The present application is based on and claims priority from U.S. provisional patent application No. 63/486,974 filed on 25 at 2 months 2023, which provisional patent application is incorporated herein by reference in its entirety.
The present application comprises a sequence listing submitted as an electronic text file, named "P38277-WO PCT application sequence listing. Xml", having a byte size of 12,306 bytes and created at 2024, 1, 25. The information contained in this electronic document is incorporated by reference herein in its entirety, as specified in 37CFR ≡1.52 (e) (5).
Detailed Description
Provided herein are methods and compositions for detecting vulvovaginal candidiasis (VVC). For example, primers and probes are provided that can bind to specific genes of a VVC related candida species to determine the presence or absence of the VVC related candida species in a sample (such as a biological sample). In some embodiments, multiplex nucleic acid amplification may be performed to allow detection of VVC related candida species and bacterial vaginosis- (BV) -related bacteria in a single assay. Detection of BV-related bacteria and the VVC-related species Candida krusei and Candida glabrata can be performed using compositions and methods as disclosed in U.S. patent publication No. U.S.2022/0205020A1, which is incorporated herein by reference in its entirety.
The methods of the invention may comprise performing at least one cycling step comprising amplifying one or more portions of a nucleic acid molecule gene target from a sample using one or more pairs of primers. As used herein, "primer" refers to an oligonucleotide primer that specifically anneals to a target gene in candida species and initiates DNA synthesis therefrom under appropriate conditions, thereby producing the corresponding amplification product. Each of the primers in question anneals to a target within or adjacent to the corresponding 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 of the target gene nucleic acids are present in the sample, one or more amplification products are produced, whereby the presence of one or more of the target gene amplification products is indicative of the presence of candida species in the sample. The amplification product should contain a nucleic acid sequence complementary to one or more detectable probes of the target gene. "probe" as used herein refers to an oligonucleotide probe that specifically anneals to a nucleic acid sequence encoding a target gene. Each cycling step includes an amplification step, a hybridization step, and a detection step, wherein the sample is contacted with one or more detectable probes to detect the presence or absence of candida species in the sample.
As used herein, the term "amplification" refers to the process of synthesizing a nucleic acid molecule that is complementary to one or both strands of a template nucleic acid molecule. Amplifying a nucleic acid molecule typically includes denaturing a template nucleic acid, annealing a primer to the template nucleic acid at a temperature below the melting temperature of the primer, and enzymatically extending from the primer to produce an amplified product. Amplification typically requires the presence of deoxyribonucleoside triphosphates, DNA polymerase (e.g.Taq) and suitable buffers and/or cofactors (e.g., mgCl2 and/or KCl) for optimizing polymerase activity.
The term "primer" as used herein is known to the person skilled in the art and refers to an oligomeric compound capable of "priming" DNA synthesis by a template dependent DNA polymerase, mainly to oligonucleotides, but also to modified oligonucleotides, i.e. e.g. the 3 '-end of the oligonucleotide provides a free 3' -OH group to which an additional "nucleotide" can be attached by the template dependent DNA polymerase, thereby establishing a 3 'to 5' phosphodiester bond, wherein deoxynucleoside triphosphates are used and thereby liberate pyrophosphate. Thus, there is no fundamental distinction between "primers", "oligonucleotides" or "probes" other than the intended function that may be possible.
The term "hybridization" refers to the annealing of one or more probes to an amplification product. Hybridization conditions generally include temperatures below the melting temperature of the probe but avoiding non-specific hybridization of the probe.
The term "5' to 3' nuclease activity" refers to the activity of a nucleic acid polymerase, typically associated with nucleic acid strand synthesis, whereby nucleotides are removed from the 5' end of the nucleic acid strand.
The term "thermostable polymerase" refers to a thermostable polymerase, i.e., an enzyme that catalyzes the formation of primer extension products complementary to a template and that does not irreversibly denature when subjected to elevated temperatures for the time required to effect denaturation of double-stranded template nucleic acids. Typically, 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 from Thermus flavus (T. Ruber), thermus thermophilus (T. Thermophilus), thermus aquaticus (T. Aquaticus), thermus lactis (T. Lactius), thermus rhodochrous (T. Rubens), bacillus stearothermophilus (Bacillus stearothermophilus) and Thermus flammulina (Methanothermus fervidus). However, polymerases that are not thermostable may also be used in a PCR assay, provided that the enzyme is supplemented.
The term "complementary sequence thereof" refers to a nucleic acid that is of the same length as a given nucleic acid and is fully complementary thereto.
When used with respect to a nucleic acid, the term "extension" or "elongation" refers to the incorporation of additional nucleotides (or other similar molecules) into the nucleic acid. For example, the nucleic acid is optionally extended by a biocatalyst incorporating the nucleotide, such as a polymerase that typically adds the nucleotide at the 3' end of the nucleic acid.
In the context of two or more nucleic acid sequences, the term "identical" or "percent identity" refers to two or more sequences or subsequences that are the same or have a specified percentage of identical nucleotides, when compared and aligned for maximum correspondence (e.g., as measured using one of the sequence comparison algorithms available to the skilled artisan or by visual inspection). Exemplary algorithms suitable for determining percent sequence identity and sequence similarity are BLAST programs described in, for example, 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. Enzyme.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,, each of which is incorporated herein by reference.
"Modified nucleotide" in the context of an oligonucleotide refers to a change in which at least one nucleotide of the oligonucleotide sequence is replaced with a different nucleotide, thereby providing the oligonucleotide with the desired properties. Exemplary modified nucleotides that may be substituted in the oligonucleotides described herein include, 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-propargylamin-dC, C5-propargylamin-dU, C7-propargylamin-dA, C7-propargylamin-dG, 7-deaza-2-deoxyxanthosine, pyrazolopyrimidine analogs, pseudo-dU, nitropyrrole, nitroindole, 2 '-O-methylribose-U, 2' -O-methylribose-C, N-ethyl-dC, N6 -methyl-dA, N6 -benzyl-dA, N4 -benzyl-dC, N6 -p-tert-butyl-p-benzyl-dA, N4 -tert-butyl-p-benzyl dA, and the like. Many other modified nucleotides that may be substituted in an oligonucleotide are mentioned herein or otherwise known in the art. In certain embodiments, the modified nucleotide substitution modifies the melting temperature (Tm) of the oligonucleotide relative to the melting temperature of the corresponding unmodified oligonucleotide. To further illustrate, in some embodiments, certain modified nucleotide substitutions can reduce non-specific nucleic acid amplification (e.g., minimize primer dimer formation, etc.), increase yield of intended target amplicon, and the like. Examples of these types of nucleic acid modifications are described, for example, in U.S. Pat. No. 6,001,611 (incorporated herein by reference).
Detection of candida species
Nucleic acid amplification may be performed, as described herein, to determine the presence, absence, and/or level of candida species in a sample. Some candida species are known to be associated with VVC, including, but not limited to, candida albicans, candida tropicalis, candida dublinii, candida parapsilosis, candida krusei, and candida glabrata. The detection of the VVC-related candida species may be performed simultaneously with the detection of the BV-related bacterial species. Many bacteria are known to be associated with BV, including but not limited to Lactobacillus species such as Lactobacillus crispatus (Lactobacillus crispatus, L. Cristatus), lactobacillus jensenii (Lactobacillus jensenii, L. Jensenii) and Lactobacillus griseus (Lactobacillus gasseri, L. Gasser), gardnerella vaginalis (G. Vaginalis), altophan vaginalis, megasphaera type 1 (Megasphaera-1) and BVAB-2. In some embodiments, the presence, absence, and/or level of VVC-related candida species and BV-related bacteria are determined by using methods known in the art (such as DNA amplification) to detect one or more target genes for each of the target organisms. In some embodiments, multiplex PCR may be performed to detect the presence, absence, or level of each of the target candida species, and may include detecting BV-related bacteria simultaneously. In some embodiments, multiplex PCR is performed to detect the presence, absence and/or level of each of target VVC-related candida species and BV-related bacteria including Lactobacillus crispatus, lactobacillus jensenii, gardnerella vaginalis, altobosa vaginalis, type 1 megaball, and BVAB-2. In some embodiments, the VVC related candida species are candida albicans, candida tropicalis, candida dublinii, candida parapsilosis, candida krusei, and candida glabrata.
In another embodiment, nucleic acid amplification may be performed in the same sample to determine the presence, absence and/or level of Trichomonas Vaginalis (TV). Compositions and methods for rapidly detecting the presence or absence of trichomonas vaginalis (Trichomonas vaginalis) in a biological or non-biological sample are described in U.S. patent application publication No. 2017/0342508, which is incorporated herein by reference in its entirety.
Each of the target VVC related candida species can be detected using a separate channel in DNA amplification. In some cases, it may be desirable to use a single fluorescent channel to detect the presence, absence, and/or level of two or more of the VVC related candida species. In some embodiments, such combinations can reduce the amount of reagents required to perform an experiment and provide an accurate qualitative indication from which determination of VVC related candida species can be assessed. Without being bound by any particular theory, it is believed that the use of a combination of markers may increase the sensitivity and specificity of the assay. In some embodiments, separate fluorescence channels are used to detect the presence, absence, and/or level of each of candida species (e.g., candida albicans, candida tropicalis, candida dublin, candida parapsilosis).
Oligonucleotides (e.g., amplification primers and probes) capable of specifically hybridizing (e.g., under standard nucleic acid amplification conditions, e.g., standard PCR conditions, and/or stringent hybridization conditions) to a target gene region or its complementary sequence in a VVC-related candida species are provided. In some embodiments, amplification of a target gene region of an organism in a sample (e.g., a vaginal swab sample) may be indicative of the presence, absence, and/or level of the organism in the sample.
The target gene region may vary. In some embodiments, oligonucleotides (e.g., amplification primers and probes) capable of specifically hybridizing to a target gene region in an organism (e.g., under standard nucleic acid amplification conditions, such as standard PCR conditions, and/or stringent hybridization conditions) are provided.
In some embodiments, 18s ribosomal RNA (18 s rRNA) of candida species ribosomal RNA (rRNA) genes is used as a target gene for DNA amplification to detect the presence, absence, and/or level of VVC-related candida species in a sample. In some embodiments, 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-related candida species in a sample. In some embodiments, the VVC related candida species include candida albicans, candida tropicalis, candida dublinii, and candida parapsilosis (collectively referred to as candida species). In some embodiments, the VVC related candida species is candida albicans, candida tropicalis, candida dublinii, candida parapsilosis, or a combination thereof. Examples of oligonucleotides capable of specifically hybridizing to the 18s rRNA or ITS1 region of candida species are provided in table 1.
TABLE 1 primers and probes for detection of candida species
In one embodiment, the above-described set of primers and probes are used in order to provide detection of candida species associated with colpitis in a biological sample suspected of containing such candida species. The set of primers and probes may comprise or consist of primers and probes specific for the nucleic acid sequence of the candida target gene, comprising or consisting of the nucleic acid sequences of SEQ ID NOs 1 to 5 and 7 to 15. In another embodiment, the primers and probes of the target gene comprise or consist of functionally active variants of any of the primers and probes of SEQ ID NOS 1 to 5 and 7 to 15.
Functionally active variants of any of the primers and/or probes of SEQ ID NOS 1 to 5 and 7 to 15 can be identified by using the primers and/or probes in the disclosed methods. Functionally active variants of the primers and/or probes of any one of SEQ ID NOs 1 to 5 and 7 to 15 relate to primers and/or probes that provide similar or higher specificity and sensitivity in the described methods or kits compared to the corresponding sequences of SEQ ID NOs 1 to 5 and 7 to 15.
For example, a variant may differ from the sequences of SEQ ID NOS: 1 to 5 and 7 to 15 by one or more nucleotide additions, deletions or substitutions (e.g., one or more nucleotide additions, deletions or substitutions at the 5 'end and/or 3' end of the corresponding sequences of SEQ ID NOS: 1 to 5 and 7 to 15). As described above, the primer (and/or probe) may be chemically modified, i.e., the primer and/or probe may comprise modified nucleotides or non-nucleotide compounds. The probe (or primer) is then a modified oligonucleotide. "modified nucleotides" (or "nucleotide analogs") differ from the natural "nucleotides" in some modification but still consist of a base or base-like compound, a pentose or pentose-like compound, a phosphate moiety or a phosphate-like moiety, or a combination thereof. For example, a "tag" may be attached to the base portion of a "nucleotide" to thereby obtain a "modified nucleotide". The natural base in a "nucleotide" can also be replaced by, for example, 7-deazapurine, whereby a "modified nucleotide" is also obtained. The term "modified nucleotide" or "nucleotide analogue" is used interchangeably in the present application. "modified nucleosides" (or "nucleoside analogs") differ from natural nucleosides in some modification in a manner as outlined above for "modified nucleotides" (or "nucleotide analogs").
Oligonucleotides, including modified oligonucleotides and oligonucleotide analogs, that amplify nucleic acid molecules encoding a target gene can be designed using, for example, computer programs such as OLIGO (Molecular Biology Insights inc., cascades, colo.). When designing oligonucleotides for use as amplification primers, important features include, but are not limited to, amplification products of appropriate size to facilitate detection (e.g., by electrophoresis), similar melting temperatures of members of a pair of primers, and the length of each primer (i.e., the primers need to be long enough to anneal specifically to the sequence and initiate synthesis, but not so long that fidelity is reduced during oligonucleotide synthesis). Typically, the oligonucleotide primer is 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, the oligonucleotide primer is 40 nucleotides or less in length.
In addition to a set of primers, these methods may use one or more probes to detect the presence or absence of a target candida species. The term "probe" refers to a synthetically or biologically produced nucleic acid (DNA or RNA) that, by design or selection, comprises a particular nucleotide sequence that allows them to specifically (i.e., preferentially) hybridize to a "target nucleic acid", in this case a target gene nucleic acid, under defined predetermined stringency. "probe" may be referred to as a "detection probe" meaning that it detects a target nucleic acid.
In some embodiments, the target gene probe may be labeled with at least one fluorescent label. In one embodiment, the target gene probe may 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.
The design of the oligonucleotides for use as probes may be performed in a manner similar to the design of primers. Embodiments may use a single probe or a pair of probes to detect the amplification product. According to embodiments, the probes used may comprise at least one label and/or at least one quencher moiety. As with the primers, the probes generally 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. The oligonucleotide probes are typically 15 to 40 (e.g., 16, 18, 20, 21, 22, 23, 24, or 25) nucleotides in length.
The construct may include vectors each containing one of a target gene primer and a probe nucleic acid molecule. The construct may be used, for example, as a control template nucleic acid molecule. Suitable vectors are commercially available and/or are produced by recombinant nucleic acid techniques conventional in the art. The target gene nucleic acid molecule may be obtained, for example, by chemical synthesis, direct cloning from candida species or by PCR amplification.
In addition to target gene nucleic acid molecules, constructs suitable for use in the methods generally include sequences encoding selectable markers (e.g., antibiotic resistance genes) for selection of a desired construct and/or transformant, as well as origins of replication. The choice of vector system will generally depend on several factors including, but not limited to, the choice of host cell, replication efficiency, selectivity, inducibility and ease of recovery.
Constructs containing target gene nucleic acid molecules can be propagated in host cells. As used herein, the term host cell is intended to include both prokaryotes and eukaryotes, such as yeast, plant and animal cells. Prokaryotic hosts may include E.coli (E.coli), salmonella typhimurium (Salmonella typhimurium), serratia marcescens (SERRATIA MARCESCENS), and Bacillus subtilis (Bacillus subtilis). Eukaryotic hosts include yeasts such as Saccharomyces cerevisiae (S.cerevisiae), schizosaccharomyces pombe (S.pombe), pichia pastoris (Pichia pastoris), mammalian cells such as COS cells or Chinese Hamster Ovary (CHO) cells, insect cells and plant cells such as Arabidopsis thaliana (Arabidopsisthaliana) and tobacco (Nicotiana tabacum). The construct may be introduced into the host cell using any technique known to one of ordinary skill 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 acids into host cells. In addition, naked DNA can be delivered directly to cells (see, e.g., U.S. Pat. nos. 5,580,859 and 5,589,466).
As also discussed above, the fluorescence detectable probe may be designed with or labeled with any suitable combination of donor and acceptor moieties. Examples of 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., cy 5). It will be appreciated that while the detectable probes described in the examples herein include specific combinations of donor and acceptor moieties, alternative moieties may be reasonably substituted without significantly affecting the utility of a particular detectable probe for detecting amplification products. In particular, the nucleic acid sequences of the detectable probes disclosed herein may be suitably combined with different donor and acceptor moieties in the same or substantially the same configuration. Thus, the disclosed detectable probe sequences should not be considered limited to use with the particular donor and acceptor moieties shown in this example.
Polymerase Chain Reaction (PCR)
Conventional PCR techniques are disclosed in U.S. Pat. Nos. 4,683,202, 4,683,195, 4,800,159 and 4,965,188. PCR typically employs two oligonucleotide primers that bind to a selected nucleic acid template (e.g., DNA or RNA). Primers useful in some embodiments include oligonucleotides capable of acting as a point of initiation of nucleic acid synthesis within the target NG gene nucleic acid sequences described. The primer may be purified from the restriction digest by conventional methods, or it may be synthetically produced. For maximum efficiency in amplification, the primers are preferentially single stranded, but the primers may be double stranded. The double stranded primer is first denatured (i.e., treated) to separate the strands. One method of denaturing the double stranded nucleic acid is by heating.
If the template nucleic acid is double stranded, the two strands must be separated before it can be used as a template in PCR. Strand separation may be accomplished by any suitable denaturing method, including physical, chemical, or enzymatic methods. One method of isolating nucleic acid strands involves heating the nucleic acid until a substantial portion thereof is denatured (e.g., greater than 50%, 60%, 70%, 80%, 90%, or 95% denatured). The heating conditions required to denature the template nucleic acid will depend, for example, on the buffer salt concentration and the length and nucleotide composition of the nucleic acid being denatured, but will typically be in the range of about 90 ℃ to about 105 ℃ for a period of time, depending on the characteristics of the reaction 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).
If the double stranded template nucleic acid is denatured by heating, the reaction mixture is cooled to a temperature that promotes annealing of each primer to the target sequence on the described target NG gene nucleic acid molecule. The temperature used for annealing is typically from about 35 ℃ to about 65 ℃ (e.g., from about 40 ℃ to about 60 ℃; from about 45 ℃ to about 50 ℃). The annealing time may be 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 for extension from the annealed primer to produce a product complementary to the template nucleic acid. The temperature should be sufficient to synthesize an extension product from each primer that anneals to the nucleic acid template, but not so high as to denature the extension product from its complementary template (e.g., the temperature used for extension is typically in the range of about 40 ℃ to about 80 ℃ (e.g., about 50 ℃ to about 70 ℃; about 60 ℃)). The extension time may be about 10 seconds to about 5 minutes (e.g., about 30 seconds to about 4 minutes; about 1 minute to about 3 minutes; about 1 minute 30 seconds to about 2 minutes).
PCR assays may use nucleic acids, such as RNA or DNA (cDNA). The template nucleic acid need not be purified, and may be a small portion of a complex mixture, such as nucleic acid contained in human cells. Nucleic acid molecules can be extracted from biological samples by conventional techniques, such as those in Diagnostic Molecular Microbiology: PRINCIPLES AND Applications (Persing et al, ed., 1993,American Society for Microbiology,Washington D.C). Nucleic acids may be obtained from a number of sources, such as plasmids or natural sources, including bacteria, yeast, protozoan viruses, organelles, or higher organisms, such as plants or animals.
The oligonucleotide primers are combined with PCR reagents under reaction conditions that induce primer extension. For example, the chain extension reaction typically includes 50mM KCl, 10mM Tris-HCl (pH 8.3), 15mM MgCl2, 0.001% (w/v) gelatin, 0.5-1.0. Mu.g of original denatured template DNA, 50pmol per oligonucleotide primer, 2.5U Taq polymerase, and 10% DMSO. The reaction typically comprises 150 to 320 μm of each of dATP, dCTP, dTTP, dGTP or one or more analogues thereof.
The newly synthesized strands form double-stranded molecules that can be used in subsequent reaction steps. The steps of strand separation, annealing, and extension can be repeated as many times as necessary to produce the desired number of amplification products corresponding to the target nucleic acid molecule. The limiting factors in the reaction are the amount of primer, thermostable enzyme and nucleoside triphosphate present in the reaction. Preferably at least one cycle of steps (i.e. denaturation, annealing and extension) is repeated. For use in detection, the number of cycling steps will depend on, for example, the nature of the sample. If the sample is a complex mixture of nucleic acids, then more cycling steps will be required to amplify the target sequence sufficient for detection. Typically, the cycling step is repeated at least about 20 times, but may be repeated as many as 40, 60, or even 100 times.
Fluorescence Resonance Energy Transfer (FRET)
FRET techniques (see, e.g., U.S. Pat. nos. 4,996,143, 5,565,322, 5,849,489, and 6,162,603) are based on the concept that when a donor fluorescent moiety and a corresponding acceptor fluorescent moiety are located within a distance of each other, energy transfer occurs between the two fluorescent moieties, which energy transfer can be visualized or otherwise detected and/or quantified. When the donor is excited by optical radiation having a suitable wavelength, the donor generally transfers energy to the acceptor. The receptor typically re-emits the transferred energy in the form of optical radiation having a different wavelength. In certain systems, non-fluorescent energy can be transferred between donor and acceptor moieties by a biomolecule that includes a substantially non-fluorescent donor moiety (see, e.g., U.S. patent No. 7,741,467).
In one example, an oligonucleotide probe may contain a donor fluorescent moiety and a corresponding quencher, which may or may not be fluorescent, and which dissipates 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 fluorescent emission from the donor fluorescent moiety is quenched by the acceptor moiety. During the extension step of the polymerase chain reaction, the probes bound to the amplified products are cleaved by 5 'to 3' nuclease activity, e.g., taq polymerase, such that the fluorescent emission of the donor fluorescent moiety is no longer quenched. Exemplary probes for this purpose are described, for example, in U.S. Pat. nos. 5,210,015, 5,994,056, and 6,171,785. Common donor-acceptor pairs include FAM-TAMRA pairs. Commonly used quenchers are DABCYL and TAMRA. Common dark quenchers include BlackHole QuenchersTM(BHQ)(Biosearch Technologies,Inc.,Novato,Cal.)、Iowa BlackTM(Integrated DNA Tech.,Inc.,Coralville,Iowa)、BlackBerryTMQuencher 650(BBQ-650)(Berry&Assoc.,Dexter,Mich.).
In another example, two oligonucleotide probes (each containing a fluorescent moiety) can hybridize to an amplification product at specific positions determined by complementarity of the oligonucleotide probes to the target nucleic acid sequence. After hybridization of the oligonucleotide probe to the amplification product nucleic acid at the appropriate position, a FRET signal is generated. The hybridization temperature may be in the range of about 35 ℃ to about 65 ℃ for about 10 seconds to about 1 minute.
The fluorescence analysis can be performed using, for example, a photon counting epifluorescence microscope system (containing appropriate dichroic mirrors and filters for monitoring the fluorescence emission of a specific range), a photon counting photomultiplier system, or a fluorometer. Excitation may 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 suitably filtered to excite in the desired range to initiate energy transfer or to allow direct detection of fluorophores.
As used herein with respect to the donor and the corresponding acceptor moiety, "corresponding" refers to an acceptor fluorescent moiety or dark quencher having an absorbance spectrum that overlaps with the emission spectrum of the donor fluorescent moiety. The maximum wavelength of the emission spectrum of the acceptor fluorescent moiety should be at least 100nm greater than the maximum wavelength of the excitation spectrum of the donor fluorescent moiety. Thus, an efficient non-radiative energy transfer can be produced between them.
The fluorescence donor and corresponding acceptor moieties are typically chosen for (a) efficient Forster energy transfer, (b) large final Stokes shift (> 100 nm), (c) shift the emission as much as possible to the red portion of the visible spectrum (> 600 nm), and (d) shift the 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 excitation maximum near the laser line (e.g., helium-cadmium 442nm or argon 488 nm), has a high extinction coefficient, high quantum yield, and its fluorescence emission well overlaps with the excitation spectrum of the corresponding acceptor fluorescent moiety. The corresponding acceptor fluorescent moiety can be selected to have a high extinction coefficient, high quantum yield, good overlap of its excitation with the emission of the donor fluorescent moiety, and emission in the red portion (> 600 nm) of the visible spectrum.
Representative donor fluorescent moieties that can be used with the various acceptor fluorescent moieties in FRET techniques include fluorescein, B-phycoerythrin, 9-acridinyl isothiocyanate, fluorescein VS, 4-acetamido-4 ' -isothiocyanatostilbene-2, 2' -disulfonic acid, 7-diethylamino-3- (4 ' -isothiocyanatophenyl) -4-methylcoumarin, succinimidyl 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, tetramethyl rhodamine 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. The donor and acceptor fluorescent moieties can be obtained, for example, from Molecular Probes (binding City, oreg.) or SIGMA CHEMICAL Co. (St. Louis, mo.).
The donor and acceptor fluorescent moieties may be attached to the appropriate probe oligonucleotide by a linker arm. The length of each linker arm is important because the linker arm affects the distance between the donor and acceptor fluorescent moieties. The length of the linker arm is in angstromsThe distance in units from the nucleotide base to the fluorescent moiety. Typically, the linker arm is aboutTo aboutThe linker arm may be of the kind described in WO 84/030885. WO 84/0308185 also discloses methods for attaching a linker arm to a specific nucleotide base, and for attaching a fluorescent moiety to a linker arm.
The acceptor fluorescent moiety, such as LC Red 640, may be combined with an oligonucleotide containing an amino linker (e.g., C6-phosphoramidite available from ABI (Foster City, calif.) or GLEN RESEARCH (Sterling, VA)) to produce, for example, an LC Red 640-labeled oligonucleotide. Frequently used linkers for coupling a donor fluorescent moiety, such as fluorescein, to an oligonucleotide include thiourea linkers (FITC-derived, e.g., fluorescein-CPG's from GLEN RESEARCH or ChemGene (Ashland, mass.), amide linkers (fluorescein-NHS-ester derived, such as CX-fluorescein-CPG from biogex (San Ramon, calif)), or 3' -amino-CPGs that require coupling of a fluorescein-NHS-ester after oligonucleotide synthesis.
Detection by real-time PCR
The present disclosure provides methods for detecting the presence or absence of bacterial and fungal target organisms in a biological sample or a non-biological sample. The provided method avoids the problems of sample contamination, false negatives and false positives. These methods comprise performing at least one cycling step comprising amplifying a portion of a target nucleic acid molecule from a sample using one or more pairs of primers and a FRET detection step. The plurality of cycling steps is performed, preferably in a thermal cycler. The method can be performed using primers and probes that detect the presence of the target organism, and detecting the target gene indicates the presence of the target organism in the sample.
As described herein, labeled hybridization probes using FRET techniques may be used to detect amplification products. FRET format utilizationTechniques to detect the presence or absence of amplification products, and thus the presence or absence of CA.The technology utilizes a single strand hybridization probe labeled with, for example, a fluorescent dye and a quencher, which may or may not be fluorescent. When the first fluorescent moiety is excited with light of a suitable wavelength, the absorbed energy is transferred to the second fluorescent moiety or dark quencher according to the FRET principle. The second fluorescent moiety is typically a quencher molecule. During the annealing step of the PCR reaction, the labeled hybridization probes bind to the target DNA (i.e., amplification product) and are degraded during the subsequent extension phase by 5 'to 3' nuclease activity, such as Taq polymerase. Thus, the fluorescent moiety and the quencher moiety become spatially separated from each other. Thus, upon excitation of the first fluorescent moiety in the absence of a quencher, fluorescent emission from the first fluorescent moiety may be detected. For example, ABI7700 Sequence detection System (Applied Biosystems) useTechniques, and are suitable for performing the methods described herein for detecting the presence or absence of NG in a sample.
Molecular beacons that bind FRET can also be used to detect the presence of amplification products using real-time PCR methods. Molecular beacon technology uses hybridization probes labeled with a first fluorescent moiety and a second fluorescent moiety. The second fluorescent moiety is typically a quencher, and the fluorescent label is typically located at each end of the probe. Molecular beacon technology uses probe oligonucleotides with sequences that allow the formation of secondary structures (e.g., hairpins). As a result of secondary structure formation within the probe, the two fluorescent moieties are spatially close when the probe is in solution. After hybridization to the target nucleic acid (i.e., amplification product), the secondary structure of the probe is destroyed and the fluorescent moieties become separated from each other, allowing detection of the emission of the first fluorescent moiety upon excitation with light of the appropriate wavelength.
Another common form of FRET technology is the use of two hybridization probes. Each probe can be labeled with a different fluorescent moiety and is typically designed to hybridize in close proximity to each other in the target DNA molecule (e.g., amplification product). A donor fluorescent moiety, such as fluorescein, is at 470nmThe light source of the instrument is activated. During FRET, luciferin transfers its energy to acceptor fluorescent moieties, e.g-Red 640 (LC Red 640) orRed705 (LC Red 705). The acceptor fluorescent moiety then emits light of longer wavelength, consisting ofAnd detecting by an optical detection system of the instrument. Efficient FRET occurs only when the fluorescent moiety is in direct local proximity and when the emission spectrum of the donor fluorescent moiety overlaps with the absorption spectrum of the acceptor fluorescent moiety. The intensity of the emitted signal may be related to the number of original target DNA molecules (e.g., the number of CA genomes). If amplification of the target nucleic acid occurs and amplification products are produced, the hybridization step produces a detectable signal based on FRET between the members of the probe pair.
Typically, the presence of FRET indicates the presence of a target organism in the sample, and the absence of FRET indicates the absence of a target organism in the sample. However, insufficient sample collection, delayed transport, improper transport conditions, or the use of certain collection swabs (calcium alginate or aluminum shafts) are conditions that can affect the success and/or accuracy of the test results. Using the methods disclosed herein, detection of FRET within, for example, 45 cycles indicates the presence of a target organism.
Representative biological samples that can be used to practice these methods include, but are not limited to, vaginal swabs, fecal specimens, blood specimens, skin swabs, nasal swabs, wound swabs, blood cultures, skin and soft tissue infections. Methods for collection and storage of biological samples are known to those skilled in the art. The biological sample may be processed (e.g., by nucleic acid extraction methods and/or kits known in the art) to release the target nucleic acid, or in some cases, the biological sample may be contacted directly with the PCR reaction components and appropriate oligonucleotides.
Melting curve analysis is an additional step that may be included in the cycle curve. Melting curve analysis is based on the fact that DNA melts at a characteristic temperature called melting temperature (Tm), which is defined as the temperature at which half of the DNA duplex separates into single strands. The melting temperature of DNA is primarily dependent on its nucleotide composition. Thus, DNA molecules rich in G and C nucleotides have a higher Tm than DNA molecules with abundant a and T nucleotides. By detecting the temperature of the signal loss, 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 of the probe from the amplified product can confirm the presence or absence of the target organism in the sample.
The control sample may also be cycled during each thermocycler run. The positive control sample can amplify a target nucleic acid control template (an amplification product different from the target gene) using, for example, a control primer and a control probe. Positive control samples can also be amplified, for example, with plasmid constructs containing target nucleic acid molecules. Such plasmid controls can be amplified internally (e.g., within a sample) or in a separate sample run in parallel with the patient sample using the same primers and probes as used to detect the intended target. Such controls are an indicator of success or failure of amplification, hybridization, and/or FRET reactions. Each thermocycler run may also include a negative control, e.g., lack of 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 be readily determined, for example, the ability of primers to anneal and initiate extension with sequence specificity, and the ability of probes to hybridize with sequence specificity and to undergo FRET.
In one embodiment, the method includes the step of avoiding contamination. Enzymatic methods utilizing uracil-DNA glycosylase are described, for example, in U.S. Pat. nos. 5,035,996, 5,683,896 and 5,945,313 to reduce or eliminate contamination between one thermocycler run and the next.
These methods can be practiced using conventional PCR methods that incorporate FRET techniques. In one embodiment, use is made ofAnd (3) an instrument. The following patent applications describe, for exampleThe real-time PCR used in the techniques is WO 97/46707, WO 97/46714 and WO 97/46712.
Except forIn addition to the instrumentation, there are various instrumentation for performing rapid and accurate PCR binding to the resulting nucleic acid products. Such an instrument can enable absolute or relative quantification of target nucleic acids, as well as post-PCR analysis of amplified nucleic acids by melting curve analysis.
Some instruments are configured to detect a target nucleic acid by exciting a fluorophore (such as a donor fluorescent moiety) attached to a probe and subsequently measuring the resulting emitted fluorescent signal. The apparatus for detecting a target nucleic acid by fluorescence excitation and measuring an emitted fluorescent signal may comprise a plurality of 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 from ROCHEThe 480 instrument includes a set of five excitation filters (450, 483, 523, 558 and 615 nm) and six emission filters (500, 533, 568, 610, 640 and 670 nm).
The individual excitation and emission filters can be freely combined to achieve accurate measurement of the best excitation and emission fluorescent signal of the fluorophore. The excitation-emission filter pairs may be used alone for monochromatic applications or in sequential combination for polychromatic applications. It will be appreciated that the choice of channel for analysis will depend, at least in part, on the fluorescent dye used in the experiment.
The PC workstation may be used for operation and the Windows NT operating system may be used. When the machine places the capillaries in sequence on the optical unit, a signal from the sample can be obtained. The software may display the fluorescent signal in real time immediately after each measurement. The fluorescence acquisition time is 10-100 milliseconds (msec). After each cycling step, the quantitative display of fluorescence versus cycle number can be updated continuously for all samples. The generated data may be stored for further analysis.
Instead of FRET, a double stranded DNA binding dye such as a fluorescent DNA binding dye (e.g.,Green orGold (Molecular Probes)) to detect the amplified product. Upon interaction with the duplex, such fluorescent DNA binding dyes emit a fluorescent signal upon excitation with light of the appropriate wavelength. Double stranded DNA binding dyes (such as nucleic acid) intercalating dyes may also be used. When using double strand DNA binding dyes, melting curve analysis is typically performed to confirm the presence of amplified products.
It should be understood that embodiments of the present disclosure are not limited by the configuration of one or more commercially available instruments.
Article/kit
Embodiments of the present disclosure further provide articles, compositions, or kits for detecting bacterial and fungal organisms associated with vaginal disease. The article of manufacture may include primers and probes for detecting the target gene, as well as suitable packaging materials. The composition may include primers for amplifying the target gene. In certain embodiments, the composition may further comprise a probe for detecting the target gene. Representative primers and probes for detecting a target organism are capable of hybridizing to a target nucleic acid molecule. In addition, the kit may also include reagents and materials required for DNA immobilization, hybridization, and detection, such as solid supports, buffers, enzymes, and DNA standards, in suitable packages. Methods of 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.
The article of manufacture may also include one or more fluorescent moieties for labeling probes, or alternatively, 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 probes. Examples of suitable FRET donor fluorescent moieties and corresponding acceptor fluorescent moieties are provided above.
The article of manufacture may further comprise packaging instructions or packaging labels having thereon instructions for using the primers and probes to detect a target organism in a sample. The articles and compositions may additionally include reagents (e.g., buffers, polymerases, cofactors, or anti-contamination reagents) for practicing the methods disclosed herein. Such reagents may be specific to one of the commercially available instruments described herein.
Embodiments of the present disclosure will be further described in the following examples, which do not limit the scope of the invention described in the claims.
Examples
The following examples, tables and figures are provided to aid in understanding the subject matter, the true scope of which is set forth in the appended claims. It will be appreciated that modifications to the procedures set forth can be made without departing from the spirit of the invention.
Example 1 PCR assay reagents and conditions
Using4800 Systems orReal-time PCR detection of target bacteria and candida species was performed by a 6800/8800 systems platform (Roche Molecular Systems, inc., plaasanton, CA). The final concentration of the amplification reagents is shown in table 2 below:
TABLE 2 PCR amplification reagents
Table 3 shows typical temperature profiles for PCR amplification reactions:
TABLE 3 PCR temperature curve
The Pre-PCR procedure involves initial denaturation and incubation at 55 ℃,60 ℃ and 65 ℃ to reverse transcribe the RNA template. Incubation at three temperatures has the beneficial effect that at lower temperatures slightly mismatched target sequences (such as genetic variants of an organism) are transcribed as well, while at higher temperatures the formation of RNA secondary structures is inhibited, thus making transcription more efficient. The PCR cycle was split into two measurements, with one step set up (binding annealing and extension) applied for both measurements. The first 5 cycles at 55 ℃ allow for increased inclusion by pre-amplifying slightly mismatched target sequences, while the 45 cycles of the second measurement provide for increased specificity by using an annealing/extension temperature of 58 ℃. Example 2 amplification and detection of candida species
Oligonucleotide primers and probes are designed to specifically detect candida species from branches associated with candidal vaginitis, which are candida albicans, dublin candida, candida tropicalis, and candida parapsilosis (collectively referred to as candida species). These oligonucleotides target conserved regions in the ribosomal DNA genes at the 18s rRNA end of the ITS1 region, fig. 1 shows rRNA genomic tissues of various candida species, with 18s, 5.8s and 28s genes mutated (expressed as percent of identification) on the right side relative to candida albicans. The rDNA gene is present in 50 to 200 copies per genome, which allows for very sensitive detection of candida species. Table 4 shows the expected hybridization of the primers and probes of the invention to 18s rRNA or ITS1 target regions of Candida albicans, candida dubliniensis, candida tropicalis and Candida parapsilosis by computer sequence analysis.
TABLE 4 lists of primers and probes used in various PCR assays
In table 4, "x" represents the expected detection of candida species from a hybridization-based computer analysis.
The list of performance of the PCR assays using various combinations of forward and reverse primers and the assays in Ct values are shown in tables 5 and 6, respectively.
TABLE 5 lists of primers and probes used in various PCR assays
TABLE 6 PCR Performance data
The PCR assay was performed using plasmid test sequences encoding the ribosomal DNA genes of Candida albicans, candida parapsilosis, candida tropicalis, and Candida dubliniensis. Three levels of plasmid template (10-fold dilution) and buffer as negative control (negative) were tested:
l1:100,000 copies/PCR, L2:10,000 copies/PCR, L3:1,000 copies/PCR.
EXAMPLE 3 multiplex PCR assay
Multiplex PCR single well assays were performed using four different detection channels, and simultaneously detect three BV-related bacteria lactobacillus species, gardnerella vaginalis, atomyces vaginalis, and candida species (including candida krusei and candida glabrata). The first channel detects 16s rRNA from Gardnerella vaginalis. The second channel detects the D-LDH gene of the Lactobacillus species. The third channel detects a variety of candida species including candida krusei and candida glabrata 18s rRNA and ITS1. Fourth channel detects tufA gene of atopoella vaginalis. Various primers and probes for use in multiplex assays for amplifying and detecting gardnerella vaginalis, lactobacillus species, atopoella vaginalis, candida krusei and candida glabrata have been disclosed in U.S. patent publication No. us 2022/0205020A1, which is incorporated herein by reference in its entirety. The selected combination sets of primers and probes are shown in table 7.
TABLE 7 primers and probes for multiplex BV-CV determination
In table 7, < t-bb_da > = tert-butylbenzyl dA, < COU > = COU dye, < Q > = quencher, < t-bb_dc > = tert-butylbenzyl dC, < FAM > = FAM dye, < JA270> = JA270 dye, < HEX > = HEX dye, < pdU > = 5-propynyldu, and Sp = C3 spacer.
Although the foregoing has been described in some detail for purposes of clarity and understanding, it will be clear to one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the application. 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 disclosure are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent application, and/or other document was individually indicated to be incorporated by reference for all purposes.