WO2012030856A2 - Compositions, methods and reaction mixtures for the detection of xenotropic murine leukemia virus-related virus - Google Patents

Abstract

The present invention relates to the detection of infectious agents, more specifically to the detection of XMRV. Compositions, methods, reaction mixtures and kits are described for the detection of XMRV by using in vitro nucleic acid amplification techniques.

Description

COMPOSITIONS, METHODS AND REACTION MIXTURES FOR THE DETECTION OF XENOTROPIC MURINE LEUKEMIA VIRUS-RELATED VIRUS.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. provisional application 61/378, 158, filed August 30, 2010, the entirety of which is hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to the detection of infectious agents, more specifically to the detection of XMRV. Compositions, methods, reaction mixtures and kits are described for the detection of XMRV by using in vitro nucleic acid amplification techniques.

BACKGROUND

Xenotropic murine leukemia virus-related virus (XMRV) is an enveloped retrovirus having an 8.3kb genome that is a dimer of linear, positive-sense, single stranded RNA. Transmission routes for XMRV are uncertain, but there is growing concern that the virus has entered and is transmitted through the blood supply. Other suspected routes include sexual transmission and respiratory transmission.

Chronic Fatigue Syndrome (CFS) patients are often infected with XMRV. Also, XMRV is associated with some cases of prostate cancer. Despite these correlations by some, others have been unable to reproducibly detect XMRV in CFS and prostate cancer patients. Thus, there is doubt and debate in the relevant fields as to whether XMRV is present in the blood of CFS patients or is associated with prostate cancers. Uncertainty surrounding this virus and its impact on human health has caused a disjointed approach on whether and how best to manage XMRV infection. Some countries have gone as far as to disallow CFS patients from donating blood, while others continue to allow donations from these patients. Disallowing donation reduces the amount of available much needed blood for transfusions. This is highly problematic if XMRV is not related to CFS. Allowing CFS patients to continue to donate blood, though, could also continue the spread of a virus associated to these disorders. Thus, there is a need to accurately determine the presence of XMRV in a sample so as to allow for a determination as to whether this virus correlates with any disease states. A sensitive, specific and reproducible diagnostic method is needed.

SUMMARY OF THE INVENTION The present invention relates to the detection of infectious agents, more specifically to the detection of XMRV. Compositions, methods, reaction mixtures and kits are described for the detection of XMRV by using in vitro nucleic acid amplification techniques.

One embodiment provides a method for the amplification and identification of an XMRV from a sample comprising the steps of: contacting a sample suspected of containing XMRV with at least two amplification oligomers for generating an amplicon, wherein each of said at least two amplification oligomers is from 10 to about 50 nucleobases in length and wherein the pair are respectively configured to specifically hybridize to regions within a target sequence of XMRVselected from the group consisting of from residue 4669 to residue 4738 and from 4777 to residue 4831 of SEQ ID NO:85, or from residue 7801 to residue 7835 and from residue 7873 to residue 7998 of SEQ ID NO:85; providing conditions sufficient for generating an amplicon from an XMRV target nucleic acid present in said sample using said amplification oligomers; andproviding conditions for detecting said amplicon and determining whether said sample contained XMRV target nucleic acid.

In one aspect, the at least two amplification oligomers comprises a target hybridizing sequence that contains a sequence selected from the group consisting of SEQ ID NOS:86-93. In another aspect, the amplification oligomer pair comprises a first amplification oligomer comprising a target binding sequence containing SEQ ID NO:89 and a second amplification oligomer comprising a target binding sequence containing SEQ ID NO:86, 87 or 88. In another aspect, the amplification oligomer pair comprises a first amplification oligomer comprising a target binding sequence containing SEQ ID NO:91 and a second amplification oligomer comprising a target binding sequence containing SEQ ID NO:92 or 93. In another aspect, the amplification oligomer pair comprises a first amplification oligomer comprising a target binding sequence containing one of SEQ ID NOS:25-36 and a second amplification oligomer comprising a target binding sequence containing one of SEQ ID NOS:50-57. In another aspect, the amplification oligomer pair comprises a first amplification oligomer comprising a target binding sequence containing one of SEQ ID NOS:37-48 and a second amplification oligomer comprising a target binding sequence containing one of SEQ ID NOS:58-65. In another aspect, the amplification oligomer pair is one of SEQ ID NOS:1 -12 and one of SEQ ID NOS:50-57. In another aspect, the amplification oligomer pair is one of SEQ ID NOS:13-24 and one of SEQ ID NOS:58-65.

In one aspect, the amplicon is detected in real-time. In one aspect, the amplicon is detected at the end of the amplification reaction. In one aspect, the amplicon is detected using a method such as sequencing, mass spectrometry, detection probe based detection or other known technique. Detection probe based detection includes, but is not limited to, chemiluminescent labeled detection probe oligomer, or fluorophore:quencher labeled detection probe oligomers. In one aspect, the amplicon is detected using a detection probe oligomer. In one aspect, the detection probe oligomer is labeled with a chemiluminescent compound. In one aspect, the detection probe oligomer is labeled with an AE compound.

In one aspect, the amplification reaction is substantially isothermal. In one aspect, the amplification reaction is PCR. In one aspect, the amplification reaction is TMA.

In one aspect, the sample is human blood donated for transfusion into an individual. In another aspect, the sample is human blood donated for use by a human blood bank. In one aspect, the is human blood.

One embodiment provides for a multiplex amplification and identification of an XMRV from a sample comprising the steps of: contacting a sample suspected of containing XMRV with at least two pair of amplification oligomers for generating separate amplicons from an XMRV target nucleic acid, wherein each of amplification oligomers is from 10 to about 50 nucleobases in length and wherein a first amplification oligomer pair is configured to specifically hybridize to regions within a target sequence of XMRV consisting of from residue 4669 to residue 4738 and from 4777 to residue 4831 of SEQ ID NO:85, and wherein a second amplification oligomer pair is configured to specifically hybridize to regions within a target sequence of XMRV consisting of from residue 7801 to residue 7835 and from residue 7873 to residue 7998 of SEQ ID NO:85; providing conditions sufficient for generating amplicons from an XMRV target nucleic acid present in said sample using said amplification oligomers; and providing conditions for detecting said amplicon and determining whether said sample contained XMRV target nucleic acid.

In one aspect, one of said amplification oligomers comprises a target hybridizing sequence that contains a sequence selected from the group consisting of SEQ ID NOS:86-93. In another aspect, the first amplification oligomer pair comprises a first amplification oligomer comprising a target binding sequence containing SEQ ID NO:89 and a second amplification oligomer comprising a target binding sequence containing SEQ ID NO:86, 87 or 88. In another aspect, the second amplification oligomer pair comprises a first amplification oligomer comprising a target binding sequence containing SEQ ID NO:91 and a second amplification oligomer comprising a target binding sequence containing SEQ ID NO:92 or 93. In another aspect, the first amplification oligomer pair comprises a first amplification oligomer comprising a target binding sequence containing one of SEQ ID NOS:25-36 and a second amplification oligomer comprising a target binding sequence containing one of SEQ ID NOS:50-57. In another aspect, the second amplification oligomer pair comprises a first amplification oligomer comprising a target binding sequence containing one of SEQ ID NOS:37-48 and a second amplification oligomer comprising a target binding sequence containing one of SEQ ID NOS:58-65. In another aspect, the first amplification oligomer pair is one of SEQ ID NOS:1-12 and one of SEQ ID NOS:50-57. In another aspect, the second amplification oligomer pair is one of SEQ ID NOS:13-24 and one of SEQ ID NOS:58-65.

In one aspect, amplicons are detected in real-time. In one aspect, amplicons are detected at the end of the amplification reaction. In one aspect, amplicon are detected using a method such as sequencing, mass spectrometry, detection probe based detection or other known technique. Detection probe based detection includes, but is not limited to, chemiluminescent labeled detection probe oligomer, or fluorophore:quencher labeled detection probe oligomers. In one aspect, detection uses a detection probe oligomer for one or both amplicon species generated by the first amplification oligomer pair and the second amplification oligomer pair. In one aspect, a detection probe oligomer is labeled with a chemiluminescent compound. In one aspect, the detection probe oligomer is labeled with an AE compound.

In one aspect, the amplification reaction is substantially isothermal. In one aspect, the amplification reaction is PCR. In one aspect, the amplification reaction is TMA.

In one aspect, the sample is human blood donated for transfusion into an individual. In another aspect, the sample is human blood donated for use by a human blood bank. In one aspect, the is human blood.

One embodiment provides one or more compositions in any one of the methods steps described herein. Compositions include amplification oligomers, target capture oligomers, detection probe oligomers and amplification products. Compositions can be included in a kit. One embodiment is a kit containing one of the compositions described herein. In one aspect, the kit is for use with screening blood for blood banking. In one embodiment, there is provided a reaction mixture containing one or more compositions for use in any one of the method steps described herein. Reaction mixtures can contain one or more of the compositions described herein, including amplification oligomers, target capture oligomers, detection probe oligomers and amplification products.

DETAILED DESCRIPTION OF THE INVENTION

To aid in understanding aspects of the disclosure, some terms used herein are described more detail. All other scientific and technical terms used herein have the same meaning commonly understood by those skilled in the relevant art, such as may be provided in Dictionary of Microbiology and Molecular Biology, 2nd ed. (Singleton et al., 1994, John Wiley & Sons, New York, NY), The Harper Collins Dictionary of Biology (Hale & Marham, 1991 , Harper Perennial, New York, NY), and references cited herein. Unless mentioned otherwise, the techniques employed or contemplated herein are standard methods well known to a person of ordinary skill in the art of molecular biology.

It is to be noted that the term "a" or "an" entity refers to one or more of that entity; for example, "a nucleic acid," 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.

Sample. A "sample" or "specimen", including "biological" or "clinical" samples may contain or may be suspected of containing XMRV or components thereof, such as nucleic acids or fragments of nucleic acids. A sample may be a complex mixture of components. Samples include "biological samples" which include any tissue or material derived from a living or dead mammal or organism, including, e.g., blood, plasma, serum, blood cells, saliva, and mucous, cerebrospinal fluid (to diagnose XMRV infections of the central nervous system) and samples - such as biopsies - from or derived from genital lesions, anogenital lesions, oral lesions, mucocutanoeus lesions, skin lesions and ocular lesions or combinations thereof. Samples may also include samples of in vitro cell culture constituents including, eg., conditioned media resulting from the growth of cells and tissues in culture medium. The sample may be treated to physically or mechanically disrupt tissue or cell structure to release intracellular nucleic acids into a solution which may contain enzymes, buffers, salts, detergents and the like, to prepare the sample for analysis. In one step of the methods described herein, a sample is provided that is suspected of containing at least a XMRV target nucleic acid. Accordingly, this step excludes the physical step of obtaining the sample from a subject.

Nucleic acid. The term "nucleic acid" refers to a multimeric compound comprising two or more covalently bonded nucleosides or nucleoside analogs having nitrogenous heterocyclic bases, or base analogs, where the nucleosides are linked together by phosphodiester bonds or other linkages to form a polynucleotide. Nucleic acids include RNA, DNA, or chimeric DNA-RNA polymers or oligonucleotides, and analogs 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 (in "peptide nucleic acids" or PNAs, see PCT No. WO 95/32305), phosphorothioate linkages, methylphosphonate linkages, or combinations thereof. Sugar moieties of the nucleic acid may be either ribose or deoxyribose, or similar compounds having known substitutions, e.g., 2' methoxy substitutions and 2' halide substitutions (e.g., 2'-F). Nitrogenous bases may be conventional bases (A, G, C, T, U), analogs thereof (e.g., inosine, 5-methylisocytosine, isoguanine; The Biochemistry of the Nucleic Acids 5-36, Adams et al., ed., 1 1^th ed., 1992, Abraham et al., 2007, BioTechniques 43: 617-24), which include derivatives of purine or pyrimidine bases (e.g., N⁴-methyl deoxygaunosine, deaza- or aza-purines, deaza- or aza- pyrimidines, pyrimidine bases having substituent groups at the 5 or 6 position, purine bases having an altered or replacement substituent at the 2, 6 and/or 8 position, such as 2-amino-6- methylaminopurine, 0⁶-methylguanine, 4-thio-pyrimidines, 4-amino-pyrimidines, 4- dimethylhydrazine-pyrimidines, and 0⁴-alkyl-pyrimidines, and pyrazolo-compounds, such as unsubstituted or 3-substituted pyrazolo[3,4-d]pyrimidine; US Pat. Nos. 5,378,825, 6,949,367 and PCT No. WO 93/13121 ). Nucleic acids may include "abasic" residues in which the backbone does not include a nitrogenous base for one or more residues (US Pat. No. 5,585,481 ). A nucleic acid may comprise only conventional sugars, bases, and linkages as found in RNA and DNA, or may include conventional components and substitutions (e.g., conventional bases linked by a 2' methoxy backbone, or a nucleic acid including a mixture of conventional bases and one or more base analogs). Nucleic acids may include "locked nucleic acids" (LNA), in which one or more nucleotide monomers have a bicyclic furanose unit locked in an RNA mimicking sugar conformation, which enhances hybridization affinity toward complementary sequences in single-stranded RNA (ssRNA), single-stranded DNA (ssDNA), or double-stranded DNA (dsDNA) (Vester et al., 2004, Biochemistry 43(42):13233-41 ). Nucleic acids may include modified bases to alter the function or behavior of the nucleic acid, e.g., addition of a 3'-terminal dideoxynucleotide to block additional nucleotides from being added to the nucleic acid. Synthetic methods for making nucleic acids in vitro are well known in the art although nucleic acids may be purified from natural sources using routine techniques.

Polynucleotide. The term "polynucleotide" denotes a nucleic acid chain. Throughout this application, nucleic acids are designated by the 5'-terminus to the 3'-terminus. Standard nucleic acids, e.g., DNA and RNA, are typically synthesized "3'-to-5'," i.e., by the addition of nucleotides to the 5'-terminus of a growing nucleic acid.

Nucleotide. As referred to herein, a "nucleotide" is a subunit of a nucleic acid consisting of a phosphate group, a 5-carbon sugar and a nitrogenous base. The 5-carbon sugar found in RNA is ribose. In DNA, the 5-carbon sugar is 2'-deoxyribose. The term also includes analogs of such subunits, such as a methoxy group at the 2' position of the ribose (2'-0-Me, or 2' methoxy). As used herein, methoxy oligonucleotides containing "T" residues have a methoxy group at the 2' position of the ribose moiety, and a uracil at the base position of the nucleotide.

Non-nucleotide unit. The term "non-nucleotide unit" is a unit that does not significantly participate in hybridization of a polymer. Such units must not, for example, participate in any significant hydrogen bonding with a nucleotide, and would exclude units having as a component one of the five nucleotide bases or analogs thereof.

Target nucleic acid. The term "target nucleic acid" refers to 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 that may be amplified. Typical target nucleic acids include virus genomes, bacterial genomes, fungal genomes, plant genomes, animal genomes, rRNA, tRNA, or mRNA from viruses, bacteria or eukaryotic cells, mitochondrial DNA, or chromosomal DNA. In the instant disclosure, target nucleic acids are nucleic acids from XMRV, or amplification products thereof. In one aspect, the target nucleic acid is RNA from XMRV. In another aspect, the target nucleic acid is an amplification product generated from an XMRV nucleic acid. The amplification product can be generated using any amplification method; PCR and TMA being two non-limiting examples. The amplification product target nucleic acid can be either single stranded or double stranded. Double stranded target nucleic acids can be DNA:DNA, DNA:RNA or RNA:RNA.

Target sequence. The term "target sequence" refers to the particular nucleotide sequence of the target nucleic acid that is to be amplified. Where the target nucleic acid is originally single- stranded, the term "target sequence" will also refer to the sequence complementary to the target sequence as present in the target nucleic acid. Where the target nucleic acid is originally double-stranded, the term "target sequence" refers to both the sense (+) and antisense (-) strands. In choosing a target sequence, the skilled artisan will understand that a sequence should be chosen so as to distinguish between unrelated or closely related target nucleic acids.

The terms "target(s) a sequence" or "target(s) a target nucleic acid" as used herein in reference to a region of XMRV nucleic acid refers to a process whereby an oligonucleotide stably hybridizes to the target sequence in a manner that allows for amplification and/or detection as described herein. In one embodiment, the oligonucleotide is complementary to the targeted XMRV nucleic acid sequence and contains no mismatches. In another embodiment, the oligonucleotide is complementary but contains 1 ; or 2; or 3; or 4; or 5 mismatches with the targeted XMRV nucleic acid sequence. Preferably, the oligonucleotide that stably hybridizes to the XMRV nucleic acid sequence includes at least 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 30, 35, 40, 45 or 50 nucleotides complementary to the target sequence. It is understood that at least 10 and as many as 50 is an inclusive range such that 10, 50 and each whole number there between are included. The term "configured to target a sequence" as used herein means that the target hybridizing region of an amplification oligonucleotide is designed to have a polynucleotide sequence that could target a sequence of the referenced XMRV region. Such an amplification oligonucleotide is not limited to targeting that sequence only, but is rather useful as a composition, in a kit or in a method for targeting a XMRV target nucleic acid, as is described herein. The term "configured to" denotes an actual arrangement of the polynucleotide sequence configuration of the amplification oligonucleotide target hybridizing sequence.

Isolated. The term "isolated" means that a nucleic acid is taken from its natural milieu, but the term does not connote any degree of purification.

Fragment. This term as used herein in reference to the XMRV targeted nucleic acid sequence refers to a piece of contiguous nucleic acid. In certain embodiments, the fragment includes contiguous nucleotides from an XMRV target nucleic acid, wherein the number of contiguous nucleotides in the fragment are less than that for the entire POL gene or LTR gene.

Region. The term "region" refers to a portion of a nucleic acid wherein said portion is smaller than the entire nucleic acid. For example, when the nucleic acid in reference is an oligonucleotide promoter provider, the term "region" may be used refer to the smaller promoter portion of the entire oligonucleotide. Similarly, and also as example only, when the referenced nucleic acid is a target nucleic acid, the term "region" may be used to refer to a smaller area of the nucleic acid.

Oligonucleotide. "Oligonucleotide" may be used interchangeably with "oligomer and "oligo" and refers to a nucleic acid having generally more than 5 nucleotide (nt) residues, and less than 1 ,000 nucleotide (nt) residues. This range includes all encompassed whole numbers. It is understood that this range is exemplary only. Oligonucleotides may be purified from naturally occurring sources, or may be synthesized using any of a variety of well known enzymatic or chemical methods. The term oligonucleotide does not denote any particular function to the reagent; rather, it is used generically to cover all such reagents described herein. An oligonucleotide may serve various different functions. For example, it may function as a primer if it is specific for and capable of hybridizing to a complementary strand and can further be extended in the presence of a nucleic acid polymerase, it may provide a promoter if it contains a sequence recognized by an RNA polymerase and allows for transcription (eg., a T7 provider), and it may function to prevent hybridization or impede primer extension if appropriately situated and/or modified.

As used herein, an oligonucleotide having a nucleic acid sequence "comprising" or "consisting of" or "consisting essentially of" a sequence selected from a group of specific sequences means that the oligonucleotide, as a basic and novel characteristic, is capable of stably hybridizing to a nucleic acid having the exact complement of one of the listed nucleic acid sequences of the group under stringent hybridization conditions. An exact complement includes the corresponding DNA or RNA sequence.

Corresponds. As used herein, a nucleic acid "corresponds" to a specified nucleic acid if the nucleic acid is 100% identical or complementary to the specified nucleic acid.

Substantially corresponding to. As used herein, a nucleic acid "substantially corresponding to" a specified nucleic acid sequence means that the referred to oligonucleotide is sufficiently similar to the reference nucleic acid sequence such that the oligonucleotide has similar hybridization properties to the reference nucleic acid sequence in that it would hybridize with the same target nucleic acid sequence under stringent hybridization conditions. Substantially corresponding nucleic acids vary by at least one nucleotide from the specified nucleic acid. This variation may be stated in terms of a percentage of identity or complementarity between the nucleic acid and the specified nucleic acid. Thus, nucleic acid substantially corresponds to a reference nucleic acid sequence if these percentages of base identity or complementarity are from less than 100% to about 80%. In preferred embodiments, the percentage is at least about 85%. In more preferred embodiments, this percentage is at least about 90%; in other preferred embodiments, this percentage is at least about 95%, 96%, 97%, 98% or 99%. One skilled in the art will understand that the recited ranges include all whole and rational numbers of the range (e.g., 92% or 92.377%).

Blocking moiety. As used herein, a "blocking moiety" is a substance used to "block" the 3'- terminus of an oligonucleotide or other nucleic acid so that it cannot be efficiently extended by a nucleic acid polymerase.

Amplification oligomer. An "amplification oligomer", which may also be called an "amplification oligonucleotide" is an oligomer, at least the 3'-end of which is complementary to a target nucleic acid ("target hybridizing sequence"), and which hybridizes to a target nucleic acid, or its complement, and participates in a nucleic acid amplification reaction. An example of an amplification oligomer is a "primer" that hybridizes to a target nucleic acid and contains a 3' OH end that is extended by a polymerase in an amplification process. Another example of an amplification oligomer is a "promoter-based amplification oligomer," which comprises a target hybridizing sequence, and a promoter sequence for initiating transcription by an appropriate polymerase. Promoter-based amplification oligomers may or may not be extended by a polymerase in a primer-based extension depending upon whether or not the 3' end of the target hybridizing sequence is modified to prevent primer-based extension (e.g., a 3' blocked end). A promoter-based amplification oligonucleotide comprising a target hybridizing region that is not modified to prevent primer-based extension is referred to as a "promoter-primer." A promoter- based amplification oligonucleotide comprising a target hybridizing region that is modified to prevent primer-based extension is referred to as a "promoter-provider." Size ranges for amplification oligonucleotides include those comprising target hybridizing regions that are about 10 to about 70 nt long - such as about 10 to about 60 nt long, about 10 to about 50 nt long, about 10 to about 40 nt long, about 10 to about 30 nt long or about 10 to about 25 nt long or about 15 to 25 nt long. Preferred sizes of amplification oligomers include those comprising target hybridizing regions that are about 18, 19, 20, 21 , 22 or 23 nt long. An amplification oligomer may optionally include modified nucleotides or analogs that are not complementary to target nucleic acid in a strict A:T/U, G:C sense. Such modified nucleotides or analogs are herein considered mismatched to their corresponding target sequence. For some embodiments, the preferred amount of amplification oligomer per reaction is about 10, 15 or 20 pmoles.

Oligomers not intended for primer-based extension by a nucleic acid polymerase may include a blocker group that replaces the 3ΌΗ to prevent the enzyme-mediated extension of the oligomer in an amplification reaction. For example, blocked amplification oligomers and/or detection probes present during amplification may not have functional 3ΌΗ and instead include one or more blocking groups located at or near the 3' end. In some embodiments a blocking group near the 3' end and may be 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. Many different chemical groups may be used to block the 3' end, e.g., alkyl groups, non-nucleotide linkers, alkane-diol dideoxynucleotide residues, and cordycepin.

Promoter. The term "promoter" refers to a specific nucleic acid sequence that is recognized by a DNA-dependent RNA polymerase ("transcriptase") as a signal to bind to the nucleic acid and begin the transcription of RNA at a specific site. Promoters include, SP6 promoters, T3 promoters and T7 promoters, to name a few.

Promoter-provider. As used herein, a "promoter-provider" or "provider" refers to an oligonucleotide comprising first and second regions, and which is modified to prevent the initiation of DNA synthesis from its 3'-terminus. The "first region" of a promoter-provider oligonucleotide comprises a base sequence which hybridizes to a DNA template, where the hybridizing sequence is situated 3', but not necessarily adjacent to, a promoter region. The target-hybridizing portion of a promoter oligonucleotide is typically at least 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 30, 35, 40 or 45 nucleotides in length, and may extend up to 50 or more nucleotides in length. The "second region" comprises a promoter sequence for an RNA polymerase. A promoter-provider oligonucleotide is configured so that it is incapable of being extended by an RNA- or DNA-dependent DNA polymerase, (e.g., reverse transcriptase), preferably by comprising a blocking moiety at its 3'-terminus as described above. This modification differentiates promoter providers from promoter primers. Preferably, the promoter portion of a promoter primer or provider is a promoter for a DNA-dependent RNA polymerase from E. coli and bacteriophages T7, T3, and SP6, though other promoters or modified version thereof can be used as well.

Terminating oligonucleotide. As used herein, a "terminating oligonucleotide" or "blocker oligonucleotide" is an oligonucleotide comprising a base sequence that is complementary to a region of the target nucleic acid in the vicinity of the 5'-end of the target sequence, so as to "terminate" primer extension of a nascent nucleic acid that includes a priming oligonucleotide, thereby providing a defined 3'-end for the nascent nucleic acid strand.

Amplification. This refers to any known procedure for obtaining multiple copies of a target nucleic acid sequence or its complement or fragments thereof. The multiple copies may be referred to as amplicons or amplification products. Amplification of "fragments" refers to production of an amplified nucleic acid that contains less than the complete target nucleic acid or its complement, eg., produced by using an amplification oligonucleotide that hybridizes to, and initiates polymerization from, an internal position of the target nucleic acid. Known amplification methods include both thermal cycling and isothermal amplification methods. For some embodiment, isothermal amplification methods are preferred. Replicase-mediated amplification, polymerase chain reaction (PCR), ligase chain reaction (LCR), strand- displacement amplification (SDA), and transcription-mediated or transcription-associated amplification are non-limiting examples of nucleic acid amplification methods. Replicase- mediated amplification uses self-replicating RNA molecules, and a replicase such as QB- replicase (eg., US Pat. No. 4,786,600). PCR amplification uses a DNA polymerase, pairs of primers, and thermal cycling to synthesize multiple copies of two complementary strands of dsDNA or from a cDNA (eg., US Pat. Nos. 4,683, 195, 4,683,202, and 4,800, 159). LCR amplification uses four or more different oligonucleotides to amplify a target and its complementary strand by using multiple cycles of hybridization, ligation, and denaturation (eg., US Pat. No. 5,427,930 and US Pat. No. 5,516,663). SDA uses a primer that contains a recognition site for a restriction endonuclease and an endonuclease that nicks one strand of a hemimodified DNA duplex that includes the target sequence, whereby amplification occurs in a series of primer extension and strand displacement steps (eg., US Pat. No. 5,422,252; US Pat. No. 5,547,861 ; and US 5,648,21 1 ). Preferred embodiments use an amplification method suitable for the amplification of RNA target nucleic acids, such as transcription mediated amplification (TMA) or NASBA, but it will be apparent to persons of ordinary skill in the art that oligomers disclosed herein may be readily used as primers in other amplification methods. Transcription associated amplification. This method of amplification, also referred to herein as "transcription mediated amplification" (TMA) refers to nucleic acid amplification that uses an RNA polymerase to produce multiple RNA transcripts from a nucleic acid template. These methods generally employ an RNA polymerase, a DNA polymerase, deoxyribonucleoside triphosphates, ribonucleoside triphosphates, and a template complementary oligonucleotide that includes a promoter sequence, and optionally may include one or more other oligonucleotides. TMA methods are embodiments of amplification methods used for amplifying and detecting XMRV target sequences as described herein. Variations of transcription associated amplification are well known in the art as previously disclosed in detail (eg., US Pat. Nos. 4,868,105; 5, 124,246; 5, 130,238; 5,399,491 ; 5,437,990; 5,554,516; and 7,374,885; and PCT Pub. Nos. WO 88/01302; WO 88/10315 and WO 95/03430). The person of ordinary skill in the art will appreciate that the disclosed compositions may be used in amplification methods based on extension of oligomer sequences by a polymerase.

Real-time TMA. As used herein, the term "real-time TMA" refers to single-primer transcription- mediated amplification ("TMA") of target nucleic acid that is monitored by real-time detection means.

Amplicon. This term, which is used interchangeably with "amplification product", refers to the nucleic acid molecule generated during an amplification procedure that is complementary or homologous to a sequence contained within the target sequence. These terms can be used to refer to a single strand amplification product, a double strand amplification product or one of the strands of a double strand amplification product.

Probe. A probe, also known as a "detection probe" or "detection oligonucleotide" are terms referring to a nucleic acid oligomer that hybridizes specifically to a target sequence in a nucleic acid, or in an amplified nucleic acid, under conditions that promote hybridization to allow detection of the target sequence or amplified nucleic acid. Probes may be DNA, RNA, analogs thereof or combinations thereof and they may be labeled or unlabeled. A probe is generally configured to specifically hybridize to a smaller nucleic acid sequence within a larger target sequence by standard base pairing. A probe may comprise target-specific sequences and other sequences that contribute to the three-dimensional conformation of the probe (e.g., US Pat. Nos. 5, 1 18,801 ; 5,312,728; 6,849,412; 6,835,542; 6,534,274; and 6,361 ,945; and US Pub. No. 20060068417). Exemplary probe types include, nucleic acid probes, AE-labeled nucleic acid probes, molecular beacons, molecular torches, molecular switches, taqman probes, hairpin probes, and other well-known configurations. In a preferred embodiment, the detection probe comprises a 2' methoxy backbone which can result in a higher signal being obtained. Stable. By "stable" or "stable for detection" is meant that the temperature of a reaction mixture is at least 2.deg.C below the melting temperature of a nucleic acid duplex.

Label. As used herein, a "label" refers to a moiety or compound joined directly or indirectly to a probe that is detected or leads to a detectable signal. Direct labelling can occur through bonds or interactions that link the label to the probe, including covalent bonds or non-covalent interactions, e.g. hydrogen bonds, hydrophobic and ionic interactions, or formation of chelates or coordination complexes. Indirect labelling can occur through use of a bridging moiety or "linker" such as a binding pair member, an antibody or additional oligomer, which is either directly or indirectly labeled, and which may amplify the detectable signal. Labels include any detectable moiety, such as a radionuclide, ligand (e.g., biotin, avidin), enzyme or enzyme substrate, reactive group, or chromophore (e.g., dye, particle, or bead that imparts detectable color), luminescent compound (e.g., bioluminescent, phosphorescent, or chemiluminescent labels), chemiluminescent compounds, e.g., acridinium ester ("AE") compounds that include standard AE and derivatives (e.g., US Pat. Nos. 5,656,207, 5,658,737, and 5,639,604), quencher or fluorophore. Labels may be detectable in a homogeneous assay in which bound labeled probe in a mixture exhibits a detectable change different from that of an unbound labeled probe, e.g., instability or differential degradation properties. A "homogeneous detectable label" can be detected without physically removing bound from unbound forms of the label or labeled probe (e.g., US Pat. Nos. 5,283, 174, 5,656,207, and 5,658,737). Synthesis and methods of attaching labels to nucleic acids and detecting labels are well known (e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), Chapter 10; US Pat. Nos. 5,658,737, 5,656,207, 5,547,842, 5,283, 174, and 4,581 ,333). More than one label, and more than one type of label, may be present on a particular probe, or detection may use a mixture of probes in which each probe is labeled with a compound that produces a detectable signal (e.g., US Pat. Nos. 6, 180,340 and 6,350,579).

Capture oligonucleotide. As used herein, a "capture oligonucleotide," "target capture oligonucleotide" or "capture probe" refers to a nucleic acid oligomer that specifically hybridizes to a target sequence in a target nucleic acid by standard base pairing and joins to a binding partner on an immobilized probe to capture the target nucleic acid to a support. One example of a capture oligomer includes an oligonucleotide comprising two binding regions: a target hybridizing sequence and an immobilized probe-binding region. A variation of this example, the two regions may be present on two different oligomers joined together by one or more linkers. Another embodiment of a capture oligomer the target hybridizing sequence is a sequence that includes random or non-random poly-GU, poly-GT, or poly U sequences to bind non-specifically to a target nucleic acid and link it to an immobilized probe on a support. (PCT Pub No. WO 2008/016988). The immobilized probe binding region can be a nucleic acid sequence, referred to as a tail. Tails include a substantially homopolymeric tail (T₀4A1₀-40), that bind to a complementary immobilized sequence attached to the support particle or support matrix. Thus, a non-limiting example of preferred nucleic acid tails can in some embodiments include about 10 to 40 nucleotides (e.g., A₁₀ to A₄₀), or of about 14 to 33 nt (e.g., T₃A₁₄ to T₃A₃₀). Another example of a capture oligomer comprises two regions, a target hybridizing sequence and a binding pair member that is not a nucleic acid sequence.

Immobilized oligonucleotide. As used herein, an "immobilized oligonucleotide", "immobilized probe" or "immobilized nucleic acid" refers to a nucleic acid binding partner that joins a capture oligomer to a support, directly or indirectly. An immobilized probe joined to a support facilitates separation of a capture probe bound target from unbound material in a sample. One embodiment of an immobilized probe is an oligomer joined to a support that facilitates separation of bound target sequence from unbound material in a sample. Supports may include known materials, such as matrices and particles free in solution, which may be made of nitrocellulose, nylon, glass, polyacrylate, mixed polymers, polystyrene, silane, polypropylene, metal, or other compositions, of which one embodiment is magnetically attractable particles. Supports may be monodisperse magnetic spheres (e.g., uniform size ± 5%), to which an immobilized probe is joined directly (via covalent linkage, chelation, or ionic interaction), or indirectly (via one or more linkers), where the linkage or interaction between the probe and support is stable during hybridization conditions.

Complementary. By "complementary" is meant that the nucleotide sequences of similar regions of two single-stranded nucleic acids, or to different regions of the same single-stranded nucleic acid have a nucleotide base composition that allow the single-stranded regions to hybridize together in a stable double-stranded hydrogen-bonded region under stringent hybridization or amplification conditions. Sequences that hybridize to each other may be completely complementary or partially complementary to the intended target sequence by standard nucleic acid base pairing (e.g. G:C, A:T or A:U pairing). By "sufficiently complementary" is meant a contiguous sequence that is capable of hybridizing to another sequence by hydrogen bonding between a series of complementary bases, which may be complementary at each position in the sequence by standard base pairing or may contain one or more residues that are not complementary by standard A:T/U and G:C pairing, or are modified nucleotides such as abasic residues, modified nucleotides or nucleotide analogs. Sufficiently complementary contiguous sequences typically are at least 80%, or at least 90%, complementary to a sequence to which an oligomer is intended to specifically hybridize (a %- complementarity range includes all whole and rational numbers of the range). Sequences that are "sufficiently complementary" allow stable hybridization of a nucleic acid oligomer with its target sequence under appropriate hybridization conditions, even if the sequences are not completely complementary. When a contiguous sequence of nucleotides of one single-stranded region is able to form a series of "canonical" hydrogen-bonded base pairs with an analogous sequence of nucleotides of the other single-stranded region, such that A is paired with U or T and C is paired with G, the nucleotides sequences are "completely" complementary.

Preferentially hybridize. By "preferentially hybridize" is meant that under stringent hybridization assay conditions, an oligonucleotide hybridizes to its target sequences, or replicates thereof, to form stable oligonucleotide: target sequence hybrid, while at the same time formation of stable oligonucleotide: non-target sequence hybrid is minimized. For example, a probe oligonucleotide preferentially hybridizes to a target sequence or replicate thereof to a sufficiently greater extent than to a non-target sequence, to enable one having ordinary skill in the art to accurately detect the RNA replicates or complementary DNA (cDNA) of the target sequence formed during the amplification. Appropriate hybridization conditions are well known in the art for probe, amplification, target capture, blocker and other oligonucleotides, may be predicted based on sequence composition, or can be determined by using routine testing methods (e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 2^nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) at §§ 1.90-1 .91 , 7.37-7.57, 9.47-9.51 and 1 1 .47-1 1 .57, particularly §§ 9.50-9.51 , 1 1 .12-1 1 .13, 1 1 .45-1 1 .47 and 1 1.55-1 1 .57).

Nucleic acid hybrid. By "nucleic acid hybrid" or "hybrid" or "duplex" is meant a nucleic acid structure containing a double-stranded, hydrogen-bonded region wherein each strand is complementary to the other, and wherein the region is sufficiently stable under stringent hybridization conditions to be detected by means including, but not limited to, chemiluminescent or fluorescent light detection, autoradiography, or gel electrophoresis. Such hybrids may comprise RNA:RNA, RNA:DNA, or DNA:DNA duplex molecules.

Sample preparation. This refers to any steps or methods that treat a sample for subsequent amplification and/or detection of XMRV nucleic acids present in the sample. The target nucleic acid may be a minority component in the sample. Sample preparation may include any known method of isolating or concentrating components, such as viruses or nucleic acids using standard microbiology methods. Sample preparation may include physical disruption and/or chemical lysis of cellular components to release intracellular components into a substantially aqueous or organic phase and removal of debris, such as by using filtration, centrifugation or adsorption. Sample preparation may include use of a nucleic acid oligonucleotide that selectively or non-specifically captures a target nucleic acid and separates it from other sample components (eg., as described in US Pat. No. 6, 1 10,678 and PCT Pub. No. WO 2008/016988). Separating, purifying. These terms mean 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 removes at least 70%, or at least 80%, or at least 95% of the target nucleic acid from other sample components. Ranges of %-purity include all whole and rational numbers of the range.

DNA-dependent DNA polymerase. As used herein, a "DNA-dependent DNA polymerase" is an enzyme that synthesizes a complementary DNA copy from a DNA template. Examples are DNA polymerase I from E. coli, bacteriophage T7 DNA polymerase, or DNA polymerases from bacteriophages T4, Phi-29, M2, or T5. DNA-dependent DNA polymerases may be the naturally occurring enzymes isolated from bacteria or bacteriophages or expressed recombinantly, or may be modified or "evolved" forms which have been engineered to possess certain desirable characteristics, e.g., thermostability, or the ability to recognize or synthesize a DNA strand from various modified templates. All known DNA-dependent DNA polymerases require a complementary primer to initiate synthesis. It is known that under suitable conditions a DNA- dependent DNA polymerase may synthesize a complementary DNA copy from an RNA template. RNA-dependent DNA polymerases typically also have DNA-dependent DNA polymerase activity.

DNA-dependent RNA polymerase. As used herein, a "DNA-dependent RNA polymerase" or "transcriptase" is an enzyme that synthesizes multiple RNA copies from a double-stranded or partially double-stranded DNA molecule having a promoter sequence that is usually double- stranded. The RNA molecules ("transcripts") are synthesized in the 5'-to-3' direction beginning at a specific position just downstream of the promoter. Examples of transcriptases are the DNA-dependent RNA polymerase from E. coli and bacteriophages T7, T3, and SP6.

RNA-dependent DNA polymerase. As used herein, an "RNA-dependent DNA polymerase" or "reverse transcriptase" ("RT") is an enzyme that synthesizes a complementary DNA copy from an RNA template. All known reverse transcriptases also have the ability to make a complementary DNA copy from a DNA template; thus, they are both RNA- and DNA-dependent DNA polymerases. RTs may also have an RNAse H activity. A primer is required to initiate synthesis with both RNA and DNA templates.

Selective RNAse. As used herein, a "selective RNAse" is an enzyme that degrades the RNA portion of an RNA:DNA duplex but not single-stranded RNA, double-stranded RNA or DNA. An exemplary selective RNAse is RNAse H. Enzymes possessing the same or similar activity as RNAse H may also be used. Selective RNAses may be endonucleases or exonucleases. Most reverse transcriptase enzymes contain an RNAse H activity in addition to their polymerase activities. However, other sources of the RNAse H are available without an associated polymerase activity. The degradation may result in separation of RNA from a RNA:DNA complex. Alternatively, a selective RNAse may simply cut the RNA at various locations such that portions of the RNA melt off or permit enzymes to unwind portions of the RNA. Other enzymes that selectively degrade RNA target sequences or RNA products of the present invention will be readily apparent to those of ordinary skill in the art.

Specificity. The term "specificity," in the context of an amplification system, is used herein to refer to the characteristic of an amplification 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).

Sensitivity. 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.

Relative fluorescence unit. As used herein, the term "relative fluorescence unit" ("RFU") is an arbitrary unit of measurement of fluorescence intensity. RFU varies with the characteristics of the detection means used for the measurement.

Specifically described herein are compositions, reaction mixtures, kits and methods useful in the identification of XMRV from a sample. XMRV target nucleic acids are separated from other sample components using target capture compositions, mixtures and methods described herein. Using the target capture compositions, mixtures and methods described herein, any target nucleic acid present in a sample is removed and then can be further assayed to determine its presence, or, optionally, specific identification. The further assay methods can be any known in the art that provide a desired determination of the presence or identification of a target nucleic acid. Captured target nucleic acid can be sequenced, amplified, analyzed by mass spectrometry, or otherwise assayed.

RNA target nucleic acids are typically converted to DNA and are then amplified as double stranded DNA target nucleic acids. This step is referred to as reverse transcription, and it uses an RNA-dependent DNA polymerase or reverse transcriptase ("RT"), which is an enzyme that synthesizes a complementary DNA copy from an RNA template. All known reverse transcriptases also have the ability to make a complementary DNA copy from a DNA template; thus, they are both RNA- and DNA-dependent DNA polymerases. RTs may also have an RNAse H activity. A primer is required to initiate synthesis with both RNA and DNA templates.

Amplification assays include PCR, wherein the captured target nucleic acid is contacted with a primer pair and a polymerase. (U.S. Pat. Nos. 4,683,202; 4,683,195; 4,800, 159; and 4,965,188). In its simplest form, .PCR is an in vitro method for the enzymatic synthesis of specific nucleic acid sequences, using two oligonucleotide primers that hybridize to opposite strands and flank the region of interest in the target nucleic acid. A repetitive series of reaction steps involving template denaturation, primer annealing, and the' extension of the annealed primers by polymerase results in the exponential accumulation of a specific fragment whose termini are defined by the 5' ends of the primers. PCR is capable of producing a selective enrichment of a specific DNA sequence by a factor of 10. sup.9. The PCR method is also described in Saiki et al., 1985, Science 230:1350. A variation on the PCR reaction or RT-PCR reaction is real time PCR. A common real-time PCR method is taqman PCR (US Pat. Nos. 5,210,015 and 5,538,848), though other methods are well known in the art. Compositions, reaction mixtures and methods described herein are useful for PCR amplification of XMRV target nucleic acids. Preferably, the PCR reaction is an RT-PCR amplification reaction.

Amplification assays also include isothermal amplification assays wherein the target nucleic acid is amplified under a substantially constant temperature. This is in contract to the series of temperature cycles used in PCR. Transcription Mediated Amplification (TMA) is an isothermal nucleic acid amplification that uses an RNA polymerase to produce multiple RNA transcripts from a target nucleic acid. TMA generally employs RNA polymerase and DNA polymerase activities, deoxyribonucleoside triphosphates, ribonucleoside triphosphates, and a promoter- based amplification oligomer, and optionally may include one or more other oligonucleotides, including "helper" or "blocker" oligomers. Variations of transcription-associated amplification are well known in the art and described in detail elsewhere (see U.S. Patent Nos. 5,399,491 and 5,554,516 to Kacian et al., 5,437,990 to Burg et al., 5, 130,238 to Malek et al., 4,868, 105 and 5, 124,246 to Urdea et al., PCT No. WO 93/22461 by Kacian et al., PCT Nos. WO 88/01302 and WO 88/10315 by Gingeras et al., PCT No. WO 94/03472 by McDonough et al., and PCT No. WO 95/03430 by Ryder et al.). The procedures of U.S. Patent Nos. 5,399,491 and 5,554,516 are preferred amplification embodiments.

Following sample preparation, amplification of an XMRV target was achieved by using amplification oligomers that define the 5' and 3' ends of the target sequence amplified by in vitro enzyme-mediated nucleic acid synthesis to generate an amplicon. TMA methods, substantially as described in U.S. Patent Nos. 5,399,491 and 5,554,516, produce a large number of amplification products (RNA transcripts) that can be detected. Preferred embodiments of the method used mixtures of amplification oligomers in which at least one promoter primer is combined with at least one primer.

In one embodiment, an amplification reaction is performed using at least one pair of amplification oligomers. Amplification oligomer pairs comprise target binding sequences. In some aspects, the target binding sequences are optionally combined with an additional nucleic acid region. In these aspects, the additional region of nucleic acids are arranged 5' to the target binding sequences. Two or more pairs of amplification oligomers are used for multiplex amplification reactions.

One or more of the target binding regions making up the amplification oligomers, include those that are from about 10 to about 50 nucleobases in length and are configured to specifically hybridize to a region within a target sequence of XMRV, wherein said region is from residue 4669 to residue 4738, or from 4777 to residue 4831 , or from residue 7801 to residue 7835, or from residue 7873 to residue 7998 of SEQ ID NO:85 (GenBank Accession Number EF185282.1 Gl: 121 104176 entered at NCBI on January 10, 2007). Exemplary target binding sequences include those that contain SEQ ID NOS:86 through 93. Exemplary target binding sequences include those consisting essentially of SEQ ID NOS:25-48 and 50-65. Exemplary amplification oligomer pairs include those configured to generate from SEQ ID NO:85, an amplicon of 200 nucleobases in length or less and containing a nucleotide sequence corresponding to residues 4738 to 4777 or residues 7835 to 7873 of SEQ ID NO:85. Exemplary multiplex amplification oligomers include those configured to generate from SEQ ID NO:85, at least two amplification products each of 200 nucleobases or less in length, wherein at least one contains a nucleotide sequence corresponding to residues 4738 to 4777 of SEQ ID NO:85, or wherein at least one contains a nucleotide sequence corresponding to residues 7835 to 7873 of SEQ ID NO:85, or wherein one contains a nucleotide sequence corresponding to residues 4738 to 4777 of SEQ ID NO:85 and one contains a nucleotide sequence corresponding to residues 7835 to 7873 of SEQ ID NO:85. Amplification oligomer pairs include, primer pairs, a primer member and a promoter- based amplification oligomer member, and tagged amplification oligomers.

In one embodiment of amplification oligomer combinations comprises a primer oligomer member and a promoter-based oligomer member. Preferably, a promoter-based amplification oligomer is a promoter primer comprising a 5' RNA polymerase promoter sequence and a 3' target binding sequence. RNA polymerase promoter sequences are known in the art to include, but not be limited to, sp6 RNA polymerase promoter sequences, T3 RNA polymerase promoter sequences and T7 RNA polymerase promoter sequences. In the preferred embodiments, a promoter primer comprises a 5' T7 RNA polymerase promoter sequence and a 3' target binding sequence. Most preferably, the 5' T7 RNA polymerase promoter sequence is SEQ ID NO:49.

In one embodiment, the 3' target binding sequence of a promoter-based amplification oligomer is from about 10 to about 70 nucleobases in length and comprises a nucleic acid sequence that is configured to specifically hybridize to a region within a target sequence of an XMRV nucleic acid, wherein said region is from residue 4777 to residue 4831 or from residue 7873 to residue 7998 of SEQ ID NO:85 (GenBank Accession Number EF185282.1 Gl:121 104176 entered at NCBI on January 10, 2007). Exemplary promoter based amplification oligomers comprise a target binding sequence that contains SEQ ID NOS:86, 87, 88, 92 or 93. Exemplary promoter based amplification oligomer target hybridizing sequences are those that are substantially identical to SEQ ID NOS:25 through 48. Exemplary promoter based amplification oligomers are those that are substantially identical to SEQ ID NOS:1-24. Moreover, it is recognized that insert sequences can be included with any of the promoter-based oligomer members of the current invention.

In one embodiment, the amplification oligomer combination comprises at least one primer amplification oligomer member. Primer amplification oligomers have a length that is from about 10 nucleobases to about 50 nucleobases, and have a nucleotide composition configured to specifically hybridize with XMRV to generate a detectable amplification product when used in an amplification reaction of the current invention. Primer target binding sequences include those that are configured to specifically hybridize all or a portion of a region of a target sequence of a XMRV, wherein said region corresponds to from residue 4669 to residue 4738 or from residue 7730 to residue 7756 or from residue 7801 to residue 7835 of GenBank Accession Number EF185282.1 Gl : 121 104176. Exemplary primers comprise a target binding sequence that contains SEQ ID NOS:89, 90 or 91. Exemplary primers comprise target hybridizing sequences are those that are substantially identical to SEQ ID NOS:50 through 65. Moreover, it is recognized that 5' tag sequences can be included with any of the primer oligomer members of the current invention. 5" tag sequences are sequences that are configured to not hybridize with a target nucleic acid. Tag sequences are often incorporated into amplification products to serve a function, such as primer binding sites for subsequent rounds of amplification, or other function.

Amplifying the target nucleic acid by TMA produces many strands of nucleic acid amplification product from a single copy of target nucleic acid, thus permitting detection of the target using detecting probes that hybridize to the sequences of the amplification product. Generally, the reaction mixture includes the target nucleic acid and at least two amplification oligomers comprising at least one primer, at least one promoter primer, reverse transcriptase and RNA polymerase activities, nucleic acid synthesis substrates (deoxyribonucleoside triphosphates and ribonucleoside triphosphates) and appropriate salts and buffers in solution to produce multiple RNA transcripts from a nucleic acid template. Briefly, a promoter-primer hybridizes specifically to a portion of the target sequence. Reverse transcriptase that includes RNase H activity creates a first strand cDNA by 3' extension of the promoter-primer. The cDNA is hybridized with a primer downstream from the promoter primer and a new DNA strand is synthesized from the 3' end of the primer using the reverse transcriptase to create a dsDNA having a functional promoter sequence at one end. RNA polymerase binds to dsDNA at the promoter sequence and transcribes multiple transcripts or amplicons. These amplicons are further used in the amplification process, serving as a template for a new round of replication, to ultimately generate large amounts of single-stranded amplified nucleic acid from the initial target sequence (e.g., 100 to 3,000 copies of RNA synthesized from a single template). The process uses substantially constant reaction conditions (i.e., substantially isothermal).

TMA reactions are also performed using a combination of amplification oligomers, wherein said combination comprises at least two promoter primer oligomer members and at least two primer members. One combination of amplification oligomers for performing a multiplex amplification reaction comprises an amplification oligomer pair targeting the polymerase gene of XMRV and an amplification oligomer pair targeting the LTR gene of XMRV. One combination of amplification oligomer for multiplex TMA includes a first pair of amplification oligomers configured to generate from SEQ ID NO:85 an amplification product containing a nucleotide sequence corresponding to from residue 4738 to residue 4777, and a second pair of amplification oligomers configured to generate from SEQ ID NO:85 an amplification product containing a nucleotide sequence corresponding to from residue 7835 to 7873. Amplification oligomers are preferably of a length less than 200 nucleobases.

EXAMPLES FOR XMRV PATENT APPLICATION.

Example 1 : Reagents for TMA-based assays

Unless otherwise specified, reagents commonly used in the TMA-based assays described herein include the following. Sample transport reagent: 1 10 mM lithium lauryl sulfate (LLS), 15 mM NaH₂P0₄, 15 mM Na₂HP0₄, 1 mM EDTA, 1 mM EGTA, pH 6.7. Lysis buffer: 790 mM HEPES, 230 mM succinic acid, 10% (w/v) LLS, and 680 mM LiOH monohydrate. Target Capture Reagent (TCR): lysis buffer containing 250 . micro. g/ml of paramagnetic particles (0.7- 1.05 micron particles, Sera-Mag™ MG-CM) with (dT)₁₄ oligomers covalently bound thereto and one or more target capture oligomers. Wash Solution: 10 mM HEPES, 150 mM NaCI, 6.5 mM NaOH, 1 mM EDTA, 0.3% (v/v) ethanol, 0.02% (w/v) methylparaben, 0.01 % (w/v) propylparaben, and 0.1 % (w/v) sodium lauryl sulfate, pH 7.5. Amplification reagent: typical 100 μΙ amplification reactions use 75 μΙ of an amplification reagent mixture containing 10-12 mM Tris Base, 13-15 mM Tris-HCI, 22-25.5 mM MgCI2, 22-25.5 mM KCI, 2-4.5% glycerol, 0.03 to 0.09 mM Zn-acetate (dihydrate), 0.5-1.0 mM each of dATP, dCTP, dGTP, and dTTP, 5 to 10 mM each of ATP, CTP, GTP, and UTP, pH 7) and 25 μΙ of an enzyme reagent mixture (600 to 900 U of T7 RNA polymerase, 1000-1400 U of reverse transcriptase from Moloney Murine Leukemia Virus (MMLV-RT), 15 to 18 mM HEPES (free acid, dihydrate), 50-100 mM N-acety-L-cysteine, EDTA, Na-azide, 20 to 23 mM Tris base, 50 to 60 mM KCI, 18-20% (v/v) anhydrous glycerol, 10-1 1 % (v/v) TRITON® X-102, and 150 to 180 mM trehalose (dihydrate), pH 7), preferably mixed with the captured target nucleic acid retained on the solid particles. Probe Reagent for AE-labeled probes: typically includes 100 mM succinate, 2% (w/v) LLS, 230 mM LiOH (monohydrate), 15 mM 2,2'-dithiodipyridine (ALDRITHIOL-2), 1 .2 M LiCI, 20 mM EDTA, 20 mM EGTA, 3 % (v/v) absolute ethanol, brought to about pH 4.7 with LiOH, and the selection reagent used for hydrolyzing the label on unbound probe included 600 mM boric acid, 182 mM NaOH, 1 % (v/v) TRITON® X-100. Signals are detected as relative light units (RLU) using a luminometer (e.g., LEADER™ 450HC+, Gen-Probe Incorporated, San Diego, CA). Detection Reagents for AE labels are Detect Reagent I: 1 mM nitric acid and 32 mM H₂0₂, and Detect Reagent II: 1 .5 M NaOH (see US Pat. Nos. 5,283,174, 5,656,744, and 5,658,737).

Example 2: Detection of XMRV using POL specific primers on in vitro transcribed RNA

The purpose of this experiment was to test the POL specific primers for their ability to detect and amplify in vitro transcribed XMRV RNA. Primers consisting of SEQ ID Nos. 50 to 57 (Non- T7) and SEQ ID Nos. 1 to 12 (T7) were tested.

The amplification mixture used contained 5 pmol of T7 primer and 5 pmol of nonT7 primer per reaction. 10 copies of in vitro transcribed XMRV RNA (SEQ ID NO:96) was used per reaction along with a no target control. 200 microlitres of oil was added to each tube and vortexed, followed by incubation at 62.deg.C for 10 minutes followed by 41 .5.deg.C for 20 minutes. 25 microlitres of enzyme was added to each tube, hand shaken and the incubation continued at 41 .5.deg. C for 50 minutes. 100 microlitres of probe reagent (AE labeled SEQ ID No. 66 at 7.5E6 RLU/100ul of hybridizing reagent) was added, vortexted and incubated at 62.deg.C for 20 minutes. 250 microlitres of selection reagent was then added and the tube vortexed, followed by incubation at 62.deg.C for 10 minutes. The results are shown below in Tables 1A and 1 B, representing the RLU obtained for each reaction Positive results are indicated in bold:

Table 1A: Amplification Oligomer Combinations.

Table 1 B: Amplification Oligomer Combinations.

96 different primer combinations were tested in total. The low RLU for the combinations of SEQ ID Nos. 7 to 12 and SEQ ID Nos. 50 to 57 for the 0 copies/reaction was due to the absence of auto-detection reagent. Around half of the combinations tested successfully detect as low as 10 copies per reaction.

Example 3: Specificity test using total RNA from human fetal skin using POL specific primers To investigate potential non-specificity of the primer combinations, 50 ng/reaction of total RNA from Human Fetal Skin (Agilent Technologies, Catalog # 540185) was spiked into amplification reactions containing XMRV IVT (SEQ ID NO:96). This assay was set-up and performed generally as described in Example 2.

The results for this experiment are set forth below in Table 2:

Table 2: Specificity Testing in Total RNA from Human Fetal Skin (RLU).

NT7 (column 1 ) 1 = SEQ ID No. 50; NT7 2 = SEQ ID No. 51 ; NT7 3 = SEQ ID No. 52; NT7 4 = SEQ ID No. 53; NT7 5 = SEQ ID No. 54; NT7 6 = SEQ ID No. 55; NT7 7 = SEQ ID No. 56; NT7 8 = SEQ ID No. 57.

T7 (row 1 ) 1 = SEQ ID No. 1 ; T7 2 = SEQ ID No. 2; T7 3 = SEQ ID No. 3; T7 4 = SEQ ID No. 4; T7 5 = SEQ ID No. 5; T7 6 = SEQ ID No. 6; T7 7 = SEQ ID No. 7; T7 8 = SEQ ID No. 8; T7 9 = SEQ ID No. 9; T7 10 = SEQ ID No. 10; T7 1 1 = SEQ ID No. 1 1 ; T7 12 = SEQ ID No. 12.

All 96 different primer combinations were tested for specificity against total RNA from Human Fetal Skin cells. Evaluating the RLUs obtained for each reaction, no obvious cross reaction was seen for any of the combinations that were tested.

Example 4: Further evaluation of the combination of POL specific primers SEQ ID No. 50 and SEQ ID No. 2

An amplification system comprising SEQ ID No. 50 as the non-T7 primer and SEQ ID No. 2 as the T7 primer together with SEQ ID No. 66 as the probe was selected to investigate the sensitivity of the system. This assay was set-up and performed generally as described in Example 2. The experimental results revealed that this amplification system could detect in vitro transcribed RNA from XMRV down to 16 copies per reaction with 100% positive rate; 8 copies per reaction with 90% positive rate; 4 copies per reaction with 60% positive rate; 2 copies per reaction with 40% positive rate; and 1 copy per reaction with 20% positive rate. The results were obtained without any optimisation of the system and were averaged from 10 different trials per reaction.

Example 5: Further evaluation of POL specific primer combinations

The aim of this Example was to further evaluate various selected XMRV POL primer combinations for their ability to amplify 4 copies per reaction of in vitro transcribed XMRV nucleic acid.

The amplification mixture used contained 5 pmol of T7 primer and 5pmol of nonT7 primer per reaction. 200 microlitres of oil was added to each tube and vortexed, followed by incubation at 62.deg.C for 10 minutes followed by 41.5.deg.C for 20 minutes. 25 microlitres of enzyme was added to each tube, hand shaken and the incubation continued at 41 .5.deg. C for 50 minutes. 100 microlitres of each of probe reagent (SEQ ID No. 66 at 7.5E6 RLU/100ul of hybridizing reagent) was added, vortexted and incubated at 62.deg.C for 20 minutes. 250 microlitres of selection reagent was then added and the tube vortexed, followed by incubation at 62.deg.C for 10 minutes.

Results are summarized in the following Table 3. Each combination of primers was tested 10 times and the RLUs highlighted indicate a positive result.

NT7 1 = SEQ ID No. 50; NT7 2 = SEQ ID No. 51 ; NT7 3 = SEQ ID No. 52; NT7 4 = SEQ ID No. 53; NT7 5 = SEQ ID No. 54; NT7 6 = SEQ ID No. 55; NT7 7 = SEQ ID No. 56; NT7 8 = SEQ ID No. 57.

T7 1 = SEQ ID No. 1 ; T7 2 = SEQ ID No. 2; T7 4 = SEQ ID No. 4; T7 5 = SEQ ID No. 5; T7 6 = SEQ ID No. 6; T7 7 = SEQ ID No. 7; T7 8 = SEQ ID No. 8; T7 9 = SEQ ID No. 9.

From these results it was concluded that the highest amplification was achieved using SEQ ID No. 50 and SEQ ID No. 1 (combination 1 ); SEQ ID No. 55 and SEQ ID No. 2 (combination 12); SEQ ID No. 57 and SEQ ID No. 2 (combination 14); and SEQ ID No. 57 and SEQ ID No. 4 (combination 15). Each of these amplifications showed an 80% positive detection rate for 4 copies/reaction.

Example 6: Capture probe analysis for XMRV POL assay

Four different target capture probes were investigated for use in the XMRV POL assay. Each reaction was performed 20 times and the results that are highlighted indicate positive amplification. Target capture was performed substantially as follows (described in detail in US Patent No. 6, 1 10,678). XMRV IVT was spiked into target capture reagent containing HEPES, LiOH, lithium lauryl sulfate (LLS), succinate, one of capture probes SEQ ID NOS:68-71 or no capture probe as a control, and magnetic particles attached to a poly-dT14 immobilization probe. Target capture hybridization occured in this reaction mixture by incubating the mixture at a first temperature (60.deg. C), allowing the capture oligomer to bind specifically to its complementary target sequence in XMRV. Then, the mixture was cooled to 40.deg. C or lower (e.g., room temperature) to allow the 3' tail of the capture oligomer to hybridize to its complementary oligomer on the particle. Following the second hybridization, the mixture was treated to separate the solid support with its bound complex of nucleic acids from the other components in the mixture, e.g., by using magnetic separation. Generally, separation employed a rack containing a magnet to pull the magnetic particles with bound nucleic acid complexes to the side of the tube. Then the supernatant was removed and the bound complexes on the particles were washed with a washing buffer containing HEPES, NaOH, EDTA, absolute ethanol, methyl paraben, propyl paraben, NaCI, and sodium dodecyl sulfate (SDS) by suspending the magnetic particles in washing buffer, separating particles to the tube side, and removing the supernatant. Amplification was performed as is generally described in Example 2.

The results are shown in the following Table 4: Table 4: Capture Probe Analysis (RLU)

The RLU Cut-off for a positive result was set at 100,000.

Based on these results, SEQ ID No. 70 and SEQ ID No. 71 were deemed to give the best performance, resulting in 95% and 85% positive results, respectively.

Example 7: Detection of XMRV using LTR specific primers

To further improve assay sensitivity and to reduce the risk of decreased sensitivity due to mutations at the POL amplification region, a second XMRV amplification targeting LTR region was investigated.

Initial studies began with 12 T7 primers (SEQ ID Nos. 13 to 24) and 8 non-T7 primers (SEQ ID Nos. 58 to 65). The amplification mixture used contained 5 pmol of T7 primer and 5 pmol of nonT7 primer per reaction. Target nucleic acid was an XMRV IVT (SEQ ID NO:95). 200 microlitres of oil was added to each tube and vortexed, followed by incubation at 62.deg.C for 10 minutes followed by 41 .5.deg.C for 20 minutes. 25 microlitres of enzyme was added to each tube, hand shaken and the incubation continued at 41.5.deg. C for 50 minutes. 100 microlitres of each of probe reagent (AE-labeled SEQ ID No. 67 at 7.5E6 RLU/100ul of hybridizing reagent) was added, vortexted and incubated at 62.deg.C for 20 minutes. 250 microlitres of selection reagent was then added and the tube vortexed, followed by incubation at 62.deg.C for 10 minutes. Reverse transcription was carried out for 10 minutes. Reactions were carried out using 10 copies of in vitro transcribed RNA and a 0 copy per reaction as a control.

The results are shown in the following tables 5A and 5B. Those results which are not highlighted indicate a positive result:

Table 5A: Amplification Oligomer Combinations

Table 5B: Amplification Oligomer Combinations

NT7 1 = SEQ ID No. 58; NT7 2 = SEQ ID No. 59; NT7 3 = SEQ ID No. 60; NT7 4 = SEQ ID No. 61 ; NT7 5 = SEQ ID No. 62; NT7 6 = SEQ ID No. 63; NT7 7 = SEQ ID No. 64; NT7 8 = SEQ ID No. 65.

T7 1 = SEQ ID No. 13; T7 2 = SEQ ID No.14; T7 3 = SEQ ID No.15; T7 4 = SEQ ID No. 16; T7 5 = SEQ ID No. 17; T7 6 = SEQ ID No. 18; T7 7 = SEQ ID No. 19; T7 8 = SEQ ID No. 20; T7 9 = SEQ ID No. 21 ; T7 10 = SEQ ID No. 22; T7 1 1 = SEQ ID No. 23; T7 12 = SEQ ID No. 24.

The results of this experiment indicated that about half of the primer combinations tested could potentially detect 10 copies of in vitro transcribed XMRV RNA per reaction. Following the results of these studies, seven T7 primers (SEQ ID Nos. 17, 18, 19, 21 , 22, 23 and 24) and four non-T7 primers (SEQ ID Nos. 58, 59, 60 and 61 ) were selected for further study. In this further study, all 28 amplifications tested 100% positive at 10 copies per reaction. After sensitivity testing four primer combinations: SEQ ID No. 61 and SEQ ID No. 23; SEQ ID No. 60 and SEQ ID No. 21 ; SEQ ID No. 59 and SEQ ID No. 22; and SEQ ID No. 58 and SEQ ID No. 21 were successfully tested further to 4 copies of in vitro transcribed RNA per reaction.

Example 8: Selection of one LTR primer combination for the detection of XMRV using in vitro transcribed RNA

Based on the results from previous experiments, one primer combination comprising SEQ ID No. 61 and SEQ ID No. 23 was tested further using various copies of in vitro transcribed XMRV RNA (SEQ ID NO:95).

The amplification mixture used contained 5 pmol of T7 primer and 5 pmol of nonT7 primer per reaction. 200 microlitres of oil was added to each tube and vortexed, followed by incubation at 62.deg.C for 10 minutes followed by 41.5.deg.C for 20 minutes. 25 microlitres of enzyme was added to each tube, hand shaken and the incubation continued at 41 .5.deg. C for 50 minutes. 100 microlitres of each of probe reagent (SEQ ID No. 67) at 5.0E6 RLU/100ul of hybridizing reagent) was added, vortexted and incubated at 62.deg.C for 20 minutes. 250 microlitres of selection reagent was then added and the tube vortexed, followed by incubation at 62.deg.C for 10 minutes. Reactions were carried out using 0, 1 , 2, 4, 8 and 16 copies of in vitro transcribed RNA and a 0 copy control.

The results are summarised in the Table 6 below. Table 6: Sensitivit Data using LTR Oli omer

The primer combination detected 16 copies with a 100% positive rate; 8 copies with a 100% positive rate; 4 copies with a 100% positive rate; 2 copies with a 75% positive rate; and 1 copy with a 50% positive rate. Example 9: Capture probe analysis for XMRV LTR assay

Various LTR region XMRV target capture oligonucleotides were screened using the same primer combination and generally the same amplification and detection conditions as in Example 8 (Amplification oligomers SEQ ID No. 61 and SEQ ID No. 23; detection probe oligomer SEQ ID NO:67). Four different target capture oligonucleotides were screened (SEQ ID No. 80; SEQ ID No. 79; SEQ ID No. 77; and SEQ ID No. 78). Samples contained either 0 copies/reaction of XMRV or they contained 4 copies/reaction or 8 copies/reaction of SEQ ID NO:95. Eight replicates of each condition was tested. SEQ ID NO:77 showed 0% detection of the eight 0 copies/reaction, and showed 100% detection of the eight 4 copies/reaction and 100% detection of the eight 8 copies/reaction. SEQ ID NO:79 showed 0% detection of the eight 0 copies/reaction, and showed 87.5% detection of the eight 4 copies/reaction and 100% detection of the eight 8 copies/reaction. SEQ ID NO:80 showed 0% detection of the eight 0 copies/reaction, and showed 75% detection of the eight 4 copies/reaction and 100% detection of the eight 8 copies/reaction. SEQ ID NO:78 showed 0% detection of the eight 0 copies/reaction, and showed 50% detection of the eight 4 copies/reaction and 100% detection of the eight 8 copies/reaction.

From the results of this experiment, it was concluded that the target capture oligonucleotide (SEQ ID No. 77) gave the best results in this experiment.

Example 10: LTR and POL primer multiplex XMRV assay

The purpose of this experiment was to test the POL specific primers and the LTR specific primers in a multiplex amplification reaction for their ability to detect and amplify in vitro transcribed XMRV RNA (SEQ ID NOS:95 and 96).

The POL specific amplification oligomers were SEQ ID NOS:2 and 50. The LTR specific amplification oligomers were SEQ ID NOS:2 and 21 . Detection probes were SEQ ID NOS:66 and 67. Target capture oligomers were SEQ ID NOS:70 and 77. Target capture, amplification and detection reactions were carried out as generally described above. Amplification reactions containes either 8 copies per reaction of SEQ ID NO:95 and 8 copies per reaction of SEQ ID NO:96 (8 replicates tested) or contained no XMRV (2 replicates tested). All eight positive reactions provided very high RLU signals, showing that the duplex reaction worked well at detecting the in vitro transcripts for XMRV. This duplex reaction was then tested in an XMRV viral lysates. Using the above duplex capture, amplification and detection oligomers, the sensitivity of the duplex system was evaluated. These experiments used an XMRV lysate from 22RV1 prostate carcinoma cells as the target nucleic acid (ATCC catalog # CRL-2505). An initial experiment demonstrated that the duplex system was 100% positive at about 5 c/mL lysate (data not shown). Two further experiments looked at sensitivity using six and twenty replicates per lysate level, respectively. Uniplex reactions and duplex reactions were performed as generally described herein. Table 7 summarizes the results. For uniplex L (LTR), uniplex P (POL) and duplex L + P (LTR + POL) amplification reactions the positivity rate for about 4 c/mL lysate was between 90 and 100 %.

Table 7: Viral Lysate Detection

Exemplary Sequences

SEQ ID Sequence 5' to 3'

NO:

9 AATTTAATACGACTCACTATAGGGAGATGTAGTAGGGGCTTTCTTCTCTGTCGA

10 AATTTAATACGACTCACTATAGGGAGAGTAGTAGGGGCTTTCTTCTCTGTCGA

11 AATTTAATACGACTCACTATAGGGAGACATGTAGTAGGGGCTTTCTTCTCTGTCG

12 AATTTAATACGACTCACTATAGGGAGAGTAGGGGCTTTCTTCTCTGTCG

13 AATTTAATACGACTCACTATAGGGAGAgcccaggtgcctgactacag

14 AATTTAATACGACTCACTATAGGGAGAgcccaggtgcctgactaca

15 AATTTAATACGACTCACTATAGGGAGAggcccaggtgcctgactac

16 AATTTAATACGACTCACTATAGGGAGagtttcgctttatctgaggacc

17 AATTTAATACGACTCACTATAGGGAGAgttagtttcgctttatctgaggac

18 AATTTAATACGACTCACTATAGGGAGAgttgttagtttcgctttatctgagga

19 AATTTAATACGACTCACTATAGGGAGAgactttccagaaactgttgtta

20 AATTTAATACGACTCACTATAGGGAGAaactgaggtgggactttcca

21 AATTTAATACGACTCACTATAGGGAGacttgaaactgaggtgggactttcc

22 AATTTAATACGACTCACTATAGGGAGAgtatttcccggtcttttggggaacttg

23 AATTTAATACGACTCACTATAGGGAGAgtatttcccggtcttttggggaactt

24 AATTTAATACGACTCACTATAGGGAGAgtatttcccggtcttttggggaact

25 GTGCCTTCATCTTTTGAGGGCTC

26 GGAGTGCCTTCATCTTTTGAGGGCTC

27 GGAGTGCCTTCATCTTTTGAGG

28 GTCGAGGAGTGCCTTCATCTTTTGAGG

29 GTCGAGGAGTGCCTTCATCTTTTGAG

30 CTGTCGAGGAGTGCCTTCATCTTTTGAG

31 GTAGTAGGGGCTTTCTTCTCTGTCGAG

32 GTAGGGGCTTTCTTCTCTGTCGAG

33 TGTAGTAGGGGCTTTCTTCTCTGTCGA

34 GTAGTAGGGGCTTTCTTCTCTGTCGA

35 CATGTAGTAGGGGCTTTCTTCTCTGTCG

36 GTAGGGGCTTTCTTCTCTGTCG

37 gcccaggtgcctgactacag

38 gcccaggtgcctgactaca

39 ggcccaggtgcctgactac

40 gtttcgctttatctgaggacc

41 gttagtttcgctttatctgaggac

42 gttgttagtttcgctttatctgagga

43 gactttccagaaactgttgtta

44 aactgaggtgggactttcca

45 acttgaaactgaggtgggactttcc

46 gtatttcccggtcttttggggaacttg

47 gtatttcccggtcttttggggaactt

48 gtatttcccggtcttttggggaact

49 AATTTAATACGACTCACTATAGGGAGA

50 ATGCCCGATCAGTCCGTG

51 TGATGCCCGATCAGTCCGTG

52 GATGCCCGATCAGTCCGT

53 TGATGCCCGATCAGTCCG

54 TGTGATGCCCGATCAGTCCG

55 GTGATGCCCGATCAGTCC

56 TGGGTCCTACAAGGCAAAC

57 AGCCACCTACAATCAGACA

58 CAAAAGTTACAAGGAAGTTTAATTAAAGAATAA SEQ ID Sequence 5' to 3'

NO:

59 CAAAAGTTACAAGGAAGTTTAATTAAAGAATA

60 CTCAAAAGTTACAAGGAAGTTTAAT

61 CTCAAAAGTTACAAGGAAGTTTAA

62 GCTTAGCACGCTAGCTAC

63 GGCTTAGCACGCTAGCTA

64 CACCATAAGGCTTAGCACG

65 CACCATAAGGCTTAGCAC

66 ACUCCCUACACAGACUCAC

67 CUGAAUAACACUGGGACAGG

68 UGGCUUUGCUGGCAUUUACTTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

69 UGCCCCAAUUUUGGCUUUGCUGGCATTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

70 UCCUUUUGUCUGAUUGUAGGUGGCTTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

71 AGUUCCCGUAGUCUUUUGAGAUCTTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

72 TTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

73 UGGCUUUGCUGGCAUUUAC

74 UGCCCCAAUUUUGGCUUUGCUGGCA

75 UCCUUUUGUCUGAUUGUAGGUGGC

76 AGUUCCCGUAGUCUUUUGAGAUC

77 GGGCUAGGACUGGGGAGCATTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

78 GAGUGUGGAGUUCUUACCCCUTTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

79 CUGGUACUUUUCCAUGCCTTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

80 GUUUACUGUAGCUAGCGUGCUAAGTTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

81 GGGCUAGGACUGGGGAGCA

82 GAGUGUGGAGUUCUUACCCCU

83 CUGGUACUUUUCCAUGCC

84 GUUUACUGUAGCUAGCGUGCUAAG

85 See GenBank Accession Number EF185282.1 GI : 121104176 entered at NCBI on January 10, 2001

86 CTCAAAAGATGAAGGCAC

87 CCTCAAAAGATGAAGGCAC

88 TCGACAGAGAAGAAAGCCCCTAC

89 ATGCCCGATCAGTCC

90 GCTTAGCAC

91 CAAAAGTTACAAGGAAGTTTAA

92 GTAGTCAGGCACCTGGGC

93 GTCCTCAGATAAAGCGAAAC

94 TGCCAGCAAAGCCA

SEQ ID NOS: 1-24 are T7 promoter based amplification oligomers; SEQ ID NOS:25-48 and 50- 65 are amplification oligomer target binding sequences; SEQ ID NO:49 is a T7 promoter sequence; SEQ ID NOS:66-67 are probe target binding sequences; SEQ ID NOS:68-71 and 77- 80 are target capture oligomers; SEQ ID NO:72 is a capture region of a target capture oligomer; SEQ ID NOS:73-76 and 81-84 are target binding sequences for target capture oligomers; SEQ ID NOS: 86-93 are consensus sequences for amplification oligomer target binding sequences; SEQ ID NO: 94 is a consensus sequence for target capture oligomer target binding sequences.

XMRV IVT sequence (LTR) SEQ ID NO:95 GGGCGAAUUGGGUACCGAUAUUGGAGAUGGUUGCCGCUCUCCCGGGGGAAGAAAAAGGACAAGACUAUAUGAUUUCU AUGUUUGCCCCGGUCAUACUGUAUUAACAGGGUGUGGAGGGCCGAGAGAGGGCUACUGUGGCAAAUGGGGAUGUGAG ACCACUGGACAGGCAUACUGGAAGCCAUCAUCAUCAUGGGACCUAAUUUCCCUUAAGCGAGGAAACACUCCUAAGGG UCAGGGCCCCUGUUUUGAUUCCUCAGUGGGCUCCGGUAGCAUCCAGGGUGCCACACCGGGGGGUCGAUGCAACCCCC UAGUCCUAGAAUUCACUGACGCGGGUAAAAGGGCCAGCUGGGAUGCCCCCAAAACAUGGGGACUAAGACUGUAUCGA UCCACUGGGGCCGACCCGGUGACCCUGUUCUCUCUGACCCGCCAGGUCCUCAAUGUAGGGCCCCGCGUCCCCAUUGG GCCUAAUCCCGUGAUCACUGAACAGCUACCCCCCUCCCAACCCGUGCAGAUCAUGCUCCCCAGGCCUCCUCGUCCUC CUCCUUCAGGCGCGGCCUCUAUGGUGCCUGGGGCUCCCCCGCCUUCUCAACAACCUGGGACGGGAGACAGGCUGCUA AACCUGGUAGAAGGAGCCUACCAAGCCCUCAACCUCACCAGUCCCGACAAAACCCAAGAGUGCUGGCUGUGUCUAGU AUCGGGACCCCCCUACUACGAAGGGGUGGCCGUCCUAGGUACUUACUCCAACCAUACCUCUGCCCCGGCUAACUGCU CCGUGACCUCCCAACACAAGCUGACCCUGUCCGAAGUGACCGGGCAGGGACUCUGCAUAGGAGCAGUUCCCAAAACC CAUCAGGCCCUGUGUAAUACCACCCAGAAGACGAGCGACGGGUCCUACUAUUUGGCCUCUCCCGCCGGGACCAUUUG GGCUUGCAGCACCGGGCUCACUCCCUGUCUAUCUACUACUGUGCUUAACUUAACCACUGAUUACUGUGUCCUGGUUG AACUCUGGCCAAAGGUAACCUACCACUCCCCUAAUUAUGUUUAUGGCCAGUUUGAAAAGAAAACUAAAUAUAAAAGA GAGCCGGUGUCAUUAACUCUGGCCCUGCUGUUGGGAGGACUUACUAUGGGCGGCAUAGCUGCAGGAGUUGGAACAGG GACUACAGCCCUAGUGGCCACCAAACAAUUCGAGCAGCUCCAGGCAGCCAUACAUACAGACCUUGGGGCCUUAGAAA AAUCAGUCAGUGCCCUAGAAAAGUCUCUGACCUCGUUGUCUGAGGUGGUCCUACAGAACCGGAGGGGAUUAGAUCUA CUGUUCCUAAAAGAAGGAGGAUUAUGUGCUGCCCUAAAAGAAGAAUGCUGUUUUUACGCGGACCACACUGGCGUAGU AAGAGAUAGCAUGGCAAAGCUAAGAGAAAGGUUAAACCAGAGACAAAAAUUGUUCGAAUCAGGACAAGGGUGGUUUG AGGGACUGUUUAACAGGUCCCCAUGGUUCACGACCCUGAUAUCCACCAUUAUGGGCCCUCUGAUAGUACUUUUAUUA AUCCUACUCUUCGGACCCUGUAUUCUCAACCGCUUGGUCCAGUUUGUAAAAGACAGAAUUUCGGUAGUGCAGGCCCU GGUUCUGACCCAACAGUAUCACCAACUCAAAUCAAUAGAUCCAGAAGAAGUGGAAUCACGUGAAUAAAAGAUUUUAU UCAGUUUCCAGAAAGAGGGGGGAAUGAAAGACCCCACCAUAAGGCUUAGCACGCUAGCUACAGUAACGCCAUUUUGC AAGGCAUGGAAAAGUACCAGAGCUGAGUUCUCAAAAGUUACAAGGAAGUUUAAUUAAAGAAUAAGGCUGAAUAACAC UGGGACAGGGGCCAAACAGGAUAUCUGUAGUCAGGCACCUGGGCCCCGGCUCAGGGCCAAGAACAGAUGGUCCUCAG AUAAAGCGAAACUAACAACAGUUUCUGGAAAGUCCCACCUCAGUUUCAAGUUCCCCAAAAGACCGGGAAAUACCCCA AGCCUUAUUUAAACUAACCAAUCAGCUCGCUUCUCGCUUCUGUACCCGCGCUUUUUGCUCCCCAGUCCUAGCCCUAU AAAAAAGGGGUAAGAACUCCACACUCGGCGCGCCAGUCAUCCGAUAGACUGAGAAGCUUGAUAUCGAAUUCCUGCAG CCCGGGGGAUCCACUAGUUCUAGAGCGGCC

XMRV IVT sequence (POL) SEQ ID NO:96

GGGCGAAUUGGGUACCGAUACAGACCGGGUUCAGUUCGGACCGGUGGUGGCCCUCAACCCGGCCACCCUGCUCCCCC UACCGGAAAAGGAAGCCCCCCAUGACUGCCUCGAGAUCUUGGCUGAGACGCACGGAACCAGACCGGACCUCACGGAC CAGCCCAUCCCAGACGCUGAUUACACUUGGUACACAGAUGGAAGCAGCUUCCUACAAGAAGGACAACGGAGAGCUGG AGCAGCGGUGACUACUGAGACCGAGGUAAUCUGGGCGAGGGCUCUGCCGGCUGGAACAUCCGCCCAACGAGCCGAAC UGAUAGCACUCACCCAAGCCUUAAAGAUGGCAGAAGGUAAGAAGCUAAAUGUUUACACUGAUAGCCGCUAUGCCUUC GCCACGGCCCAUGUCCAUGGAGAAAUAUAUAGGAGGCGAGGGUUGCUGACCUCAGAAGGCAGAGAAAUUAAAAACAA GAACGAGAUCUUGGCCUUGCUAAAAGCUCUCUUUCUGCCCAAACGACUUAGUAUAAUUCACUGUCCAGGACAUCAAA AAGGAAACAGUGCUGAGGCCAGAGGCAACCGUAUGGCAGAUCAAGCAGCCCGAGAGGCAGCCAUGAAGGCAGUUCUA GAAACCUCUACACUCCUCAUAGAGGACUCAACCCCGUAUACGCCUCCCCAUUUCCAUUACACCGAAACAGAUCUCAA AAGACUACGGGAACUGGGAGCCACCUACAAUCAGACAAAAGGAUAUUGGGUCCUACAAGGCAAACCUGUGAUGCCCG AUCAGUCCGUGUUUGAACUGUUAGACUCCCUACACAGACUCACCCAUCUGAGCCCUCAAAAGAUGAAGGCACUCCUC GACAGAGAAGAAAGCCCCUACUACAUGUUAAACCGGGACAGAACUAUCCAGUAUGUGACUGAGACCUGCACCGCCUG UGCCCAAGUAAAUGCCAGCAAAGCCAAAAUUGGGGCAGGGGUGCGAGUACGCGGACAUCGGCCAGGCACCCAUUGGG AAGUUGAUUUCACGGAAGUAAAGCCAGGACUGUAUGGGUACAAGUACCUCCUAGUGUUUGUAGACACCUUCUCUGGC UGGGUAGAGGCAUUCCCGACCAAGCGGGAAACUGCCAAGGUCGUGUCCAAAAAGCUGUUAGAAGACAUUUUUCCGAG AUUUGGAAUGCCGCAGGUAUUGGGAUCUGAUAACGGGCCUGCCUUCGCCUCCCAGGUAAGUCAGUCAGUGGCCGAUU UACUGGGGAUCGAUUGGAAGUUACAUUGUGCUUAUAGACCCCAGAGUUCAGGACAGGUAGAAAGAAUGAAUAGAACA AUUAAGGAGACUUUGACCAAAUUAACGCUUGCAUCUGGCACUAGAGACUGGGUACUCCUACUCCCCUUAGCCCUCUA CCGAGCCCGGAAUACUCCGGGCCCCCACGGACUGACUCCGUAUGAAAUUCUGUAUGGGGCACCCCCGCCCCUUGUCA AUUUUCAUGAUCCUGAAAUGUCAAAGUUAACUAAUAGUCCCUCUCUCCAAGCUCACUUACAGGCCCUCCAAGCAGUA CAACAAGAGGUCUGGAAGCCGCUGGCCGCUGCUUAUCAGGACCAGCUAGAUCAGCCAGUGAUACCACACCCCUUCCG UGUCGGUGACGCCGUGUGGGUACGCCGGCACCAGACUAAGAACUUAGAACCUCGCUGGAAAGGACCCUACACCGUCC UGCUGACAACCCCCACCGCUCUCAAAGUAGACGGCAUCUCUGCGUGGAUACACGCCGCUCACGUAAAGGCGGCGACA ACUCCUCCGGCCGGAACAGCAUGGAAAGUCCAGCGUUCUCAAAACCCCUUAAAGAUAAGAUUAACCCGUGGGGCCCC CUGAUAAUUAUGGGGAUCUUGGUGAGGGCAGGAGCCUCAGUACAACGUGACAGCCCUCACCAGGUCUUUAAUGUCAC UUGGAAAAUUACCAACCUAAUGACAGGACAAACAGCUAAUGCUACCUCCCUCCUGGGGACGAUGACAGACACUUUCC CUAAACUAUAUUUUGACUUGUGUGAUUUAGUUGGAGACAACUGGGAUGACCCGGAACCCGAUAUUGGAGAUGGUUGC CGCUCUCCCGGGGGAAGAAAAAGGACAAGACUAUAUGAUUUCUAUGUUUGCCCCGGUCAUACUGUAUUAACAGGGUG UGGAGGGCCGAGAGAGGGCUACUGUGGCAAAUGGGGAUGUGAGACCACUGGACAGGCAUACUGGAAGCCAUCAUCGG AAUU

Claims

Claims We claim:

1. A method for the amplification and identification of an XMRV from a sample comprising the steps of: a. contacting a sample suspected of containing XMRV with at least two amplification oligomers for generating an amplicon, wherein each of said at least two amplification oligomers is from 10 to about 50 nucleobases in length and wherein the pair are respectively configured to specifically hybridize to regions within a target sequence of XMRVselected from the group consisting of from residue 4669 to residue 4738 and from 4777 to residue 4831 of SEQ ID NO:85, or from residue 7801 to residue 7835 and from residue 7873 to residue 7998 of SEQ ID NO:85; b. providing conditions sufficient for generating an amplicon from an XMRV target nucleic acid present in said sample using said amplification oligomers from step a; c. providing conditions for detecting said amplicon and determining whether said sample contained XMRV target nucleic acid.

2. The method of claim 1, wherein one of said at least two amplification oligomers comprises a target hybridizing sequence that contains a sequence selected from the group consisting of SEQ ID NOS: 86-93.

3. The method of claim 1, wherein said amplification oligomer pair comprises a first amplification oligomer comprising a target binding sequence containing SEQ ID NO: 89 and a second amplification oligomer comprising a target binding sequence containing SEQ ID NO: 86, 87 or 88.

4. The method of claim 1, wherein said amplification oligomer pair comprises a first amplification oligomer comprising a target binding sequence containing SEQ ID NO:91 and a second amplification oligomer comprising a target binding sequence containing SEQ ID NO:92 or 93.

5. The method of claim 1, wherein said amplification oligomer pair comprises a first amplification oligomer comprising a target binding sequence containing one of SEQ ID NOS:25- 36 and a second amplification oligomer comprising a target binding sequence containing one of SEQ ID NOS:50-57.

6. The method of claim 1, wherein said amplification oligomer pair comprises a first amplification oligomer comprising a target binding sequence containing one of SEQ ID NOS:37- 48 and a second amplification oligomer comprising a target binding sequence containing one of SEQ ID NOS:58-65.

7. The method of claim 1, wherein said amplification oligomer pair is one of SEQ ID NOS: l-12 and one of SEQ ID NOS:50-57.

8. The method of claim 1, wherein said amplification oligomer pair is one of SEQ ID NOS: 13-24 and one of SEQ ID NOS:58-65.

9. The method of any one of claim 1-8, wherein said amplicon is detected in real-time.

10. The method of any one of claims 1-8, wherein said amplicon is detected using a detection probe oligomer.

11. The method of claim 10, wherein said detection probe oligomer is labeled with a chemiluminescent compound.

12. The method of Any one of claims 1-11, wherein said amplification reaction is substantially isothermal.

13. The method of any one of claims 1-12, wherein said sample is human blood donated for transfusion into an individual.

14. The method of any one of claims 1-12, wherein said sample is human blood donated for use by a human blood bank.

15. The method of any one of claims 1-12, wherein said sample is human blood.

16. A method for the multiplex amplification and identification of an XMRV from a sample comprising the steps of: a. contacting a sample suspected of containing XMRV with at least two pair of amplification oligomers for generating separate amplicons from an XMRV target nucleic acid, wherein each of amplification oligomers is from 10 to about 50 nucleobases in length and wherein a first amplification oligomer pair is configured to specifically hybridize to regions within a target sequence of XMRV consisting of from residue 4669 to residue 4738 and from 4777 to residue 4831 of SEQ ID NO:85, and wherein a second amplification oligomer pair is configured to specifically hybridize to regions within a target sequence of XMRV consisting of from residue 7801 to residue 7835 and from residue 7873 to residue 7998 of SEQ ID NO:85; b. providing conditions sufficient for generating amplicons from an XMRV target nucleic acid present in said sample using said amplification oligomers from step a; c. providing conditions for detecting said amplicon and determining whether said sample contained XMRV target nucleic acid.

17. The method of claim 16, wherein one of said amplification oligomers comprises a target hybridizing sequence that contains a sequence selected from the group consisting of SEQ ID NOS:86-93.

18. The method of claim 16, wherein said first amplification oligomer pair comprises a first amplification oligomer comprising a target binding sequence containing SEQ ID NO: 89 and a second amplification oligomer comprising a target binding sequence containing SEQ ID NO: 86, 87 or 88.

19. The method of claim 16, wherein said second amplification oligomer pair comprises a first amplification oligomer comprising a target binding sequence containing SEQ ID NO:91 and a second amplification oligomer comprising a target binding sequence containing SEQ ID NO: 92 or 93.

20. The method of claim 16, wherein said first amplification oligomer pair comprises a first amplification oligomer comprising a target binding sequence containing one of SEQ ID NOS:25- 36 and a second amplification oligomer comprising a target binding sequence containing one of SEQ ID NOS:50-57.

21. The method of claim 16, wherein said second amplification oligomer pair comprises a first amplification oligomer comprising a target binding sequence containing one of SEQ ID NOS:37-48 and a second amplification oligomer comprising a target binding sequence containing one of SEQ ID NOS:58-65.

22. The method of claim 16, wherein said first amplification oligomer pair is one of SEQ ID NOS: l-12 and one of SEQ ID NOS:50-57.

23. The method of claim 16, wherein said second amplification oligomer pair is one of SEQ ID NOS: 13-24 and one of SEQ ID NOS:58-65.

24. The method of any one of claim 16-23, wherein said amplicon is detected in real-time.

25. The method of any one of claims 16-23, wherein said amplicon is detected using a detection probe oligomer for one or both amplicon species generated by the first amplification oligomer pair and the second amplification oligomer pair.

26. The method of claim 25, wherein a detection probe oligomer is labeled with a chemiluminescent compound.

27. The method of any one of claims 16-26, wherein said amplification reaction is substantially isothermal.

28. The method of any one of claims 16-27, wherein said sample is human blood donated for transfusion into an individual.

29. The method of any one of claims 16-27, wherein said sample is human blood donated for use to a human blood bank.

30. The method of any one of claims 16-27, wherein said sample is human blood.

31. A composition used in any one of the methods in claims 1-30.

32. A primer member used in any one of the methods in claims 1-30.

33. A promoter based amplification oligomer used in any one of the methods in claims 1-30.

34. A kit containing one or more compositions for use in any one of claims 1-30.

35. A reaction mixture containing one or more compositions for use in any one of claims 1- 30.