Compositions and methods for detecting Candida species

Abstract

A method for rapid detection of the presence or absence of vulvovaginal candidiasis (VVC)-associated Candida species in biological or non-biological samples is described. The method may include performing amplification, hybridization, and detection steps. Additionally, primers and probes targeting specific genes and kits designed for the detection of VVC-associated Candida species are provided.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 486,974, filed February 25, 2023, which is incorporated by reference in its entirety.

[0002] Sequence Listing Reference This application contains a Sequence Listing that has been submitted as an electronic text file entitled "P38277-WO PCT filing seq_listing.xml," which is 12,306 bytes in size and was created on January 25, 2024. The information contained in this electronic file is hereby incorporated by reference in its entirety pursuant to 37 CFR § 1.52(e)(5).

[0003] The present disclosure relates to the field of molecular diagnostics, and more particularly to the detection of various fungal strains associated with Candida vaginitis. [Background technology]

[0004] Vaginitis accounts for as much as 50% of all gynecological visits in the United States and is a leading cause of healthcare costs. Bacterial vaginosis (BV), vulvovaginal candidiasis (VVC), and infectious vaginitis due to trichomoniasis account for up to 90% of these cases (2). Unlike trichomoniasis, both BV and VVC are caused by several pathogens. In the case of VVC, Candida albicans overgrowth predominates, but other Candida species, including Candida glabrata, can contribute as well. BV can be difficult to diagnose because the etiology involves decreased levels of Lactobacillus bacteria, along with increased concentrations of BV-associated bacteria, such as Gardnerella vaginalis, Mobiluncus species, and Atopobium vaginae.

[0005] Various diagnostic methods are available to identify the underlying cause of vaginitis. In clinicians' offices, a combination of pH, potassium hydroxide (KOH) testing, and microscopy of fresh samples of vaginal secretions is routinely used, despite their relatively poor performance. In the case of BV, diagnosis often relies on the use of either clinical Amsel criteria or Gram staining and Nugent's score (considered the gold standard for diagnosing BV). Examination of wet mounts with KOH preparations and / or vaginal cultures for Candida is the most common diagnostic tool for VVC. Although highly sensitive and specific nucleic acid amplification tests (NAATs) are recommended for the detection of Trichomonas vaginalis, testing of wet mount preparations remains commonly used in clinical settings. However, the use of non-molecular methods is associated with several barriers, including a lack of clinic facilities, subjectivity of the clinical endpoints used, and inconsistent use among physicians, a lack of appropriate training in microscopy, and the overall low sensitivity of the tests. Diagnosis of the underlying cause of vaginitis is further complicated by the common symptomatology reported for BV, VVC, and trichomoniasis, the incidence of mixed or coinfections, and the recurrence of vaginal symptoms.

[0006] Therefore, there is a need to develop more efficient and rapid methods for detecting vulvovaginal candidiasis and bacterial vaginosis, for example, methods that allow for the detection of both vaginal disorders in a single assay in order to effectively deliver appropriate treatment to patients. Summary of the Invention

[0007] In one aspect, the present disclosure provides a method for detecting vulvovaginal candidiasis (VVC)-associated Candida species in a biological sample, including Candida albicans, Candida tropicalis, Candida dubliniensis, and Candida parapsilosis (collectively referred to as Candida spp.).

[0008] As used herein, the method includes performing an amplification step, which includes contacting a sample with a primer set to produce an amplification product if nucleic acid from VVC-associated Candida is present in the sample; performing a hybridization step, which includes contacting each amplification product with one or more detectable probes; and detecting the presence of each amplification product, wherein the presence of the amplification product indicates the presence of VVC-associated Candida in the sample. The primer set for producing amplification products from Candida species includes one or more forward primers comprising or consisting of an oligonucleotide sequence selected from SEQ ID NOs: 1 to 5 or any combination thereof, one or more reverse primers comprising or consisting of an oligonucleotide sequence selected from SEQ ID NOs: 7 to 14 or any combination thereof, and one or more detectable probes comprising or consisting of the oligonucleotide sequence of SEQ ID NO: 15 or its complement. In some embodiments, a primer set for producing an amplification product from Candida species comprises a forward primer comprising or consisting of the oligonucleotide sequence of SEQ ID NO: 1, at least first and second reverse primers (wherein the first reverse primer comprises or consists of an oligonucleotide sequence selected from SEQ ID NOs: 7-9, and the second reverse primer comprises or consists of an oligonucleotide sequence selected from SEQ ID NOs: 10-14), and one or more detectable probes comprising or consisting of the oligonucleotide sequence of SEQ ID NO: 15.In some embodiments, the primer set for producing an amplification product derived from Candida species includes at least a first and a second forward primer (the first forward primer comprises or consists of an oligonucleotide sequence selected from SEQ ID NOS: 2-5), at least a first and a second reverse primer (the first reverse primer comprises or consists of an oligonucleotide sequence selected from SEQ ID NOS: 7-9, and the second reverse primer comprises or consists of an oligonucleotide sequence selected from SEQ ID NOS: 10-14), and one or more detectable probes comprising or consisting of the oligonucleotide sequence of SEQ ID NOS: 15. In other embodiments, the hybridizing step includes contacting the amplification product with a detectable probe labeled with a donor fluorescent moiety and a corresponding acceptor moiety, and the detecting step includes detecting the presence or absence of fluorescence resonance energy transfer (FRET) between the donor fluorescent moiety and the acceptor moiety of the probe, where the presence or absence of fluorescence indicates the presence or absence of VVC-associated Candida in the sample. In further embodiments, the amplifying step uses a polymerase enzyme with 5'-3' nuclease activity. In some embodiments, the "contacting" step further comprises contacting the biological sample and the primer with a DNA polymerase, a plurality of free nucleotides including adenine, thymine, cytosine, and guanine, and / or a buffer to produce a reaction mixture. The nucleic acid extracted from the biological sample may comprise or consist of double-stranded DNA. The reaction mixture may optionally further comprise divalent cations, monovalent cation potassium ions, one or more detectably labeled probes, and / or any combination thereof.In some embodiments, the step of "generating amplicons" includes (a) heating the reaction mixture to a first predetermined temperature for a first predetermined time to separate the strands of double-stranded DNA present in the biological sample or nucleic acid, (b) cooling the reaction mixture to a second predetermined temperature for a second predetermined time under conditions that allow the primers to hybridize to their complementary sequences and the DNA polymerase to extend the primers, and (c) repeating steps (a) and (b) at least 10-12 times. In some embodiments, steps (a) and (b) are repeated at least 15, 20, 22, or 25 times.

[0009] In another aspect, a kit for detecting vulvovaginal candidiasis (VVC)-associated Candida species in a sample is provided, comprising at least one forward primer comprising or consisting of an oligonucleotide sequence selected from the group consisting of SEQ ID NOS: 1-5, or any combination of SEQ ID NOS: 1-5; at least one reverse primer comprising or consisting of an oligonucleotide sequence selected from the group consisting of SEQ ID NOS: 7-14, or any combination of SEQ ID NOS: 7-14; and a detectably labeled probe comprising or consisting of the oligonucleotide sequence of SEQ ID NOS: 15 or its complement, wherein the detectably labeled oligonucleotide sequence is configured to hybridize to an amplicon generated by the forward and reverse primers. In some embodiments, the detectably labeled oligonucleotide sequence comprises a donor fluorescent moiety and a corresponding acceptor moiety. In some embodiments, the kit further comprises nucleoside triphosphates, a nucleic acid polymerase, and a buffer necessary for nucleic acid polymerase function. In still other embodiments, at least one of the oligonucleotide sequences comprises at least one modified nucleotide. In some embodiments, VVC-associated Candida species include Candida albicans, Candida tropicalis, Candida dubliniensis, and Candida parapsilosis (collectively referred to as Candida species). In some embodiments, the kit includes primers and probes capable of hybridizing to the 18s ribosomal RNA (18s rRNA) of Candida species, comprising or consisting of an oligonucleotide sequence selected from SEQ ID NOs: 1 and 15. In some embodiments, the kit includes primers and probes capable of hybridizing to the internal transcribed spacer 1 (ITS1) between the 18s rRNA gene and the 5.8s rRNA gene of Candida species, comprising or consisting of an oligonucleotide sequence selected from SEQ ID NOs: 2-5 and 7-14.

[0010] In some embodiments, the sample is a biological sample. In some embodiments, the biological sample is collected from the urethra, penis, anus, throat, cervix, or vagina. In some embodiments, the biological sample is DNA, RNA, or total nucleic acid extracted from a clinical specimen.

[0011] Additionally, the present disclosure provides oligonucleotides comprising a nucleic acid having at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, or 95%) to one of SEQ ID NOs: 1-5 and 7-15, the oligonucleotide having 100 or fewer nucleotides, or its complement. Generally, these oligonucleotides may be primer nucleic acids, probe nucleic acids, etc., in these embodiments. In some embodiments, the oligonucleotide comprises at least one modified nucleotide, e.g., to alter nucleic acid hybridization stability compared to unmodified nucleotides. In some embodiments, the at least one modified nucleotide is N 6 -benzyl-dA,N 4 -benzyl-dC,N 6 -para-tert-butyl-benzyl-dA,N 4The oligonucleotide is selected from the group consisting of -para-tert-butyl-benzyl-dC, and 2'-O-methyl-rU. Optionally, the oligonucleotide comprises at least one label and / or at least one quencher moiety. In some embodiments, the oligonucleotide comprises at least one conservatively modified variation. A "conservatively modified variation" or simply "conservative variation" of a particular nucleic acid sequence refers to a nucleic acid that encodes the same or essentially the same amino acid sequence, or, if the nucleic acid does not encode an amino acid sequence, an essentially identical sequence. Those skilled in the art will recognize that individual substitutions, deletions, or additions that alter, add, or delete a single amino acid or a small percentage of amino acids (typically less than 5%, more typically less than 4%, 2%, or 1%) in the encoded sequence are "conservatively modified variations," where the alteration results in the deletion, addition, or substitution of an amino acid with a chemically similar amino acid. In some embodiments, at least one of the first and second target gene primers and the detectable target gene probe comprises at least one modified nucleotide.

[0012] In another aspect, the Candida species and Candida krusei in the sample are and detecting the presence of each amplification product, wherein the presence of the amplification product indicates the presence of Candida spp. and at least one selected from the group consisting of Candida krusei and Candida glabrata, Lactobacillus spp., Gardnerella vaginalis, and Atopobium vaginae in the sample. In one embodiment, a primer set for producing an amplification product from Lactobacillus species comprises a forward primer comprising or consisting of the oligonucleotide sequence of SEQ ID NO: 19, a reverse primer comprising or consisting of the oligonucleotide sequence of SEQ ID NO: 20, and one or more detectable probes, one of which comprises or consists of the oligonucleotide sequence of SEQ ID NO: 21. In another embodiment, a primer set for producing an amplification product from Gardnerella vaginalis comprises a forward primer comprising or consisting of the oligonucleotide sequence of SEQ ID NO: 16, a reverse primer comprising or consisting of the oligonucleotide sequence of SEQ ID NO: 17, and one or more detectable probes, one of which comprises or consists of the oligonucleotide sequence of SEQ ID NO: 18 and one of which comprises or consists of the oligonucleotide sequence of SEQ ID NO: 31.In another embodiment, a primer set for producing an amplification product from Atopobium vaginae comprises a forward primer comprising or consisting of the oligonucleotide sequence of SEQ ID NO: 22, a reverse primer comprising or consisting of the oligonucleotide sequence of SEQ ID NO: 23, and one or more detectable probes, one of which comprises or consists of the oligonucleotide sequence of SEQ ID NO: 24 and one of which comprises or consists of the oligonucleotide sequence of SEQ ID NO: 32. In another embodiment, a primer set for producing an amplification product from Candida krusei comprises a forward primer comprising or consisting of the oligonucleotide sequence of SEQ ID NO: 25, a reverse primer comprising or consisting of the oligonucleotide sequence of SEQ ID NO: 26, and one or more detectable probes, one of which comprises or consists of the oligonucleotide sequence of SEQ ID NO: 27. In another embodiment, a primer set for producing an amplification product from Candida glabrata comprises a forward primer comprising or consisting of the oligonucleotide sequence of SEQ ID NO: 28, a reverse primer comprising or consisting of the oligonucleotide sequence of SEQ ID NO: 29, and one or more detectable probes comprising or consisting of the oligonucleotide sequence of SEQ ID NO: 30.

[0013] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.Methods and materials similar or equivalent to those described herein can be used in the practice or testing of this subject, and suitable methods and materials are described below.In addition, materials, methods, and examples are only illustrative and are not intended to be limiting.All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.In case of conflict, the present specification, including definitions, will prevail.

[0014] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the drawings and detailed description, and from the claims. [Brief explanation of the drawings]

[0015] [Figure 1] Genomic organization of ribosomal RNA genes in Candida albicans, Candida dubliniensis, Candida tropicalis, Candida parapsilosis, Candida krusei, and Candida glabrata is shown. Sequence homology of 18s rRNA, 5.8s rRNA, and 25s rRNA of Candida species to Candida albicans is shown on the right. DETAILED DESCRIPTION OF THE INVENTION

[0016] Methods and compositions for detecting vulvovaginal candidiasis (VVC) are provided herein. For example, primers and probes capable of binding to specific genes of VVC-associated Candida species are provided to determine the presence or absence of VVC-associated Candida species in a sample, such as a biological sample. In some embodiments, multiplex nucleic acid amplification can be performed to enable the detection of VVC-associated Candida species and bacterial vaginosis (BV)-associated bacteria in a single assay. Detection of BV-associated bacteria and the VVC-associated species Candida krusei and Candida glabrata is described in U.S. Patent Application Publication No.

[0017] This can be carried out using the compositions and methods disclosed in US2022 / 0205020A1, which is incorporated herein by reference in its entirety.

[0018] The methods of the present invention may include performing at least one cycling step, which involves amplifying one or more portions of a nucleic acid molecule gene target from a sample using one or more primer pairs. As used herein, "primer(s)" refers to oligonucleotide primers that specifically anneal to a target gene in Candida species and initiate DNA synthesis therefrom under appropriate conditions to produce a respective amplification product. Each of the considered primers anneals to a target within or adjacent to a respective target nucleic acid molecule such that at least a portion of each amplification product contains a nucleic acid sequence corresponding to the target. If one or more target gene nucleic acids are present in the sample, one or more amplification products will be produced, and therefore the presence of one or more target gene amplification products indicates the presence of Candida species in the sample. The amplification products must contain nucleic acid sequences complementary to one or more detectable probes of the target gene. As used herein, "probe(s)" refers to oligonucleotide probes that specifically anneal to a nucleic acid sequence encoding the target gene. Each cycling step includes an amplification step, a hybridization step, and a detection step, in which the sample is contacted with one or more detectable probes to detect the presence or absence of Candida species in the sample.

[0019] The term "amplifying" as used herein refers to the process of synthesizing a nucleic acid molecule complementary to one or both strands of a template nucleic acid molecule. Amplifying a nucleic acid molecule typically involves denaturing the template nucleic acid, annealing a primer to the template nucleic acid at a temperature below the melting temperature of the primer, and enzymatically extending the primer to generate an amplification product. Amplification typically requires the presence of deoxyribonucleoside triphosphates, a DNA polymerase enzyme (e.g., Platinum® Taq), and an appropriate buffer and / or cofactors for optimal activity of the polymerase enzyme (e.g., MgCl2 and / or KCl).

[0020] The term "primer," as used herein, is known to those skilled in the art and refers to an oligomeric compound, primarily oligonucleotides, but also modified oligonucleotides that can "prime" DNA synthesis by a template-dependent DNA polymerase, i.e., for example, an oligonucleotide whose 3'-terminus provides a free 3'-OH group, whereby a deoxynucleoside triphosphate is used to establish a 3'-5' phosphodiester bond to which further "nucleotides" can be attached by the template-dependent DNA polymerase, thereby releasing pyrophosphate. Thus, there is no fundamental difference between a "primer," an "oligonucleotide," or a "probe," except perhaps in their intended function.

[0021] The term "hybridizing" refers to the annealing of one or more probes to an amplification product. Hybridization conditions typically include a temperature below the melting temperature of the probe, but which avoids non-specific hybridization of the probe.

[0022] The term "5' to 3' nuclease activity" refers to the activity of a nucleic acid polymerase typically associated with nucleic acid chain synthesis whereby nucleotides are removed from the 5' end of a nucleic acid chain.

[0023] The term "thermostable polymerase" refers to a polymerase enzyme that is thermostable, catalyzes the formation of primer extension products complementary to a template, and does not irreversibly denature when exposed to elevated temperatures for the time required to effect denaturation of the double-stranded template nucleic acid. Generally, synthesis is initiated at the 3' end of each primer and proceeds in the 5' to 3' direction along the template strand. Thermostable polymerases have been isolated, for example, from Thermus flavus, T. ruber, T. thermophilus, T. aquaticus, T. lacteus, T. rubens, Bacillus stearothermophilus, and Methanothermus fervidus. Nevertheless, polymerases that are not thermostable can also be used in PCR assays if the enzyme is replenished.

[0024] The term "complement thereof" refers to a nucleic acid that is the same length as and exactly complementary to a given nucleic acid.

[0025] The term "extension" or "lengthening" when used with respect to nucleic acids refers to when additional nucleotides (or other similar molecules) are incorporated into a nucleic acid. For example, a nucleic acid is optionally extended by a biocatalyst that incorporates a nucleotide, e.g., a polymerase that typically adds a nucleotide to the 3' end of the nucleic acid.

[0026] The terms "identical" or percent "identity" in the context of two or more nucleic acid sequences refer to two or more sequences or subsequences that are identical or have a specified percentage of identical nucleotides when compared or aligned for maximum correspondence as determined, for example, using one of the sequence comparison algorithms available to those of skill in the art or by visual inspection. Exemplary algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST programs, see, e.g., Altschul et al. (1990) "Basic local alignment search tool," J. Mol. Biol. 215:403-410; Gish et al. (1993) "Identification of protein coding regions by database similarity search," Nature Genet. 3:266-272; Madden et al. (1996) "Applications of network BLAST server," Meth. Enzymol. 266:131-141; Altschul et al. (1997) "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs," Nucleic Acids Res. 25:3389-3402; and Zhang et al. (1997) "PowerBLAST: A new network BLAST application for interactive or automated sequence analysis and annotation," Genome Res. 7:649-656, which are incorporated herein by reference.

[0027] " Modified nucleotide " in the context of oligonucleotide refers to the change in which at least one nucleotide of oligonucleotide sequence is replaced by a different nucleotide, which provides the oligonucleotide with desired properties.The exemplary modified nucleotide that can be substituted in the oligonucleotide described herein includes, for example, C5-methyl-dC, C5-ethyl-dC, C5-methyl-dU, C5-ethyl-dU, 2,6-diaminopurine, C5-propynyl-dC, C5-propynyl-dU, C7-propynyl-dA, C7-propynyl-dG, C5-propargylamino-dC, C5-propargylamino-dU, C7-propargylamino-dA, C7-propargylamino-dG, 7-deaza-2-deoxyxanthosine, pyrazolopyrimidine analogues, pseudo-dU, nitropyrrole, nitroindole, 2'-0-methylriboU, 2'-0-methylriboC, N4-ethyl-dC, N 6 -methyl-dA,N 6 -benzyl-dA,N 4 -benzyl-dC,N 6 -para-tert-butylbenzyl-dA,N 4 -para-tert-butyl-benzyl-dC, etc. Many other modified nucleotides that can be substituted in oligonucleotides are mentioned herein or known in the art. In certain embodiments, the modified nucleotide substitution modifies the melting temperature (Tm) of the oligonucleotide compared to the melting temperature of the corresponding unmodified oligonucleotide. To further explain, some modified nucleotide substitutions can, in some embodiments, reduce non-specific nucleic acid amplification (e.g., minimize primer-dimer formation, etc.), increase the yield of the intended target amplicon, etc. Examples of these types of nucleic acid modifications are described, for example, in U.S. Patent No. 6,001,611, which is incorporated herein by reference.

[0028] Detection of Candida species As described herein, nucleic acid amplification can be performed to determine the presence, absence, and / or level of Candida species in a sample. Several Candida species, including but not limited to, C. albicans, C. tropicalis, C. dubliniensis, C. parapsilosis, C. krusei, and C. glabrata, are known to be associated with VVC. Detection of VVC-associated Candida species can be performed simultaneously with detection of BV-associated bacterial species. Many bacteria known to be associated with BV include, but are not limited to, Lactobacillus species (e.g., Lactobacillus crispatus (L. crispatus), Lactobacillus jensenii (L. jensenii), and Lactobacillus gasseri (L. gasseri)), Gardnerella vaginalis (G. vaginalis), Atopobium vaginae, Megasphaera type 1 (Megasphaera-1), and BVAB-2. In some embodiments, the presence, absence, and / or levels of VVC-associated Candida species and BV-associated bacteria are determined by detecting one or more target genes for each target organism using methods known in the art, such as DNA amplification. In some embodiments, multiplex PCR can be performed to detect the presence, absence, or levels of each of the target Candida species, which may include simultaneous detection of BV-associated bacteria. In some embodiments, multiplex PCR is performed to detect the presence, absence, and / or levels of target VVC-associated Candida species and each of BV-associated bacteria, including L. crispatus, L. jensenii, G. vaginalis, Atopobium vaginae, Megasphaera type 1, and BVAB-2. In some embodiments, the VVC-associated Candida species are C. albicans, C. tropicalis, C. dubliniensis, C. parapsilosis, C. krusei, and C. glabrata.

[0029] In another embodiment, nucleic acid amplification can be performed on the same sample to determine the presence, absence, and / or levels of Trichomonas vaginalis (TV). Compositions and methods for rapid detection of the presence or absence of Trichomonas vaginalis in biological or non-biological samples are described in U.S. Patent Application Publication No. 2017 / 0342508, which is incorporated herein by reference in its entirety.

[0030] Each target VVC-associated Candida species can be detected using a separate channel in the DNA amplification. In some cases, it may be desirable to use a single fluorescent channel to detect the presence, absence, and / or levels of two or more VVC-associated Candida species. Such a combination, in some embodiments, can reduce the amount of reagents required to perform an experiment and provide an accurate qualitative metric by which the determination of a VVC-associated Candida species can be assessed. Without being bound by theory, it is believed that the use of combined markers can increase the sensitivity and specificity of the assay. In some embodiments, separate fluorescent channels are used to detect the presence, absence, and / or levels of each Candida species (e.g., C. albicans, C. tropicalis, C. dubliniensis, C. parapsilosis).

[0031] Oligonucleotides (e.g., amplification primers and probes) are provided that can specifically hybridize (e.g., under standard nucleic acid amplification conditions, e.g., under standard PCR conditions and / or stringent hybridization conditions) to target gene regions or their complements in VVC-associated Candida species. Amplification of the target gene region of an organism in a sample (e.g., a vaginal swab sample) can, in some embodiments, indicate the presence, absence, and / or level of the organism in the sample.

[0032] The target gene region can vary. In some embodiments, oligonucleotides (e.g., amplification primers and probes) are provided that can specifically hybridize (e.g., under standard nucleic acid amplification conditions, e.g., under standard PCR conditions and / or stringent hybridization conditions) to the target gene region in an organism.

[0033] In some embodiments, the 18s ribosomal RNA (18s rRNA) of the Candida species ribosomal RNA (rRNA) gene is used as a target gene for DNA amplification to detect the presence, absence, and / or level of VVC-associated Candida species in a sample. In some embodiments, the ITS1 between the 18s rRNA gene and the 5.8s rRNA gene of Candida species is used as a target region for DNA amplification to detect the presence, absence, and / or level of VVC-associated Candida species in a sample. In some embodiments, VVC-associated Candida species include C. albicans, C. tropicalis, Candida dubliniensis, and C. parapsilosis (collectively, Candida species). In some embodiments, VVC-associated Candida species are C. albicans, C. tropicalis, C. dubliniensis, C. parapsilosis, or a combination thereof. Examples of oligonucleotides capable of specifically hybridizing to the 18s rRNA or ITS1 region of Candida species are shown in Table 1. [Table 1]

[0034] In one embodiment, the primer sets and probes described above are used to provide for the detection of Candida species associated with vaginitis in biological samples suspected of containing such Candida species. The primer sets and probes can comprise or consist of primers and probes specific to the nucleic acid sequences of Candida target genes comprising or consisting of the nucleic acid sequences of SEQ ID NOS: 1-5 and 7-15. In another embodiment, the primers and probes for the target genes comprise or consist of functionally active variants of any of the primers and probes of SEQ ID NOS: 1-5 and 7-15.

[0035] Functionally active variants of any of the primers and / or probes of SEQ ID NOS: 1-5 and 7-15 can be identified by using the primers and / or probes in the disclosed methods. Functionally active variants of any of the primers and / or probes of SEQ ID NOS: 1-5 and 7-15 relate to primers and / or probes that provide similar or greater specificity and sensitivity in the described methods or kits compared to the respective sequences of SEQ ID NOS: 1-5 and 7-15.

[0036] Variants can vary from the sequences of SEQ ID NOS: 1-5 and 7-15 by the addition, deletion, or substitution of one or more nucleotides, such as the addition, deletion, or substitution of one or more nucleotides at the 5' and / or 3' ends of each of the sequences of SEQ ID NOS: 1-5 and 7-15. As detailed above, primers (and / or probes) can be chemically modified, i.e., the primers and / or probes can contain modified nucleotides or non-nucleotide compounds. Thus, the probes (or primers) are modified oligonucleotides. A "modified nucleotide" (or "nucleotide analog") differs from a natural "nucleotide" by some modification but still consists of a base or base-like compound, a pentofuranosyl sugar or pentofuranosyl sugar-like compound, a phosphate moiety or phosphate-like moiety, or a combination thereof. For example, a "label" can be attached to the base portion of a "nucleotide" to thereby obtain a "modified nucleotide." A natural base in a "nucleotide" can also be replaced with, for example, a 7-deazapurine, thereby obtaining a "modified nucleotide." The terms "modified nucleotide" or "nucleotide analog" are used interchangeably in this application. A "modified nucleoside" (or "nucleoside analog") differs from a natural nucleoside by some modification, as outlined above for "modified nucleotides" (or "nucleotide analogs").

[0037] Oligonucleotides, including modified oligonucleotides and oligonucleotide analogs, that amplify nucleic acid molecules encoding target genes can be designed using computer programs such as OLIGO (Molecular Biology Insights Inc., Cascade, Colorado). Important features when designing oligonucleotides to be used as amplification primers include, but are not limited to, an appropriate size for the amplification product to facilitate detection (e.g., by electrophoresis), similar melting temperatures for the members of the pair of primers, and the length of each primer (i.e., primers must be long enough to anneal and initiate synthesis with sequence specificity, but not so long that fidelity is reduced during oligonucleotide synthesis). Typically, oligonucleotide primers are 8 to 50 nucleotides in length (e.g., 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50 nucleotides in length). In some embodiments, oligonucleotide primers are 40 nucleotides or less in length.

[0038] In addition to the primer set, the method can use one or more probes to detect the presence or absence of the target Candida species. The term "probe" refers to a synthetically or biologically produced nucleic acid (DNA or RNA) that contains a specific nucleotide sequence that, by design or selection, allows it to specifically (i.e., preferentially) hybridize to the target gene nucleic acid under a defined, predetermined stringency. The "probe" can also be referred to as a "detection probe," which means that it detects the target nucleic acid.

[0039] In some embodiments, the disclosed target gene probe can be labeled with at least one fluorescent label. In one embodiment, the target gene probe can be labeled with a donor fluorescent moiety (e.g., a fluorescent dye) and a corresponding acceptor moiety (e.g., a quencher). In one embodiment, the probe comprises or consists of a fluorescent moiety, and the nucleic acid sequence comprises or consists of SEQ ID NO: 15.

[0040] Oligonucleotides used as probes can be designed in a similar manner to primer design. In embodiments, a single probe or a pair of probes may be used for detecting amplification products. Depending on the embodiment, the probe(s) used may contain at least one label and / or at least one quencher moiety. Like primers, probes typically have similar melting temperatures, and the length of each probe must be sufficient for sequence-specific hybridization to occur, but not so long that fidelity is reduced during synthesis. Oligonucleotide probes are generally 15 to 40 (e.g., 16, 18, 20, 21, 22, 23, 24, or 25) nucleotides in length.

[0041] The construct can comprise a vector, each containing one of target gene primer and probe nucleic acid molecules.The construct can be used, for example, as a control template nucleic acid molecule.Suitable vectors for use are commercially available and / or produced by recombinant nucleic acid technology methods routine in the art.Target gene nucleic acid molecules can be obtained, for example, by chemical synthesis, direct cloning from Candida species, or PCR amplification.

[0042] Constructs suitable for use in the present methods typically contain, in addition to the target gene nucleic acid molecule, a sequence encoding a selection marker (e.g., an antibiotic resistance gene) for selecting the desired construct and / or transformant, as well as an origin of replication. The choice of vector system usually depends on several factors, including, but not limited to, the choice of host cell, replication efficiency, selectability, inducibility, and ease of recovery.

[0043] Constructs containing target gene nucleic acid molecules can be propagated in host cells. As used herein, the term host cell is meant to include prokaryotic and eukaryotic organisms, such as yeast, plant, and animal cells. Prokaryotic hosts include Escherichia coli (E. coli), Salmonella typhimurium, Serratia marcescens, and Bacillus subtilis. Eukaryotic hosts include yeasts such as S. cerevisiae, S. pombe, and Pichia pastoris; mammalian cells such as COS cells or Chinese hamster ovary (CHO) cells; insect cells; and plant cells such as Arabidopsis thaliana and Nicotiana tabacum. The construct can be introduced into host cells by any technique commonly known to those skilled in the art.For example, calcium phosphate precipitation, electroporation, heat shock, lipofection, microinjection and virus-mediated nucleic acid transfer are common methods for introducing nucleic acid into host cells.In addition, naked DNA can be directly delivered into cells (for example, U.S. Patent No. 5,580,859 and U.S. Patent No. 5,589,466).

[0044] Also, as discussed above, fluorescent detectable probes can be designed or labeled with any suitable combination of donor and acceptor moieties. Exemplary donor fluorescent moieties suitable for labeling detectable probes according to the present disclosure include, but are not limited to, coumarin dyes, fluorescein dyes (e.g., FAM), rhodamine dyes (e.g., JA270; see U.S. Patent No. 6,184,379), hexachlorofluorescein dyes (e.g., HEX), and cyanine dyes (e.g., Cy5). While the detectable probes described in the Examples herein contain specific combinations of donor and acceptor moieties, it will be understood that alternative moieties can be reasonably substituted without significantly affecting the utility of a particular detectable probe for detecting amplification products. Specifically, the nucleic acid sequences of the detectable probes disclosed herein can be suitably combined with different donor and acceptor moieties of the same or essentially the same composition. Thus, the disclosed detectable probe sequences should not be considered limited to use with the specific donor and acceptor moieties shown in the Examples.

[0045] polymerase chain reaction (PCR) Conventional PCR techniques are disclosed in U.S. Patent Nos. 4,683,202, 4,683,195, 4,800,159, and 4,965,188. PCR typically uses two oligonucleotide primers that bind to a selected nucleic acid template (e.g., DNA or RNA). Primers useful in some embodiments include oligonucleotides that can act as initiation points for nucleic acid synthesis within the target NG gene nucleic acid sequence described. Primers can be purified from restriction digests by conventional methods or produced synthetically. While primers are preferably single-stranded for maximum efficiency in amplification, primers can also be double-stranded. Double-stranded primers are first denatured, i.e., treated to separate the strands. One method for denaturing double-stranded nucleic acids is by heating.

[0046] If the template nucleic acid is double-stranded, it is necessary to separate the two strands before it can be used as a template in PCR. Strand separation can be achieved by any suitable denaturing method, including physical, chemical, or enzymatic means. One method for separating nucleic acid strands involves heating the nucleic acid until it is significantly denatured (e.g., greater than 50%, 60%, 70%, 80%, 90%, or 95% denatured). The heating conditions required to denature the template nucleic acid depend, for example, on the buffer salt concentration and the length and nucleotide composition of the nucleic acid to be denatured, but typically range from about 90°C to about 105°C for a period of time, depending on reaction characteristics such as temperature and nucleic acid length. Denaturation is typically carried out for about 30 seconds to 4 minutes (e.g., 1 minute to 2 minutes 30 seconds, or 1.5 minutes).

[0047] Once the double-stranded template nucleic acid has been denatured by heat, the reaction mixture is cooled to a temperature that promotes annealing of each primer to its target sequence on the target NG gene nucleic acid molecule. Annealing temperatures are typically about 35°C to about 65°C (e.g., about 40°C to about 60°C, about 45°C to about 50°C). Annealing times can range from about 10 seconds to about 1 minute (e.g., about 20 seconds to about 50 seconds, about 30 seconds to about 40 seconds). The reaction mixture is then adjusted to a temperature that promotes or optimizes polymerase activity, i.e., a temperature sufficient to allow extension of the annealed primers to generate products complementary to the template nucleic acid. The temperature must be sufficient to synthesize an extension product from each primer annealed to the nucleic acid template, but not so high as to denature the extension product from its complementary template (e.g., temperatures for extension generally range from about 40°C to about 80°C (e.g., about 50°C to about 70°C, about 60°C)). Extension times can be from about 10 seconds to about 5 minutes (e.g., about 30 seconds to about 4 minutes, about 1 minute to about 3 minutes, or about 1 minute 30 seconds to about 2 minutes).

[0048] PCR assays can use nucleic acids such as RNA or DNA (cDNA). The template nucleic acid does not need to be purified and can be a minor fraction of a complex mixture, such as the nucleic acids contained in human cells. Nucleic acid molecules can be extracted from biological samples by conventional techniques, such as those described in Diagnostic Molecular Microbiology: Principles and Applications (Persing et al. (eds), 1993, American Society for Microbiology, Washington DC). Nucleic acids can be obtained from any number of sources, such as plasmids, or natural sources, including bacteria, yeast, protozoan viruses, organelles, or higher organisms such as plants or animals.

[0049] Oligonucleotide primers are combined with PCR reagents under reaction conditions conducive to primer extension. For example, a chain extension reaction typically contains 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 15 mM MgCl, 0.001% (w / v) gelatin, 0.5-1.0 μg of proto-denatured template DNA, 50 pmol of each oligonucleotide primer, 2.5 U of Taq polymerase, and 10% DMSO. The reaction typically contains 150-320 μM each of dATP, dCTP, dTTP, dGTP, or one or more analogs thereof.

[0050] The newly synthesized strands form double-stranded molecules that can be used in subsequent steps of the reaction. The steps of strand separation, annealing, and extension can be repeated as many times as necessary to produce the desired amount of amplification product corresponding to the target nucleic acid molecule. The limiting factors of the reaction are the amount of primers, thermostable enzyme, and nucleoside triphosphates present in the reaction. The cycling steps (i.e., denaturation, annealing, and extension) are preferably repeated at least once. For detection applications, the number of cycling steps depends, for example, on the nature of the sample. If the sample is a complex mixture of nucleic acids, more cycling steps will be required to amplify the target sequence sufficiently for detection. Generally, the cycling steps are repeated at least about 20 times, but may be repeated 40, 60, or 100 times.

[0051] Fluorescence Resonance Energy Transfer (FRET) FRET technology (e.g., U.S. Pat. Nos. 4,996,143, 5,565,322, 5,849,489, and 6,162,603) is based on the concept that when a donor fluorescent moiety and a corresponding acceptor fluorescent moiety are positioned within a certain distance from each other, energy transfer occurs between two fluorescent moieties that can be visualized or otherwise detected and / or quantified. Typically, when the donor is excited by light radiation of a suitable wavelength, it transfers energy to the acceptor. Typically, the acceptor re-emits the transferred energy in the form of light radiation of a different wavelength. In certain systems, non-fluorescent energy can be transferred between the donor and acceptor moieties via a biomolecule containing a substantially non-fluorescent donor moiety (see, e.g., U.S. Pat. No. 7,741,467).

[0052] In one example, an oligonucleotide probe can contain a donor fluorescent moiety and a corresponding quencher, which may or may not be fluorescent, that dissipates the transferred energy in a form other than light. When the probe is intact, energy transfer typically occurs between the donor and acceptor moieties, such that the fluorescence emission from the donor fluorescent moiety is quenched by the acceptor moiety. During the extension step of the polymerase chain reaction, the probe bound to the amplification product is cleaved, for example, by the 5' to 3' nuclease activity of Taq polymerase, so that the fluorescence emission of the donor fluorescent moiety is no longer quenched. Exemplary probes for this purpose are described, for example, in U.S. Patent Nos. 5,210,015, 5,994,056, and 6,171,785. Commonly used donor-acceptor pairs include the FAM-TAMRA pair. Commonly used quenchers are DABCYL and TAMRA. Commonly used dark quenchers include BlackHole Quenchers™ (BHQ) (Biosearch Technologies, Inc., Novato, CA), Iowa Black™ (Integrated DNA Tech., Inc., Coralville, IA), and BlackBerry® Quencher 650 (BBQ-650) (Berry & Assoc, Dexta, MI).

[0053] In another example, two oligonucleotide probes, each containing a fluorescent moiety, can hybridize to an amplification product at a specific position determined by the complementarity of the oligonucleotide probe to the target nucleic acid sequence. When the oligonucleotide probe hybridizes to the nucleic acid of the amplification product at the appropriate position, a FRET signal is generated. The hybridization temperature can range from about 35°C to about 65°C, and the hybridization time can be from about 10 seconds to about 1 minute.

[0054] Fluorescence analysis can be performed, for example, using a photon-counting epifluorescence microscope system (equipped with appropriate dichroic mirrors and filters to monitor fluorescence emission over a specific range), a photon-counting photomultiplier system, or a fluorometer. Excitation to initiate energy transfer or allow direct detection of the fluorophore can be performed using an argon ion laser, a high-intensity mercury (Hg) arc lamp, a fiber optic light source, or other high-intensity light source appropriately filtered for excitation of the desired range.

[0055] As used herein, "corresponding" refers to an acceptor fluorescent moiety or dark quencher that has an absorbance spectrum that overlaps with the emission spectrum of the donor fluorescent moiety. The wavelength maximum of the emission spectrum of the acceptor fluorescent moiety must be at least 100 nm greater than the wavelength maximum of the excitation spectrum of the donor fluorescent moiety. Thus, efficient non-radiative energy transfer can occur between them.

[0056] Fluorescent donor moieties and corresponding acceptor moieties are generally selected for (a) efficient Förster energy transfer, (b) a large final Stokes shift (>100 nm), (c) a shift of emission as far as possible into the red portion of the visible spectrum (>600 nm), and (d) a shift of emission to a wavelength higher than the Raman water fluorescence emission produced by excitation at the donor excitation wavelength. For example, a donor fluorescent moiety can be selected that has its maximum excitation wavelength near a laser line (e.g., helium-cadmium 442 nm or argon 488 nm), a high extinction coefficient, a high quantum yield, and good overlap of its fluorescence emission with the excitation spectrum of the corresponding acceptor fluorescent moiety. The corresponding acceptor fluorescent moiety can be selected to have a high extinction coefficient, a high quantum yield, good excitation overlap with the emission of the donor fluorescent moiety, and emission in the red portion of the visible spectrum (>600 nm).

[0057] Representative donor fluorescent moieties that can be used with various acceptor fluorescent moieties in FRET technology include fluorescein, Lucifer Yellow, B-phycoerythrin, 9-acridine isothiocyanate, Lucifer Yellow VS, 4-acetamido-4'-isothio-cyanatostilbene-2,2'-disulfonic acid, 7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin, succinyl 1-pyrenebutyrate, and 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid derivatives. Representative acceptor fluorescent moieties include LC Red 640, LC Red 705, Cy5, Cy5.5, Lissamine rhodamine B sulfonyl chloride, tetramethylrhodamine isothiocyanate, rhodamine x isothiocyanate, erythrosine isothiocyanate, fluorescein, diethylenetriamine pentaacetate, or other chelates of lanthanide ions (e.g., europium or terbium), depending on the donor fluorescent moiety used. Donor and acceptor fluorescent moieties can be obtained, for example, from Molecular Probes (Junction City, Oregon) or Sigma Chemical Co. (St. Louis, Missouri).

[0058] The donor and acceptor fluorescent moieties can be attached to the appropriate probe oligonucleotide via linker arms. The length of each linker arm is important because it affects the distance between the donor and acceptor fluorescent moieties. The length of the linker arm can be the angstrom (Å) distance from the nucleotide base to the fluorescent moiety. Typically, the linker arm is about 10 Å to about 25 Å. The linker arm can be of the type described in WO 84 / 03285. WO 84 / 03285 also discloses methods for attaching the linker arm to a specific nucleotide base and for attaching the fluorescent moiety to the linker arm.

[0059] Acceptor fluorescent moieties such as LC Red 640 can be combined with oligonucleotides containing amino linkers (e.g., C6-aminophosphoramidites available from ABI (Foster City, CA) or Glen Research (Sterling, VA)) to produce, for example, LC Red 640-labeled oligonucleotides. Commonly used linkers for attaching donor fluorescent moieties such as fluorescein to oligonucleotides include thiourea linkers (derived from FITC, e.g., Fluorescein-CPG from Glen Research or ChemGene (Ashland, MA)), amide-linkers (derived from fluorescein-NHS-esters, such as CX-Fluorescein-CPG from BioGenex (San Ramon, CA)), or 3'-amino-CPG, which requires attachment of the fluorescein-NHS-ester after oligonucleotide synthesis.

[0060] Detection by real-time PCR The present disclosure provides a method for detecting the presence or absence of bacterial and fungal target organisms in a biological or non-biological sample. The provided method avoids the problems of sample contamination, false negatives, and false positives. The method includes at least one cycling step, which involves amplifying a portion of a target nucleic acid molecule from the sample using one or more primer pairs, and a FRET detection step. The multiple cycling steps are preferably performed in a thermocycler. The method can be performed using primers and probes to detect the presence of the target organism, and detection of the target gene indicates the presence of the target organism in the sample.

[0061] As described herein, amplification products can be detected using labeled hybridization probes that utilize FRET technology. One FRET format utilizes TaqMan® technology to detect the presence or absence of amplification products, and therefore the presence or absence of CA. TaqMan® technology utilizes a single-stranded hybridization probe labeled with a fluorescent dye and a quencher, which may or may not be fluorescent. When the first fluorescent moiety is excited with light of the appropriate wavelength, the absorbed energy is transferred to a second fluorescent moiety or dark quencher according to the principles of FRET. The second moiety is typically a quencher molecule. During the annealing step of the PCR reaction, the labeled hybridization probe binds to the target DNA (i.e., the amplification product) and is subsequently degraded during the extension step by, for example, the 5' to 3' nuclease activity of Taq polymerase. As a result, the fluorescent moiety and the quencher moiety are spatially separated from each other. As a result, upon excitation of the first fluorescent moiety in the absence of the quencher, fluorescent emission from the first fluorescent moiety can be detected. By way of example, the ABI PRISM® 7700 Sequence Detection System (Applied Biosystems) uses TaqMan® technology and is suitable for carrying out the methods described herein for detecting the presence or absence of NG in a sample.

[0062] Molecular beacons combined with FRET can also be used to detect the presence of amplification products using real-time PCR. Molecular beacon technology uses a hybridization probe labeled with a first fluorescent moiety and a second fluorescent moiety. The second fluorescent moiety is generally a quencher, and fluorescent labels are typically placed at each end of the probe. Molecular beacon technology uses a probe oligonucleotide with a sequence that allows for secondary structure formation (e.g., a hairpin). As a result of the formation of the secondary structure within the probe, both fluorescent moieties are spatially close together when the probe is in solution. After hybridization to the target nucleic acid (i.e., the amplification product), the secondary structure of the probe is disrupted, separating the fluorescent moieties from each other, allowing the emission of the first fluorescent moiety to be detected after excitation with light of the appropriate wavelength.

[0063] Another common form of FRET technology utilizes two hybridization probes. Each probe can be labeled with a different fluorescent moiety and is generally designed to hybridize close to each other within a target DNA molecule (e.g., an amplification product). A donor fluorescent moiety, such as fluorescein, is excited at 470 nm by the LightCycler® instrument's light source. During FRET, fluorescein transfers its energy to an acceptor fluorescent moiety, such as LightCycler®-Red640 (LC Red640) or LightCycler®-Red705 (LC Red705). The acceptor fluorescent moiety then emits light of a longer wavelength, which is detected by the LightCycler® instrument's optical detection system. Efficient FRET can only occur when the fluorescent moieties are in direct local proximity and the emission spectrum of the donor fluorescent moiety overlaps with the absorption spectrum of the acceptor fluorescent moiety. The intensity of the emitted signal can be correlated with the number of original target DNA molecules (e.g., the number of CA genomes). When amplification of the target nucleic acid occurs and an amplification product is produced, the hybridizing step results in a detectable signal based on FRET between the members of the probe pair.

[0064] Generally, the presence of FRET indicates the presence of target organisms in the sample, and the absence of FRET indicates the absence of target organisms in the sample.However, insufficient specimen collection, delayed transport, improper transport conditions, or the use of specific collection swabs (calcium alginate or aluminum shafts) are all conditions that can affect the success and / or accuracy of test results.Using the method disclosed herein, for example, detection of FRET within 45 cycle steps indicates the presence of target organism(s).

[0065] The representative biological samples that can be used in the implementation of this method include, but are not limited to, vaginal swabs, fecal specimens, blood specimens, skin swabs, nasal swabs, wound swabs, blood cultures, and skin and soft tissue infections.The collection and preservation methods of biological samples are known to those skilled in the art.Biological samples can be processed (for example, by nucleic acid extraction methods and / or kits known in the art) to release target nucleic acid, or in some cases, biological samples can be directly contacted with PCR reaction components and suitable oligonucleotides.

[0066] Melting curve analysis is an additional step that can be included in the cycle profile. Melting curve analysis is based on the fact that DNA melts at a characteristic temperature called the melting temperature (Tm), which is defined as the temperature at which one half of a DNA duplex separates into single strands. The melting temperature of DNA depends primarily on its nucleotide composition. Thus, DNA molecules rich in G and C nucleotides have a higher Tm than DNA molecules rich in A and T nucleotides. By detecting the temperature at which the signal is lost, the melting temperature of the probe can be determined. Similarly, by detecting the temperature at which the signal is generated, the annealing temperature of the probe can be determined. The melting temperature(s) of the probe from the amplification product can be used to confirm the presence or absence of the target organism(s) in the sample.

[0067] During each thermocycler run, control samples can also be cycled. A positive control sample can amplify a target nucleic acid control template (other than the amplification product of the listed target gene) using, for example, control primers and a control probe. A positive control sample can also amplify, for example, a plasmid construct containing the target nucleic acid molecule. Such a plasmid control can be amplified internally (e.g., within the sample) or in a separate sample run alongside the patient sample, using the same primers and probes used to detect the intended target. Such controls are indicators of the success or failure of the amplification, hybridization, and / or FRET reaction. Each thermocycler run can also include, for example, a negative control lacking target template DNA. The negative control can measure contamination. This ensures that the system and reagents do not produce false-positive signals. Thus, control reactions can easily determine, for example, the ability of primers to anneal with sequence specificity and initiate elongation, and the ability of probes to hybridize with sequence specificity and allow FRET to occur.

[0068] In one embodiment, the method includes a step to avoid contamination, for example, an enzymatic method utilizing uracil-DNA glycosylase is described in U.S. Patent Nos. 5,035,996, 5,683,896, and 5,945,313 to reduce or eliminate contamination between one thermocycler run and the next.

[0069] This method can be carried out using conventional PCR combined with FRET technology. In one embodiment, a LightCycler® instrument is used. The following patent applications describe real-time PCR used with LightCycler® technology: WO 97 / 46707, WO 97 / 46714 and WO 97 / 46712.

[0070] In addition to the LightCycler® instrument, various instruments exist for performing rapid and accurate PCR in combination with detection of the resulting nucleic acid product. Such instruments can enable absolute or relative quantification of target nucleic acids, as well as post-PCR analysis of amplified nucleic acids by melting curve analysis.

[0071] Some instruments are configured to detect target nucleic acids by exciting a fluorophore, such as a donor fluorescent moiety attached to a probe, and then measuring the resulting emitted fluorescent signal. Instruments for detecting target nucleic acids by fluorescent excitation and measuring the emitted fluorescent signal can include multiple excitation and emission filters. The inclusion of multiple excitation and emission filters allows for the detection of different target nucleic acids in separate channels. For example, ROCHE's LightCycler® 480 instrument includes a filter set consisting of five excitation filters (450, 483, 523, 558, and 615 nm) and six emission filters (500, 533, 568, 610, 640, and 670 nm).

[0072] Individual excitation and emission filters can be freely combined to allow optimal excitation of fluorophores and accurate measurement of emitted fluorescent signals. Excitation-emission filter pairs can be used alone for monochromatic applications or in sequential combinations for multichromatic applications. It will be understood that the choice of channel for analysis will depend, at least in part, on the fluorochromes used in the experiment.

[0073] The LightCycler® can be operated using a PC workstation and utilizes the Windows® NT operating system. Signals from samples are acquired as the machine sequentially positions capillaries on the optical unit. The software can display the fluorescence signal in real time immediately after each measurement. Fluorescence acquisition times range from 10 to 100 milliseconds (msec). After each cycling step, a quantitative display of fluorescence versus cycle number can be continuously updated for all samples. The generated data can be saved for further analysis.

[0074] As an alternative to FRET, double-stranded DNA-binding dyes, such as fluorescent DNA-binding dyes (e.g., SYBR® Green or SYBR® Gold (Molecular Probes)), can be used to detect amplification products. Upon interaction with double-stranded nucleic acids, such fluorescent DNA-binding dyes emit a fluorescent signal after excitation with light of an appropriate wavelength. Double-stranded DNA-binding dyes, such as nucleic acid intercalating dyes, can also be used. When using double-stranded DNA-binding dyes, a melting curve analysis is usually performed to confirm the presence of amplification products.

[0075] It is understood that embodiments of the present disclosure are not limited by the configuration of one or more commercially available devices.

[0076] Manufactured Products / Kits Embodiments of the present disclosure further provide articles of manufacture, compositions, or kits for detecting bacterial and fungal organisms associated with vaginitis. The articles of manufacture may include primers and probes used to detect target genes, along with appropriate packaging. The compositions may include primers used to amplify the target genes. In certain embodiments, the compositions may also include probes for detecting the target genes. Representative primers and probes for detecting target organism(s) may hybridize to target nucleic acid molecules. Furthermore, the kits may also include appropriately packaged reagents and materials necessary for DNA immobilization, hybridization, and detection, such as solid supports, buffers, enzymes, and DNA standards. Methods for designing primers and probes are disclosed herein, and representative examples of primers and probes that amplify and hybridize to target nucleic acid molecules are provided.

[0077] The article of manufacture may also include one or more fluorescent moieties for labeling the probe, or the probes provided with the kit may be labeled. For example, the article of manufacture may include donor and / or acceptor fluorescent moieties for labeling the probe. Examples of suitable FRET donor fluorescent moieties and corresponding acceptor fluorescent moieties are provided above.

[0078] The product may also include a package insert or packaging label with instructions for using the primers and probes to detect target organisms in a sample.The product and composition may further include reagents (e.g., buffers, polymerase enzymes, cofactors, or agents for preventing contamination) for carrying out the methods disclosed herein.Such reagents may be specific to one of the commercially available instruments described herein.

[0079] Embodiments of the present disclosure are further described in the following examples, which do not limit the scope of the claimed invention. [Example]

[0080] The following examples, tables and figures are provided to aid the understanding of the subject matter, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.

[0081] Example 1: PCR assay reagents and conditions Real-time PCR detection of target bacteria and Candida species was performed using either the cobas® 4800 system or the cobas® 6800 / 8800 system platform (Roche Molecular Systems, Inc. Pleasanton, CA). The final concentrations of amplification reagents are shown in Table 2 below: [Table 2]

[0082] Table 3 shows a typical thermal profile used in a PCR amplification reaction: [Table 3]

[0083] The pre-PCR program included incubations at 55°C, 60°C, and 65°C for initial denaturation and reverse transcription of the RNA template. The three-temperature incubation has the advantageous effect that even slightly mismatched target sequences (e.g., genetic variants of an organism) are transcribed at lower temperatures, while the higher temperatures suppress the formation of RNA secondary structures, thus resulting in more efficient transcription. PCR cycling was divided into two runs, each using a single-step setup (combined annealing and extension). The first five cycles at 55°C allow comprehensive amplification by preamplifying slightly mismatched target sequences, while the second run (45 cycles) increases specificity by using an annealing / extension temperature of 58°C.

[0084] Example 2: Amplification and detection of Candida species Oligonucleotide primers and probes were designed for the specific detection of Candida species from the clade associated with Candida vaginitis: Candida albicans, Candida dubliniensis, Candida tropicalis, and Candida parapsilosis (collectively referred to as Candida species). These oligonucleotides target a conserved region within the ribosomal DNA gene located at the end of the 18s rRNA, the ITS1 region. Figure 1 shows the rRNA genome organization of various Candida species, with variation (expressed as percent identity) in the 18s, 5.8s, and 28s genes relative to Candida albicans shown on the right. rDNA genes are present at 50–200 copies per genome, allowing for highly sensitive detection of Candida species. Table 4 shows the predicted hybridization of the primers and probes of the present invention to target regions of 18s rRNA or ITS1 of C. albicans, C. dubliniensis, C. tropicalis, and C. parapsilosis by in silico sequence analysis. [Table 4]

[0085] In Table 4, an "x" represents the expected detection of Candida species from the hybridization-based in silico analysis.

[0086] A list of PCR assays using various combinations of forward and reverse primers, and the performance of the assays expressed as Ct values, are shown in Tables 5 and 6, respectively. [Table 5-1] [Table 5-2] [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4]

[0087] The PCR assay was tested using plasmids encoding ribosomal DNA gene sequences from C. albicans, C. parapsilosis, C. tropicalis, and C. dubliniensis. Three levels of plasmid template (10-fold dilutions) and buffer as a negative control (neg.) were tested: L1: 100,000 copies / PCR; L2: 10,000 copies / PCR; L3: 1,000 copies / PCR.

[0088] Example 3: Multiplex PCR assay A multiplex PCR single-well assay for simultaneous detection of three BV-associated bacteria, Lactobacillus spp., Gardnerella vaginalis, and Atopobium vaginae, as well as Candida spp., including Candida krusei and Candida glabrata, was performed using four different detection channels. The first channel detects the 16s rRNA of Gardnerella vaginalis. The second channel detects the D-LDH gene of Lactobacillus spp. The third channel detects multiple Candida species, including the 18s rRNA and ITS1 of Candida spp., Candida krusei, and Candida glabrata. The fourth channel detects the tufA gene of Atopobium vaginae. Many of the primers and probes used in the multiplex assay for the amplification and detection of Gardnerella vaginalis, Lactobacillus spp., Atopobium vaginae, Candida krusei, and Candida glabrata are disclosed in U.S. Patent Application Publication No. US2022 / 0205020A1, which is incorporated herein by reference in its entirety. Selected primer sets and probe combinations are shown in Table 7. [Table 7]

[0089] In Table 7,<t-bb_dA> = t-butylbenzyl dA, <cou>=COU pigment, <q>= quencher,<t-bb_dC> = t-butylbenzyl dC, <fam>=FAM pigments, <ja270>=JA270 pigment, <hex>=HEX pigment, <pdu>= 5-propynyl dU, and Sp = C3 spacer.

[0090] Although the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be apparent to those skilled in the art from a reading of this disclosure that various changes in form and detail may be made therein without departing from the true scope of the invention. For example, all of the techniques and devices described above may be used in various combinations. All publications, patents, patent applications, and / or other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, and / or other document was individually indicated to be incorporated by reference for all purposes.< / pdu> < / hex> < / fam> < / q> < / cou>

Claims

1. 1. A method for detecting vulvovaginal candidiasis (VVC)-associated Candida species in a sample, wherein the VVC-associated Candida species include Candida albicans, Candida tropicalis, Candida dubliniensis, and Candida parapsilosis (collectively referred to as Candida spp.), the method comprising: - carrying out an amplification step, which comprises contacting said sample with a primer set to produce an amplification product if nucleic acid from said VVC-associated Candida species is present in said sample; - carrying out a hybridization step, which comprises contacting each amplification product with one or more detectable probes, and - detecting the presence of each amplification product, the presence of said amplification product indicating the presence of said VVC-associated Candida species in said sample; Including, 10. A method according to claim 9, wherein the primer set for producing an amplification product derived from Candida species comprises one or more forward primers comprising an oligonucleotide sequence selected from SEQ ID NOs: 1-5 and one or more reverse primers comprising an oligonucleotide sequence selected from SEQ ID NOs: 6-14, and one of the one or more detectable probes comprises the oligonucleotide sequence of SEQ ID NO: 15 or its complement.

2. 2. The method of claim 1, wherein the primer set for producing an amplification product derived from Candida species comprises a forward primer comprising the oligonucleotide sequence of SEQ ID NO: 1 and at least a first and a second reverse primer, wherein the first reverse primer comprises the oligonucleotide sequence of SEQ ID NO: 6 and the second reverse primer comprises the oligonucleotide sequence of SEQ ID NO:

7.

3. 3. The method of claim 1, wherein the hybridizing step comprises contacting the amplification product with the detectable probe labeled with a donor fluorescent moiety and a corresponding acceptor moiety, and the detecting step comprises detecting the presence or absence of fluorescence resonance energy transfer (FRET) between the donor fluorescent moiety and the acceptor moiety of the probe, wherein the presence or absence of fluorescence indicates the presence or absence of the VVC-associated Candida species in the sample.

4. The method according to any one of claims 1 to 3, wherein the amplification step uses a polymerase enzyme having 5'-3' nuclease activity.

5. The method of any one of claims 1 to 4, wherein at least one of the oligonucleotide sequences comprises at least one modified nucleotide.

6. 6. The method of claim 1, further comprising detecting at least one of Candida krusei, Candida glabrata, Lactobacillus spp., Gardnerella vaginalis, and Atopobium vaginae in the sample.

7. 1. A kit for detecting vulvovaginal candidiasis (VVC)-associated Candida species in a sample, wherein the VVC-associated Candida species include Candida albicans, Candida tropicalis, Candida dubliniensis, and Candida parapsilosis (collectively referred to as Candida spp.), the kit comprising: - at least one forward primer comprising an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 5; - at least one reverse primer comprising an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 6 to 14; a detectably labeled probe comprising the oligonucleotide sequence of SEQ ID NO: 15 or its complement, wherein the detectably labeled oligonucleotide sequence is configured to hybridize to an amplicon generated by said at least one forward primer and said at least one reverse primer; Includes a kit.

8. The kit of claim 7 , wherein the detectably labeled probe comprises a donor fluorescent moiety and a corresponding acceptor moiety.

9. The kit according to any one of claims 7 to 8, further comprising a nucleoside triphosphate, a nucleic acid polymerase, and a buffer necessary for the function of the nucleic acid polymerase.

10. The kit according to any one of claims 7 to 9, wherein at least one of the oligonucleotide sequences comprises at least one modified nucleotide.

11. 1. A method for simultaneously detecting vulvovaginal candidiasis (VVC)-associated Candida species and at least one member selected from the group consisting of Candida krusei and Candida glabrata, Lactobacillus spp., Gardnerella vaginalis, and Atopobium vaginae in a sample, wherein the VVC-associated Candida species include Candida albicans, Candida tropicalis, Candida dubliniensis, and Candida parapsilosis (collectively referred to as Candida spp.), the method comprising: - performing an amplifying step comprising contacting the sample with a primer set to produce a plurality of amplification products when nucleic acid from Candida spp. and at least one selected from the group consisting of Candida krusei and Candida glabrata, Lactobacillus spp., Gardnerella vaginalis, and Atopobium vaginae is present in the sample; - carrying out a hybridization step comprising contacting said amplification products with one or more detectable probes, and - detecting the presence of said amplification product, wherein the presence of said amplification product indicates the presence in said sample of Candida spp. and at least one member of the group selected from Candida krusei and Candida glabrata, Lactobacillus spp., Gardnerella vaginalis and Atopobium vaginae, Including, the primer set for producing the first subset of amplification products from Candida spp. comprises one or more forward primers comprising an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 1-5 and one or more reverse primers comprising an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 6-14, and one of the one or more detectable probes comprises the oligonucleotide sequence of SEQ ID NO: 15 or its complement; and the primer set for producing the second subset of amplification products from Lactobacillus spp. comprises a forward primer comprising the oligonucleotide sequence of SEQ ID NO: 19 and a reverse primer comprising the oligonucleotide sequence of SEQ ID NO: 20, and one of the one or more detectable probes comprises the oligonucleotide sequence of SEQ ID NO: 21; and / or the primer set for producing the third subset of amplification products from Gardnerella vaginalis comprises a forward primer comprising the oligonucleotide sequence of SEQ ID NO: 16 and a reverse primer comprising the oligonucleotide sequence of SEQ ID NO: 17, one of the one or more detectable probes comprises the oligonucleotide sequence of SEQ ID NO: 18, and one of the one or more detectable probes comprises the oligonucleotide sequence of SEQ ID NO: 31; and / or the primer set for producing a fourth subset of the amplification products from Atopobium vaginae comprises a forward primer comprising the oligonucleotide sequence of SEQ ID NO: 22 and a reverse primer comprising the oligonucleotide sequence of SEQ ID NO: 23, one of the one or more detectable probes comprises the oligonucleotide sequence of SEQ ID NO: 24, and one of the one or more detectable probes comprises the oligonucleotide sequence of SEQ ID NO: 32; and / or the primer set for producing the fifth subset of amplification products from Candida krusei comprises a forward primer comprising the oligonucleotide sequence of SEQ ID NO: 25 and a reverse primer comprising the oligonucleotide sequence of SEQ ID NO: 26, and one of the one or more detectable probes comprises the oligonucleotide sequence of SEQ ID NO: 27; and / or the primer set for producing a sixth subset of the amplification products from Candida glabrata comprises a forward primer comprising the oligonucleotide sequence of SEQ ID NO:28 and a reverse primer comprising the oligonucleotide sequence of SEQ ID NO:29, and one of the one or more detectable probes comprises the oligonucleotide sequence of SEQ ID NO:

30.

12. The method of claim 11, wherein the primer set for producing the first subset of amplification products derived from Candida species comprises a forward primer comprising the oligonucleotide sequence of SEQ ID NO: 1 and at least first and second reverse primers, wherein the first reverse primer comprises the oligonucleotide sequence of SEQ ID NO: 6 and the second reverse primer comprises the oligonucleotide sequence of SEQ ID NO: 7.

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