Multiplex panel for detecting rotavirus a / b / c nucleic acids

Abstract

Described herein are compositions, methods, and kits for detecting diarrhea causing pathogens from porcine samples. One embodiment described herein is primer pairs and probes for multiplex polymerase chain reaction (PCR) based assays for the detection of diarrhea causing pathogens, such as Rotavirus A, Rotavirus B, and Rotavirus C. Other embodiments include methods and kits for detecting diarrhea causing pathogens.

Description

[0001] MULTIPLEX PANEL FOR DETECTING ROTAVIRUS A / B / C NUCLEIC ACIDS

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to United States Provisional Patent Application No. 63 / 557,248, titled “MULTIPLEX PANEL FOR DETECTING ROTAVIRUS A / B / C NUCLEIC ACIDS,” filed February 23, 2024, the entirety of which is incorporated herein by reference.

[0004] INCORPORATION BY REFERENCE

[0005] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0006] REFERENCE TO SEQUENCE LISTING

[0007] This application was filed with a Sequence Listing XML in ST.26 XML format accordance with 37 C.F.R. § 1.831. The Sequence Listing XML file submitted in the USPTO Patent Center, “TP387462WO1.xml,” was created on January 31 , 2025, contains 182 sequences, has a file size of 230,162 bytes, and is incorporated by reference in its entirety into the specification.

[0008] FIELD OF THE INVENTION

[0009] The invention relates to nucleic acid-based kits and in vitro methods for determining the presence or absence of diarrhea causing pathogens in a sample, the diarrhea causing pathogens including the following: Rotavirus A, B, and C.

[0010] BACKGROUND OF THE INVENTION

[0011] Rotaviruses (Reoviridae) are one of the most prevalent etiological agents causing gastroenteritis and severe diarrhea in young pigs. Transmission occurs via the fecal-oral route and new infections can occur through direct exposure to infected feces or through contact with contaminated environments including water, feed, transportation vehicles, and housing. Diarrhea is most frequently observed in pigs at 1-4 weeks of age. Although common in young pigs, the disease is rarely fatal unless there are complications owing to concurrent infections or environmental stress. Although rarely fatal, disease severity can increase with concurrent infection or environmental stressors, which can further delay weight gain and time-to-market. As a result, there is a significant economic impact associated with rotavirus (over $230-350 M in the U.S.). Adult pigs become resistant to clinical disease as their immune system matures. Of the 9 known rotavirus species, Rotavirus A, B, and C (hereinafter alternatively referred to as RVA, RVB, and RVC, respectively) are the most important species associated with diarrhea in piglets.

[0012] Reverse transcription quantitative polymerase chain reaction (also known as reverse transcription real-time PCR) “RT-qPCR” is the most widely used method for the identification of rotavirus in porcine derived samples. Rotaviruses are double-stranded RNA (dsRNA) viruses with linear genomes comprising 11 gene segments generally expressing 12 or 13 proteins depending on species. RNA viruses depend on RNA-dependent RNA polymerase (RdRp) to replicate their genome during infection. The poor fidelity of RdRps results in numerous mutations between members of the same RNA virus species. The nature of the rotavirus genome, consisting of dsRNA, greatly reduces the efficiency of RT-qPCR because reverse transcriptase mediates the synthesis of DNA from ssRNA templates. Rotavirus dsRNA must be denatured to create ssRNA template for efficient reverse transcription, which is commonly done by including a heat-denaturation step. Samples must be heat-denatured prior to RT-qPCR because reverse transcriptase is heat-labile. The high genetic variability within and between rotavirus species and the resulting lack of conserved consensus sequences make assay design for rotavirus difficult, and many RT-qPCR methods show reduced coverage of known rotavirus isolates. Molecular detection of rotavirus must account for this complexity.

[0013] Both modified live virus and killed rotavirus vaccines are available, primarily for Rotavirus A. Most RVB and RVC strains do not replicate in cell culture, which is a major impediment for studying RVB and RVC and designing assays for detection. To date, it has been difficult to develop assays for rotavirus. The rotavirus genomes demonstrate high sequence variability, the available sequence differs greatly between the different gene segments, and many sequences are poorly annotated. It is believed that no commercial assay exists which can detect and speciate RVA, RVB, and RVC in a single multiplex qPCR assay design.

[0014] Accordingly, there is a need for a multiplex qPCR assay that can detect Rotavirus A / B / C from a single well with higher sensitivity, faster run times, limited to no cross- reactivity, and more reliable outcomes. The need is solved by the presently disclosed method that is directed to the detection of the presence or absence of Rotavirus A, Rotavirus B, and Rotavirus C in a multiplex qPCR assay.

[0015] SUMMARY OF THE INVENTION

[0016] Disclosed herein is an in vitro method for determining the presence or absence of at least one of Rotavirus A, Rotavirus B, and Rotavirus C in a sample. An exemplary embodiment of the method comprises the steps of: (a) creating a reaction mixture containing the sample and a primer mixture comprising: at least one primer that specifically amplifies a portion of Rotavirus A genome; at least one primer that specifically amplifies a portion of Rotavirus B genome; at least one primer that specifically amplifies a portion of Rotavirus C; and (b) subjecting the reaction mixture to reaction conditions suitable to amplify targeted nucleic acids, thereby generating one or more amplicons; wherein the presence or absence of at least one amplicon in the sample indicates the presence or absence of Rotavirus A, Rotavirus B, and Rotavirus C in the sample. In some embodiments, the reaction mixture further comprises at least one control primer that specifically amplifies a portion of the Xeno™ genome.

[0017] Another embodiment described herein is a method for determining the presence or absence of Rotavirus A, Rotavirus B, and Rotavirus C in a sample. The method comprises the steps of: (a) creating a reaction mixture containing the sample and a primer mixture comprising: at least one primer selected from SEQ ID NO: 1-52 that specifically amplifies a portion of Rotavirus A genome; at least one primer selected from SEQ ID NO: 64-120 that specifically amplifies a portion of Rotavirus B genome; and at least one primer selected from SEQ ID NO: 139-171 that specifically amplifies a portion of Rotavirus C genome; and (b) subjecting the reaction mixture to reaction conditions suitable to amplify targeted nucleic acids, thereby generating one or more amplicons; wherein the presence or absence of at least one amplicon in the sample indicates the presence or absence of Rotavirus A, Rotavirus B, or Rotavirus C in the sample.

[0018] In an exemplary embodiment of the method, the generating of the at least one amplicon includes performing PCR.

[0019] In an exemplary embodiment of the method, the at least one amplicon is one selected from an amplicon produced using at least one or more primers selected from SEQ ID NO: 1-6 and 37-42 for the detection of Rotavirus A and comprising at least one sequence selected from 53-55 and / or 62-63; an amplicon produced using at least one or more primers selected from SEQ ID NO: 64-79 and 109-114 for the detection of Rotavirus B and comprising at least one sequence selected from SEQ ID NO: 121-128 and / or 129-130; and / or an amplicon produced using at least one or more primers selected from SEQ ID NO: 139 and / or 164 for the detection of Rotavirus C and comprising the sequence SEQ ID NO: 172.

[0020] In an exemplary embodiment of the method, the reaction mixture further comprises probes specific for the at least one amplicon.

[0021] In an exemplary embodiment of the method, the reaction mixture further comprises probes specific for the at least one amplicon and suitable for use with the primer mixture, the probes comprising: a probe specific for Rotavirus A having a sequence selected from SEQ ID NO: 53-55 and 62-63; a probe specific for Rotavirus B having a sequence selected from SEQ ID NO: 121- 130; or a probe specific for Rotavirus C of SEQ ID NO: 172.

[0022] In an exemplary embodiment of the method, the reaction mixture further contains a control sample and at least one control primer and, optionally, a control probe, that specifically amplify the target nucleic acid of the control sample.

[0023] In an exemplary embodiment of the method, the at least one control primer comprises a Xeno™ primer sequence selected from SEQ ID NO: 180-181 and the Xeno™ control probe sequence of SEQ ID NO: 182.

[0024] In an exemplary embodiment of the method, the probe(s) each comprise a fluorescent reporter.

[0025] In an exemplary embodiment of the method, the probe(s) each comprise a quencher.

[0026] In an exemplary embodiment of the method, each probe(s) is labeled at or near the 5'-end with a dye selected from ABY, FAM, VIC or CY5.

[0027] In an exemplary embodiment of the method, each probe(s) is labeled at the 3-end with a quencher selected from NFQ-MGB, QSY7, QSY21 , Eclipse, BHQ, and DFQ.

[0028] In an exemplary embodiment of the method, the probe(s) specific for amplicons generated using one or more primers for the detection of Rotavirus A are labeled with ABY; the probe(s) specific for amplicons generated using one or more primers for the detection of Rotavirus B are labeled with FAM; and the probe(s) specific for amplicons generated using one or more primers for the detection of Rotavirus C are labeled with VIC.

[0029] In an exemplary embodiment of the method, the probe(s) specific for amplicons generated using one or more primers for the detection of Xeno™ are labeled with CY5.

[0030] In an exemplary embodiment of the composition, the composition comprises the following sequences: at least one primer specific for Rotavirus A selected from SEQ ID NO: 1-52; at least one primer specific for Rotavirus B selected from SEQ ID NO: 64-120; and at least one primer specific for Rotavirus C selected from SEQ ID NO: 139-171.

[0031] In an exemplary embodiment of the composition, further comprising probes specific for amplicons produced using the primers specific for Rotavirus A, Rotavirus B, and Rotavirus C. Such probes include: a probe specific for Rotavirus A having a sequence selected from SEQ ID NO: 53-63; a probe specific for Rotavirus B produced using a sequence selected from SEQ ID NO: 121-138; or a probe specific for Rotavirus C selected from SEQ ID NO: 172-179.

[0032] In an exemplary embodiment of the composition, the composition further includes at least one control primer selected from SEQ ID NO: 180-181 ; and, optionally, a control probe with a sequence of SEQ ID NO: 182, that specifically amplifies the target nucleic acid of a control sample.

[0033] In one aspect, at least one probe contains a fluorescent reporter.

[0034] In one aspect, at least one probe contains a quencher.

[0035] In one aspect, at least one probe is labeled at or near the 5'-end with a dye selected from ABY, FAM, VIC, or CY5.

[0036] In one aspect, at least one probe is labeled at the 3'-end with a quencher selected from NFQ-MGB, QSY7, QSY21, Eclipse, BHQ, and DFQ.

[0037] In one aspect, at least one probe specific for amplicons generated using one or more primers for Rotavirus A are labeled with ABY; the probes specific for amplicons generated using one or more primers for Rotavirus B are labeled with FAM; and the probes specific for amplicons generated using one or more primers for Rotavirus C are labeled with VIC.

[0038] In an exemplary embodiment of the method, the probes specific for amplicons generated using one or more control primers for Xeno™ are labeled with CY5.

[0039] One embodiment described herein is a multiplex panel of forward primers, reverse primers, and probes for detecting rotavirus nucleic acids in a sample, including Rotavirus A, Rotavirus B, and Rotavirus C.

[0040] Another embodiment described herein is a multiplex panel of the forward primers, reverse primers, and probes described above.

[0041] Another embodiment described herein is a kit for determining the presence or absence of Rotavirus A, Rotavirus B, or Rotavirus C in a sample comprising any of the compositions described herein. In one aspect, the kit comprises any of the multiplex panels of forward primers, reverse primers, and probes for detecting rotavirus nucleic acids in a sample. In one embodiment, the kit further comprises a one or more control primers, and, optionally, a control probe, that specifically amplify the target nucleic acid of a control sample.

[0042] In another embodiment, the kit comprises a multiplex panel of forward primers, reverse primers, and probes as described above. In an embodiment, the primers are selected from SEQ ID NO: 1-6 and 37-42; and the probes are selected from SEQ ID NO: 53-55. In another embodiment, the primers are selected from SEQ ID NO: 27-36 and 49-52; and the probes are selected from SEQ ID NO: 62-63. In another embodiment, the primers are selected from SEQ ID NO: 64-79 and 109-114; and the probes are selected from SEQ ID NO: 121-128. In another embodiment, the primers are selected from SEQ ID NO: 80-88; and the probes are selected from SEQ ID NO: 129-130. In another embodiment, the primers are selected from SEQ ID NO: 139 and 164; and the probe is SEQ ID NO: 172. In another embodiment, the primers are selected from SEQ ID NO: 7-22 and 43; and the probes are selected from SEQ ID NO: 56-58. In another embodiment, the primers are selected from SEQ ID NO: 24 and 45; and the probes are selected from SEQ ID NO: 60. In another embodiment, the primers are selected from SEQ ID NO: 25-26 and 46-48; and the probes are selected from SEQ ID NO: 61. In another embodiment, the primers are selected from SEQ ID NO: 89-104 and 115-116; and the probes are selected from SEQ ID NO: 131-132. In another embodiment, the primers are selected from SEQ ID NO: 103 and 117; and the probes are selected from SEQ ID NO: 133-136. In another embodiment, the primers are selected from SEQ ID NO: 106-107 and 118; and the probes are selected from SEQ ID NO: 137. In another embodiment, the primers are selected from SEQ ID NO: 108 and 119-120; and the probes are selected from SEQ ID NO: 138. In another embodiment, the primers are selected from SEQ ID NO: 140-142 and 165-166; and the probes are selected from SEQ ID NO: 173. In another embodiment, the primers are selected from SEQ ID NO: 143-146 and 167-168; and the probes are selected from SEQ ID NO: 174-177. In another embodiment, the primers are selected from SEQ ID NO: 147-162 and 169-170; and the probes are selected from SEQ ID NO: 178. In another embodiment, the primers are selected from SEQ ID NO: 163 and 171 ; and the probes are selected from SEQ ID NO: 179.

[0043] DESCRIPTION OF THE DRAWINGS

[0044] FIG. 1 shows an exemplary amplification plot depicting the detection of RVA, RVB, RVC, and the internal positive control (I PC) in a single reaction mixture.

[0045] FIG. 2A shows a graph depicting the detection of Rotavirus A on 4 different real-time PCR instruments: an Applied Biosystems™ QuantStudio™ 5 0.1 mL Real-Time PCR system, an Applied Biosystems™ QuantStudio™ 5 0.2mL Real-Time PCR system, an Applied Biosystems™ 7500 Fast Real-Time PCR system with SDS version 1.5 software, and an Applied Biosystems™ 7500 Fast Real-Time PCR system with SDS version 2.3 software.

[0046] FIG. 2B shows a graph depicting the detection of Rotavirus B on 4 different real-time PCR instruments: an Applied Biosystems™ QuantStudio™ 5 0.1 mL Real-Time PCR system, an Applied Biosystems™ QuantStudio™ 5 0.2mL Real-Time PCR system, an Applied Biosystems™ 7500 Fast Real-Time PCR system with SDS version 1.5 software, and an Applied Biosystems™ 7500 Fast Real-Time PCR system with SDS version 2.3 software.

[0047] FIG. 2C shows a graph depicting the detection of Rotavirus C on 4 different real-time PCR instruments: an Applied Biosystems™ QuantStudio™ 5 0.1 mL Real-Time PCR system, an Applied Biosystems™ QuantStudio™ 5 0.2mL Real-Time PCR system, an Applied Biosystems™ 7500 Fast Real-Time PCR system with SDS version 1.5 software, and an Applied Biosystems™ 7500 Fast Real-Time PCR system with SDS version 2.3 software.

[0048] FIG. 3A shows an amplification plot for the double-stranded RNA control for RVA and RVC and single-stranded RNA control for RVB and Xeno™ with heat denaturation.

[0049] FIG. 3B shows an amplification plot for the double-stranded RNA control for RVA and RVC and single-stranded RNA control for RVB and Xeno™ without heat-denaturation.

[0050] FIG. 4 shows a graph depicting the Cq values of RVA, RVB, and RVC tested individually (left bar) and combined with four rotavirus negative samples in a pool (right bar).

[0051] FIG. 5A shows a graph depicting the serial 10-fold dilution of RVA control RNA run in the presence (left bar) and absence (right bar) of the other two rotavirus species each present at 107copies / reaction for each dilution of RVA.

[0052] FIG. 5B shows a graph depicting the serial 10-fold dilution of RVB control RNA run in the presence (left bar) and absence (right bar) of the other two rotavirus species each present at 107copies / reaction for each dilution of RVB.

[0053] FIG. 5C shows a graph depicting the serial 10-fold dilution of RVC control RNA run in the presence (left bar) and absence (right bar) of the other two rotavirus species each present at 107copies / reaction for each dilution of RVC.

[0054] DETAILED DESCRIPTION

[0055] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of biochemistry, molecular biology, immunology, microbiology, genetics, cell and tissue culture, and protein and nucleic acid chemistry described herein are well known and commonly used in the art. In case of conflict, the present disclosure, including definitions, will control. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the embodiments and aspects described herein.

[0056] As used herein, the terms “amino acid,” “nucleotide,” “oligonucleotide”, “polynucleotide,” “oligonucleotide primer (or primer)”, “oligonucleotide probe (or probe)”, “vector,” “polypeptide,” and “protein” have their common meanings as would be understood by a biochemist of ordinary skill in the art. Standard single letter nucleotides (A, C, G, T, U) and standard single letter amino acids (A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y) are used herein.

[0057] As used herein, the terms such as “include,” “including,” “contain,” “containing,” “having,” and the like mean “comprising.” The present disclosure also contemplates other embodiments “comprising,” “consisting essentially of,” and “consisting of” the embodiments or elements presented herein, whether explicitly set forth or not.

[0058] As used herein, the term “a,” “an,” “the” and similar terms used in the context of the disclosure (especially in the context of the claims) are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context. In addition, “a,” “an,” or “the” means “one or more” unless otherwise specified.

[0059] As used herein, the term “or” can be conjunctive or disjunctive.

[0060] As used herein, the term “and / or” refers to both the conjunctive and disjunctive.

[0061] As used herein, the term “substantially” means to a great or significant extent, but not completely.

[0062] As used herein, the term “about” or “approximately” as applied to one or more values of interest, refers to a value that is similar to a stated reference value, or within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, such as the limitations of the measurement system. In one aspect, the term “about” refers to any values, including both integers and fractional components that are within a variation of up to ± 10% of the value modified by the term “about.” Alternatively, “about” can mean within 3 or more standard deviations, per the practice in the art. Alternatively, such as with respect to biological systems or processes, the term “about” can mean within an order of magnitude, in some embodiments within 5-fold, and in some embodiments within 2-fold, of a value. As used herein, the symbol means “about” or “approximately.”

[0063] All ranges disclosed herein include both end points as discrete values as well as all integers and fractions specified within the range. For example, a range of 0.1-2.0 includes 0.1 , 0.2, 0.3, 0.4 . . . 2.0. If the end points are modified by the term “about,” the range specified is expanded by a variation of up to ±10% of any value within the range or within 3 or more standard deviations, including the end points.

[0064] As used herein, the terms “control,” or “reference” are used herein interchangeably. A “reference” or “control” level may be a predetermined value or range, which is employed as a baseline or benchmark against which to assess a measured result. “Control” also refers to control experiments.

[0065] As used herein, the term “subject” refers to an animal. Typically, the subject is a mammal. A subject also refers to primates (e.g., humans, male or female; infant, adolescent, or adult), nonhuman primates, rats, mice, rabbits, pigs, cows, sheep, goats, horses, dogs, cats, fish, birds, and the like. In one embodiment, the subject is a primate. In one embodiment, the subject is a human. As used herein, a subject is “in need of treatment” if such subject would benefit biologically, medically, or in quality of life from such treatment. A subject in need of treatment does not necessarily present symptoms, particular in the case of preventative or prophylaxis treatments.

[0066] As used herein, the terms “inhibit,” “inhibition,” or “inhibiting” refer to the reduction or suppression of a given biological process, condition, symptom, disorder, or disease, or a significant decrease in the baseline activity of a biological activity or process.

[0067] As used herein, “treatment” or “treating” refers to prophylaxis of, preventing, suppressing, repressing, reversing, alleviating, ameliorating, or inhibiting the progress of biological process including a disorder or disease, or completely eliminating a disease. A treatment may be either performed in an acute or chronic way. The term “treatment” also refers to reducing the severity of a disease or symptoms associated with such disease prior to affliction with the disease. “Repressing” or “ameliorating” a disease, disorder, or the symptoms thereof involves administering a cell, composition, or compound described herein to a subject after clinical appearance of such disease, disorder, or its symptoms. “Prophylaxis of” or “preventing” a disease, disorder, or the symptoms thereof involves administering a cell, composition, or compound described herein to a subject prior to onset of the disease, disorder, or the symptoms thereof. “Suppressing” a disease or disorder involves administering a cell, composition, or compound described herein to a subject after induction of the disease or disorder thereof but before its clinical appearance or symptoms thereof have manifest.

[0068] The polynucleotides described herein may include variants that have substitutions, deletions, and / or additions that can involve one or more nucleotides. Some embodiments described herein include nucleic acid molecules comprising polynucleotides having nucleotide sequences about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, and more preferably at least about 95-99% or 100% identical to (a) nucleotide sequences of SEQ ID NO: 1-182 and capable of being used as primers or probes as described herein; or (b) nucleotide sequences capable of hybridizing to the complement of any of the nucleotide sequences of SEQ ID NO: 1-182 and capable of being used as primers or probes as described herein.

[0069] As used herein, a nucleotide having a nucleotide sequence at least 90-99% “identical” to a reference nucleotide sequence indicates that the nucleotide sequence is identical to the reference sequence except that the nucleotide sequence can include up to about 10-to-1 point mutations, additions, or deletions per each 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a nucleotide having a nucleotide sequence at least 90-99% identical to a reference nucleotide sequence, up to 10% of the nucleotides in the reference sequence can be deleted, added, or substituted with another nucleotide, or a number of nucleotides up to 10% of the total nucleotides in the reference sequence can be inserted into the reference sequence. These mutations of the reference sequence can occur at the 5'- or 3'-terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence. These may include standard nucleotides, modified nucleotides, fluorescent dyes, linkers, or other modifications.

[0070] As described above, two or more polynucleotide sequences can be compared by determining their percent identity. The percent identity of two sequences is generally described as the number of exact matches between two aligned sequences divided by the length of the shorter sequence and multiplied by 100. Alignment for nucleic acid sequences can be performed using the global alignment method of Needleman and Wunsch, J. Mol. Biol. 48 (3): 443-453 (1970) or the local homology algorithm of Smith and Waterman, Adv. Appt. Math. 2: 482-489 (1981).

[0071] Described herein are primers, primer sets, and probes designed for target sequences and compatible for use in a multiplex qPCR determining the presence of one or more gastrointestinal pathogens. Multiplex PCR presents a challenge for quantitation of the pathogen DNA: the different amplicons compete for the same PCR reaction components (such as e.g., DNA polymerase and MgCh) and this can compromise the quantitative comparison between samples. It is known in the art that there is bias in the amplification efficiencies between different template amounts or lengths so that e.g., short amplicons are favored in the expense of longer ones. At the same time, undesired cross-reactions of multiplex set oligo combinations must be avoided. Finding suitable primer and probe sequences for the detection of a diverse group of pathogenic microbes is far from trivial especially when designing multiplex set ups. Described herein are primer sets and probes for detecting the presence or absence of each of Rotavirus A, Rotavirus B, and Rotavirus C. The selected primers and probes of the assay do not cross-react with other closely-related pathogens. The disclosed detection method and assay may contain an internal process control, such as Xeno™ Internal Positive Control RNA (or DNA) by Applied Biosystems™, and ROX as a normalization reference. In addition, the assay may have a real time PCR positive control, comprising RNA templates for RVA, RVB, RVC, and Xeno created in vitro from artificial templates, for example. In such example, the templates are combined into a single reaction tube to be used as a positive control. Additionally, the assay may have a "negative control" (no-template control (NTC)), such as (nuclease free) water because water contains no templates for RVA, RVB, RVC, or Xeno and therefore should result in no amplification. The probes that target genomic regions of different pathogens utilize three distinct fluorophores, while a fourth fluorophore is designated for the process control detection. Therefore, a 4-plex qPCR assay is described herein, wherein the detection takes place in a single-well reaction, to meet the speed and high-throughput needs for rapid gastrointestinal pathogen testing.

[0072] When an exemplary “embodiment” or a particular “assay” is described herein, it will be understood that the features of that embodiment may be applicable to a composition (e.g., the particular physical components of an assay such as primers and / or probes), a kit (e.g., primers and / or probes and additional buffers, reagents, etc.), or a method (e.g., a process for detecting target nucleic acids) as appropriate. For simplicity, embodiments are presented by describing “assays,” but it will be understood that the associated methods for the assays, and the compositions for carrying out the assays (primers, probes, and primer sets), are also intended to be part of this disclosure.

[0073] One embodiment described herein is a method for determining the presence or absence of Rotavirus A, Rotavirus B, or Rotavirus C, in a sample is provided, the method comprising the steps of: (a) creating a reaction mixture containing the sample and a primer set; wherein the primer set comprises: at least one primer that specifically amplifies a portion of Rotavirus A genome; at least one primer that specifically amplifies a portion of Rotavirus B genome; at least one primer that specifically amplifies a portion of Rotavirus C genome; and (b) subjecting the reaction mixture to reaction conditions suitable to amplify targeted nucleic acids, thereby generating one or more amplicons; wherein the presence or absence of at least one amplicon in the sample indicates the presence or absence of Rotavirus A, Rotavirus B, or Rotavirus C in the sample. In some embodiments, the primer set further comprises at least one control primer that specifically amplifies a portion of the Xeno™ internal positive control template. In some embodiments, the primers are used with probes.

[0074] Another embodiment described herein is a method for determining the presence or absence of Rotavirus A, Rotavirus B, or Rotavirus C in a sample is provided, the method comprising the steps of: (a) creating a reaction mixture containing the sample and at least three primer pair sets; wherein the primer pair set comprises: at least one primer selected from SEQ ID NO: 1-36 and 37-52 that specifically amplifies a portion of the Rotavirus A genome; at least one primer selected from SEQ ID NO: 64-108 and 109-120 that specifically amplifies a portion of the Rotavirus B genome; and at least one primer selected from SEQ ID NO: 139-163 and 164-171 that specifically amplifies a portion of the Rotavirus C genome; and (b) subjecting the reaction mixture to reaction conditions suitable to amplify targeted nucleic acids, thereby generating one or more amplicons; wherein the presence or absence of at least one amplicon in the sample indicates the presence or absence of Rotavirus A, Rotavirus B, or Rotavirus C in the sample. In one embodiment, the method and / or assay comprises a panel comprising one or more primers and one or more probes for the simultaneous detection of the presence or absence of one or more of Rotavirus A, Rotavirus B, and Rotavirus C in a sample. In one aspect, the previous recited methods have one or more primer pairs and one or more probes for the detection of a control sample of Xeno™.

[0075] One embodiment described herein is a multiplex panel for the detection of the presence or absence of one or more of Rotavirus A, Rotavirus B, and Rotavirus C in a sample. In one aspect, the multiplex panel comprises at least one forward primer, at least one reverse primer, and at least one probe for each organism. As an exemplary aspect, for detecting Rotavirus A, a primer pair and probe set could include: a forward primer such as SEQ ID NO: 1 , a reverse primer such as SEQ ID NO: 37, and a probe such as SEQ ID NO: 53. Optionally, the multiplex panel comprises a control, comprising at least one forward primer, at least one reverse primer, and a probe for the Xeno™ control template (SEQ ID NO: 180-182).

[0076] In another embodiment, the method and / or assay comprises one or more primer pairs and one or more probes for the detection of the presence or absence of one or more of Rotavirus A, Rotavirus B, and Rotavirus C in a sample. Specifically, an embodiment of the method and / or assay comprises the following primers and probes for the detection of Rotavirus A: SEQ ID NO: 1-6, 37-42, and 53-55. In another embodiment, the primers and probes for the detection of Rotavirus A are SEQ ID NO: 7-22, 43, and 56-58. In another embodiment, the primers and probes for the detection of Rotavirus A are SEQ ID NO: 23, 44, and 59. In another embodiment, the primers and probes for the detection of Rotavirus A are SEQ ID NO: 24, 45, and 60. In another embodiment, the primers and probes for the detection of Rotavirus A are SEQ ID NO: 25-26, 46- 48, and 61 . In another embodiment, the primers and probes for the detection of Rotavirus A are SEQ ID NO: 27-36, 49-52, and 62-63.

[0077] Specifically, an embodiment of the method and / or assay comprises the following primers and probes for the detection of Rotavirus B: SEQ ID NO: 64-79, 109-114, and 121-128. In another embodiment, the primers and probes for the detection of Rotavirus B are SEQ ID NO: 80-88 and 129-130. In another embodiment, the primers and probes for the detection of Rotavirus B are SEQ ID NO: 89-104, 115-116, and 131-132. In another embodiment, the primers and probes for the detection of Rotavirus B are SEQ ID NO: 105, 117, and 133-136. In another embodiment, the primers and probes for the detection of Rotavirus B are SEQ ID NO: 106-107, 118, and 137. In another embodiment, the primers and probes for the detection of Rotavirus B are SEQ ID NO: 108, 119-120, and 138. Specifically, an embodiment of the method and / or assay comprises the following primers and probes for the detection of Rotavirus C are: SEQ ID NO: 139, 164, and 172. In another embodiment, the primers and probes for the detection of Rotavirus C are SEQ ID NO: 140-142, 165-166, and 173. In another embodiment, the primers and probes for the detection of Rotavirus C are SEQ ID NO: 143-146, 167-168, and 174-177. In another embodiment, the primers and probes for the detection of Rotavirus C are SEQ ID NO: 147-162, 169-170, and 178. In another embodiment, the primers and probes for the detection of Rotavirus C are SEQ ID NO: 163, 171 , and 179.

[0078] A primer / probe mix can be generated by mixing any combination of the above primers and probes for Rotavirus A, Rotavirus B, and / or Rotavirus C. An exemplary embodiment of a multiplex panel for the detection of Rotavirus A, Rotavirus B, and / or Rotavirus C includes the following primers and probes: SEQ ID NO: 1-6, 27-36, 37-42, 49-52, 53-55, 62-63, 64-79, 80-88, 109-114, 121-128, 129-130, 139, 164, and 172. This exemplary panel may also include the primers and probe for the Xeno™ internal positive control template: SEQ ID NO: 180-182. An alternative exemplary embodiment of a multiplex panel for the detection of Rotavirus A, Rotavirus B, and / or Rotavirus C includes the following primers and probes: SEQ ID NO: 7-23, 43-44, 56-59, 89-105, 115-117, 131-136, 140-146, 165-168, and 173-177. An alternative exemplary embodiment of a multiplex panel for the detection of Rotavirus A, Rotavirus B, and / or Rotavirus C includes the following primers and probes: SEQ ID NO: 24-26, 45-48, 60-61 , 106-108, 118-120, 137-138, 147- 163, 169-171, and 178-179.

[0079] Sample or Specimen

[0080] Described herein are compositions, kits, and method for the detection (i.e. , the presence or absence) of diarrhea-causing pathogens including Rotavirus A, Rotavirus B, and / or Rotavirus C in a specimen or a sample. DNA is extracted from one or more specimen / sample, multiplied using real-time amplification, and detected using specific primers and a fluorescent reporter dye probe for Rotavirus A, Rotavirus B, and Rotavirus C. As will be appreciated by those in the art, the specimen / sample may comprise any number of things, including, but not limited to, feces, fecal swabs, rectal swabs, feedback, fluid, homogenate, oral fluid, processing fluid, tissue composite, tissue cultures, intestinal tissue, environmental samples (such as water, feed, and fomites), research samples, purified samples (e g., purified genomic DNA and RNA), raw samples (bacteria, virus, genomic DNA, etc.), and possible vaccines. As will be appreciated by those in the art, any experimental manipulation can be performed on the sample before analysis. In some embodiments, the specimen / sample type for diagnosis of diarrhea-causing rotaviruses is a feces sample, fecal swab, or a rectal swab.

[0081] If required, nucleic acid from the sample / specimen is isolated using known techniques. For example, the sample / specimen may be treated to lyse the cells, using known lysis buffers, sonication, electroporation, etc., with purification occurring as needed, as will be appreciated by those in the art. In addition, the reactions outlined herein may be accomplished in a variety of ways, as will be appreciated by those in the art. Components of the reaction may be added simultaneously, or sequentially, in any order, with preferred embodiments outlined below. In addition, the reaction may include a variety of other reagents that may be included in the assays. These include reagents like salts, buffers, neutral proteins, e.g., albumin, detergents, etc., which may be used to facilitate optimal hybridization and detection, and / or reduce non-specific or background interactions. Reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., may be used, depending on the sample / specimen preparation methods and purity of the target diarrhea-causing rotaviruses.

[0082] In some embodiments, total nucleic acid extraction from a feces sample, a fecal swab, a rectal swab, etc. is performed. Nucleic acids may be isolated and purified from the specimen / sample using a nucleic acid isolation, such as, e.g., the MagMAX™ CORE Nucleic Acid Purification Kit by Applied Biosystems™. Nucleic acid extraction may be performed via an automated process using, e.g., the Kingfisher™ Flex Purification System. For RNA viruses, the RNA is reverse transcribed into cDNA. The cDNA and genomic DNA (from DNA viruses) are then subjected for amplification using the currently disclosed composition, method, or kit.

[0083] Process Control, Control Template, Control Plasmid, or Control Sequence

[0084] In addition, the disclosed kit and method of detection may further include a process control (“process control” is herein referred to, interchangeably, as “control template”, “control plasmid”, or “control sequence”). Controls are treated and tested in parallel with target pathogen and are used to generate a predetermined expected result. When the expected result is reported, one or more aspects of the diagnostic test are confirmed to be working as intended, enabling the user of to verify the diagnostic test as valid. The process control may be Xeno™ Internal Positive Control RNA or DNA by Applied Biosystems™. Xeno™ Internal Positive Control RNA is a transcript and DNA by Applied Biosystems™ is a plasmid. Xeno™ Internal Positive Control RNA and DNA are each a unique artificial sequence that is not representative of any RNA nor DNA sequence, respectively, from any living organisms (i.e. , an artificial sequence not found in nature). Preferably, the process control can function as a positive control for lysis, purification and amplification within the cartridges described herein.

[0085] One exemplified process control is the Xeno™ Internal Positive Control RNA by Applied Biosystems™. The process control may be supplied in a quantity of 1 x 104copies / uL or 5 x 103copies / uL. The sample preparation protocol recommends adding 2 uL of this reagent to the lysis buffer prior to sample lysis.

[0086] Positive Control

[0087] In addition, the disclosed method of detection may further include a positive control to determine the validity of the assay. The positive control is a mixture of RNA transcripts (also referred to as “RNA templates” or “templates”) of the target pathogens. The RNA transcripts are prepared by taking DNA plasmids and performing a transcription reaction to create large quantities of RNA transcripts. In an exemplary embodiment, a positive control is a mixture of RNA transcripts of RVA, RVB, and / or RVC. In another embodiment, the positive control mixture includes RNA transcripts of RVA, RVB, RVC, and / or Xeno™ RNA. Such positive controls are stored between -30 C to -10 C. In some embodiments the positive control mixture is provided in 8 uL per reaction.

[0088] Negative Control

[0089] Additionally, the disclosed method of detection may further include a negative control to determine the validity of the assay. In an embodiment, the negative control is a no-template control (NTC), such as nuclease-free water. In some embodiments the NTC is provided in 8 uL per reaction. Such negative control is stored between -30 C to -10 C.

[0090] Reaction Mixture

[0091] The terms “reaction mixture,” “amplification mixture,” or “PCR mixture” as used herein refer to a mixture of components necessary to amplify at least one amplicon from nucleic acid templates. The mixture may comprise nucleotides (dNTPs, such as A, C, G, T, and / or U), a thermostable polymerase, primers, and a plurality of nucleic acid templates. The mixture may further comprise a buffer. As used herein, “buffer” refers to a solution that can include multiple components such buffers that maintain a specific pH (e.g., Tris HCI), mono and divalent salts (e.g., NaCI, KOI, (NH4)2SC>4, MgCL, MgSC ), cofactors, detergents (e.g., Polysorbate 20 (Tween™ 20), t-octylphenoxy polyethoxyethanol (Triton™ X-100), octylphenoxy polyethoxyethanol (Nonidet™ P-40)), and other additives (e.g., glycerol, polyethylene glycol, betaine, formamide, dimethyl sulfoxide, dithiothreitol, tetramethylammonium chloride, bovine serum albumin, gelatin, inter alia). The working concentration range of each component is known in the art and can be optimized as needed. An exemplary RT-qPCR reaction mixture comprises: 2 uL the target rotavirus primer and probe mixture; 10 uL of the VetMAX™ Rotavirus Master Mix, resulting in a total volume of 12 uL per reaction. Typically, the RT-qPCR reaction mixture is dispensed into a PCR microtube or plate and 8 uL of sample or NTC (nuclease-free water) is added to reach a final reaction volume of 20 uL. The reaction mixture is mixed by vortexing, and either capped, sealed, or overlayed with mineral oil or silicone oil to prevent evaporation.

[0092] Amplification

[0093] 'Amplification” as used herein refers to the use of any amplification procedures to increase the concentration of a particular nucleic acid sequence within a mixture of nucleic acid sequences.

[0094] In one embodiment and as describe more fully herein, a sequence from a sample is amplified to produce a secondary target (e.g., an amplicon) that is detected, as outlined herein.

[0095] Amplification involves the amplification (replication) of the sequence to be detected, such that the number of copies of the sequence is increased. Suitable amplification techniques include, but are not limited to, the polymerase chain reaction (PCR), strand displacement amplification (SDA), transcription mediated amplification (TMA) and nucleic acid sequence-based amplification (NASBA).

[0096] In one embodiment, the amplification technique is PCR. The polymerase chain reaction (PCR) is well known and involves the use of primer extension combined with thermal cycling to amplify a target sequence. As used herein, “PCR”, unless specifically defined, refers to either single-plex or multiplex PCR assays, and can be real time or quantitative PCR (wherein detection occurs during amplification), end-point PCR (when detection occurs at the end amplification), or reverse transcription PCR, including but not limited to, “reverse transcription-quantitative polymerase chain reaction” or RT-qPCR", “real-time PCR” or “quantitative PCR” or “qPCR”, “digital PCR” or “dPCR”, “reverse transcriptase PCR” or “RT-PCR”, “multiplex PCR”, “nested PCR”, “hot start PCR”, “long-range PCR”, “assembly PCR”, “asymmetric PCR”, “in situ PCR,” “single-cell PCR,” or “fast-cycling PCR,” among others.

[0097] An exemplary PCR amplification typically consists of 25-35 cycles of: a denaturing step at 94 °C for 30-45 seconds; an annealing step at 55 °C for 20-60 seconds; and an extension step at 72 °C for 30-90 seconds. The amplification may be preceded by an initial denaturation step at 94 °C for 2-3 minutes. The amplification may be followed by a final extension step at 72 °C for 10 min, and then cooled to 4 °C and maintained at this temperature until the samples are removed from the thermocycler.

[0098] Example embodiments of amplification are not limited to PCR. For instance, signal amplification, single base extension (SBE) or minisequencing, oligonucleotide ligation amplification (OLA) and / or rolling-circle amplification can be used for amplification. In an embodiment, amplification can include OLA followed by RCA.

[0099] A skilled person would be knowledgeable of the real-time PCR systems that can be used for a multiplex detection using several dyes, or nucleic acid amplification assays as described herein. Exemplary systems include a real-time quantitative PCR (qPCR) instrument, including for example a QuantStudio™ Real-Time PCR system, such as the QuantStudioTM5 Real-Time PCR System (QS5), QuantStudio™ 7 Real-Time PCR System (QS7), QuantStudio™ 12K Flex System (QS12K), QuantStudio™ DX Real-Time PCR System (QS Dx or QS5 Dx), or a 7500 Real-Time PCR system, such as the 7500 Fast Dx system, all from Applied Biosystems™ - a Thermo Fisher Scientific brand.

[0100] Amplicon

[0101] The terms “amplified product” or “amplicon” refer to a fragment of DNA amplified by a polymerase using a pair of primers in an amplification method such as PCR.

[0102] Probe

[0103] “Probe” as used herein, is a non-extendable oligonucleotide attached to a fluorescent reporter dye and a quencher moiety.

[0104] Primer

[0105] “Primer” as used herein can refer to more than one primer and refers to an oligonucleotide, whether occurring naturally or produced synthetically, which is capable of acting as an initiationpoint for primer extension synthesis when placed under conditions that produce a primer extension product which is complementary to a nucleic acid strand, e.g., in the presence of nucleotides and an agent for polymerization such as DNA polymerase, at a suitable temperature for a sufficient amount of time and in the presence of a buffering agent. Such conditions can include, for example, the presence of at least four different deoxyribonucleoside triphosphates (such as A, C, G, and T) and a polymerization-inducing agent such as DNA polymerase or reverse transcriptase, in a suitable buffer, and at a suitable temperature. In some embodiments, the primer may be single-stranded for maximum efficiency in amplification. The primers herein are selected to be substantially complementary to the different strands of each specific sequence to be amplified. This means that the primers must be sufficiently complementary to hybridize with their respective strands. A non-complementary nucleotide fragment may be attached to the 5'-end of the primer, with the remainder of the primer sequence being complementary, or partially complementary, to the target region of the target nucleic acid. Commonly, the primers are complementary, except when non-complementary nucleotides may be present at a predetermined sequence location, such as a primer terminus as described.

[0106] The complement of a nucleic acid sequence as used herein refers to an oligonucleotide which, when aligned with the nucleic acid sequence such that the 5 -end of one sequence is paired with the 3 -end of the other, is in “antiparallel association.” Complementarity need not be perfect; stable duplexes may contain mismatched base pairs or unmatched bases.

[0107] Dyes, Detectable Labels, or Fluorescent Labels

[0108] The primers and / or probes described herein may further comprise a dye, fluorescent label, or other detectable label. It should be appreciated that when using multiple fluorescent or detectable labels, particularly in a multiplex format, each fluorescent or detectable label preferably differs in its spectral properties from the other detectable labels used therewith such that the labels may be distinguished from each other, or such that together the fluorescent or detectable labels emit a signal that is not emitted by either fluorescent or detectable label alone. Exemplary fluorescent or detectable labels include, for instance, a fluorescent dye or fluorophore (e.g., a chemical group that can be excited by light to emit fluorescence or phosphorescence), “acceptor dyes” capable of quenching a fluorescent signal from a fluorescent donor dye, and the like, as described above. Suitable fluorescent or detectable labels may include, for example, fluoresceins (e.g., 5-carboxy-2,7-dichlorofluorescein; 5-carboxyfluorescein (5-FAM); 5-hydroxy tryptamine (5- HAT); 6-JOE; 6-carboxyfluorescein (6-FAM); Mustang Purple, VIC, ABY, JUN; FITC; 6-carboxy- 4',5'-dichloro-2',7'-dimethoxy-fluorescein (JOE)); 6-carboxy-1 ,4-dichloro-2',7'-dichloro-fluorescein (TET); 6-carboxy-1 ,4-dichloro-2',4',5',7'-tetra-chlorofluorescein (HEX); Alexa Fluor fluorophores (e.g., 350, 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 635, 647, 660, 680, 700, 750); BODIPY fluorophores (e.g., 492 / 515, 493 / 503, 500 / 510, 505 / 515, 530 / 550, 542 / 563, 558 / 568, 564 / 570, 576 / 589, 581 / 591, 630 / 650-X, 650 / 665-X, 665 / 676, FL, FL ATP, Fl-Ceramide, R6G SE, TMR, TMR-X conjugate, TMR-X, SE, TR, TR ATP, TR-X SE), Cascade Blue, Cascade Yellow; Cy™ dyes (e.g., 3, 3.18, 3.5, 5, 5.18, 5.5, 7), cyan GFP, cyclic AMP Fluorosensor (FiCRhR), fluorescent proteins (e.g., green fluorescent protein (e.g., GFP, EGFP), blue fluorescent protein (e.g., BFP, EBFP, EBFP2, Azurite, mKalamal), cyan fluorescent protein (e.g., ECFP, Cerulean, CyPet), yellow fluorescent protein (e.g., YFP, Citrine, Venus, YPet), FRET donor / acceptor pairs (e g., fluorescein / fluorescein, fluorescein / tetramethylrhodamine, lAEDANS / fluorescein, EDANS / dabcyl, BODIPY FL / BODIPY FL, Fluorescein / QSY7 and QSY9), LysoTracker and LysoSensor (e.g., LysoTracker Blue DND-22, LysoTracker Blue-White DPX, LysoTracker Yellow HCK-123, LysoTracker Green DND-26, LysoTracker Red DND-99, LysoSensor Blue DND-167, LysoSensor Green DND-189, LysoSensor Green DND-153, LysoSensor Yellow / Blue DND-160, LysoSensor Yellow / Blue 10,000 MW dextran), Oregon Green (e.g., 488, 488-X, 500, 514); rhodamines (e.g., 110, 123, B, B 200, BB, BG, B extra, 5- carboxytetram ethyl rhodamine (5-TAMRA), 5 GLD, 6-Carboxyrhodamine 6G, Lissamine, Lissamine Rhodamine B, Phallicidin, Phalloidine, Red, Rhod-2, ROX (6-carboxy-X-rhodamine), 5-ROX (carboxy-X-rhodamine), Sulphorhodamine B can C, Sulphorhodamine G Extra, TAMRA (6-carboxytetramethyl-rhodamine), Tetramethylrhodamine (TRITC), Texas Red, Texas Red-X, among others as would be known to those of skill in the art.

[0109] Exemplary fluorescent labels include but are not limited to ABY, FAM, VIC, or CY5. In one exemplary multiplex dye scheme, the fluorescent labels include ABY, FAM, VIC, or CY5. In another exemplary multiplex dye scheme, the fluorescent labels include FAM, HEX (or JOE or VIC), ABY (or PET or NED or TAMRA) and Cy5 (or LIZ or Alexa Fluor 647) dyes. Exemplary dye schemes are shown in Table 1 .

[0110] Table 1. Exemplary Dyes and Quenchers for Multiplex Primers and Probes

[0111] Target Reporter Quencher

[0112] RVA ABY QSY™ 7

[0113] RVB FAM NFQ-MGB

[0114] RVC VIC NFQ-MGB

[0115] Xeno CY5 Eclipse™

[0116] Referring to Table 1 , the probes specific for amplicons generated using one or more primers for Rotavirus A are labeled with ABY; the probes specific for amplicons generated using one or more primers for Rotavirus B are labeled with FAM; the probes specific for amplicons generated using one or more primers for Rotavirus C are labeled with VIC; and the probes specific for amplicons generated using one or more primers for Xeno™ are labeled with Cy5.

[0117] Other detectable labels may be used in addition to or as an alternative to labelled probes. For example, primers can be labeled and used to both generate amplicons and to detect the presence (or concentration) of amplicons generated in the reaction, and such may be used in addition to or as an alternative to labeled probes described herein. As a further example, primers may be labeled and utilized as described in Nazarenko et al., Nucleic Acids Res. 30(9): e37 (2002), Hayashi et al., Nucleic Acids Res. 17(9): 3605 (1989), and / or Neilan et al., Nucleic Acids Res. 25(14): 2938-2939 (1997). Those of skill in the art are capable of utilizing the PCR processes (and associated probe and primer design techniques) described in Zhu et al., Biotechniques (4): 317-325 (2020).

[0118] In some embodiments, the primers and / or probes may further comprise a quencher. Suitable quenchers include but are not limited to NFQ-MGB (nonfluorescent quencher-minor groove binder), MGB, QSY (e.g., QSY7 and QSY21), Eclipse™, BHQ (Black Hole Quencher) and DFQ (Dark Fluorescent Quencher). In an exemplary embodiment, the quencher is NFQ-MGB. In a further exemplary embodiment, the probes specific for amplicons generated using one or more primers for Rotavirus A are labeled with QSY (e.g., QSY7), the probes specific for amplicons generated using one or more primers for Rotavirus B are labeled with NFQ-MGB, the probes specific for amplicons generated using one or more primers for Rotavirus C are labeled with NFQ- MGB, and the probes specific for amplicons generated using one or more primers for Xeno™ control are labeled with Eclipse™.

[0119] Detector probes may be digestion probes, or TaqMan™ probes, wherein, for example, a fluorophore is bound to the 5’ end of a probe and a quencher is bound to the 3’ end of the probe, such that digestion of the probe by the thermostable polymerase during amplification results in separation of the fluorophore from the quencher causing an increase in signal from the fluorophore. Detector probes may also include sulfonate derivatives of fluorescein dyes with SO3 instead of the carboxylate group, phosphoramidite forms of fluorescein, phosphoramidite forms of Cy5.

[0120] Any of these systems and detectable labels, as well as many others, may be used to detect amplified target nucleic acids. In some embodiments, intercalating labels can be used such as ethidium bromide, SYBR Green I, SYBR GreenER, and PicoGreen (all products of Applied Biosystems - a brand of Thermo Fisher Scientific), thereby allowing visualization in realtime, or end point, of an amplification product in the absence of a detector probe. In some embodiments, real-time visualization may include both an intercalating detector probe and a sequence-based detector probe. In some embodiments, the detector probe is at least partially quenched when not hybridized to a complementary sequence in the amplification reaction and is at least partially unquenched when hybridized to a complementary sequence in the amplification reaction. In some embodiments, probes may further comprise various modifications such as a minor groove binder (MGB) to further provide desirable thermodynamic characteristics. In some embodiments, the amplicon is labeled by incorporation of, or hybridization to labeled primer. In some embodiments, the amplicon is labeled by hybridization to a labeled probe. In some embodiments, the amplicon is labeled by binding of a DNA-binding dye. In some embodiments, the dye may be a single-strand DNA binding dye. In other embodiments, the dye may be a double-stranded DNA binding dye. In other embodiments, the amplicon is labeled via polymerization or incorporation of labeled nucleotides in a template-dependent (or templateindependent) polymerization reaction. This can be part of the amplifying step or alternatively the labeled nucleotide can be added after amplifying is completed. The labeled amplicon (or labeled derivative thereof) can be detected using any suitable method such as, for example, electrophoresis, hybridization-based detection (e.g., microarray, molecular beacons, and the like), chromatography, NMR, and the like.

[0121] In one exemplary embodiment, the labeled amplicon is detected using qPCR. In some embodiments, a plurality of different amplicons is formed, and optionally labeled, within a single reaction volume via a single amplification reaction. For example, a multiplex reaction (e.g., 4- plex) carried out in a single tube or reaction vessel (e.g., “single-tube” or” l-tube” or “single-vessel” reaction) can produce a plurality of amplicons that are labeled. In some embodiments, the plurality of amplicons can be differentially labeled. In some embodiments, each of the plurality of amplicons produced during amplification is labeled with a different label.

[0122] Assay Mixture

[0123] The terms “assay mixture” or “assay mix” or “assay composition,” as used herein, include mixture containing the primer-probe pairs described above that are used in PCR.

[0124] Another aspect provided herein is a method of detecting or quantifying a target nucleic acid molecule in a sample by polymerase chain reaction (PCR), such as by quantitative real-time polymerase chain reaction (qPCR). In one embodiment, the method includes: (i) contacting a sample comprising one or more target nucleic acid molecules with (a) at least one probe, such as those described herein, being sequence specific for the target nucleic acid molecule, where the at least one probe undergoes a detectable change in fluorescence upon amplification of the one or more target nucleic acid molecules; and with (b) at least one oligonucleotide primer pair; (ii) incubating the mixture of step (i) with a DNA polymerase under conditions sufficient to amplify one or more target nucleic acid molecules; and (iii) detecting the presence or absence or quantifying the amount of the amplified target nucleic acid molecules by measuring fluorescence of the probe. In some embodiments, the DNA polymerase comprises 5'-exonuclease activity. In some other embodiments, the DNA polymerase is a Thermus aquaticus (Taq) DNA polymerase. In some embodiments, the probe is a hydrolysis probe, such as a TaqMan™ probe.

[0125] Another aspect provided herein is a kit for PCR, such as quantitative real-time polymerase chain reaction (qPCR) and reverse transcription polymerase chain reaction (RT-PCR). In an exemplary embodiment, the kit includes the assay mixture, the process control, and the positive control each described above, as well as a multiplex master mix.

[0126] In an embodiment, the multiplex master mix is a RT-qPCR mix that provides for sensitive, reproducible detection of at least four different target pathogens in a single multiplex reaction. In an embodiment, the multiplex master mix may include an enzyme (for instance, DNA polymerase), a thermostable enzyme, enzyme cofactors, deoxynucleotide triphosphates (dNTPs) including dUTP, an enzyme inhibitor (for instance, RNase inhibitor), a dye and / or a buffer agent. In an exemplary embodiment, the multiplex master mix can be, for instance, VetMAX™ Rotavirus Master Mix or VetMAX™ Fast Multiplex Master Mix by Applied Biosystems - a brand of Thermo Fisher Scientific.

[0127] In an embodiment, the master mix may be concentrated. For instance, the master mix may be provided at a 2x concentration. In some embodiments, the master mix is prepared such that it requires more than a 2x concentration prior to use in PCR, e.g., 4x concentration.

[0128] In some embodiments, the kit also includes instructions for conducting the PCR, and one or more of the following: a buffering agent, deoxynucleotide triphosphates (dNTPs), an organic solvent, an enzyme, enzyme cofactors, and an enzyme inhibitor. In another embodiment, the kit for PCR comprises the described dye and / or quencher moiety, instructions for conjugating or labeling the dye and / or quencher moiety to a biomolecule, such as an oligonucleotide, instructions for conducting the PCR, and one or more of the following: a buffering agent, deoxynucleotide triphosphates (dNTPs), an organic solvent, an enzyme, enzyme cofactors, and an enzyme inhibitor.

[0129] In some embodiments, the systems, compositions, methods, and devices used for nucleic acid amplification comprise a “point-of-service” (PCS) system. In some embodiments, samples may be collected and / or analyzed at a “point-of-care” (POC) location. In some embodiments, analysis at a POC location typically does not require specialized equipment and has rapid and easy-to-read visual results. In some embodiments, analysis can be performed in the field and / or by a lay person not having specialized skills.

[0130] In certain embodiments, for example, the analysis of a small-volume clinical sample may be completed using a POS system in a short period of time (e.g., within hours or minutes). Optionally, a POS system is utilized at a location that is capable of providing a service (e g., testing, monitoring, treatment, diagnosis, guidance, sample collection, and other services) at or near the site or location of the porcine subject. A service may be a veterinary service, or it may be a non-veterinary service. In some situations, a POS system provides a service at a predetermined location, such as a commercial swine operation, a porcine farm, a veterinary clinic, a veterinarian’s office, an outdoor triage tent, a makeshift veterinary hospital, etc. A POS system can include one or more point of service devices, such as a portable virus / pathogen detector. In some embodiments, a POS system is a point of care system. In some embodiments, the POS system is suitable for use by non-specialized workers or personnel, such as farmers and commercial operators.

[0131] In certain embodiments, a POC system is utilized at a location at which veterinary-related care (e.g., treatment, testing, monitoring, diagnosis, counseling, etc.) is provided. A POC may be, e.g., at a commercial swine operation, a porcine farm, a veterinary clinic, a veterinarian’s office, an outdoor triage tent, a makeshift veterinary hospital, etc. A POC system is a system which may aid in, or may be used in, providing such veterinary-related care, and may be located at or near the site or location of the porcine or the porcine’s owner.

[0132] In certain embodiments, a POS system is configured to accept a sample obtained from a porcine subject at the associated POS location. In some embodiments, a POS system is further configured to analyze the sample at the POS location. In some embodiments, the sample is a small volume sample. In some embodiments, the sample is analyzed in a short period of time. In some embodiments, the short period of time is determined with respect to the time at which sample analysis began. In some embodiments, the short period of time is determined with respect to the time at which the sample was inserted into a device for the analysis of the sample. In some embodiments, the short period of time is determined with respect to the time at which the sample was obtained from the subject.

[0133] In some embodiments, a POS system or a POC system can include the amplificationbased methods, compositions and kits disclosed herein, including any of the described assays and / or assay panels. Such assays are contemplated for use with both thermal cycling amplification workflows and protocols, such as in PCR, as well as isothermal amplification workflows and protocols, such as in LAMP.

[0134] Notably, in some embodiments, the nucleic acid amplification protocol can be configured for rapid processing (e.g., in less than about 45 minutes) and high throughput, allowing for a minimally invasive method to quickly screen large numbers of porcine in a scalable way. This can be particularly useful to perform testing for large commercial operations or for epidemiological purposes. The disclosed embodiments can also beneficially provide a lower cost sample collection system and method using a low-cost collection device. This eliminates the requirements for swabs, buffers, virus transmission media (or other specialized transport medium), and the like. Overall, such embodiments allow for a less expensive assay that can be accomplished more quickly from sample collection through result generation.

[0135] It will be apparent to one of ordinary skill in the relevant art that suitable modifications and adaptations to the compositions, formulations, methods, processes, and applications described herein can be made without departing from the scope of any embodiments or aspects thereof. The compositions and methods provided are exemplary and are not intended to limit the scope of any of the specified embodiments. All of the various embodiments, aspects, and options disclosed herein can be combined in any variations or iterations. The scope of the compositions, formulations, methods, and processes described herein include all actual or potential combinations of embodiments, aspects, options, examples, and preferences herein described. The exemplary compositions and formulations described herein may omit any component, substitute any component disclosed herein, or include any component disclosed elsewhere herein. The ratios of the mass of any component of any of the compositions or formulations disclosed herein to the mass of any other component in the formulation or to the total mass of the other components in the formulation are hereby disclosed as if they were expressly disclosed. Should the meaning of any terms in any of the patents or publications incorporated by reference conflict with the meaning of the terms used in this disclosure, the meanings of the terms or phrases in this disclosure are controlling. Furthermore, the foregoing discussion discloses and describes merely exemplary embodiments. All patents and publications cited herein are incorporated by reference herein for the specific teachings thereof.

[0136] EXAMPLES

[0137] Example 1

[0138] Multiplex Detection of 4 Nucleic Acid Targets

[0139] Rotavirus A, B, C and Xeno™ primer pairs and probes were amplified with the rotavirus RNA control. The rotavirus RNA control contains a mixture of four RNA templates: an RNA template for RVA, an RNA template for RVB, an RNA template for RVC, and an RNA template for Xeno™ Internal Positive Control. The RNA control was heat denatured at 95°C for 3 minutes prior to amplification on an Applied Biosystems™ QuantStudio™ 50.1 mL Real-Time PCR system (the reaction was run in triplicate). The result from amplification is shown in FIG. 1. Each target is detected by a different fluorescent dye demonstrated by amplification curves. The results show all four RNA templates amplified in the same reaction mixture and visualized individually based on the fluorescent dye assigned to the probe.

[0140] Example 2

[0141] Primer / Probe Mix Design

[0142] Initial tests of an exemplary primer and probe mixture were conducted on extracted porcine samples. Rotavirus A, B, and C primer pairs and probes (SEQ ID NO: 1-6, 27-36, 37-42, 49-52, 53-55, 62-63, 64-79, 80-88, 109-114, 121-128, 129-130, 139, 164, and 172) were amplified with extracted nucleic acids using 4 different real-time PCR instruments: an Applied Biosystems™ QuantStudio™ 5 0.1mL Real-Time PCR system, an Applied Biosystems™ QuantStudio™ 5 0.2mL Real-Time PCR system, an Applied Biosystems™ 7500 Fast Real-Time PCR system with SDS version 1.5 software, and an Applied Biosystems™ 7500 Fast Real-Time PCR system with SDS version 2.3 software. The results from the four different instrument runs are shown in FIG. 2A (RVA), FIG. 2B (RVB), and FIG. 2C (RVC).

[0143] Example 3

[0144] Primer / Probe Mix Design

[0145] Exemplary primer and probe mixtures (PPMs) were tested individually and in combinations on extracted porcine samples. The extracted porcine samples were also tested with “reference” primer and probe mixtures described in the art (consisting of one reaction mixture for detecting RVA and RVC and another reaction mixture for detecting RVB). Rotavirus A, B, and C primer pairs and probes were amplified with extracted nucleic acids using the QuantStudio™ 5 0.1 mL Real-Time PCR System. Results from the reference served as a baseline. The results show improved detection with most of the newly designed Test Primer / Probe Mixtures 1 and 2 (Test PPM1 and Test PPM2) compared to the Reference Primer / Probe Mixtures, and even better detection when Test Primer / Probe Mixtures 1 and 2 were combined (Test PPM1 + PPM2) (Tables 2a-2c).

[0146] Table 2a. Results from testing 96 porcine fecal samples for detection of RVA

[0147] Description Reference Test PPMI Test PPM2 Test PPM1 + PPM2

[0148] Positive 57 69 68 70

[0149] Suspect 0 4 2 4

[0150] Negative 39 23 26 22

[0151] Table 2b. Results from testing 192 porcine fecal samples for detection of RVB Description Reference Test PPMI Test PPM2 Test PPM1 + PPM2

[0152] Positive 76 95 90 97

[0153] Suspect 12 12 10 13

[0154] Negative 104 85 92 82

[0155] Table 2c. Results from testing 48 porcine fecal samples for detection of RVC

[0156] Description Reference Test PPMI Test PPM2 Test PPM1 + PPM2

[0157] Positive 28 26 29 35

[0158] Suspect 12 12 10 13

[0159] Negative 8 10 9 0

[0160] Example 4

[0161] Primer / Probe Mixture Analytical Specificity The rotavirus primer / probe mixture of Example 1 showed no potential for amplifying nonrotavirus sequences within GeneBank, indicating high analytical specificity for rotavirus in situ. In vitro studies with the rotavirus primer / probe mix of Example 1 showed no false positive detection with a panel of porcine samples infected with non-rotavirus pathogens associated with porcine enteric disease, as depicted in Table 3. These results indicate the rotavirus primer / probe mix of Example 1 is highly specific for rotavirus. Univ16S is a universal real-time PCR assay that detects bacterial DNA based on the 16S gene sequence and was included in this example to demonstrate the presence of microbial nucleic acid in the sample.

[0162] Table 3. Exclusion panel consisting of non-rotavirus enteric pathogens

[0163] Agent positive for: Sample type Univ16S* RVA RVB RVC Xeno

[0164] Porcine sapovirus (Cq=15.7) Fecal swab 13.2 Und Und Und 29.2

[0165] Porcine sapovirus (Cq=32.3) Fecal swab 10.4 Und Und Und 29.9

[0166] Porcine sapovirus (Cq=13.4) Fecal swab 5.2 Und Und Und 30.9

[0167] Porcine sapovirus (Cq=20.3) Fecal swab 5.2 Und Und Und 31.2

[0168] Porcine sapovirus (Cq=28, 6) Fecal swab 4.5 Und Und Und 31.0

[0169] Lawsonia (Cq=16.2) Feces 11.2 Und Und Und 30.0

[0170] Lawsonia (Cq=16.2) Feces 5.0 Und Und Und 30.5

[0171] Lawsonia (Cq= 16.4) Feces 9.5 Und Und Und 30.1

[0172] Brachyspira hyodysenteriae Media 6.8 Und Und Und 29.7

[0173] Salmonella choleraesuis Media <3 Und Und Und 29.9

[0174] Salmonella typhimurium Media <3 Und Und Und 29.2

[0175] Clostridium perfringens Media 4.5 Und Und Und 29.3 Clostridium difficile Media 4.4 Und Und Und 29.5

[0176] PEDV positive Environmental 4.9 Und Und Und 30.1

[0177] PDCoV positive Environmental 5.0 Und Und Und 29.9

[0178] TGEV positive Environmental 5.0 Und Und Und 29.7

[0179] Example 5

[0180] Single Template Performance Analysis

[0181] The analytical sensitivity of the rotavirus primer / probe mix of Example 1 was determined using dilutions of the rotavirus RNA control samples. The concentration of each rotavirus RNA control was determined using digital PCR and each rotavirus control was tested separately. For this study, a Cq value below 40 was considered positive. At each concentration, 20 replicates were tested. The limit of detection (LCD) was defined as the lowest concentration to give a minimum of 19 out of 20 replicates testing positive (95% of replicates test positive). The analytical sensitivity for RVA, RVB, and RVC was 25 copies, 40 copies, and 10 copies, respectively, as shown in Table 4. For reference, UND denotes: undetected up to 40 cycles. Cq Range is the Cq range for samples with Cq values. Cq Avg is the Cq average for samples with Cq values.

[0182] Table 4. Level of Detection for RVA, RVB, and RVC

[0183] Species copies / rxn Positive Cq Range Cq Avg LOD

[0184] 10 12 37.6 - 39.7 38.4

[0185] RVA 25 20 35.4 - 37.7 36.3 25

[0186] 50 20 34.1 - 35.8 34.6

[0187] 100 19 32.7 - 33.8 33.1

[0188] 5 0 UND UND

[0189] RVB 20 18 38.0 - 39.7 38.9 40

[0190] 40 20 36.3 - 38.4 37.3

[0191] 80 20 36.1 - 37.0 36.4

[0192] 5 13 37.1 - 40.0 38.3

[0193] RVC 10 20 35.7 - 38.1 36.7 10

[0194] 20 20 35.2 - 36.8 35.7

[0195] 40 20 34.2 - 35.3 24.7

[0196] Example 6

[0197] Rotavirus RNA Control

[0198] Double-stranded RNA is not an efficient template for MMLV-derived RNA-dependent RNA polymerase (RdRp), the enzyme most used for in vitro reverse transcription prior to real-time PCR. The rotavirus RNA control comprises double-stranded RNA for RVA and RVC and singlestranded RNA for RVB and Xeno™. The rotavirus RNA control was tested with and without heat- denaturation (FIGs. 3A and 3B). FIGs. 3A-3B shows amplification plots for the disclosed controls with heat denaturation (A), and without heat-denaturation (B). Amplification of RVB and Xeno™ are equivalent with and without heat-denaturation, but RVC shows weak detection and RVA shows no detection when the control is not heat denatured. The amplification plots show the threshold settings for the 4 amplified targets. In this way, the RNA controls served four purposes: (1) to confirm samples were heat-denatured prior to being added to the PCR reaction mix, (2) to confirm the PCR reaction was prepared correctly, (3) to demonstrate the PCR enzymes and reagents were active, and (4) to set the threshold for data analysis using the procedure known as control-based threshold (CBT).

[0199] Example 7

[0200] Sample Pooling

[0201] The rotavirus primer / probe mix of Example 1 was used on sample pools containing five samples with one rotavirus-positive porcine sample mixed with 4 negative porcine samples in equal volumes prior to extraction. Each rotavirus-positive sample was tested separately and in a pool of 5 samples (FIG. 4). The results show amplification of rotavirus-positive sample in both single and pooled samples; however, the pooled sample gives a higher Cq value due to dilution. A rotavirus-positive sample in a pool of 5 shows an average Cq increase of 3.0 compared to the same sample run individually. Pooling samples may result in no amplification in pools containing a single rotavirus-positive sample with an individual Cq > 35.

[0202] Example 8

[0203] Co-Infected Samples

[0204] It is common for animal derived samples to contain multiple rotavirus isolates, including different species of rotavirus. A potential concern for multiplex real-time PCR is the possibility that targets present at high concentrations will deplete shared PCR substrates and obscure the amplification of targets present at much lower levels. To evaluate competitive inhibition of the primer / probe mix of Example 1, each rotavirus control template was diluted over 7 logs and each log dilution was tested in the presence and absence of the other two rotavirus species control templates present at 107copies / rxn (FIGs. 5A-5C). FIGs. 5A, 5B, and 5C depict the results for Rotavirus A, Rotavirus B, and Rotavirus C, respectively. The results from the competitive inhibition study shows only a small impact on the Cq value of each rotavirus species across most dilutions in the presence of high concentrations of the other two rotavirus species. Amplification was observed for the minor rotavirus species down to 100 copies even when the major rotavirus species were presence at 107copies. Amplification of RVA and RVC was not observed at 10 copies when the other two rotaviruses were present at 107copies.

[0205] Example 9

[0206] Exemplary Test Sample Preparation

[0207] A porcine sample is obtained. Samples typically include feces, fecal swabs, rectal swabs, feedback, fluid, homogenate, oral fluid, processing fluid, tissue composite, intestinal tissue, environmental samples, and other. The sample is processed to isolate genomic DNA. Alternatively frozen samples can be stored at -80°C on dry ice until processed to extract nucleic acid for RT-qPCR. Frozen samples are thawed on ice.

[0208] Control Sample Preparation

[0209] The Xeno™ Internal Positive Control RNA by Applied Biosystems™ is used to verify the efficacy of the sample preparation and the absence of inhibitors in the real-time PCR reaction. The Xeno™ Internal Positive Control RNA is added to MagMAX™ CORE Lysis Solution prior to combining the Lysis Solution with either the porcine test sample or the negative extraction control. Xeno™ Internal Positive Control RNA is available at 10,000 copies / uL and 2 uL is added per extraction reaction.

[0210] Real-time PCR Reaction Preparation

[0211] Genomic DNA samples are thawed or placed on ice. All reagents are gently vortexed, briefly centrifuged to collect the liquid at the bottom of the container and kept on ice until use. Total nucleic acid was extracted from porcine samples using the MagMAX CORE Nucleic Acid Purification Kit automated on the KingFisher Flex Purification System following the Complex Workflow as described in the MagMAX CORE User Guide. Xeno™ Control RNA was added to a lysis solution to confirm successful extraction and to evaluate presence of PCR inhibitors that might have been co-extracted with nucleic acid. The extracted nucleic acid was used immediately in RT-qPCR testing and stored at -20°C for up to 12 months for later use, if needed.

[0212] All extracted nucleic acid samples and controls were heat-denatured to allow for efficient RNA transcription prior to PCR amplification. The samples and control were heat-denatured at 95°C for 3 minutes, allowed to cool on ice, and briefly centrifuged prior to combining with the RT- qPCR reaction mix.

[0213] A pre-mix was prepared by combining the master mix and primer / probe mix on ice (Table 5). The volume of each component in the pre-mix was multiplied by the number of samples and controls tested, with an overage included (approximately -10%) to account for pipetting imprecision. Three controls were included in each RT-qPCR run; (1) a positive control, (2) a notemplate control (NTC) consisting of water, and (3) a negative extraction control (NEC) prepared by the extraction of 1XPBS (NEC contained Xeno™ that was added to the lysis buffer).

[0214] Table 5. PCR Multiplex Master Mix and Primer / Probes

[0215] Component Volume per sample or control

[0216] Multiplex Master Mix 10.00 pL

[0217] Multiplex Primer / Probe Panel 2.00 pL

[0218] Total Volume 12.00 pL

[0219] Twelve microliters of the RT-qPCR pre-mix was aliquoted to wells of a 96-well PCR plate on ice. To each of these aliquots was added eight microliters of test sample or control, as listed in Table 7.

[0220] Table 7. Samples and Controls Added to RT-qPCR pre-mix

[0221] Reaction type Component Vol. per rxn

[0222] Test sample Denatured Sample RNA 8 uL

[0223] NTC Nuclease-free Water 8 uL

[0224] NEC Mock-purified sample 8 uL

[0225] RVA / RVB / RVC / Xeno™ RNA control Denatured RNA control 8 uL

[0226] The 96-well PCR plate was sealed with optical adhesive film, vortexed for 5-15 seconds, and centrifuged in a plate centrifuge at 2000 rpm for 1 minute to collect solution to the bottom of the plate.

[0227] Samples were run on the 7500 Fast Real-Time PCR System, the QuantStudio 5 Real- Time PCR System, 96-well, 0.1 mL, and the QuantStudio 5 Real-Time PCR System, 96-well, 0.2 mL using conditions listed in Table 8. The real-time PCR instruments were run under two different Run Modes, Fast and Standard as described in Tables 9 and 10, respectively.

[0228] Table 8. Instrument Setup Utilized

[0229] Reaction type Component

[0230] Experiment Type Standard Curve

[0231] Reaction Volume 20 uL

[0232] Passive Reference ROX (included in Master Mix)

[0233] Table 9. Fast run mode thermal cycling conditions utilized

[0234] Stage Description Step Reps. Temp. Reverse transcription RNA cDNA 1 1 50°C

[0235] Enzyme activation Activate Taq Pol 2 1 95°C

[0236] PCR Template denaturation 3a 40 95°C

[0237] Template 3b 60°C annealing / extension

[0238] Table 10. Standard run mode thermal cycling conditions utilized

[0239] Stage Description Step Reps. Temp.

[0240] Reverse transcription RNA cDNA 1 1 48°C

[0241] Enzyme activation Activate Taq Pol 2 1 95°C

[0242] PCR Template denaturation 3a 40 95°C

[0243] Template 3b 60°C annealing / extension

Claims

CLAIMSWhat is claimed:

1. A method for simultaneously determining the presence or absence of Rotavirus A, Rotavirus B, and Rotavirus C in a sample, the method comprising the steps of:(a) creating a reaction mixture containing the sample and a primer mixture comprising: at least one primer that specifically amplifies a portion of Rotavirus A genome; at least one primer that specifically amplifies a portion of Rotavirus B genome; and at least one primer that specifically amplifies a portion of Rotavirus C genome; and(b) subjecting the reaction mixture to reaction conditions suitable to amplify targeted nucleic acids, thereby generating at least one amplicon when the targeted nucleic acids are present in the sample; wherein the presence or absence of at least one amplicon in the sample indicates the presence or absence of Rotavirus A, Rotavirus B, and Rotavirus C in the sample.

2. The method of claim 1 , wherein: the at least one primer specific for Rotavirus A is selected from SEQ ID NO: 1-52; the at least one primer specific for Rotavirus B is selected from SEQ ID NO: 64-120; and the at least one primer specific for Rotavirus C is selected from SEQ ID NO: 139-171.

3. The method of claim 1 or 2, wherein the generating of the at least one amplicon comprises performing PCR.

4. The method of any one of claims 1-3, wherein the at least one amplicon is one selected from: an amplicon specific for Rotavirus A produced using at least one primer selected from SEQ ID NO: 1-6 and 37-42, and a sequence selected from SEQ ID NO: 53-55; an amplicon specific for Rotavirus B produced using at least one primer selected from SEQ ID NO: 64-79 and 109-114, and a sequence selected from SEQ ID NO: 121- 128; and / or an amplicon specific for Rotavirus C produced using at least one primer selected from SEQ ID NO: 139 and 164, and a sequence selected from SEQ ID NO: 172.

5. The method any one of claims 1—4, wherein the reaction mixture further comprises probes specific for the at least one amplicon.

6. The method of any one of claims 1-5, wherein the reaction mixture further comprises probes specific for the at least one amplicon and suitable for use with the primer mixture, the probes comprising: a probe specific for Rotavirus A having a sequence selected from SEQ ID NO: 53-55 and 62-63; a probe specific for Rotavirus B having a sequence selected from SEQ ID NO: 121-130; or a probe specific for Rotavirus C having a sequence of SEQ ID NO: 172.

7. The method of any one of claims 1-6, wherein the reaction mixture further contains a control sample and at least one control primer; and, optionally, a control probe, that specifically amplifies a target nucleic acid of the control sample.

8. The method of claim 7, wherein the at least one control primer is selected from SEQ ID NO: 180-181; and the control probe comprises a sequence of SEQ ID NO: 182.

9. The method of any one of claims 1-8, wherein the probe comprises a fluorescent reporter.

10. The method of claim 9, wherein the probe comprises a quencher.

11. The method of claim 9 or 10, wherein the probe is labeled at or near the 5'-end with a dye selected from ABY, FAM, VIC, or CY5.

12. The method of any one of claims 9-11 , wherein the probe is labeled at or near the 3' end with a quencher selected from NFQ-MGB, QSY7, QSY21 , BHQ, Eclipse, or DFQ.

13. The method of claim 11 or 12, wherein: the probe specific for amplicons generated using one or more primers for Rotavirus A is labeled with ABY; the probe specific for amplicons generated using one or more primers for Rotavirus B is labeled with FAM; andthe probe specific for amplicons generated using one or more primers for Rotavirus C is labeled with VIC.

14. The method of any one of claims 9-13, wherein the probe specific for amplicons generated from a control sample using one or more control primers labeled with CY5.

15. A composition for simultaneously determining the presence or absence of Rotavirus A, Rotavirus B, and Rotavirus C in a sample, comprising: at least one primer that specifically amplifies a portion of Rotavirus A genome; at least one primer that specifically amplifies a portion of Rotavirus B genome; and at least one primer that specifically amplifies a portion of Rotavirus C genome.

16. The composition of claim 15, wherein: the at least one primer specific for Rotavirus A is selected from SEQ ID NO: 1-52; the at least one primer specific for Rotavirus B is selected from SEQ ID NO: 64-120; and the at least one primer specific for Rotavirus A is selected from SEQ ID NO: 139-171.

17. The composition of claim 15 or 16, further comprising a probe specific for amplicons produced using the at least one primer selected from Rotavirus A, Rotavirus B, and Rotavirus C.

18. The composition of any one of claims 15-17, wherein the probe suitable for use with specific primers are selected from: a probe specific for Rotavirus A having a sequence selected from SEQ ID NO: 53-63; a probe specific for Rotavirus B having a sequence selected from SEQ ID NO: 121-138; or a probe specific for Rotavirus C having a sequence selected from SEQ ID NO: 172-179.

19. The composition of any one of claims 15-18, further comprising at least one control primer; and, optionally, a control probe, that specifically amplifies the target nucleic acid of a control sample.

20. The composition of claim 19, wherein the at least one control primer is selected from SEQ ID NO: 180-181 ; and the control probe comprises a sequence of SEQ ID NO: 182.

21. The composition of any one of claims 15-20, wherein the probe contains a fluorescent reporter.

22. The composition of claim 21 , wherein the probe contains a quencher.

23. The composition of any one of claims 21 or 22, wherein the probe is labeled at or near the 5' end with a dye selected from ABY, FAM, VIC, or CY5.

24. The composition of any one of claims 22-23, wherein the probe is labeled at or near the 3' end with a quencher selected from NFQ-MGB, QSY7, QSY21 , BHQ, Eclipse, or DFQ.

25. The composition of any one of claims 17-24, wherein: the probe specific for amplicons generated using one or more primers for Rotavirus A is labeled with ABY; the probe specific for amplicons generated using one or more primers for Rotavirus B is labeled with FAM; and the probe specific for amplicons generated using one or more primers for Rotavirus C is labeled with VIC.

26. The composition of any one of claims 19-25, wherein the probe specific for amplicons generated using control primers are labeled with CY5.

27. A kit for simultaneously determining the presence or absence of Rotavirus A, Rotavirus B, and Rotavirus C in a sample, comprising the compositions of any one of claims 15-18.

28. The kit of claim 27, further comprising the compositions of any one of claims 19-26.

29. The kit of claim 27 or 28, wherein the primers are selected from SEQ ID NO: 1-6 and 37- 42; and the probe is selected from SEQ ID NO: 53-55.

30. The kit of claim 27 or 28, wherein the primers are selected from SEQ ID NO: 7-22 and 43; and the probe is selected from SEQ ID NO: 56-58.

31. The kit of claim 27 or 28, wherein the primers are selected from SEQ ID NO: 24 and 45; and the probe is selected from SEQ ID NO: 60.

32. The kit of claim 27 or 28, wherein the primers are selected from SEQ ID NO: 25-26 and 46-48; and the probe comprises a sequence of SEQ ID NO: 61.

33. The kit of claim 27 or 28, wherein the primers are selected from SEQ ID NO: 27-36 and 49-52; and the probe is selected from SEQ ID NO: 62-63.

34. The kit of claim 27 or 28, wherein the primers are selected from SEQ ID NO: 64-79 and 109-114; and the probe is selected from SEQ ID NO: 121-128.

35. The kit of claim 27 or 28, wherein the primers are selected from SEQ ID NO: 80-88; and the probes are selected from SEQ ID NO: 129-130.

36. The kit of claim 27 or 28, wherein the primers are selected from SEQ ID NO: 89-104 and 115-116; and the probe is selected from SEQ ID NO: 131-132.

37. The kit of claim 27 or 28, wherein the primers are selected from SEQ ID NO: 103 and 117; and the probe is selected from SEQ ID NO: 133-136.

38. The kit of claim 27 or 28, wherein the primers are selected from SEQ ID NO: 106-107 and 118; and the probe comprises a sequence of SEQ ID NO: 137.

39. The kit of claim 27 or 28, wherein the primers are selected from SEQ I D NO: 108 and 119- 120; and the probe comprises a sequence of SEQ ID NO: 138.

40. The kit of claim 27 or 28, wherein the primers are selected from SEQ I D NO: 139 and 164; and the probe comprises a sequence of SEQ ID NO: 172.

41. The kit of claim 27 or 28, wherein the primers are selected from SEQ ID NO: 140-142 and 165-166; and the probes are selected from SEQ ID NO: 173.

42. The kit of claim 27 or 28, wherein the primers are selected from SEQ ID NO: 143-146 and 167-168; and the probes are selected from SEQ ID NO: 174-177.

43. The kit of claim 27 or 28, wherein the primers are selected from SEQ ID NO: 147-162 and 169-170; and the probes are selected from SEQ ID NO: 178.

44. The kit of claim 27 or 28, wherein the primers are selected from SEQ I D NO: 163 and 171 ; and the probes are selected from SEQ ID NO: 179.

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