Compositions and methods for detecting viral pathogens in a sample

Abstract

To provide compositions and methods for detecting a viral pathogen in a sample.SOLUTION: The present disclosure relates to amplification primers, hybridization assay probes, compositions containing such primers and probes, and related reagents, kits, and methods that can be used to analyze samples for the presence of influenza A virus, influenza B virus, respiratory syncytial virus type A, and / or respiratory syncytial virus type B target nucleic acids.SELECTED DRAWING: None

Description

[Technical Field]

[0001] background CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. § 119 of U.S. Provisional Application No. 62 / 476,659, filed March 24, 2017, the contents of which are incorporated herein by reference.

[0002] The present disclosure relates to the field of biotechnology. More particularly, the present disclosure relates to compositions, including kits and reagents, and methods for analyzing samples to detect viral pathogens, particularly influenza virus and respiratory syncytial virus. [Background technology]

[0003] Influenza is an acute respiratory disease in humans caused by infection with influenza (Flu) viruses, primarily types A and B. Influenza type A viruses are further classified into subtypes based on two major surface protein antigens, hemagglutinin (H) and neuraminidase (N). Influenza type B viruses are not classified into subtypes. Influenza viruses are RNA viruses in the family Orthomyxoviridae. Influenza types A and B (Flu A and Flu B, respectively) are each separate genera containing one species and numerous subspecies.

[0004] Influenza epidemics occur annually worldwide. Flu types A and B circulate within populations, with type A usually predominating. These annual epidemics are due in part to antigenic variation in the virus's H and N surface proteins. Influenza is transmitted primarily through droplets (coughing or sneezing). Symptoms occur on average 1–2 days after exposure and include fever, chills, headache, fatigue, cough, and coryza. Gastrointestinal symptoms, such as nausea, vomiting, and diarrhea, may occur, primarily in children. Influenza complications include pneumonia, which can cause increased morbidity and mortality in children, the elderly, and immunocompromised populations. In the United States, influenza is estimated to result in more than 200,000 hospitalizations and up to 36,000 deaths annually. Influenza epidemics or pandemics are rare. Three influenza pandemics occurred in the 20th century: in 1918, 1958, and 1968, each of which caused millions of deaths worldwide. Other animals can also develop influenza, including pigs, horses, and birds.

[0005] Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infections in infants and children. Like influenza, RSV is an RNA virus. It belongs to the Orthopneumovirus genus in the Paramyxoviridae family. Two subgroups of RSV, A and B, exist, distinguished based on antigenic and surface protein differences. Most annual epidemics contain a mixture of RSV A and RSV B, but one subgroup may dominate throughout a season. RSV infection can cause severe respiratory illness in people of all ages but is more common in children, the elderly, and immunocompromised populations. RSV can infect up to 80% of children under the age of 1 year. Bronchiolitis and pneumonia are major clinical complications in infants and young children, resulting in an estimated 51,000 to 82,000 hospitalizations in the United States each year. RSV infection is also an important cause of severe respiratory illness and a significant number of deaths among older adults, with hospitalizations due to RSV pneumonia costing an estimated $150-680 million annually.

[0006] Given the morbidity, mortality, and economic costs associated with influenza and RSV infections, there is a clear need for improved detection of these pathogens. The present disclosure addresses this and other needs. Summary of the Invention [Means for solving the problem]

[0007] [Mode for Carrying Out the Invention] The present disclosure provides compositions, including kits and reagents, and methods for in vitro diagnostic analysis of influenza A virus (Flu A), influenza B virus (Flu B), respiratory syncytial virus A (RSV A), or respiratory syncytial virus B (RSV B) nucleic acids in a sample. The in vitro diagnostic analysis preferably utilizes polymerase chain reaction (PCR), although other in vitro assay methodologies are contemplated for use with the compositions of the present disclosure. A particularly useful in vitro assay for Flu A, Flu B, RSV A, or RSV B target nucleic acids is a reverse transcription PCR assay, since these target nucleic acids are RNA viruses. Advantageously, an in vitro amplification assay can be performed simultaneously with an in vitro detection assay (real-time PCR). Therefore, a particularly useful and convenient in vitro assay for Flu A, Flu B, RSV A, or RSV B target nucleic acids is a real-time reverse transcription PCR assay.

[0008] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise. Thus, for example, a reference to "an oligonucleotide" includes a plurality of oligonucleotides, etc. The conjunction "or" should be construed in an inclusive sense, i.e., equivalent to "and / or," unless an inclusive sense is inappropriate in the context.

[0009] Temperatures, concentrations, times, etc. stated in this disclosure are preceded by an implicit "about," whereby small, insubstantial deviations are understood to be within the scope of the teachings herein. In general, the term "about" indicates insubstantial variations in the amounts of components of a composition that do not significantly affect the activity or stability of the composition. All ranges should be construed as including the endpoints unless there is a clear exclusion, such as "excluding the endpoints," thus, for example, "in the range of 10 to 15" includes the values ​​10 and 15, and all values ​​and partial values ​​(where applicable) therebetween.

[0010] Unless otherwise stated, embodiments herein that recite various components "comprising" are also contemplated as "consisting of" or "consisting essentially of" the recited components; embodiments herein that recite various components "consisting of" are also contemplated as "comprising" or "essentially consisting of" the recited components; and embodiments herein that recite various components "consisting essentially of" are also contemplated as "consisting of" or "comprising" the recited components (this interchangeability does not apply to the use of these terms in the claims). "Consisting essentially of" means that additional components, compositions, or method steps that do not substantially alter the basic and novel characteristics of the compositions and methods described herein may be included in those compositions or methods. Such characteristics include the ability to detect a target nucleic acid present in a sample with specificity that distinguishes the target nucleic acid from other known respiratory pathogens. Any component, composition, or method step that has a substantial effect on the basic and novel characteristics of the present disclosure would deviate from this term.

[0011] The term "complement" refers to a nucleic acid molecule that contains a contiguous nucleotide sequence that is complementary to a contiguous nucleic acid sequence of another nucleic acid molecule (for standard nucleotides, A:T, A:U, C:G). For example, 5'-AACTGUC-3' is the complement of 5'-TTGACAG-3'. Two nucleic acid sequences are "fully complementary" if their respective contiguous nucleic acid sequences are at least 70% complementary (see, e.g., Sambrook, et al., Molecular Cloning, A Laboratory Manual, 2002). nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).

[0012] "Perfectly matched" in the context of a nucleic acid duplex means that the polynucleotide or oligonucleotide strands forming the duplex form a double-stranded structure or region of double-stranded structure with each other such that all nucleotides (or nucleotide analogs) in each strand in the duplex (i.e., hybridized) region undergo Watson-Crick base pairing with nucleotides in the other strand. The term also encompasses pairing of nucleoside analogs, e.g., deoxyinosine, nucleosides containing 2-aminopurine bases, etc. Conversely, a "mismatch" in a nucleic acid duplex means that one or more nucleotide pairs in the duplex are unable to undergo Watson-Crick base pairing.

[0013] "Substantially homologous," "substantially corresponding," or "substantially corresponding" means that a nucleic acid molecule contains a contiguous nucleic acid sequence that is at least 70% homologous to a contiguous nucleic acid sequence in another nucleic acid molecule.

[0014] A "sample" or "biological sample" is any tissue or polynucleotide-containing material obtained from a human, animal, or environmental sample that may contain a target nucleic acid. Biological samples include peripheral blood, mucus, plasma, serum, saliva, cerebrospinal fluid, urine, or other bodily fluids, bone marrow, or other organs, biopsied tissue, or other materials of biological origin, as well as solutions or compositions containing biological materials, such as bronchial lavage fluid. Samples can be obtained from numerous sources, including clinical sources where samples are taken to determine the presence or absence of a target nucleic acid in the sample, thereby providing a diagnosis to the patient. Samples are chemically and / or mechanically treated to disrupt tissue or cellular structure, thereby releasing intracellular components into solution.

[0015] As used herein, the term "nucleotide" is defined to include nucleotides and nucleosides, including deoxyribonucleotides (e.g., dATP, dCTP, dGTP, dTTP), ribonucleotides (e.g., rATP, rCTP, rGTP, rUTP), and analogs thereof. A nucleotide contains a purine or pyrimidine base glycosidically linked to a ribose or deoxyribose sugar and a phosphate group attached to the ribose or deoxyribose sugar. A nucleoside contains a purine or pyrimidine base glycosidically linked to a ribose or deoxyribose sugar, but lacks the phosphate residue present in a nucleotide. As used herein, nucleotides and nucleosides refer to monomers of DNA or RNA, respectively. (See, e.g., Kornberg and Baker, DNA Replication, 2001). nd Ed. (Freeman, San Francisco, 1992).

[0016] The term "analog" with respect to a compound refers to a compound that has a similar structure to that of another compound but differs from that compound in that one or more different atoms, functional groups, or substructures have been removed or replaced with one or more other atoms, functional groups, or substructures. In the context of a nucleotide or nucleoside, an analog refers to a compound that, like the nucleotide / nucleoside of which it is an analog, can be incorporated into nucleic acid molecules (e.g., primers, probes, and / or amplification products). Nucleotide / nucleoside analogs are generally added to synthetic oligonucleotides (e.g., primers and probes) using phosphoramidite chemistry methods and devices. Nucleotide / nucleoside analogs are generally added to amplification products by including the analog in a reaction mixture where a suitable polymerase, e.g., DNA polymerase, incorporates the analog into the amplification product. Nucleotide / nucleoside (hereinafter "nucleotide") analogs include synthetic nucleotides with modified base moieties and / or modified sugar moieties and / or modified phosphate groups (see, e.g., Scheit, Nucleotide Analogues (John Wiley, New York, 1980; Uhlman and Peyman, Chemical Reviews, 90:543-584 (1990). Such analogs include synthetic nucleosides designed to enhance binding properties, reduce complexity, increase specificity, etc.

[0017] "DNA" refers to deoxyribonucleic acid, i.e., a polymer of deoxyribonucleotides joined by phosphodiester bonds. DNA can be single-stranded (ssDNA) or double-stranded (dsDNA) and can contain both single-stranded and double-stranded (or "duplex") regions. "RNA" refers to ribonucleic acid, i.e., a polymer of ribonucleotides joined by phosphodiester bonds. RNA can be single-stranded (ssRNA) or double-stranded (dsRNA) and can contain both single-stranded and double-stranded (or "duplex") regions. Single-stranded DNA (or regions thereof) and ssRNA can hybridize to form a double-stranded complex (or region) if they are sufficiently complementary. "RNA equivalent," "DNA equivalent," "RNA equivalent base," and "DNA equivalent base" refer to RNA and DNA molecules that have similar complementary base-pair hybridization properties. RNA and DNA equivalents have different sugar moieties (i.e., ribose versus deoxyribose) and may differ, for example, by the presence of uracil in RNA and thymine in DNA. Because the equivalents have the same degree of complementarity to a particular sequence, differences between RNA and DNA equivalents do not contribute to differences in homology (or sequence identity).

[0018] The terms "polynucleotide" and "oligonucleotide" (used interchangeably herein) refer to a polymeric compound containing two or more linked RNA nucleotides, DNA nucleotides, RNA nucleotide analogs, DNA nucleotide analogs, or combinations thereof. A polynucleotide may be present in a linked sequence of nucleotides and may contain other molecules that do not interfere with hybridization of the polynucleotide with a second molecule having a complementary sequence. For example, a polynucleotide often contains two or more linked nucleotides at one end of a linker molecule and two or more linked nucleotides at the second end of the linker molecule, similar to a molecular torch configuration. A polynucleotide is preferably a polymeric chain of 10 to 200 contiguous nucleotides. Polynucleotides may be purified from naturally occurring sources but are preferably synthesized using any of a variety of well-known enzymatic or chemical methods. Whenever an oligonucleotide (or other nucleic acid) is represented by a sequence of letters such as "ATGCUCTG," it is understood that the nucleotides are in left-to-right 5'-3' orientation unless otherwise specified, and that unless otherwise noted, "A" means adenosine (dATP / rATP) or an analog thereof, "C" means cytidine (dCTP / rCTP) or an analog thereof, "G" means guanosine (dGTP / rGTP) or an analog thereof, "U" means uracil (rUTP) or an analog thereof, and "T" means thymidine (dTTP) or an analog thereof. Typically, oligonucleotides of the present disclosure contain the four naturally occurring nucleotides, although they may also contain unnatural nucleotide analogs.

[0019] A "probe" is an oligonucleotide that specifically hybridizes to a target nucleic acid sequence in a nucleic acid, preferably an amplified nucleic acid, under conditions that promote hybridization, forming a detectable hybrid. Probe oligonucleotides contain one or more of a contiguous nucleotide sequence, a sequence that hybridizes to the target, a sequence that does not hybridize to the target, a detectable label, a linker, and a nucleotide analog. Probes preferably have an oligonucleotide length of about 10 to 100 contiguous nucleotides. Certain preferred specific probes have target-hybridizing sequences ranging in length from 12 to 87, 10 to 20, 13 to 37, or 17 to 23 nucleotides. Probe sequences may comprise RNA, DNA, analogs, and combinations thereof. The "backbone" of a probe may be composed of various linkages known in the art, including one or more sugar-phosphodiester linkages, peptide-nucleic acid linkages (PNAs), phosphorothioate linkages, methylphosphonate linkages, or combinations thereof. The sugar moiety of the probe can be ribose or deoxyribose, or similar compounds with known substitutions, such as 2'-O-methylribose and 2'-halide substitutions (e.g., 2'-O-Me or 2'-F). Nucleotide analogs incorporated into the probe oligonucleotide sequence include inosine or "I," 5-Me-dC, isoguanine, other derivatives of purine or pyrimidine bases, or abasic residues (e.g., nucleoside residues (see, e.g., The Biochemistry of the Nucleic Acids, pages 5-36, Adams, et al., ed., 11 th ed., 1992, PCT Application Publication No. WO 93 / 13121). Generally, the target nucleic acid sequence of a probe refers to a sequence contained within an amplified nucleic acid molecule that specifically hybridizes to at least a portion of a probe oligonucleotide using standard hydrogen bonding.

[0020] Probe may comprise target-specific sequence, and optionally other sequence that is not hybridized with target (for example, not hybridized with the nucleic acid to be detected).Such not hybridized with target sequence can include, for example, promoter sequence, restriction endonuclease recognition site, or the sequence that contributes to the three-dimensional conformation of probe (for example, see U.S. Patent No. 5,118,801 and U.S. Patent No. 5,312,728).Probe that shows at least some degree of self-complementarity includes molecular torch and molecular beacon.

[0021] A "molecular torch" can be designed to contain distinct self-complementary regions (designated a "target-hybridizing sequence domain" and a "target closing domain") that are linked by a linking region and hybridize to each other under predetermined hybridization assay conditions. When exposed to denaturing conditions, the two complementary regions of the molecular torch (which may be fully or partially complementary) melt, allowing the target-hybridizing sequence domain to hybridize with the target nucleic acid sequence when returned to the predetermined hybridization assay conditions. Molecular torches are designed such that the target-hybridizing sequence domain favors hybridization with the target nucleic acid sequence over the target closing domain. The target-hybridizing sequence domain and target-closing domain of the molecular torch contain interactive labels (e.g., fluorescers / quenchers) arranged to produce a different signal when the molecular torch self-hybridizes as opposed to hybridizing with the target nucleic acid sequence, thereby allowing for the detection of probe:target duplexes in a test sample in the presence of unhybridized probes having functional labels attached to the probes. Molecular torches are described, for example, in U.S. Patent No. 6,361,945.

[0022] "Molecular beacons" can be designed to have a target-hybridizing sequence, an affinity pair (or nucleic acid arm) that keeps the probe in a closed conformation in the absence of the target nucleic acid sequence, and a label pair that interacts when the probe is in the "closed" conformation. Hybridization of the target-hybridizing sequence of the molecular beacon with its intended target nucleic acid sequence separates the members of the affinity pair, thereby transitioning the probe to an "open" conformation. The transition to the "open" conformation can be detected by a decrease in the interaction of the label pair. Molecular beacons are described, for example, in U.S. Pat. No. 5,925,517.

[0023] A probe may optionally contain a detectable label, which may be attached to the end of the probe or to the interior of the probe. The terms "label" and "detectable label" are used interchangeably herein and refer to one or more atoms that can be specifically detected to indicate the presence of a substance attached to the one or more atoms. A label may be a directly detectable primary label or a secondary label that can be indirectly detected, for example, by direct or indirect interaction with the primary label. A label may be directly or indirectly linked to a polynucleotide probe. Labels include dyes, particles, chromophores (e.g., atoms or molecules that impart a detectable color), combined fluorescent energy transfer labels, electrophores, redox-active moieties (e.g., transition metals), enzymes, haptens, light-emitting compounds (e.g., bioluminescent, phosphorescent, or chemiluminescent moieties), fluorophores, mass labels, and radiolabels. Labels and associated detection methods are well known (see, e.g., U.S. Patent No. 6,627,748 (B1); Styer and Haugland, (1967), Proc. Natl. Acad. Sci. USA 98:719; U.S. Patent No. 5,591,578; U.S. Patent No. 5,491,063; U.S. Patent No. 5,201,015).

[0024] The term "fluorophore" means a fluorescent compound that can re-emit light upon excitation with light. Fluorophores include, for example, fluorescent lanthanide complexes (including those of europium and terbium), fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methylcoumarin, pyrene, malachite green, Cy3, Cy5, CalFluor Red™, CalFluor Orange™, stilbene, Quasar dyes (e.g., Quasar 570, Quasar 670, Quasar 705), Lucifer Yellow, Cascade Blue™, Texas Red, Alexa dyes, phycoerythrin, Bodipy, and others known in the art (see, e.g., Haugland, Molecular Probes Handbook (Eugene, OR), 6th Edition; The Synthegen catalog (Houston, Tex.); Lakowicz, Principles of Fluorescence Spectroscopy, 2nd Ed., Plenum Press New York, 1999). York (1999), and WO98 / 59066).

[0025] The term "quencher" is used to refer to a molecule that absorbs light. Quenchers are generally used in combination with luminescent labels, such as fluorophores, to absorb the emitted light when in close proximity to the fluorophore. Quenchers are well known in the art, and include, for example, Black Hole Quencher™ (or BHQ™, BHQ-1™, or BHQ-2™), Blackberry Quencher, Dabcyl, QSY, and Tamra™ compounds, to name a few.

[0026] " Homogeneous detectable label " refers to a label that is attached to probe oligonucleotide and can be detected without physically removing the label or the hybridized form of the labeled probe from the unhybridized form.Examples of homogeneous labels are detailed in, for example, U.S. Patent Nos. 5,283,174, 6,150,097, 5,201,015, 5,656,207 and 5,658,737.

[0027] The linear probe, molecular torch, and beacon are preferably labeled with an interactive pair of detectable labels. Examples of detectable labels that are preferred as members of an interactive pair of detectable labels are those that interact with each other through FRET or non-FRET energy transfer mechanisms. Fluorescence resonance energy transfer (FRET) involves the non-radiative transfer of an energy quantum from an absorption site to its utilization site in the molecule or molecular system through a resonant interaction between chromophores over a distance significantly longer than the interatomic distance, without conversion to thermal energy and without dynamic collision between the donor and acceptor moieties. The "donor" is the moiety that first absorbs and then transfers energy, and the "acceptor" is the moiety to which energy is subsequently transferred. In addition to FRET, there are at least three other "non-FRET" energy transfer processes by which excitation energy can be transferred from a donor to an acceptor molecule.

[0028] When the two labels of a donor / acceptor pair are kept close enough that the energy emitted by one label can be accepted or absorbed by the second label, regardless of whether it is a FRET or non-FRET mechanism, the two labels are said to be in an "energy transfer relationship" with each other.This is the case, for example, when a molecular beacon or a molecular torch is kept in a "closed" state by forming a stem duplex, and the fluorescence emission from the fluorophore attached to one arm of the probe is quenched by the quencher moiety on the opposite arm.This is also the case, for example, when a linear probe is labeled with a fluorophore and a quencher at a distance along the linear probe, and the fluorescence emission from the attached fluorophore is quenched by the attached quencher.In these cases, the spatial separation of the fluorophore and the quencher molecule (for example, by "opening" the molecular torch or beacon, or by hydrolysis of the linear probe molecule) can be achieved.

[0029] Examples of donor / acceptor pairs include fluorescein / tetramethylrhodamine, IAEDANS / fluorescein, EDANS / DABCYL, coumarin / DABCYL, fluorescein / fluorescein, BODIPY FL / BODIPY FL, fluorescein / DABCYL, Lucifer Yellow / DABCYL, BODIPY / DABCYL, eosin / DABCYL, erythrosine / DABCYL, tetramethylrhodamine / DABCYL, CalOrange / BHQ1, CalRed / BHQ2, FAM / BHQ1, Quasar / BHQ2, Texas Red / DABCYL, CY5 / BH1, CY5 / BH2, CY3 / BH1, CY3 / BH2, and fluorescein / QSY7 dye. Labels are available from companies such as LGC Biosearch Technologies (Petaluma, CA), Glen Research (Sterling, VA), Integrated DNA Technologies (Skokie, IL), and Thermo Fisher (Waltham, MA).

[0030] Synthetic techniques and methods for attaching labels to nucleic acids and detecting labels are well known in the art (e.g., Sambrook, et al., Molecular Cloning, A Laboratory Manual,2 nd (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), Chapter 10; U.S. Patent Nos. 5,658,737, 5,656,207, 5,547,842, 5,283,174, and 4,581,333; and European Patent Application No. 0747706). The probe may optionally contain a fluorophore and a quencher. As with detectable moieties within the probe sequence, the nucleotide residues of the probe that bind to the target nucleic acid sequence need not be strictly contiguous.

[0031] An "amplification primer" or "primer" is an optionally modified oligonucleotide that can hybridize to a target nucleic acid sequence or its complement and participate in a nucleic acid amplification reaction. A primer oligonucleotide contains one or more of a contiguous nucleotide sequence, a sequence that hybridizes to the target, a sequence that does not hybridize to the target, a linker, and a nucleotide analog. A primer preferably has an oligonucleotide length of about 10 to 100 contiguous nucleotides. The primer sequence may comprise RNA, DNA, analogs, and combinations thereof. The "backbone" of a primer may be composed of various linkages known in the art, including one or more sugar-phosphodiester linkages, peptide-nucleic acid linkages (PNAs), phosphorothioate linkages, methylphosphonate linkages, or combinations thereof. The sugar portion of a primer may be ribose or deoxyribose, or similar compounds with known substitutions, such as 2'-O-methylribose and 2'-halide substitutions (e.g., 2'-O-Me or 2'-F). Nucleotide analogs incorporated into the primer oligonucleotide sequence may include inosine or "I", 5-Me-dC, isoguanine, other derivatives of purine or pyrimidine bases, or abasic residues (e.g., nucleoside residues). The target nucleic acid sequence of a primer generally refers to both a sequence contained within the genetic information of the organism to be detected and a sequence contained within the amplified nucleic acid molecule that specifically hybridizes with at least a portion of the primer oligonucleotide using standard hydrogen bonding. The primer hybridizes to the target nucleic acid sequence and has a 3' end that can be extended by a DNA polymerase to incorporate nucleotides complementary to the target nucleic acid sequence and generate its double-stranded portion.

[0032] "Capture oligonucleotide" refers to at least one nucleic acid oligonucleotide that allows for linkage of a target nucleic acid to an immobilized oligonucleotide by base pair hybridization (preferably resulting in an immobilized probe:capture oligonucleotide:target nucleic acid complex). A capture oligonucleotide preferably contains two binding regions, usually contiguous on the same oligonucleotide: a target nucleic acid binding region and an immobilized probe binding region; however, capture oligonucleotides of the present invention may contain target nucleic acid binding regions and immobilized probe binding regions present on two different oligonucleotides linked by one or more linkers. For example, the immobilized probe binding region is present on a first oligonucleotide, and the target nucleic acid binding region is present on a second oligonucleotide, with the two different oligonucleotides linked by hydrogen bonds to a linker that is a third oligonucleotide containing a sequence that specifically hybridizes to the sequences of the first and second oligonucleotides. The target-hybridizing region of the capture probe can be specific for the target nucleic acid (e.g., sufficiently complementary to the target nucleic acid sequence) or non-specific for the target nucleic acid. One target capture system containing a capture oligonucleotide is described in U.S. Patent Nos. 6,110,678 and 9,051,601.

[0033] "Immobilized probe" or "immobilized nucleic acid" refers to a nucleic acid that directly or indirectly links a capture oligonucleotide to an immobilized support. An immobilized probe is an oligonucleotide linked to a solid support that facilitates separation of bound target nucleic acid from unbound material in a sample.

[0034] The term "solid substrate" refers to any suitable medium present in a solid phase to which an antibody or agent can be covalently or non-covalently bound or immobilized.

[0035] "Separating" or "purifying" or "isolating" means that one or more components of a biological sample are removed from one or more other components of the sample. Sample components generally include nucleic acids in an aqueous phase, which may also include other substances, such as proteins, carbohydrates, lipids, and labeled probes. Preferably, the separating, isolating, or purifying step removes at least about 70%, more preferably at least about 90%, and even more preferably at least about 95% of the other components present in the sample.

[0036] "Homogeneous assay" refers to a detection procedure that does not require physical separation of hybridized from unhybridized probes before determining the degree of specific probe hybridization. Exemplary homogeneous assays can use molecular beacons or other self-reporting probes that emit a fluorescent signal when hybridized to an appropriate target nucleic acid sequence, chemiluminescent acridinium ester labels that can be selectively destroyed by chemical means unless present in the hybrid duplex, and other homogeneous detectable labels familiar to those skilled in the art.

[0037] "Amplification" refers to an in vitro procedure for obtaining multiple copies of a target nucleic acid sequence, its complement, or fragments thereof.

[0038] "Amplicon" refers to DNA or RNA that is the product of a nucleic acid amplification or replication process. Amplicons can be formed using a variety of methods, such as polymerase chain reaction (PCR), ligase chain reaction (LCR), and transcription-associated amplification (e.g., TMA).

[0039] The term "multiplex PCR" refers to a PCR reaction characterized by the generation of two or more different amplification products or amplicons by using two or more pairs of amplification primers in the same PCR reaction.

[0040] The term "multicolor" RT-PCR refers to a real-time PCR assay characterized in that one or more distinct amplification products or amplicons generated in multiplex PCR or monoplex PCR (using only one pair of amplification primers) are detected using distinguishably labeled hybridization probes.

[0041] "Target nucleic acid" or "target" refers to a nucleic acid containing a target nucleic acid sequence. As described herein, target nucleic acids include Flu A nucleic acids, Flu B nucleic acids, RSV A nucleic acids, and RSV B nucleic acids. A "target nucleic acid sequence" (also referred to as a "target nucleotide sequence," "target sequence," "target region," or "target nucleic acid molecule") refers to a specific deoxyribonucleotide or ribonucleotide molecule or nucleotide sequence that comprises all or a portion of the nucleotide sequence of a single-stranded nucleic acid molecule, and complementary deoxyribonucleotide or ribonucleotide sequences thereto.

[0042] "Transcription-associated amplification" refers to any type of nucleic acid amplification that uses an RNA polymerase to produce multiple RNA transcripts from a nucleic acid template. One example of a transcription-associated amplification method, called "transcription-mediated amplification" (TMA), generally uses an RNA polymerase, a DNA polymerase, deoxyribonucleoside triphosphates, ribonucleoside triphosphates, and a promoter-template complementary oligonucleotide, which may optionally include one or more similar oligonucleotides. Variations of TMAs are well known in the art and are described, for example, in U.S. Pat. Nos. 5,437,990, 5,399,491, 5,554,516, 5,130,238, 4,868,105, and 5,124,246, PCT Publication Nos. WO 93 / 22461, WO 88 / 01302, WO 88 / 10315, WO 94 / 03472, and WO 95 / 03430. [Summary of the Invention]

[0043] The present disclosure provides compositions, including kits and reagents, and methods for in vitro diagnostic analysis of influenza A virus (Flu A), influenza B virus (Flu B), respiratory syncytial virus A (RSV A), or respiratory syncytial virus B (RSV B) nucleic acid in a sample. The in vitro diagnostic analysis preferably utilizes polymerase chain reaction (PCR), although other in vitro assay methodologies are contemplated for use with the compositions of the present disclosure. Flu A, Flu B, RSV A, or RSV B nucleic acid are not included in the in vitro diagnostic analysis. A particularly useful in vitro assay for use with Flu A, Flu B, RSV A, or RSV B target nucleic acids is a reverse transcription PCR assay, since these target nucleic acids are RNA viruses. Advantageously, an in vitro amplification assay can be performed simultaneously with an in vitro detection assay (real-time PCR). Thus, a particularly useful and convenient in vitro assay for use with Flu A, Flu B, RSV A, or RSV B target nucleic acids is a real-time reverse transcription PCR assay.

[0044] In one aspect, the sample is a biological sample. In one aspect, the biological sample is a clinical sample. In another aspect, the sample is, for example, a swab sample from a nasopharyngeal (NP) swab specimen obtained from a patient. In some embodiments, the compositions and methods of the present invention can be used to aid in the differential diagnosis of Flu A, Flu B, and RSV A and RSV B infections. A negative result does not exclude the infection. Conversely, a positive result does not exclude bacterial infection or co-infection with other viruses. A final diagnosis of respiratory virus infection may also be obtained by taking into account the use of additional laboratory tests and clinical symptoms.

[0045] One aspect provides nucleic acid molecules that are hybridization assay probes useful for detecting target nucleic acid sequences of Flu A, Flu B, RSV A, or RSV B. Preferably, such probe molecule species comprise a probe sequence that is substantially complementary to a probe target nucleic acid sequence within a viral genome or amplicon generated therefrom that is targeted for detection. In a preferred embodiment, the probe target nucleic acid sequence consists of about 17 to about 100 contiguous bases contained within the targeted viral genome (or amplicon generated therefrom). Preferably, the probe molecule is up to about 100 nucleotide residues in length, with lengths of between about 20 and 60 nucleotide residues being particularly preferred.

[0046] In the context of Flu A, in some preferred embodiments, a probe of the invention preferably comprises a sequence designated FA1-F, FA1-G, FA1-H, FA1-I, FA1*-J, FA1*-K, FA1-L, FA1-M, FA1-N, FA1*-O, FA1*-P, or FA1*-Q. In other embodiments, a probe sequence of the invention preferably comprises a sequence designated FA2-R, FA2-S, FA2-T, FA2*-U, or FA2*-V (SEQ ID NOS: 6-22). In particularly preferred embodiments, two probes, one from each of the aforementioned groups, are used in tandem to target two different regions of the Flu A genome or an amplification product generated therefrom.

[0047] In the context of Flu B, in a preferred embodiment, the probe sequences of the invention are preferably: FB-B, FB-B!, FB-C, FB-C!, FB-D, FB-D!, FB-E, FB-E!, FB-F, FB-F!, FB-G, FB-G!, FB-H, FB-H!, FB-I, FB-I!, FB-J, FB-K, FB-K! ... :FB-J!, Sequence Name:FB-K, Sequence Name:FB-K!, Sequence Name:FB-L, Sequence Name:FB-L!, Sequence Name:FB-M, Sequence Name:FB-M!, Sequence Name:FB-N, Sequence Name:FB-N!, Sequence Name:FB-O, Sequence Name:FB-O!, Sequence Name:FB-Q, Sequence Name:FB-R, Sequence Name:FB-S, Sequence Name:FB-T, Sequence Name:FB-U, Sequence Name:FB-V, Sequence Name:FB*-W, or Sequence Name:FB*-X (SEQ ID NOs: 30 to 57 and 59 to 66).

[0048] In the context of RSV A, in a preferred embodiment, the probe sequences of the present invention are preferably RA-A, RA-E, RA-F, RA-G, RA-H, RA-J, RA-J!, RA-K, RA-K!, RA-L, RA-L!, RA-M, RA-M!, RA-O, RA-P, RA-Q, RA*-W, and RA*-X (SEQ ID NOs: 71, 75-78, 80-87, 89-91, 97, and 98).

[0049] In the context of RSV B, in a preferred embodiment, the probe sequences of the present invention are preferably SEQ ID NO: RB-D, SEQ ID NO: RB-E, SEQ ID NO: RB-V, SEQ ID NO: RB-V!, SEQ ID NO: RB-W, SEQ ID NO: RB-W!, SEQ ID NO: RB-X, SEQ ID NO: RB-X!, SEQ ID NO: RB-Y, and SEQ ID NO: RB-Y! (SEQ ID NOs: 102, 103, and 107-114).

[0050] Preferably, the probe species are labeled, and optionally distinguishably labeled so that any one probe species can be distinguished from other probe species in a multiplex detection assay. Distinguishable labeling can be achieved using two or more detectable labels, for example, a chemiluminescent moiety, a fluorophore moiety, and both a fluorophore moiety and a quencher moiety.

[0051] Another aspect of the present disclosure relates to nucleic acid molecules that are amplification primers designed for use in in vitro amplification of a target nucleic acid sequence. A related aspect of the disclosure relates to pairs of such primers that can be used to amplify a desired amplicon containing the target nucleic acid sequence. These primers include a first Flu A primer pair, a second Flu A primer pair, a Flu A primer pair that can be used to amplify a Flu A target nucleic acid region that is different from the Flu A target nucleic acid region that can be amplified using the first Flu A primer pair, and a Flu A primer pair that can be used to amplify a Flu A target nucleic acid region that is different from the Flu A target nucleic acid region that can be amplified using the first Flu A primer pair. The primer pairs include one or more of a Flu A primer pair, a Flu B primer pair, an RSV A primer pair, and an RSV B primer pair, which include a first and a second primer that can be used to generate a corresponding amplicon for Flu A, Flu B, RSV A, and / or RSV B if a viral pathogen is present in the biological sample being tested.

[0052] Typically, a primer pair includes a first primer containing a priming nucleotide sequence that is substantially complementary to a first target nucleic acid sequence of a viral genome, i.e., a portion of the viral genome to be amplified. Preferably, the first and second target nucleic acid sequences of the target nucleic acid are separated by at least 10 nucleotides, preferably about 50 to 1,000 nucleotides, and each of them preferably consists of about 17 to about 100 consecutive bases of the viral genome to be detected. In some embodiments, one or more of the primers in one or more primer pairs further includes a primer upstream region having a nucleotide sequence that is not complementary to the target nucleotide sequence of that primer.

[0053] A preferred first primer for generating a first Flu A amplicon has a priming nucleotide sequence designated FA1-A or FA1-W. A preferred second primer useful with such a first primer has a priming nucleotide sequence designated FA1-Y or FA1-AB. SEQ ID NOs: 1, 23, 25, and 28.

[0054] Preferred first primers for generating the second Flu A amplicon have a priming nucleotide sequence of FA2-B, FA2-C, FA2-D, FA2-E, or FA2-X. Preferred second primers useful with such first primers have a priming nucleotide sequence of FA2-Z or FA2-AA. SEQ ID NOs: 2, 5, 24, 26, and 27.

[0055] Preferred first primers for generating Flu B amplicons have the priming nucleotide sequences FB-A or FB-Y. Preferred second primers useful with such first primers have the priming nucleotide sequences FB-Z, FB-AA, and FB-AB. SEQ ID NOS: 29 and 67-70.

[0056] Preferred first primers for generating RSV A amplicons have a priming nucleotide sequence of SEQ ID NO: RA-I or SEQ ID NO: RA-N. Preferred second primers useful with such first primers have a priming nucleotide sequence of SEQ ID NO: RA-B, SEQ ID NO: RA-C, SEQ ID NO: RA-D, SEQ ID NO: RA-R, SEQ ID NO: RA-S, SEQ ID NO: RA-T, SEQ ID NO: RA-U, and SEQ ID NO: RA-V. SEQ ID NO: 79, 88, 72-74, and 92-96.

[0057] Preferred first primers for generating RSV B amplicons have priming nucleotide sequences of SEQ ID NOs: RB-A, RB-B, RB-C, and RB-U. Preferred second primers useful with such first primers have priming nucleotide sequences of SEQ ID NOs: RB-F, RB-G, RB-U, and RB-Z. SEQ ID NOs: 99-101, 104-106, and 115.

[0058] In some preferred embodiments, the probes and / or primers contain one or more methylated cytosine bases.

[0059] Another related aspect of the present disclosure relates to compositions containing the above-described probes, primers, and primer pairs. Such compositions include dry compositions or liquid compositions. Dry compositions include lyophilized reagents containing one or more of the primers and probes.

[0060] Another aspect of the present disclosure relates to kits containing primers and / or probes. Such kits may also include salts, enzymes, dNTPs, dRTPs, other substrates, and / or instructions for use of such materials. The primers, probes, salts, enzymes, dNTPs, rNTPs, and / or other substrates in the kits may be in dry or aqueous form.

[0061] Another aspect of the present disclosure relates to a reagent containing a primer and / or a probe. Such a reagent may also contain salts, enzymes, dNTPs, rNTPs, and / or other substrates. The primers, probes, salts, enzymes, dNTPs, rNTPs, and / or other substrates in the reagent may be in dry or aqueous form.

[0062] Yet another aspect of the present disclosure relates to methods of using the primers and probes described above to analyze a sample to determine whether the sample contains one or more of a Flu A target nucleic acid, a Flu B target nucleic acid, a RSV A target nucleic acid, and a RSV B target nucleic acid.The above and other objects, features, and advantages of the compositions and methods of the present invention will become apparent from the following detailed description and claims. DETAILED DESCRIPTION OF THE INVENTION

[0063] Described herein are compositions, including kits and reagents, and methods for selectively detecting nucleic acids of various viral pathogens in a sample, specifically influenza A (Flu A), influenza B (Flu B), respiratory syncytial virus A (RSV A), and respiratory syncytial virus B (RSV B). These compositions and methods can be used, for example, in diagnostic applications, to screen clinical samples, nasopharyngeal samples, bronchoalveolar samples, donated blood and blood products, or other tissues that may contain one or more of these pathogenic organisms.

[0064] As will be apparent, any primer and probe sequence specific to Flu A, Flu B, RSV A, RSV B, and / or other pathogenic viral targets may be used as a primer or probe in any suitable primer / probe-based in vitro nucleic acid amplification method suitable for amplifying the target nucleic acid of interest. Furthermore, it is understood that oligonucleotides having the sequences described herein can perform other functions in assays for detecting viral target nucleic acids. For example, a probe can be used as a primer (e.g., as one member of a primer pair), and a primer can be used as a probe in alternative detection assays.

[0065] Amplification primers are useful as components of uniplex or multiplex amplification reactions in which amplicon species can be generated from target-specific primers in a reaction mixture. Multiplex amplification reactions include primer pairs for amplifying two or more of Flu A, Flu B, RSV A, and RSV B, or additionally include primers for one or more of Flu A, Flu B, RSV A, and RSV B and one or more additional targets (e.g., human metapneumovirus, rhinovirus, adenovirus, parainfluenza virus, and / or Bordetella).

[0066] Useful amplification methods related to the present disclosure include polymerase chain reaction (PCR), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), and amplification methods using self-replicating polynucleotide molecules and replicative enzymes such as MDV-1 RNA and Q-β enzyme. Methods for implementing these various amplification techniques can be found in U.S. Patent Nos. 4,965,188, 5,399,491, 5,455,166, and 5,472,840, European Patent Application Publication No. EP 0525882, and Lizardi et al., BioTechnology 6:1197 (1988), respectively. In a particularly preferred embodiment, the nucleic acid sequences of Flu A, Flu B, RSV A, and RSV B are amplified using real-time PCR (RT-PCR).

[0067] Due to the lack of sequence conservation among respiratory virus strains (particularly Flu A) and to accommodate mismatches / mutations between primers or probes and the corresponding target nucleic acid sequence within the viral target nucleic acid, in some preferred embodiments, degenerate bases and non-Watson-Crick (NWC) base pairing can be included in primer or probe oligonucleotides. An NWC position within an oligonucleotide refers to a position where the oligonucleotide is configured to hybridize with at least one target nucleic acid sequence via non-Watson-Crick pairing, e.g., GU, GT, or GA (G or any of U, T, and A can be the base within the oligonucleotide). In some embodiments, the NWC position is configured to hybridize via a wobble (GU or GT) or purine-purine (GA) pair. In some embodiments, if one or more degenerate bases are identified within the target nucleic acid sequence for a single primer or probe, multiple primer or probe species can be synthesized to encompass all base combinations.

[0068] Guidelines useful for designing amplification primers and probes with desirable characteristics are known in the art and described herein. The optimal site for amplifying and detecting RSV B, RSV A, and RSV B nucleic acids contains two and preferably three conserved regions, each longer than about 15 bases and all spatially separated from each other within a region of about 1,000 bases, preferably about 500 bases, and even more preferably about 200 bases, of the contiguous sequence of the target nucleic acid. The degree of amplification observed with a set of primers depends on several factors, including the ability of the primers to hybridize with their complementary sequences and the ability of the primers to be extended by enzymes. Because the degree and specificity of the hybridization reaction are affected by many factors, manipulation of these factors determines the precise sensitivity and specificity of a particular oligonucleotide, regardless of whether it is perfectly complementary to its target. The effects of varying assay conditions are known in the art; see, for example, U.S. Patent No. 5,840,488.

[0069] Amplification primers and probes should be positioned to minimize the stability of oligonucleotide:non-target (e.g., nucleic acids with sequence similar to the target nucleic acid) and oligonucleotide:oligonucleotide (e.g., primer-dimers and self-complementary) nucleic acid hybrids. Amplification primers and detection probes are preferably capable of distinguishing between target and non-target sequences. When designing primers and probes, the melting temperature (T) of the oligonucleotide:target compared to the oligonucleotide:non-target and oligonucleotide:oligonucleotide should be taken into consideration. m The difference in Λ / Λ values ​​must be large enough to support oligonucleotide:target hybridization, and preferably avoids long homopolymer tracts and high GC content to reduce spurious primer extension.

[0070] As is known, nucleic acid hybridization involves the association of two complementary nucleic acid single strands to form a hydrogen-bonded duplex. If one of the two strands is fully or partially involved in a hybrid, it is implied that that strand is no longer able to participate in the formation of new hybrids. The rate and extent of hybridization can be greatly increased by designing primers and probes so that a significant portion of the sequence of interest is single-stranded. When the target is in double-stranded form (as in the case of PCR products), denaturation prior to hybridization is usually required.

[0071] Primers useful for performing amplification reactions can have different lengths to accommodate the presence of extraneous sequences that are not involved in target binding and may not substantially affect the amplification or detection procedure. For example, promoter-primers useful for performing amplification reactions according to the present disclosure have at least a minimal sequence that hybridizes to the desired target nucleic acid sequence and a promoter sequence located upstream of that minimal sequence. However, inserting a sequence between the target-binding sequence and the promoter sequence can change the length of the primer without impairing its usefulness in amplification reactions. Furthermore, the length of the amplification primers and detection probes is a matter of choice, as long as the sequences of these oligonucleotides meet the minimum essential requirements for hybridizing to the desired complementary target sequence.

[0072] Hybridization assay probes useful for detecting Flu A, Flu B, RSV A, and RSV B nucleic acid sequences include a base sequence that is substantially complementary to a selected target nucleic acid sequence (or an amplicon representing the corresponding region and its adjacent or surrounding region) in the genome of Flu A, Flu B, RSV A, or RSV B. Such probes may optionally have additional bases outside the target nucleic acid region, which may or may not be complementary to the nucleic acid of Flu A, Flu B, RSV A, or RSV B.

[0073] Preferred probes are sufficiently homologous to the target nucleic acid to hybridize under stringent hybridization conditions corresponding to the designed amplification and detection reactions. For example, in PCR, extension and detection reactions are performed so that oligonucleotides hybridize to their target nucleic acid sequences at a reaction temperature of approximately 60°C. Salt concentration also affects the hybridization of oligonucleotides to their target nucleic acid sequences. Exemplary salt concentrations range from approximately 0.6 to 0.9 M. Preferred salts include lithium chloride, but other salts, such as sodium chloride and sodium citrate, can also be used in the hybridization solution. Examples of high-stringency hybridization conditions are provided by 0.48 M sodium phosphate buffer, 0.1% sodium dodecyl sulfate, and 1 mM each of EDTA and EGTA, or 0.6 M LiCl, 1% lithium lauryl sulfate, 60 mM lithium succinate, and 10 mM each of EDTA and EGTA. Those skilled in the art are familiar with preparing solutions for nucleic acid hybridization.

[0074] Probes according to the present disclosure have sequences complementary to or corresponding to preselected target regions of the particular viral target nucleic acid to which the probe is directed. Preferred probes are in the range of 10-100 nucleotides in length and have probe sequences that include a base sequence that hybridizes to the target, along with any base sequence that is not complementary to the nucleic acid to be detected.

[0075] Amplification of nucleic acids by polymerase chain reaction (PCR) is a fundamental technique in molecular biology, typically requiring sample preparation, amplification, and product analysis. While these steps are typically performed sequentially, amplification and analysis can be performed simultaneously. DNA dyes or fluorescent probes can be added to the PCR mixture before amplification and used to analyze PCR products during amplification. Sample analysis is performed simultaneously with amplification in the same tube within the same instrument. Such combined techniques eliminate the need to remove samples from their closed containers for further analysis, reducing sample handling, saving time, and significantly reducing the risk of product contamination in subsequent reactions. The concept of combining amplification and product analysis has become known as "real-time" PCR (RT-PCR). See, for example, U.S. Patents 6,174,670 and 8,137,616. In real-time PCR, PCR product formation is monitored during each PCR cycle. Amplification is typically measured in a thermocycler equipped with additional equipment for generating and detecting signals from labels attached to probe oligonucleotide species during the amplification reaction. Many such devices are known in the art for performing multiplexed diagnostic assays using three, four, or more distinguishably labeled hybridization probes in one reaction vessel.

[0076] As is known, there are various formats for probe-based real-time detection of amplified DNA in multiplex assays, common examples of which include the "Taqman" probe system, molecular beacons and torches, single-labeled probes, and FRET hybridization probes.

[0077] In the Taqman probe format, single-stranded hybridization probes for a given target are labeled with a donor / acceptor pair of detectable labels. When the donor (e.g., a fluorophore moiety) is excited with light of the appropriate wavelength, the absorbed energy is transferred to the acceptor (e.g., a quencher moiety) according to the principle of FRET. The hybridization probe binds to the target DNA during the annealing step of the PCR reaction cycle and is subsequently degraded by the 5'-3' exonuclease activity of Taq polymerase during the extension phase. As a result, the excited donor and acceptor moieties are spatially separated, which prevents quenching of the signal (e.g., fluorescence emission) from the donor detected by the instrument. See, e.g., U.S. Patent No. 5,538,848.

[0078] Molecular beacons and torch formats also typically include hybridization probes labeled with donor / acceptor pairs, with the donor and acceptor moieties located at opposite ends of the probe. Often, the secondary structure of the probe, which involves hybridization of complementary regions at both ends of the probe, results in both the donor and acceptor moieties (e.g., fluorophore and quencher moieties) being spatially close in solution. After hybridizing the target-hybridizing region of the probe to the desired target nucleic acid sequence, the donor and acceptor moieties are separated from each other so that the donor moiety can be excited with light of an appropriate wavelength and its emission can be measured. See, for example, U.S. Patent No. 5,118,801.

[0079] In the single-labeled probe (SLP) format, a single oligonucleotide is labeled with a single fluorescent dye at either the 5' or 3' end. Various designs are available for oligonucleotide labeling, including G-quenching probes and nitroindole-dequenching probes. In G-quenching embodiments, a fluorescent dye is attached to the 5' or 3' end of the oligonucleotide via a C. When two Gs are located on opposite ends of the target strand from a C at position 1 of a complementary oligonucleotide probe, fluorescence is significantly reduced upon hybridization of the probe to the target. In nitroindole-dequenching embodiments, a fluorescent dye is attached to a nitroindole at the 5' or 3' end of the oligonucleotide, which reduces the fluorescent signal from free (e.g., unhybridized) probe molecules. Due to the dequenching effect, fluorescence increases upon hybridization of the probe to the target DNA.

[0080] Multiplex assays using FRET hybridization probes to detect target nucleic acids are particularly useful in homogeneous hybridization assays (see, e.g., Matthews and Kricka, Analytical Biochemistry, vol. 169 (1988), pp: 1-25). In particular, the FRET hybridization probe format can be used in RT-PCR to detect amplified target DNA species.

[0081] In addition to PCR and real-time PCR, FRET hybridization probes can also be used in melting curve analysis. In such assays, the target nucleic acid is first amplified in a typical PCR reaction using appropriate amplification primers. The hybridization probe is either already present during the amplification reaction or added later. After the PCR reaction is completed, the temperature of the sample is gradually increased, and fluorescence is detected as long as the hybridization probe remains bound to the target DNA. At the melting temperature, the hybridization probe molecules are released from their complementary target sequence, and the fluorescent signal quickly decreases to background levels. This decrease is monitored on a fluorescence versus temperature-time plot so that the first derivative value at which the maximum fluorescence decrease is observed can be determined.

[0082] In some preferred embodiments, RT-PCR is used to amplify and detect multiple target DNA sequences in multiplex assays. Such methods involve providing a composition or reaction mixture containing nucleic acids from biological samples, probes, primers, and suitable polymerase activity for catalyzing amplification; subjecting the reaction mixture to a thermal cycling protocol so that multiple target sequences are amplified; and monitoring the hybridization of each probe molecule species (e.g., FRET hybridization probe pair) at least once after multiple amplification cycles. In embodiments where the target nucleic acid of the virus to be detected is composed of one or more RNA molecules, such methods usually involve first converting RNA into DNA (e.g., "complementary" DNA or "cDNA") by utilizing reverse polymerase activity.

[0083] In such multiplexed embodiments, the composition or reaction mixture typically includes at least two, preferably three to five, and most preferably four pairs of detection probes, each pair of probes preferably including a FRET donor moiety and a FRET acceptor moiety, and also includes multiple reagents, including one or more of a buffer designed for PCR, dNTPs, a template-dependent DNA polymerase (preferably a thermostable DNA polymerase), and a reverse transcriptase.

[0084] During or after the amplification process is complete, the reaction is monitored to detect stable hybridization between one or more of the distinguishably labeled probe species present in the reaction and its corresponding target nucleic acid sequence (carried by amplicons generated using primer pairs corresponding to the particular viral (or other) pathogen being detected). Based on whether the donor moiety from each of the different donor / acceptor pairs is detected, it can be determined whether the biological sample contains Flu A, Flu B, RSV A and / or RSV B, and / or other pathogens as targeted in a particular assay.

[0085] Certain preferred kits will include one or more of the following: probes, primers, capture oligonucleotides, internal standard oligonucleotides, other ancillary oligonucleotides, buffers, dNTPs, DNA polymerase, reverse transcriptase, and instructions (or links to websites providing such instructions) for using the components of the kit.

[0086] The following examples are provided to illustrate certain disclosed embodiments and should not be construed as limiting the scope of the disclosure in any way.

[0087] General Reagents and Methods. Unless otherwise indicated, amplifications were performed using an ABI 7500 FAST® instrument. Viral isolates used as targets or controls for amplification were stored in appropriate media, e.g., Micro Test M4 media (Remel Inc., Cat. No. R12500), Micro Test M5 Viral Transport Medium (Remel Inc., Cat. No. R12515), Micro Test M6 Viral Transport Medium (Remel Inc., Cat. No. R12516), Micro Test M5 Viral Transport Medium (Remel Inc., Cat. No. R12517), Micro Test M6 Viral Transport Medium (Remel Inc., Cat. No. R12519), Micro Test M4 Viral Transport Medium (Remel Inc., Cat. No. R12519), Micro Test M5 Viral Transport Medium (Remel Inc., Cat. No. R12519), Micro Test M6 ... Inc., Catalog No. R12530), MicroTest M4RT Viral Transport Medium (Remel Inc., Catalog No. R12505), or Copan Universal Transport Medium (Copan Diagnostics Inc., Catalog No. 330C). Nucleic acid was extracted from viral isolates using a nonspecific target capture method as described in U.S. Patent Application Publication No. 2013 / 0209992.

[0088] PCR reaction mixtures were typically assembled as follows: 19.05 uL Supermix (Promega GoTaqA® Supermix), 0.35 uL MMLV Reverse Transcriptase (35 U), 0.6 uL GoTaq MDX Hotstart Taq (3 U), 5 uL nucleic acids (primers, probe, and target in appropriate dilutions), total reaction volume = 25 uL (Promega, Madison, WI; New England Biolabs, Ipswich, MA; Sigma-Aldrich, St. Louis, MO; Thermo Fisher, Waltham, MA, etc.).

[0089] Example 1 Multiplex RT-PCR assay for detecting Flu A, Flu B, RSV A, and RSV B This example describes a representative RT-PCR assay based on Taqman reagents for the detection and differentiation of influenza A virus, influenza B virus, and respiratory syncytial virus types A and B in biological samples.

[0090] Here, the process begins with, for example, collecting a nasopharyngeal swab specimen from a symptomatic human patient. Unless the sample is to be analyzed immediately, the sample is preferably placed in a sealable container (e.g., an RNase / DNase-free 1.5 mL polypropylene microcentrifuge tube) along with an appropriate volume of viral transport medium (VTM, e.g., Remel, Inc., Copan Diagnostics, Inc., or Becton, Dickinson and Co.). A Universal Internal Control (UIC) is then preferably also added to the sample to monitor for any inhibitors that may be present in the sample.

[0091] Next, the nucleic acid in the sample is isolated using, for example, the MagNA Pure LC System (Roche) and MagNA Pure Total Nucleic Acid Isolation Kit (Roche, Cat. No. 03038505001) or the NucliSENS easyMAG System (bioMerieux) and Automated Magnetic Extraction Reagents (bioMerieux).The purified nucleic acid is then added to a reaction mixture together with a thermostable DNA polymerase and reverse transcriptase.The reaction mixture contains oligonucleotide primer pairs and target-specific oligonucleotide probes for Flu A, Flu B, RSV A, and RSV B, respectively, as well as Taq DNA polymerase, dNTPs (dATP, dCTP, dGTP, dTTP (or dUTP)), MgCl2, and a buffer containing stabilizers and bovine serum albumin.M-MLV reverse transcriptase can be used for reverse transcription of the viral genome, and an RNase inhibitor (e.g., RNase inhibitor II) can also be included to protect RNA from degradation. Various control nucleic acids can also be included. Such controls can be, for example, non-infectious RNA of a specific viral sequence transcribed in vitro and / or non-infectious plasmid DNA containing the control sequence. If necessary, two different sets of amplification primers and probes targeting different genomic regions of the virus to be detected can be used for any given target genome, particularly in cases where genetic diversity between strains may exist, such as in the case of Flu A, and detection based on a single region may therefore be insufficient to ensure accurate analysis. The amplification primers of the various primer pairs are complementary to highly conserved regions of the genetic sequences of these respiratory syncytial viruses. Each probe species is dual-labeled with a distinguishable reporter dye and quencher.

[0092] Reverse transcription of RNA into cDNA and subsequent amplification of the DNA is performed using, for example, Cepheid This process can be performed on a SmartCycler II (Cepheid, Sunnyvale, CA). For each viral genome to be detected, a probe species for the target viral genome (or region thereof) specifically anneals to a target nucleotide sequence (e.g., a specific region of the Flu A genome) in a target nucleic acid molecule, followed by primer extension and amplification. The Taqman reagents cleave the probe using the 5'-3' exonuclease activity of Taq polymerase, thereby separating the reporter dye from the quencher. This results in an increase in fluorescent signal upon excitation by a light source. With each cycle, additional reporter dye molecules are cleaved from each probe, further increasing the fluorescent signal. The amount of fluorescence at any given cycle depends on the number of amplification products (amplicons) present at that time. Fluorescence intensity is monitored during each PCR cycle by a real-time instrument.

[0093] Example 2 Amplification and detection of Flu A, Flu B, RSV A, and RSV B in clinical samples Nasopharyngeal (NP) swab specimens and remaining lower respiratory tract (LRT) specimens from individuals showing signs and / or symptoms of respiratory tract infection were analyzed with a multiplex real-time PCR assay using primers and probes for the amplification and detection of target nucleic acids for Flu A, Flu B, RSV A, and RSV B. NP swab and LRT samples were analyzed using Panther Tested in the Fusion Flu A / B / RSV assay.

[0094] For this example, 2930 residual NP swab specimens were used. The specimens were processed to release nucleic acids. Briefly, residual NP swab specimens were received in Remel transport medium (Thermo Fisher, Waltham, MA). A 500 μl aliquot of transport medium from each specimen was mixed with lysis solution (710 μl) in a separate Panther Fusion Lysis Tube (Hologic, Marlborough, MA). After incubation, 360 μl of the lysed specimen was mixed with 450 μl of target nucleic acid isolation reagent containing capture oligonucleotides and a solid support. The target nucleic acid isolation reaction was performed using a Panther Fusion instrument (Hologic, Marlborough, MA) as generally described in U.S. Patent Nos. 6,110,678 and 9,051,601. The target nucleic acids isolated from each clinical specimen were then eluted from the capture reaction into 50 μl of eluate, resulting in 2930 sample conditions, each corresponding to one NP swab specimen. Nucleic acid amplification and detection reactions were set up as follows: 5 μl of each sample condition was added to wells of a multiplex plate. 20 μl of rehydrated real-time PCR reaction mixture was also contained in the wells. The dried PCR reaction mixture was rehydrated using 24 μl of buffer containing magnesium salt. The components of this real-time PCR reaction mixture were as described above and further included primers and probes having the nucleotide sequences shown as SEQ ID NOS: 5, 7, 12, 18, 23, 25-27, 64, 67, 68, 75, 79, 92, 101, 102, and 115. The probe for detecting the Flu A amplification product was labeled with FAM / BHQ1, the probe for detecting the Flu B amplification product was labeled with CalRed / BHQ2, and the probes for detecting the RSV A and RSV B amplification products were labeled with CalOrange / BHQ1 (labeled with LGC). (Available from Biosearch Technologies, Petaluma, CA). Each sample condition was added independently to a PCR reaction microtube. Control wells included an internal standard, a positive control, and a negative control.

[0095] Each PCR reaction microtube was then placed in a Panther Fusion instrument (Hologic, Marlborough, MA), and each well was analyzed for the presence or absence of one or more of the target nucleic acids. Of the 2,930 NP swab specimens, 61 gave inconsistent results and were therefore considered invalid and excluded from evaluation. 189 / 2,869 (6.6%) were positive for Flu A target nucleic acid; 55 / 2,869 (1.9%) were positive for Flu B target nucleic acid; and 365 / 2,869 (12.7%) were positive for RSV. were positive for RSV A and / or RSV B.

[0096] A similar assay was performed using residual lower respiratory tract (LRT) specimens, except that 250 μl of LRT specimen was mixed with 250 μl of lysis solution, and then 360 μl of this mixed solution was used for the target nucleic acid reaction. For this example, 144 residual LRT specimens were used. The specimens were generally processed as described above in this example (specimen lysis, nucleic acid isolation, amplification, and detection). Of the 144 LRT specimens, 4 gave inconsistent results or were not tested and were therefore considered invalid and excluded from the evaluation results. 3 / 140 (2.1%) were positive for Flu A target nucleic acid. 0 / 140 (0.0%) were positive for Flu B target nucleic acid. 1 / 140 (0.7%) were positive for RSV A and / or RSV B.

[0097] These results indicate that the assay is highly sensitive and specific for detecting target nucleic acids from NP swab specimens. These results also indicate that the assay is highly sensitive for detecting Flu A target nucleic acids, but for LRT specimens, sensitivity to Flu B and RSV A and B target nucleic acids could not be determined. These results indicate that the assay has high specificity for Flu A, Flu B, RSV A, and RSV B target nucleic acids.

[0098] Example 3 Exemplary Oligonucleotide Sequences Table 1 shows a number of primer and probe sequences that are useful as compositions, in kits, as diagnostic reagents, and / or in methods for amplifying or detecting one or more of Flu A, Flu B, RSV-A, and RSV-B. Table 1 below shows only the nucleotide sequences. It will be understood that these sequences may further include detectable labels, sugar modifications (e.g., 2'-methoxy), base modifications (e.g., methylated bases), and other chemical moieties not represented within the sequence of consecutive symbols shown. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7]

[0099] All of the articles, devices, systems, and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the devices, systems, and methods of the present disclosure have been described with reference to preferred embodiments, it will be apparent to those skilled in the art that variations can be applied to the articles and methods without departing from the spirit and scope of the invention. All such variations and equivalents apparent to those skilled in the art, whether now existing or later developed, are deemed to be within the spirit and scope of the present disclosure. It will also be understood that computer-based embodiments of the present disclosure can be implemented using any suitable hardware and software.

[0100] All patents, patent applications, and publications mentioned in this specification are indicative of the level of those skilled in the art to which this disclosure pertains. All patents, patent applications, and publications are herein incorporated by reference in their entireties for all purposes to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Examples of embodiments of the present invention include the following: (Item 1) The method comprises: identifying one or more of a plurality of target nucleic acid molecular species associated with pathogens that may be present in a biological sample; A kit for analyzing (a) a first Flu A amplicon if Flu A is present in said biological sample; a first Flu A primer pair for generating and a second Flu A primer, (i) a first Flu A primer that is substantially complementary to a first Flu A target nucleic acid sequence; The nucleotide sequence is about 18 to about 100 consecutive bases in length, and is selected from SEQ ID NO: 1 and SEQ ID NO: 23. and comprising an oligonucleotide sequence selected from the group consisting of: (ii) a second Flu A primer substantially complementary to a second Flu A target nucleic acid; The mer is about 18 to about 100 consecutive bases in length, and is SEQ ID NO: 25 and SEQ ID NO: 28 a first Flu A primer comprising an oligonucleotide sequence selected from the group consisting of: versus, and / or (b) a second Flu A amplicon, if Flu A is present in said biological sample; a second Flu A primer pair for generating a third Flu A primer and a fourth Flu A primer, (i) a third Flu A primer that is substantially complementary to the third Flu A target nucleic acid sequence; The nucleotide sequence is about 19 to about 100 consecutive bases in length, and is selected from the group consisting of SEQ ID NOs: 2 to 5 and 24. an oligonucleotide sequence selected from the group consisting of: (ii) a fourth Flu A primer substantially complementary to the fourth Flu A target nucleic acid sequence; The primer has a length of about 20 to about 100 consecutive bases and is selected from SEQ ID NOs: 26 and 27. a second Flu A primer pair comprising an oligonucleotide sequence selected from the group consisting of: and / or (c) generating a Flu B amplicon if Flu B is present in the biological sample; a Flu B primer pair for detecting a nucleotide sequence comprising a first Flu B primer and a second Flu B primer; Contains Flu B primers (i) a first Flu B primer that is substantially complementary to a first Flu B target nucleic acid sequence; The nucleotide sequence is about 22 to about 100 consecutive bases in length and is composed of SEQ ID NOs: 29 and 67. an oligonucleotide sequence selected from the group consisting of: (ii) a second Flu B primer that is substantially complementary to a second Flu B target nucleic acid sequence; The primer has a length of about 21 to about 100 consecutive bases and is represented by SEQ ID NOs: 68 to 70. a Flu B primer pair comprising an oligonucleotide sequence selected from the group or (d) generating an RSV A amplicon if RSV A is present in the biological sample; A RSV A primer pair for detecting RSV A, comprising a first RSV A primer and a second RSV A primer Contains RSV A primers (i) a first RSV A (R) that is substantially complementary to the first RSV A target nucleic acid sequence; SV A) The primer has a length of about 26 to about 100 consecutive bases and is SEQ ID NO: 79 and and 88, (ii) a second RSV A primer substantially complementary to a second RSV A target nucleic acid sequence; The primer has a length of about 22 to about 100 consecutive bases, and is represented by SEQ ID NOs: 72 to 74 and 9. RSV A primer comprising an oligonucleotide sequence selected from the group consisting of 2 to 96 -vs. and / or (e) generating an RSV B amplicon if RSV B is present in the biological sample; A RSV B primer pair for detecting RSV B, comprising a first RSV B primer and a second RSV B primer Contains RSV B primers (i) the first RSV B (RSV B) primer is a first RSV B target nucleic acid; and is substantially complementary to the nucleic acid sequence of SEQ ID NO: 1, and is about 17 to about 100 consecutive bases long. comprising an oligonucleotide sequence selected from the group consisting of 99 to 101 and 106; (ii) the second RSV B primer substantially matches the second RSV B target nucleic acid sequence; and is approximately 17 to 100 consecutive bases long. RSV B promoter comprising an oligonucleotide sequence selected from the group consisting of: 6 and 115. Kit, including Reimer Vs. (Item 2) The kit further comprises a probe molecular species for each primer pair included in the kit; (a) if the kit includes a first Flu A primer pair, the probe molecule is , the first Flu A probe is substantially complementary to the target nucleic acid sequence, and has about 17 to about 100 and an oligonucleotide having a length of consecutive bases selected from the group consisting of SEQ ID NOs: 6 to 17. Contains a code array, and / or (b) if the kit includes a second Flu A primer pair, the probe species is , a second Flu A probe substantially complementary to the target nucleic acid sequence, and having about 17 to about 100 and an oligonucleotide having a contiguous base length of 18 to 22, and / or (c) When the kit includes a Flu B primer pair, the probe molecular species is Fl u B probe substantially complementary to the target nucleic acid sequence and having about 17 to about 100 consecutive bases and comprising an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 30 to 66. and / or (d) if the kit includes an RSV A primer pair, the probe species is RS VA probe is substantially complementary to the target nucleic acid sequence and has about 17 to about 100 consecutive bases. base length, and SEQ ID NOs: 71, 75-78, 80-87, 89-91, 97 and 98 and / or comprising an oligonucleotide sequence selected from the group consisting of (e) if the kit includes an RSV B primer pair, the probe species is RSV The probe is substantially complementary to the target nucleic acid sequence and has a length of about 17 to about 100 consecutive bases. and an oligonucleotide selected from the group consisting of SEQ ID NOs: 102, 103, and 107 to 114. Item 1. The kit according to item 1, comprising a nucleotide sequence. (Item 3) one or more of the primers in one or more of the primer pairs a primer having a nucleotide sequence that is not complementary to the target nucleotide sequence of the primer Item 1, the kit further comprising an upstream region. (Item 4) Each probe molecule is labeled, and any one probe molecule is labeled with another probe molecule. 3. The kit of item 2, optionally identifiably labeled so as to be distinguishable from the species. (Item 5) Each probe molecule contains a chemiluminescent moiety, a fluorophore moiety, a quencher moiety, and a fluorophore moiety. one or more detectors selected from the group consisting of both a fluorophore moiety and a quencher moiety 5. The kit of item 4, wherein the kit is identifiably labeled with an emissive label. (Item 6) One or more of the probe species is detectably labeled with a donor / acceptor label pair. Item 3. The kit according to item 2, (Item 7) 3. The method of claim 2, wherein each probe molecule is up to about 60 nucleotide residues in length. tt. (Item 8) Multiple primer pairs, optionally a first Flu A primer pair, a second Flu A primer pair, Primer pair, Flu B primer pair, RSV A primer pair and RSV B primer pair 2. The kit according to item 1, comprising a mer pair. (Item 9) One primer and / or at least one probe detects one or more methylation sequences. 3. The kit according to item 2, containing tosine base. (Item 10) Reagents for carrying out a nucleic acid amplification reaction, and optionally, a method for carrying out nucleic acid amplification Item 1, further comprising instructions for using the primer pair species and the reagent. Kit included. (Item 11) At least one primer is present in the kit as a lyophilized reagent. The kit according to any one of items 1 to 10. (Item 12) At least one probe is present in the kit as a lyophilized reagent. 12. The kit according to any one of 1 to 11. (Item 13) The kit comprises one of a reverse transcriptase, a DNA polymerase, a buffer, and dNTPs. Item 13. The kit according to Item 11 or 12, comprising the above. (Item 14) Reverse transcriptase, DNA polymerase, buffer, and dNTPs were each lyophilized. Item 14. The kit according to item 13, wherein the drug is present in the kit. (Item 15) 15. The method according to any one of items 11 to 14, further comprising a rehydration reagent comprising a salt, preferably a magnesium salt. The kit according to any one of claims 1 to 4. (Item 16) Item 1, comprising at least one primer, a reverse transcriptase, a DNA polymerase, , a buffer, and dNTPs. (Item 17) Item 17. The reaction mixture of item 16, further comprising at least one probe of item 2. (Item 18) 18. The reaction mixture according to item 17, wherein the reaction mixture is a lyophilized composition. (Item 19) The biological samples were influenza A (Flu A), influenza B (Flu B), Respiratory syncytial virus type A (RSV A), and / or respiratory syncytial virus type B ( A method for determining whether a patient contains RSV (Respiratory Syncytial Virus B), comprising: (a) Probe: A probe that is used to bind a target nucleic acid molecule from the biological sample to form a target duplex. Two or more distinguishably labeled probe molecular species are subjected to stringent hybridization. contacting under conditions (i) distinguishably determining whether the biological sample contains Flu A; A labeled first Flu A probe species and / or a distinguishably labeled second (A) using a Flu A probe species, The probe species is substantially complementary to the first Flu A probe target nucleic acid sequence and is about It has a length of 17 to about 100 consecutive bases and is selected from the group consisting of SEQ ID NOs: 6 to 17. (B) the distinguishably labeled second Flu A promoter; The probe molecular species is substantially complementary to the second Flu A probe target nucleic acid sequence and is about 17 a length of about 100 consecutive bases, selected from the group consisting of SEQ ID NOs: 18 to 22 and / or (ii) determining whether the biological sample contains Flu B; B probe substantially complementary to the target nucleic acid sequence and having a length of about 17 to about 100 consecutive bases and comprising an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 30 to 66. using distinguishably labeled Flu B probe species, and / or (iii) determining whether the biological sample contains RSV A; A probe is substantially complementary to the target nucleic acid sequence and has about 17 to about 100 consecutive bases. and consisting of SEQ ID NOs: 71, 75-78, 80-87, 89-91, 97 and 98. A distinguishably labeled RSV A primer comprising an oligonucleotide sequence selected from the group consisting of: Use lobe molecular species, and / or (iv) detecting an RSV protein to determine whether the biological sample contains RSV B; The probe is substantially complementary to the target nucleic acid sequence and has a length of about 17 to about 100 consecutive bases. and an oligonucleotide selected from the group consisting of SEQ ID NOs: 102, 103, and 107 to 114. Contacting the RSV B probe using a distinctly labeled RSV B probe species containing a nucleic acid sequence. and (b) If stable in step (a), the distinguishably labeled probe:target duplex Detecting whether a chain is formed and, if so, (c) detecting the first, second, third, fourth, and / or fifth distinguishable labels; and / or based on whether the biological sample is Flu A, Flu B, RSV A, and / or or determining that the antibody contains RSV B. (Item 20) The nucleic acid molecule derived from the biological sample, when present in the sample, is selected from Flu A, Flu F, amplification products corresponding to lu B, RSV A, and / or RSV B, optionally 20. The method of item 19, comprising a precon. (Item 21) The amplification product comprises carrying out a nucleic acid amplification reaction using one or more primer pairs. and wherein the one or more primer pairs are produced via a nucleic acid amplification process comprising: (a) a first Flu A amplicon if Flu A is present in said biological sample; a first Flu A primer pair for generating and a second Flu A primer, (i) a first Flu A primer that is substantially complementary to a first Flu A target nucleic acid sequence; The nucleotide sequence is about 18 to about 100 consecutive bases in length, and is selected from SEQ ID NO: 1 and SEQ ID NO: 23. and comprising an oligonucleotide sequence selected from the group consisting of: (ii) a second Flu A primer substantially complementary to a second Flu A target nucleic acid; The mer is about 18 to about 100 consecutive bases in length, and is SEQ ID NO: 25 and SEQ ID NO: 28 a first Flu A primer comprising an oligonucleotide sequence selected from the group consisting of: versus, and / or (b) a second Flu A amplicon, if Flu A is present in said biological sample; a second Flu A primer pair for generating a third Flu A primer and a fourth Flu A primer, (i) a third Flu A primer that is substantially complementary to the third Flu A target nucleic acid sequence; The nucleotide sequence is about 19 to about 100 consecutive bases in length, and is selected from the group consisting of SEQ ID NOs: 2 to 5 and 24. an oligonucleotide sequence selected from the group consisting of: (ii) a fourth Flu A primer substantially complementary to the fourth Flu A target nucleic acid sequence; The primer has a length of about 20 to about 100 consecutive bases and is selected from SEQ ID NOs: 26 and 27. a second Flu A primer pair comprising an oligonucleotide sequence selected from the group consisting of: and / or (c) generating a Flu B amplicon if Flu B is present in the biological sample; a Flu B primer pair for detecting a nucleotide sequence comprising a first Flu B primer and a second Flu B primer; Contains Flu B primers (i) a first Flu B primer that is substantially complementary to a first Flu B target nucleic acid sequence; The nucleotide sequence is about 22 to about 100 consecutive bases in length and is composed of SEQ ID NOs: 29 and 67. an oligonucleotide sequence selected from the group consisting of: (ii) a second Flu B primer that is substantially complementary to a second Flu B target nucleic acid sequence; The primer has a length of about 21 to about 100 consecutive bases and is represented by SEQ ID NOs: 68 to 70. a Flu B primer pair comprising an oligonucleotide sequence selected from the group or (d) generating an RSV A amplicon if RSV A is present in the biological sample; A RSV A primer pair for detecting RSV A, comprising a first RSV A primer and a second RSV A primer Contains RSV A primers (i) a first RSV A (R) that is substantially complementary to the first RSV A target nucleic acid sequence; SV A) The primer has a length of about 26 to about 100 consecutive bases and is SEQ ID NO: 79 and and 88, (ii) a second RSV A primer substantially complementary to a second RSV A target nucleic acid sequence; The primer has a length of about 22 to about 100 consecutive bases, and is represented by SEQ ID NOs: 72 to 74 and 9. RSV A primer comprising an oligonucleotide sequence selected from the group consisting of 2 to 96 -vs. and / or (e) generating an RSV B amplicon if RSV B is present in the biological sample; A RSV B primer pair for detecting RSV B, comprising a first RSV B primer and a second RSV B primer Contains RSV B primers (i) the first RSV B (RSV B) primer is a first RSV B target nucleic acid; and is substantially complementary to the nucleic acid sequence of SEQ ID NO: 1, and is about 17 to about 100 consecutive bases long. comprising an oligonucleotide sequence selected from the group consisting of 99 to 101 and 106; (ii) the second RSV B primer substantially matches the second RSV B target nucleic acid sequence; and is approximately 17 to 100 consecutive bases long. RSV B promoter comprising an oligonucleotide sequence selected from the group consisting of: 6 and 115. Item 21. The method according to item 20, wherein the rimer pair is a rimer pair. (Item 22) 20. The method of claim 19, wherein the biological sample comprises a clinical specimen. (Item 23) 23. The method of claim 22, wherein the clinical specimen is a nasopharyngeal specimen. (Item 24) 23. The method of claim 22, wherein the clinical specimen is a bronchoalveolar specimen. (Item 25) 23. The method of claim 22, wherein the specimen is a lower respiratory tract specimen. (Item 26) one or more of the primers in one or more of the primer pairs a primer having a nucleotide sequence that is not complementary to the target nucleotide sequence of the primer 22. The method of claim 21, further comprising an upstream region. (Item 27) Each distinguishable label comprises a chemiluminescent moiety, a fluorophore moiety, a quencher moiety, and a detectable molecule selected from the group consisting of both a fluorophore moiety and a quencher moiety Item 19. The method according to item 19, wherein the label is a label. (Item 28) One or more of the probe species is detectably labeled with a donor / acceptor label pair. Item 19. The method according to Item 19, (Item 29) 20. The method of claim 19, wherein each probe species is up to about 60 nucleotide residues in length. method. (Item 30) Multiple primer pairs, optionally a first Flu A primer pair, a second Flu A primer pair, Primer pair, Flu B primer pair, RSV A primer pair, and RSV B primer pair. 23. The method of claim 22, comprising a primer pair. (Item 31) One primer and / or at least one probe detects one or more methylation sequences. 23. The method of claim 22, containing a tosine base. (Item 32) a composition further comprising one nucleic acid molecule, each independently: (a) generating a first Flu A amplicon when Flu A is present in a biological sample; a first Flu A primer pair for generating a first Flu A primer pair, and a second Flu A primer, (i) a first Flu A primer that is substantially complementary to a first Flu A target nucleic acid sequence; The nucleotide sequence is about 18 to about 100 consecutive bases in length, and is selected from SEQ ID NO: 1 and SEQ ID NO: 23. and comprising an oligonucleotide sequence selected from the group consisting of: (ii) a second Flu A primer substantially complementary to a second Flu A target nucleic acid; The mer is about 18 to about 100 consecutive bases in length, and is SEQ ID NO: 25 and SEQ ID NO: 28 a first Flu A primer comprising an oligonucleotide sequence selected from the group consisting of: versus, and / or (b) a second Flu A amplicon, if Flu A is present in said biological sample; a second Flu A primer pair for generating a third Flu A primer and a fourth Flu A primer, (i) a third Flu A primer that is substantially complementary to the third Flu A target nucleic acid sequence; The nucleotide sequence is about 19 to about 100 consecutive bases in length, and is selected from the group consisting of SEQ ID NOs: 2 to 5 and 24. an oligonucleotide sequence selected from the group consisting of: (ii) a fourth Flu A primer substantially complementary to the fourth Flu A target nucleic acid sequence; The primer has a length of about 20 to about 100 consecutive bases and is selected from SEQ ID NOs: 26 and 27. a second Flu A primer pair comprising an oligonucleotide sequence selected from the group consisting of: and / or (c) generating a Flu B amplicon if Flu B is present in the biological sample; a Flu B primer pair for detecting a nucleotide sequence comprising a first Flu B primer and a second Flu B primer; Contains Flu B primers (i) a first Flu B primer that is substantially complementary to a first Flu B target nucleic acid sequence; The nucleotide sequence is about 22 to about 100 consecutive bases in length and is composed of SEQ ID NOs: 29 and 67. an oligonucleotide sequence selected from the group consisting of: (ii) a second Flu B primer that is substantially complementary to a second Flu B target nucleic acid sequence; The primer has a length of about 21 to about 100 consecutive bases and is represented by SEQ ID NOs: 68 to 70. a Flu B primer pair comprising an oligonucleotide sequence selected from the group or (d) generating an RSV A amplicon if RSV A is present in the biological sample; A RSV A primer pair for detecting RSV A, comprising a first RSV A primer and a second RSV A primer Contains RSV A primers (i) a first RSV A (R) that is substantially complementary to the first RSV A target nucleic acid sequence; SV A) The primer has a length of about 26 to about 100 consecutive bases and is SEQ ID NO: 79 and and 88, (ii) a second RSV A primer substantially complementary to a second RSV A target nucleic acid sequence; The primer has a length of about 22 to about 100 consecutive bases, and is represented by SEQ ID NOs: 72 to 74 and 9. RSV A primer comprising an oligonucleotide sequence selected from the group consisting of 2 to 96 -vs. and / or (e) generating an RSV B amplicon if RSV B is present in the biological sample; A RSV B primer pair for detecting RSV B, comprising a first RSV B primer and a second RSV B primer Contains RSV B primers (i) the first RSV B (RSV B) primer is a first RSV B target nucleic acid; and is substantially complementary to the nucleic acid sequence of SEQ ID NO: 1, and is about 17 to about 100 consecutive bases long. comprising an oligonucleotide sequence selected from the group consisting of 99 to 101 and 106; (ii) the second RSV B primer substantially matches the second RSV B target nucleic acid sequence; and is approximately 17 to 100 consecutive bases long. RSV B promoter comprising an oligonucleotide sequence selected from the group consisting of: 6 and 115. Reimer Vs. (f) a first Flu A probe target nucleic acid sequence substantially complementary to the first Flu A probe target nucleic acid sequence, 100 consecutive bases in length, and an oligonucleotide selected from the group consisting of SEQ ID NOs: 6 to 17. a probe molecule species comprising a nucleic acid sequence, and / or (g) a second Flu A probe substantially complementary to the target nucleic acid sequence, the second Flu A probe having a length of about 17 to about 1 100 consecutive bases in length, and an oligonucleotide selected from the group consisting of SEQ ID NOs: 18 to 22. a probe molecular species, including a nucleotide sequence; and / or (h) a Flu B probe having substantially complementary to the target nucleic acid sequence and having about 17 to about 100 amino acids; and an oligonucleotide having a contiguous base length of 30 to 66. a probe molecule species containing a nucleotide sequence, and / or (i) substantially complementary to the RSV A probe target nucleic acid sequence, and and SEQ ID NOs: 71, 75 to 78, 80 to 87, 89 to 91, 97, and and 98, a probe molecule species comprising an oligonucleotide sequence selected from the group consisting of and / or (j) a nucleic acid sequence substantially complementary to the RSV probe target nucleic acid sequence and having about 17 to about 100 consecutive sequences; and the base length is selected from the group consisting of SEQ ID NOs: 102, 103, and 107 to 114. a probe molecule species comprising an oligonucleotide sequence to be detected; (k) by a nucleic acid amplification process utilizing the Flu A primer pair described in part (a). Influenza A (Flu A) amplicons generated by (l) by a nucleic acid amplification process utilizing the Flu A primer pair described in part (b); Influenza A (Flu A) amplicons generated by (m) by a nucleic acid amplification process utilizing the Flu B primer pair described in part (c). Influenza B (Flu B) amplicons generated by (n) by a nucleic acid amplification process utilizing the RSV A primer pair described in part (d). Respiratory syncytial virus type A (RSV A) amplicons generated by (o) by a nucleic acid amplification process utilizing the RSV B primer pair described in part (e). Respiratory syncytial virus type B (RSV B) amplicons produced by The composition is selected from the group consisting of: (Item 33) Flu A target nucleic acid, Flu B target nucleic acid, RSV A target nucleic acid, and RSV B target nucleic acid in the sample. 1. A method for determining the presence or absence of a VB target nucleic acid, or a combination thereof, comprising: (A) contacting a sample with the combination of amplification oligomers described in item 1; (B) performing an in vitro nucleic acid amplification reaction to generate an amplification product; to identify the Flu A target nucleic acid, the Flu B target nucleic acid, and the RSV A target nucleic acid in the sample. A target nucleic acid, and any of the RSV B target nucleic acids amplify the target nucleic acid. and (C) detecting the amplification product; This determines the presence or absence of the target nucleic acid in the sample. (Item 34) The detection step (C) is configured to hybridize to the amplification product. 34. The method of claim 33, carried out using a releasably labeled probe. (Item 35) The detectably labeled probe is detectable by a donor / acceptor label pair. 35. The method according to item 34, wherein the method is labeled. (Item 36) The detection step (C) detects the presence of a specific primer pair for each primer pair used to generate the amplification product. is carried out using a detectably labeled probe species that (i) for the first Flu A primer pair, the probe molecular species is A probe is substantially complementary to the target nucleic acid sequence and has about 17 to about 100 consecutive bases. and comprising an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 6 to 17. and / or (ii) for the second Flu A primer pair, the probe molecular species is a second Flu A primer pair; u A probe substantially complementary to the target nucleic acid sequence and having about 17 to about 100 consecutive bases. and comprising an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 18 to 22. and / or (iii) For the Flu B primer pair, the probe molecular species is a Flu B primer The probe is substantially complementary to the target nucleic acid sequence and has a length of about 17 to about 100 consecutive bases. , comprising an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 30 to 66; and / or (iv) For RSV A primer pairs, the probe species is RSV A probe The nucleic acid sequence is substantially complementary to the target nucleic acid sequence and is about 17 to about 100 consecutive bases in length. Selected from the group consisting of SEQ ID NOs: 71, 75-78, 80-87, 89-91, 97 and 98 and / or (v) for the RSV B primer pair, the probe species is an RSV probe target It is substantially complementary to the nucleic acid sequence, has a length of about 17 to about 100 consecutive bases, and has SEQ ID NO: Oligonucleotide sequences selected from the group consisting of Nos. 102, 103, and 107 to 114 Item 34. The method of item 33, including a column. (Item 37) The detectably labeled probe species is associated with a donor / acceptor label pair. 37. The method of claim 36, wherein each of the two is detectably labeled. (Item 38) The detectably labeled probe molecular species are each detectable by their distinguishable labels. The method according to item 36 or 37, wherein the (Item 39) The detectably labeled Flu A probe species is detected by FAM / BHQ-1. or the detectably labeled Flu B probe species is C detectably labeled with alRed / BHQ-2 or The RSV A probe species was detectably labeled with CalOrange / BHQ-1. or the detectably labeled RSV B probe species is detectably labeled with e / BHQ-1, or a combination thereof Item 36. The method according to item 36. (Item 40) Implementing one or more steps of the method according to one of items 19 to 31 or 33 to 39 A system for implementing the above. (Item 41) Item 41. The system of item 40, wherein the system is an automated system.

Claims

[Claim 1] The invention described in this specification.