Compositions and methods for amplifying, detecting or quantifying human cytomegalovirus

Abstract

To provide compositions and methods for amplifying, detecting or quantifying a human cytomegalovirus.SOLUTION: Oligomer nucleotides, compositions, methods, kits and uses are provided for detecting or quantifying a Human Cytomegalovirus virus 1 (CMV (human herpesvirus 5, HHV5)) nucleic acid, e.g., using nucleic acid amplification and hybridization assays. Multiphase amplification of a CMV target sequence is also described. The oligomer nucleotides, compositions, methods, kits and uses can be used to amplify and / or detect the UL56 gene of CMV.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 720,658, filed August 21, 2018, which is incorporated herein by reference.

[0002] Sequence Listing The sequence listing set forth in the application 535372_SeqListing_ST25.txt is 17 kilobytes in size, was created on August 21, 2019, and is incorporated herein by reference. [Background technology]

[0003] Human cytomegalovirus (CMV, also known as human herpesvirus 5 (HHV5)) is part of a larger family of viruses that includes herpes simplex virus (HSV), varicella-zoster virus (VZV), and Epstein-Barr virus (EBV). CMV is an enveloped, double-stranded DNA virus that causes infection in humans. CMV is a common virus that can infect almost anyone. It is so common that nearly all adults in developing countries and 50% to 85% of adults in the United States are infected. CMV is transmitted from person to person through bodily fluids such as blood, saliva, urine, semen, vaginal fluid, and breast milk. Like other herpesviruses, CMV establishes lifelong latency that can reactivate intermittently. No cure exists. In immunocompetent hosts, CMV infection is generally asymptomatic and self-limiting.

[0004] CMV infection is a cause of concern in pregnant women, infants, and immunocompromised individuals. Active CMV infection during pregnancy can transmit the virus to infants. CMV infection is a significant cause of morbidity and mortality, especially in people with weakened immune systems due to organ transplants. However, drug treatments may be useful in treating newborns and people with weakened immune systems.

[0005] In solid organ transplant (SOT) recipients, CMV transmitted from the donor (D) organ to the recipient (R) can cause primary infection in CMV-seronegative SOT recipients (R-) or reinfection in CMV-seropositive SOT recipients (R+). In D- / R+ SOT recipients, impairment of CMV-specific immunity due to immunosuppression can lead to reactivation of endogenous latent CMV. Because D- / R+ SOT recipients lack preexisting host immunity, they are at high risk for developing CMV disease, while R+ recipients constitute an intermediate-risk group (Razonable 2013). Once infected, CMV cannot be eradicated from the body due to its tendency for lifelong latency. Therefore, the goal of CMV therapy in SOT patients is to prevent the indirect effects of CMV infection on the graft and / or the development of CMV disease by suppressing viral replication. Viral load testing is the primary method for diagnosing active disease due to CMV infection and has become a routine component of transplant recipient care (Rychert J., et.al. 2014).

[0006] Testing is important in pregnant women and those with weakened or compromised immune systems. Current diagnostic tests look for anti-CMV antibodies. However, such tests require multiple tests for accuracy, and individuals must be symptomatic. Additional diagnostic tests include culture, PCR, and the CMV pp65 antigenemia assay. The CMV pp65 antigenemia assay, which quantifies the number of CMV-infected leukocytes in peripheral blood, has been used to detect and monitor CMV infection in immunocompromised patients.

[0007] Thus, there is a need for compositions and methods that allow for the sensitive and specific detection and quantification of CMV. The present disclosure is directed to meeting these needs, providing other benefits, or at least providing the public with a useful choice. Summary of the Invention [Means for solving the problem]

[0008] Amplification oligomers, nucleic acids, methods, compositions, and kits are described for detecting and / or quantifying human cytomegalovirus (CMV) in a sample or amplifying CMV UL56 gene sequences. The amplification oligomers include forward primers, reverse primers, promoter primers (e.g., T7 primers), non-promoter primers (e.g., NT7 primers), helper oligomers, and displacer oligomers. Probe oligomers and target capture oligomers (TCOs) are also described, which facilitate detection of the amplified sequences and isolation of CMV nucleotide sequences from a sample, respectively. The method involves amplifying viral nucleic acids to detect CMV target sequences in a sample. The method can advantageously provide highly sensitive detection of CMV.

[0009] The amplification oligomers can be used to amplify, detect, and / or quantify CMV sequences using any nucleic acid amplification method known in the art. Nucleic acid amplification methods may use thermal cycling or may be isothermal. Nucleic acid amplification methods known in the art include, but are not limited to, polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR), nucleic acid sequence-based amplification (NASBA), replicase-mediated amplification (including Qβ-replicase-mediated amplification), ligase chain reaction (LCR), strand displacement amplification (SDA), isothermal transcription-associated amplification, and polyphase isothermal transcription-associated amplification.

[0010] The described amplification oligomers can be used to amplify CMV sequences. The amplified CMV sequence, the amplicon, comprises all or a portion of SEQ ID NO: 1 and / or its complement. The amplification oligomers are configured to amplify and optionally detect a CMV UL56 gene amplicon comprising all or a portion of SEQ ID NO: 1 and / or its complement. In some embodiments, the amplicon comprises SEQ ID NO: 51 and / or its complement and / or SEQ ID NO: 53 and / or its complement. The amplicon can be DNA or RNA. Various methods in the art can be used to detect CMV amplicons.

[0011] In some embodiments, the forward primer or non-promoter primer comprises 19-31 contiguous nucleic acid bases having at least 80% identity to the nucleotide sequence present in SEQ ID NO:2. In some embodiments, the non-promoter primer is an amplification oligonucleotide that specifically binds downstream of the promoter primer end to its target sequence in the cDNA product of promoter primer extension. The promoter primer is combined with the non-promoter primer to form an amplification pair, which are configured to together amplify a portion of the target nucleic acid. In some embodiments, the forward primer or non-promoter primer comprises the nucleotide sequence of SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:19. The forward primer or non-promoter primer can hybridize to SEQ ID NO:79 and initiate DNA or RNA polymerization. Exemplary forward primers and non-promoter primers are provided in Table 1B.

[0012] In some embodiments, the reverse primer or promoter primer comprises 21 to 40 consecutive nucleic acid bases having at least 80% identity to the nucleotide sequence present in SEQ ID NO:3. In some embodiments, the reverse primer or promoter primer comprises the nucleotide sequence of SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, or SEQ ID NO:47. The reverse primer or promoter primer can hybridize to SEQ ID NO:80 and initiate DNA or RNA polymerization. Exemplary reverse primers and promoter primers are provided in Table 1B.

[0013] An RNA polymerase promoter sequence can be added to any of the forward and / or reverse primers described to form a promoter primer. The RNA polymerase primer sequence is operably linked to the 5' end of the forward or reverse primer described. In some embodiments, the RNA polymerase promoter sequence is linked to the 5' end of the reverse primer. The RNA polymerase promoter sequence is selected from the group consisting of T7 The promoter primer may be, but is not limited to, an RNA polymerase promoter sequence. The T7 RNA polymerase promoter sequence may contain the nucleotide sequence of SEQ ID NO: 78. A promoter primer having a T7 polymerase promoter sequence is referred to as a T7 primer. In some embodiments, the promoter primer or T7 primer comprises the nucleotide sequence of SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, or SEQ ID NO: 46.

[0014] In some embodiments, a helper oligomer promotes or enhances hybridization of a forward primer to a template nucleotide sequence. Similarly, in some embodiments, a displacer oligomer promotes or enhances hybridization of a reverse primer to a template nucleic acid sequence. Exemplary helper and displacer oligomers are provided in Table 1B. Promotion or enhancement of primer hybridization to a template can promote or enhance amplification of a target nucleotide sequence. When used to promote hybridization of a forward and / or reverse primer, the helper oligomer and displacer oligomer can be blocked. When blocked, the helper or displacer oligomer cannot prime polymerization from its 3' end. In some embodiments, the helper and / or displacer oligomer can be a forward primer or a reverse primer. In some embodiments, the described helper and / or displacer oligomer can have an RNA polymerase promoter sequence linked to the 5' end of the helper or displacer oligomer to form a promoter primer. In some embodiments, the helper oligomer comprises SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19. In some embodiments, the displacer oligomer comprises SEQ ID NO: 25, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 6, SEQ ID NO: 41, or SEQ ID NO: 12. Exemplary helper and displacer oligomers are provided in Table 1B.

[0015] The described probe oligomers (also referred to as detection oligomers) can be used to detect CMV amplicons. In some embodiments, the probe oligomer comprises 24-35 consecutive nucleobases having at least 90% identity to the nucleotide sequence present in SEQ ID NO:4. In some embodiments, the probe oligomer comprises 24-35 consecutive nucleobases that hybridize to SEQ ID NO:81. In some embodiments, the probe oligomer comprises the nucleotide sequence of SEQ ID NO:51, SEQ ID NO:52, or SEQ ID NO:57, wherein one or more uracil nucleotides can be substituted for thymine nucleotides. In some embodiments, the probe oligomer contains a hairpin. The hairpin can comprise 4-5 nucleobases complementary to each other at the 5' and 3' ends of the probe oligomer. Exemplary probe oligomers are provided in Table 1C. The probe oligomer can have one or more modified nucleotides. For any of the probe oligomers described, one or more nucleotides in the probe oligomer can be substituted with ribonucleotides, 2'-O-methylribonucleotides, or a combination of ribonucleotides and 2'-O-methylribonucleotides. In some embodiments, the probe oligomer can have 1, 2, 3, 4, 5, 6, 7, or more thymidines substituted for uridine. In some embodiments, all thymidines in the probe oligomer can be substituted with uridine. In some embodiments, the probe oligomer can have 1, 2, 3, 4, 5, 6, 7, or more uridines substituted for thymidines. In some embodiments, all uridines in the probe oligomer can be substituted with thymidines. In some embodiments, one or more of the uridines are 2'-O-methylribonucleotides. In some embodiments, all of the uridines are 2'-O-methylribonucleotides.

[0016] The probe oligomer can contain one or more detectable markers or labels. The detectable marker can be, but is not limited to, a fluorescent molecule. The fluorescent molecule can be attached to the 5' or 3' end of the probe oligomer, or anywhere along the oligomer. In some embodiments, the probe oligomer can be a molecular beacon or torch. The probe oligomer can contain a fluorescent molecule attached to the 5' end of the probe oligomer and a quencher attached to the 3' end of the probe oligomer, or the fluorescent molecule can be attached to the 3' end of the probe oligomer and the quencher can be attached to the 5' end of the probe oligomer.

[0017] The described target capture oligomers (TCOs) can be used to capture or isolate target CMV sequences from a sample. The CMV TCOs contain a target-specific (TS) nucleotide sequence that hybridizes (i.e., is complementary to) a region of a target nucleotide sequence within the CMV. In some embodiments, the TCO TS sequence contains a 10-35 nucleotide sequence that has at least 90%, at least 95%, or 100% complementarity with a nucleotide sequence present in the target nucleic acid and hybridizes to a region within the target nucleic acid sequence (the TCO binding site). In some embodiments, the TCO TS sequence is 20-30 nucleotides in length. In some embodiments, the TCO TS sequence is 22-26 nucleotides in length and has at least 90% complementarity with a nucleotide sequence present in the target nucleic acid. The TCO TS and TCO binding site can be fully complementary or have one or more mismatches. The TCO contains an immobilized capture probe binding region that binds to an immobilized capture probe (e.g., via a specific binding pair interaction). Members of a specific binding pair (or binding partner) are moieties that specifically recognize and bind to each other. The members are sometimes referred to as a first binding pair member (BPM1) and a second binding pair member (BPM2), which represent the various moieties that specifically bind together. Specific binding pairs are exemplified by, for example, a receptor and its ligand, an enzyme and its substrate, a cofactor or coenzyme, an antibody or Fab fragment and its antigen or ligand, a sugar and a lectin, biotin and streptavidin or avidin, a ligand and a chelator, a protein or amino acid and its specific binding metal (such as histidine and nickel), substantially complementary polynucleotide sequences containing fully or partially complementary sequences, and complementary homopolymer sequences. Specific binding pairs can be naturally occurring (e.g., an enzyme and a substrate), synthetic (e.g., a synthetic receptor and a synthetic ligand), or a combination of a naturally occurring BPM and a synthetic BPM. In some embodiments, both the TS sequence and the immobilized capture probe binding region are nucleic acid sequences.The TS sequence and the capture probe binding region may be covalently linked to each other or may be on different oligonucleotides connected by one or more linkers. In some embodiments, the capture probe binding region comprises a polyA sequence, a polyT sequence, or a polyT-polyA sequence. In some embodiments, the polyT-polyA sequence is (dT)3(dA). 30 One or more TCOs may be used in the target capture and / or amplification reaction. The one or more TCOs may bind to the same target sequence or different target sequences. The target sequences may be derived from the same gene or different genes and / or from the same organism or different organisms. In some embodiments, the CMV TCO comprises the nucleotide sequence of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:43, or SEQ ID NO:45, or a nucleic acid sequence having at least 90% identity to SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:43, or SEQ ID NO:45. In some embodiments, the CMV TCO containing a polyA sequence comprises SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:42, or SEQ ID NO:44, or a nucleic acid sequence having at least 90% identity to SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:42, or SEQ ID NO:44. Exemplary probe oligomers are provided in Table ID. The TCO may have one or more modified nucleotides. For any of the TCOs described, one or more cytidines in the TCO can be substituted for 5'-methyl dC. The TCO can have 1, 2, 3, 4, 5, 6, 7 or more cytidines substituted for 5'-methyl dC. In some embodiments, all cytidines in the TCO can be substituted for 5'-methyl dC.

[0018] In some embodiments, an amplification oligomer, detection oligomer, or TCO contains one or more modified nucleotides. The oligomer may have 1, 2, 3, 4, 5, 6, 7, 8, or more modified nucleotides. In some embodiments, more than 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the nucleotides are modified. Modified nucleotides include nucleotides with modified nucleobases. Modified nucleobases include, but are not limited to, synthetic and natural nucleobases, 5-substituted pyrimidines, 5'-methylcytosine, 6-azapyrimidines, N-2, N-6, and O-6 substituted purines. Modified nucleotides also include nucleotides with modified bases, including, but not limited to, 2'-modified nucleotides (including, but not limited to, 2'-O-methyl nucleotides and 2'-halogen nucleotides such as 2'-fluoro nucleotides). Modified nucleotides also include nucleotides with modified linkages, such as, but not limited to, phosphorothioate linkages. In some embodiments, an amplification oligomer contains two or more modified nucleotides. The two or more modified nucleotides may have the same or different modifications. In some embodiments, any of the described oligomers can contain one or more 5'-methylcytosines. The oligomer can have one, two, three, four, five, or more 5'-methylcytosines. In some embodiments, all cytosine nucleotides in the described oligomers are 5'-methylcytosine modified nucleotides. In some oligomers, the use of 5'-methyl-2' deoxycytosine bases can increase duplex stability by increasing the Tm of each 5'-methyl-2' deoxycytosine incorporated into the oligomer by about 0.5°C to 1.3°C compared to the corresponding unmethylated oligomer.

[0019] The described amplification oligomers can be used to amplify CMV UL56 sequences. In some embodiments, the described amplification oligomers can be used to amplify CMV UL56 sequences using a thermal cycling reaction, such as polymerase chain reaction (PCR). In some embodiments, the described amplification oligomers can be used to amplify CMV UL56 sequences using an isothermal reaction, such as transcription-mediated amplification (TMA). Transcription-mediated amplification can be monophasic or multiphasic (e.g., biphasic). Other nucleic acid amplification methods that can utilize the described amplification oligomers include, but are not limited to, nucleic acid sequence-based amplification (NASBA), replicase-mediated amplification, ligase chain reaction (LCR), strand displacement amplification (SDA), and reverse transcriptase PCR (RT-PCR). A forward or helper oligomer can be combined with a reverse or displacer oligomer to form an amplification pair. Any of the described forward or helper oligomers can be combined with any of the described reverse or displacer oligomers to form an amplification pair. In some embodiments, the first amplification oligomer (forward primer) and the second amplification oligomer (reverse primer) are configured to amplify a CMV UL56 amplicon at least about 56, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, or at least about 95 nucleotides in length.

[0020] In some embodiments, the described oligomers can be used in single-phase or multi-phase (e.g., biphasic) transcription-mediated amplification. In multi-phase amplification, at least a portion of the target nucleic acid sequence is subjected to a first-phase amplification reaction under conditions that do not support exponential amplification of the target nucleic acid sequence. The first-phase amplification reaction produces a first amplification product, which is then subjected to a second-phase amplification reaction under conditions that allow exponential amplification of the first amplification product, thereby producing a second amplification product. Compared to single-phase formats, multi-phase amplification provides improved sensitivity and precision at the lower limit of analyte concentration. Multi-phase amplification can provide improved precision and shorten detection times.

[0021] In some embodiments, polyphasic amplification of a CMV target nucleic acid sequence comprises: a) contacting a sample containing or suspected of containing a CMV target nucleic acid sequence with a target capture mixture, the target capture mixture comprising an RNA polymerase promoter-containing oligonucleotide (promoter primer), and optionally a target capture oligomer (TCO) and / or a displacer oligomer to form a pre-amplification hybrid; b) isolating the pre-amplified hybrids; c) contacting the pre-amplified hybrid with a phase 1 amplification mixture, wherein the phase 1 amplification mixture comprises a non-RNA polymerase promoter-containing oligonucleotide (non-promoter primer), optionally a helper oligomer, a reverse transcriptase, an RNA polymerase, dNTPs, and NTPs, and wherein the phase 1 amplification mixture lacks at least one component necessary for exponential amplification; d) amplifying at least a portion of the target nucleic acid sequence of the pre-amplified hybrid in a substantially isothermal transcription-associated amplification reaction under conditions that support linear amplification to form a first amplification product; e) contacting the first amplification product with a second-phase amplification mixture, wherein the second-phase amplification mixture comprises an RNA polymerase promoter-containing oligonucleotide or at least one component required for exponential amplification that is absent from the first-phase amplification mixture; f) exponentially amplifying the first amplification product in a substantially isothermal transcription-associated amplification reaction to produce a second amplification product; g) detecting the second amplification product. In some embodiments, the second phase amplification mixture contains a detection oligomer. In some embodiments, one or more of any of the oligomers may be used in the reaction, such as one or more TCOs, one or more promoter primers, one or more non-promoter primers, one or more displacer oligomers, one or more helper oligomers, and / or one or more probe oligomers.

[0022] In some embodiments, the pre-amplification hybrid comprises a target nucleic acid hybridized to a promoter primer. In some embodiments, the pre-amplification hybrid comprises a target nucleic acid hybridized to one or more TCOs and a promoter primer. In some embodiments, the pre-amplification hybrid comprises a target nucleic acid hybridized to one or more TCOs, a promoter primer, and optionally a displacer oligomer. In some embodiments, isolating the pre-amplification hybrid comprises capturing the pre-amplification hybrid using a solid support. In some embodiments, the solid support comprises an immobilized capture probe. The solid support can be, but is not limited to, a magnetically attractable particle. In some embodiments, isolating the pre-amplification hybrid comprises removing the promoter primer that is not hybridized to the target nucleic acid.

[0023] In some embodiments, during the first phase of the isothermal transcription-associated amplification reaction, a promoter primer specifically bound to the target nucleic acid of the target sequence is extended by reverse transcriptase (RT) using the target nucleic acid as a template to create a cDNA copy. The cDNA is then used as a template to enzymatically extend a non-promoter primer to generate double-stranded DNA. The double-stranded DNA then serves as a template for RNA transcription from the RNA polymerase promoter provided by the promoter primer. The non-promoter primer then binds to the RNA and is extended by reverse transcriptase to produce a first amplification product. In the absence of additional promoter primers, exponential amplification does not occur. The first amplification product is then contacted with a second phase amplification mixture to initiate exponential second phase amplification.

[0024] In some embodiments, the phase 1 and phase 2 isothermal transcription-associated amplification reactions each comprise an RNA polymerase and a reverse transcriptase, hi some embodiments, the reverse transcriptase comprises endogenous RNase H activity.

[0025] In some embodiments, a composition suitable for use in the first phase of multiphase amplification of CMV comprises (a) an optional TCO, (b) a promoter primer hybridized to a first portion of the CMV target nucleic acid sequence, (c) an optional displacer oligomer hybridized to a portion of the CMV target nucleic acid sequence, (d) a non-promoter primer, (e) optionally a helper oligomer, and (f) additional components necessary for amplification of the target nucleic acid during the linear first phase of amplification reaction, but lacking at least one component necessary for exponential amplification of the target nucleic acid sequence. In some embodiments, the component lacking at least one component necessary for exponential amplification is the additional (free) promoter primer. In some embodiments, the first phase of amplification lacks a promoter primer that is not hybridized to the target nucleic acid in the pre-amplification hybrid. The additional components may include one or more of an RNA-dependent DNA polymerase, an RNA polymerase, dNTPs, NTPs, a buffer, and a salt.

[0026] In some embodiments, compositions suitable for use in phase 2 or subsequent phase amplification of multiphase amplification of CMV include (a) a first amplification product, (b) a promoter primer, (c) a non-promoter primer, and (d) other necessary components required for amplification of a target nucleic acid during an exponential phase 2 amplification reaction. The additional components may include one or more of an RNA-dependent DNA polymerase, an RNA polymer, dNTPs, NTPs, a buffer, and salts.

[0027] In some embodiments, there is provided a method for polyphasic amplification and / or detection of CMV, comprising: (a) contacting a sample containing or suspected of containing a CMV target nucleic acid with a promoter primer specific for a first portion of the target nucleic acid sequence under conditions that permit hybridization of the promoter primer to a first portion of the target nucleic acid sequence, thereby generating a pre-amplification hybrid comprising the promoter primer and the target nucleic acid sequence; (b) isolating the pre-amplified hybrids by target capture onto a solid support followed by washing to remove any promoter primer that did not hybridize to the first portion of the target nucleic acid sequence in step (a); (c) amplifying in a first-phase substantially isothermal transcription-associated amplification reaction mixture at least a portion of the target nucleic acid sequence of the pre-amplified hybrid isolated in step (b) under conditions that support linear amplification thereof but not exponential amplification thereof (i.e., the first-phase amplification reaction mixture lacks at least one component necessary for exponential amplification of the first amplification product), thereby resulting in a reaction mixture comprising a first amplification product; (d) combining the reaction mixture containing the first amplification product with at least one component required for exponential amplification of the first amplification product but absent from the reaction mixture containing the first amplification product to produce a second-phase amplification reaction mixture; (e) exponentially amplifying the first amplification product in the second phase amplification mixture in a substantially isothermal transcription-associated amplification reaction to produce a second amplification product; (f) optionally detecting the second amplification product.

[0028] In some embodiments, at least one component required for exponential amplification of the first amplification product includes a primer-promoter (e.g., a promoter-primer in addition to the promoter-primer hybridized to the target nucleic acid and isolated as part of the pre-amplification hybrid). In some embodiments, the first amplification product of step (c) is a cDNA molecule having the same polarity as the target nucleic acid sequence in the sample, and the second amplification product of step (e) is an RNA molecule. The second amplification product can be detected using a sequence-specific detection probe. The sequence-specific detection probe can be, but is not limited to, a stereo-sensitive probe that generates a detectable signal when hybridized to the second amplification product. In some embodiments, the sequence-specific detection probe of step (f) is a fluorescently labeled sequence-specific hybridization probe. The detecting can be performed at regular time intervals. In some embodiments, the detecting is performed in real time. In some embodiments, detecting the second amplification product includes quantifying the target nucleic acid sequence in the sample using a linear calibration curve.

[0029] In some embodiments, a target enhancer reagent (TER) is added to the sample prior to the addition of the TCO or target capture mixture. In some embodiments, the TER comprises 1.68 M lithium hydroxide (LiOH). The amount of TER to combine with the sample can be determined empirically. TER can be added to provide a final LiOH concentration in the sample of 50-350 mM. The sample can be added to the TER, or the TER can be added to the sample.

[0030] Compositions and kits for amplifying, detecting, and / or quantifying CMV are described. In some embodiments, the described compositions and kits provide direct, rapid, specific, and / or sensitive CMV detection. The compositions and kits can include one or more of the described amplification oligomers, probe oligomers, and / or TCOs. In some embodiments, the compositions or kits include at least one forward primer and at least one reverse primer. In some embodiments, the compositions or kits include at least one NT7 primer and at least one T7 primer. The compositions or kits can further include at least one probe oligomer. The compositions or kits can further include at least one TCO. The compositions or kits can further include one or more helper oligomers and / or displacer oligomers. The composition or kit may further comprise any one or more of capture beads, target capture reagent, target capture wash solution, target enhancer reagent, amplification reagent (lyophilized pellet), amplification reagent reconstitution solution, enzyme reagent (lyophilized pellet), enzyme reagent reconstitution solution, promoter reagent (lyophilized pellet), promoter reagent reconstitution solution, positive calibrator, CMV positive control nucleic acid, negative control nucleic acid, nucleotide triphosphates, DNA polymerase, RNA polymerase, reverse transcriptase, sample transport medium, and instructions for use.

[0031] A method for amplifying, detecting, and / or quantifying a target CMV sequence is described, comprising contacting a sample containing or suspected of containing CMV with at least two amplification oligomers to amplify a target region of CMV, wherein the at least two amplification oligomers comprise the forward and reverse primers described above, each of which hybridizes to the UL56 gene of CMV. An in vitro nucleic acid amplification reaction is performed in which the CMV target nucleic acid present in the sample is used as a template to generate an amplification product. In some embodiments, the forward and reverse primers each hybridize to SEQ ID NO: 1 or its complement. In some embodiments, the forward and reverse primers amplify an amplicon comprising SEQ ID NO: 51 or its complement.

[0032] In some embodiments, the method further comprises detecting the presence or absence of an amplification product, thereby indicating the presence or absence of CMV in the sample. The amplification product is detected using a probe oligomer. The described probe oligomer can be used in an amplification reaction to detect and / or quantify CMV in a sample.

[0033] In some embodiments, quantification of CMV in samples can be used to aid in the management of solid organ transplant recipients. In patients undergoing anti-CMV therapy, serial CMV DNA measurements can be used to assess viral response to treatment. CMV viral load information can also be used to diagnose CMV disease in transplant patients.

[0034] In some embodiments, the described oligonucleotides, compositions, and methods are suitable for use in amplifying and / or detecting CMV in a multiplex multiphase reaction. Multiplex multiphase reactions can be used to detect CMV as well as one or more other target sequences and / or organisms. DETAILED DESCRIPTION OF THE INVENTION

[0035] A.Definition Before describing the present teachings in detail, it is to be understood that the present disclosure is not limited to particular compositions or process steps, which may vary. As used herein and in the appended claims, it should be noted that the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to "an oligomer" includes a plurality of oligomers, etc. The conjunction "or" should be construed in an inclusive sense, i.e., equivalent to "and / or," unless an inclusive sense is unwarranted in the context.

[0036] All patents, applications, published applications, and other publications referenced herein are incorporated by reference in their entirety. To the extent that a definition set forth in this section contradicts or otherwise conflicts with a definition set forth in a patent, application, published application, or other publication incorporated herein by reference, the definition set forth in this section shall take precedence over the definition incorporated herein by reference.

[0037] Unless otherwise apparent from the context, any element, embodiment, step, feature, or aspect of the invention can be practiced in combination with any other.

[0038] It is understood that there is an implicit "about" before temperatures, concentrations, times, etc. discussed in this disclosure so that minor and negligible deviations are within the scope of the teachings herein. Generally, the term "about" refers to slight variations in the amount of a composition component that do not significantly affect the activity or stability of the composition. All ranges should be interpreted as including the endpoints unless expressly excluded, such as "excluding the endpoint." Thus, for example, "within 10 to 15" includes the values ​​10 and 15. Additionally, the use of "comprise," "comprises," "comprising," "contain," "contains," "containing," "include," "includes," and "including" is not intended to be limiting. It should be understood that both the summary and detailed description above are exemplary and explanatory only, and not limiting of the teachings. To the extent that the scope of any material incorporated by reference conflicts with the explicit content of this disclosure, the explicit content controls.

[0039] Throughout this specification and claims, approximation language can be applied to modify any quantitative or qualitative expression that can be permissibly varied without resulting in a change in the basic function to which it relates. Thus, a value modified by a term such as "about" or "approximately" is not limited to a particular exact value, but may include values ​​that differ from the particular value. In some embodiments, about or approximately indicates a slight variation and / or a variation of less than 5%.

[0040] Unless otherwise stated, embodiments herein that recite various components "comprising" are also contemplated as "consisting of" or "consisting essentially of" the recited components, and embodiments herein that recite various components "consisting of" are also contemplated as "comprising" or "essentially consisting of," and embodiments herein that recite various components "consisting essentially of" are also contemplated as "consisting of" or "comprising" the recited components (this interchangeability does not apply to the use of these terms in the claims). "Consisting essentially of" means that additional component(s), composition(s), or method step(s) that do not substantially alter the basic and novel characteristics of the compositions and methods described herein may be included in those compositions or methods. Such characteristics include the ability to detect CMV nucleic acid sequences present in a sample with a specificity that distinguishes CMV nucleic acid from other known pathogens, and with a sensitivity capable of detecting about 1 to 100 copies of virus within about 45 minutes of initiating an amplification reaction to generate amplified viral sequences that are optionally detected.

[0041] A "sample," "specimen," "biological sample," "biological specimen," "clinical sample," or "clinical specimen" is any sample containing or suspected of containing an analyte of interest, e.g., a nucleic acid (e.g., a target nucleic acid) of a microorganism, virus, gene, or the like, or a component thereof, including a nucleic acid sequence in or derived from the analyte. A "sample" may contain or be suspected of containing CMV, or a component thereof, such as a nucleic acid or a fragment of a nucleic acid. A sample may be from any source, such as, but not limited to, a biological specimen, a clinical specimen, and an environmental source. A sample may be a complex mixture of components. Samples include, for example, "biological samples" including any tissue or material from a living or dead mammal or organism, including blood, plasma, serum, blood cells, saliva, and mucous membranes, cerebrospinal fluid (for diagnosing CMV infection of the central nervous system), as well as samples such as biopsies from or derived from genital lesions, anogenital lesions, oral lesions, mucocutaneous lesions, skin lesions, and ocular lesions, or a combination thereof. Biological samples also include, but are not limited to, respiratory tissue, exudates (e.g., bronchoalveolar lavage fluid), sputum, tracheal aspirates, lymph nodes, gastrointestinal tissue, feces, urine, urogenital fluids, and biopsied cells or tissues. Samples may also include samples of in vitro cell culture components, including, for example, conditioned medium resulting from the growth of cells and tissues in culture medium. Samples may be treated to prepare samples for analysis by physically or mechanically disrupting tissue or cellular structures to release intracellular nucleic acids into solutions that may contain enzymes, buffers, salts, detergents, etc. Examples of environmental samples include, but are not limited to, water, ice, soil, slurry, debris, biofilms, atmospheric particles, and aerosols. Samples may also include samples of in vitro cell culture components, including, for example, conditioned medium resulting from the growth of cells and tissues in culture medium. Samples may also be processed specimens or materials, such as those obtained from processing samples by using filtration, centrifugation, sedimentation, or adherence to media such as matrices or supports.Other treatments of the sample may include, but are not limited to, physical or mechanical disruption of tissues, cell aggregates, or cells to release intracellular components, including nucleic acids, into a solution that may contain other components such as enzymes, buffers, salts, detergents, etc.

[0042] The term "contacting" means bringing two or more components together. Contacting can be achieved by mixing all components in a fluid or semi-fluid mixture. Contacting can also be achieved when one or more components physically contact one or more other components on a solid surface, such as a solid tissue section or substrate.

[0043] "Nucleic acid" and "polynucleotide" refer to polymeric compounds containing nucleosides or nucleoside analogs with nitrogenous heterocyclic bases or base analogs that are linked together to form polynucleotides, including polymers that are conventional RNA, DNA, mixed RNA-DNA, and analogs thereof. The nucleic acid "backbone" can be composed of various linkages, including sugar-phosphodiester linkages, peptide-nucleic acid linkages ("peptide nucleic acids" or PNA, PCT No. WO 95 / 32305), phosphorothioate linkages, methylphosphonate linkages, or combinations thereof. The sugar portion of the nucleic acid can be ribose, deoxyribose, or similar compounds with substitutions, e.g., 2' methoxy or 2' halide substitutions. The nitrogenous bases can be conventional bases (A, G, C, T, U), their analogs (e.g., inosine or others, see The Biochemistry of the Nucleic Acids, vol. 5-36, Adams et al., eds., 11 th ed., 1992), purine or pyrimidine derivatives (e.g., N 4 -methyldeoxyguanosine, deazapurines or azapurines, deazapyrimidines or azapyrimidines, pyrimidine bases with a substituent at the 5- or 6-position, purine bases with a substituent at the 2-, 6-, or 8-position, 2-amino-6-methylaminopurine, O 6-methylguanine, 4-thio-pyrimidine, 4-amino-pyrimidine, 4-dimethylhydrazine-pyrimidine, and O 4 -alkyl-pyrimidines (U.S. Pat. No. 5,378,825 and PCT No. WO93 / 13121). Nucleic acids may contain one or more "abasic" residues, where the backbone does not contain a nitrogenous base at one or more positions in the polymer (U.S. Pat. No. 5,585,481). Nucleic acids may contain only conventional RNA or DNA sugars, bases, and linkages, or may contain both conventional components and substitutions (e.g., conventional bases with 2' methoxy linkages, or polymers containing conventional bases and one or more base analogs). Nucleic acids include "locked nucleic acids" (LNAs), analogs containing one or more LNA nucleotide monomers with bicyclic furanose units locked into RNA that mimic the sugar configuration, which enhances hybridization affinity to complementary RNA and DNA sequences (Vester and Wengel, 2004, Biochemistry 43(42):13233-41). Nucleic acids may contain modified bases that alter the function or behavior of the nucleic acid, for example, the addition of 3'-terminal dideoxyribonucleotides that block the addition of additional nucleotides to the nucleic acid. Examples of oligomers that can affect the stability of a hybridization complex include PNA oligomers, oligomers containing 2'-methoxy or 2'-fluoro substituted RNA, or oligomers that affect the overall charge, charge density, or steric association of the hybridization complex, including oligomers containing charged linkages (e.g., phosphorothioates) or neutral groups (e.g., methylphosphonates). When referring to a range of lengths for an oligonucleotide, amplicon, or other nucleic acid, it is understood that the range includes all integers (e.g., a length of 19 to 25 contiguous nucleotides includes 19, 20, 21, 22, 23, 24, and 25).

[0044] A "target nucleic acid" or "target" is a nucleic acid containing a target nucleic acid sequence. A "target nucleic acid sequence," "target sequence," or "target region" is a specific deoxyribonucleotide or ribonucleotide sequence comprising a nucleotide sequence of a target organism, such as CMV, that is to be amplified. The target sequence or its complement contains a sequence that hybridizes to capture oligonucleotides, amplification oligomers, and / or detection oligomers used to amplify and / or detect the target nucleic acid. The target nucleic acid may contain other sequences other than the target sequence that may not be amplified. The target nucleic acid may be DNA or RNA and may be either single-stranded or double-stranded. The target nucleic acid may be, but is not limited to, a genomic nucleic acid, a transcribed nucleic acid such as rRNA, or a nucleic acid derived from a genomic nucleic acid or a transcribed nucleic acid.

[0045] Sequence identity can be determined by using algorithms such as BESTFIT, FASTA, and TFASTA in Wisconsin Genetics Software Package Release 7.0 (Genetics Computer Group, 575 Science Dr., Madison, Wis.) to align sequences using default gap parameters, and by using inspection and best alignment (i.e., the highest sequence similarity percentage across the entire comparison window).The sequence identity percentage is calculated by comparing two optimally aligned sequences across the entire comparison window, determining the number of positions where identical residues occur in both sequences, resulting in the number of matched positions, and dividing the number of matched positions without counting gaps within the comparison window (i.e., window size) by the total number of matched and mismatched positions, and multiplying the result by 100 to obtain the sequence identity percentage.Unless otherwise indicated, the comparison window between two sequences is defined by the total length of the shorter of the two sequences.

[0046] The term "complementarity" refers to the ability of a polynucleotide to form hydrogen bond(s) (hybridize) with another polynucleotide sequence, either by conventional Watson-Crick or other non-conventional methods. The percent complementarity indicates the percentage of bases in a contiguous strand in a first nucleic acid sequence that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 are 50%, 60%, 70%, 80%, 90%, and 100% complementary). The percent complementarity is calculated in a similar manner to the percent identity.

[0047] Exemplary portions of CMV sequences are provided in Table 1A (for brevity, the complete CMV genome known in the art is not included). Unless otherwise indicated, "hybridizing to a CMV nucleic acid" includes hybridizing to either the sense or antisense strand of a CMV nucleic acid, or to RNA transcribed from a genomic sequence.

[0048] In some embodiments, an amplification oligomer, probe oligomer, or TCO can contain one or more modified nucleotides. An amplification oligomer can have 1, 2, 3, 4, 5, 6, or more modified nucleotides. Modified nucleotides include nucleotides with modified nucleobases. Modified nucleobases include, but are not limited to, synthetic and natural nucleobases, 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines. Modified nucleotides also include nucleotides with modified bases, including, but not limited to, 2'-modified nucleotides (including, but not limited to, 2'-O-methyl nucleotides and 2'-halogen nucleotides such as 2'-fluoro nucleotides). A "C residue" includes methylated (5-methylcytosine) and unmethylated cytosine, unless the context indicates otherwise.

[0049] "RNA and DNA equivalents" refer to RNA and DNA molecules that have essentially the same complementary base pair hybridization properties. RNA and DNA equivalents have different sugar moieties (i.e., ribose versus deoxyribose) and may differ by the presence of uracil in RNA and thymine in DNA. Because equivalents have the same degree of complementarity to a particular sequence, differences between RNA and DNA equivalents do not contribute to differences in homology. Unless otherwise indicated, reference to CMV nucleic acid includes CMV RNA and its DNA equivalents.

[0050] An "oligomer," "oligonucleotide," or "oligo" is a polymer composed of two or more nucleoside or nucleobase subunits linked together. Oligonucleotides can be DNA and / or RNA and their analogs. In some embodiments, oligomers range in size from 5 to 15 nt lower limit and 50 to 500 nt upper limit. In some embodiments, oligomers range in size from 10 to 100 nucleobases, 10 to 90 nucleobases, 10 to 80 nucleobases, 10 to 70 nucleobases, or 10 to 60 nucleobases. In some embodiments, oligomers range in size from about 5 to 15, 16, 17, 18, 19, or 20 nucleobases lower limit and about 50 to 100 nucleobases upper limit. In some embodiments, oligomers range in size from about 10 to 21 nucleobases lower limit and about 22 to 100 nucleobases upper limit. The oligomers do not consist of wild-type chromosomal DNA or its in vivo transcription products. They can be synthetically produced using any known in vitro chemical or enzymatic method and can be purified after synthesis using standard methods, such as high-performance liquid chromatography (HPLC). Oligomers described include RNA polymerase promoter-containing oligomers (also called promoter primers, e.g., T7 primers), non-RNA polymerase promoter-containing oligomers (also called non-T7 primers, NT7 primers, or non-promoter primers), probe oligomers (also called detection oligomers or detection probes, probes, or torches), target capture oligomers (TCOs), forward primers, reverse primers, helper oligomers, and displacer oligomers.

[0051] An "immobilized capture probe" provides a means for binding a TCO to a solid support. In some embodiments, the immobilized capture probe contains a base sequence recognition molecule bound to the solid support, which facilitates separation of the bound target polynucleotide from unbound materials. Any known solid support may be used, such as matrices and particles that are free in solution. For example, the solid support may be nitrocellulose, nylon, glass, polyacrylate, mixed polymers, polystyrene, silane polypropylene, and magnetically attractable particles. In some embodiments, the support comprises monodisperse (i.e., uniform in size ± about 5%) magnetic spheres. The immobilized capture probe may be bound to the solid support directly (e.g., via covalent bonds or ionic interactions) or indirectly. Common examples of useful solid supports include magnetic particles or beads.

[0052] The term "target capture" refers to the selective separation or isolation of a target nucleic acid from other components of a sample mixture, such as cellular fragments, organelles, proteins, lipids, carbohydrates, or other nucleic acids. A target capture system can specifically and selectively separate a given target nucleic acid from other sample components (e.g., by using a sequence specific for the target nucleic acid of interest, such as a TCO TS sequence), or it can nonspecifically and selectively separate a target nucleic acid from other sample components by using other features of the target (e.g., a physical property of the target nucleic acid that distinguishes it from other sample components that do not exhibit that physical characteristic). Target capture methods and compositions have been described in detail previously (U.S. Patent Nos. 6,110,678 and 6,534,273, and U.S. Publication No. 2008 / 0286775A1). In some embodiments, target capture utilizes a solution-phase TCO and a support-bound immobilized capture probe to form a complex with the target nucleic acid and separate the captured target from other components.

[0053] The terms "separating," "isolating," or "purifying" generally refer to the removal of one or more components of a mixture, such as a sample, from one or more other components in the mixture. Sample components generally include nucleic acids in an aqueous solution phase, which may include cellular fragments, proteins, carbohydrates, lipids, and other nucleic acids. In some embodiments, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the target nucleic acid is separated or removed from other components in the mixture.

[0054] "Nucleic acid amplification" or "amplification" refers to any in vitro procedure that produces multiple copies of a target nucleic acid sequence or its complementary sequence or a fragment thereof (i.e., an amplified sequence that contains less than the entire target nucleic acid). Examples of nucleic acid amplification procedures include transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), and others (e.g., U.S. Pat. Nos. 5,399,491, 5,554,516, 5,437,990, 5,130,238, 4,868,105, and 5,124,246), replicase-mediated amplification (e.g., U.S. Pat. No. 4,786,600), polymerase chain reaction (PCR) (e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159), ligase chain reaction (LCR) (e.g., European Patent Application No. 0320308), and transcription-related methods such as strand displacement amplification (SDA) (e.g., U.S. Pat. No. 5,422,252). Replicase-mediated amplification uses self-replicating RNA molecules and a replicase such as Qβ-replicase. PCR amplification uses DNA polymerase, primers, and thermal cycling steps to synthesize multiple copies of two complementary strands of DNA or cDNA. LCR amplification uses at least four separate oligonucleotides to amplify a target and its complementary strand by using multiple cycles of hybridization, ligation, and denaturation. SDA uses primers containing recognition sites for restriction endonucleases to nick one strand of a semi-modified DNA duplex containing the target sequence, followed by amplification in a series of primer extension and strand displacement steps. While certain embodiments use PCR or TMA, it will be apparent to those skilled in the art that the oligomers disclosed herein can easily be used as primers in other amplification methods.

[0055] Transcription-associated amplification uses DNA polymerase, RNA polymerase, deoxyribonucleoside triphosphates, ribonucleoside triphosphates, promoter-containing oligonucleotides, and optionally other oligonucleotides, to ultimately generate multiple RNA transcripts from a nucleic acid template (see U.S. Pat. Nos. 5,399,491 and 5,554,516 to Kacian et al., U.S. Pat. No. 5,437,990 to Burg et al.). (This is described in detail in PCT Nos. WO88 / 01302 and WO88 / 10315 to Gingeras et al., U.S. Patent No. 5,130,238 to Malek et al., U.S. Patent Nos. 4,868,105 and 5,124,246 to Urdea et al., PCT No. WO94 / 03472 to McDonough et al., and PCT No. WO95 / 03430 to Ryder et al., each of which is incorporated herein by reference.) The method of using TMA has been previously described in detail (U.S. Patent Nos. 5,399,491 and 5,545,516, each of which is incorporated herein by reference).

[0056] The term "substantially isothermal amplification" refers to an amplification reaction that is performed at a substantially constant temperature. The isothermal portion of the reaction can be preceded or followed by one or more steps at variable temperatures, such as a first denaturation step and a final heat inactivation or cooling step. It is understood that this definition does not exclude small variations in temperature, but rather is used to distinguish isothermal amplification techniques from other amplification techniques known in the art that essentially rely on "cycling temperatures" to generate amplification products. Isothermal amplification differs from PCR, for example, in that PCR relies on heating followed by denaturation cycles by primer hybridization and polymerization at low temperatures.

[0057] An "amplicon" or "amplification product" is a nucleic acid molecule produced in a nucleic acid amplification reaction and derived from a target nucleic acid. The amplicon or amplification product contains a target nucleic acid sequence that may be the same as or in the opposite orientation to the target nucleic acid.

[0058] "Amplification oligomer" refers to an oligonucleotide that hybridizes to a target nucleic acid or its complement and participates in a nucleic acid amplification reaction. An amplification oligomer can be a primer, forward primer, reverse primer, promoter primer, non-promoter primer, helper oligomer, or displacer oligomer. In some embodiments, an amplification oligomer contains at least about 10 contiguous bases, and optionally at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 contiguous bases, that are complementary to a region of the target nucleic acid sequence or its complementary strand. The contiguous bases can be at least about 80%, at least about 90%, at least 95%, or fully complementary to the target sequence to which the amplification oligomer binds. In some embodiments, an amplification oligomer contains 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 contiguous bases that are at least 80%, at least 90%, at least 95%, or 100% complementary to a region of the target nucleic acid sequence or its complementary strand. In some embodiments, an amplification oligomer contains additional 3' or 5' sequence that is not complementary to the target nucleic acid sequence. Those skilled in the art will understand that recited ranges include all integers and rational numbers within the range (e.g., 92% or 98.377%). Certain amplification oligomers are about 10 to about 60 bases in length and may optionally contain modified nucleotides. In some embodiments, the primer may contain at least one methylated cytosine and / or at least one 2'-modified nucleotide.

[0059] "Single-phase amplification" refers to an amplification reaction in which all components necessary for nucleic acid amplification are present in the reaction mixture at the start of amplification.

[0060] "Linear amplification" refers to an amplification mechanism designed to generate an increase in target nucleic acid that is linearly proportional to the amount of target nucleic acid in the reaction. For example, a transcription-associated reaction can be used to generate multiple RNA copies from a DNA target, and the increase in copy number can be described by a linear factor (e.g., starting copies of template × n). In some embodiments, the linear amplification of the first phase of a multiphase amplification procedure increases the starting number of target nucleic acid strands or their complements by at least 10-fold, at least 100-fold, or at least 1,000-fold before the start of the second phase amplification reaction. An example of a linear amplification system is "T7-based Linear Amplification of DNA" (TLAD, see Liu et al., BMC Genomics, 4:Art. No. 19, May 9, 2003). Other methods are disclosed herein. Thus, the term "linear amplification" refers to an amplification reaction that does not result in exponential amplification of the target nucleic acid sequence. The term "linear amplification" does not refer to methods that simply generate a single copy of a nucleic acid strand, such as transcription of an RNA molecule into a single cDNA molecule, as in reverse transcription.

[0061] "Exponential amplification" refers to nucleic acid amplification designed to generate an increase in target nucleic acid that is geometrically proportional to the amount of target nucleic acid in the reaction. For example, PCR generates one DNA strand for every original target strand and every synthesized strand present. Similarly, transcription-associated amplification generates multiple RNA transcripts for every original target strand and every subsequently synthesized strand. Amplification is exponential because the synthesized strands are used as templates in subsequent rounds of amplification. An amplification reaction does not need to actually generate an exponentially increasing amount of nucleic acid to be considered exponential, as long as the amplification reaction is designed to generate such an increase.

[0062] "Primer" refers to an oligomer that hybridizes to a template nucleic acid and has a 3' end that is extended by polymerization. A primer can be optionally modified, for example, by including a 5' region that is not complementary to the target sequence. Such modifications can include the addition of functions such as tags, promoters, or other sequences used or useful in manipulating or amplifying the primer or target oligonucleotide. In the context of transcription-mediated amplification, a primer modified with a 5' promoter sequence can be referred to as a "promoter-primer." Those skilled in the art of molecular biology or biochemistry will understand that an oligomer that can function as a primer can be modified to include a 5' promoter sequence and then function as a promoter-primer, and similarly, any promoter-primer can function as a primer regardless of its 5' promoter sequence.

[0063] In the cyclic amplification method of detecting amplicons in real time, the term "threshold cycle" (Ct) is a measure of the time of the appearance of a signal associated with the amplification of a target, and is generally 10 times the standard deviation of the normalized reporter signal. When amplification reaches the "threshold cycle", it is generally considered that there is a positive amplification product of the sequence to which the probe binds. The identity of the amplification product can then be determined by methods known to those skilled in the art, such as gel electrophoresis, nucleic acid sequencing, and other such well-known methods.

[0064] As used herein, the term "relative fluorescence unit" ("RFU") is a unit of measurement for fluorescence intensity. RFU varies with the properties of the detection means used in the measurement and can be used as a measure to compare relative intensities between samples and controls. The analytical sensitivity (limit of detection or LoD) is expressed as the 50% median tissue culture infective dose (TCID 50 / ml) is determined by the TCID 50 / ml is the amount of pathogen that produces pathological changes in 50% of the seeded cell cultures.

[0065] "Detection probe" or "probe" refers to an oligomer that specifically hybridizes to a target sequence, including an amplification sequence, under conditions that promote nucleic acid hybridization for the detection of a target nucleic acid. Detection can be either direct (i.e., a probe that hybridizes directly to the target) or indirect (i.e., a probe that hybridizes to an intermediate structure that links the probe to the target). The target sequence of a probe generally refers to the specific sequence within a larger sequence to which the probe specifically hybridizes. Detection probes can include complementary (target-specific) sequences and non-complementary (not target-complementary) sequences. Such non-target-complementary sequences can include sequences that impart desired secondary or tertiary structures, such as hairpin structures, that can be used to facilitate detection and / or amplification. (See, e.g., U.S. Pat. Nos. 5,118,801, 5,312,728, 5,925,517, 6,150,097, 6,849,412, 6,835,542, 6,534,274, and 6,361,945, and U.S. Patent Application Publication Nos. 2006 / 0068417A1 and 2006 / 0194240A1.) The complementary and non-complementary sequences may be contiguous or connected by a linker. In some embodiments, the linker is C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, C26, C27, C28, C29, C30, C31, C32, C33, C34, C35, C36, C37, C38, C39, C40, C41, C42, C43, C44, C45, C46, ​​C47, C48, C49, C50, C51, C52, C53, C54, C55, C56, C57, C58, C59, C60, C61, C62, C63, C64, C65, C66, C67, C68, C69, C70, C71, C72, C73, C74, C75, C76, C77, C78, ​​C79, C80, C81, C82, C83, C84, C85 10 , C 11 , C 12 , C 13 , C 14 , C 15 , or C 16 In some embodiments, the linker is a C9 linker. The detection oligomer may be RNA, DNA, contain one or more modified nucleotides, or a combination thereof. In some embodiments, the detection oligomer contains one or more 2' methoxy ribonucleotides. In some embodiments, the detection oligomer contains all 2' methoxy ribonucleotides. Probes of defined sequences can be produced by techniques known to those skilled in the art, such as by chemical synthesis and in vitro or in vivo expression from recombinant nucleic acid molecules.

[0066] Detection can be achieved using single-stranded nucleic acid torches present during target amplification and hybridizing to the amplicons in real time. Each torch has a fluorophore and a quencher. The torch contains complementary regions at each end. These complementary regions bind to each other to form a "closed" torch. In the closed configuration, the fluorophore and quencher are in close proximity, and the fluorophore signal is quenched. That is, it does not emit a detectable signal when excited by light. However, when the torch binds to a complementary target, the complementary regions within the torch are forced apart to form an "open" torch. In the open configuration, the fluorophore and quencher are no longer in close proximity, and the fluorophore signal is detectable upon excitation (i.e., no longer quenched). The amplicon-torch binding results in the separation of the quencher from the fluorophore, allowing the fluorophore to be excited in response to light stimulation and emit a signal at a specific wavelength. Torches are present during amplification and may bind to complementary amplicons as they are generated in real time. As more amplicons are generated, more torches bind, generating more signal. The signal eventually reaches a detectable level above background, eventually reaching a point where all available torches bind to the amplicons and the signal reaches a maximum. At the beginning of amplification, and when the copy number of the amplified sequence is low, most of the probe oligomers are closed (the 3' and 5' ends are base-paired, quenching the fluorescent signal). During amplification, as more probe oligomers bind to the target sequence, the 3' and 5' ends of the probe oligos separate, resulting in increased fluorescence (decreased fluorescence quenching). After further amplification, the fluorescent signal approaches a maximum.

[0067] As used herein, "label" or "detectable label" refers to a moiety or compound directly or indirectly attached to the probe to be detected or to produce a detectable signal. Direct attachment can use covalent or non-covalent interactions (e.g., hydrogen bonds, hydrophobic or ionic interactions, and chelate or coordinate complex formation), while indirect attachment can use a bridging moiety or linker (e.g., via an antibody or additional oligonucleotide), which amplifies the detectable signal. Any detectable moiety may be used, such as radionuclides, ligands such as biotin or avidin, enzymes, enzyme substrates, reactive groups, chromophores such as dyes or particles that impart detectable color (e.g., latex or metal beads), luminescent compounds (e.g., bioluminescent, phosphorescent, or chemiluminescent compounds), and fluorescent compounds (i.e., fluorophores). Fluorophores include those that absorb light in the range of approximately 495-650 nm and emit light in the range of approximately 520-670 nm, including, but not limited to, FAM™, TET™, CAL FLUOR™ (orange or red), QUASAR™, fluorescein, hexochlorofluorescein (HEX), rhodamine, carboxy-X-rhodamine (ROX), tetramethylrhodamine, IAEDANS, EDANS, DABCYL, coumarin, BODIPY FL, Lucifer Yellow, eosin, erythrosine, Texas Red, ROX, CY dyes (such as CY5), cyanine 5.5 (Cy5.5), and those known as fluorescein / QSY7 dye compounds. Fluorophores may be used in conjunction with a quencher molecule, which absorbs light when in close proximity to the fluorophore, thereby reducing background fluorescence. Such quenchers are well known in the art and include, but are not limited to, BLACK HOLE QUENCHER™ (or BHQ™™, including but not limited to Black Hole Quencher-2 (BHQ2)) or TAMRA™ compounds.Certain embodiments include "homogeneous detectable labels" that are detectable in a homogeneous system in which bound labeled probes in a mixture exhibit a detectable change compared to unbound labeled probes, allowing for label detection without physically removing hybridized labeled probes from unhybridized labeled probes (e.g., U.S. Pat. Nos. 5,283,174, 5,656,207, and 5,658,737). Certain homogeneous detectable labels include chemiluminescent compounds, including acridinium ester ("AE") compounds, such as the well-known standard AE or AE derivatives (U.S. Pat. Nos. 5,656,207, 5,658,737, and 5,639,604). The method of synthesizing the label, the method of binding the label to nucleic acid, and the method of detecting the signal from the label are well known (for example, Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) at Chapter 10, and U.S. Patent Nos. 5,658,737, 5,656,207, 5,547,842, 5,283,174 and 4,581,333, and European Patent Application No. 0747706).The specific method of binding AE compounds to nucleic acid is known (for example, see U.S. Patent Nos. 5,585,481 and 5,639,604, column 10, line 6 to column 11, line 3, and Example 8). Specific AE labeling locations are the central region of the probe and near the region of A / T base pairs, the 3' or 5' end of the probe, or at or near mismatch sites with known sequences that the probe should not detect compared to the desired target sequence. Other detectably labeled probes include TaqMan™ probes, molecular torches, and molecular beacons. TaqMan™ probes contain donor and acceptor labels, and fluorescence is detected when the probe is enzymatically degraded during amplification to release the fluorophore from the presence of the quencher.Molecular torches and beacons exist in open and closed configurations, the closed configuration quenches the fluorophore, and the open position separates the fluorophore from the quencher, allowing fluorescence. Hybridization to a target opens the otherwise closed probe.

[0068] "Hybridization" or "hybridizing" refers to the ability of two fully or partially complementary nucleic acid strands to come together in a parallel or antiparallel orientation under specific hybridization assay conditions to form a stable structure having a double-stranded region. The two constituent strands of this double-stranded structure, sometimes called a hybrid, are held together by hydrogen bonds. These hydrogen bonds are most commonly formed between nucleotides containing the bases adenine and thymine or uracil (A and T or U) or cytosine and guanine (C and G) on a single nucleic acid strand, although base pairing can also occur between bases that are not members of these "canonical" pairs. Non-canonical base pairing is well known in the art. (See, e.g., R.L.P. Adams et al., The Biochemistry of the Nucleic Acids (11th ed. 1992).)

[0069] "Preferentially hybridize" means that under stringent hybridization conditions, an amplification or detection probe oligomer can hybridize to its target nucleic acid to form a stable oligomer:target hybrid, but does not form a sufficient number of stable oligomer:non-target hybrids. Amplification and detection oligomers that hybridize preferentially to a target nucleic acid are useful for amplifying and detecting the target nucleic acid, but not non-target nucleic acids, particularly in phylogenetically closely related organisms. Thus, the oligomer hybridizes to a sufficiently higher degree than non-target nucleic acids, allowing one skilled in the art to amplify and / or accurately detect the presence (or absence) of nucleic acids derived from specific influenza viruses, as needed. Generally, reducing the degree of complementarity between an oligonucleotide sequence and its target sequence will reduce the degree or rate of hybridization of the oligonucleotide to its target region. However, including one or more non-complementary nucleosides or nucleobases can enhance the ability of an oligonucleotide to discriminate against non-target organisms.

[0070] Preferential hybridization can be measured using techniques known in the art and described herein, such as in the Examples provided below. In some embodiments, there is at least a 10-fold difference, at least a 20-fold difference, at least a 50-fold difference, at least a 100-fold difference, at least a 200-fold difference, at least a 500-fold difference, or at least a 1,000-fold difference between the target hybridization signal and the non-target hybridization signal in the test sample. In some embodiments, the non-target hybridization signal in the test sample is below background signal level.

[0071] "Stringent hybridization conditions" or "stringent conditions" refer to conditions that allow an oligomer to hybridize preferentially to a target nucleic acid (such as an HPIV nucleic acid) but not to nucleic acids derived from closely related non-target nucleic acids. While the definition of stringent hybridization conditions remains constant, the actual reaction environment that can be used for stringent hybridization can vary depending on factors including the GC content and length of the oligomer, the degree of similarity between the oligomer sequence and non-target nucleic acid sequences that may be present in the test sample, and the target sequence. Hybridization conditions include temperature and the composition of the hybridization reagents or solutions. Exemplary hybridization assay conditions for amplifying and / or detecting target nucleic acids derived from one or more CMV strains with oligomers of the present disclosure correspond to a temperature of approximately 60°C when the salt concentration is within the range of approximately 0.6-0.9M. Specific hybridization assay conditions are described in the Examples section below. Other acceptable stringent hybridization conditions can be readily ascertained by one of ordinary skill in the art.

[0072] By "competes under stringent conditions for hybridization to CMV nucleic acid" with a referenced oligomer is meant that the oligomer substantially reduces binding of the referenced oligomer to its target CMV sequence under stringent conditions, or the competing oligomer, when provided in excess, is capable of reducing binding of the referenced oligomer by about 20%, 30%, 40%, 50% or more at subsaturating concentrations, or the Tm of the competing oligomer is higher than or within about 5°C, 4°C, 3°C, 2°C, or 1°C of the Tm of the referenced oligomer with respect to the target. Suitable oligonucleotide competition assay conditions and procedures are known in the art.

[0073] "Assay conditions" means conditions that allow stable hybridization of an oligonucleotide to a target nucleic acid. Assay conditions do not require preferential hybridization of an oligonucleotide to a target nucleic acid.

[0074] Although sequences do not need to be completely complementary, sequences are "sufficiently complementary" if they allow stable hybridization of two nucleic acid sequences, such as a stable hybrid between a probe and a target sequence. That is, a "sufficiently complementary" sequence hybridizes to another sequence by hydrogen bonding between a subset series of complementary nucleotides using standard base pairing (e.g., G:C, A:T, or A:U), but the two sequences may contain one or more residues that are not complementary (including abasic positions), as long as the entire sequence is under suitable hybridization conditions to form a stable hybridization complex. Sufficiently complementary sequences may be at least about 80%, at least about 90%, or completely complementary in the sequences that hybridize to each other. Suitable hybridization conditions are well known to those skilled in the art and can be predicted based on sequence composition or can be empirically determined by using routine tests (e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 2002). nd ed. §§1.90-1.91, 7.37-7.57, 9.47-9.51, and 11.47-11.57, especially §§9.50-9.51, 11.12-11.13, 11.45-11.47, and 11.55-11.57).

[0075] In some embodiments, oligomers, such as helper oligomers or displacer oligomers, are blocked. A blocked, or "non-extendable," oligomer contains a blocking moiety at or near its 3' end that prevents a polymerase from extending the nascent nucleic acid chain (i.e., the oligomer is blocked). In some embodiments, the blocking group near the 3' end is within five residues of the 3' end and is large enough to restrict polymerase binding to the oligomer. In some embodiments, the blocking group is covalently attached to the 3' end. Many different chemical groups can be used to block the 3' end, including alkyl groups, non-nucleotide linkers, alkane-diol dideoxyribonucleotide residues, and cordycepin. Further examples of blocking moieties include 3'-deoxynucleotides (e.g., 2',3'-dideoxynucleotides); 3'-phosphorylated nucleotides; fluorophores, quenchers, or other labels that prevent extension; inverted nucleotides (e.g., linked to the preceding nucleotide through a 3'-to-3' phosphodiester, optionally with an exposed 5'-OH or phosphate); or proteins or peptides attached to the oligonucleotide to prevent further extension of the nascent nucleic acid chain by a polymerase. Non-extendable oligonucleotides of the present disclosure can be at least 10 bases in length and can be extended up to 15, 20, 25, 30, 35, 40, 50, or more nucleotides in length. Non-extendable oligonucleotides containing a detectable label can be used as probes. In some embodiments, the helper oligomer or displacer oligomer is blocked (i.e., non-extendable or contains a blocking moiety).

[0076] In particular, references in the claims to "the sequence of SEQ ID NO: X" refer to the base sequence set forth in the corresponding sequence listing unless otherwise indicated, and do not require identity of the backbone (e.g., RNA, 2'-O-Me RNA, or DNA) or base modifications (e.g., methylation of cytosine residues).

[0077] A "degenerate" position in an oligomer refers to a position where two or more base pairs are present in a population of oligomers. For example, a nucleotide can be represented as C or Y, which represents T / U. Oligomers with degenerate positions can be synthesized by providing a mixture of nucleotide precursors corresponding to the desired degenerate combination in the synthesis step where introduction of the degenerate position is desired.

[0078] A "non-Watson-Crick" (NWC) position in an oligomer refers to a position at which the oligomer is configured to hybridize to at least one nucleic acid sequence by non-Watson-Crick pairing, such as GU, GT, or GA (either G or U / T / A can be the base in the oligomer). In some embodiments, the NWC position is configured to hybridize via a wobble (GU or GT) or purine-purine (GA) pair.

[0079] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Common definitions can be found in technical books related to the field of molecular biology, such as the Dictionary of Microbiology. and Molecular Biology, 2nd ed. (Singleton et al., 1994, John Wiley & Sons, New York, NY) or The Harper Collins Dictionary of Biology (Hale & Marham, 1991, Harper Perennial, New York, NY).

[0080] B. Oligomer CMV target regions containing the regions to be amplified are shown in Table 1A. Amplification oligomers suitable for amplifying CMV target regions can be found in Table 1B. The amplification oligomers contain nucleotide sequences present in the forward (Fwd) primer / helper region, reverse (Rev) primer region, and / or displacer region and hybridize to the forward (Fwd) primer / helper, reverse (Rev) primer, and displacer complementary (Compl.) regions shown in Table 1A. The probe oligomers contain nucleotide sequences present in the probe region and hybridize to the primer complementary (Compl.) regions shown in Table 1A. Probe oligomers suitable for detecting CMV amplicons can be found in Table 1C. TCOs suitable for capturing CMV nucleic acids can be found in Table 1D. Exemplary T7 promoter sequences can be found in Table 1E. [Table 1A] [Table 1B-1] [Table 1B-2] [Table 1C] [Table 1D] [Table 1E]

[0081] The described amplification oligomers are configured to specifically hybridize to CMV UL56 gene nucleic acid. In some embodiments, the amplification oligomer has a target hybridizing region of about 19-40 bases in length, or about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 bases in length. In some embodiments, the oligomer includes, in addition to the target hybridizing region, a second sequence region, such as a T7 RNA polymerase promoter, which may be located 5′ of the target hybridizing region. In some embodiments, the oligomer does not include a second sequence region.

[0082] In some embodiments, the amplification oligomer comprises any of the sequences in Table 1B. In some embodiments, the amplification oligomer consists of any of the sequences in Table 1B. In some embodiments, the amplification oligomer comprises an oligomer that competes with any of the sequences in Table 1B for binding to a CMV target nucleic acid under stringent conditions. The CMV target nucleic acid can be, but is not limited to, SEQ ID NO: 1 or its complement. Any of the listed forward or non-promoter primers can be combined with any of the listed reverse or promoter primers to form an amplification oligomer pair (amplification oligomer combination). Similarly, any of the helper oligomers, displacer oligomers, or probe oligomers can be combined with any amplification oligomer pair. In some embodiments, the first amplification oligomer (e.g., forward primer) and second amplification oligomer (e.g., reverse primer) are configured to amplify a CMV UL56 amplicon at least about 56, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, or at least about 95 nucleotides in length. In some embodiments, the first amplification oligomer (e.g., forward primer) and second amplification oligomer (e.g., reverse primer) are configured to amplify a CMV UL56 amplicon 56-340, 56-312, 56-252, or 56-227, 95-340, 95-312, 95-252, or 95-227 nucleotides in length. In some embodiments, the first amplification oligomer (e.g., a forward primer) and the second amplification oligomer (e.g., a reverse primer) are configured to amplify a CMV UL56 amplicon that is 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides in length.

[0083] In some embodiments, the forward primer or non-promoter primer comprises 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 19-31 contiguous nucleic acid bases that have at least 80% or at least 90% identity to a 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 19-31 nucleotide sequence present in SEQ ID NO: 2. In some embodiments, the forward primer or non-promoter primer comprises 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 19-31 contiguous nucleic acid bases having a nucleotide sequence present in SEQ ID NO: 2. In some embodiments, the forward primer or non-promoter primer comprises the nucleotide sequence of SEQ ID NO: 10 or SEQ ID NO: 11. In some embodiments, the forward primer or non-promoter primer comprises or consists of a nucleotide sequence selected from the group consisting of SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19. In some embodiments, the forward primer or non-promoter primer comprises a nucleotide sequence having 90% identity to SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:19. In some embodiments, the forward primer or non-promoter primer can hybridize to SEQ ID NO:79 and initiate DNA or RNA polymerization. In some embodiments, the forward primer or non-promoter primer comprises an oligomer that can compete with any of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:19 for hybridization to SEQ ID NO:79.

[0084] In some embodiments, the reverse primer or promoter primer comprises 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or 21-40 consecutive nucleobases that have at least 80% or at least 90% identity to a 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or 21-40 nucleotide sequence present in SEQ ID NO:3. In some embodiments, the reverse primer or promoter primer comprises 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or 21-40 consecutive nucleic acid bases having the nucleotide sequence present in SEQ ID NO:3. In some embodiments, the reverse primer or promoter primer comprises the nucleotide sequence of SEQ ID NO:23, SEQ ID NO:24, or SEQ ID NO:25. In some embodiments, the reverse primer or promoter primer comprises or consists of a nucleotide sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:41, or SEQ ID NO:47. In some embodiments, the reverse primer or promoter primer comprises a nucleotide sequence having 90% identity to SEQ ID NO:6, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:41, or SEQ ID NO:47. In some embodiments, the reverse primer or promoter primer can hybridize to SEQ ID NO: 80 and initiate polymerization of DNA or RNA. In some embodiments, the reverse primer or promoter primer comprises an oligomer that can compete with any of SEQ ID NO: 6, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 41, or SEQ ID NO: 47 for hybridization to SEQ ID NO: 80.

[0085] In some embodiments, an RNA polymerase promoter sequence can be added to any of the forward and / or reverse primers described to form a promoter primer. The RNA polymerase primer sequence is operably linked to the 5' end of the forward or reverse primer. The RNA polymerase promoter sequence can be, but is not limited to, a T7, T3, or SP6 RNA polymerase promoter sequence. A T7 RNA polymerase promoter sequence can contain the nucleotide sequence of SEQ ID NO:78. In some embodiments, the promoter primer comprises or consists of the nucleotide sequence of SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, or SEQ ID NO:46.

[0086] In some embodiments, the helper oligomer promotes or enhances hybridization of the forward primer to the template nucleotide sequence. In some embodiments, the displacer oligomer promotes or enhances hybridization of the reverse primer to the template nucleic acid sequence. Promotion or enhancement of hybridization of the primer to the template can promote or enhance amplification of the target nucleotide sequence. In some embodiments, the helper oligomer and / or displacer oligomer can be blocked (i.e., non-extendable). When blocked, the helper and / or displacer oligomer cannot prime polymerization from the 3' end. For example, a helper / displacer oligomer can be made non-extendable by 3'-phosphorylation, having a 3'-terminal 3'-deoxynucleotide (e.g., a terminal 2',3'-dideoxynucleotide), having a 3'-terminal inverted nucleotide (e.g., the last nucleotide is inverted so that it is attached to the penultimate nucleotide by a 3'-to-3' phosphodiester bond or its analog (such as phosphorothioate)), or having an attached fluorophore, quencher, or other label that interferes with extension (possibly, but not necessarily, attached through the 3' position of the terminal nucleotide). For any of the described helper oligomers, one or more nucleotides in the helper oligomer can be modified. In some embodiments, the helper oligomer contains a 3'-inverted (reverse polarity) nucleotide. In some embodiments, the inverted nucleotide is an inverted dC.

[0087] In some embodiments, the helper oligomer comprises 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 19-31 consecutive nucleobases that have at least 80% or at least 90% identity to a 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 19-31 nucleotide sequence present in SEQ ID NO: 2. In some embodiments, the helper oligomer comprises 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 19-31 consecutive nucleobases having a nucleotide sequence present in SEQ ID NO: 2. In some embodiments, the helper oligomer comprises the nucleotide sequence of SEQ ID NO: 10 or SEQ ID NO: 19. In some embodiments, the helper oligomer comprises or consists of a nucleotide sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19. In some embodiments, the helper oligomer comprises a nucleotide sequence having 90% identity to SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19. In some embodiments, the helper oligomer comprises an oligomer that can compete with any of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19 for hybridization to SEQ ID NO: 79.

[0088] In some embodiments, the displacer oligomer comprises 21, 22, 23, 24, 25, or 21-27 consecutive nucleobases that have at least 90% identity to 21, 22, 23, 24, 25, 26, 27, or 21-27 nucleotides present in SEQ ID NO:5. In some embodiments, the displacer oligomer comprises 21, 22, 23, 24, 25, 26, 27, or 21-27 consecutive nucleobases having a nucleotide sequence present in SEQ ID NO:5. In some embodiments, the displacer oligomer comprises the nucleotide sequence of SEQ ID NO:25, SEQ ID NO:12, or SEQ ID NO:41. In some embodiments, the displacer oligomer comprises or consists of a nucleotide sequence selected from the group consisting of SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:6, SEQ ID NO:41, and SEQ ID NO:12. In some embodiments, the displacer oligomer comprises a nucleotide sequence having 90% identity to SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:6, SEQ ID NO:41, or SEQ ID NO:12. In some embodiments, the displacer oligomer comprises an oligomer that can compete with any of SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:6, SEQ ID NO:41, or SEQ ID NO:12 for hybridization to SEQ ID NO:82. For any of the described displacer oligomers, one or more nucleotides in the displacer oligomer may be modified. In some embodiments, the displacer oligomer contains a 3' inverted (reverse polarity) nucleotide. In some embodiments, the inverted nucleotide is an inverted dC.

[0089] In some embodiments, the helper or displacer oligomer can be a forward primer or a reverse primer. In some embodiments, the described helper or displacer oligomer can have an RNA polymerase promoter sequence linked to the 5' end of the helper or displacer oligomer to form a promoter primer.

[0090] In some embodiments, oligomers are provided that include a detectable label(s). Such oligomers can be used as probes (probe oligomers). The probe oligomers are used to detect the presence or absence of CMV amplification products generated using the described amplification oligomers.

[0091] The probe oligomer can be used to detect a CMV amplicon, i.e., the probe oligomer hybridizes to the CMV amplicon. The CMV amplicon can be generated using any of the described amplification oligomers. In some embodiments, the probe oligomer comprises 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 24-35 consecutive nucleobases that have at least 90% identity to a 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 24-35 nucleotide sequence present in SEQ ID NO:4. In some embodiments, the probe oligomer comprises 24-35 consecutive nucleobases having a nucleotide sequence present in SEQ ID NO:4. In some embodiments, the probe oligomer comprises 24-35 consecutive nucleobases that hybridize to SEQ ID NO:81. In some embodiments, the probe oligomer comprises the nucleotide sequence of SEQ ID NO:51 or SEQ ID NO:52, wherein one or more uracil nucleotides can be substituted for thymine nucleotides. In some embodiments, the probe oligomer comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:21, SEQ ID NO:26, or SEQ ID NO:39. In some embodiments, the probe oligomer contains a hairpin. In some embodiments, 4 to 5 nucleobases at the 5' and 3' ends of the probe oligomer are complementary to each other. In some embodiments, the probe oligomer comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, and SEQ ID NO:70. In some embodiments, the probe oligomer comprises a nucleobase sequence having at least 90% identity to SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, or SEQ ID NO:70.

[0092] In some embodiments, the detectable label is a non-nucleotide label. Suitable labels include compounds that emit detectable light signals, such as fluorophores or luminescent (e.g., chemiluminescent) compounds that can be detected in a homogeneous mixture. Two or more labels and two or more types of labels may be present on a particular probe, or detection may rely on the use of a mixture of probes, each of which is labeled with a compound that generates a different detectable signal (see, for example, U.S. Patent Nos. 6,180,340 and 6,350,579, each of which is incorporated herein by reference). Labels can be attached to probes by various means, including covalent bonding, chelation, and ionic interactions, but in some embodiments, the labels are covalently attached. For example, in some embodiments, the detection probe has an attached chemiluminescent label, such as, for example, an acridinium ester (AE) compound (see, e.g., U.S. Patent Nos. 5,185,439, 5,639,604, 5,585,481, and 5,656,744). A label, such as a fluorescent label or a chemiluminescent label, can be attached to the probe via a non-nucleotide linker (see, e.g., U.S. Patent Nos. 5,585,481, 5,656,744, and 5,639,604). In some embodiments, the detection oligomer comprises a base spacer between the 5' end of the oligonucleotide and the label.

[0093] In some embodiments, the probe (e.g., comprising a fluorescent label) further comprises a second label that interacts with the first label. For example, the second label can be a quencher. Such probes can be used, for example, in TaqMan™ assays, in which the probe is hybridized to a target or amplicon, followed by nucleolysis by a polymerase that contains 5'-3' exonuclease activity, resulting in the release of the fluorescent label, and thereby an increase in fluorescence, or fluorescence that is independent of interaction with the second label.

[0094] In some applications, one or more probes exhibiting at least some degree of self-complementarity are used to facilitate detection of probe:target duplexes in a test sample without first requiring removal of unhybridized probes prior to detection. Some embodiments of such detection probes include probes that form a configuration maintained by intramolecular hybridization, such as a configuration commonly referred to as a hairpin. Suitable hairpin probes include "molecular torches" (also called torches) (see, e.g., U.S. Pat. Nos. 6,849,412, 6,835,542, 6,534,274, and 6,361,945) and "molecular beacons" (see, e.g., U.S. Pat. Nos. 5,118,801 and 5,312,728). The spacer (or linker) can be an alkyl group. In some embodiments, the torch contains a 5-6 nucleotide sequence at its 3' end that is complementary to and can hybridize with a 5-6 nucleotide sequence at its 5' end. In some embodiments, a 5-6 nucleotide sequence at the 3' end that is complementary to and can hybridize with the 5-6 nucleotides at the 5' end is linked to the torch via a linker. In some embodiments, the linker is a C1-16 linker. In some embodiments, the linker is a C9 linker. Molecular torches are designed so that the target binding domain promotes hybridization to the target sequence more than the target closing domain. The target binding domain and target closing domain of the molecular torch contain interactive labels (e.g., fluorescent / quencher) positioned such that a different signal is generated when the molecular torch self-hybridizes as opposed to when the molecular torch hybridizes to the target nucleic acid, thereby enabling detection of a probe:target duplex in a test sample in the presence of an unhybridized probe having an associated viable label. In some embodiments, the torch contains a fluorescent molecule attached to the 5' end and a quencher attached to the 3' end. Alternatively, the fluorescent molecule may be attached to the 3' end of the torch and the quencher may be attached to the 5' end of the detection oligomer.

[0095] Examples of interactive donor / acceptor label pairs that may be used in connection with the present disclosure, without attempting to distinguish between FRET and non-FRET pairs, include, but are not limited to, fluorescein / tetramethylrhodamine, IAEDANS / fluorescein, EDANS / DABCYL, coumarin / DABCYL, fluorescein / fluorescein, BODIPY FL / BODIPY FL, fluorescein / DABCYL, CalRed-610 / BHQ-2, Lucifer Yellow / DABCYL, Quasar750 / BHQ-2, BODIPY / DABCYL, eosin / DABCYL, erythrosine / DABCYL, tetramethyl-rhodamine / DABCYL, Texas Red / DABCYL, CY5 / BHQ1, CY5 / BHQ2, CY3 / BHQ1, CY3 / BH2, and fluorescein / QSY7 dyes. Those skilled in the art will understand that when the donor and acceptor dyes are different, energy transfer can be detected by the appearance of sensitized fluorescence of the acceptor or quenching of donor fluorescence. Non-fluorescent acceptors such as DABCYL and QSY7 dyes advantageously eliminate the potential problem of background fluorescence resulting from direct (i.e., non-sensitized) acceptor excitation. Exemplary fluorophore moieties that can be used as one member of a donor-acceptor pair include fluorescein, ROX, and CY dyes (e.g., CY5). Exemplary quencher moieties that can be used as the other member of a donor-acceptor pair include DABCYL, Blackberry, and BLACK HOLE QUENCHER moieties available from Glen Research (Sterling, VA), Berry & Associates, Inc. (Dexter, Mich.), and Biosearch Technologies, Inc. (Novato, Calif.).

[0096] In some embodiments, the labeled oligomer (e.g., probe) is non-extendable (i.e., blocked). For example, the labeled oligomer can be made non-extendable by 3'-phosphorylation, having a 3'-terminal 3'-deoxynucleotide (e.g., a terminal 2',3'-dideoxynucleotide), a 3'-terminal inverted nucleotide (e.g., the last nucleotide is inverted so that it is attached to the penultimate nucleotide by a 3'-to-3' phosphodiester bond or its analog (e.g., phosphorothioate)), or having an attached fluorophore, quencher, or other label that interferes with extension (possibly, but not necessarily, attached via the 3' position of the terminal nucleotide). In some embodiments, the 3'-terminal nucleotide is unmethylated.

[0097] In some embodiments, it may be desirable to isolate the target nucleic acid sequence prior to the first phase of amplification. To this end, the sample can be contacted with a target capture oligomer (TCO) under conditions that allow hybridization of the TCO to a portion of the target nucleic acid sequence (the TCO binding site). In some embodiments, the target nucleic acid is captured directly on the solid support, for example, by interaction with an immobilized capture probe. In some embodiments, the target nucleic acid is captured on the solid support as a member of a molecular complex (pre-amplification hybrid), and the TCO crosslinks the target nucleic acid and the immobilized capture probe. In some embodiments, the solid support comprises a plurality of magnetic or magnetizable particles or beads that can be manipulated using a magnetic field. The step of isolating the target nucleic acid sequence may include washing the TCO:target nucleic acid sequence hybrid to remove undesirable components that may interfere with subsequent amplification. The step of isolating the target nucleic acid sequence may also include washing the TCO:target nucleic acid sequence hybrid to substantially remove excess promoter primer that is not hybridized to the target nucleic acid.

[0098] In some embodiments, the step of isolating the target nucleic acid sequence comprises contacting the sample with the promoter primer and the TCO under conditions that allow hybridization of the promoter primer and the TCO to the target nucleic acid sequence. The portion of the target sequence targeted by the promoter primer may be different (e.g., non-overlapping) from the portion targeted by the TCO. The portion of the target sequence targeted by the promoter primer may completely or partially overlap, or may be identical to, the portion targeted by the TCO.

[0099] In some embodiments, one or more TCOs, one or more promoter primers, and optionally one or more displacer oligomers are provided in a target capture reagent (TCR mixture). One or more promoter primers and optionally one or more displacer oligomers can be hybridized to one or more target nucleic acid sequences to form pre-amplification hybrids (together with the TCO(s)) and isolated along with one or more target nucleic acid sequences during the target capture step. One advantage of this method is that by hybridizing promoter primer(s) to target nucleic acid sequence(s) during target capture, the captured nucleic acid can be washed to remove sample components, including unhybridized oligomers. In a multiplex amplification reaction, removing unhybridized promoter primers allows the first phase of amplification to occur without interference from excess promoter primers, thereby substantially reducing or eliminating a problem common in multiplex reactions. In a single-phase multiplex amplification reaction, primers may interfere with each other. Excess primers more easily misprime (hybridize to non-target nucleic acids) in single-plex and multiplex reactions. Mispriming is a greater concern in multiplex reactions, where different organisms each have their own unique rRNA and oligonucleotides. Polyphase amplification addresses these issues by hybridizing promoter primers to their intended targets under stringent conditions and then washing away excess promoter primers. The resulting 1:1 primer / target ratio present in the first phase of polyphase amplification can boost the population of target nucleic acids to a level that allows subsequent addition of excess primers while reducing the level of mispriming or the impact of any mispriming on amplification.

[0100] Any of the described oligomers can contain at least one modified nucleotide. The modified nucleotide can be, but is not limited to, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, or a 5'-methylcytosine. In some embodiments, the 2'-O-methyl modified nucleotide is a 2'-OMe ribonucleotide. In some embodiments, the oligomer contains two or more modified nucleotides. In some embodiments, all of the nucleotides in the oligomer are modified. The two or more modified nucleotides can be the same or different. In some embodiments, any of the described oligomers can contain one or more 5'-methylcytosines. The oligomer can have 1, 2, 3, 4, 5, 6, 7, or more 5'-methylcytosines. In some embodiments, all cytosine nucleotides in the oligomer are 5'-methylcytosine modified nucleotides. The oligomer can have 1, 2, 3, 4, 5, 6, 7, or more 2'-OMe ribonucleotides. In some embodiments, all nucleotides in the oligomer are 2'-OMe ribonucleotides. In some embodiments, thymidine nucleotides can be substituted for uridine nucleotides. In some embodiments, all thymidine nucleotides can be substituted for uridine nucleotides. In some oligomers, 5'-methyl-2' deoxycytosine bases can be used to increase duplex stability by increasing the Tm of each 5'-methyl-2' deoxycytosine incorporated into the oligonucleotide (relative to the corresponding unmethylated oligomer) by about 0.5°C to 1.3°C.

[0101] C. Polyphase Amplification Disclosed are methods that use aspects of an isothermal amplification system, commonly referred to as "transcription-associated amplification," to amplify a target sequence by generating multiple transcripts from a nucleic acid template. Such methods generally use one or more amplification oligonucleotides, one of which provides an RNA polymerase promoter sequence, deoxyribonucleoside triphosphates (dNTPs), ribonucleoside triphosphates (NTPs), and enzymes with RNA polymerase and DNA polymerase activity to generate a functional promoter sequence near the target sequence and then transcribe the target sequence from the promoter (e.g., U.S. 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 Publication Nos. WO1988 / 001302, WO1988 / 010315, and WO1995 / 003430). Examples include transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), and self-sustained sequence replication (3SR).

[0102] To aid in understanding some embodiments disclosed herein, we briefly summarize the TMA method previously described in detail (e.g., U.S. Patent Nos. 5,399,491, 5,554,516, and 5,824,518). In TMA, a target nucleic acid containing a sequence to be amplified is provided as a single-stranded nucleic acid (e.g., ssRNA or ssDNA). Any conventional method for converting a double-stranded nucleic acid (e.g., dsDNA) to a single-stranded nucleic acid can be used. A promoter primer (e.g., a T7 primer) specifically binds to the target nucleic acid at its target sequence, and reverse transcriptase (RT) extends the 3' end of the promoter primer using the target strand as a template to create a cDNA copy, resulting in an RNA:cDNA duplex. RNase activity (e.g., RNase H of the RT enzyme) digests the RNA of the RNA:cDNA duplex. A second primer (e.g., a non-promoter primer or NT7 primer) specifically binds to its target sequence in the cDNA downstream of the promoter-primer end. RT then synthesizes a new DNA strand by extending the 3' end of the second primer using the cDNA as a template, creating a dsDNA containing a functional promoter sequence. RNA polymerase specific to the functional promoter initiates transcription, producing multiple (e.g., 100-1000) RNA transcripts (amplified copies or amplicons) complementary to the initial target strand. A second primer specifically binds to its target sequence in each amplicon, and RT creates cDNA from the amplicon RNA template, generating an RNA:cDNA duplex. RNase digests the RNA:cDNA double-stranded amplicon RNA, the target-specific sequence of the promoter primer binds to its complementary sequence in the newly synthesized DNA, and RT extends the 3' end of the promoter primer and the 3' end of the cDNA to create dsDNA containing a functional promoter to which RNA polymerase binds and transcribes additional amplicons complementary to the target strand. An autocatalytic cycle using these steps repeatedly during the reaction produces amplification of the initial target sequence.Amplicons can be detected during amplification (real-time detection) or at the end of the reaction (end-point detection) by using probes that specifically bind to sequences contained in the amplicons. Detection of a signal from the bound probe indicates the presence of the target nucleic acid in the sample.

[0103] A method for amplifying and / or detecting CMV using a multiphase amplification procedure is described. The method involves amplifying a CMV target nucleic acid sequence in a sample, including the following steps: first, subjecting the target nucleic acid sequence to a first-phase amplification reaction under conditions that do not support exponential amplification of the target nucleic acid sequence; the first-phase amplification reaction produces a first amplification product, which is then subjected to a second-phase amplification reaction under conditions that allow exponential amplification of the first amplification product, thereby producing a second amplification product.

[0104] In some embodiments, the portion of the target sequence targeted by the promoter primer (promoter primer binding site) can be different (e.g., non-overlapping) from the portion targeted by the TCO (if used). The promoter primer binding site may completely or partially overlap, or may be identical to, the TCO binding site. In some embodiments, the amplified region of the target sequence partially or completely overlaps with the target capture binding site. In some embodiments, the amplified region of the target sequence does not overlap with the target capture binding site.

[0105] In some embodiments, prior to the first amplification step, the sample is contacted with one or more promoter primers under conditions that allow the promoter primers to hybridize to a portion of the target nucleic acid sequence in the sample. The RNA polymerase promoter sequence of the promoter primer is recognized by an RNA polymerase, such as T7 RNA polymerase. The one or more promoter primers may target the same or different target nucleic acid sequences. The different target nucleic acid sequences may be derived from the same organism or different organisms.

[0106] The phase 1 amplification reaction is performed under conditions that do not support exponential amplification of the target nucleic acid sequence. In some embodiments, the phase 1 amplification reaction is a linear amplification reaction. The phase 1 amplification reaction typically generates about 2-fold to about 10,000-fold amplification. In some embodiments, the phase 1 amplification reaction generates about 10-fold to about 10,000-fold amplification of the target nucleic acid sequence. In some embodiments, the phase 1 amplification reaction is substantially isothermal, i.e., it does not involve the thermal cycling characteristic of PCR and other common amplification techniques. The phase 1 amplification reaction can be performed at 43±2°C, 43±1°C, 42±1°C, 42±0.5°C, 43±0.5°C, 44±0.5°C, 41-45°C, or 42-44°C.

[0107] In some embodiments, the phase 1 amplification reaction involves contacting the target nucleic acid sequence with a phase 1 amplification reaction mixture (e.g., an AMP or AMP1 mixture) that supports linear amplification of the target nucleic acid sequence and lacks at least one component required for its exponential amplification. In some embodiments, the at least one component required for its exponential amplification is an additional or excess promoter-primer. In some embodiments, the AMP or AMP1 reaction mixture includes one or more amplification enzymes. The one or more amplification enzymes can be, but are not limited to, a DNA polymerase, an RNA polymerase, or a combination thereof. The DNA polymerase can be, but is not limited to, an RNA-dependent DNA polymerase (reverse transcriptase), a DNA-dependent DNA polymerase, or a combination thereof. In some embodiments, the AMP or AMP1 mixture includes a ribonuclease (RNase), such as RNase H or a reverse transcriptase with RNase H activity. In some embodiments, the AMP or AMP1 mixture includes a reverse transcriptase with RNase H activity and an RNA polymerase. The RNA polymerase can be, but is not limited to, T7 RNA polymerase. In some embodiments, the AMP or AMP1 mixture contains one or more non-RNA polymerase promoter-containing amplification oligonucleotides (e.g., non-promoter primers (i.e., NT7 primers)). The one or more non-promoter primers may target the same target nucleic acid sequence or different target nucleic acid sequences. The different target nucleic acid sequences may be from the same organism or different organisms. In some embodiments, the AMP or AMP1 mixture includes one or more non-promoter primer(s), an RNA polymerase, ribonucleotide triphosphates (NTPs), and deoxyribonucleotide triphosphates (dNTPs). The AMP or AMP1 mixture may further contain other components, including, but not limited to, buffers, dNTPs, NTPs, and salts.

[0108] In some embodiments, the first phase amplification reaction is unable to support an exponential amplification reaction due to the absence of one or more components necessary for exponential amplification, the presence of an agent that inhibits exponential amplification, and / or the temperature of the reaction mixture is not conducive to exponential amplification. Without limitation, the absence of one or more components necessary for exponential amplification and / or inhibitors and / or reaction conditions can be selected from any of the following: amplification oligonucleotides (e.g., promoter primers, non-promoter primers, or combinations thereof), enzymes (e.g., polymerases such as RNA polymerases), nucleases (e.g., exonucleases, endonucleases, cleavase, RNase, phosphorylase, glycosylase, etc.), enzyme cofactors, chelators (e.g., EDTA or EGTA), ribonucleotide triphosphates (NTPs), deoxyribonucleotide triphosphates (dNTPs), Mg, salts, buffers, enzyme inhibitors, blocking oligonucleotides, pH, temperature, salt concentration, and any combination thereof. In some cases, the missing component may be indirectly involved, such as an agent that reverses the effect of an inhibitor of exponential amplification present in the first phase reaction. In some embodiments, the missing component(s) is a promoter-primer (an additional promoter-primer beyond the promoter-primer hybridized to the target nucleic acid as part of the pre-amplification hybrid).

[0109] The amplification reaction of Phase 2 (or a later phase, if three or more phases are present) is performed under conditions that allow for exponential amplification of the target nucleic acid sequence. In some embodiments, the amplification reaction of Phase 2 is an exponential amplification reaction. In some embodiments, the amplification reaction of Phase 2 is a substantially isothermal reaction, such as, for example, a transcription-associated amplification reaction or a strand displacement amplification reaction. In some embodiments, the amplification reaction of Phase 2 is a transcription-mediated amplification (TMA) reaction. In some embodiments, the amplification reaction of Phase 2 is performed at 43±2°C, 43±1°C, 42±1°C, 42±0.5°C, 43±0.5°C, 44±0.5°C, 41-45°C, or 42-44°C.

[0110] In some embodiments, the second (or later) phase of amplification involves combining and contacting the first amplification product with a second-phase amplification reaction mixture (e.g., a PRO or AMP2 mixture) that supports exponential amplification of the target nucleic acid sequence. Thus, the second-phase amplification reaction mixture typically contains, at a minimum, one or more components necessary for exponential amplification that are absent from the first-phase amplification reaction mixture. In some embodiments, the second-phase amplification reaction mixture contains one or more components selected from amplification oligonucleotides (such as promoter primers), reverse transcriptase, polymerase, nuclease, phosphorylase, enzyme cofactors, chelators, ribonucleotide triphosphates (NTPs), deoxyribonucleotide triphosphates (dNTPs), Mg, optimal pH, optimal temperature, salts, and combinations thereof. The polymerase can be, but is not limited to, an RNA-dependent DNA polymerase (e.g., reverse transcriptase), a DNA-dependent DNA polymerase, a DNA-dependent RNA polymerase, and combinations thereof. In some embodiments, the phase 2 amplification reaction mixture comprises an RNase, such as RNase H or a reverse transcriptase with RNase H activity. In some embodiments, the phase 2 amplification reaction mixture comprises a promoter primer, a reverse transcriptase with RNase H activity, and / or an RNA polymerase. In some embodiments, the phase 2 amplification reaction mixture further comprises a detection oligomer. The detection oligomer can be, but is not limited to, a torch or a molecular beacon.

[0111] In some embodiments, the target capture reagent (TCR) contains one or more TCOs, one or more T7 promoter primers, and optionally one or more displacer oligomers; the AR (AMP or AMP1) reagent contains a buffer, dNTPs, NTPs, salts, one or more non-T7 primers, and optionally one or more helper oligomers; the promoter (PR or AMP2) reagent contains a buffer, dNTPs, NTPs, salts, detergents, one or more T7 promoter primers, and one or more torch oligonucleotides; and the enzyme (ENZ) reagent contains a buffer, detergents, chelators, reverse transcriptase, and DNA polymerase.

[0112] In some embodiments, the described methods further include contacting the second amplification product with a bolus of one or more amplification components selected from, but not limited to, an amplification oligonucleotide (promoter primer or non-promoter primer), a reverse transcriptase (e.g., a reverse transcriptase with RNase H activity), a polymerase (e.g., an RNA polymerase), a nuclease, a phosphorylase, an enzyme cofactor, a chelator, ribonucleotide triphosphates (NTPs), deoxyribonucleotide triphosphates (dNTPs), Mg, a salt, and combinations thereof. This additional step can provide a boost to the second phase of the amplification reaction, as some of the amplification reaction components may be depleted.

[0113] The present method can be used to detect and / or quantify CMV target nucleic acid sequences in biological samples. The second-phase amplification reaction can be a quantitative amplification reaction. Methods for detecting second amplification products are also described. Detection and / or quantification of second amplification products can be performed using various detection techniques known in the art. Detection and / or quantification can be achieved, for example, by using a detection probe, a sequencing reaction, electrophoresis, mass spectrometry, melting curve analysis, or a combination thereof. In some embodiments, the second amplification product is detected and / or quantified using a detection probe. The detection probe can be, but is not limited to, a molecular torch (such as those described in U.S. Pat. No. 6,534,274), a molecular beacon, a hybridization switch probe, or a combination thereof. In some embodiments, detection and / or quantification can be performed in real time. The detection probe can be included in the first and / or second-phase amplification reactions with substantially equal success. A detector probe can be provided in the first and / or second phase amplification reaction mixture (e.g., the AMP or AMP1 mixture and / or the PRO or AMP2 mixture). In some embodiments, the PRO mixture contains a detector probe. The detector probe can include a torch.

[0114] D. Compositions and Kits The present disclosure provides oligomers, compositions, and kits useful for amplifying, detecting, and / or quantifying CMV in a sample. The oligomers, compositions, and kits can be used in thermal cycling and isothermal amplification methods, as well as single-phase and / or multi-phase amplification methods. In some embodiments, any combination of oligomers described herein can be provided in a kit.

[0115] Also described are reaction mixtures for determining the presence or absence, or quantifying the amount, of CMV target nucleic acid in a sample.

[0116] In some embodiments, a reaction mixture according to the present disclosure comprises at least one of the following: a combination of oligomers (amplification pairs), and optionally a helper oligomer and / or displacer oligomer described herein for amplifying a CMV UL56 gene target nucleic acid, and a detection probe oligomer described herein for determining the presence or absence of a CMV amplification product. In some embodiments, the various reaction mixtures comprise one or more of a target capture (TCR) mixture, an amplification (AR or AMP1) mixture, a promoter (PR or AMP2) mixture, and an enzyme (ENZ) mixture. The reaction mixtures may independently comprise one or more of a promoter primer (e.g., a T7 primer), a non-promoter primer (NT7 oligonucleotide), a helper oligomer, a displacer oligomer, a TCO, a detection oligomer, a reverse transcriptase, an RNA polymerase, dNTPs, NTPs, buffers, salts, and combinations thereof, as described herein for amplifying and / or detecting a CMV target nucleic acid in a sample. The kit can include, for example, one or more of the TER, TCR, AMP1 (AR) mix, and / or AMP2 (PR) mix, each described herein.

[0117] In some embodiments, the kit includes one or more control oligonucleotides, including, but not limited to, a control TCO, a control promoter primer, a control non-promoter primer, a control detection oligomer, and combinations thereof. The kit may include oligonucleotides for amplification and detection of CMV, or may include oligonucleotides for amplification and detection of CMV and one or more other organisms.

[0118] The compositions, kits, and / or reaction mixtures may further include several optional components, such as, for example, target capture probes (including, but not limited to, poly-(K) capture probes and poly-(A)-containing capture probes described in US2013 / 0209992, which is incorporated herein by reference). In some embodiments, the kits, compositions, or reaction mixture(s) further contain one or more of the following: enzyme(s) (e.g., a thermostable DNA polymerase, a reverse transcriptase, and / or an RNA polymerase), a positive control nucleic acid, a negative control nucleic acid, a control nucleic acid, dNTPs (e.g., dATP, dTTP, dGTP, and dCTP), NTPs (e.g., ATP, UTP, GTP, and CTP), Cl, MgCl, potassium acetate, buffer, BSA, sucrose, trehalose, DMSO, betaine, formamide, glycerol, polyethylene glycol, non-ionic surfactants, ammonium ions, EDTA, and other reagents or buffers suitable for isothermal amplification and / or detection. The DNA polymerase can be, but is not limited to, a reverse transcriptase. The buffer can be, but is not limited to, Tris-HCl and Tris-acetate. The non-ionic detergent can be, but is not limited to, Tween-20 and Triton X-100. The reaction mixture can contain amplification oligomers for only one target region of the CMV genome, or for multiple CMV target regions. Additionally, for reaction mixtures that include a detection probe along with a combination of amplification oligomers, the selection of amplification oligomers and detection probe oligomers for the reaction mixture will be linked by a common target region (i.e., the reaction mixture will contain a probe that binds to a sequence that can be amplified by the combination of amplification oligomers in the reaction mixture).

[0119] In some embodiments, the reaction mixture includes KCl. In some embodiments, the KCl concentration is about 50 mM. In some embodiments, the KCl concentration is greater than about 50 mM, e.g., about 60-150 mM, about 75-125 mM, about 80-120 mM, about 85-115 mM, or about 90-110 mM. In some embodiments, the KCl concentration is 55-65, 65-75, 75-85, 85-95, 95-105, 105-115, 115-125, 125-135, or 135-145 mM, each of the above being in mM and optionally modified by "about." In some embodiments, a composition according to the present disclosure includes KCl, e.g., at any of the concentrations described above. In some embodiments, a method according to the present disclosure includes performing an amplification reaction in the presence of KCl, e.g., at any of the concentrations described above.

[0120] In some embodiments, the described oligomers for amplification and / or detection of CMV have a shelf life of at least 3 months, at least 6 months, at least 9 months, at least 12 months, at least 15 months, at least 18 months, or at least 24 months from the date of manufacture.

[0121] In some embodiments, oligomers are provided, for example, in a kit or composition. The oligomers generally include a target hybridizing region configured to specifically hybridize to, for example, CMV nucleic acid. While oligomers of different lengths and base compositions can be used to amplify CMV nucleic acid, in some embodiments, oligomers of the present disclosure have a target hybridizing region of about 19-40 bases in length, or about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 bases in length. In some embodiments, the oligomer includes, in addition to the target hybridizing region, a second sequence region, such as a T7 RNA polymerase promoter, which may be located 5′ of the target hybridizing region. In some embodiments, the oligomer does not include a second sequence region.

[0122] In some embodiments, pairs of oligomers are provided, where one oligomer is configured to hybridize to the sense strand of a CMV nucleic acid and the other is configured to hybridize to the antisense strand of a CMV nucleic acid. Such oligomers include primer pairs for PCR, transcription-mediated amplification, or other forms of amplification known in the art.

[0123] In some embodiments, one or more oligomers, such as a primer pair, or a primer pair and a third oligomer that is optionally labeled (e.g., for use as a probe), are configured to hybridize to the CMV UL56 gene. In some embodiments, one or more oligomers, such as a primer pair, or a primer pair and a third oligomer that is optionally labeled (e.g., for use as a probe), are configured to hybridize to a CMV sequence represented by SEQ ID NO: 1 and / or its complement. In some embodiments, one or more internal control probe oligomers are also provided.

[0124] In some embodiments, one or more oligomers comprise a degenerate position. In some embodiments, the described oligomers comprise a degenerate position. In some embodiments, one or more oligomers comprise a non-Watson-Crick (NWC) position. In some embodiments, the oligomers comprise an NWC position. Exemplary NWC positions include U residues in the various exemplary oligomers of Tables 1A-E.

[0125] In some embodiments, one or more oligomers in a set, kit, composition, or reaction mixture comprise a methylated cytosine (e.g., 5-methylcytosine). In some embodiments, the oligomer contains 1, 2, 3, 4, 5, or more methylated cytosines. In some embodiments, at least about half of the cytosines in the oligomer are methylated. In some embodiments, all or substantially all (e.g., all but one or two) of the cytosines in the oligomer are methylated. In some embodiments, the cytosines at the 3' end or within 2, 3, 4, or 5 bases of the 3' end are unmethylated.

[0126] In some embodiments, the composition or kit includes a probe oligomer containing a torch or beacon. Each torch has a fluorophore and a quencher, such as 6'-carboxy-X-rhodamine (ROX) with an acridine quencher for IC torches, and fluorescein (FAM) with a Dabsyl quencher for CMV. The fluorophores associated with CMV and IC targets emit light at different wavelengths, allowing these targets to be distinguished from each other.

[0127] Additional components or reaction mixtures, compositions, and / or kits include, but are not limited to, capture beads, target capture reagents, target capture wash solutions, target enhancer reagents, amplification reagents (lyophilized cakes), amplification reagent reconstitution solutions, enzyme reagents (lyophilized cakes), enzyme reagent reconstitution solutions, promoter reagents (lyophilized cakes), promoter reagent reconstitution solutions, positive calibrators, CMV positive control nucleic acids, negative control nucleic acids, and / or sample transport media. In certain embodiments, the kits further comprise a set of instructions for practicing a method according to the present disclosure, which instructions may be associated with a package insert and / or packaging of the kit or its components.

[0128] Any method disclosed herein should also be understood as a disclosure of the corresponding use of the materials involved in the method for the purposes of the method. Any of the oligomers comprising CMV sequences, as well as any combinations (e.g., kits and compositions) comprising such oligomers, should also be understood as disclosed for use in detecting and / or quantitating CMV or amplifying CMV UL56 gene sequences, and for use in preparing compositions for detecting and / or quantitating CMV or amplifying CMV UL56 gene sequences.

[0129] E. Methods for amplifying, detecting, and / or quantifying CMV Methods for detecting and / or quantifying CMV or amplifying CMV UL56 gene sequences using one or more of the above oligomers, compositions, or kits are described.

[0130] Broadly speaking, the method can include one or more of the following components: target capture, in which CMV nucleic acid (e.g., from a sample such as a clinical sample) is annealed to a TCO; isolation, e.g., washing, to remove material not associated with the capture oligomer; amplification; and amplicon detection, e.g., amplicon quantification, which can be performed in real time with amplification. Certain embodiments involve each of the above steps. Certain embodiments involve exponential amplification, optionally with a preceding linear amplification step. Certain embodiments involve exponential amplification and amplicon detection. Certain embodiments involve any two of the above-listed components. Certain embodiments involve any two of the immediately above-listed components, e.g., washing and amplification, or amplification and detection.

[0131] In some embodiments, the amplification comprises: (1) contacting the sample with at least two oligomers to amplify a CMV nucleic acid target region corresponding to the CMV target UL56 gene nucleic acid, wherein the oligomers comprise at least two amplification oligomers described above (e.g., one or more primers oriented in the sense direction and one or more primers oriented in the antisense direction for exponential amplification); (2) performing an in vitro nucleic acid amplification reaction in which any CMV target nucleic acid present in the sample is used as a template to generate an amplification product; and (3) detecting the presence or absence of the amplification product, thereby determining the presence or absence of CMV in the sample or quantifying the amount of CMV nucleic acid in the sample.

[0132] Detection methods according to the present disclosure can further include obtaining a sample to be subjected to subsequent steps of the method. In certain embodiments, "obtaining" the sample to be used includes, for example, receiving the sample at a testing facility or other location where one or more steps of the method are performed, and / or retrieving the sample from a location within the facility where one or more steps of the method are performed (e.g., from a storage facility or other repository).

[0133] In certain embodiments, the method further includes purifying the CMV target nucleic acid from other components in the sample prior to amplification, e.g., prior to the capture step. Such purification may include methods to separate and / or concentrate organisms contained in the sample from other sample components, or to remove or degrade non-nucleic acid sample components, e.g., proteins, carbohydrates, salts, lipids, etc. In some embodiments, DNA in the sample is degraded, e.g., with DNase, and optionally removing or inactivating the DNase or removing the degraded DNA.

[0134] In some embodiments, purifying the target nucleic acid involves capturing the target nucleic acid and specifically or non-specifically separating the target nucleic acid from other sample components. Non-specific target capture methods may involve selective precipitation of the nucleic acid from a substantially aqueous mixture, attachment of the nucleic acid to a support that is washed to remove other sample components, or other means of physically separating the nucleic acid from a mixture containing CMV nucleic acid and other sample components.

[0135] Target capture typically occurs in a solution-phase mixture containing one or more TCOs that hybridize to the CMV target sequence under hybridization conditions. In embodiments involving TCOs, the CMV-target:TCO complex is captured by adjusting the hybridization conditions so that the TCO tail hybridizes to the immobilized probe. Certain embodiments use a particulate solid support, such as paramagnetic beads. In some embodiments, a promoter primer is present during capture. Hybridization conditions are adjusted to allow isolation and purification of the pre-amplified hybrid.

[0136] Isolation can follow capture, separating the complex on the solid support from other sample components. Isolation can be achieved by any suitable technique, for example, by washing the support associated with the CMV-target sequence one or more times (e.g., two or three times) to remove other sample components and / or unbound oligomers. In embodiments using a particulate solid support such as paramagnetic beads, the particles associated with the CMV target can be suspended in a wash solution and recovered from the wash solution by magnetic attraction. To limit the number of handling steps, the CMV target nucleic acid can be amplified by simply mixing the CMV target sequence in the complex on the support with amplification oligomers and proceeding to the amplification step.

[0137] Exponential amplification of the CMV target sequence utilizes an in vitro amplification reaction that uses at least two amplification oligomers flanking the target region to be amplified. In some embodiments, at least the first (forward) and second (reverse) oligomers are used to amplify the target sequence. The amplification reaction can be thermal cycling or isothermal. Suitable amplification methods include, but are not limited to, replicase-mediated amplification, polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), and transcription-mediated or transcription-associated amplification (TMA).

[0138] The detection step can be performed using any of a variety of known techniques for detecting signals specifically associated with the amplified target sequence, such as by hybridizing the amplification product with a labeled detection probe and detecting a signal generated from the labeled probe (including, in some embodiments, from a label released from the probe after hybridization). In some embodiments, the labeled probe includes a second moiety, such as a quencher or other moiety that interacts with the first label, as discussed above. The detection step can also provide additional information about the amplified sequence, such as all or part of its nucleic acid sequence. Detection can be performed after the amplification reaction is complete or can be performed simultaneously with the amplification of the target region, for example, in real time. In some embodiments, the detection step allows for homogeneous detection, e.g., detecting hybridized probes without removing unhybridized probes from the mixture (see, e.g., U.S. Pat. Nos. 5,639,604 and 5,283,174). In some embodiments, the nucleic acid is associated with a surface that produces a physical change, such as a detectable charge. Amplified nucleic acids can be detected by concentrating them in or on a matrix and detecting the nucleic acids or their associated dyes (e.g., intercalators such as ethidium bromide or SYBR Green), or by detecting an increase in a dye associated with the nucleic acids in the solution phase. Other detection methods include using nucleic acid detection probes configured to specifically hybridize to sequences in the amplification product and detect the presence of a probe:product complex, or by using a complex of probes that can amplify a detectable signal associated with the amplification product (e.g., U.S. Pat. Nos. 5,424,413, 5,451,503, and 5,849,481, each of which is incorporated herein by reference). Directly or indirectly labeled probes that specifically associate with the amplification product provide a detectable signal indicative of the presence of the target nucleic acid in the sample. In particular, the amplification product contains a sequence in or complementary to the CMV UL56 gene, and the probe binds directly or indirectly to a sequence contained in the amplification product to indicate the presence of CMV nucleic acid in the test sample.

[0139] In some embodiments that detect amplification products near or at the end of the amplification step, a linear detection probe may be used to provide a signal indicating hybridization of the probe to the amplification product. One example of such detection uses a luminescently labeled probe that hybridizes to the target nucleic acid. The luminescent label is then hydrolyzed from the unhybridized probe. Detection is performed by chemiluminescence using a luminometer (see, e.g., International Patent Application Publication No. WO 89 / 002476). In some embodiments that use real-time detection, the detection probe may be, for example, a hairpin probe such as a molecular beacon, a molecular torch, or a hybridization switch probe that is labeled with a reporter moiety that is detected when the probe binds to the amplification product. Such a probe may include a target-hybridizing sequence and a non-target-hybridizing sequence.

[0140] In some embodiments, detection is performed at time intervals. Detection can be performed by measuring fluorescence at regular time intervals. The time intervals can be, but are not limited to, 1 to 60 seconds, 1 to 120 seconds, 1 to 180 seconds, 1 to 240 seconds, or 1 to 300 seconds. In some embodiments, the time intervals are 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 seconds. When detection is performed at regular time intervals, each interval is referred to as a cycle. Detection can be performed over 20 to 240 cycles, 30 to 210 cycles, 40 to 180 cycles, 50 to 150 cycles, or 60 to 120 cycles. For example, detection every 30 seconds over 60 minutes constitutes 120 cycles. Detection can occur at the beginning or end of a cycle. Detection can also be performed continuously.

[0141] Embodiments of the compositions and methods described herein can be further understood by the following examples. The method steps used in the examples are described herein, and the following information more specifically describes typical reagents and conditions used in the methods. Other reagents and conditions that do not substantially affect the process or results may be used, so long as the guidance provided in the above description is followed. Furthermore, the disclosed methods and compositions may be performed in systems that perform one or more steps (e.g., pipetting, mixing, incubation, etc.) manually or in an automated device, or may be used in any type of known device (e.g., test tubes, multi-tube unit devices, multi-well devices such as 96-well microtiter plates, etc.).

[0142] F. List of Embodiments 1. A kit for amplifying a target region of nucleic acid derived from a human cytomegalovirus (CMV) UL56 gene sequence, the kit comprising: (a) a forward primer comprising 19 to 31 contiguous nucleic acid bases having at least 90% identity to a 19 to 31 nucleotide sequence present in SEQ ID NO:2; and (b) a reverse primer comprising 21 to 40 contiguous nucleic acid bases having at least 90% identity to a 21 to 40 nucleotide sequence present in SEQ ID NO:3. 2. The kit of embodiment 1, wherein the forward primer, the reverse primer, or both the forward and reverse primers comprise at least one modified nucleotide. 3. The kit of embodiment 2, wherein the modified nucleotides comprise 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, or 5'-methylcytosine. 4. The kit of any one of embodiments 1 to 3, wherein the forward primer comprises the nucleobase sequence of SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 19. 5. The kit of any of embodiments 4, wherein the forward primer is a non-promoter primer comprising the nucleobase sequence of SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:19. 6. The kit of any one of embodiments 1 to 5, wherein the reverse primer comprises the nucleobase sequence of SEQ ID NO:23, SEQ ID NO:24, or SEQ ID NO:25. 7. The kit of embodiment 6, wherein the reverse primer comprises the nucleobase sequence of SEQ ID NO:6, SEQ ID NO:23, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:41, SEQ ID NO:47. 8. The kit of any one of embodiments 1-4 or 6-7, wherein the RNA polymerase promoter sequence is linked to the 5' end of the forward primer or the reverse primer. 9. The kit of embodiment 8, wherein the RNA polymerase promoter sequence is a T7 RNA polymerase promoter sequence. 10. The kit of embodiment 9, wherein the T7 RNA polymerase promoter sequence comprises the nucleotide sequence of SEQ ID NO: 78. 11. The kit of embodiment 10, wherein the reverse primer comprises the nucleobase sequence of SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, or SEQ ID NO:46. 12. The kit of any one of embodiments 1-10, wherein the forward primer comprises SEQ ID NO: 11 and the reverse primer comprises SEQ ID NO: 23. 13. The kit of any one of embodiments 1 to 12, further comprising a probe oligomer. 14. The kit of embodiment 13, wherein the probe oligomer (a) comprises the nucleobase sequence of SEQ ID NO: 51 or SEQ ID NO: 52, wherein one or more uracil nucleotides may be substituted for thymine nucleotides, or (b) comprises a nucleotide sequence comprising 24 to 35 consecutive nucleobases that hybridize to SEQ ID NO: 81. 15. The kit of embodiment 14, wherein the probe oligomer comprises at least one modified nucleotide. 16. The probe oligomer of embodiment 15, wherein the modified nucleotide comprises a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, or a 5'-methylcytosine. 17. The kit of any one of embodiments 14-16, wherein the probe oligomer comprises the nucleobase sequence of SEQ ID NO:21, SEQ ID NO:26, SEQ ID NO:39, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71. 18. The kit of any one of embodiments 14 to 17, wherein the probe oligomer contains a detectable label. 19. The kit of embodiment 18, wherein the detectable label comprises a fluorescent molecule. 20. The kit of embodiment 19, wherein the fluorescent molecule is attached to the 5' or 3' end of the probe oligomer. 21. The kit of any one of embodiments 14-20, wherein the probe oligomer contains 4-5 nucleobases at the 3' end of the probe oligomer that are complementary to 4-5 nucleobases at the 5' end of the probe oligomer. 22. The kit of embodiment 21, wherein the fluorescent molecule is attached to the 5' end of the probe oligomer and the quencher is attached to the 3' end of the probe oligomer, or the fluorescent molecule is attached to the 3' end of the probe oligomer and the quencher is attached to the 5' end of the probe oligomer. 23. The kit of embodiment 22, wherein the probe oligomer comprises the nucleobase sequence of SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, or SEQ ID NO:70. 24. The kit of any one of embodiments 14 to 22, wherein the forward primer comprises SEQ ID NO: 11, the reverse primer comprises SEQ ID NO: 23, and the probe oligonucleotide comprises SEQ ID NO: 53. 25. The kit of any one of embodiments 1 to 24, further comprising a helper oligomer comprising 19 to 31 consecutive nucleobases having at least 90% identity to a 19 to 31 nucleotide sequence present in SEQ ID NO:2. 26. The kit of embodiment 25, wherein the helper oligomer is blocked. 27. The kit of embodiment 25 or 26, wherein the helper oligomer comprises the nucleotide sequence of SEQ ID NO: 10 or SEQ ID NO: 19. 28. The kit of embodiment 27, wherein the helper oligomer comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19. 29. The kit of any one of embodiments 1-28, further comprising a displacer oligomer comprising 21 to 27 consecutive nucleobases having at least 90% identity to a 21 to 25 nucleotide sequence present in SEQ ID NO:5. 30. The kit of embodiment 29, wherein the displacer oligomer comprises the nucleotide sequence of SEQ ID NO: 12, SEQ ID NO: 25, or SEQ ID NO: 41. 31. The kit of embodiment 30, wherein the displacer oligomer comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:41, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:86, SEQ ID NO:87, and SEQ ID NO:88. 32. The kit of any one of embodiments 1 to 31, further comprising a target capture oligomer (TCO) comprising the nucleotide sequence of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:43, or SEQ ID NO:45. 33. The kit of embodiment 32, wherein the TCO contains a moiety that allows for isolation of the TCO. 34. The kit of embodiment 33, wherein the portion comprises a polyA nucleotide sequence. 35. The kit of embodiment 33, wherein the moiety comprises (dT)3(dA)30. 36. The kit of embodiment 35, wherein the TCO comprises the nucleotide sequence of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 42, or SEQ ID NO: 44. 37. A kit according to any one of embodiments 32 to 36, wherein the kit comprises a first TCO comprising the nucleotide sequence of SEQ ID NO: 42 and a second TCO comprising the nucleotide sequence of SEQ ID NO: 44. 38. The kit of any one of embodiments 1-37, further comprising one or more of a target capture reagent, a target capture wash solution, a target enhancer reagent, an amplification reagent, an enzyme reagent, a promoter reagent, a CMV positive control nucleic acid, a negative control nucleic acid, a sample transport medium, a reverse transcriptase, an RNA polymerase, dNTPs, NTPs, a buffer, and a positive and / or negative control sample. 39. A method for amplifying a target region of nucleic acid derived from a human cytomegalovirus (CMV) UL56 gene sequence present in a sample, comprising: (a) contacting the sample with a forward primer and a reverse primer configured to amplify a CMV UL56 amplicon, wherein the forward primer comprises 19 to 31 contiguous nucleobases having at least 90% identity to a 19 to 31 nucleotide sequence present in SEQ ID NO:2 and the reverse primer comprises 21 to 40 contiguous nucleobases having at least 90% identity to a 21 to 40 nucleotide sequence present in SEQ ID NO:3; (b) exposing the sample to conditions sufficient to amplify the target region, thereby producing an amplification product. 40. The method of embodiment 39, wherein the forward primer and / or the reverse primer comprises at least one modified nucleotide. 41. The method of embodiment 40, wherein at least one modified nucleotide comprises a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, or a 5'-methylcytosine. 42. The method of any one of embodiments 39-41, wherein the forward primer comprises the nucleobase sequence of SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 19, and the reverse primer comprises the nucleobase sequence of SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 47. 43. The method of embodiment 42, wherein the forward primer comprises the nucleobase sequence of SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:19, and the reverse primer comprises the nucleobase sequence of SEQ ID NO:6, SEQ ID NO:23, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:41, SEQ ID NO:47. 44. The method of embodiment 43, wherein a T7 RNA polymerase promoter sequence is linked to the 5' end of the reverse primer. 45. The method of embodiment 44, wherein the reverse primer comprises the nucleobase sequence of SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, or SEQ ID NO:46. 46. ​​The method of any one of embodiments 39-43, wherein the forward primer comprises SEQ ID NO: 11 and the reverse primer comprises SEQ ID NO: 23. 47. The method of any one of embodiments 39-46, further comprising detecting the presence or absence of an amplification product. 48. The method of embodiment 47, wherein detecting the presence or absence of the amplification product utilizes a probe oligomer that specifically hybridizes to the amplification product. 49. The method of embodiment 48, wherein the probe oligomer comprises the nucleobase sequence of SEQ ID NO: 51 or SEQ ID NO: 52, wherein one or more uracil nucleotides may be substituted for thymine nucleotides, or (b) comprises a nucleotide sequence comprising 24 to 35 consecutive nucleobases that hybridize to SEQ ID NO: 81. 50. The method of embodiment 49, wherein the probe oligomer comprises the nucleobase sequence of SEQ ID NO:21, SEQ ID NO:26, SEQ ID NO:39, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71. 51. The method of any one of embodiments 48-50, wherein the probe oligomer contains 4-5 nucleobases at the 3' end of the probe oligomer that are complementary to 4-5 nucleobases at the 5' end of the probe oligomer. 52. The method of embodiment 51, wherein the fluorescent molecule is attached to the 5' end of the probe oligomer and the quencher is attached to the 3' end of the probe oligomer, or the fluorescent molecule is attached to the 3' end of the probe oligomer and the quencher is attached to the 5' end of the probe oligomer. 53. The method of embodiment 52, wherein the probe oligomer comprises the nucleobase sequence of SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, or SEQ ID NO:70. 54. The method of any one of embodiments 39-44 and 46-53, wherein the forward primer comprises SEQ ID NO: 11, the reverse primer comprises SEQ ID NO: 23, and the probe oligonucleotide comprises SEQ ID NO: 53. 55. The method of any one of embodiments 39-54, wherein amplifying comprises a thermal cycling reaction. 56. The method of embodiment 55, wherein the thermal cycling reaction comprises a polymerase chain reaction (PCR). 57. The method of any one of embodiments 39-54, wherein amplifying comprises an isothermal nucleic acid amplification reaction. 58. The method of embodiment 57, wherein the isothermal nucleic acid amplification reaction comprises transcription-mediated amplification (TMA). 59. The method of any one of embodiments 39 to 54, wherein amplifying comprises nucleic acid sequence-based amplification, replicase-mediated amplification, Qβ-replicase-mediated amplification, ligase chain reaction (LCR), or strand displacement amplification (SDA). 60. The method of any one of embodiments 47-59, wherein detecting the presence or absence of an amplified CMV UL56 amplicon further comprises quantifying the amplified CMV UL56 amplicon. 61. The method of embodiment 60, wherein quantifying the amplified CMV UL56 amplicon comprises monitoring production of the CMV amplicon. 62. The method of any one of embodiments 47 to 61, wherein the detecting and / or quantifying is analyzed in real time. 63. A method for quantifying a human cytomegalovirus (CMV) UL56 gene target nucleic acid sequence in a sample, comprising: (a) contacting a sample with at least one target capture oligomer (TCO) comprising the nucleobase sequence of SEQ ID NO: 43 or SEQ ID NO: 45 and a first promoter primer comprising the nucleobase sequence of SEQ ID NO: 47 under conditions that allow hybridization of the at least one TCO and the first promoter primer to a CMV UL56 gene target nucleic acid sequence, thereby generating a pre-amplification hybrid comprising a target nucleic acid sequence hybridized to each of the at least one TCO and the first promoter primer; (b) isolating the pre-amplified hybrids by target capture onto a solid support followed by washing to remove any first promoter primer that did not hybridize to the CMV UL56 gene target nucleic acid sequence in step (a); (c) amplifying at least a portion of the CMV UL56 gene target nucleic acid sequence of the pre-amplified hybrid isolated in step (b) in a phase 1 amplification reaction mixture comprising a non-promoter primer comprising the nucleobase sequence of SEQ ID NO: 19 under conditions that support linear amplification thereof but not exponential amplification thereof in a phase 1 substantially isothermal transcription-associated amplification reaction, thereby resulting in a reaction mixture comprising a first amplification product, wherein the first amplification product is not a template for nucleic acid synthesis during the phase 1 substantially isothermal transcription-associated amplification reaction; (d) combining the first amplification product with a second-phase amplification reaction mixture comprising a second promoter primer comprising the nucleobase sequence of SEQ ID NO:47 and a probe oligomer comprising the nucleobase sequence of SEQ ID NO:57, and performing exponential amplification of the first amplification product in a second-phase substantially isothermal transcription-associated amplification reaction in the second-phase amplification reaction mixture, thereby synthesizing a second amplification product; (f) detecting synthesis of a second amplification product in the second phase amplification reaction mixture using the probe oligomer at regular time intervals; (g) using the results of step (f) to quantify the target nucleic acid sequence in the sample. 64. The method of embodiment 63, wherein at least one TCO comprises a first TCO comprising the nucleobase sequence of SEQ ID NO: 43 and a second TCO comprising the nucleobase sequence of SEQ ID NO: 45. 65. The method of embodiment 63 or 64, wherein the first and second promoter primers each comprise a 5' promoter sequence of an RNA polymerase. 66. The method of embodiment 65, wherein the RNA polymerase is T7 RNA polymerase. 67. The method of any one of embodiments 63-67, wherein the solid support comprises an immobilized capture probe. 68. The method of embodiment 67, wherein the solid support is a magnetically attractable particle. 69. The method of any one of embodiments 63-68, wherein the first-phase and second-phase isothermal transcription-associated amplification reactions each comprise an RNA polymerase and a reverse transcriptase, and the reverse transcriptase comprises endogenous RNase H activity. 70. A method according to any one of embodiments 63 to 69, wherein the first amplification product of step (c) is a cDNA molecule having the same polarity as the target nucleic acid sequence in the sample, and the second amplification product of step (d) is an RNA molecule. 71. The method of any one of embodiments 63 to 70, wherein the first probe oligomer in step (d) is a configuration-enhanced sensitivity probe that generates a detectable signal when hybridized to the second amplification product. 72. The method of any one of embodiments 63 to 71, wherein the probe oligomer of step (d) is a fluorescently labeled sequence-specific hybridization probe. 73. The method of any one of embodiments 64 to 72, wherein the first TCO comprises the nucleobase sequence of SEQ ID NO: 42, the second TCO comprises the nucleobase sequence of SEQ ID NO: 44, the first and second promoter primers each comprise the nucleobases of the sequence of SEQ ID NO: 46, and the probe oligomer comprises the nucleobase sequence of SEQ ID NO: 56. 74. The method of any one of embodiments 63-73, wherein the phase 1 amplification reaction mixture and / or the phase 2 amplification reaction mixture further comprises a helper oligomer and / or a displacer oligomer. 75. The method of embodiment 74, wherein the helper oligomer is 19 to 31 nucleobases in length and comprises the nucleobase sequence of SEQ ID NO: 14, and the displacer oligomer is 21 to 27 nucleobases in length and comprises the nucleobase sequence of SEQ ID NO: 41. 76. The method of embodiment 74 or 75, wherein the helper oligomer, the displacer oligomer, or both the helper oligomer and the displacer oligomer are blocked. [Example]

[0143] Example 1. CMV Amplification. Various combinations of salt and oligomer concentrations for CMV were evaluated to determine the optimal amplification conditions. PPR (primer-probe containing reconstitution buffer) mixtures were made by mixing primers, probes, KCl, and MgCl mixtures. The following mixtures were made and used for CMV DOE: [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8]

[0144] PPR mixtures 1-12 were vortexed, spun down, and 250 µL of oil was added to the top, spun down again, and then loaded into the instrument. PPR mixture 13 was also spun down, but with 400 µL of oil on top instead of 250 µL.

[0145] CMV plasmid was diluted to 1000 cp / rxn for testing in the PPR mixture in section 1. CMV plasmid was diluted to 1000 cp / rxn in STM by doing the following: [Table 1-9]

[0146] 34 ml was aliquoted into 30 tubes. All 30 tubes were processed in a Panther Fusion system (Hologic, Inc., San Diego, CA) with two extractions per tube and three replicates per extraction (n = 12 for PPR1-12 and n = 36 for PPR13). Data were analyzed using DevTool (4.9.8.0) with the following parameters: [Table 1-10] [Table 1-11] [Table 1-12] [Table 1-13] [Table 1-14]

[0147] Signal to noise remains highest with high salt and high oligomers. Reduction of oligomers only slightly affects signal to noise. In some embodiments, salt was >4 mM.

[0148] Conclusion: Ct and baseline show significant differences between primers + Mg and primers + probe, and RFU between primers + Mg and probe + Mg. All show a lack of compatibility greater than 0.0001, with the lowest RFU at 0.0412. The most desirable option is 1 μM primers, 0.8 μM probe, and 6 mM MgCl. However, lower MgCl also shows very good results. Because lower salt increases RFU and background, JPM likely estimates 6 mM as optimal based on background alone. However, fewer primers and probes show similar Ct, but each 0.2 μM difference results in a 20% decrease in RFU. The data suggest that Ct is too similar across all conditions, since Ct accounts for only 57% of the variability, while RFU and baseline account for 90% and 94%, respectively.

[0149] Example 2: Limit of detection of CMV plasmid. CMV plasmid was evaluated in serum to determine the limit of detection (LoD). Sample lysis was performed using 360 μL of sample combined with 450 μL of target capture reagent and 126 μL of target enhancer reagent. After incubation, the mixture was washed with target capture wash reagent and eluted in a final volume of 50 μL. A CMV PPR was made to determine the LoD of the plasmid in serum. [Table 2-1]

[0150] Two tubes were prepared. One was a reconstitution tube with 1200 μL of PPR mixture and 400 μL of oil added on top. The other was a reconstitution tube with 850 μL of PPR mixture added and 350 μL of oil added on top. All tubes were spun down again before loading into the instrument. CMV plasmid was diluted to three concentrations in serum and tested with the PPR mixture from section 1. CMV plasmid was diluted to 100, 10, and 1 cp / rxn in pooled serum by doing the following: [Table 2-2] [Table 2-3]

[0151] Pooled serum samples consisted of 3 ml each of serum 051 to 056. STM with 1 mg / ml proteinase K was prepared by adding 400 μL of 20 mg / mL stock ProK solution to 7600 μL of STM.

[0152] 1230 μL of each concentration of CMV plasmid was added to two tubes containing 1230 μL of STM:ProK (solution transport medium:proteinase K) mixture from 2.2. The remaining volume was not dissolved and stored at -70°C as needed. Each tube was processed with 10 PCR replicates (three PCR replicates for three extractions and four PCR replicates for the fourth) at each concentration (n=20). A negative control was generated consisting of 390 μL of STM:ProK mixture and 390 μL of serum pool. One extraction was processed with three PCR replicates per extraction. All samples were processed and data was analyzed using DevTools (4.9.8.0) with the following parameters: [Table 2-4]

[0153] Conclusion: The LoD of CMV plasmid in pooled serum is 10 cp / rxn, or 277.78 cp / ml in a 95% lysed sample tube. Plasmids have not been tested in serum by the TMA CMV team. [Table 2-5] [Table 2-6]

[0154] Example 3: CMV Preliminary Virus Detection Limits in Serum and Plasma Detection limits were determined for CMV virus (TCID50 / ml) in serum and plasma at 1 log increments and an STM ratio of 1:0.2 as described in Example 2.

[0155] CMV PPR was prepared to determine the LoD of virus in serum and plasma. The PPR mixture was as follows: [Table 3-1]

[0156] Two tubes were prepared. One was a reconstitution tube with 1100 μL of PPR mixture and 400 μL of oil added on top. The other was a reconstitution tube with 1000 μL of PPR mixture added and 400 μL of oil added on top. All tubes were spun down again before loading into the device. CMV virus was diluted to five concentrations in serum and plasma and tested with the PPR mixture from section 1. CMV was diluted to 10,000, 1,000, 100, 10, and 1 TCID50 / ml in pooled serum and pooled plasma by doing the following: [Table 3-2] [Table 3-3]

[0157] Pooled serum samples each consisted of 3 ml of serum, and pooled plasma samples each consisted of 3 ml of plasma. Concentrations are based on a BioFire LoD of 100 TCID50 / ml. STM with 2.6 mg / ml proteinase K was prepared by doing the following: [Table 3-4]

[0158] To ensure that ProK did not remain in the STM for too long, this mixture was made immediately before the samples were ready to be lysed. 800 μL of each concentration of CMV plasmid in both serum and plasma was added to one tube containing 185 μL of STM:ProK mixture. Each tube was processed with two extractions and three PCRs. Negative controls consisting of 115 μL of STM:ProK mixture and 500 μL of serum and plasma pools were made separately. One extraction was processed with three PCR replicates per extraction. All samples were processed on a Panther Fusion system (Hologic, Inc. San Diego). Data were analyzed using DevTools (4.9.8.0) with the following parameters: [Table 3-5] [Table 3-6] [Table 3-7]

[0159] of serum (i.e., 1 x 10 1 or 10) resulted in a nearly 1-log difference between extractions. Because only one tube was processed, all extractions were from the same tube. This is an unusual occurrence. Plasma at the same concentration yielded the expected results. All other samples also yielded similar results between extraction / PCR replicates.

[0160] Conclusion: The preliminary LoD indicates 100% detection of approximately 10 TCID50 / ml in both serum and plasma. Since there was a delay in the Ct, it appears that the plasma had some inhibition issues or resulted in degradation of the virus itself. Also, the serum result of 1E1 TCID50 / ml showed a high standard deviation. Further analysis at half-log increments will help determine the true LoD of CMV virus in serum and plasma.

[0161] Example 4. CMV Preliminary Virus Detection Limits in Serum and Plasma. The detection limits were determined for CMV virus (TCID50 / ml) in serum and plasma at half-log increments and an STM ratio of 1:0.2 as described in Example 2. CMV PPRs were made to determine the LoD of virus in serum and plasma. The following PPR mixtures were made: [Table 4-1]

[0162] Five tubes were prepared. Four were reconstituted with 1200 μL of PPR mixture and 400 μL of oil added on top. The other was reconstituted with 400 μL of PPR mixture and 250 μL of oil added on top. All tubes were spun down again before loading into the device. CMV virus was diluted to four concentrations in serum and plasma and tested with the PPR mixture from section 1. CMV was diluted to 31.6, 10, 3.16, and 1 TCID20 / ml in pooled serum and pooled plasma by doing the following: [Table 4-2] [Table 4-3]

[0163] The pooled serum samples each consisted of 3 ml of serum and the pooled plasma samples each consisted of 3 ml of plasma. STM with 2.6 mg / ml proteinase K was prepared by doing the following. [Table 4-4]

[0164] To ensure that ProK did not remain in the STM for too long, this mixture was made immediately before the samples were ready to be thawed. 1400 μL of each concentration of CMV plasmid in both serum and plasma was added to two tubes containing 325 μL of STM:ProK mixture. Each tube was processed with three extractions and three PCR replicates, with one extraction per PCR replicate. Two tubes were processed, resulting in n=20 per concentration. Separate negative controls were made, consisting of 115 μL of STM:ProK mixture and 500 μL of serum and plasma pools. One extraction was processed with three PCR replicates per extraction. All samples were processed on a Panther Fusion system. Data was analyzed using DevTools with the following parameters: [Table 4-5] [Table 4-6] [Table 4-7] [Table 4-8]

[0165] Of the 20 PCR replicates, 6 were very early compared to the average, and all 6 were from the same tube (extraction 1 and 2).

[0166] Conclusions: The LoD for CMV in plasma and serum is somewhat variable. Differences in Ct exist between the two matrices; plasma may have inhibitors that prevent 100% CMV detection. Plasma has an LoD of 3.16 TCID50 / ml, or 135.9 cp / ml per BioFire. Serum exhibits an LoD of 1 TCID50 / ml, or 43 cp / ml per BioFire.

[0167] Example 5. Analysis of Analyte-Specific Reagent CMV Reactivity and ZeptoMetrix CMV Control. CMV reactivity with four strains of CMV was assessed using the current CMV PCR oligo set. All CMV isolates were tested at a 10x LoD of the original strain. CMV controls from Zeptometrix (Franklin, MA) were analyzed to determine assay sensitivity in cp / ml from whole virus and also to help determine the range for the same strains in TCID50 / ml. The following CMV PPRs were prepared: [Table 5-1]

[0168] One reconstitution tube was prepared with 1200 μL of PPR mixture and 400 μL of oil, and the other with 700 μL of PPR mixture and 300 μL of oil on top. All tubes were spun down and then loaded into the Panther Fusion system. CMV virus isolates were diluted to 1E1.5 TCID50 / ml in plasma and processed in the PantherFusion system. [Table 5-2]

[0169] CMV virus stock was diluted to 1E1.5 TCID50 / ml (10x LoD for AD-169 strain) by doing the following (in plasma): [Table 5-3A] [Table 5-3B] [Table 5-3C] [Table 5-3D]

[0170] ProK was added to PBS at 3 mg / ml (for a final 0.5 mg / ml in the sample) by doing the following. [Table 5-4]

[0171] 800 μL of each virus sample at 31.6 TCID50 / ml was added to 160 μL of PBS:ProK mixture and mixed by pipetting up and down. Inactivated virus stocks (cp / ml) from Zeptometrix were also analyzed to determine assay sensitivity. Stocks were diluted in PBS by doing the following: [Table 5-5]

[0172] A negative control was processed and consisted of a 500 μL plasma pool with 100 μL of PBS:ProK mix. All samples except the negative control were processed with two extractions (three PCR replicates per extraction). The negative control was processed with one extraction and three PCR replicates. All samples were processed on the Panther Fusion system using the following sequence file: [Table 5-6]

[0173] Data analysis was performed according to the following parameters: [Table 5-7] [Table 5-8] [Table 5-9] [Table 5-10]

[0174] Conclusion: The strain that emerged very early had the lowest concentration in TCID50 / ml. This isolate only reached a low titer after several weeks. However, it resulted in an early Ct. For all isolates, the true LoD for each was less than 1 TCID50 / ml, lower than that of the AD-169 strain. The LoD for inactivated virus NATtrol in PBS ranged from 100 to 10 cp / ml, which is the theoretical limit of PCR. The TCID50 / ml for the virus strains ranged from 3.16 TCID50 / ml to 1 TCID50 / ml. The BioFire ranged from 43 and 136 cp / ml. The PCR efficiency on the instrument produced a slope of 3.4 and an R2 of >0.98.

[0175] Example 6. CMV specificity. CMV specificity was assessed based on the concentration closest possible to 1E+06 cp / ml based on CMV titer conversion. CMV PPRs were prepared. The following CMV PPRs were prepared: [Table 6-1]

[0176] One reconstitution tube was prepared with 1000 μL of PPR mixture and 400 μL of oil on top. All tubes were spun down before loading into the Panther Fusion system. Eight panels of SD-AJH-000263 were tested on the Panther Fusion system (one extraction and three PCR replicates per extraction). A positive control was tested and consisted of 1000 cp / ml of CMV plasmid. [Table 6-2]

[0177] A negative control was run, consisting of 600 μL of STM. Both controls were run with one extraction and one PCR replicate to determine if PPR was performed correctly. All samples were run on the Panther Fusion system using the following sequence file: [Table 6-3]

[0178] Data analysis was performed according to the following parameters: [Table 6-4] [Table 6-5] [Table 6-6]

[0179] Panels with high cell / cp counts showed delayed IC Ct.

[0180] Conclusions: All panels were negative for CMV and positive for IC.

[0181] Example 7. Analysis of CMV-positive and CMV-negative plasma clinical samples. Fifty CMV-positive and fifty CMV-negative plasma samples were evaluated to determine the performance of the CMV PCR assay. Specimens were tested in 1:0.2 PBS with 10x TCO and 0.5 mg / ml ProK. The following PPR mixtures were prepared: [Table 7-1]

[0182] For the four PPRs, 1200 μL was added to the reconstitution tube and 400 μL of oil was added on top. [Table 7-2]

[0183] For the four PPRs, 1200 μL was added to the reconstitution tube and 400 μL of oil was added on top. Fifty CMV-negative and fifty CMV-positive clinical plasma samples were tested with each PPR mixture. Additional 10x TCO and 3 mg / ml ProK were added to PBS, and 100 μL was added to the labeled tubes: 0.666 mg per L, 0.000666 mg / ml, 0.0002997 mg / reaction, 360 μL sample input, 0.0003 mg in sample / reaction, and 0.0030 mg of 10x sample / reaction. [Table 7-3]

[0184] The negative run contained 600 μL of PBS. The positive control consisted of CMV plasmid in PBS spiked at 50 cp / rxn.

[0185] Step 1. Initial Dilution Stock concentration = 1.00 x 10 7 Final concentration=1.00×10 5 Stock volume (µL) = 10 PBS μL = 990 Final volume (μL) 1000

[0186] Step 2. CMV Calibrator in STM, Required Concentration (Calculation) Starting concentration (cp / ml)=1.00×10 5 Test volume per 5 μL rxn (cp) = 50 cp / mL in "sample" tube = 1388.89 Starting volume (μL)=22 μL of PBS = 1578 Final volume (μL)=1600

[0187] All clinical specimens were mixed by adding 500 μL to a tube containing 2.1 to 100 μL of PBS / ProK / TCO mixture and mixing by inverting three times. Samples were tested according to the following table. [Table 7-4]

[0188] 25 positive and 25 negative samples were processed on one instrument each. Samples were processed on the PantherFusion system using the following DNA thermal cycling conditions: [Table 7-5]

[0189] Data analysis was performed according to the following parameters: [Table 7-6]

[0190] The discordant samples, along with one high positive and one low positive, were processed for CMV TMA testing. [Table 7-7] [Table 7-8-1] [Table 7-8-2]

[0191] NOTE: Multi-core vs. single-core resulted in differences in RED677 background and therefore overall RFU values. NOTE: CMV_Neg22 resulted in an aberrant curve, possibly indicating a base pair mismatch in the probe. [Table 7-9-1] [Table 7-9-2] [Table 7-10]

[0192] NOTE: CMV112 may result in an aberrant curve, indicating a base pair mismatch in the probe. [Table 7-11] [Table 7-12]

[0193] Note: All samples that were negative using the COBAS TaqMan® CMV Test ("Roche" or "Roche Test") (Roche Diagnostics, North America) and positive by CMV PCR were positive by CMV TMA quantification. These values, along with low and high positivity, returned a strong correlation between TTime and Ct for the two assays. TTime is the term used to describe the amplification time when the sample signal value exceeds a threshold signal value (typically a predetermined background signal value) during the sample amplification and detection reaction.

[0194] Conclusions: In an initial study of 50 Roche test-negative plasma samples, 44 / 50 were positive for CMV. Six discrepancies were negative by PCR (80%). Six discrepancies were confirmed as positive by CMV TMA quantification, which showed a strong correlation between TTime and Ct values. This gives 100% specificity when the six specimens are removed from the data set.

[0195] 40 / 50 samples were positive by CMV PCR, but all 50 samples were positive for CMV by Roche testing. Ten specimens were tested by CMV TMA quantification, and eight were low positive, below the theoretical limit (and therefore not positive in the initial test). The other two were also negative by CMV TMA quantification. The eight that were positive were well below the IU / ml given by Roche (1-3 logs lower). The CMV PCR assay shows promising results with the ability to pick up specimens in the 5-10 cp / rxn range and 100% specificity.

[0196] Example 8. CMV Oligo Screening. Various combinations of CMV oligomer designs were evaluated using CMV plasmids. The designs were based on the CMV TMA oligomer design. CMV oligomers were screened and tested in various combinations. The following PPR mixtures were made: [Table 8-1-1] [Table 8-1-2] [Table 8-1-3]

[0197] 550 μL of the PPR mixture was added to eight separate reconstitution tubes, and 250 μL of oil was added on top of each. All tubes were spun down again before loading into the instrument.

[0198] CMV plasmid was tested at three concentrations in STM with PPR mixture: CMV plasmid was diluted to 1000, 100, and 10 cp / rxn by doing the following: [Table 8-2]

[0199] 1.34 ml of each concentration was added to four tubes. One extraction from each tube was processed with three PCR replicates per extraction. A negative control was also run, consisting of 2.9 ml of STM only. N=3 per extraction. All samples were processed on the Panther Fusion system. Data was analyzed using DevTool with the following parameters: [Table 8-3] [Table 8-4] [Table 8-5-1] [Table 8-5-2]

[0200] Conclusion: Results show 100% detection up to 10 cp / rxn for all oligo sets. The best combination includes SEQ ID NO: 11 and SEQ ID NO: 23. Both probes show good results, with SEQ ID NO: 53 showing much higher RFU and a higher signal-to-noise ratio at 1000 cp / rxn, and SEQ ID NO: 53 showing higher RFU at 100 and 10 cp / rxn. SEQ ID NO: 13, SEQ ID NO: 23, and SEQ ID NO: 53 also show good results at all concentrations, but begin to lag at 10 cp / rxn compared to the SEQ ID NO: 11 / SEQ ID NO: 23 combination.

[0201] Example 9. CMV Quantification. The CMV quantification assay is an in vitro nucleic acid amplification test for quantifying cytomegalovirus (CMV) DNA and / or RNA in a sample, including, but not limited to, a biological sample such as human plasma. The CMV quantification assay can be combined with an automated detection system. The CMV quantification assay can be used to aid in the management of solid organ transplant recipients. In patients receiving anti-CMV therapy, serial DNA measurements can be used to assess viral response to treatment.

[0202] In some embodiments, the CMV quantification assay is a transcription-mediated amplification test with real-time detection. This assay is used to detect and / or quantify CMV in a sample and can be combined with a detection system. The detection system can be an instrument that provides automated sample processing, amplification, detection, data reduction for quantification, and amplicon inactivation.

[0203] Reagents: Controls and calibrators are optionally provided in separate boxes. Details of the reagents, calibrators, and controls provided in each assay kit box are detailed in Table 9A below. Each kit contains three lyophilized materials: an amplification reagent, a promoter reagent, and an enzyme reagent. These are reconstituted by the user using reconstitution reagents specific to each lyophilized reagent. Each kit includes a target capture reagent (TCR) and a target enhancer reagent (TER) provided in liquid format, while the remaining reagents are lyophilized. Calibrators and additional controls may be provided separately. [Table 9A]

[0204] Exemplary reagents include:

[0205] "Sample transport medium" or "STM" is a phosphate buffer (pH 6.7) containing EDTA, EGTA, and lithium lauryl sulfate (LLS).

[0206] "Target capture reagent" or "TCR" refers to a (dT) 14 It is a HEPES buffer (pH 6.4) containing lithium chloride and EDTA with 250 μg / ml of magnetic particles (1 micron Sera-Mag™ MG-CM particles, Seradyn, Inc. Indianapolis, Ind.) to which oligonucleotides are covalently attached. In some embodiments, the TCR contains one or more TCOs, and / or one or more T7 primers, and optionally one or more displacer oligomers.

[0207] The "target capture wash solution" or "TC wash solution" is HEPES buffer (pH 7.5) containing sodium chloride, EDTA, 0.3% (v / v) absolute ethanol, 0.02% (w / v) methylparaben, 0.01% (w / v) propylparaben, and 0.1% (w / v) sodium lauryl sulfate.

[0208] The "amplification reagent" or "AR" is a HEPES buffer (pH 7.7) containing magnesium chloride, potassium chloride, the four deoxyribonucleotide triphosphates (dATP, dCTP, dGTP, and dTTP), and the four ribonucleotide triphosphates (rATP, rCTP, rGTP, and rUTP). Primers and / or probes may be added to the reaction mixture in the amplification reagent or added separately from the reagent (primer-free amplification reagent).

[0209] When used in an amplification or preamplification reaction mixture, the "enzyme reagent" or "ER" is a HEPES buffer (pH 7.0) containing MMLV reverse transcriptase (RT), T7 RNA polymerase, salts, and cofactors.

[0210] The "Target Enhancer Reagent" (TER) solution contains 1.6N LiOH.

[0211] Procedure: The CMV quantification assay uses real-time monitoring of transcription-mediated amplification (TMA) to quantify CMV virus. The assay targets the UL56 gene of CMV. The amount of virus in a sample is determined by comparing the signal to that generated from a known concentration of viral DNA (calibrator). In addition, controls are run every 24 hours to ensure the validity of the test. The assay is performed using a detection system that employs three basic processing steps: 1) Target capture, where viruses are lysed, hybridized to magnetic particles, and separated from the sample components. 2) Amplification of targets using TMA with simultaneous collection of fluorescent signals. 3) Processing of the signals to generate quantitative results for each sample.

[0212] During target capture, viral DNA is isolated from the sample by treating with detergent to solubilize viral envelope, denature proteins, and release viral genomic DNA. If present in the test sample, the capture oligonucleotide hybridizes to the highly conserved region of CMV DNA. The hybridized target binds to magnetic particles, which are then separated from the sample using a magnetic field.

[0213] Target amplification occurs isothermally via transcription-mediated amplification, using two enzymes, T7 RNA polymerase and reverse transcriptase, to exponentially generate RNA from the captured DNA through cycles of forward and reverse transcription.

[0214] Detection is achieved using single-stranded nucleic acid torches that exist during target amplification and hybridize to amplicons in real time.Each torch has a fluorophore and a quencher.When the torch is not hybridized to the amplicon, the quencher is close to the fluorophore and suppresses fluorescence.Amplicon-torch binding results in the separation of the quencher from the fluorophore, which allows the fluorophore to be excited in response to light stimulation and emits a signal at a specific wavelength.

[0215] Table 9B shows processing steps that can be used in a CMV quantification assay. [Table 9B]

[0216] Assay Processing: Real-time detection and quantification was performed using a fluorometer.

[0217] The target capture reagent (TCR), optionally combined with a target enhancer reagent (TER), lyses the CMV and facilitates capture of the released CMV DNA onto magnetic particles. The lithium lauryl sulfate present in the TCR is used to lyse the virus. The CMV DNA released by this process is captured onto magnetic particles using CMV-specific oligonucleotides or "capture oligos" (also referred to as "target capture oligos"). The TCR may also contain an internal calibrator / internal control (IC), which is a sequence of DNA unrelated to CMV. The IC is processed with the target in the same tube and serves as both an internal control and an internal calibrator for the test.

[0218] Step 1. Add 60 μL of TER to each tube, followed by 500 μL of sample. Add 400 μL of TCR to each reaction tube. The TCR buffer contains 10 g / 100 mL of lithium lauryl sulfate for virus lysis, magnetic particles for target capture, and IC and CMV-specific oligonucleotides for amplification. Mix the fluids to ensure uniformity of the mixture.

[0219] Step 2. Samples are optionally incubated in a 43.7°C transfer incubator to pre-warm the samples before transferring them to a 64°C high temperature incubator.

[0220] Step 3. The sample is then transferred to a high-temperature incubator set at 64°C. During incubation at 64°C, the CMV is destroyed and the genomic DNA is released. Several oligonucleotides are present in the TCR. The first of these is the T7 promoter primer, which is complementary to the target and incorporates the T7 promoter region. Due to the length of this oligonucleotide, it is less affected by mismatches within the target sequence, thereby improving the detection of equivalent genotypes. A displacer primer is also present, which serves to open the double-stranded DNA and ensure binding of the T7 primer to the target.

[0221] Step 4. Return the sample to the transition incubator to begin the cooling process. The TCR contains capture oligonucleotides and magnetic beads conjugated to poly-T oligonucleotides. The capture oligonucleotides have sequences complementary to the target that allow them to capture the target, and a 30-base poly-A tail that allows them to hybridize to the poly-T oligonucleotides on the magnetic beads. During the initial cooling step and continuing through step 5, the target and IC are captured on the magnetic particles.

[0222] Step 5. Cool the sample in a chiller gradient (17 °C to 19 °C) to allow for tighter binding between the CMV and IC targets and the magnetic beads.

[0223] Step 6. The sample is moved to a magnetic parking station, where it is subjected to a magnet that attracts the magnetic particles to the side of the tube before entering the magnetic washing station.

[0224] Step 7. The sample is then moved to a magnetic washing station where potential interfering substances are removed from the reaction by washing the magnetic particles. A magnet briefly moves the magnetic particles to the side of the tube containing the sample, and the liquid is removed. A wash buffer is added, and the process is repeated to ensure removal of potentially interfering substances. The sample containing the purified magnetic beads is then moved to the amplification loading station for reagent addition.

[0225] Amplification and Signal Detection: Amplification creates multiple copies of a target so that it can be more easily detected. This is accomplished using TMA technology (patents incorporated by reference). Amplification, promoter, and enzyme reagents are used to initiate and sustain amplification, and the product is detected in real time. These reagents can be lyophilized reagents that are reconstituted before use. The reconstituted amplification reagent is a buffer and contains non-T7 oligonucleotides specific for CMV and IC. Blocked helper oligomers are also present to aid in binding of the non-T7 oligonucleotides to the target. It also contains the raw materials necessary for amplicon construction.

[0226] The CMV quantification assay uses two phases of amplification: linear and exponential. The second phase, exponential amplification, is achieved using a promoter reagent. The reconstituted promoter reagent is a buffer containing T7 oligonucleotides for the CMV and IC targets. The promoter reagent also contains the materials necessary to assemble copies of the RNA amplicon, along with target-specific torches that detect amplified CMV or IC in real time. The reconstituted enzyme reagent is a buffer and contains two enzymes that initiate and sustain amplification of both the CMV and IC targets.

[0227] Amplification: First, a promoter primer binds to the target DNA or RNA of the sample. A displacement primer can also be used to improve promoter primer binding. Reverse transcriptase then extends the promoter primer or the promoter primer and displacement primer to create single-stranded DNA. Single-stranded DNA with the promoter primer is created. A forward primer then binds to the ssDNA, and RNA polymerase extends the strand. The single RNA strand serves as a template for multiple copies of the RNA.

[0228] Step 8. Add amplification reagent (50 µL / test) to the sample and mix in the amplification filling station.

[0229] Step 9. Move the samples to a 43.7°C transition incubator to increase the temperature of the samples.

[0230] Step 10. Return the sample to the amplification loading station where the enzyme reagent (25 μL / test) is added.

[0231] Step 11. The sample is moved to an amplification incubator set at 42.7°C. The sample remains in this incubator for 5 minutes, during which the first round of amplification is initiated. The T7 initiation primer is complementary to the CMV target and also contains a promoter sequence for T7 RNA polymerase. The reverse transcriptase present in the enzyme reagent binds to the T7 initiation primer-target complex and initiates the generation of complementary DNA from the CMV target. The reverse transcriptase also initiates a displacement reaction using a displacer oligomer to generate single-stranded DNA from the CMV target. A similar reaction occurs simultaneously for the IC. This single-stranded DNA incorporates the T7 promoter region. The amplification reagent also contains a non-T7 primer that binds to the complementary DNA (cDNA) and initiates the creation of double-stranded DNA. This is then used by the RNA polymerase from the enzyme reagent to make multiple copies of the RNA. This RNA is then converted to a single-stranded DNA amplicon by reverse transcriptase using the non-T7 primer. This phase is known as the linear amplification or enrichment phase.

[0232] Step 12. After the 5 minute linear amplification phase, the sample is returned to the amplification loading station where promoter reagent is added and mixed.

[0233] Step 13. Return the samples to the amplification incubator for additional rounds of amplification. The promoter reagent (25 μL / test) contains additional CMV and IC T7 primers. The addition of the promoter reagent initiates and sustains additional rounds of exponential amplification of the CMV and IC targets. The amplification incubator also incorporates a fluorometer that measures the fluorescent signal generated by the amplification. The addition of a second T7 promoter primer initiates the generation of a complementary strand of DNA to the single-stranded cDNA created in the previous step. This is then used by RNA polymerase to create multiple copies of RNA. The internal control uses a mechanism similar to CMV to create cDNA from the RNA target and double-stranded DNA.

[0234] The promoter reagent also includes the torch mentioned above.

[0235] Signal and Result Processing: Both the CMV target and IC signals are processed by first performing baseline subtraction. Baseline subtraction estimates the baseline for each curve and then removes that level of fluorescence from each data point. As a result, the baseline for each curve starts at the same level.

[0236] The data are then scaled (normalized) so that all samples have the same maximum fluorescence. The resulting curves are then analyzed by standard curve-fitting algorithms.

[0237] After baseline subtraction and normalization are complete, the TTime for each curve can be calculated. TTime is the time (on the x-axis) at which the normalized fluorescent signal (y-axis) emerges from the background signal. This is set by a predetermined cutoff. The TTime for each reaction is calculated for both the target and the corresponding IC curve.

[0238] To correct for individual variations, the CMV TTime is divided by the IC TTime to generate a "ratio." CMV TTime is inversely proportional to the CMV concentration in the initial specimen. CMV curves generated by samples ranging from 100 IU / mL to 1E8 IU / mL are separated into separate curves. Because the IC target concentration for each reaction is constant, the IC TTime remains relatively constant. Minor competition between the CMV and IC amplification systems means that the IC TTime increases slightly, but in a predictable manner. Calibrators of known CMV concentrations are then used to generate a calibration curve, using the ratio of CMV and IC TTime, plotting the TTime ratio against the target concentration. Once the calibration curve is established, the concentration of CMV in unknown samples can be calculated by comparing the resulting ratio to the calibration curve.

[0239] A saved calibration curve. The calibration curve is linear and the slope of the assay is negative. This calibration curve can be generated for each reagent lot. The equation for the calibration curve is established, and the point where the line intersects the x-axis is determined by extrapolation.

[0240] Before generating results, each reagent kit is calibrated by running three replicates of a calibrator. Two positive controls, one at a low concentration and one at a higher concentration, are present, and a negative control is used. The calibrator contains synthetic CMV DNA in a buffer solution at a predefined concentration. Due to the linear nature of the calibration curve, a combination of the user-run calibrator and the x-intercept of the calibration curve is used to generate a calibration curve specific to the reagent kit.

[0241] Results Reporting: Results can be calculated using information generated from calibrator and control samples.

[0242] Example 10. Polyphasic (biphasic) amplification / detection.

[0243] "Sample transport medium" or "STM" is a phosphate buffer (pH 6.7) containing EDTA, EGTA, and lithium lauryl sulfate (LLS).

[0244] The "target capture reagent" or "TCR" is a HEPES buffer (pH 6.4) containing lithium chloride and EDTA with 125 μg / ml of magnetic particles (1 micron SERA-MAG™ MG-CM particles, Seradyn, Inc. Indianapolis, IN) to which a (dT)14 oligonucleotide is covalently attached. The TCR contains multiple oligos, which may include one or more TCOs, one or more T7 primers, and one or more displacers. In some embodiments, the TCR contains one or more displacer oligomers.

[0245] The "target capture wash solution" or "TC wash solution" is HEPES buffer (pH 7-8, pH 7.5±5, or pH 7.5) containing sodium chloride, EDTA, 0.3% (v / v) absolute ethanol, 0.02% (w / v) methylparaben, 0.01% (w / v) propylparaben, and 0.1% (w / v) sodium lauryl sulfate.

[0246] The "amplification reagent" or "AR" is a Tris buffer solution (pH 7-8, pH 7.5±5, or pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 76, pH 7.7, pH 7.9, or pH 8) containing magnesium chloride, potassium chloride, the four deoxyribonucleotide triphosphates (dATP, dCTP, dGTP, and dTTP), and the four ribonucleotide triphosphates (NTP: ATP, CTP, GTP, and UTP). One or more primers, helper oligomers, displacer oligomers, and / or probe oligomers may be added to the reaction mixture via the amplification reagent. In some embodiments, one or more primers, helper oligomers, displacer oligomers, and / or probe oligomers may be added to the reaction mixture separately from the amplification reagent. In some embodiments, for the first phase amplification reaction, the amplification reagent may contain one or more non-promoter primers and one or more helper oligomers. In some embodiments, for a second phase amplification reaction, the amplification reagents may contain one or more promoter primers, one or more displacer oligomers, and one or more probe oligomers.

[0247] The "promoter reagent" or PR is a Tris buffer solution containing magnesium chloride, potassium chloride, the four deoxyribonucleotide triphosphates (dATP, dCTP, dGTP, and dTTP), and the four ribonucleotide triphosphates (NTPs: ATP, CTP, GTP, and UTP). Some of the primers, helpers, and probes may be added to the reaction mixture through the promoter reagent.

[0248] When used in an amplification or preamplification reaction mixture, "enzyme reagent" or "ENZ" is a HEPES-buffered solution (pH 6.5-8, pH 7.0±5, or pH 6.5, pH 6.6, pH 6.7, pH 6.8, pH 6.9, pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 76, pH 7.7, pH 7.9, or pH 8) containing MMLV reverse transcriptase (RT), T7 RNA polymerase, salts, and cofactors.

[0249] The "Target Enhancer Reagent" (TER) is an alkaline solution containing 1.68 M LiOH lithium hydroxide.

[0250] The T7 primer hybridizes to the target sequence during target capture, followed by removal of excess T7 primer during a wash step prior to the first amplification reaction. In some embodiments, the TCO hybridizes to the target sequence during target capture. In some embodiments, the displacer oligomer hybridizes to the target sequence during target capture. Excess TCO and / or displacer oligomer may also be removed during a wash step prior to the first amplification reaction.

[0251] During the first amplification phase (AMP1), an oligo containing an NT7 primer and, optionally, a helper, is introduced along with all the necessary amplification and enzyme reagents, except for an additional T7 primer. In the presence of reverse transcriptase, the T7 primer hybridized to the captured target is extended to create a cDNA copy. The NT7 primer then hybridizes to the cDNA, extending it and filling in the promoter region of the T7 primer, creating an active double-stranded DNA template. T7 polymerase then generates multiple RNA transcripts from the template. The NT7 primer then hybridizes to the RNA transcript and extends it, creating a promoterless cDNA copy of the target RNA template. The RNA strand is degraded by the RNase activity of the reverse transcriptase. Because no free T7 primers are available in the first-phase amplification mixture, the reaction does not proceed further. Phase 2 begins with the addition of additional oligos, which may include T7 primers, non-T7 primers, and optionally helper and detection oligonucleotides, thus initiating exponential amplification and detection of the cDNA pool generated in Phase 1.

[0252] For multiplex amplification and detection, use one or more of TCO, T7 primer, NT7 primer, torch oligonucleotide, and optionally one or more of displacer and helper.Oligonucleotide can amplify one or more different sequences in the same target nucleic acid, or can amplify sequences in different target nucleic acids, or a combination thereof.Different target nucleic acids can be derived from the same organism or from different organisms.

[0253] A. Exemplary Experimental Protocol 1:

[0254] Plate Settings:

[0255] In some embodiments, four different plates are set up for use with two automated KingFisher devices. 1. Plate 1 (TCR plate) contains the sample. Add target capture reagent (e.g., 100 μL) to this plate. TCO and T7 primer, and optionally a displacer oligomer, hybridize to the target nucleic acid (e.g., 400 μL sample). Use a magnet and magnetic beads (capture probes on a solid support) to capture the TCO:target nucleic acid:T7 primer:(optional displacer oligomer) (pre-amplified hybrid). In the case of single-phase TMA, the T7 primer may not be present in the TCR mixture. In some embodiments, the sample is added to the TER, followed by the TCR containing the TCO, and in biphasic amplification, the T7 primer. In some embodiments, the TER is added to the sample, followed by the TCR containing the TCO, and in biphasic amplification, the T7 primer. In some embodiments, the TER may be added to the mixture of the TCR and the sample. The mixture of these reagents may be incubated at a higher temperature for a period of time so that the TCO, T7 primer, and optionally the displacer oligomer hybridize to the target nucleic acid in the sample. The TCO also hybridizes to the magnetic beads. The target nucleic acid with the hybridized TCO, T7 primer, and optional displacer oligomer (pre-amplification hybrid) is captured by separating the magnetic beads using a magnet. The mixture of the sample and TCR is then removed from the tube, and the beads are washed twice with Aptima wash buffer. 2. Plate 2 is a deep-well plate and holds 200-500 µL / well of APTIMA wash buffer. Aptima wash buffer contains detergent and alcohol, which is used to wash captured targets (pre-amplified hybrids). 3. Plate 3 contains 200-500 µL / well of APTIMA wash buffer and is used to provide a second wash of captured targets (pre-amplification hybrids). 4. Plate 4 contains 50 μL / well of AMP or AMP1 reagent. In some embodiments, the AMP or AMP1 reagent contains buffer, salt, dNTP, NTP, and one or more NT7 primers, and optionally and / or one or more helper oligomers.

[0256] Target capture and isolation: In some embodiments, the sample is first contacted with a target enhancer reagent. In the case of biphasic TMA, TCO(s), T7 primer(s), and optionally a displacer oligomer are added to a sample containing or suspected of containing a target nucleic acid. In the case of single-phase TMA, TCO(s) are added to a sample containing or suspected of containing a target nucleic acid. In some embodiments, if present, the T7 primer is added at a ratio of about 1 T7 primer to 1 target nucleic acid. The TCO, T7 primer, and displacer oligomer are incubated with the target nucleic acid for a period of time to allow hybridization of these oligomers to the target nucleic acid to form a pre-amplification hybrid. The pre-amplification hybrid is then captured and purified, and excess or unhybridized oligomers are removed. The pre-amplification hybrid is then isolated using magnetic particles with a binding partner, such as poly(dT), in the case of TCO. 1. Plate 1 (TCR plate) is placed in a heat block and heated to 62°C for 20-30 minutes, followed by incubation at a lower temperature (e.g., 23°C) for 20 minutes to 2 hours. In some embodiments, the TCR plate is covered with a 65°C lid to prevent condensation from forming on top of the wells. The captured pre-amplified hybrids are then transferred to Plate 2. 2. After the first wash (approximately 10 minutes), add a deep-well comb / magnet cover to plate 2 to capture the pre-amplified hybrids. The captured pre-amplified hybrids are transferred to plate 3. 3. After the second wash, a small comb (magnetic cover) is added to plate 3 to capture the pre-amplified hybrids. The washed pre-amplified hybrids are captured and moved to plate 4. The fourth plate is moved to a thermal cycler for real-time isothermal amplification and detection.

[0257] Biphasic transcription-mediated amplification and real-time detection.

[0258] Phase 1 Amplification: AMP reagents containing NT7 primer(s), enzyme, dNTPs, NTPs, and optionally one or more helper oligomer(s) (AMP1 mixture) are added to the purified target nucleic acid containing the pre-amplified hybrid. The mixture is incubated for a period of time to allow the formation of a first amplification product. 1. The AMP1 plate containing the NT7 primer, optionally helper oligomers, and purified target nucleic acid is incubated with the hybridized T7 primer at approximately 42-44°C for 5-15 minutes. 2. Add 25 μL of ENZ mix containing reverse transcriptase and T7 RNA polymerase, seal and mix, and incubate at approximately 42-44°C for 5 minutes.

[0259] Phase 2 amplification: A promoter reagent (AMP2) containing a T7 primer and optionally a probe oligomer such as torch is added to the first amplification product and incubated for a period of time to allow the formation of a second amplification product. In some embodiments, one or more helper oligomer(s) and more non-T7 primers are added during phase 2 amplification. Add 3.25 μL of AMP2 (also called PR) mixture to each well, seal, and mix. In some embodiments, the AMP2 mixture contains buffer, salt, detergent, dNTP, NTP, one or more T7 primers, torch probe(s), and optionally more non-T7 primers and / or helper oligomer(s). 4. Reaction program: 120 cycles of 30 seconds at 42-43°C, with label detection (collection) at the end of each cycle.

[0260] Detection: The amplification of the target nucleic acid sequence is detected in real time by recording the fluorescent signal from the detector oligonucleotide at regular intervals.

[0261] B. Exemplary Experimental Protocol 2:

[0262] Target capture and isolation: In some embodiments, the sample is first contacted with a target enhancer reagent. In the case of biphasic TMA, TCO(s), T7 primer(s), and optionally a displacer oligomer are added to a sample containing or suspected of containing a target nucleic acid. In the case of single-phase TMA, TCO(s) are added to a sample containing or suspected of containing a target nucleic acid. The TCO, T7 primer, and displacer oligomer are incubated with the target nucleic acid for a period of time to allow hybridization of these oligos to the target nucleic acid to form a pre-amplification hybrid. The pre-amplification hybrid is then captured and purified, and excess or unhybridized oligos are removed. The pre-amplification hybrid is then isolated using magnetic particles with binding partners, such as poly(dT), in the case of TCO.

[0263] The mixture of sample, TCR, and optionally TER is heated to 60-65°C for 20-30 minutes, followed by incubation at a lower temperature for 20 minutes to 2 hours. The pre-amplified hybrids are captured using a magnet to separate the magnetic beads to which they hybridize. The sample and reagent mixture is removed from the tube. The magnetic beads are washed 1-2 times by adding wash buffer to the tube, mixing, and then incubating it with a magnet to separate the magnetic beads. After the beads are captured, the wash buffer is removed from each tube.

[0264] Biphasic transcription-mediated amplification and real-time detection.

[0265] Phase 1 amplification: AMP reagents containing NT7 primer(s), enzyme, dNTPs, NTPs, and optionally one or more helper oligomer(s) (AMP1 mixture) are added to the purified target nucleic acid containing the pre-amplified hybrid. The mixture is incubated for a period of time to allow the formation of a first amplification product. a. The AMP1 mixture containing the NT7 primer, optionally helper oligomers, and purified target nucleic acid (pre-amplified hybrid) is incubated at about 42-44°C for 5-15 minutes. b. Add 25 µL of ENZ mix containing reverse transcriptase, T7 RNA polymerase, mix, and incubate at approximately 42-44 °C for 5 min.

[0266] Second Phase Amplification: A promoter reagent containing a T7 primer and a probe oligomer, such as a torch, is added to the first amplification product and incubated for a period of time to allow the formation of a second amplification product. In some embodiments, one or more helper oligomer(s) and more non-T7 primers are added during the second phase of amplification. a. Add 25 μL of AMP2 (also referred to as PR) mixture to each tube and mix. In some embodiments, the AMP2 mixture contains buffer, salt, detergent, dNTP, NTP, one or more T7 primers, torch probe(s), and optionally more non-T7 primers and / or helper oligomer(s).

[0267] Reaction program: Incubate at 42-43°C for 30-60 minutes, with label detection (collection) performed at approximately 30-second intervals.

[0268] Detection: The amplification of the target nucleic acid sequence is detected in real time by recording the fluorescent signal from the detector oligonucleotide at regular intervals.

[0269] Example 11. Real-time biphasic TMA CMV assay from plasma samples. Target capture was achieved using a TCO against either the UL56 gene of CMV (SEQ ID NOS: 42 and 44). Two TCR (target capture mixture) formulations, A (containing 679 mM LiOH) and B (453 mM LiOH), were added to the plasmid and plasma sample. For some reactions, 100 μL or 200 μL of target enhancer reagent (TER) was added to the sample during the target capture phase. In some embodiments, the amount of TER used is 25-200 μL. In some embodiments, the amount of TER used is 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, or 200 μL. In some embodiments, the amount of LiOH used in the target capture phase is 50-350 mM. In some embodiments, the amount of LiOH in the target capture step is about 50 mM, about 75 mM, about 100 mM, about 125 mM, about 150 mM, about 175 mM, about 200 mM, about 225 mM, about 250 mM, about 275 mM, about 300 mM, about 325 mM, or about 350 mM.

[0270] TCO(s) were added to 1x TCR buffer (400µL / reaction). In some samples, an internal control target nucleic acid was also added. For biphasic TMA reactions, the indicated amount of T7 primer was added.

[0271] For single-phase TMA, T7 primer, NT7 primer, and Torchi oligo were added to AMP buffer to form the AMP reagent, and 75 μL of AMP reagent was added to each sample.

[0272] For biphasic TMA, NT7 oligo was added to AMP buffer to form the AMP1 reagent (AR), and T7 oligo and Torchi oligo were added to AMP buffer to form the AMP2 (PR) reagent. After target capture, 50 μL of AMP1 reagent was added to each sample.

[0273] For single-phase TMA, 25 μL of ENZ was added to each test, and the samples were mixed at 1400 RPM for 1 minute. The reactions were incubated at 43°C for 10 minutes.

[0274] For biphasic TMA, 25 μL of ENZ was added to each test, and the samples were mixed at 1400 RPM for 1 minute. AMP1 reactions were incubated at 43°C for 5 minutes. 25 μL of AMP2 was then added to each sample, and the plate was mixed at 1400 RPM for 1 minute. The samples were then incubated at 43°C, and fluorescence was measured in real time.

[0275] Each experiment included a control reaction containing the internal control oligomer listed in Table 11-3. [Table 11-1] [Table 11-2] [Table 11-3] [Table 11-4] [Table 11-5]

[0276] Summary: Using the indicated oligonucleotides, CMV UL56 was readily detected in both plasmid and plasma samples when 200 μL of Formulation B TCR with TER was used. CMV UL56 was readily detected in plasma samples when 200 μL of Formulation A TCR with TER was used. The performance in detecting UL56 was not as good.

[0277] Example 12. CMV Detection Limit. The above experiments demonstrated a limit of detection (LOD) of 109 IU / mL to 250 IU / mL for CMV. Various parameters, including the incorporation of viral load buffer and enzyme, as well as salt, oligo, and dNTP / NTP concentrations, were varied to improve the detection of CMV in the samples. Adjusting various parameters improved the LOD to 37 IU / mL and the limit of quantification (LOQ) to 40 IU / mL. Additionally, faster CVM TTimes were observed with improved conditions.

[0278] These studies included 1 × 10 2 ~1×10 7 Plasmids at 30 copies / mL were used as calibrators. Some studies analyzed amplification of a CMV UL56 plasmid panel at 30 copies / mL. Other studies analyzed amplification of cultured viruses diluted to approximately 70, 30, 10, and 3 copies / mL in processed plasma (Part No. BI0052). The exact values ​​have not been definitively determined. Nevertheless, these low-concentration virus panels allowed us to compare the sensitivity of the various conditions tested. [Table 12-1] [Table 12-2]

[0279] The addition of 7.5% DMSO in the AMP reagent resulted in improved sensitivity. The addition of DMSO resulted in higher sensitivity, faster CMV TTime, and less variability (Tables 12-5 and 12-6).

[0280] pH titration studies were performed with HEPES / trehalose buffer formulations. Results showed that a higher AMP1 pH improved sensitivity. Higher pH of AMP1 and AMP2 decreased sensitivity compared to increasing pH of AMP1 alone. A pH of 8.5 (increasing Tris base from 11.4 to 22 mM) was selected for further evaluation.

[0281] In some reactions, increasing MgCL2 in the AMP reagent also improved sensitivity.

[0282] Based on the initial studies, further optimization was performed as described below. To further improve CMV detection, additional TCOs were tested, as were displacer and helper NT7 oligos in the AMP buffer. Two displacer oligos (SEQ ID NO: 12 and SEQ ID NO: 41) and three helper NT7 oligos (SEQ ID NO: 14, SEQ ID NO: 17, and SEQ ID NO: 18) were identified that improved CMV detection and precision (i.e., reduced standard deviation). [Table 12-3] [Table 12-4] [Table 12-5] [Table 12-6] [Table 12-7]

[0283] Although both displacers improved sensitivity, the displacer of SEQ ID NO: 41 provided a greater increase in sensitivity than the displacer of SEQ ID NO: 12. The results are shown in Table 12-5.

[0284] The addition of the helper NT7 oligo improved the accuracy of CMV by lowering the standard deviation log copies of CMV quantification. The helper NT7 oligo of SEQ ID NO: 14 showed the lowest standard deviation. The results are shown in Table 12-6.

[0285] CMV detection was further tested using a combination of the improvements identified above, including a lower amount of TER / LiOH and the addition of a displacer and NT7 helper oligonucleotide. These conditions were then run in parallel with the original assay conditions (Aptima assay reagents). The results are shown in Table 12-7.

[0286] As shown in Table 12-8, sensitivity was further increased using newly synthesized oligonucleotides with increased purity. [Table 12-8]

[0287] A significant improvement in sensitivity was observed when TER was added to the sample before the addition of TCR. TER was added to the sample tube or well first, followed by the sample. After mixing, TCR was added to the TER-treated sample. The results are shown in Table 12-9. [Table 12-9] [Table 12-10]

[0288] Conclusion: Sensitivity and TTime were improved using the above conditions. With this new formulation and order of TER addition, an LOD of 37 IU / mL and an LOQ of 40 IU / mL were achieved. In some embodiments, the composition contains CMV detection oligonucleotides (TCO, T7 primer, NT7 primer, displacer oligonucleotide, helper oligonucleotide, and torch oligonucleotide) and internal control oligonucleotides (TCO, T7 primer, NT7 primer, and torch oligonucleotide).

[0289] Example 13. Torch Specifications. Eleven torches were designed and tested to eliminate gradients and optimize precision and positivity. [Table 13-1] [Table 13-2] [Table 13-3]

[0290] Of those tested, torches SEQ ID NO:20, SEQ ID NO:60, and SEQ ID NO:70 showed a gradient in negative samples. Torch SEQ ID NO:54 had a lower RFU range and lower positivity for R2356 than torch SEQ ID NO:20. All oligos were further tested with mutant CMV sequences to ensure they were accurately quantified in the presence of mutations. Torches SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, and SEQ ID NO:68 showed lower sensitivity for detection of mutant 4. A summary of the results can be found in Table 13-4. [Table 13-4]

[0291] Torches (SEQ ID NO: 56) and (SEQ ID NO: 58) did not produce any slope for negative samples in the FAM background subtraction curve (CMV target channel). Torches SEQ ID NO: 56 and SEQ ID NO: 58 also showed improved positivity with the mutant4 panel, comparable recovery, and improved sensitivity with all panels. The present invention provides, for example, the following items. (Item 1) 1. A kit for amplifying a target region of nucleic acid derived from a human cytomegalovirus (CMV) UL56 gene sequence, comprising: (a) a forward primer comprising 19 to 31 consecutive nucleic acid bases that have at least 90% identity to a 19 to 31 nucleotide sequence present in SEQ ID NO:2; and (b) a reverse primer comprising 21 to 40 consecutive nucleic acid bases that have at least 90% identity to a 21 to 40 nucleotide sequence present in SEQ ID NO:3. (Item 2) 2. The kit of claim 1, wherein the forward primer, the reverse primer, or both the forward primer and the reverse primer comprise at least one modified nucleotide. (Item 3) 3. The kit of claim 2, wherein the modified nucleotide comprises a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, or a 5'-methylcytosine. (Item 4) 4. The kit according to any one of Items 1 to 3, wherein the forward primer comprises the nucleic acid sequence of SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 19. (Item 5) 5. The kit of any of items 4, wherein the forward primer is a non-promoter primer comprising the nucleobase sequence of SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:19. (Item 6) 6. The kit according to any one of Items 1 to 5, wherein the reverse primer comprises the nucleic acid sequence of SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25. (Item 7) 7. The kit of item 6, wherein the reverse primer comprises the nucleobase sequence of SEQ ID NO: 6, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 41, or SEQ ID NO: 47. (Item 8) 8. The kit according to any one of items 1 to 4 or 6 to 7, wherein an RNA polymerase promoter sequence is linked to the 5' end of the forward primer or the reverse primer. (Item 9) 9. The kit according to item 8, wherein the RNA polymerase promoter sequence is a T7 RNA polymerase promoter sequence. (Item 10) 10. The kit of item 9, wherein the T7 RNA polymerase promoter sequence comprises the nucleotide sequence of SEQ ID NO: 78. (Item 11) 11. The kit of item 10, wherein the reverse primer comprises the nucleobase sequence of SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, or SEQ ID NO:46. (Item 12) 11. The kit according to any one of Items 1 to 10, wherein the forward primer comprises SEQ ID NO: 11 and the reverse primer comprises SEQ ID NO: 23. (Item 13) 13. The kit according to any one of items 1 to 12, further comprising a probe oligomer. (Item 14) Item 14. The kit of Item 13, wherein the probe oligomer (a) comprises the nucleobase sequence of SEQ ID NO: 51 or SEQ ID NO: 52, wherein one or more uracil nucleotides may be substituted for thymine nucleotides, or (b) comprises a nucleotide sequence comprising 24 to 35 consecutive nucleobases that hybridize to SEQ ID NO: 81. (Item 15) 15. The kit of item 14, wherein the probe oligomer comprises at least one modified nucleotide. (Item 16) 16. The probe oligomer of item 15, wherein the modified nucleotide comprises a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, or a 5'-methylcytosine. (Item 17) 17. The kit according to any one of Items 14 to 16, wherein the probe oligomer comprises the nucleic acid sequence of SEQ ID NO: 21, SEQ ID NO: 26, SEQ ID NO: 39, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, or SEQ ID NO: 71. (Item 18) 18. The kit according to any one of items 14 to 17, wherein the probe oligomer contains a detectable label. (Item 19) 19. The kit of claim 18, wherein the detectable label comprises a fluorescent molecule. (Item 20) 20. The kit of item 19, wherein the fluorescent molecule is attached to the 5' or 3' end of the probe oligomer. (Item 21) 21. The kit according to any one of items 14 to 20, wherein the probe oligomer contains 4 to 5 nucleobases at the 3' end of the probe oligomer that are complementary to 4 to 5 nucleobases at the 5' end of the probe oligomer. (Item 22) 22. The kit of claim 21, wherein a fluorescent molecule is attached to the 5' end of the probe oligomer and a quencher is attached to the 3' end of the probe oligomer, or a fluorescent molecule is attached to the 3' end of the probe oligomer and a quencher is attached to the 5' end of the probe oligomer. (Item 23) 23. The kit of item 22, wherein the probe oligomer comprises the nucleobase sequence of SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, or SEQ ID NO:70. (Item 24) 23. The kit according to any one of Items 14 to 22, wherein the forward primer comprises SEQ ID NO: 11, the reverse primer comprises SEQ ID NO: 23, and the probe oligonucleotide comprises SEQ ID NO: 53. (Item 25) 25. The kit of any one of items 1 to 24, further comprising a helper oligomer comprising 19 to 31 consecutive nucleobases having at least 90% identity to a 19 to 31 nucleotide sequence present in SEQ ID NO:2. (Item 26) 26. The kit of item 25, wherein the helper oligomer is blocked. (Item 27) 27. The kit of item 25 or 26, wherein the helper oligomer comprises the nucleotide sequence of SEQ ID NO: 10 or SEQ ID NO: 19. (Item 28) 28. The kit of item 27, wherein the helper oligomer comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19. (Item 29) 29. The kit of any one of items 1 to 28, further comprising a displacer oligomer comprising 21 to 27 consecutive nucleobases having at least 90% identity to a 21 to 25 nucleotide sequence present in SEQ ID NO:5. (Item 30) 30. The kit of item 29, wherein the displacer oligomer comprises the nucleotide sequence of SEQ ID NO: 12, SEQ ID NO: 25, or SEQ ID NO: 41. (Item 31) 32. The kit of claim 30, wherein the displacer oligomer comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:41, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:86, SEQ ID NO:87, and SEQ ID NO:88. 32. The kit of any one of items 1 to 31, further comprising a target capture oligomer (TCO) comprising the nucleotide sequence of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 43, or SEQ ID NO: 45. (Item 33) 33. The kit of item 32, wherein the TCO contains a moiety that allows for isolation of the TCO. (Item 34) 34. The kit of item 33, wherein the portion comprises a polyA nucleotide sequence. (Item 35) The moiety is (dT)3(dA) 30 Item 34. The kit according to Item 33, comprising: (Item 36) 36. The kit of item 35, wherein the TCO comprises the nucleotide sequence of SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:42, or SEQ ID NO:44. (Item 37) 37. The kit according to any one of items 32 to 36, wherein the kit comprises a first TCO comprising the nucleotide sequence of SEQ ID NO: 42 and a second TCO comprising the nucleotide sequence of SEQ ID NO: 44. (Item 38) 38. The kit of any one of items 1-37, further comprising one or more of a target capture reagent, a target capture wash solution, a target enhancer reagent, an amplification reagent, an enzyme reagent, a promoter reagent, a CMV positive control nucleic acid, a negative control nucleic acid, a sample transport medium, a reverse transcriptase, an RNA polymerase, dNTPs, NTPs, a buffer, and a positive and / or negative control sample. (Item 39) 1. A method for amplifying a target region of nucleic acid derived from a human cytomegalovirus (CMV) UL56 gene sequence present in a sample, comprising: (a) contacting the sample with a forward primer and a reverse primer configured to amplify a CMV UL56 amplicon, wherein the forward primer comprises 19 to 31 contiguous nucleobases having at least 90% identity to a 19 to 31 nucleotide sequence present in SEQ ID NO:2 and the reverse primer comprises 21 to 40 contiguous nucleobases having at least 90% identity to a 21 to 40 nucleotide sequence present in SEQ ID NO:3; (b) exposing the sample to conditions sufficient to amplify the target region, thereby producing an amplification product. (Item 40) 40. The method of claim 39, wherein the forward primer and / or the reverse primer comprises at least one modified nucleotide. (Item 41) 41. The method of claim 40, wherein the at least one modified nucleotide comprises a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, or a 5'-methylcytosine. (Item 42) 42. The method of any one of Items 39 to 41, wherein the forward primer comprises the nucleobase sequence of SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 19, and the reverse primer comprises the nucleobase sequence of SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 47. (Item 43) 43. The method of claim 42, wherein the forward primer comprises the nucleobase sequence of SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:19, and the reverse primer comprises the nucleobase sequence of SEQ ID NO:6, SEQ ID NO:23, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:41, SEQ ID NO:47. (Item 44) 44. The method of claim 43, wherein a T7 RNA polymerase promoter sequence is linked to the 5' end of the reverse primer. (Item 45) 45. The method of claim 44, wherein the reverse primer comprises the nucleobase sequence of SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, or SEQ ID NO:46. (Item 46) 44. The method of any one of Items 39 to 43, wherein the forward primer comprises SEQ ID NO: 11 and the reverse primer comprises SEQ ID NO: 23. (Item 47) 47. The method of any one of items 39 to 46, further comprising detecting the presence or absence of the amplification product. (Item 48) 48. The method of claim 47, wherein detecting the presence or absence of the amplification product utilizes a probe oligomer that specifically hybridizes to the amplification product. (Item 49) 49. The method of Item 48, wherein the probe oligomer comprises the nucleobase sequence of SEQ ID NO: 51 or SEQ ID NO: 52, wherein one or more uracil nucleotides may be substituted for thymine nucleotides, or (b) comprises a nucleotide sequence comprising 24 to 35 consecutive nucleobases that hybridize to SEQ ID NO: 81. (Item 50) 50. The method of claim 49, wherein the probe oligomer comprises the nucleobase sequence of SEQ ID NO:21, SEQ ID NO:26, SEQ ID NO:39, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71. (Item 51) 51. The method of any one of items 48 to 50, wherein the probe oligomer contains 4 to 5 nucleobases at the 3' end of the probe oligomer that are complementary to 4 to 5 nucleobases at the 5' end of the probe oligomer. (Item 52) Item 52. The method of item 51, wherein a fluorescent molecule is attached to the 5' end of the probe oligomer and a quencher is attached to the 3' end of the probe oligomer, or a fluorescent molecule is attached to the 3' end of the probe oligomer and a quencher is attached to the 5' end of the probe oligomer. (Item 53) 53. The method of claim 52, wherein the probe oligomer comprises the nucleobase sequence of SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, or SEQ ID NO:70. (Item 54) 54. The method of any one of Items 39 to 44 and 46 to 53, wherein the forward primer comprises SEQ ID NO: 11, the reverse primer comprises SEQ ID NO: 23, and the probe oligonucleotide comprises SEQ ID NO: 53. (Item 55) 55. The method of any one of items 39 to 54, wherein the amplifying comprises a thermal cycling reaction. (Item 56) 56. The method of claim 55, wherein the thermal cycling reaction comprises a polymerase chain reaction (PCR). (Item 57) 55. The method of any one of items 39 to 54, wherein the amplifying comprises an isothermal nucleic acid amplification reaction. (Item 58) 58. The method of claim 57, wherein the isothermal nucleic acid amplification reaction comprises transcription-mediated amplification (TMA). (Item 59) 55. The method of any one of items 39 to 54, wherein the amplifying comprises nucleic acid sequence-based amplification, replicase-mediated amplification, Qβ-replicase-mediated amplification, ligase chain reaction (LCR), or strand displacement amplification (SDA). (Item 60) 60. The method of any one of items 47 to 59, wherein detecting the presence or absence of the amplified CMV UL56 amplicon further comprises quantifying the amplified CMV UL56 amplicon. (Item 61) 61. The method of claim 60, wherein quantifying the amplified CMV UL56 amplicon comprises monitoring production of the CMV amplicon. (Item 62) 62. The method of any one of items 47 to 61, wherein the detecting and / or quantifying is analyzed in real time. (Item 63) 1. A method for quantifying a human cytomegalovirus (CMV) UL56 gene target nucleic acid sequence in a sample, comprising: (a) contacting the sample with at least one target capture oligomer (TCO) comprising the nucleobase sequence of SEQ ID NO: 43 or SEQ ID NO: 45 and a first promoter primer comprising the nucleobase sequence of SEQ ID NO: 47 under conditions that allow hybridization of the at least one TCO and first promoter primer to the CMV UL56 gene target nucleic acid sequence, thereby generating pre-amplification hybrids comprising target nucleic acid sequences hybridized to each of the at least one TCO and the first promoter primer; (b) isolating the pre-amplified hybrids by target capture onto a solid support followed by washing to remove any of the first promoter primer that did not hybridize to the CMV UL56 gene target nucleic acid sequence in step (a); (c) amplifying at least a portion of the CMV UL56 gene target nucleic acid sequence of the pre-amplified hybrid isolated in step (b) in a phase 1 amplification reaction mixture comprising a non-promoter primer comprising the nucleobase sequence of SEQ ID NO: 19 under conditions that support linear amplification but not exponential amplification in a phase 1 substantially isothermal transcription-associated amplification reaction, thereby resulting in a reaction mixture comprising a first amplification product, wherein the first amplification product is not a template for nucleic acid synthesis during the phase 1 substantially isothermal transcription-associated amplification reaction; (d) combining the first amplification product with a second-phase amplification reaction mixture comprising a second promoter primer comprising the nucleobase sequence of SEQ ID NO:47 and a probe oligomer comprising the nucleobase sequence of SEQ ID NO:57, and performing exponential amplification of the first amplification product in a second-phase substantially isothermal transcription-associated amplification reaction in the second-phase amplification reaction mixture, thereby synthesizing a second amplification product; (f) detecting synthesis of the second amplification product in the second phase amplification reaction mixture using the probe oligomer at regular time intervals; (g) quantifying the target nucleic acid sequence in the sample using the results of step (f). (Item 64) 64. The method of claim 63, wherein the at least one TCO comprises a first TCO comprising the nucleobase sequence of SEQ ID NO: 43 and a second TCO comprising the nucleobase sequence of SEQ ID NO: 45. (Item 65) 65. The method of item 63 or 64, wherein the first and second promoter primers each comprise a 5' promoter sequence of an RNA polymerase. (Item 66) 66. The method of claim 65, wherein the RNA polymerase is T7 RNA polymerase. (Item 67) 68. The method of any one of items 63 to 67, wherein the solid support comprises an immobilized capture probe. (Item 68) 68. The method of claim 67, wherein the solid support comprises magnetically attractable particles. (Item 69) 69. The method of any one of items 63 to 68, wherein the phase 1 and phase 2 isothermal transcription-associated amplification reactions each comprise an RNA polymerase and a reverse transcriptase, and the reverse transcriptase comprises endogenous RNase H activity. (Item 70) 70. The method of any one of Items 63 to 69, wherein the first amplification product of step (c) is a cDNA molecule having the same polarity as the target nucleic acid sequence in the sample, and the second amplification product of step (d) is an RNA molecule. (Item 71) 71. The method of any one of items 63 to 70, wherein the probe oligomer of step (d) is a configuration-sensitive probe that generates a detectable signal when hybridized to the second amplification product. (Item 72) 72. The method of any one of items 63 to 71, wherein the probe oligomer of step (d) is a fluorescently labeled sequence-specific hybridization probe. (Item 73) 73. The method of any one of Items 64 to 72, wherein the first TCO comprises the nucleobase sequence of SEQ ID NO: 42, the second TCO comprises the nucleobase sequence of SEQ ID NO: 44, the first and second promoter primers each comprise the nucleobase sequence of SEQ ID NO: 46, and the probe oligomer comprises the nucleobase sequence of SEQ ID NO: 56. (Item 74) 74. The method of any one of items 63 to 73, wherein the phase 1 amplification reaction mixture and / or the phase 2 amplification reaction mixture further comprises a helper oligomer and / or a displacer oligomer. (Item 75) 75. The method of Item 74, wherein the helper oligomer is 19 to 31 nucleobases in length and comprises the nucleobase sequence of SEQ ID NO: 14, and the displacer oligomer is 21 to 27 nucleobases in length and comprises the nucleobase sequence of SEQ ID NO: 41. (Item 76) 76. The method of claim 74 or 75, wherein the helper oligomer, the displacer oligomer, or both the helper oligomer and the displacer oligomer are blocked.

Claims

[Claim 1] The invention described in the present specification.

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