Methods and compositions for amplifying and / or detecting rotavirus nucleic acids

Abstract

Disclosed are nucleic acid oligomers, including amplification oligomers and detection probes, for detection of rotavirus target nucleic acid. Also disclosed are methods of specific nucleic acid amplification and detection using the disclosed oligomers, as well as corresponding formulations, reaction mixtures, and kits. Methods of synthesizing the nucleic acid oligomers are also disclosed.

Description

METHODS AND COMPOSITIONS FOR AMPLIFYING AND / OR DETECTINGROTAVIRUS NUCLEIC ACIDSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 688,793, filed August 29, 2024, which is incorporated by reference herein in its entirety.REFERENCE TO SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML Copy, created on June 13, 2025, is named“GPR_9910PC_Seq_Listing_ST26” and is 91,577 bytes in size.BACKGROUND

[0003] Acute diarrhea from gastrointestinal (GI) infections is the leading cause of outpatient visits, hospitalizations, and loss of quality of life, with an estimated global impact of 500 million illnesses and 230,000 deaths annually. Most GI infections from bacteria, viruses, and parasites present similar symptoms, but successful treatment is dependent on accurate pathogen identification. For identification of parasites, microscopic testing is often used, but is laborious and results in inconclusive or inaccurate diagnoses. Clinicians now rely on rapid and accurate molecular diagnostics to correctly identify the causative organism, which leads to optimal infection control and appropriate treatment.

[0004] Rotavirus is the leading cause of acute gastroenteritis in the world. Studies estimate that approximately 200,000 people die annually from infection. Although the infection rate has decreased with the advent of vaccines, infections are still common throughout the world.

[0005] There is a need to efficiently and sensitively detect the presence of rotavirus in samples, including biological specimens to provide diagnostic and prognostic information to physicians treating patients suffering from, or suspected of suffering from, viral gastroenteritis or related disorders.SUMMARY

[0006] In some aspects, the present disclosure provides a composition or kit for determining the presence or absence of rotavirus in a sample. The composition or kit generally includes a set of oligonucleotides comprising an amplification oligomer set capable of amplifying a target region of a rotavirus target nucleic acid, wherein the amplification oligomer set comprises first and second amplification oligomers respectively comprising first and second target-hybridizing sequences, wherein (a) the first targethybridizing sequence is selected from (i) a sequence that is from 19 to 22 contiguous nucleotides contained in the sequence of SEQ ID NO:43 and that includes at least the sequence of SEQ ID NO:42, including from 0 to 18 nucleotide analogs, (ii) a sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO:20 or SEQ ID NO:30, including from 0 to 18 nucleotide analogs, and (iii) a sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:26, or SEQ ID NO:27, including from 0 to 18 nucleotide analogs; and (b) the second target-hybridizing sequence is selected from (i) a sequence that is from 18 to 20 contiguous nucleotides contained in the sequence of SEQ ID NO:53 and that includes at least the sequence of SEQ ID NO:52, including from 0 to 18 nucleotide analogs, (ii) a sequence that is from 18 to 23 contiguous nucleotides contained in the sequence of SEQ ID NO: 55 and that includes at least the sequence of SEQ ID NO: 54, including from 0 to 18 nucleotide analogs, and (iii) a sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO:29, including from 0 to 18 nucleotide analogs.

[0007] In some embodiments, the first target-hybridizing sequence is selected from (i) a sequence that is from 19 to 22 contiguous nucleotides contained in the sequence of SEQ ID NO:43 and that includes at least the sequence of SEQ ID NO:42, including from 0 to 18 nucleotide analogs, and (ii) a sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO:20 or SEQ ID NO:30, including from 0 to 18 nucleotide analogs. In some such embodiments, the first target-hybridizing sequence is SEQ ID NO:20 or SEQ ID NO:30, including from 0 to 18 nucleotide analogs.

[0008] In other embodiments of a composition or kit, the first target-hybridizing sequence is a sequence having at least 90% sequence identity with the nucleotide sequenceshown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:26, or SEQ ID NO:27, including from 0 to 18 nucleotide analogs. In some such embodiments, the first targethybridizing sequence is SEQ ID NO:46, including from 0 to 18 nucleotide analogs (e.g., a first target-hybridizing sequence of SEQ ID NO:7, SEQ ID NO:26, or SEQ ID NO:27, including from 0 to 18 nucleotide analogs). In other such embodiments, the first targethybridizing sequence is SEQ ID NO:8 or SEQ ID NO:9, including from 0 to 18 nucleotide analogs.

[0009] In some embodiments of a composition or kit as above, the second targethybridizing sequence is selected from (i) a sequence that is from 18 to 20 contiguous nucleotides contained in the sequence of SEQ ID NO:53 and that includes at least the sequence of SEQ ID NO: 52, including from 0 to 18 nucleotide analogs, and (ii) a sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO:29, including from 0 to 18 nucleotide analogs; in some such variations, the second target-hybridizing sequence is SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO:29, including from 0 to 18 nucleotide analogs. In other embodiments, the second target-hybridizing sequence is selected from (i) a sequence that is from 18 to 23 contiguous nucleotides contained in the sequence of SEQ ID NO:55 and that includes at least the sequence of SEQ ID NO: 54, including from 0 to 18 nucleotide analogs, and (ii) a sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 14, SEQ ID NO:15, SEQ ID NO: 16, or SEQ ID NO:29, including from 0 to 18 nucleotide analogs; in some such variations, the second targethybridizing sequence is SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO:29, including from 0 to 18 nucleotide analogs.

[0010] In certain embodiments of a composition or kit as above, the set of oligonucleotides further includes at least one detection probe comprising a targethybridizing sequence configured to specifically hybridize to a rotavirus target sequence amplifiable by the first and second amplification oligomers. In some such embodiments, the at least one detection probe target-hybridizing sequence is selected from (i) a sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 10 or SEQ ID NO: 11; (ii) a sequence that is from 29 to 30 contiguous nucleotides contained in the sequence of SEQ ID NO:49 and that includes at least the sequence of SEQ ID NO:48, including from 0 to 18 nucleotide analogs; and (iii) a sequence having at least90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 19 or SEQ ID NO:28, including from 0 to 18 nucleotide analogs. In other, non-mutually exclusive variations, the at least one detection probe further includes (i) a detectable label, and / or (ii) a blocking moiety at or near the 3’ terminus. In certain variations wherein the at least one detection probe includes the detectable label, the detectable label is a fluorescent or chemiluminescent label. In some variations, the detectable label is a fluorescent label and the at least one detection probe further includes a non-fluorescent quencher.

[0011] In some embodiments, the set of oligonucleotides comprises at least first and second detection probes, wherein each of the first and second detection probes comprises a target-hybridizing sequence configured to specifically hybridize to a rotavirus target sequence amplifiable by the first and second amplification oligomers. In some such variations, each of the first and second detection probe target-hybridizing sequences is independently selected from (i) a sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 10 or SEQ ID NO: 11; (ii) a sequence that is from 29 to 30 contiguous nucleotides contained in the sequence of SEQ ID NO:49 and that includes at least the sequence of SEQ ID NO:48, including from 0 to 18 nucleotide analogs; and (iii) a sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 19 or SEQ ID NO:28, including from 0 to 18 nucleotide analogs. In other, non-mutually exclusive variations, each of the first and second detection probes further includes (i) a detectable label, and / or (ii) a blocking moiety at or near the 3’ terminus. In certain variations wherein each of the first and second detection probes includes the detectable label, the detectable label is a fluorescent or chemiluminescent label. In some variations, the detectable label is a fluorescent label and each of the first and second detection probes further includes a non-fluorescent quencher.

[0012] In another aspect, the present disclosure provides an oligonucleotide for determining the presence or absence of rotavirus in a sample, wherein said oligonucleotide comprises a nucleotide sequence selected from SEQ ID NOs:7-16 and 19-32, including from 0 to 18 nucleotide analogs. In some embodiments, the 3’ end of the oligonucleotide is attached to a solid support.

[0013] In other aspects, the present disclosure provides a reaction mixture for determining the presence or absence of rotavirus in a sample. In some embodiments, the reaction mixture includes a set of oligonucleotides as specified above. In other, non-mutually exclusive embodiments, the reaction mixture includes an oligonucleotide comprising a nucleotide sequence selected from SEQ ID NOs:7-16 and 19-32, including from 0 to 18 nucleotide analogs

[0014] In another aspect, the present disclosure provides a method for determining the presence or absence of rotavirus in a sample. The method generally includes the following steps: (1) contacting a sample, the sample suspected of containing rotavirus, with an amplification oligomer set capable of amplifying a target region of rotavirus target nucleic acid, wherein the amplification oligomer set includes first and second amplification oligomers respectively comprising first and second target-hybridizing sequences, wherein (a) the first target-hybridizing sequence is selected from (i) a sequence that is from 19 to 22 contiguous nucleotides contained in the sequence of SEQ ID NO:43 and that includes at least the sequence of SEQ ID NO:42, including from 0 to 18 nucleotide analogs, (ii) a sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO:20 or SEQ ID NO:30, including from 0 to 18 nucleotide analogs, and a sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:26, or SEQ ID NO:27, including from 0 to 18 nucleotide analogs; and (b) the second target-hybridizing sequence is selected from (i) a sequence that is from 18 to 20 contiguous nucleotides contained in the sequence of SEQ ID NO:53 and that includes at least the sequence of SEQ ID NO:52, including from 0 to 18 nucleotide analogs, (ii) a sequence that is from 18 to 23 contiguous nucleotides contained in the sequence of SEQ ID NO: 55 and that includes at least the sequence of SEQ ID NO: 54, including from 0 to 18 nucleotide analogs, and (iii) a sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO:29, including from 0 to 18 nucleotide analogs; (2) performing an in vitro nucleic acid amplification reaction, wherein any rotavirus target nucleic acid present in the sample is used as a template for generating an amplification product corresponding to the rotavirus target region; and (3) detecting the presence or absence of the amplification product, thereby determining the presence or absence of rotavirus in the sample. In some embodiments, the detecting step (3) includes contacting the in vitro nucleic acid amplification reaction with at least one detection probe comprising a target-hybridizing sequence configured to specifically hybridize to the amplification product.

[0015] In yet another aspect, the present disclosure provides a method for synthesizing an oligonucleotide, wherein the oligonucleotide includes a nucleotide sequence selected from SEQ ID NOs:7-16 and 19-32, including from 0 to 18 nucleotide analogs. The method generally includes the following steps: (a) obtaining a solid support comprising at least one nucleobase residue, wherein the at least one nucleobase residue is covalently bound at a 3’ position to the solid support; (b) coupling a 5’ position of the nucleobase residue furthest from the solid support to a 3’ position of another nucleobase residue; (c) repeating step (b) at least 16 additional times, thereby generating at least 18 contiguous nucleobase residues coupled to the solid support; and (d) cleaving the at least 18 contiguous nucleobase residues generated in step (c), thereby obtaining the oligonucleotide.

[0016] Representative embodiments of these aspects are further set forth below. Embodiments

[0017] Embodiment 1. A composition or kit for determining the presence or absence of rotavirus in a sample, said composition or kit comprising a set of oligonucleotides comprising: an amplification oligomer set capable of amplifying a target region of a rotavirus target nucleic acid, wherein the amplification oligomer set comprises first and second amplification oligomers respectively comprising first and second targethybridizing sequences, wherein(a) the first target-hybridizing sequence is selected from the group consisting of a sequence that is from 19 to 22 contiguous nucleotides contained in the sequence of SEQ ID NO:43 and that includes at least the sequence of SEQ ID NO:42, including from 0 to 18 nucleotide analogs, a sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO:20 or SEQ ID NO:30, including from 0 to 18 nucleotide analogs, and a sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO:26, or SEQ ID NO:27, including from 0 to 18 nucleotide analogs; and(b) the second target-hybridizing sequence is selected from the group consisting ofa sequence that is from 18 to 20 contiguous nucleotides contained in the sequence of SEQ ID NO:53 and that includes at least the sequence of SEQ ID NO: 52, including from 0 to 18 nucleotide analogs, a sequence that is from 18 to 23 contiguous nucleotides contained in the sequence of SEQ ID NO: 55 and that includes at least the sequence of SEQ ID NO: 54, including from 0 to 18 nucleotide analogs, and a sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO:29, including from 0 to 18 nucleotide analogs.

[0018] Embodiment 2. The composition or kit of Embodiment 1, wherein the first target-hybridizing sequence is selected from the group consisting of a sequence that is from 19 to 22 contiguous nucleotides contained in the sequence of SEQ ID NO:43 and that includes at least the sequence of SEQ ID NO:42, including from 0 to 18 nucleotide analogs, and a sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO:20 or SEQ ID NO:30, including from 0 to 18 nucleotide analogs.

[0019] Embodiment s. The composition or kit of Embodiment 2, wherein the first target-hybridizing sequence is SEQ ID NO:20 or SEQ ID NO:30, including from 0 to 18 nucleotide analogs.

[0020] Embodiment 4. The composition or kit of Embodiment 1, wherein the first target-hybridizing sequence is a sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO:26, or SEQ ID NO:27, including from 0 to 18 nucleotide analogs.

[0021] Embodiment 5. The composition or kit of Embodiment 4, wherein the first target-hybridizing sequence is SEQ ID NO:46, including from 0 to 18 nucleotide analogs.

[0022] Embodiment 6. The composition or kit of Embodiment 5, wherein the first target-hybridizing sequence is SEQ ID NO:7, SEQ ID NO:26, or SEQ ID NO:27, including from 0 to 18 nucleotide analogs.

[0023] Embodiment 7. The composition or kit of Embodiment 4, wherein the first target-hybridizing sequence is SEQ ID NO:8 or SEQ ID NO:9, including from 0 to 18 nucleotide analogs.

[0024] Embodiment 8. The composition or kit of Embodiment 4, wherein the first target-hybridizing sequence is SEQ ID NO:56, SEQ ID NO:57, or SEQ ID NO:58.

[0025] Embodiment 9. The composition or kit of any one of Embodiments 1 to 8, wherein the second target-hybridizing sequence is selected from the group consisting of a sequence that is from 18 to 20 contiguous nucleotides contained in the sequence of SEQ ID NO:53 and that includes at least the sequence of SEQ ID NO: 52, including from 0 to 18 nucleotide analogs, and a sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO:29, including from 0 to 18 nucleotide analogs.

[0026] Embodiment 10. The composition or kit of Embodiment 9, wherein the second target-hybridizing sequence is SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO:29, including from 0 to 18 nucleotide analogs.

[0027] Embodiment 11. The composition or kit of Embodiment 10, wherein the second target-hybridizing sequence is SEQ ID NO:61 or SEQ ID NO:62.

[0028] Embodiment 12. The composition or kit of any one of Embodiments 1 to 8, wherein the second target-hybridizing sequence is selected from the group consisting of a sequence that is from 18 to 23 contiguous nucleotides contained in the sequence of SEQ ID NO: 55 and that includes at least the sequence of SEQ ID NO: 54, including from 0 to 18 nucleotide analogs, and a sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO:29, including from 0 to 18 nucleotide analogs.

[0029] Embodiment 13. The composition or kit of Embodiment 12, wherein the second target-hybridizing sequence is SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO:29, including from 0 to 18 nucleotide analogs.

[0030] Embodiment 14. The composition or kit of Embodiment 13, wherein the second target-hybridizing sequence is SEQ ID NO:63, SEQ ID NO:64, or SEQ ID NO:65.

[0031] Embodiment 15. The composition or kit of any one of Embodiments 1 to 14, wherein the set of oligonucleotides further comprises at least one detection probe comprising a target-hybridizing sequence configured to specifically hybridize to a rotavirus target sequence amplifiable by the first and second amplification oligomers.

[0032] Embodiment 16. The composition or kit of Embodiment 15, wherein the at least one detection probe target-hybridizing sequence is selected from the group consisting of a sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 10 or SEQ ID NO: 11; a sequence that is from 29 to 30 contiguous nucleotides contained in the sequence of SEQ ID NO:49 and that includes at least the sequence of SEQ ID NO:48, including from 0 to 18 nucleotide analogs; and a sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 19 or SEQ ID NO:28, including from 0 to 18 nucleotide analogs.

[0033] Embodiment 17. The composition or kit of Embodiment 16, wherein the at least one detection probe target-hybridizing sequence is SEQ ID NO:61 or SEQ ID NO:62.

[0034] Embodiment 18. The composition or kit of any one of Embodiments 12 to 17, wherein the at least one detection probe further comprises(i) a detectable label, and / or(ii) a blocking moiety at or near the 3’ terminus.

[0035] Embodiment 19. The composition or kit of Embodiment 18, wherein the at least one detection probe comprises the detectable label.

[0036] Embodiment 20. The composition or kit of Embodiment 19, wherein the detectable label is a fluorescent or chemiluminescent label.

[0037] Embodiment 21. The composition or kit of Embodiment 19, wherein the detectable label is a fluorescent label and the at least one detection probe further comprises a non-fluorescent quencher.

[0038] Embodiment 22. The composition or kit of any one of Embodiments 1 to 14, wherein the set of oligonucleotides further comprises at least first and second detection probes, wherein each of the first and second detection probes comprises a targethybridizing sequence configured to specifically hybridize to a rotavirus target sequence amplifiable by the first and second amplification oligomers.

[0039] Embodiment 23. The composition or kit of Embodiment 22, wherein each of the first and second detection probe target-hybridizing sequences is independently selected from the group consisting of a sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 10 or SEQ ID NO: 11; a sequence that is from 29 to 30 contiguous nucleotides contained in the sequence of SEQ ID NO:49 and that includes at least the sequence of SEQ ID NO:48, including from 0 to 18 nucleotide analogs; and a sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 19 or SEQ ID NO:28, including from 0 to 18 nucleotide analogs.

[0040] Embodiment 24. The composition or kit of Embodiment 22 or 23, wherein each of the first and second detection probes further comprises (i) a detectable label, and / or (ii) a blocking moiety at or near the 3’ terminus.

[0041] Embodiment 25. The composition or kit of Embodiment 24, wherein each of the first and second detection probes comprises the detectable label.

[0042] Embodiment 26. The composition or kit of Embodiment 25, wherein the detectable label is a fluorescent or chemiluminescent label.

[0043] Embodiment 27. The composition or kit of Embodiment 25, wherein the detectable label is a fluorescent label and each of the first and second detection probes further comprises a non-fluorescent quencher.

[0044] Embodiment 28. An oligonucleotide for determining the presence or absence of rotavirus in a sample, wherein said oligonucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs:7-16 and 19-32, including from 0 to 18 nucleotide analogs.

[0045] Embodiment 29. The oligonucleotide of Embodiment 28, wherein the nucleotide sequence is selected from the group consisting of SEQ ID NOs: 56-65.

[0046] Embodiment 30. The oligonucleotide of Embodiment 28 or 29, wherein the 3’ end of said oligonucleotide is attached to a solid support.

[0047] Embodiment 31. A reaction mixture for determining the presence or absence of rotavirus in a sample, said reaction mixture comprising a set of oligonucleotides as specified in any one of Embodiments 1 to 27.

[0048] Embodiment 32. A reaction mixture for determining the presence or absence of rotavirus in a sample, said reaction mixture comprising the oligonucleotide of Embodiment 28 or 29.

[0049] Embodiment 33. A method for determining the presence or absence of rotavirus in a sample, the method comprising:(1) contacting a sample, said sample suspected of containing rotavirus, with an amplification oligomer set capable of amplifying a target region of rotavirus target nucleic acid, wherein the amplification oligomer set comprises first and second amplification oligomers respectively comprising first and second target-hybridizing sequences, wherein(a) the first target-hybridizing sequence is selected from the group consisting of a sequence that is from 19 to 22 contiguous nucleotides contained in the sequence of SEQ ID NO:43 and that includes at least the sequence of SEQ ID NO:42, including from 0 to 18 nucleotide analogs, a sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO:20 or SEQ ID NO:30, including from 0 to 18 nucleotide analogs, and a sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO:26, or SEQ ID NO:27, including from 0 to 18 nucleotide analogs; and(b) the second target-hybridizing sequence is selected from the group consisting of a sequence that is from 18 to 20 contiguous nucleotides contained in the sequence of SEQ ID NO:53 and that includes at least thesequence of SEQ ID NO: 52, including from 0 to 18 nucleotide analogs, a sequence that is from 18 to 23 contiguous nucleotides contained in the sequence of SEQ ID NO: 55 and that includes at least the sequence of SEQ ID NO: 54, including from 0 to 18 nucleotide analogs, and a sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 29, including from 0 to 18 nucleotide analogs;(2) performing an in vitro nucleic acid amplification reaction, wherein any rotavirus target nucleic acid present in the sample is used as a template for generating an amplification product corresponding to the rotavirus target region; and(3) detecting the presence or absence of the amplification product, thereby determining the presence or absence of rotavirus in the sample.

[0050] Embodiment 34. The method of Embodiment 33, wherein the detecting step (3) comprises contacting the in vitro nucleic acid amplification reaction with at least one detection probe comprising a target-hybridizing sequence configured to specifically hybridize to the amplification product.

[0051] Embodiment 35. A method for synthesizing an oligonucleotide, comprising the steps of:(a) obtaining a solid support comprising at least one nucleobase residue, wherein the at least one nucleobase residue is covalently bound at a 3’ position to the solid support;(b) coupling a 5’ position of the nucleobase residue furthest from the solid support to a 3’ position of another nucleobase residue;(c) repeating step (b) at least 16 additional times, thereby generating at least 18 contiguous nucleobase residues coupled to the solid support; and(d) cleaving the at least 18 contiguous nucleobase residues generated in step (c), thereby obtaining the oligonucleotide,wherein the oligonucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs:7-16 and 19-32, including from 0 to 18 nucleotide analogs.

[0052] Embodiment 36. The method of claim 35, wherein the nucleotide sequence is selected from the group consisting of SEQ ID NOs: 56-65

[0053] These and other aspects and embodiments will become evident upon reference to the following detailed description.DEFINITIONS

[0054] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art pertinent to the methods and compositions described. As used herein, the following terms and phrases have the meanings ascribed to them unless specified otherwise.

[0055] The terms “a,” “an,” and “the” include plural referents, unless the context clearly indicates otherwise. For example, “a nucleic acid” as used herein is understood to represent one or more nucleic acids. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.

[0056] When a value is expressed as “about” X or “approximately” X, the stated value of X will be understood to be accurate to ±10%.

[0057] All ranges are to be interpreted as encompassing the endpoints in the absence of express exclusions such as “not including the endpoints”; thus, for example, “from 0 to 18” includes the values 0 and 18.

[0058] “Sample” includes any specimen that may contain rotavirus, including components thereof, such as nucleic acids or fragments of nucleic acids. Samples include “biological samples” which include any tissue or material derived from a living or dead human, including, for example, stool, blood, plasma, serum, blood cells, saliva, mucous, and cerebrospinal fluid. The biological sample may be treated to physically or mechanically disrupt tissue or cell structure, thus releasing intracellular components into a solution which may further contain enzymes, buffers, salts, detergents, and the like, which are used to prepare a biological sample for analysis. Also, samples may include processed samples such as samples in which one or more components have been concentrated or purified. Processed samples include, e.g., those obtained from passing samples over orthrough a filtering device, or following centrifugation, or by adherence to a medium, matrix, or support.

[0059] A “nucleotide” as used herein is a subunit of a nucleic acid consisting of a phosphate group, a 5-carbon sugar, and a nitrogenous base (also referred to herein as “nucleobase”). The 5-carbon sugar found in RNA is ribose. In DNA, the 5-carbon sugar is 2’ -deoxyribose.

[0060] “Nucleic acid” and “polynucleotide” refer to a multimeric compound comprising nucleotides and / or nucleotide analogs linked together to form a biopolymer. The biopolymers include conventional RNA, conventional DNA, mixed RNA-DNA, and nucleotide-analog-containing versions thereof. A nucleic acid “backbone” may be made up of a variety of linkages, including one or more of sugar-phosphodiester linkages, peptide-nucleic acid bonds (“peptide nucleic acids” or PNA), phosphorothioate linkages, methylphosphonate linkages, or combinations thereof. Sugar moieties of a nucleic acid may be ribose, deoxyribose, or similar compounds with substitutions, e.g., analogs with a methoxy, fluoro or halide group at the 2’ position of the ribose (also referred to herein as “2’-0-Me” or “2’-methoxy” or 2’-fluoro, or “2’-halide”). Nitrogenous bases may be conventional bases, adenine (A), uracil (U), guanine (G), thymine (T), and cytosine (C), and analogs thereof (e.g., inosine, 5 methyl 2’ deoxy cytosine (“5-methyl cytosine”) (5mC) , isoguanine, 5-(l-propynyl)-2’- deoxycytidine (pdC), or 5-(l-propynyl)-2’-deoxyuridine (pdU)). As used in the present disclosure, pdC is considered a cytosine analogue; and pdU is considered a thymine analogue. Nucleic acids may include one or more “abasic” residues where the backbone includes no nitrogenous base for position(s) of the polymer.

[0061] By “RNA and DNA equivalents” is meant RNA and DNA molecules having essentially the same complementary base pair hybridization properties. RNA and DNA equivalents have different sugar moieties (z.e., ribose versus deoxyribose) and may differ by the presence of uracil in RNA and thymine in DNA. The differences between RNA and DNA equivalents do not contribute to differences in homology because the equivalents have the same degree of complementarity to a particular sequence. By “DNA / RNA chimeric” is meant a nucleic acid comprising both DNA and RNA nucleotides. One example of a DNA / RNA chimeric is a DNA oligomer wherein all thymine (T) nucleobase residues are replaced with uracil (U). Unless the context clearlydictates otherwise, reference to a rotavirus nucleic acid includes the RNA and DNA equivalents and DNA / RNA chimerics thereof.

[0062] The phrase “including from 0 to 18 nucleotide analogs,” as used herein following reference to one or more nucleotide sequences by SEQ ID NO, means that the referenced sequence(s) include equivalents of each sequence having from 0 to 18 nucleotide analogs (also referred to herein as “modified nucleotides”). By “equivalents having from 0 to 18 nucleotide analogs” is meant oligonucleotides that (i) have from 0 to 18 nucleotide analogs substituting conventional nucleotides within the reference sequence and (ii) have essentially the same complementary base pair hybridization properties as the reference sequence. Exemplary modified nucleotides are shown in Table 41, infra.

[0063] “Oligomer,” “oligonucleotide,” or “oligo” refers to a nucleic acid of generally less than 1,000 nucleotides (nt), including those in a size range having a lower limit of about 5 nt and an upper limit of about 500 to 900 nt. Some particular embodiments are oligonucleotides in a size range with a lower limit of about 5 to 15, 16, 17, 18, 19, or 20 nt and an upper limit of about 50 to 600 nt, and other particular embodiments are in a size range with a lower limit of about 10 to 20 nt and an upper limit of about 22 to 100 nt. Oligonucleotides may be purified from naturally occurring sources but may be synthesized by using any well-known enzymatic or chemical method. Oligomers may be referred to by a functional name (e.g., detection probe, primer, or promoter primer) but those skilled in the art will understand that such terms refer to oligomers.

[0064] A “target nucleic acid” as used herein is a nucleic acid comprising a target sequence to be amplified. Target nucleic acids may be DNA or RNA and may be either single-stranded or double-stranded. The target nucleic acid may include other sequences besides the target sequence, which may not be amplified.

[0065] The term “target region” or “target nucleic acid region” as used herein refers to the particular nucleotide sequence of the target nucleic acid that is to be amplified and / or detected. The “target region” includes the complexing sequences to which oligonucleotides (e.g., priming oligonucleotides and / or promoter oligonucleotides) complex during an amplification processes (e.g., PCR, TMA). Unless the context clearly dictates otherwise, where the target nucleic acid is originally single-stranded, the term “target region” will also refer to the sequence complementary to the “target region” aspresent in the target nucleic acid, and where the target nucleic acid is originally doublestranded, the term “target region” refers to both the sense (+) and antisense (-) strands.

[0066] The term “target sequence” or “target nucleic acid sequence” as used herein refers to the particular nucleotide sequence of the target nucleic acid to which oligonucleotides (e.g., priming oligonucleotides, detection probes, or capture probes) complex during amplification and / or detection of the target nucleic acid.

[0067] “Target-hybridizing sequence” or “target-specific sequence” is used herein to refer to the portion of an oligomer that is configured to hybridize with a target nucleic acid sequence. Preferably, the target-hybridizing sequences are configured to specifically hybridize with a target nucleic acid sequence. Target-hybridizing sequences may be 100% complementary to the portion of the target sequence to which they are configured to hybridize, but not necessarily. Target-hybridizing sequences may also include inserted, deleted and / or substituted nucleotide residues relative to a target sequence.

[0068] “Non-target-specific sequence” or “non-target-hybridizing sequence” as used herein refers to a region of an oligomer sequence, wherein said region does not stably hybridize with a target sequence under standard hybridization conditions. Oligomers with non-target-specific sequences include, but are not limited to, promoter primers, promoter providers, target capture oligomers, torches, and molecular beacons.

[0069] The term “target a sequence,” as used herein in reference to a region of a rotavirus nucleic acid, refers to a process whereby an oligonucleotide hybridizes to a target region in a manner that allows for amplification and detection as described herein. In one embodiment, the oligonucleotide is complementary with the targeted rotavirus nucleic acid sequence and contains no mismatches. In another embodiment, the oligonucleotide is complementary but contains 1, 2, 3, 4, or 5 mismatches with the targeted rotavirus nucleic acid sequence.

[0070] The term “configured to” denotes an actual arrangement of the polynucleotide sequence configuration of a referenced oligonucleotide target-hybridizing sequence. For example, amplification oligomers that are configured to generate a specified amplicon from a target nucleic acid region have polynucleotide sequences that hybridize to the target region and can be used in an amplification reaction to generate the amplicon. Also, as an example, oligonucleotides that are configured to specifically hybridize to atarget region have a polynucleotide sequence that specifically hybridizes to the referenced sequence under stringent hybridization conditions.

[0071] The term “configured to specifically hybridize to” as used herein means that the target-hybridizing region of an amplification oligonucleotide, detection probe, or other oligonucleotide is designed to have a polynucleotide sequence that could target a sequence of the referenced rotavirus target region. The oligonucleotide is designed to function as a component of an assay for amplification and detection of rotavirus target nucleic acid from a sample, and therefore is designed to target rotavirus nucleic acid in the presence of other nucleic acids commonly found in testing samples. “Specifically hybridize to” does not mean exclusively hybridize to, as some small level of hybridization to non-target nucleic acids may occur, as is understood in the art. Rather, “specifically hybridize to” means that the oligonucleotide is configured to function in an assay to primarily hybridize the target so that an accurate detection of target nucleic acid in a sample can be determined.

[0072] An “amplification oligonucleotide” or “amplification oligomer” is an oligonucleotide that hybridizes to a target nucleic acid and participates in a nucleic acid amplification reaction, e.g., serving as a primer. Amplification oligomers can have 3’ ends that are extended by polymerization as part of the nucleic acid amplification reaction. Amplification oligomers can alternatively have 3’ ends that are not extended by polymerization, but provide a component that facilitates nucleic acid amplification, e.g., a promoter sequence joined 5’ to the target hybridizing sequence of the amplification oligomer. Such an amplification oligomer is referred to as a promoter provider. Amplification oligomers that provide both a 3’ target hybridizing region that is extendable by polymerization and a 5’ promoter sequence are referred to as promoter primers. Amplification oligomers may be optionally modified to include 5’ non-target hybridizing regions such as tags, promoters (as mentioned), or other sequences used or useful for manipulating or amplifying the primer or target oligonucleotide.

[0073] “Nucleic acid amplification” refers to any in vitro procedure that produces multiple copies of a target nucleic acid sequence, or its complementary sequence, or fragments thereof (ie., an amplified sequence containing less than the complete target nucleic acid). Examples of nucleic acid amplification procedures include transcription associated methods, such as transcription-mediated amplification (TMA), nucleic acidsequence-based amplification (NASBA) and others (e.g., U.S. Patent Nos. 5,399,491, 5,554,516, 5,437,990, 5,130,238, 4,868,105, and 5,124,246), and polymerase chain reaction (PCR) (e.g., U.S. Patent Nos. 4,683,195, 4,683,202, and 4,800,159).

[0074] By “amplicon” or “amplification product” is meant a nucleic acid molecule generated in a nucleic acid amplification reaction and which is derived from a target nucleic acid. An amplicon or amplification product contains a target nucleic acid region that may be of the same or opposite sense as the target nucleic acid.

[0075] “Detection probe oligomer,” “detection probe,” or “probe” refers to an oligomer that hybridizes specifically to a target nucleic acid region, including an amplified product, under conditions that promote nucleic acid hybridization, for detection of the target nucleic acid. Detection may either be direct (i.e., probe hybridized directly to the target) or indirect (i.e., a probe hybridized to an intermediate structure that links the probe to the target). A probe’s target sequence generally refers to the specific sequence within a larger sequence which the probe hybridizes specifically. A detection probe may include target-specific sequence(s) and non-target-specific sequence(s). Such non-target-specific sequences can include sequences which will confer a desired secondary or tertiary structure, such as a hairpin structure, which can be used to facilitate detection and / or amplification.

[0076] By “stringent hybridization conditions,” or “stringent conditions” is meant conditions permitting an oligomer to preferentially hybridize to a target nucleic acid region and not to nucleic acid derived from a closely related non-target nucleic acid (i.e., conditions permitting an oligomer to hybridize to its target sequence to form a stable oligomertarget hybrid, but not form a sufficient number of stable oligomernon-target hybrids, so as to allow for amplification and / or detection of target nucleic acids but not non-targeted organisms). While the definition of stringent hybridization conditions does not vary, the actual reaction environment that can be used for stringent hybridization may vary depending upon factors including the GC content and length of the oligomer, the degree of similarity between the oligomer sequence and sequences of non-target nucleic acids that may be present in the test sample, and the target sequence. Hybridization conditions include the temperature and the composition of the hybridization reagents or solutions. Stringent hybridization conditions are readily ascertained by those having ordinary skill in the art.

[0077] “Label” or “detectable label” refers to a moiety or compound joined directly or indirectly to a probe that is detected or leads to a detectable signal. Direct joining may use covalent bonds or non-covalent interactions (e.g., hydrogen bonding, hydrophobic or ionic interactions, and chelate or coordination complex formation) whereas indirect joining may use a bridging moiety or linker (e.g., via an antibody or additional oligonucleotide(s), which may amplify a detectable signal). Any detectable moiety may be used, e.g., radionuclide, ligand such as biotin or avidin, enzyme, enzyme substrate, reactive group, chromophore such as a dye or particle (e.g. , latex or metal bead) that imparts a detectable color, luminescent compound (e.g., bioluminescent, phosphorescent, or chemiluminescent compound such as an acridinium ester (“AE”) compound), and fluorescent compound (z.e., fluorophore). Embodiments of fluorophores include those that absorb light in the range of about 495 to 690 nm and emit light in the range of about 520 to 705 nm, which include those known as HEX, FAM™, TET™, CAL FLUOR™ (Orange or Red), and QUASAR™ compounds. Fluorophores may be used in combination with a quencher molecule that absorbs light when in close proximity to the fluorophore to diminish background fluorescence. Such quenchers are well known in the art and include, e.g, BLACK HOLE QUENCHER™ (or BHQ™) or TAMRA™ compounds. Particular embodiments include a “homogeneous detectable label” that is detectable in a homogeneous system in which bound labeled probe in a mixture exhibits a detectable change compared to unbound labeled probe, which allows the label to be detected without physically removing hybridized from unhybridized labeled probe (e.g., US Pat. Nos. 5,283,174, 5,656,207, and 5,658,737). Particular homogeneous detectable labels include chemiluminescent compounds, including acridinium ester (“AE”) compounds, such as standard AE or AE derivatives, which are well known (US Pat. Nos. 5,656,207, 5,658,737, and 5,639,604). Methods of synthesizing labels, attaching labels to nucleic acid, and detecting signals from labels are well known (e.g, Sambrook el al. , Molecular Cloning, A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) at Ch. 10, and US Pat. Nos. 5,658,737, 5,656,207, 5,547,842, 5,283,174, and 4,581,333, and EP Pat. App. 0 747 706). Particular methods of linking an AE compound to a nucleic acid are known (e.g., US Pat. No. 5,585,481 and US Pat. No. 5,639,604, see column 10, line 6 to column 11, line 3, and Example 8). Particular AE labeling positions are a probe’s central region and near a region of A / T base pairs, at a probe’s 3’ or 5’terminus, or at or near a mismatch site with a known sequence that is the probe should not detect compared to the desired target sequence. Other detectably labeled probes include, e.g., TaqMan™ probes, molecular torches, and molecular beacons. TaqMan™ probes include a donor and acceptor label wherein fluorescence is detected upon enzymatically degrading the probe during amplification in order to release the fluorophore from the presence of the quencher. Molecular torches and beacons exist in open and closed configurations wherein the closed configuration quenches the fluorophore and the open position separates the fluorophore from the quencher to allow fluorescence. Hybridization to target opens the otherwise closed probes.

[0078] A “non-extendable” oligomer includes a blocking moiety at or near its 3’- terminus to prevent extension. A blocking group near the 3’ end is in some embodiments within five residues of the 3’ end and is sufficiently large to limit binding of a polymerase to the oligomer. In other embodiments, a blocking group is covalently attached to the 3’ terminus. Suitable blocking groups include, e.g., alkyl groups, non-nucleotide linkers, alkane-diol dideoxynucleotide residues, cordycepin, 3 ’-deoxy nucleotides, 3’- phosphorylated nucleotides, inverted nucleotides, proteins, peptides, and labels such as fluorophores or quenchers.

[0079] References, particularly in the embodiments, to “the sequence of SEQ ID N0:X” refer to the sequence of nucleotides and / or nucleotide analogs linked together to form a biopolymer. Reference to a sequence by SEQ ID NO does not connote the identity of the backbone (e.g., RNA, 2’-0-Me RNA, or DNA) or any nucleobase modifications (e.g., methylation of cytosine residues (“5MeC”)) unless the context clearly dictates otherwise. In some instances, the sequence of a SEQ ID NO is followed by the statement “including from [x-y] nucleotide analogs”; it is understood that the nucleotide analogs may be substitutions within the sequence of the SEQ ID NO. Unless the context clearly dictates otherwise, reference to a sequence by SEQ ID NO includes reference to its complementary sequence (e.g., reference to the sequence 5’-ttagc-3’ includes reference to the sequence 5’- gctaa-3’).

[0080] “Separating” or “purifying” means that one or more components of a sample are removed or separated from other sample components. Sample components include target nucleic acids usually in a generally aqueous solution phase, which may also include cellular fragments, proteins, carbohydrates, lipids, and other nucleic acids.“ Separating” or “purifying” does not connote any degree of purification. Typically, separating or purifying removes at least 70%, or at least 80%, or at least 95% of the target nucleic acid from other sample components.

[0081] The term “non-linear surfactant,” as used herein, means a surfactant having a branched chain structure. A non-linear surfactant may include one or more ring structures, which may be, for example, in a principal chain and / or in one or more branched chains. Exemplary non-linear surfactants include polysorbate 20, polysorbate 40, polysorbate 60, and digitonin. In certain variations, the non-linear surfactant is non-ionic.

[0082] The term “specificity,” in the context of an amplification and / or detection system, is used herein to refer to the characteristic of the system which describes its ability to distinguish between target and non-target sequences dependent on sequence and assay conditions. In terms of nucleic acid amplification, specificity generally refers to the ratio of the number of specific amplicons produced to the number of side-products (e.g., the signal-to-noise ratio). In terms of detection, specificity generally refers to the ratio of signal produced from target nucleic acids to signal produced from non-target nucleic acids.

[0083] The term “sensitivity” is used herein to refer to the precision with which a nucleic acid amplification reaction can be detected or quantitated. The sensitivity of an amplification reaction is generally a measure of the smallest copy number of the target nucleic acid that can be reliably detected in the amplification system, and will depend, for example, on the detection assay being employed, and the specificity of the amplification reaction, e.g., the ratio of specific amplicons to side-products.DETAILED DESCRIPTION

[0084] Provided herein are compositions, kits, and methods for amplifying and / or detecting target nucleic acid from rotavirus. Preferably, the samples are biological samples. The compositions, kits, and methods provide oligonucleotide sequences that target rotavirus gene sequences or their complementary sequences. Such oligonucleotides may be used as amplification oligonucleotides, which may include primers, promoter primers, blocked oligonucleotides, and promoter provider oligonucleotides, whose functions have been described previously (see, e.g., US Patent Nos. 4,683,195; 4,683,202; 4,800,159; 5,399,491; 5,554,516; 5,824,518; and 7,374,885; each incorporated by reference herein).Other oligonucleotides may be used as probes for detecting amplified sequences or for capture of a rotavirus target nucleic acid.

[0085] The methods provide for the sensitive and specific detection of rotavirus nucleic acids. The methods include performing nucleic acid amplification of a target region of rotavirus and detecting an amplified product by, for example, specifically hybridizing the amplified product with one or more nucleic acid detection probes that provide a signal to indicate the presence of rotavirus in the sample. The amplification step includes contacting the sample with one or more rotavirus-specific amplification oligomers specific for a target sequence in rotavirus target nucleic acid. Particularly suitable target nucleic acids include regions within segment 11 of the rotavirus genome such as, for example, the NSP5 gene for phosphoprotein (see, e.g., GenBank Accession No. LC763221.1, which shows an exemplary reference sequence for the segment 11 NSP5 gene of human rotavirus A). Nucleic acid amplification is performed to produce an amplification product corresponding to one or more of the rotavirus target nucleic acid, if present in the sample, wherein the amplification reaction synthesizes additional copies of the target sequence or its complement by using at least one nucleic acid polymerase and the one or more amplification oligomers to produce the copies from a template strand (e.g., by extending the sequence from a primer using the template strand). One embodiment for detecting the amplification product uses a hybridizing step that includes contacting the amplified product with at least one detection probe oligomer specific for a sequence amplified by the selected amplification oligomers, e.g., a sequence contained in the target sequence flanked by a pair of selected amplification oligomers.

[0086] In some aspects, oligonucleotides are provided, e.g., in a kit or composition. Oligonucleotides generally comprise a target-hybridizing region, e.g., configured to hybridize specifically to a target nucleic acid of rotavirus. While oligonucleotides of different lengths and base composition may be used for amplifying target nucleic acids, in some embodiments, oligonucleotides in this disclosure have targethybridizing regions from about 10 to about 60 bases in length, from about 14 to about 50 bases in length, from about 14 to about 40 bases in length, from about 14 to about 35 bases in length, from about 15 to about 30 bases in length, or from about 16 to about 30 bases in length. In some embodiments, an oligonucleotide comprises a second region of sequence in addition to the target-hybridizing region, such as a promoter, which can be located 5’ ofthe target-hybridizing region. In some embodiments, an oligonucleotide does not comprise a second region of sequence.

[0087] In some embodiments, a set of oligonucleotides comprising a combination of two or more oligonucleotides are provided, e.g., in a kit or composition, such as an amplification oligomer (e.g., primer) pair or an amplification oligomer pair and a third oligonucleotide that is optionally labeled (e.g., for use as a probe), wherein the oligonucleotides are configured to hybridize to a target nucleic acid of rotavirus. In some embodiments a third oligonucleotide that is optionally labeled, the set of oligonucleotides further includes at least a fourth oligonucleotide that is optionally labeled (e.g., for use as a probe).

[0088] In some embodiments, one or more oligonucleotides comprise a nonWatson Crick (NWC) position. In some embodiments, a rotavirus amplification oligomer, a rotavirus amplification oligomer pair, and / or a rotavirus probe comprises a NWC position, such as a position that includes inosine.

[0089] In some embodiments, one or more oligonucleotides comprise a position comprising 5-methylcytosine. In some embodiments, a rotavirus amplification oligomer, a rotavirus amplification oligomer pair, and / or a rotavirus probe comprises a position comprising 5-methylcytosine.

[0090] In some embodiments, one or more oligonucleotides comprise a position comprising propyne dU. In some embodiments, a rotavirus amplification oligomer, a rotavirus amplification oligomer pair, and / or a rotavirus probe comprises a position comprising propyne dU.

[0091] Exemplary oligomers targeting rotavirus target nucleic acid in accordance with the present disclosure are shown in Table 40. Exemplary amplification oligomer pairs and optional third and fourth oligomers (e.g., detection probe) are set forth (by SEQ ID NO) in the following Table 1.Table 1. Exemplary Oligonucleotide Sets

[0092] Exemplary oligomers containing one or more nucleotide analogues (“modified oligomers”) are set forth by SEQ ID NO in the following Table 2 below.Table 2. Exemplary Modified Oligomers* “Corresponds to” means that the modified oligomer is an example of an oligomer containing one or more nucleotide analogues relative to this SEQ ID NO.

[0093] In some embodiments, an oligonucleotide is provided that comprises a label. Such an oligonucleotide can be used as a detection probe. In some embodiments, the labeled oligonucleotide has a sequence corresponding to a SEQ ID NO listed in the Oligonucleotide 3 or Oligonucleotide 4 column of Table 1. In some embodiments, the label is a non-nucleotide label. Suitable labels include compounds that emit a detectable light signal, e.g, fluorophores or luminescent (e.g, chemiluminescent) compounds that can be detected in a homogeneous mixture. More than one label, and more than one type of label,may be present on a particular probe, or detection may rely on using a mixture of probes, in which each probe is labeled with a compound that produces a detectable signal (see. e.g., US Pat. Nos. 6,180,340 and 6,350,579, each incorporated by reference herein). Labels may be attached to a probe by various means including covalent linkages, chelation, and ionic interactions, but in some embodiments the label is covalently attached. For example, in some embodiments, a detection probe has an attached chemiluminescent label such as, e.g., an acridinium ester (AE) compound (see, e.g., US Pat. Nos. 5,185,439; 5,639,604; 5,585,481; and 5,656,744). A label, such as a fluorescent or chemiluminescent label, can be attached to the probe by a non-nucleotide linker (see, e.g., US Pat. Nos. 5,585,481; 5,656,744; and 5,639,604). In some embodiments, the label may include one or more of Quasar670, CalRed610, CalOrange560, fluorescein, ROX, FAM, and HEX.

[0094] In some embodiments, a detection probe (e.g., comprising a fluorescent label) further comprises a second label that interacts with the first label. For example, the second label can be a quencher. In some embodiments, the second label may include one or both of BHQ-1 and BHQ-2. Such probes can be used, e.g., in TaqMan™ assays, where hybridization of the probe to a target or amplicon followed by nucleolysis by a polymerase comprising 5 ’-3’ exonuclease activity results in liberation of the fluorescent label and thereby increased fluorescence, or fluorescence independent of the interaction with the second label.

[0095] In some applications, one or more detection probes exhibiting at least some degree of self-complementarity are used to facilitate detection of probe:target duplexes in a test sample without first requiring the removal of unhybridized probe prior to detection. Specific embodiments of such detection probes include, for example, probes that form conformations held by intramolecular hybridization, such as conformations generally referred to as hairpins. Suitable hairpin probes include a “molecular torch” (see, e.g., US Pat. Nos. 6,849,412; 6,835,542; 6,534,274; and 6,361,945) and a “molecular beacon” (see, e.g., US Pat. No. 5,118,801 and U.S. Pat. No. 5,312,728). Molecular torches include distinct regions of self-complementarity (coined “the target-binding domain” and “the target-closing domain”) which are connected by a joining region (e.g., a -(aUOUOjs- linker) and which hybridize to one another under predetermined hybridization assay conditions. When exposed to an appropriate target or denaturing conditions, the two complementary regions (which may be fully or partially complementary) of the moleculartorch melt, leaving the target-binding domain available for hybridization to a target sequence when the predetermined hybridization assay conditions are restored. Molecular torches are designed so that the target-binding domain favors hybridization to the target sequence over the target-closing domain. The target-binding domain and the target-closing domain of a molecular torch include interacting labels (e.g., fluorescent / quencher) positioned so that a different signal is produced when the molecular torch is self-hybridized as opposed to when the molecular torch is hybridized to a target nucleic acid, thereby permitting detection of probe:target duplexes in a test sample in the presence of unhybridized probe having a viable label associated therewith.

[0096] Examples of interacting donor / acceptor label pairs that may be used in connection with the disclosure, making no attempt to distinguish FRET from non-FRET pairs, include fluorescein / tetramethylrhodamine, lAEDANS / fluorescein, EDANS / DABCYL, coumarin / DABCYL, fluorescein / fluorescein, BODIPY FL / BODIPY FL, fluorescein / DABCYL, lucifer yellow / DABCYL, BODIPY / DABCYL, eosine / DABCYL, erythrosine / DABCYL, tetramethylrhodamine / DABCYL, Texas Red / DABCYL, CY5 / BHQ-1, CY5 / BHQ-2, CY3 / BHQ-1, CY3 / BHQ-2 and fluorescein / QSY7 dye. Those having an ordinary level of skill in the art will understand that when donor and acceptor dyes are different, energy transfer can be detected by the appearance of sensitized fluorescence of the acceptor or by quenching of donor fluorescence. Non-fluorescent acceptors such as DABCYL and the QSY7 dyes advantageously eliminate the potential problem of background fluorescence resulting from direct (ie., non-sensitized) acceptor excitation. Exemplary fluorophore moi eties that can be used as one member of a donor-acceptor pair include fluorescein, ROX, the ATTO dyes, the DY dyes, and the CY dyes. Exemplary quencher moieties that can be used as another member of a donor-acceptor pair include DABCYL, BlackBerry Quencher, and the Black Hole Quencher moieties.

[0097] In some embodiments, a labeled oligonucleotide (e.g., probe) is non- extendable. For example, the labeled oligomer can be rendered non-extendable by 3’- phosphorylation, having a 3 ’-terminal 3 ’-deoxy nucleotide (e.g., a terminal 2’,3’- dideoxynucleotide), having a 3 ’-terminal inverted nucleotide (e.g., in which the last nucleotide is inverted such that it is joined to the penultimate nucleotide by a 3’ to 3’ phosphodiester linkage or analog thereof, such as a phosphorothioate), or having anattached fluorophore, quencher, or other label that interferes with extension (possibly but not necessarily attached via the 3’ position of the terminal nucleotide). In some embodiments, the 3 ’-terminal nucleotide is not methylated.

[0098] Also provided by the disclosure is a reaction mixture for determining the presence or absence of a target nucleic acid of rotavirus in accordance with the methods as described herein. A reaction mixture in accordance with the present disclosure comprises at least one or more of the following: an oligonucleotide as described herein for amplification of a target nucleic acid; and an oligonucleotide (e.g., probe) as described herein for determining the presence or absence of an amplification product of the target nucleic acid. For a reaction mixture that includes a detection probe together with an amplification oligonucleotide combination, the amplification oligonucleotides and detection probe oligonucleotides for a reaction mixture are linked by a common target region (z.e., the reaction mixture will include a probe that binds to a sequence amplifiable by an amplification oligonucleotides combination of the reaction mixture).

[0099] A reaction mixture may further include a number of optional components such as, for example, capture probes, e.g., poly-(k) capture probes as described in US 2013 / 0209992, which is incorporated herein by reference, and / or poly-(R) capture probes as described in US 2020 / 0165599, which is incorporated herein by reference. For an amplification reaction mixture, the reaction mixture will typically include other reagents suitable for performing in vitro amplification such as, e.g., buffers, salt solutions, appropriate nucleotide triphosphates (e.g., dATP, dCTP, dGTP, and dTTP; and / or ATP, CTP, GTP and UTP), and / or enzymes (e.g., a thermostable DNA polymerase, or reverse transcriptase and / or RNA polymerase), and will typically include test sample components, in which a target nucleic acid may or may not be present. Suitable reagents include, for example, formulations containing lithium lauryl sulfate (LLS), sodium lauryl sulfate (SLS), NaH2PO4, Na2HPO4, EDTA, EGTA, LiOH, NaCl, KC1, MgCl2, NaOH, ethanol, methylparaben, propylparaben, trehalose, Tris Buffer, Triton X-100, paramagnetic particles, target capture oligonucleotides, HEPES, succinic acid, polymerases (e.g., DNA polymerases, reverse transcriptases), and / or RNasin.

[0100] In some embodiments, a reaction mixture comprises KC1. In some embodiments, the KC1 concentration is about 50 mM. In some embodiments, the KC1 concentration is greater than about 50 mM, e.g., about 60-150 mM, about 75-125 mM,about 80-120 mM, about 85-115 mM, or about 90-110 mM. In some embodiments, the KC1 concentration is about 55-65 mM, about 65-75 mM, about 75-85 mM, about 85-95 mM, about 95-105 mM, about 105-115 mM, about 115-125 mM, about 125-135 mM, or about 135-145 mM. In some embodiments, a composition according to the disclosure comprises KC1, e.g., at any of the foregoing concentrations. In some embodiments, a method according to the disclosure comprises performing an amplification reaction in the presence of KC1, e.g., at any of the foregoing concentrations.

[0101] In some embodiments, a reaction mixture comprises a non-linear surfactant such as, for example, polysorbate 20. In certain variations, the non-linear surfactant (e.g., polysorbate 20) is present in the reaction mixture at a concentration of from about 0.001% to about 0.025% (v / v) or from about 0.0015% to about 0.015% (v / v). In certain embodiments, a reaction mixture comprises a-cyclodextrin. In certain variations, the a-cyclodextrin is present at a concentration from about 1.0 mg / mL to about 10 mg / mL, from about 3.0 mg / mL to about 9.0 mg / mL, or from about 2.0 mg / mL to about 7.0 mg / mL.

[0102] In some embodiments, a reaction mixture comprises a lyoprotectant. Exemplary lyoprotectants include glycerol; non-reducing sugars such as, e.g., sucrose, raffinose, or trehalose; and amino acids such as, e.g., glycine, arginine, or methionine. In certain variations wherein the lyoprotectant is trehalose, trehalose is present at a concentration of from about 0.1 M to about 0.2 M (e.g., about 0.15 M).

[0103] In some embodiments, a reaction mixture comprises a chelating agent. Suitable chelating agents include ethylenediaminetetraacetic acid (EDTA) and ethylene glycol-bis(P-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). In some embodiments comprising EDTA as the chelating agent, EDTA is present in the reaction mixture at a concentration of from about 0.025 mM to about 0.25 mM (e.g., at a concentration of about 0.08 mM).

[0104] Also provided by the subject disclosure are kits for practicing the methods as described herein. A kit in accordance with the present disclosure comprises at least one or more of the following: an oligonucleotide as described herein for amplification of a target nucleic acid; and an oligonucleotide (e.g., probe) as described herein for determining the presence or absence of an amplification product of the target nucleic acid. In some embodiments, any oligonucleotide combination described herein is present in the kit. The kits may further include a number of optional components such as, for example, captureprobes, e.g., poly-(k) capture probes as described in US 2013 / 0209992 and / or poly-(R) capture probes as described in US 2020 / 0165599. Other reagents that may be present in the kits include reagents suitable for performing in vitro amplification such as, e.g., buffers, salt solutions, appropriate nucleotide triphosphates (e.g., dATP, dCTP, dGTP, dTTP; and / or ATP, CTP, GTP and UTP), and / or enzymes e.g., a thermostable DNA polymerase, or a reverse transcriptase and / or RNA polymerase). Oligonucleotides as described herein may be packaged in a variety of different embodiments, and those skilled in the art will appreciate that the disclosure embraces many different kit configurations. In addition, for a kit that includes a detection probe together with an amplification oligomer combination, the amplification oligonucleotides and detection probe oligonucleotides for a reaction mixture are linked by a common target region (i.e., the reaction mixture will include a probe that binds to a sequence amplifiable by an amplification oligonucleotides combination of the reaction mixture). In certain embodiments, the kit further includes a set of instructions for practicing methods in accordance with the present disclosure, where the instructions may be associated with a package insert and / or the packaging of the kit or the components thereof.

[0105] In some embodiments of a kit as described herein, the oligonucleotides are contained in a formulation comprising at least one of a non-linear surfactant (e.g., polysorbate 20), a-cyclodextrin, a lyoprotectant (e.g. , a non-reducing sugar such as sucrose, raffinose, or trehalose, or an amino acid such as glycine, arginine, or methionine), and a chelating agent e.g., EDTA or EGTA). In some such embodiments, polysorbate 20 is present in the formulation at a concentration of from about 0.002% to about 0.05% (v / v) or about 0.003% to about 0.03% (v / v), a-cyclodextrin is present at a concentration from about 1.0 mg / mL to about 10 mg / mL or about 3.0 mg / mL to about 9 mg / mL, trehalose is present in the formulation at a concentration of from about 0.2 M to about 0.4 M e.g., about 0.26 M or about 0.3 M), and / or EDTA is present in the formulation at a concentration of from about 0.05 mM to about 0.5 mM e.g., about 0.16 mM or about 0.14 mM). In certain variations, the formulation is a lyophilized formulation. In some embodiments of a lyophilized formulation, the formulation is for reconstitution into an aqueous formulation containing polysorbate 20, trehalose, and / or EDTA at concentration(s) as specified above.

[0106] Also provided by the subject disclosure are methods for determining the presence or absence of rotavirus in a sample by, for example, using one or more of theoligonucleotides disclosed herein. Any method disclosed herein is also to be understood as a disclosure of corresponding uses of materials involved in the method directed to the purpose of the method. Any of the oligonucleotides and any combinations (e.g., kits and compositions) comprising such an oligonucleotide are to be understood as also disclosed for use in detecting rotavirus target nucleic acid and for use in the preparation of a composition for detecting rotavirus target nucleic acid.

[0107] Broadly speaking, methods can comprise one or more of the following components: target capture, in which a target nucleic acid (e.g., from a sample, such as a clinical sample) is annealed to a capture oligomer; isolation, e.g., washing, to remove material not associated with a capture oligomer; amplification; and amplicon detection, e.g., amplicon quantification, which may be performed in real time with amplification. Certain embodiments involve each of the foregoing steps. Certain embodiments involve exponential amplification, optionally with a preceding linear amplification step. Certain embodiments involve exponential amplification and amplicon detection. Certain embodiments involve any two of the components listed above. Certain embodiments involve any two components listed adjacently above, e.g., washing and amplification, or amplification and detection.

[0108] Amplifying a rotavirus target nucleic acid region utilizes an in vitro amplification reaction using at least two amplification oligomers that flank a target region to be amplified (e.g., one or more oriented in the sense direction and one or more oriented in the antisense direction for exponential amplification). Particularly suitable oligomer combinations for amplification of rotavirus target regions are described herein. Exemplary amplification oligomers for amplifying rotavirus target regions (including exemplary target-hybridizing core sequences and regions) are listed in Table 40, infra (see also exemplary modified oligomers in Table 2, supra), and particular combinations of first and second amplification oligomers for rotavirus are set forth herein (see, e.g., Table 1, supra, Embodiments section, supra, and Examples 1-3, infra (including Tables 3-11, 14, 16, and 17)).

[0109] A detection method in accordance with the present disclosure can further include the step of obtaining the sample to be subjected to subsequent steps of the method. In certain embodiments, “obtaining” a sample to be used includes, for example, receiving the sample at a testing facility or other location where one or more steps of the method areperformed, and / or retrieving the sample from a location (e.g., from storage or other depository) within a facility where one or more steps of the method are performed.

[0110] In certain embodiments, the method further includes purifying the rotavirus target nucleic acid from other components in the sample, e.g., before an amplification, such as before a capture step. Such purification may include methods of separating and / or concentrating rotavirus contained in a sample from other sample components, or removing or degrading non-nucleic acid sample components, e.g., protein, carbohydrate, salt, lipid, etc. In some embodiments, purifying the target nucleic acid includes degrading nucleic acid in the sample, e.g., with DNase, and optionally removing or inactivating the DNase or removing degraded nucleic acid.[OHl] In particular embodiments comprising a target purification step, a target nucleic acid is captured specifically or non-specifically and separated from other sample components. Non-specific target capture methods may involve selective precipitation of nucleic acids from a substantially aqueous mixture, adherence of nucleic acids to a support that is washed to remove other sample components, or other means of physically separating nucleic acids from a mixture that contains rotavirus nucleic acid and other sample components.

[0112] Target capture typically occurs in a solution phase mixture that contains one or more capture probe oligomers that hybridize to the rotavirus target nucleic acid under hybridizing conditions. For embodiments comprising a capture probe tail, the target: capture-probe complex is captured by adjusting the hybridization conditions so that the capture probe tail hybridizes to an immobilized probe. Certain embodiments use a particulate solid support, such as paramagnetic beads. Selective and non-specific target capture methods are also described, e.g., in US Patent No. 6,110,678 and International Patent Application Pub. No. WO 2008 / 016988, each incorporated by reference herein.

[0113] Isolation can follow capture, where, for example, the complex on the solid support is separated from other sample components. Isolation can be accomplished by any appropriate technique, e.g., washing a support associated with the rotavirus target nucleic acid one or more times (e.g., two or three times) to remove other sample components and / or unbound oligomer. In embodiments using a particulate solid support, such as paramagnetic beads, particles associated with the rotavirus target may be suspended in a washing solution and retrieved from the washing solution, in some embodiments by using magneticattraction. To limit the number of handling steps, the rotavirus target nucleic acid may be amplified by simply mixing the target region in the complex on the support with amplification oligomers and proceeding with amplification steps.

[0114] Exponentially amplifying a target sequence utilizes an in vitro amplification reaction using at least two amplification oligomers that flank a target region to be amplified. In some embodiments, at least one oligonucleotide as described above is provided. In some embodiments, at least two oligonucleotides as described above are provided. The amplification reaction can be cycled or isothermal. Suitable amplification methods include, for example, replicase-mediated amplification, polymerase chain reaction (PCR), ligase chain reaction (LCR), strand-displacement amplification (SDA), and transcription-mediated or transcription-associated amplification (TMA).

[0115] A detection step may be performed using any of a variety of known techniques to detect a signal specifically associated with the amplified target region, such as, e.g., by hybridizing the amplification product with a labeled detection probe and detecting a signal resulting from the labeled probe (including from label released from the probe following hybridization in some embodiments), performing electrophoresis on the sample and / or the amplification product, or determining the sequence of the amplification product. In some embodiments, the labeled probe comprises a second moiety, such as a quencher or other moiety that interacts with the first label, as discussed above. The detection step may also provide additional information on the amplified sequence, such as, e.g., all or a portion of its nucleic acid base sequence. Detection may be performed after the amplification reaction is completed or may be performed simultaneously with amplifying the target region, e.g., in real time. In one embodiment, the detection step allows homogeneous detection, e.g., detection of the hybridized probe without removal of unhybridized probe from the mixture (see, e.g., US Pat. Nos. 5,639,604 and 5,283,174). In some embodiments, the nucleic acids are associated with a surface that results in a physical change, such as a detectable electrical change. Amplified nucleic acids may be detected by concentrating them in or on a matrix and detecting the nucleic acids or dyes associated with them (e.g, an intercalating agent such as ethidium bromide or cyber green) or detecting an increase in dye associated with nucleic acid in solution phase. Other methods of detection may use nucleic acid detection probes that are configured to specifically hybridize to a sequence in the amplified product and detecting the presence of the probe:product complex, or by using a complex of probes that may amplify the detectable signal associated with the amplified products (see, e.g., US Pat. Nos. 5,424,413; 5,451,503; and 5,849,481; each incorporated by reference herein). Directly or indirectly labeled probes that specifically associate with the amplified product provide a detectable signal that indicates the presence of the target nucleic acid in the sample. In particular, the amplified product will contain a target sequence in or complementary to a target nucleic sequence of rotavirus, and a probe will bind directly or indirectly to a sequence contained in the amplified product to indicate the presence or absence of rotavirus in the tested sample.

[0116] In embodiments that detect the amplified product near or at the end of the amplification step, a linear detection probe may be used to provide a signal to indicate hybridization of the probe to the amplified product. One example of such detection uses a luminescently labeled probe that hybridizes to target nucleic acid. The luminescent label is then hydrolyzed from non-hybridized probe. Detection is performed by chemiluminescence using a luminometer. See, e.g., International Patent Application Pub. No. WO 89 / 002476, incorporated by reference herein). In other embodiments that use realtime detection, the detection probe may be a hairpin probe such as, for example, a molecular beacon, molecular torch, or hybridization switch probe that is labeled with a reporter moiety that is detected when the probe binds to amplified product (e.g., a dual -labeled hairpin probe comprising both a fluorescent label and a quenching moiety). In other embodiments for real-time detection, the detection probe is a linear oligomer such as, e.g., an oligomer labeled with both a fluorophore and a quenching moiety (e.g., a TaqMan probe). Such probes may comprise target-hybridizing sequences and non-targethybridizing sequences. Various forms of such probes have been described previously (see, e.g., US Patent Nos. 5,210,015; 5,487,972; 5,118,801; 5,312,728; 5,925,517; 6,150,097; 6,849,412; 6,835,542; 6,534,274; and 6,361,945; and US Patent Application Pub. Nos. 20060068417A1 and 20060194240A1; each incorporated by reference herein). Exemplary rotavirus-specific detection probe oligomers are listed in Tables 1 and 2, supra, and Table 40, infra, and are also set forth in the Embodiments section, supra, and Examples, infra (including, e.g., their use in combination with at least two rotavirus-specific amplification oligomers for detection of a rotavirus target nucleic acid).

[0117] Assays for detection of a rotavirus nucleic acid may optionally include a non-rotavirus internal control (IC) nucleic acid that is amplified and detected in the sameassay reaction mixtures by using amplification and detection oligomers specific for the IC sequence. IC nucleic acid sequences can be, e.g., a DNA plasmid, an RNA template sequence (e.g. , an in vitro transcript), or a synthetic nucleic acid that is spiked into a sample. Alternatively, the IC nucleic acid sequence may be a cellular component, which may be from exogenous cellular sources or endogenous cellular sources relative to the specimen. In these instances, an internal control nucleic acid is co-amplified with the rotavirus nucleic acid in the amplification reaction mixtures. The internal control amplification product and the rotavirus target region amplification product can be detected independently.

[0118] In certain embodiments, amplification and detection of a signal from an amplified IC sequence demonstrates that the assay reagents, conditions, and performance of assay steps were properly used in the assay if no signal is obtained for an intended target rotavirus nucleic acid (e.g., samples that test negative for rotavirus). An IC may also be used as an internal calibrator for the assay when a quantitative result is desired, z.e., the signal obtained from the IC amplification and detection is used to set a parameter used in an algorithm for quantitating the amount of rotavirus nucleic acid in a sample based on the signal obtained for an amplified rotavirus target region. ICs are also useful for monitoring the integrity of one or more steps in an assay. The primers and probe for the IC target sequence are configured and synthesized by using any well-known method provided that the primers and probe function for amplification of the IC target sequence and detection of the amplified IC sequence using substantially the same assay conditions used to amplify and detect the rotavirus target region(s). In certain embodiments that include a target capture-based purification step, it is preferred that a target capture probe specific for the IC target be included in the assay in the target capture step so that the IC is treated in the assay in a manner analogous to that for the intended rotavirus analyte(s) in all of the assay steps.

[0119] Methods for determining the presence or absence of rotavirus as described herein may have a detection sensitivity of, for example, from 10 to 5,000 cp / mL, from 50 to 5,000 cp / mL, from 100 to 5,000 cp / mL, from 250 to 5,000 cp / mL, from 500 to 5,000 cp / mL, from 750 to 5,000 cp / mL, from 10 to 2,500 cp / mL, from 50 to 2,500 cp / mL, from 100 to 2,500 cp / mL, from 250 to 2,500 cp / mL, from 500 to 2,500 cp / mL, from 750 to 2,500 cp / mL, from 10 to 1,500 cp / mL, from 50 to 1,500 cp / mL, from 100 to 1,500 cp / mL, from 250 to 1,500 cp / mL, from 500 to 1,500 cp / mL, or from 750 to 1,500 cp / mL (e.g., copies of IVT in Aptima® tube).

[0120] Also provided by the subject disclosure are methods for synthesizing one or more (e.g., one or more pairs) of the oligonucleotides disclosed herein, the oligonucleotides useful for determining the presence or absence of rotavirus. The method may, for example, include the steps of (a) obtaining a solid support comprising at least one nucleobase residue, wherein the at least one nucleobase residue is bound (e.g., covalently bound) at a 3’ position to the solid support; (b) coupling a 5’ position of the nucleobase residue furthest from the solid support to a 3’ position of another nucleobase residue; (c) repeating step (b) at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, or at least 28 additional times, thereby generating at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 contiguous nucleobase residues coupled to the solid support; and (d) cleaving the at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 contiguous nucleobase residues generated in step (c), thereby obtaining the oligonucleotide or oligonucleotides. In some embodiments, the oligonucleotide has a length of from 18 to 32 or from 18 to 30 contiguous nucleobase residues.

[0121] A method for synthesizing one or more of the oligonucleotides disclosed herein may be a solid phase method. For example, phosphoramidite solid-phase chemistry for joining nucleotides by phosphodiester linkages is disclosed in Caruthers et al., “Chemical Synthesis of Deoxynucleotides by the Phosphoramidite Method,” Methods EnzymoL 154:287 (1987). As another example, automated solid-phase chemical synthesis using cyanoethyl phosphoramidite precursors has been described in Barone et al., “In Situ Activation of bis-dialkylaminephosphines - a New Method for Synthesizing Deoxy oligonucleotides on Polymer Supports,” Nucleic Acids Res. 12(10):4051 (1984). As another example, U.S. Patent No. 5,449,769, titled “Method and Reagent for Sulfurization of Organophosphorous Compounds,” discloses a procedure for synthesizing oligonucleotides containing phosphorothioate linkages. In addition, U.S. Patent No. 5,811,538, titled “Process for the Purification of Oligomers,” discloses the synthesis of oligonucleotides having different linkages, including methylphosphonate linkages.Moreover, methods for the organic synthesis of oligonucleotides are described in, for example, Sambrook c / a / ., Molecular Cloning, A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) at Ch. 10.

[0122] Following synthesis and purification of a particular oligonucleotide, several different procedures may be utilized to purify and control the quality of the oligonucleotide. Suitable procedures include electrophoresis (e.g., polyacrylamide gel electrophoresis) or chromatography (e.g., high pressure liquid chromatography).

[0123] The present disclosure is further illustrated by the following non-limiting examples.Example 1: Evaluation of Oligonucleotides for Analytical Sensitivity

[0124] Different primer and probe combinations were evaluated for analytical sensitivity. Oligo combinations were tested with rotavirus A RNA sample at 100, 10, and 1 copies of the RNA in a PCR reaction (5 pL of sample added to 20 pL of reaction mixture for a total volume of 25 pL per reaction). Oligo concentrations were 400 nM for primers and 200 nM for TaqMan probes. Runs were conducted with Bio-Rad CFX instruments using the following thermal protocol: RT step, 53 °C for 5 min; denaturing step, 95 °C for 3 min; followed by 45 cycles of the cycling 2 step, 95 °C for 15 sec, 60 °C for 1 min. Results are shown in Tables 3-11 below. For each table, results are shown as Cq data in an amplification oligo matrix, with forward primers shown in vertical on the left and reverse primers shown in horizontal on the top (by SEQ ID NO).Table 3. Primer Combinations with Probe of SEQ ID NO: 10, Rotavirus Target at 100 c / rxnTable 4. Primer Combinations with Probe of SEQ ID NO: 11, Rotavirus Target at 100 c / rxnTable 5. Primer Combinations with Probe of SEQ ID NO: 10, Rotavirus Target at 10 c / rxnTable 6. Primer Combinations with Probe of SEQ ID NO: 11, Rotavirus Target at 10 c / rxnTable 7. Primer Combinations with Probe of SEQ ID NO: 10, Rotavirus Target at 1 c / rxnTable 8. Primer Combinations with Probe of SEQ ID NO: 11, Rotavirus Target at 1 c / rxnTable 9. Primer Combinations with Probes of SEQ ID NO: 19 and SEQ ID NO:28, Rotavirus Target at 100 c / rxnTable 10. Primer Combinations with Probes of SEQ ID NO: 19 andSEQ ID NO:28, Rotavirus Target at 10 c / rxnTable 11. Primer Combinations with Probes of SEQ ID NO: 19 andSEQ ID NO:28, Rotavirus Target at 1 c / rxn

[0125] As a reference, the Amplidiag VGE Rotavirus A assay was run with the corresponding template amount. The Amplidiag assay used the primers and probes shown in Table 12 below.Table 12. Amplidiag Assay Primers and Probes

[0126] The average Cqs achieved using the Amplidiag assay (Ref Cq) are shown in Table 13 below.Table 13. Amplidiag Assay Results

[0127] The best performing oligo combinations are shown in Table 14 below.Example 2: Evaluation of Oligonucleotides for Inclusivity

[0128] Different primer and probe combinations were evaluated for inclusivity with rotavirus gBlocks. Nine gBlocks were designed to represent -95% of the rotavirus A variants. The nucleotide sequences of these nine gBlocks, based on real rotavirus A segment 11 sequences, are shown in Table 15 below.Table 15. Rotavirus gBlocks for Inclusivity Testing

[0129] The primers and probes used (without reference to combinations) are shown in Table 16 below.Table 16. Primer and Probe Sequences

[0130] The primer and probe combinations tested are shown in Table 17 below.

[0131] Oligo combinations were tested with gBlock samples at 1,000, 100, and 10 copies in a PCR reaction (without RT enzyme because the gBlock targets were as DNA; 5 pL of sample added to 20 pL of reaction mixture for a total volume of 25 pL per reaction). Oligo concentrations were 400 nM for primers and 200 nM for TaqMan probes. Runs were conducted with Bio-Rad CFX instruments using the following thermal protocol: RT step, 47 °C for 5 min; denaturing step, 95 °C for 3 min; followed by 45 cycles of the cycling 2 step, 95 °C for 15 sec, 60 °C for 30 sec.

[0132] An initial screen evaluated oligo combinations 1-4. Results are shown in Tables 17-25 below.Table 17. Inclusivity Results for gBl - Oligo Combinations 1-4Table 18. Inclusivity Results for gB2 - Oligo Combinations 1-4Table 19. Inclusivity Results for gB3 - Oligo Combinations 1-4Table 20. Inclusivity Results for gB4 - Oligo Combinations 1-4Table 21. Inclusivity Results for gB5 - Oligo Combinations 1-4Table 22. Inclusivity Results for gB6 - Oligo Combinations 1-4Table 23. Inclusivity Results for gB7 - Oligo Combinations 1-4Table 24. Inclusivity Results for gB8 - Oligo Combinations 1-4Table 25. Inclusivity Results for gB9 - Oligo Combinations 1-4

[0133] gBlock gB7 produced poor amplification with oligo combinations 1-4. A second screen evaluated oligo combinations 5 and 6 (see Table [CC2]), which replaced the reverse primer of SEQ ID NO: 12 with SEQ ID NO: 16. Good performance and inclusivity were obtained with assay when SEQ ID NO: 12 was replaced with SEQ ID NO: 16. The results of this second screen are shown in Table 26-34 below.Table 26. Inclusivity Results for gBl - Oligo Combinations 5 & 6Table 27. Inclusivity Results for gB2 - Oligo Combinations 5 & 6Table 28. Inclusivity Results for gB3 - Oligo Combinations 5 & 6Table 29. Inclusivity Results for gB4 - Oligo Combinations 5 & 6Table 31. Inclusivity Results for gB6 - Oligo Combinations 5 & 6Table 32. Inclusivity Results for gB7 - Oligo Combinations 5 & 6Table 33. Inclusivity Results for gB8 - Oligo Combinations 5 & 6Table 34. Inclusivity Results for gB9 - Oligo Combinations 5 & 6Example 3: Genomic Samples Testing

[0134] Primer and probe combination number 6 from Example 2 (see Tables 16 and 17; also referred to below as “segment 11 assay” or “Sgtl l assay”) was tested using clinical stool samples as well as a dilution series of rotavirus A particles. The segment 11 assay was compared against an oligo combination comprising the following primers and probes: SEQ ID NO: 1 (forward primer), SEQ ID NO:2 (reverse primer), SEQ ID NO:3 (detection probe), and SEQ ID NO:4 (detection probe) (also referred to below as “NSP3 assay”).

[0135] Four different clinical samples were used, designated as SMP-10985, SMP-11015, SMP-11024, and SMP-11206. Clinical samples were used either as raw eluate or at a 1 : 10 dilution. Rotavirus A CFHI viral particles (Zeptometrix) were diluted 10-fold in extracted stool BG to concentrations from 10,000 to 10 c / pl. For PCR, 5 pL of sample was added to 20 pL of reaction mixture for a total volume of 25 pL per reaction.PCR reactions were conducted with Bio-Rad CFX instruments using the following thermal protocol: RT step, 47 °C for 5 min; denaturing step, 95 °C for 3 min; followed by 45 cycles of the cycling 2 step, 95 °C for 15 sec, 60 °C for 30 sec. Results are shown in Tables 35- 39 below.Table 35. Clinical Sample Results: SMP-10985 Raw Eluate and 1:10 DilutionTable 36. Clinical Sample Results: SMP-11015 Raw Eluate and 1:10 DilutionTable 38. Clinical Sample Results: SMP-11026 Raw Eluate and 1:10 Dilution

[0136] The segment 11 assay outperformed the NSP3 assay (in some cases, Cq benefits were observed; all cases showed higher end FU / yield with the Sgtl 1 assay).SEQUENCESTable 40. Exemplary Oligonucleotide SequencesTable 41. Exemplary Nucleotide Modifications and Labels

[0137] While illustrative embodiments have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.

Claims

CLAIMSThe embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A composition or kit for determining the presence or absence of rotavirus in a sample, said composition or kit comprising a set of oligonucleotides comprising: an amplification oligomer set capable of amplifying a target region of a rotavirus target nucleic acid, wherein the amplification oligomer set comprises first and second amplification oligomers respectively comprising first and second target-hybridizing sequences, wherein(a) the first target-hybridizing sequence is selected from the group consisting of a sequence that is from 19 to 22 contiguous nucleotides contained in the sequence of SEQ ID NO:43 and that includes at least the sequence of SEQ ID NO:42, including from 0 to 18 nucleotide analogs, a sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO:20 or SEQ ID NO:30, including from 0 to 18 nucleotide analogs, and a sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO:26, or SEQ ID NO:27, including from 0 to 18 nucleotide analogs; and(b) the second target-hybridizing sequence is selected from the group consisting of a sequence that is from 18 to 20 contiguous nucleotides contained in the sequence of SEQ ID NO:53 and that includes at least the sequence of SEQ ID NO: 52, including from 0 to 18 nucleotide analogs, a sequence that is from 18 to 23 contiguous nucleotides contained in the sequence of SEQ ID NO: 55 and that includes at least the sequence of SEQ ID NO: 54, including from 0 to 18 nucleotide analogs, and a sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14,SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO:29, including from 0 to 18 nucleotide analogs.

2. The composition or kit of claim 1, wherein the first target-hybridizing sequence is selected from the group consisting of a sequence that is from 19 to 22 contiguous nucleotides contained in the sequence of SEQ ID NO:43 and that includes at least the sequence of SEQ ID NO:42, including from 0 to 18 nucleotide analogs, and a sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO:20 or SEQ ID NO:30, including from 0 to 18 nucleotide analogs.

3. The composition or kit of claim 2, wherein the first target-hybridizing sequence is SEQ ID NO:20 or SEQ ID NO:30, including from 0 to 18 nucleotide analogs.

4. The composition or kit of claim 1, wherein the first target-hybridizing sequence is a sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:26, or SEQ ID NO:27, including from 0 to 18 nucleotide analogs.

5. The composition or kit of claim 4, wherein the first target-hybridizing sequence is SEQ ID NO:46, including from 0 to 18 nucleotide analogs.

6. The composition or kit of claim 5, wherein the first target-hybridizing sequence is SEQ ID NO:7, SEQ ID NO:26, or SEQ ID NO:27, including from 0 to 18 nucleotide analogs.

7. The composition or kit of claim 4, wherein the first target-hybridizing sequence is SEQ ID NO:8 or SEQ ID NO:9, including from 0 to 18 nucleotide analogs.

8. The composition or kit of any one of claims 1 to 7, wherein the second target-hybridizing sequence is selected from the group consisting ofa sequence that is from 18 to 20 contiguous nucleotides contained in the sequence of SEQ ID NO:53 and that includes at least the sequence of SEQ ID NO: 52, including from 0 to 18 nucleotide analogs, and a sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO:29, including from 0 to 18 nucleotide analogs.

9. The composition or kit of claim 8, wherein the second target-hybridizing sequence is SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO:29, including from 0 to 18 nucleotide analogs.

10. The composition or kit of any one of claims 1 to 7, wherein the second target-hybridizing sequence is selected from the group consisting of a sequence that is from 18 to 23 contiguous nucleotides contained in the sequence of SEQ ID NO: 55 and that includes at least the sequence of SEQ ID NO: 54, including from 0 to 18 nucleotide analogs, and a sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO:29, including from 0 to 18 nucleotide analogs.

11. The composition or kit of claim 10, wherein the second target-hybridizing sequence is SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO:29, including from 0 to 18 nucleotide analogs.

12. The composition or kit of any one of claims 1 to 11, wherein the set of oligonucleotides further comprises at least one detection probe comprising a targethybridizing sequence configured to specifically hybridize to a rotavirus target sequence amplifiable by the first and second amplification oligomers.

13. The composition or kit of claim 12, wherein the at least one detection probe target-hybridizing sequence is selected from the group consisting ofa sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 10 or SEQ ID NO: 11; a sequence that is from 29 to 30 contiguous nucleotides contained in the sequence of SEQ ID NO:49 and that includes at least the sequence of SEQ ID NO:48, including from 0 to 18 nucleotide analogs; and a sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 19 or SEQ ID NO:28, including from 0 to 18 nucleotide analogs.

14. The composition or kit of claim 12 or 13, wherein the at least one detection probe further comprises(i) a detectable label, and / or(ii) a blocking moiety at or near the 3’ terminus.

15. The composition or kit of claim 14, wherein the at least one detection probe comprises the detectable label.

16. The composition or kit of claim 15, wherein the detectable label is a fluorescent or chemiluminescent label.

17. The composition or kit of claim 15, wherein the detectable label is a fluorescent label and the at least one detection probe further comprises a non-fluorescent quencher.

18. The composition or kit of any one of claims 1 to 11, wherein the set of oligonucleotides further comprises at least first and second detection probes, wherein each of the first and second detection probes comprises a target-hybridizing sequence configured to specifically hybridize to a rotavirus target sequence amplifiable by the first and second amplification oligomers.

19. The composition or kit of claim 18, wherein each of the first and second detection probe target-hybridizing sequences is independently selected from the group consisting of a sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 10 or SEQ ID NO: 11; a sequence that is from 29 to 30 contiguous nucleotides contained in the sequence of SEQ ID NO:49 and that includes at least the sequence of SEQ ID NO:48, including from 0 to 18 nucleotide analogs; and a sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 19 or SEQ ID NO:28, including from 0 to 18 nucleotide analogs.

20. The composition or kit of claim 18 or 19, wherein each of the first and second detection probes further comprises(i) a detectable label, and / or(ii) a blocking moiety at or near the 3’ terminus.

21. The composition or kit of claim 20, wherein each of the first and second detection probes comprises the detectable label.

22. The composition or kit of claim 21, wherein the detectable label is a fluorescent or chemiluminescent label.

23. The composition or kit of claim 21, wherein the detectable label is a fluorescent label and each of the first and second detection probes further comprises a non-fluorescent quencher.

24. An oligonucleotide for determining the presence or absence of rotavirus in a sample, wherein said oligonucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs:7-16 and 19-32, including from 0 to 18 nucleotide analogs.

25. The oligonucleotide of claim 24, wherein the 3’ end of said oligonucleotide is attached to a solid support.

26. A reaction mixture for determining the presence or absence of rotavirus in a sample, said reaction mixture comprising a set of oligonucleotides as specified in any one of claims 1 to 23.

27. A reaction mixture for determining the presence or absence of rotavirus in a sample, said reaction mixture comprising the oligonucleotide of claim 24.

28. A method for determining the presence or absence of rotavirus in a sample, the method comprising:(1) contacting a sample, said sample suspected of containing rotavirus, with an amplification oligomer set capable of amplifying a target region of rotavirus target nucleic acid, wherein the amplification oligomer set comprises first and second amplification oligomers respectively comprising first and second target-hybridizing sequences, wherein(a) the first target-hybridizing sequence is selected from the group consisting of a sequence that is from 19 to 22 contiguous nucleotides contained in the sequence of SEQ ID NO:43 and that includes at least the sequence of SEQ ID NO:42, including from 0 to 18 nucleotide analogs, a sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO:20 or SEQ ID NO:30, including from 0 to 18 nucleotide analogs, and a sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO:26, or SEQ ID NO:27, including from 0 to 18 nucleotide analogs; and(b) the second target-hybridizing sequence is selected from the group consisting ofa sequence that is from 18 to 20 contiguous nucleotides contained in the sequence of SEQ ID NO:53 and that includes at least the sequence of SEQ ID NO: 52, including from 0 to 18 nucleotide analogs, a sequence that is from 18 to 23 contiguous nucleotides contained in the sequence of SEQ ID NO: 55 and that includes at least the sequence of SEQ ID NO: 54, including from 0 to 18 nucleotide analogs, and a sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 29, including from 0 to 18 nucleotide analogs;(2) performing an in vitro nucleic acid amplification reaction, wherein any rotavirus target nucleic acid present in the sample is used as a template for generating an amplification product corresponding to the rotavirus target region; and(3) detecting the presence or absence of the amplification product, thereby determining the presence or absence of rotavirus in the sample.

29. The method of claim 28, wherein the detecting step (3) comprises contacting the in vitro nucleic acid amplification reaction with at least one detection probe comprising a target-hybridizing sequence configured to specifically hybridize to the amplification product.

30. A method for synthesizing an oligonucleotide, comprising the steps of:(a) obtaining a solid support comprising at least one nucleobase residue, wherein the at least one nucleobase residue is covalently bound at a 3’ position to the solid support;(b) coupling a 5’ position of the nucleobase residue furthest from the solid support to a 3’ position of another nucleobase residue;(c) repeating step (b) at least 16 additional times, thereby generating at least 18 contiguous nucleobase residues coupled to the solid support; and(d) cleaving the at least 18 contiguous nucleobase residues generated in step (c), thereby obtaining the oligonucleotide, wherein the oligonucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs:7-16 and 19-32, including from 0 to 18 nucleotide analogs.

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