encoded by the RPR1 gene, the term "region" can be used to refer to a smaller section of the nucleic acid, which is targeted by one or more oligonucleotides of the present invention. As another non-limiting example, if the reference nucleic acid is an amplicon, the term region can be used to refer to a smaller nucleotide sequence that is specified for hybridization by the target hybridization sequence of the probe.

[0053] The interchangeable terms "oligomer," "oligo," and "oligonucleotide" generally refer to nucleic acids having fewer than 1,000 nucleotide (nt) residues, including polymers ranging in size from about 5 nt residues at a lower limit and about 500-900 nt residues at an upper limit. In some embodiments, oligonucleotides range in size from about 12-15 nt at a lower limit and about 50-600 nt at an upper limit, while other embodiments range from about 15-20 nt at a lower limit and about 22-100 nt at an upper limit. Oligonucleotides can be purified from naturally occurring sources or synthesized using any of a variety of well-known enzymatic or chemical methods. The term "oligonucleotide" does not denote any specific function for the reagent, but rather is used generically to encompass all such reagents described herein. Oligonucleotides can serve a variety of different functions. For example, it may function as a primer if it is specific for and allows hybridization with a complementary strand, which can be further extended in the presence of a nucleic acid polymerase; it may function as a primer and provide a promoter if it contains a sequence recognized by an RNA polymerase and allows transcription (e.g., a T7 primer); and it may function to detect a target nucleic acid if it is capable of hybridizing to a target nucleic acid, or an amplicon thereof, and further provided with a detectable moiety (e.g., an acridinium-ester compound).

[0054] As used herein, an oligonucleotide "substantially corresponding" to a particular reference nucleic acid sequence means that the oligonucleotide is sufficiently similar to the reference nucleic acid sequence such that it has similar hybridization properties to the reference nucleic acid sequence, in that it will hybridize to the same target nucleic acid sequence under stringent hybridization conditions. Those skilled in the art will understand that a "substantially corresponding oligonucleotide" may differ from the reference sequence but still hybridize to the same target nucleic acid sequence. It is also understood that a first nucleic acid corresponding to a second nucleic acid includes its RNA and DNA counterparts, including its complement, unless the context clearly dictates otherwise. This variation in nucleic acid may be described in terms of the percentage of identical bases within the sequence or the percentage of perfectly complementary bases between a probe or primer and its target sequence. Thus, in certain embodiments, an oligonucleotide "substantially corresponds" to a reference nucleic acid sequence if these percentages of base identity or complementarity are between 100% and about 80%. In preferred embodiments, the percentage is between 100% and about 85%. In more preferred embodiments, the percentage is between 100% and about 90%. In other preferred embodiments, this percentage is between 100% and about 95%. Similarly, a nucleic acid or a region of an amplified nucleic acid may be referred to herein as corresponding to a reference nucleic acid sequence. Those skilled in the art will understand the various modifications to hybridization conditions that may be required for various percentages of complementarity to permit hybridization to a specific target sequence without causing unacceptable levels of nonspecific hybridization.

[0055] An "amplification oligomer" is an oligomer, at least the 3' end of which is complementary to a target nucleic acid, hybridizes to the target nucleic acid or its complement, and participates in a nucleic acid amplification reaction. An example of an amplification oligomer is a "primer" that hybridizes to a target nucleic acid and contains a 3' OH end that is extended by a polymerase in the amplification process. Another example of an amplification oligomer is an oligomer that is not extended by a polymerase (e.g., because it has a 3' blocked end), but participates in or facilitates amplification. For example, the 5' region of an amplification oligonucleotide may contain a promoter sequence (which may be referred to as a "promoter primer" or "promoter provider") that is non-complementary to the target nucleic acid. Those skilled in the art will understand that an amplification oligomer that functions as a primer can be modified to include a 5' promoter sequence and thus function as a promoter primer. The incorporation of the 3' blocked end further modifies the promoter primer, allowing it to hybridize to the target nucleic acid and providing an upstream promoter sequence that functions to initiate transcription, but does not provide a primer for oligo extension. Such modified oligos are referred to herein as "promoter provider" oligomers. Amplification oligonucleotides range in size from about 10 to about 70 nt in length (not including any promoter sequence or poly-A tail) and contain at least about 10 contiguous bases, or even at least 12 contiguous bases (or their complementary strands) that are complementary to a region of the target nucleic acid sequence. The contiguous bases are at least 80%, or at least 90%, or fully complementary to the target sequence to which the amplification oligomer binds. Amplification oligomers can optionally contain modified nucleotides or analogs, or additional nucleotides that participate in the amplification reaction but are not complementary to or contained in the target nucleic acid or template sequence. When referring to a range for the length of an oligonucleotide, amplicon, or other nucleic acid, the range includes all integers (e.g., a length of 19 to 25 contiguous nucleotides includes 19, 20, 21, 22, 23, 24, and 25).

[0056] As used herein, a "promoter" is a specific nucleic acid sequence that binds to a nucleic acid at a specific site and is recognized by a DNA-dependent RNA polymerase ("transcriptase") as a signal to initiate transcription of RNA.

[0057] As used herein, "promoter provider" or "provider" refers to an oligonucleotide that contains a first and a second region and is modified to block the initiation of DNA synthesis from its 3' end. The "first region" of a promoter provider oligonucleotide contains a base sequence that hybridizes with a DNA template; the hybridizing sequence is located 3', but not necessarily adjacent to, the promoter region. The hybridizing portion of a promoter oligonucleotide is typically at least 10 nucleotides in length and can extend up to 50 nucleotides or more in length. The "second region" contains a promoter sequence for an RNA polymerase. The promoter oligonucleotide is engineered so that it cannot be extended by an RNA- or DNA-dependent DNA polymerase, such as a reverse transcriptase, preferably containing a blocking moiety at its 3' end, as described above. As referred to herein, a "T7 provider" is a blocked promoter provider oligonucleotide that provides an oligonucleotide sequence recognized by T7 RNA polymerase.

[0058] "Amplification" refers to any known procedure for obtaining multiple copies of a target nucleic acid sequence or its complement, or fragments thereof. The multiple copies may be referred to as amplicons or amplification products. Known amplification methods include both thermal cycling and isothermal amplification methods. In some embodiments, isothermal amplification methods are preferred. Replicase-mediated amplification, polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), and transcription-mediated or transcription-associated amplification are non-limiting examples of nucleic acid amplification methods. Replicase-mediated amplification uses self-replicating RNA molecules and a replicase such as QB-replicase (e.g., U.S. Pat. No. 4,786,600). PCR amplification uses a DNA polymerase, a primer pair, and thermal cycling to synthesize multiple copies of two complementary strands from dsDNA or cDNA (e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159). LCR amplification uses four or more different oligonucleotides to amplify a target and its complementary strand through multiple cycles of hybridization, ligation, and denaturation (e.g., U.S. Pat. Nos. 5,427,930 and 5,516,663). SDA uses a restriction endonuclease and a primer containing a recognition site for the endonuclease that nicks one strand of a hemi-modified DNA duplex containing the target sequence, thereby allowing amplification to occur in a series of primer extension and strand displacement steps (e.g., U.S. Pat. Nos. 5,422,252, 5,547,861, and 5,648,211). While preferred embodiments use amplification methods suitable for amplifying RNA target nucleic acids, such as transcription-mediated amplification (TMA) or NASBA, it will be apparent to those skilled in the art that the oligomers disclosed herein can readily be used as primers in other amplification methods.

[0059] "Transcription-associated amplification," also referred to herein as "transcription-mediated amplification" (TMA), refers to nucleic acid amplification that uses an RNA polymerase to produce multiple RNA transcripts from a nucleic acid template. These methods generally employ an RNA polymerase, a DNA polymerase, deoxyribonucleoside triphosphates, ribonucleoside triphosphates, and template-complementary oligonucleotides containing a promoter sequence, and may optionally include one or more other oligonucleotides. The TMA method is an embodiment of the amplification method used to amplify and detect HSV target sequences described herein. Variations of transcription-associated amplification are well known in the art, as previously disclosed in detail (e.g., U.S. Patent Nos. 4,868,105, 5,124,246, 5,130,238, 5,399,491, 5,437,990, 5,554,516, and 7,374,885, and PCT Publication Nos. WO88 / 01302, WO88 / 10315, and WO95 / 03430). Those skilled in the art will understand that the disclosed compositions can be used in amplification methods based on the extension of oligomeric sequences by polymerases.

[0060] As used herein, the term "real-time TMA" refers to single-primer transcription-mediated amplification ("TMA") of a target nucleic acid monitored by a real-time detection means.

[0061] The term "amplicon" is used interchangeably with "amplification product" and refers to a nucleic acid molecule produced during an amplification procedure that is complementary or homologous to a sequence contained within a target sequence. These terms may be used to refer to a single-stranded amplification product, a double-stranded amplification product, or one of the strands of a double-stranded amplification product.

[0062] "Probe," "detection probe," "detection oligonucleotide," and "detection probe oligomer" are used interchangeably herein to refer to a nucleic acid oligomer that specifically hybridizes to a target sequence in a nucleic acid or amplified nucleic acid under conditions that promote hybridization that allows for detection of the target sequence or amplified nucleic acid. Detection can be either direct (e.g., a probe that hybridizes directly to its target sequence) or indirect (e.g., a probe that is linked to its target via an intermediate molecular structure). Probes can be DNA, RNA, analogs thereof, or combinations thereof, and they can be labeled or unlabeled. The "target sequence" of a probe generally refers to a smaller nucleic acid sequence within a larger nucleic acid sequence that specifically hybridizes to at least a portion of the probe oligomer through standard base pairing. The probe may contain target-specific sequences and other sequences that contribute to the three-dimensional conformation of the probe (e.g., U.S. Patent Nos. 5,118,801, 5,312,728, 6,849,412, 6,835,542, 6,534,274, and 6,361,945, and U.S. Publication No. 2006 / 0068417). In a preferred embodiment, the detection probe contains a 2' methoxy backbone, which can provide a higher signal.

[0063] The term "TaqMan® probe" refers to a detection oligonucleotide that contains a fluorescent dye, typically on the 5' base, and a non-fluorescent quencher dye (quencher), typically on the 3' base. When illuminated, the excited fluorescent dye, rather than fluorescing, transfers energy to a nearby quencher dye molecule, resulting in a non-fluorescent substrate. During amplification, the exonuclease activity of the polymerase cleaves the TaqMan probe, separating the fluorophore from the quencher, thereby allowing an unquenched signal to be emitted from the fluorophore as an indication of amplification.

[0064] As used herein, "label" refers to a moiety or compound directly or indirectly attached to a probe that is detected or provides a detectable signal. Direct labeling can occur through a bond or interaction that links the label to the probe, including covalent or non-covalent interactions, such as hydrogen bonding, hydrophobic and ionic interactions, or the formation of chelate or coordination complexes. Indirect labeling can occur through the use of a bridging moiety or "linker," such as a binding pair member, antibody, or additional oligomer, that can be labeled either directly or indirectly and amplify the detectable signal. Labels include any detectable moiety, such as a radionuclide, a ligand (e.g., biotin, avidin), an enzyme or enzyme substrate, a reactive group, or a chromophore (e.g., a dye, particle, or bead that imparts a detectable color), a light-emitting compound (e.g., a bioluminescent, phosphorescent, or chemiluminescent label), or a fluorophore. Labels can be detectable in homogeneous assays in which bound labeled probe in a mixture exhibits a detectable change, e.g., instability or differential degradation properties, that differs from unbound labeled probe. A "homogeneous detectable label" can be detected without physically removing the bound form of the label from the unbound form or the unlabeled probe (e.g., U.S. Pat. Nos. 5,283,174, 5,656,207, and 5,658,737). Labels include chemiluminescent compounds, such as acridinium ester ("AE") compounds, including standard AEs and derivatives (e.g., U.S. Pat. Nos. 5,656,207, 5,658,737, and 5,639,604). Methods for synthesizing, attaching labels to nucleic acids, and detecting labels are well known (e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), Chapter 10; U.S. Patent Nos. 5,658,737, 5,656,207, 5,547,842, 5,283,174, and 4,581,333).More than one label and more than one type of label can be present on a particular probe, or detection can use a mixture of probes where each probe is labeled with a compound that produces a detectable signal (e.g., U.S. Pat. Nos. 6,180,340 and 6,350,579).

[0065] As used herein, structures referred to as "molecular torches" are designed to contain distinct regions of self-complementarity ("closing domains") connected by junction regions ("target binding domains") that hybridize to each other under defined hybridization assay conditions. All or a portion of the nucleotide sequence comprising the target closing domain may also function as the target binding domain. Thus, the target closing sequence may include target binding sequences, non-target binding sequences, and combinations thereof.

[0066] The terms "capture probe," "capture oligonucleotide," "target capture oligonucleotide," and "capture probe oligomer" are used interchangeably herein to refer to a nucleic acid oligomer that specifically hybridizes to a target sequence of a target nucleic acid through standard base pairing and is attached to a binding partner on an immobilized probe to capture the target nucleic acid to a support. One example of a capture oligomer comprises an oligonucleotide comprising two binding regions: a target-hybridizing sequence and an immobilized probe-binding region. In a variation of this example, the two regions may be present on two different oligomers joined together by one or more linkers. In another embodiment of a capture oligomer, the target-hybridizing sequence is a sequence comprising a random or nonrandom poly-GU, poly-GT, or poly-U sequence that nonspecifically binds to the target nucleic acid and links it to the immobilized probe on the support (see, e.g., PCT Publication No. WO2008 / 016988). The immobilized probe-binding region may be a nucleic acid sequence referred to as a tail. The tail is approximately 10-40 nucleotides (e.g., A) that binds to a complementary immobilized sequence attached to a support particle or support matrix. 10 ~A 40 ), or about 14 to 33 nt (e.g., T3A 14 ~T3A 30) in some embodiments. 0~4 A 10~40 Another example of a capture oligomer contains two regions that are not nucleic acid sequences: a target hybridizing sequence and a binding pair member.

[0067] As used herein, "immobilized oligonucleotide," "immobilized probe," or "immobilized nucleic acid" refers to a nucleic acid binding partner that directly or indirectly attaches a capture oligomer to a support. The support-attached immobilized probe facilitates separation of the capture probe-bound target from unbound material in a sample. One embodiment of an immobilized probe is an oligomer attached to a support that facilitates separation of the bound target sequence from unbound material in a sample. Supports can include known materials such as matrices and particles free in solution, which may be composed of nitrocellulose, nylon, glass, polyacrylate, mixed polymers, polystyrene, silane, polypropylene, metal, or other compositions, one embodiment of which is magnetically attractable particles. The support can be monodisperse magnetic spheres (e.g., uniform size ±5%) to which the immobilized probe is attached directly (via covalent bonding, chelation, or ionic interactions) or indirectly (via one or more linkers), and the link or interaction between the probe and the support is stable during hybridization. [Brief explanation of the drawings]

[0068] [Figure 1] Illustrated is the reference sequence (SEQ ID NO: 129) of the Candida species RPR1 gene (Candida albicans strain ATCC 90028 ribonuclease P RNA (RPR1) gene, partial sequence, found in GenBank under accession number DQ660433, GI: 110084517, 10-MAR-2007). [Figure 2]Illustrated is the reference sequence (SEQ ID NO: 131) of the Candida species RPR1 gene (Candida glabrata strain ATCC 2238 ribonuclease P RNA (RPR1) gene, partial sequence, found in GenBank under accession number DQ660434, GI: 133874753, 21-MAR-2007). [Figure 3] Illustrated is the reference sequence (SEQ ID NO: 130) of the Candida species RPR1 gene (Candida parapsilosis strain ATCC 22019 ribonuclease P RNA (RPR1) gene, partial sequence, found in GenBank under accession number DQ660436, GI: 110084520, 10 MAR-2007). DETAILED DESCRIPTION OF THE INVENTION

[0069] The present invention provides compositions, kits, and methods for determining the presence or absence of Candida species nucleic acids in a sample. Preferably, the sample is a biological sample. The compositions, kits, and methods provide oligonucleotide sequences that recognize target sequences of the RPR1 gene or non-coding RNA encoded by the RPR1 gene, including RPR1 target sequences of C. albicans, C. parapsilosis, C. dubliniensis, C. tropicalis, and / or C. glabrata, or their complementary sequences. Such oligonucleotides can be used as amplification oligonucleotides, which can include primers, promoter-primers, and promoter-provider oligonucleotides, the functions of which have been previously described (see, e.g., U.S. Patent Nos. 4,683,195, 4,683,202, 4,800,159, 5,399,491, 5,554,516, 5,824,518, and 7,374,885, each of which is incorporated herein by reference). Other oligonucleotides can be used as probes to detect the amplified sequence of Candida species or for capture of the Candida species target nucleic acid. In certain embodiments, the compositions of the invention are combinations of two or more oligomers that recognize the Candida species RPR1 target sequence (e.g., two or more amplification oligomers).

[0070] The present methods provide sensitive and specific detection of Candida species nucleic acids. In certain embodiments, the methods involve performing nucleic acid amplification of a Candida species target region and detecting the amplified product, e.g., by specifically hybridizing the amplified product with a nucleic acid detection probe that provides a signal indicative of the presence of Candida species in the sample. The amplification step involves contacting the sample with one or more amplification oligomers specific for a target sequence of the Candida species target nucleic acid to produce an amplified product if Candida species nucleic acid is present in the sample. Amplification synthesizes additional copies of the target sequence or its complement by producing copies from a template strand using at least one nucleic acid polymerase and amplification oligomers (e.g., by using the template strand to extend sequences from primers). One embodiment for detecting the amplified product uses a hybridization step that involves contacting the amplified product with at least one probe specific for a sequence amplified by selected amplification oligomers, e.g., a sequence contained in the target sequence flanking a pair of selected amplification oligomers.

[0071] The detection step can be carried out using any of a variety of known techniques to detect a signal specifically associated with the amplified target sequence, for example, by hybridizing the amplification product with a labeled detection probe and detecting the signal obtained from the labeled probe. The detection step can also provide additional information about the amplified sequence, for example, all or part of its nucleic acid sequence. Detection can be carried out after the amplification reaction is completed, or can be carried out simultaneously with the amplification of the target region, for example, in real time. In one embodiment, the detection step allows for homogeneous detection, for example, detection of hybridized probes without removing unhybridized probes from the mixture (see, e.g., U.S. Patent Nos. 5,639,604 and 5,283,174, each of which is incorporated herein by reference).

[0072] In embodiments that detect amplified products near or at the end of the amplification step, a linear detection probe may be used to provide a signal indicative of hybridization of the probe to the amplified product. One example of such detection uses a luminescently labeled probe that hybridizes to the target nucleic acid. The luminescent label is then hydrolyzed from the unhybridized probe. Detection is carried out by chemiluminescence using a luminometer. (See, e.g., International Patent Application Publication No. WO 89 / 002476, incorporated herein by reference.) In other embodiments that use real-time detection, the detection probe may be, for example, a hairpin probe such as a molecular beacon, molecular torch, or hybridization switch probe labeled with a reporter moiety that is detected when the probe binds to the amplified product. Such a probe may include a target-hybridizing sequence and a non-target-hybridizing sequence. Various forms of such probes have been previously described (see, e.g., U.S. Patent Nos. 5,118,801, 5,312,728, 5,925,517, 6,150,097, 6,849,412, 6,835,542, 6,534,274, and 6,361,945, and U.S. Patent Application Publication Nos. 2006 / 0068417A1 and 2006 / 0194240A1, each of which is incorporated herein by reference).

[0073] Preferred compositions of the invention are configured to specifically hybridize with the nucleic acid of at least one of C. albicans, C. parapsilosis, C. dubliniensis, C. tropicalis, and / or C. glabrata, and have minimal cross-reactivity with other non-Candida nucleic acids suspected of being present in a sample (e.g., other pathogens associated with vaginal infections). In certain variations, compositions (e.g., oligomer combinations) of the invention allow for the detection of a broad range of Candida species (e.g., any of C. albicans, C. dubliniensis, and C. tropicalis; any of C. albicans, C. parapsilosis, C. dubliniensis, and C. tropicalis; or any of C. albicans, C. parapsilosis, C. dubliniensis, C. tropicalis, and C. glabrata). In some aspects, the compositions of the invention are configured to specifically hybridize with nucleic acids of one or more of C. albicans, C. parapsilosis, C. dubliniensis, C. tropicalis, and C. glabrata, and are not specifically hybridizable with nucleic acids of Trichomonas vaginalis, Chlamydia trachomatis, Acinetobacter iwoffii, Actinomyces israelii, Alcaligenes faecalis, Bacteroides fragilis, Clostridium difficile, Corynebacterium genitalium, Enterobacter cloacae, Enterococcus feacalis, Escherichia coli, Bifidobacterium adolescentis, Campylobacter jejuni, Fusobacterium nucleatum, Haemophilus ducreyi, Klebsiella pneumoniae, Listeria monocytogenes, Mycoplasma hominis, Peptostreptococcus magnus, Propionibacterium acnes, Neisseria gonorrhoeae, Trichomonas vaginalis, Ureaplasma urealyticum, Ureaplasma parvum, Candida krusei, Candida lusitaniae, Prevotella bivia, Eggerthella lenta, Pseudomonas aeruginosa, Mobiluncus curtisii, Chlamydia trachomatis, Cryptococcus neoformans, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus agalactiae, Streptococcus pyogenes, Leptotrichia bucalis, Proteus vulgaris, Megaspahaera elsdenii, Atopobium vaginae, Lactobacillus acidophilus, Lactobacillus mucosae, Lactobacillus gastricus, Lactobacillus iners, Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri, and Gardnerella vaginalis. In some embodiments, the compositions of the invention are part of a multiplex system that includes multiple sets of oligomers in combination to enable the detection of any of C. albicans, C. parapsilosis, C. dubliniensis, C. tropicalis, and C. glabrata, such as the multiplex detection methods described herein.

[0074] In certain embodiments of the invention, a combination of at least two oligomers is provided for determining the presence or absence of Candida species in a sample. Typically, the combination of oligomers includes (a) first and second Candida-specific amplification oligomers for amplifying a first Candida species target region corresponding to the region of SEQ ID NO: 129 or a second Candida species target region corresponding to the region of SEQ ID NO: 130, and / or (b) first and second C. glabrata-specific amplification oligomers for amplifying a third Candida species target region corresponding to the region of SEQ ID NO: 131. In such embodiments, at least one amplification oligomer from each of oligomer sets (a) and (b) above comprises a target hybridizing sequence in a sense orientation (the "sense THS") and at least one amplification oligomer comprises a target hybridizing sequence in an antisense orientation (the "antisense THS"), wherein the sense and antisense THSs of the amplification oligomers of set (a) are each configured to specifically hybridize to a Candida species target sequence corresponding to a sequence contained within SEQ ID NO: 129 or 130, and the sense and antisense THSs of the amplification oligomers of set (b) are each configured to specifically hybridize to a Candida species target sequence corresponding to a sequence contained within SEQ ID NO: 131, and the target hybridizing sequences are selected such that the Candida sequence targeted by the antisense THS is located downstream of the Candida sequence targeted by the sense THS (i.e., at least two amplification oligomers are positioned adjacent to the target region to be amplified). In some embodiments, a first Candida species target region corresponds to a region from about nucleotide 133 or 161 to about nucleotide 259 of SEQ ID NO:129, a second Candida species target region corresponds to a region from about nucleotide 202 to about nucleotide 308 of SEQ ID NO:130, and / or a third Candida species target region corresponds to a region from about nucleotide 355 to about nucleotide 554 of SEQ ID NO:131.In some variations, the oligomer combination includes (a) a first Candida-specific amplification oligomer comprising a first Candida-specific target hybridizing sequence that substantially corresponds to the nucleotide sequence of residues 28-46 of SEQ ID NO: 9. In some variations, the oligomer combination includes (a) a second Candida-specific amplification oligomer comprising a second Candida-specific target hybridizing sequence that is (i) 15-24 contiguous nucleotides contained in the sequence of SEQ ID NO: 132 and at least the sequence of SEQ ID NO: 133, (ii) 20-23 contiguous nucleotides contained in the sequence of SEQ ID NO: 152 and at least the sequence of SEQ ID NO: 151, or (iii) a sequence that substantially corresponds to the nucleotide sequence of SEQ ID NO: 34. In some variations, the oligomer combination includes (b) a first C. glabrata-specific amplification oligomer comprising 15-24 contiguous nucleotides contained in the sequence of SEQ ID NO: 134 and a first C. glabrata-specific target hybridizing sequence, the sequence comprising at least the sequence of SEQ ID NO: 135. In some variations, the oligomer combination includes (b) a second C. glabrata-specific amplification oligomer comprising 16-21 contiguous nucleotides contained in the sequence of SEQ ID NO: 136 and a second C. glabrata-specific target hybridizing sequence, the sequence comprising at least the sequence of SEQ ID NO: 137.

[0075] In more specific embodiments of the present invention, the above-described oligomer combinations for determining the presence or absence of Candida species in a sample comprise at least one of: (A) an amplification oligomer comprising, or consisting of, the nucleotide sequence of residues 28-46 of SEQ ID NO:9; (B) an amplification oligomer comprising, or consisting of, the nucleotide sequence of SEQ ID NO:26; (C) an amplification oligomer comprising, or consisting of, the nucleotide sequence of residues 3-22 of SEQ ID NO:74; (D) an amplification oligomer comprising, or consisting of, the nucleotide sequence of SEQ ID NO:34; (E) an amplification oligomer comprising, or consisting of the nucleotide sequence of residues 28-49 of SEQ ID NO:14; and (F) an amplification oligomer comprising, or consisting of the nucleotide sequence of SEQ ID NO:12.

[0076] In certain embodiments, the amplification oligomers described herein are promoter primers or promoter providers that further comprise a promoter sequence located 5' to the target hybridizing sequence and that is non-complementary to the Candida species target nucleic acid. For example, in some embodiments of the oligomer combinations described herein for amplifying a Candida species target region, the first Candida-specific amplification oligomer (e.g., a first Candida-specific amplification oligomer comprising a first Candida-specific target hybridizing sequence substantially corresponding to the nucleotide sequence of residues 28-46 of SEQ ID NO:9) and / or the first C. glabrata-specific amplification oligomer (e.g., a first C. glabrata-specific amplification oligomer comprising a first C. glabrata-specific target hybridizing sequence that is a sequence comprising 15-24 contiguous nucleotides contained in the sequence of SEQ ID NO:134 and at least the sequence of SEQ ID NO:135) is a promoter primer that further comprises a 5' promoter sequence. In specific embodiments, the promoter sequence is a T7 RNA polymerase promoter sequence, such as, for example, the T7 promoter sequence having the sequence set forth in residues 1-27 of SEQ ID NO: 9. In specific variations, the first Candida-specific amplification oligomer is a promoter primer having the sequence set forth in SEQ ID NO: 9 and / or the first C. glabrata-specific amplification oligomer is a promoter primer having the sequence set forth in SEQ ID NO: 14.

[0077] In some embodiments, the oligomer combinations described herein further comprise a terminating oligonucleotide (also referred to herein as a "blocker" oligonucleotide) that comprises a base sequence that is substantially complementary (e.g., perfectly complementary) to a sequence contained within the target nucleic acid near the 5' end of the target region. Terminating oligomers are typically used in combination with, for example, a promoter provider amplification oligomer, such as in certain embodiments described herein with respect to transcription-mediated amplification (TMA).

[0078] In some embodiments, the oligomer combinations described herein further comprise at least one Candida-specific capture probe oligomer and / or at least one C. glabrata-specific capture probe oligomer. In certain embodiments, the Candida-specific capture probe oligomer comprises a target hybridizing sequence substantially corresponding to a sequence contained in the complement of SEQ ID NO: 129 or SEQ ID NO: 30. In certain embodiments, the C. glabrata-specific capture probe oligomer comprises a target hybridizing sequence substantially corresponding to a sequence contained in the complement of SEQ ID NO: 131. In some embodiments, the capture probe oligomer target hybridizing sequence is covalently linked to a sequence or moiety that binds to an immobilized probe. In some embodiments, the Candida-specific capture probe oligomer has (i) a target hybridizing sequence that is 16-21 contiguous nucleotides contained in the sequence of SEQ ID NO: 138 and comprises at least the sequence of SEQ ID NO: 139, or (ii) a target hybridizing sequence that is 15-25 contiguous nucleotides contained in the sequence of SEQ ID NO: 140 and comprises at least the sequence of SEQ ID NO: 141. In some embodiments, the C. glabrata-specific capture probe oligomer has a target hybridizing sequence that is 16-27 contiguous nucleotides contained within the sequence of SEQ ID NO: 142 and includes at least the sequence of SEQ ID NO: 143 or SEQ ID NO: 144. In certain embodiments, the Candida-specific capture probe target hybridizing sequence comprises or consists of a nucleotide sequence selected from (i) the nucleotide sequence of residues 1-20 of SEQ ID NO: 24 and (ii) the nucleotide sequence of residues 1-17 of SEQ ID NO: 66, and / or the C. glabrata-specific capture probe target hybridizing sequence comprises or consists of the nucleotide sequence of residues 1-26 of SEQ ID NO: 48. In more particular variations, the Candida-specific capture probe oligomer has a nucleotide sequence selected from SEQ ID NO: 24 and SEQ ID NO: 66, and / or the C. glabrata-specific capture probe oligomer has the nucleotide sequence of SEQ ID NO: 48.The oligomer combination can include at least two (e.g., three) capture probe oligomers described above. In some embodiments, the oligomer combination includes both a Candida-specific capture probe oligomer and a C. glabrata-specific capture probe oligomer described above, and in some such embodiments, the oligomer combination further includes first and second Candida-specific capture probe oligomers, each of which is a Candida-specific capture probe oligomer described above. In certain embodiments, the capture probe oligomers are provided in a target capture reaction mixture.

[0079] In certain variations, the oligomer combinations described herein further comprise at least one detection probe oligomer configured to specifically hybridize to a Candida species target sequence amplifiable using first and second amplification oligomers targeting a Candida species target region (e.g., a Candida species target region adjacent to the target hybridizing sequences of the first and second Candida-specific or C. glabrata-specific amplification oligomers described herein). In some embodiments, the oligomer combination comprises a Candida-specific detection probe that specifically hybridizes to (ai) a first Candida species target region corresponding to the region from about nucleotide 133 or 161 to about nucleotide 259 of SEQ ID NO: 129, (a-ii) a second Candida species target region corresponding to the region from about nucleotide 202 to about nucleotide 308 of SEQ ID NO: 130, or (a-iii) the exact complement of (ai) or (a-ii). In some embodiments, the oligomer combination comprises (bi) a C. glabrata-specific detection probe that specifically hybridizes to a third Candida species target region corresponding to the region from about nucleotide 355 to about nucleotide 554 of SEQ ID NO: 131, or (b-ii) the exact complement of (bi). In some embodiments, the Candida-specific detection probe comprises 18 to 22 contiguous nucleotides contained in the sequence of SEQ ID NO: 145 and a Candida-specific detection probe target hybridizing sequence comprising at least the sequence of SEQ ID NO: 146, or a target hybridizing sequence that is the exact complement of the foregoing. In some embodiments, the C. glabrata-specific detection probe includes a C. glabrata-specific detection probe target hybridizing sequence selected from (A) a sequence comprising 17 to 23 consecutive nucleotides contained in the sequence of SEQ ID NO: 147 and at least the sequence of SEQ ID NO: 148; (B) a sequence substantially corresponding to the sequence of residues 1 to 17 of SEQ ID NO: 18; (C) a sequence substantially corresponding to the sequence of residues 1 to 20 of SEQ ID NO: 21; and (D) the exact complement of any one of (A) to (C).In more specific embodiments, the Candida-specific detection probe target hybridizing sequence comprises or consists of residues 1-22 of SEQ ID NO:27, or a complete complement thereof, and / or the C. glabrata-specific detection probe target hybridizing sequence comprises or consists of residues 1-17 of SEQ ID NO:60, residues 1-23 of SEQ ID NO:45, residues 1-17 of SEQ ID NO:18, residues 1-20 of SEQ ID NO:21, or a complete complement of any of the foregoing. In specific variations of Candida-specific detection probes, the probe has the sequence of SEQ ID NO:27, or a complete complement thereof. In specific variations of C. glabrata-specific detection probes, the probe has the sequence of SEQ ID NO:60, SEQ ID NO:45, SEQ ID NO:18, SEQ ID NO:21, or a complete complement of any of the foregoing. Suitable detection probes further include DNA equivalents and DNA / RNA chimeras of any of the above. Detection probe oligomers may contain a 2'-methoxy backbone in one or more linkages of the nucleic acid backbone. In some variations, the oligomer combination comprises at least two detection probe oligomers (e.g., both the Candida-specific and C. glabrata-specific detection probes described herein). In some embodiments, at least one detection probe oligomer is provided in the amplicon detection reaction mixture.

[0080] Typically, the detection probe oligomer according to the present invention further comprises a label. Particularly suitable labels include compounds that emit a detectable light signal, such as fluorophores or luminescent (e.g., chemiluminescent) compounds that can be detected in a homogeneous mixture. Two or more labels and two or more types of labels can be present on a particular probe, or detection can rely on the use of a mixture of probes, each labeled with a compound that provides a detectable signal (see, e.g., U.S. Patent Nos. 6,180,340 and 6,350,579, each of which is incorporated herein by reference). Labels can be attached to the probe by various means, including covalent bonding, chelation, and ionic interactions, but preferably the labels are covalently attached. For example, in some embodiments, the detection probe has an attached chemiluminescent label, such as, for example, an acridinium ester (AE) compound (see, e.g., U.S. Pat. Nos. 5,185,439, 5,639,604, 5,585,481, and 5,656,744, each of which is incorporated herein by reference), which in typical variations is attached to the probe by a non-nucleotide linker (see, e.g., U.S. Pat. Nos. 5,585,481, 5,656,744, and 5,639,604, particularly column 10, line 6 through column 11, line 3, and Example 8, each of which is incorporated herein by reference). In other embodiments, the detection probe includes both a fluorescent label and a quencher, a combination that is particularly useful in fluorescence resonance energy transfer (FRET) assays. Specific variations of such detection probes include, for example, TaqMan detection probes (Roche molecular Diagnostics) and "molecular beacons" (see, e.g., Tyagi et al., Nature Biotechnol. 16:49-53, 1998; U.S. Patent Nos. 5,118,801 and 5,312,728, each of which is incorporated herein by reference).

[0081] The detection probe oligomer according to the present invention may further comprise a non-target hybridizing sequence. Specific embodiments of such detection probes include probes that form conformations held together by intramolecular hybridization, such as conformations commonly referred to as hairpins. Particularly suitable hairpin probes include "molecular torches" (see, e.g., U.S. Pat. Nos. 6,849,412, 6,835,542, 6,534,274, and 6,361,945, each of which is incorporated herein by reference) and "molecular beacons" (see, e.g., Tyagi et al., supra; U.S. Pat. Nos. 5,118,801 and 5,312,728, supra). Methods for using such hairpin probes are well known in the art.

[0082] In yet another embodiment, the detection probe is a linear oligomer that does not substantially form a conformation held together by intramolecular bonds. In a particular variation, the linear detection probe oligomer comprises a chemiluminescent compound, preferably an acridinium ester (AE) compound, as a label.

[0083] The present invention also provides the detection probe oligomers and capture probe oligomers described herein.

[0084] In another aspect, the present invention provides methods for determining the presence or absence of Candida species in a sample using the oligomer combinations described herein. Such methods generally include: (1) contacting a sample with at least one of a first amplification oligomer combination and a second oligomer combination, where (a) the first amplification oligomer combination comprises first and second Candida-specific amplification oligomers for amplifying a first Candida species target region corresponding to the region of SEQ ID NO: 129 or a second Candida species target region corresponding to the region of SEQ ID NO: 130, and (b) the second amplification oligomer combination comprises first and second C. glabrata-specific amplification oligomers for amplifying a third Candida species target region corresponding to the region of SEQ ID NO: 131; (2) performing an in vitro nucleic acid amplification reaction in which any Candida species target nucleic acid, if present in the sample, is used as a template to generate one or more amplification products corresponding to at least one of the first, second, and third target regions; and (3) detecting the presence or absence of the one or more amplification products, thereby determining the presence or absence of Candida species in the sample. Detection methods according to the invention typically further include the step of obtaining a sample to be contacted with at least two oligomers. In certain embodiments, "obtaining" a sample for use in steps (1)-(3) includes, for example, receiving the sample at a testing facility or other location where one or more steps of the method are performed and / or removing the sample from a location within the facility where one or more steps of the method are performed (e.g., from a repository or other storage location).

[0085] In certain embodiments, the method further comprises purifying the Candida species target nucleic acid from other components in the sample prior to the contacting step. Such purification may include methods for separating and / or concentrating organisms contained in the sample from other sample components. In certain embodiments, purifying the target nucleic acid comprises capturing the target nucleic acid to specifically or nonspecifically separate the target nucleic acid from other sample components. Nonspecific target capture methods may involve selective precipitation of the nucleic acid from a substantially aqueous mixture, attachment of the nucleic acid to a support that is washed to remove other sample components, or other means of physically separating the nucleic acid from a mixture containing Candida species nucleic acid and other sample components.

[0086] In some embodiments, Candida species target nucleic acids are selectively separated from other sample components by specifically hybridizing the Candida species target nucleic acid with a capture probe oligomer. The capture probe oligomer comprises a target hybridizing sequence configured to specifically hybridize with the Candida species target sequence to form a target sequence:capture probe complex that is separated from the sample components. Suitable capture probe target hybridizing sequences include those described above with respect to the Candida-specific capture probes and / or C. glabrata-specific capture probes that may be used in certain embodiments of oligomer combinations for detecting Candida species. In preferred variations, specific target capture binds the Candida species target:capture probe complex to the immobilized probe, forming a target:capture probe:immobilized probe complex that is separated from the sample and optionally washed to remove non-target sample components (see, e.g., U.S. Pat. Nos. 6,110,678, 6,280,952, and 6,534,273, each of which is incorporated herein by reference). In such variations, the capture probe oligomer further comprises a sequence or moiety that binds and attaches the capture probe, along with its bound target sequence, to an immobilized probe attached to a solid support, thereby enabling the hybridized target nucleic acid to be separated from other sample components. In some embodiments, a sample suspected of containing Candida species is contacted with both a Candida-specific capture probe oligomer and a C. glabrata capture probe oligomer; in some such embodiments, the sample is further contacted with a second Candida-specific capture probe oligomer.

[0087] In more specific embodiments, the capture probe oligomer includes a tail portion (e.g., a 3' tail) that is not complementary to the Candida species target sequence, but that specifically hybridizes to a sequence on the immobilized probe, thereby functioning as a moiety that allows the target nucleic acid to be separated from other sample components, such as previously described in U.S. Patent No. 6,110,678 (incorporated herein by reference). Any sequence can be used in the tail region, generally about 5-50 nt in length, with preferred embodiments including a tail region of about 10-40 nt (e.g., A) that binds to a complementary immobilized sequence (e.g., poly-T) attached to a solid support, e.g., a matrix or particle. 10 ~A 40 ), more preferably about 14 to 33 nt (e.g., A 14 ~A 30 or T3A 14 ~T3A 30 For example, in specific embodiments of capture probes comprising a 3' tail, the capture probe has a sequence selected from SEQ ID NO:24, SEQ ID NO:66, and SEQ ID NO:48.

[0088] Target capture typically involves hybridization under hybridizing conditions, usually a T tail sequence:immobilized probe sequence duplex. mThis occurs in a solution-phase mixture containing one or more capture probe oligomers that specifically hybridize to the Candida species target sequence at temperatures above 100°C. For embodiments including a capture probe tail, the Candida species target:capture probe complex is captured by adjusting the hybridization conditions so that the capture probe tail hybridizes to the immobilized probe, after which the entire complex on the solid support is separated from other sample components. The support with the bound immobilized probe:capture probe:Candida species target sequence can be washed one or more times to further remove other sample components. A preferred embodiment uses a particulate solid support, such as paramagnetic beads, so that the particles with the bound Candida species target:capture probe:immobilized probe complex are suspended in a wash solution and can be recovered from the wash solution, preferably using magnetic attraction. To limit the number of handling steps, the Candida species target nucleic acid can be amplified by simply mixing the Candida species target sequence of the complex on the support with amplification oligomers and proceeding with the amplification step.

[0089] Amplification of Candida species target sequences utilizes an in vitro amplification reaction that uses at least two amplification oligomers that flank the target region to be amplified. In some embodiments, the target region to be amplified corresponds to the region from about nucleotide 133 or 161 to about nucleotide 259 of SEQ ID NO: 129. In some embodiments, the target region to be amplified corresponds to the region from about nucleotide 202 to about nucleotide 308 of SEQ ID NO: 130. In some embodiments, the target region to be amplified corresponds to the region from about nucleotide 355 to about nucleotide 554 of SEQ ID NO: 131. Amplification oligomer combinations particularly suitable for amplifying these target regions are described herein. Suitable amplification methods include, for example, replicase-mediated amplification, polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), and transcription-mediated or transcription-associated amplification (TMA). Such amplification methods are well known in the art and are readily used in accordance with the methods of the present invention.

[0090] For example, some amplification methods using TMA amplification include the following steps: Briefly, a target nucleic acid containing the sequence to be amplified is provided as a single-stranded nucleic acid (e.g., ssRNA or ssDNA). Those skilled in the art will understand that conventional melting of double-stranded nucleic acids (e.g., dsDNA) can also be used to provide a single-stranded target nucleic acid. A promoter primer specifically binds to the target nucleic acid at its target sequence, and reverse transcriptase (RT) extends the 3' end of the promoter primer using the target strand as a template to create a cDNA copy of the target sequence strand, resulting in an RNA:DNA duplex. RNase digests the RNA strand of the RNA:DNA duplex, and a second primer specifically binds to its target sequence located on the cDNA strand downstream from the promoter primer end. RT synthesizes a new DNA strand by extending the 3' end of the second primer using the first cDNA template to create a dsDNA containing a functional promoter sequence. RNA polymerase specific to the promoter sequence then begins transcription to produce RNA transcripts that are approximately 100 to 1000 amplified copies ("amplicons") of the initial target strand in the reaction. Amplification continues when a second primer specifically binds to its target sequence in each amplicon and RT creates DNA copies from the amplicon RNA template, producing an RNA:DNA duplex. RNase in the reaction mixture digests the amplicon RNA from the RNA:DNA duplex, and the promoter primer specifically binds to its complementary sequence in the newly synthesized DNA. RT extends the 3' end of the promoter primer, creating dsDNA containing a functional promoter to which RNA polymerase binds to transcribe additional amplicons complementary to the target strand. This autocatalytic cycle of creating more amplicon copies is repeated during the course of the reaction, resulting in approximately one billion-fold amplification of the target nucleic acid present in the sample. The amplified products can be detected in real time during amplification or at the end of the amplification reaction by using probes that specifically bind to target sequences contained in the amplified products, and detection of a signal obtained from the bound probe indicates the presence of the target nucleic acid in the sample.

[0091] In some embodiments, the method utilizes a "reverse" TMA reaction. In such variations, the initial or "forward" amplification oligomer is a priming oligonucleotide that hybridizes to the target nucleic acid near the 3' end of the target region. Reverse transcriptase (RT) synthesizes a cDNA strand by extending the 3' end of the primer using the target nucleic acid as a template. The second or "reverse" amplification oligomer is a promoter primer or promoter provider having a target hybridizing sequence configured to hybridize with the target sequence contained within the synthesized cDNA strand. If the second amplification oligomer is a promoter primer, RT extends the 3' end of the promoter primer using the cDNA strand as a template to create a second cDNA copy of the target sequence strand, thereby creating a dsDNA containing a functional promoter sequence. Amplification then continues essentially as described above in paragraph

[0105] , with transcription initiation from the promoter sequence utilizing an RNA polymerase. Alternatively, when the second amplification oligomer is a promoter provider, a termination oligonucleotide that hybridizes with a target sequence near the 5' end of the target region is typically used to terminate the extension of the priming oligomer at the 3' end of the termination oligonucleotide, thereby providing a defined 3' end for the initial cDNA strand synthesized by extension from the priming oligomer. The target-hybridizing sequence of the promoter provider then hybridizes with the defined 3' end of the initial cDNA strand, and the 3' end of the cDNA strand is extended to add a sequence complementary to the promoter sequence of the promoter provider, resulting in the formation of a double-stranded promoter sequence. The initial cDNA strand is then used as a template for transcribing multiple RNA transcripts complementary to the initial cDNA strand, without the promoter portion, using an RNA polymerase that recognizes and initiates transcription from the double-stranded promoter. Each of these RNA transcripts is then available to serve as a template for further amplification from the first priming amplification oligomer.

[0092] Detection of amplified products can be achieved by various methods. Nucleic acids may be associated with a surface that produces a detectable physical change, such as an electrical change. Amplified nucleic acids can be detected by concentrating them in or on a matrix and detecting their associated nucleic acids or dyes (e.g., intercalators such as ethidium bromide or SYBR Green), or by detecting an increase in a dye associated with the nucleic acid in the solution phase. Other detection methods can use nucleic acid detection probes that specifically hybridize to the amplified product sequence and are configured to detect the presence of a probe:product complex, or by using a complex of probes that can amplify a detectable signal associated with the amplified product (e.g., U.S. Pat. Nos. 5,424,413, 5,451,503, and 5,849,481, each of which is incorporated herein by reference). Directly or indirectly labeled probes that specifically associate with the amplified product provide a detectable signal indicative of the presence of the target nucleic acid in the sample. Specifically, the amplified product contains a target sequence of, or complementary to, the Candida species RPR1 gene or a sequence of RNA encoded by the RPR1 gene, and the probe binds directly or indirectly to the sequence contained in the amplified product to indicate the presence of Candida species nucleic acid in the test sample.

[0093] Preferred embodiments of detection probes that hybridize to complementary amplified sequences can be DNA or RNA oligomers, or oligomers containing a combination of DNA and RNA nucleotides (also referred to herein as "RNA / DNA chimeras"), or oligomers synthesized with modified backbones, such as oligomers containing one or more 2'-methoxy-substituted ribonucleotides. Probes used to detect amplified Candida species sequences can be unlabeled, indirectly detectable (e.g., by attachment of another binding partner to a moiety on the probe), or labeled with a variety of detectable labels. Specific embodiments of detection probes suitable for use with the methods of the invention are further described herein. In some embodiments of methods for detecting Candida species sequences, for example, certain embodiments using transcription-mediated amplification (TMA), the detection probe is a linear chemiluminescently labeled probe, more preferably a linear acridinium ester (AE)-labeled probe. In other embodiments, the detection probe comprises both a fluorescent label and a quencher (e.g., a molecular torch or molecular beacon).

[0094] Oligomers not intended to be extended by a nucleic acid polymerase preferably contain a blocker group that replaces the 3' OH to prevent enzyme-mediated extension of the oligomer in an amplification reaction. For example, blocked amplification oligomers and / or detection probes present during amplification preferably do not have a functional 3' OH but instead contain one or more blocking groups located at or near the 3' end. Blocking groups near the 3' end are preferably within five residues of the 3' end and are sufficiently large to limit polymerase binding to the oligomer; other preferred embodiments contain blocking groups covalently attached to the 3' end. Many different chemical groups can be used to block the 3' end, such as alkyl groups, non-nucleotide linkers, alkane-diol dideoxynucleotide residues, and cordycepin.

[0095] An example of an oligomer that is typically blocked at its 3' end and is particularly suitable in certain embodiments using transcription-mediated amplification is a promoter provider. As described above, the promoter provider comprises a first target-hybridizing region and a second region located 5' from the first region that comprises a promoter sequence for an RNA polymerase. The promoter provider oligonucleotide is modified to prevent initiation of DNA synthesis from its 3' end, such as by including a blocker group as described above.

[0096] Another example of a typically 3'-blocked oligomer is a terminating ("blocker") oligonucleotide, as previously described above. Terminating oligomers are typically used in combination with, for example, a promoter provider amplification oligomer, such as in certain embodiments described herein with respect to transcription-mediated amplification (TMA). The terminating oligomer hybridizes to a sequence contained within the target nucleic acid near the 5' end of the target region to "terminate" primer extension of the nascent nucleic acid, including the priming oligonucleotide, thereby providing a defined 3' end of the nascent nucleic acid strand.

[0097] Other embodiments using transcription-mediated amplification utilize a promoter primer that includes a first target-hybridizing region and a second region located 5' to the first region that includes a promoter sequence for an RNA polymerase, but that is not modified to prevent initiation of DNA synthesis from its 3' end. In some embodiments, a promoter primer for use with the detection method includes (i) a Candida-specific target-hybridizing sequence substantially corresponding to the nucleotide sequence of residues 28-46 of SEQ ID NO:9, or (ii) a C. glabrata-specific target-hybridizing sequence that includes 15-24 contiguous nucleotides contained in the sequence of SEQ ID NO:134 and at least the sequence of SEQ ID NO:135. In some such embodiments, the promoter primer includes (i) a Candida-specific target-hybridizing sequence that includes or consists of the nucleotide sequence of residues 28-46 of SEQ ID NO:9, or (ii) a C. glabrata-specific target-hybridizing sequence that includes or consists of the nucleotide sequence of residues 28-49 of SEQ ID NO:14. In a more particular variation, the promoter primer for use with the detection method has the sequence shown in SEQ ID NO:9 or SEQ ID NO:14.

[0098] Assays for the detection of Candida species nucleic acids can optionally include a non-Candida species IC nucleic acid that is amplified and detected in the same assay reaction mixture by using amplification and detection oligomers specific for the internal control (IC) sequence. The IC nucleic acid sequence can be an RNA template sequence (e.g., an in vitro transcript), or a synthetic nucleic acid sequence added to the sample or the IC nucleic acid sequence can be a cellular constituent. The cellular constituent IC nucleic acid sequence can be from an exogenous or endogenous cellular source relative to the specimen. In these cases, the internal control nucleic acid is co-amplified with the Candida species nucleic acid in the amplification reaction mixture. The internal control amplification product and the Candida species target sequence amplification product can be detected independently.

[0099] Also provided by the present invention are reaction mixtures for determining the presence or absence of a Candida species target nucleic acid in a sample. Reaction mixtures according to the present invention include at least one or more of the oligomer combinations described herein for amplifying a Candida species target nucleic acid, the capture probe oligomers described herein for purifying a Candida species target nucleic acid, and the detection probe oligomers described herein for determining the presence or absence of a Candida species amplification product. The reaction mixture may further include several optional components, such as, for example, an array of capture probe nucleic acids. In the case of an amplification reaction mixture, the reaction mixture typically includes other reagents suitable for performing in vitro amplification, such as, for example, a buffer, a salt solution, appropriate nucleotide triphosphates (e.g., dATP, dCTP, dGTP, dTTP, ATP, CTP, GTP, and UTP), and / or enzymes (e.g., reverse transcriptase and / or RNA polymerase), and typically includes test sample components in which a Candida species target nucleic acid may or may not be present. Additionally, for reaction mixtures that include a detection probe along with a combination of amplification oligomers, the selection of amplification oligomers and detection probe oligomers in the reaction mixture are linked by a common target region (i.e., the reaction mixture includes a probe that binds to a sequence that can be amplified by the combination of amplification oligomers in the reaction mixture).

[0100] Kits for practicing the methods described herein are also provided by the present invention. Kits according to the present invention include at least one or more of the amplification oligomer combinations described herein for amplifying Candida species target nucleic acids, capture probe oligomers described herein for purifying Candida species target nucleic acids, and detection probe oligomers described herein for determining the presence or absence of Candida species amplification products. The kits may further include several optional components, such as an array of capture probe nucleic acids. Other reagents that may be present in the kit include reagents suitable for performing in vitro amplification, such as buffers, salt solutions, appropriate nucleotide triphosphates (e.g., dATP, dCTP, dGTP, dTTP, ATP, CTP, GTP, and UTP), and / or enzymes (e.g., reverse transcriptase and / or RNA polymerase). The oligomers described herein can be packaged in a variety of different embodiments, and those skilled in the art will understand that the present invention encompasses many different kit configurations. For example, a kit may include amplification oligomers for only one target region of the Candida species genome, or it may include amplification oligomers for multiple Candida species target regions. Additionally, for kits that include a detection probe along with a combination of amplification oligomers, the selection of amplification oligomers and detection probe oligomers in the kit are linked by a common target region (i.e., the kit includes a probe that binds to a sequence amplifiable by the combination of amplification oligomers in the kit). In certain embodiments, the kit further includes a set of instructions for practicing the methods of the present invention, which instructions may be associated with a package insert and / or the packaging of the kit or its components.

[0101] The present invention is further illustrated by the following non-limiting examples. [Example]

[0102] Example 1: Exemplary Protocol for Performing a Candida Amplification and Detection Assay One exemplary protocol for performing Candida amplification and detection reactions is as follows. (a) Reagent Preparation: The total volume of each component was determined based on the expected number of tests to be performed. The volume required for each oligo stock material was calculated and added to each reagent mix to achieve the desired final oligonucleotide concentration. (1) A total of four separate reagents were then prepared: TCR (target capture reagent: poly-T magnetic beads (magnetic beads conjugated to adT.sub.14 oligonucleotides), target capture oligos, and (optionally) T7 primers in aqueous HEPES buffer), AMP (amplification reagent: NT7 primers in a TRIS buffer solution containing salts and nucleotides), PRO (promoter reagent: T7 and Torch oligos in a TRIS buffer solution containing salts and nucleotides), and ENZ (enzyme solution: a mixture of MMLV reverse transcriptase and T7 RNA polymerase in an aqueous buffer containing glycerol). For the current example, unless otherwise noted, the following concentrations of oligonucleotides were used: 5 pmol / reaction of each T7 oligo in TCR, 15 pmol / reaction of each target capture oligo (TCO) in TCR, 15 pmol / reaction of each non-T7 oligo in AMP, 15 pmol / reaction of each T7 oligo in PRO, and 15 pmol / reaction of each Torch oligo in PRO. In preparing each reagent, the volume of the oligomer-free form of the reagent was determined based on the volume of oligonucleotide added to the reagent. The oligomer-free reagent was then dispensed, and each aliquot was brought to the maximum volume using the calculated oligomer volume.

[0103] Reactions were performed in an automated system (e.g., the Panther system (Hologic, Inc., Marlborough, MA)) in a pure system or semi-manual manner. For running reactions in an automated system such as the Panther system, the prepared reagents (TCR, AMP, PRO, ENZ) were loaded into the automated device, followed by the sample. The automated system then performed target capture, amplification, and data collection. For reactions run in a "pure system," there was no target capture step. Rather, the target nucleic acid (e.g., in vitro transcript) was added directly to the AMP mix in the microtiter plate, which then proceeded to the amplification reaction. For semi-manual reactions, the target capture and wash steps were performed as follows.

[0104] When performed using the semi-manual method, the target capture and wash steps were performed as follows: (1) AMP mix was dispensed into a 96-well PCR plate at 50 uL / well. Aptima wash buffer (e.g., Wash Solution (Hologic, Inc., Marlborough, MA, catalog number 302179)) was dispensed into a round-bottom Kingfisher deep-well plate at 500 uL / well and into a shallow Kingfisher 96-well plate at 200 uL / well. These three plates were then set aside. (2) TCR was dispensed into the deep-well plate at 100 uL / well, followed by 400 uL of sample / well. The sample-containing plate was then covered and placed on a Torrey Pines heat block (Torrey Pines Scientific, CA, catalog number IC25) and incubated at 62°C for 30 minutes. The heated plate was then slowly cooled to room temperature (20 minutes). (3) The room temperature plate was then transferred to a Kingfisher instrument for bead washing as generally described in the Aptima HCV RNA Qualitative Assay (Hologic, Inc., Cat. No. 302179) package insert. A tip comb (magnetic cover) was placed on the plate, the plate was placed in the Kingfisher instrument, and a wash program was run to collect the beads, transfer the collected beads to a deep-well wash plate, mix the plate, recapture the beads, and transfer them to a shallow-well wash plate. (4) After the wash program, the bead-containing plate was transferred to a Kingfisher instrument with a smaller comb (magnetic cover), after which the washed beads were collected and transferred / released to a plate containing AMP reagent.

[0105] The amplification and detection reactions were generally performed as follows: The plate containing the AMP reagent and sample was covered and incubated at 44°C for approximately 5 minutes using a Thermomixer® (shaker / heat block) (Eppendorf, Hamburg DE, Eppendorf model 5355). After the 5-minute incubation, 25uL of enzyme mix was added to each well, and the plate was re-covered. The covered plate was mixed at 1400 rpm for 1 minute and then incubated at 44°C for 5 minutes. After this incubation, 25uL of promoter mix (PRO) was added to each well, and the plate was mixed at 1400 rpm for 1 minute. The plate was then covered with an optical plate seal, and the sealed plate was immediately placed in a Stratagene real-time cycler (model Mx3005p, Agilent Technologies, CA) and incubated at 43°C. 150 cycles of FAM, HEX, and ATP were performed for 30 seconds each. Data was collected from each of the ROX channels, exported, and analyzed.

[0106] Example 2: Candida RT-TMA oligo screening This example illustrates assays for the amplification and detection of Candida species albicans, parapsilosis, tropicalis, and dubliniensis, and separately for the amplification and detection of Candida glabrata.

[0107] The assay consists of two sets of amplification and detection systems. The first system consists of a single broad-range T7 oligo, a pair of non-T7 oligos, and a labeled Torch oligo for amplification and detection of C. albicans, C. parapsilosis, C. tropicalis, and C. dubliniensis. The second system consists of a T7, a non-T7, and a labeled Torch oligo specific for amplification and detection of C. glabrata. The assay includes three target capture oligos (TCOs): one TCO is specific for C. glabrata, while the other two TCOs capture species detected by the broad-range amplification system. All oligos in the broad-range oligo combination and the C. glabrata-specific oligo combination are listed in Table 1. The oligomer sequences evaluated in this study are listed in Table 2. The entire amplification system can be performed in a two-phase real-time TMA assay format using separate promoters and amplification reagents. The entire amplification two-phase system involves four major steps: (i) target capture of target nucleic acids in a sample: The target capture reaction involved the general step of mixing the target nucleic acid with target capture oligomers (TCOs), poly-T magnetic beads, and T7 promoter primers (T7s). The TCOs and T7s were hybridized to the target nucleic acid, and the 3' ends of the TCOs were hybridized to the immobilized probes on the poly-T magnetic beads. The resulting hybridization complexes were then separated from other components of the sample. (ii) The separated hybridization complexes were washed to remove any remaining interfering substances, unbound target, and T7 from the sample, yielding substantially purified hybridization complexes. (iii) An initial linear amplification was then performed. The substantially purified hybridization complexes were resuspended in a linear amplification reagent containing a non-T7 primer (NT7) and the necessary reagents for amplification (i.e., reverse transcriptase with RNase activity, dNTPs, and salts). No additional T7 amplification oligomers were included in the linear amplification reagent. As a result, the linear amplification reaction yielded an initial amount of RNA transcripts as amplicons. (iv) Exponential amplification of the linear amplification products was then carried out. Briefly, after the duration of the linear amplification reaction, exponential amplification reagents were added to the reaction. The exponential amplification reagents contained T7 amplification oligomers and torch detection probe oligomers.Thus, the exponential amplification reagents provide the reaction components necessary to produce double-stranded DNA products (with double-stranded promoter sequences) from RNA transcript amplicons, thus exponentially increasing the number of double-stranded DNA molecules that produce RNA transcript amplicons. Once the RNA transcript amplicons are produced, a torch binds the amplicons and generates a detection signal. [Table 1] [Table 2-1] [Table 2-2]

[0108] Oligo selection summary The selection of oligos is primarily based on the following criteria: 1. Signal-to-noise ratio of fluorescent torches 2. Appearance of the amplification curve (if any). A sigmoidal curve usually indicates efficient amplification. 3.T time 4. Limits of detection

[0109] For simplicity, limit of detection (sensitivity) results are presented in this example: only the lowest tested concentration of in vitro transcript copies per reaction is shown in each table.

[0110] Amplification and detection were performed in a pure amplification system. A pure amplification system does not involve target capture, washing, or two-phase amplification. Instead, all amplification oligos and in vitro transcribed target nucleic acids are added to one common amplification reagent, and after an incubation period, an enzyme is added along with a torch for real-time detection. Oligo screening was performed in a pure system for simplicity. Unless otherwise specified in this example, most of the screening process was performed using a pure amplification system.

[0111] Wide range T7 oligos of SEQ ID NO:9 and SEQ ID NO:10 were each tested with various combinations of non-T7 oligos and torches. Initial testing was performed using only C. albicans in vitro transcripts (serial diluted) as the target nucleic acid. Table 3 shows the oligo combinations screened and the sensitivity results. The lowest concentration of in vitro transcript (IVT) tested was 1E4. [Table 3]

[0112] All oligo combinations showed the same detection limit (1E6), but the combination of SEQ ID NO:9 (T7) and SEQ ID NO:12 (non-T7) showed a slightly faster T time in this experiment. Varying the oligo concentration did not significantly change the sensitivity of these oligonucleotide combinations. All subsequent tests were performed using the T7 oligomer having the sequence shown in SEQ ID NO:9.

[0113] New oligo combinations were then tested to address the low RFU signal of C. parapsilosis and improve sensitivity for all Candida species targeted by this system. Table 4 shows the oligos tested in this screen. In the original screen, other Candida strains besides C. parapsilosis showed similar amplification as C. albicans. Therefore, these oligo combinations were tested with C. albicans and C. parapsilosis. [Table 4]

[0114] The oligo combination of SEQ ID NO:9, SEQ ID NO:26, and SEQ ID NO:27 significantly improved sensitivity for C. albicans, but no amplification was obtained for C. parapsilosis at the concentrations tested. Weak amplification of C. parapsilosis was observed at 1E8 IVT copies per reaction. Other Candida strains were tested using the oligo combination of SEQ ID NO:9, SEQ ID NO:26, and SEQ ID NO:27. The results are shown in Table 5. [Table 5]

[0115] As noted above, amplification of C. parapsilosis as well as other Candida species was observed using SEQ ID NO:9, SEQ ID NO:12, and SEQ ID NO:4. In further assays, SEQ ID NO:9 and SEQ ID NO:4 or SEQ ID NO:27 were tested in combination with several non-T7 oligos to determine sensitivity for amplification and detection of C. parapsilosis. Table 6 shows the full set of oligos used. The lowest tested concentration of IVT copies / reaction was 1E4 / uL. [Table 6]

[0116] The amplification and detection sensitivity of C. parapsilosis was 1E8 IVT copies / reaction when SEQ ID NO:27 was used with SEQ ID NO:9 and SEQ ID NO:12. Replacing the SEQ ID NO:27 torch with the SEQ ID NO:4 torch demonstrated a sensitivity of 1E6 IVT copies / rxn. Significantly improved sensitivity was seen for both the SEQ ID NO:4 and SEQ ID NO:27 torches when the non-T7 primer SEQ ID NO:12 was replaced with the non-T7 primer SEQ ID NO:34.

[0117] C. albicans was tested using SEQ ID NO:9, SEQ ID NO:34, and separately SEQ ID NOs:4 and 27. Amplification and detection of C. albicans was absent in these assays. However, by adding the non-T7 primer of SEQ ID NO:26 to these reactions, amplification and detection of all Candida species, including C. parapsilosis, was observed. However, amplification efficiency of C. albicans was adversely affected by the T time delay at low IVT copy levels, resulting in a one-log decrease in the limit of detection (1E4 to 1E5).

[0118] The oligo combinations of SEQ ID NO:9, SEQ ID NO:26, and SEQ ID NO:34, and SEQ ID NO:27 were then tested in an automated amplification and detection system (Panther system, Hologic, Inc.) using a two-phase amplification approach. When comparing the results of the pure and two-phase systems, no significant decrease in sensitivity was observed for any of the Candida species except for C. dubliniesis.

[0119] The combination of both non-T7 oligos in one single reaction using the two-phase system did not affect the amplification of either of the targets when compared to a two-phase reaction using the same two non-T7 oligos (SEQ ID NO:26 and SEQ ID NO:34) in individual reactions (see Tables 4, 5, and 6 for results with the oligos in individual reactions). The results for the pure system versus the two-phase system are shown in Table 7 below. Sensitivity concentrations are IVT copies / rxn. [Table 7]

[0120] Example 3: Sensitivity testing of feasibility RT-TMA reagent formulations for the amplification and detection of Candida albicans, Candida dubliniensis, Candida parapsilosis, Candida tropicalis, and Candida glabrata Analytical sensitivity was assessed using lysates from each of five Candida species. Each lysate was generated by growing the organisms in culture, quantitating them by plate count, and diluting them into sample transport medium (STM) (e.g., Cary-Blair Transport Medium (Becton-Dickenson & Co., NJ, Catalog No. 211102) and, e.g., Aptima Vaginal Swab Specimen Collection Kit (Hologic, Inc., Marlborough, MA, Catalog No. 301162)) to a normalized concentration of colony-forming units per milliliter (CFU / mL). Lysates in STM were serially diluted into STM to final concentrations of 100, 300, 1000, 3000, 10,000, and 30,000 CFU / mL.

[0121] Fifteen replicates of each concentration were used as samples in real-time transcription-mediated amplification and detection reactions (RT-TMA) on an automated amplification and detection system (Panther system (Hologic, Inc.)). The formulations were configured to amplify and detect RNAse P RNA from each of the following Candida species: a nucleic acid internal control, C. albicans, C. dublimiensis, C. parapsilosis, and C. tropicalis (collectively referred to as "broad-range"), and C. glabrata. The broad-range targets were amplified with a single T7 and two non-T7 primers and then detected with a single probe configured as a torch. C. glabrata was amplified with a single T7, a single non-T7, and a single torch oligo that was differentially labeled from the broad-range torch oligo to distinguish C. glabrata from the broad-range torch oligo. An internal control target was included along with amplification and detection oligos (not shown), and the detection oligo was differentially labeled to be distinguishable from the target. The nucleotide sequences of the Candida oligos used in the triplex reactions are shown in Table 8 below. [Table 8]

[0122] Results: Positivity was estimated as RFU above threshold at various cutoff times (in minutes). Table 9 below shows some positive replicates out of 15 using an RFU threshold of 3000 and a T time cutoff of 20 minutes for broad Candida species, and an RFU threshold of 1600 and a T time cutoff of 25 minutes for Candida glabrata. [Table 9]

[0123] Probit analysis was performed to estimate the 95% and 50% detection levels for each analyte. For C. albicans, the C50 (estimated level with a 50% probability of positivity) was 583 CFU / mL with a 95% confidence limit of 383–747, and the C95 (estimated level with a 95% probability of positivity) was 1035 CFU / mL with a 95% confidence limit of 809–1563. For C. tropicalis, the C50 was 200 (124–244) and the C95 was 309 (253–437). For C. dubliniensis, the C50 was 3142 (3000–3292) and the C95 was 3337 (3195–3495). For C. parapsilosis, the C50 was 292 (285-300) and the C95 was 310 (302-319). For C. glabrata, the C50 was 2841 (2763-2922) and the C95 was 3012 (2929-3097) CFU / mL.

[0124] Example 4: Specificity and interference testing of oligonucleotides for amplification and detection of Candida albicans, Candida dubliniensis, Candida parapsilosis, Candida tropicalis, and Candida glabrata To test the specificity of the reagent formulation from Example 3, a panel of non-target organisms was constructed in STM. Each panel member consisted of cell lysate material from one to five non-target organisms diluted in STM to a final concentration of 1 million CFU / mL. Organisms not viable at this concentration (Trichomonas vaginalis, Chlamydia trachomatis) were tested at lower concentrations (43,000 and 38,500 organisms per mL, respectively). The organisms in each sample panel were as follows: Panel 1: Acinetobacter iwoffii, Actinomyces israeliii, Alcaligenes faecalis, Bacteroides fragilis. Panel 2: Clostridium difficile, Corynebacterium genitalium, Enterobacter cloacae, Enterococcus feacalis, Escherichia coli. Panel 3: Bifidobacterium adolescentis, Campylobacter jejuni, Fusobacterium nucleatum, Haemophilus ducreyi, Klebsiella pneumoniae. Panel 4: Listeria monocytogenes, Mycoplasma hominis, Peptostreptococcus magnus, Propionibacterium acnes. Panel 5: Neisseria gonorrhoeae, Trichomonas vaginalis, Ureaplasma urealyticum, Ureaplasma Parvum. Panel 6: Candida krusei, Candida lusitaniae, Prevotella bivia, Eggerthella lenta. Panel 10: Pseudomonas aeruginosa, Mobiluncus curtisii, Chlamydia trachomatis, Cryptococcus neoformans.Panel 11: Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus agalactiae, Streptococcus pyogenes. Panel 12: Leptotrichia bucalis, Proteus vulgaris, Megaspahaera elsdenii, Atopobium vaginae. Panel 13: Lactobacillus acidophilus, Lactobacillus mucosae, Lactobacillus gastricus, Lactobacillus iners. Panel 14: Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri. Panel 15: Gardnerella vaginalis. Ten replicate reactions for each panel were run on an automated Panther system using the broad-range and C. glabrata oligonucleotide combinations shown in Table 1.

[0125] Specificity Results: No reactions were positive as judged by an RFU range above a threshold of 1600 in the fluorescence channel used for C. glabrata detection or a threshold of 3000 for the fluorescence channel used for broad-range oligo combination detection. All replicates of the internal control were positive, with an RFU range above the 1600 threshold.

[0126] To determine whether detection of Candida organisms was impaired by the presence of non-target organisms in the samples, each of the specificity panels described above was spiked with target organisms using the broad-range and C. glabrata oligonucleotide combinations shown in Table 1 before being run on the automated Panther system. The panels were spiked with either 3000 CFU / mL of C. parapsilosis or 3000 CFU / mL of C. albicans, and 3000 CFU / mL of C. glabrata. Results: All of the reactions (10 replicates per condition) were positive as judged by RFU range. No significant inhibition was observed.

[0127] Example 5: Evaluation of alternative torch designs for the detection of Candida glabrata Six alternative torch probe designs were directly compared for the detection of Candida glabrata in a two-phase amplification and detection format. Each torch was labeled with a FAM fluorophore and a Dabcyl quencher. Each reaction contained 10,000 CFU / reaction of Candida glabrata lysate diluted in STM. The cathodic control was STM without lysate. The various oligomer conditions all contained SEQ ID NOS: 12, 14, and 48, as well as one of SEQ ID NOS: 60, 18, 45, 46, 21, and 3. The nucleotide sequences of the Candida oligos used in this example are shown in Table 10 below. [Table 10]

[0128] Torch performance was assessed by performing RT-TMA amplification and detection reactions on eight replicates of each target per condition, with all reagents constant except for the identity of the Torch probe. Reactions were performed semi-manually, essentially as described above, using a Torrey Pines heat block (Torrey Pines Scientific, CA, catalog number IC25), a Kingfisher® extraction system (ThermoScientific, MA, catalog number 5400500), a Thermomixer® (shaker / heat block) (Eppendorf, Hamburg, DE, Eppendorf model 5355), and a Stratagene® real-time cycler (model Mx3005p, Agilent Technologies, Santa Clara, CA), or a Panther system (Hologic, Inc., Marlborough, MA).

[0129] The results are shown in Table 11 below. Two of the six torch probes (PR4 and PR6) showed insufficient discrimination between the non-target control and the Candida glabrata lysate target sample and were therefore unsuitable for use in the assay. Of the remaining four candidate torch probes, PR2 and PR1 had favorable performance compared to PR3 and PR5 due to lower mean T time and higher mean T slope. [Table 11]

[0130] Example 6: Alternative non-T7 primer designs for the detection of Candida species Five individual non-T7 primers were tested using RT-TMA for broad-spectrum Candida species detection. Each non-T7 primer was used at 15 pmol per reaction in the amplification mix. Common reagents were used for the TCR and promoter solutions. The TCR contained 15 pmol / rxn each of target capture oligos of SEQ ID NOs: 24 and 66, and 5 pmol / rxn of the T7 primer oligo of SEQ ID NO: 9. The promoter solution contained 15 pmol / rxn of the T7 primer of SEQ ID NO: 9 and 15 pmol / rxn of the torch oligo of SEQ ID NO: 27. The non-T7 primers compared are shown in Table 12 below. [Table 12]

[0131] The targets for amplification were in vitro transcripts containing partial RNase PR1 gene sequences from each of the following species: Candida albicans, Candida parapsilosis, Candida tropicalis, and Candida dubliniensis.

[0132] Reactions were performed semi-manually and analyzed using a Torrey Pines heat block, Kingfisher® extraction system, Thermomixer® (shaker / heat block), and Stratagene® real-time cycler essentially as described in Example 5 above.

[0133] Results: The non-T7 primer of SEQ ID NO: 73 did not detectably amplify the target. The non-T7 primer of SEQ ID NO: 74 amplified the target with a faster T time than the non-T7 primer of SEQ ID NO: 26, while the non-T7 primer of SEQ ID NO: 75 amplified C. parapsilosis with a slower T time than the non-T7 primer of SEQ ID NO: 34.

[0134] Example 7: Comparison of Candida species torches with alternative stem configurations Two alternative torch designs were functionally compared to assess the potential impact of design differences on clinical accuracy and analytical performance. The torches were designed to contain the same analyte-specific region (for detection of broad-spectrum target nucleic acids (C. albicans, C. dubliniensis, C. parapsilosis, or C. tropicalis)) and the same linker, but varied in the two 3'-most bases. Both oligonucleotides were composed of methoxy-RNA with the two 3'-most bases either CC or GG.

[0135] Two experiments were performed. In the first experiment, multiplex Candida formulations were constructed (broad-range oligo, C. glabrata oligo, and internal control oligo), but the broad-range torch oligo was omitted. The reagents were then split in half, and either SEQ ID NO:27 or SEQ ID NO:155 was added at 15 pmoles per reaction to complete each formulation. Each completed formulation was run on the Panther system against analytical (control) samples, and negative samples were pooled (remaining vaginal swab material from presumptive negative samples was pooled together and then split for equal testing with the two formulations). Data represent two pooled negative samples in 10 replicates per formulation per pool, and a positive control Candida albicans lysate at 1E4 colony-forming units per milliliter in five replicates per formulation. As shown in Table 13 below, the SEQ ID NO:155 torch produced a higher RFU range, minus the positive control background, than the SEQ ID NO:27 torch, with similar variability (%CV) from replicate to replicate. The average background signal for these reactions was 3337 RFU for the torch of SEQ ID NO: 27 and 1182 for the torch of SEQ ID NO: 155. In the pooled negative samples, the torch of SEQ ID NO: 155 produced a lower RFU range and lower replicate-to-replicate variability (%CV) than the torch of SEQ ID NO: 27. Based on these data, the torch of SEQ ID NO: 155 produced better discrimination between positive and cathodic samples. [Table 13]

[0136] In a second experiment, 50 clinical samples were run on an automated Panther system (Hologic, Inc., Marlborough, MA) using two formulations of Candida reagent (broad range reported for FAM and Candida glabrata reported for HEX). Formulation 1 used SEQ ID NO:27 (FAM torch) at 32 pmoles per reaction, and formulation 2 used SEQ ID NO:155 (FAM torch) at 10 pmoles per reaction. Otherwise, the formulations were identical. Candida glabrata (HEX) torch (SEQ ID NO:60) was present at 26 pmoles per reaction in both formulations. Each clinical sample was run in one replicate with each formulation. Background signals were similar on the HEX channel for both formulations, but formulation 1 had a substantially higher background than formulation 2. The average background signal was 5645 (FAM-Formulation 1), 638 (FAM-Formulation 2), 1457 (HEX-Formulation 1), and 1347 (HEX-Formulation 2). The distribution of background-subtracted RFU ranges from the 48 valid results is shown in Table 14 below (number of positives out of N=48 for each condition). For the HEX results, no samples yielded a range of 1250-5000 RFU, suggesting the possibility of setting an RFU range threshold to distinguish positive from negative samples. For FAM, Formulation 2 also provided robust separation between samples yielding high or low RFU ranges, whereas Formulation 1 did not. The absence of results for samples in the intermediate RFU range in this experiment suggests that SEQ ID NO:155 is a better performing torch than SEQ ID NO:27 for broad-range oligo combinations. [Table 14] array [Table 15-1] [Table 15-2] [Table 15-3] [Table 15-4]

[0137] From the foregoing, it will be understood that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. [ka]

Claims

[Claim 1] The invention described in this specification.

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