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US-20260125773-A1 - COMPOSITIONS AND METHODS FOR AMPLIFYING, DETECTING OR QUANTIFYING HUMAN CYTOMEGALOVIRUS

US20260125773A1US 20260125773 A1US20260125773 A1US 20260125773A1US-20260125773-A1

Abstract

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.

Inventors

  • Paul Darby
  • Siobhan Miick
  • Jo Ann JACKSON
  • Hee Cheol Kim
  • Amber HILLIUS
  • Ankur Shah

Assignees

  • GEN-PROBE INCORPORATED

Dates

Publication Date
20260507
Application Date
20251001

Claims (20)

  1. 1 . A kit for amplifying a target region of nucleic acid derived from a human cytomegalovirus (CMV) UL56 gene sequence comprising: (a) a target capture reagent comprising at least one target capture oligonucleotide (TCO) and a first promoter primer, wherein the first promoter primer comprises a first RNA polymerase promoter sequence linked to the 5′ end of a first target hybridizing sequence, wherein the first target hybridizing sequence is 21-40 nucleobases in length and is at least 90% identical to a 21-40 contiguous nucleotide sequence present in SEQ ID NO: 3; (b) an amplification reagent comprising a non-promoter primer, wherein the non-promoter primer comprises a second target hybridizing sequence, wherein the second target hybridizing sequence is 19-31 nucleobases in length and is at least 90% identical to a 19-31 contiguous nucleotide sequence present in SEQ ID NO:2; and (c) a promoter reagent comprising a second promoter primer and a probe oligomer, wherein the second promoter primer comprises a second RNA polymerase promoter sequence linked to the 5′ end of a third target hybridizing sequence, wherein the third target hybridizing sequence is 21-40 nucleobases in length and is at least 90% identical to a 21-40 contiguous nucleotide sequence present in SEQ ID NO:3, and wherein the probe oligomer comprises a fourth target hybridizing sequence, wherein the fourth target hybridizing sequence comprises 24-35 contiguous nucleobases that hybridizes to SEQ ID NO: 81.
  2. 2 . The kit of claim 1 , wherein the TCO at least one TCO comprises: (a) a target specific nucleotide sequence that is 20-30 nucleotides in length and a moiety that enables isolation of the TCO; (b) a target specific nucleotide sequence that is 20-30 nucleotides in length and a moiety that enables isolation of the TCO, wherein the moiety that enables isolation of the TCO comprises a poly A nucleotide sequence or (dT) 3 (dA) 30.
  3. 3 . The kit of claim 1 , wherein the at least one TCO comprises: (a) a target specific nucleotide sequence that is 20-30 nucleotides in length and comprises SEQ ID NO:43 or SEQ ID NO:45; (b) SEQ ID NO:42; (c) SEQ ID NO:44; or (d) a first TCO comprising SEQ ID NO:42 and a second TCO comprising SEQ ID NO:44.
  4. 4 . The kit of claim 1 , wherein the first target hybridizing sequence and the third target hybridizing sequence each comprise a nucleobase sequence selected from the group consisting of: SEQ ID NOs: 6, 23-25, 27, 29, 31, 33, 35, 37, 41. and 47.
  5. 5 . The kit of claim 4 , wherein the first RNA polymerase promoter sequence and the second RNA polymerase promoter sequence independently comprise: (a) a T7 RNA polymerase promoter sequence; or (b) nucleotide sequence consisting of SEQ ID NO:78.
  6. 6 . The kit of claim 5 , wherein the first promoter primer and the second promoter primer each comprises a nucleobase sequence selected from the group consisting of: SEQ ID NOs: 28, 30, 32, 34, 36, 38, 40, and 46.
  7. 7 . The kit of claim 1 , wherein the target capture reagent further comprises: (a) a solid support or a solid support and an immobilized capture probe; and/or (b) a reverse transcriptase, an RNA polymerase, or the reverse transcriptase and the RNA polymerase.
  8. 8 . The kit of claim 1 , wherein the non-promoter primer comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 10, 11, and 13-19.
  9. 9 . The kit of claim 1 , wherein the amplification reagent further comprises: a reverse transcriptase, an RNA polymerase, or the reverse transcriptase and the RNA polymerase.
  10. 10 . The kit of claim 1 , wherein: (a) the target capture reagent further comprises: (i) a displacer oligomer, or (ii) a displacer oligomer, wherein the displace oligomer is 21-27 nucleobases in length, comprises SEQ ID NO: 6, 12, 25, 33, 35, 37, or 41, and may be blocked or unblocked; and/or (b) the amplification reagent further comprises: (i) a helper oligomer, wherein the helper oligomer may be blocked or unblocked, or (ii) a helper oligomer, wherein the helper oligomer is 19-31 nucleotides in length, comprises SEQ ID NO: 10, 14, 15, 17, 18, or 19, and may be blocked or unblocked.
  11. 11 . The kit of claim 1 , wherein the probe oligomer comprises SEQ ID NO:21, 26, 39, 53, 55, 57, 59, 61, 65, 67, 69, or 71.
  12. 12 . The kit of claim 11 , wherein the probe oligomer comprises a fluorescent molecule attached to the 5′ or 3′ end of the probe oligomer.
  13. 13 . The kit of claim 12 , wherein the probe oligomer contains 4-5 nucleobases at the 3′ end of the probe oligomer that are complementary to 4-5 nucleobase at the 5′ end of the probe oligomer wherein (i) the 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 (ii) the 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.
  14. 14 . The kit of claim 13 , wherein the probe oligomer comprises SEQ ID NO:20, 22, 54, 56, 58, 60, 62, 64, 66, 68, or 70.
  15. 15 . The kit of claim 1 , wherein one or more of the first promoter primer, the non-promoter primer, and the second promoter primer, and the probe oligomer comprises one or more modified nucleotides independently selected from the group consisting of: a 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, and a 5′-methyl cytosine.
  16. 16 . The kit of claim 1 , wherein (a) the target capture reagent is provided in a liquid format; (b) the amplification reagent is lyophilized; and/or (c) the promoter reagent is lyophilized.
  17. 17 . The kit of claim 1 , wherein the kit further contains one or more of: an enzyme reagent comprising an RNA polymerase, a reverse transcriptase, or the RNA polymerase and the reverse transcriptase, a lyophilized enzyme reagent comprising an RNA polymerase, a reverse transcriptase, or the RNA polymerase and the reverse transcriptase, a target capture wash solution, a target enhancer reagent, an enzyme reagent reconstitution solution, an amplification reagent reconstitution solution, a promoter reagent reconstitution solution, a CMV positive control nucleic acid, a negative control nucleic acid, a sample transport medium, a reverse transcriptase, an RNA polymerase, dNTPs, NTPs, buffer, positive and/or negative control samples, an internal control promoter primer comprising SEQ ID NO: 50, an internal control primer comprising SEQ ID NO: 63, and internal probe oligomer comprising SEQ ID NO: 88, an internal control TCO comprising SEQ ID NO:49, and instructions for use.
  18. 18 . The kit of claim 1 , wherein: (a) the at least one target capture oligonucleotide (TCO) comprises a first TCO comprising SEQ ID NO:42 and a second TCO comprising SEQ ID NO:44, and the promoter primer comprises SEQ ID NO:46; (b) the non-promoter primer comprises SEQ ID NO:19; and (c) the second promoter primer comprises SEQ ID NO:46 and the probe oligomer comprises SEQ ID NO:56.
  19. 19 . The kit of claim 1 , wherein the target capture reagent further comprises a displacer oligomer comprising SEQ ID NO:41.
  20. 20 . A system for analyzing a sample to determine the presence or absence of a nucleic acid derived from a cytomegalovirus (CMV) UL56 gene sequence, where the system: (a) contacts the sample with a target capture reagent, wherein the target capture reagent comprises at least one target capture oligonucleotide (TCO) and a first promoter primer, wherein the first promoter primer comprises a first RNA polymerase promoter sequence linked to the 5′ end of a first target hybridizing sequence, wherein the first target hybridizing sequence is 21-40 nucleobases in length and is at least 90% identical to a 21-40 contiguous nucleotide sequence present in SEQ ID NO:3, wherein (i) CMV UL56 gene target nucleic acid sequence, if present in the sample, is hybridized to the at least one TCO and the first promoter primer to from a pre-amplification hybrid, and separated from sample components; (b) contacts the pre-amplification hybrid with an amplification reagent, wherein the amplification reagent comprises a non-promoter primer, wherein the non-promoter primer comprises a second target hybridizing sequence, wherein the second target hybridizing sequence is 19-31 nucleobases in length and is at least 90% identical to a 19-31 contiguous nucleotide sequence present in SEQ ID NO:2, wherein (i) a first phase amplification is performed to form a first amplification product, wherein the first phase amplification comprises an isothermal, transcription-associated amplification reaction of at least a portion of the CMV UL56 gene target nucleic acid sequence of the pre-amplification hybrid, (c) contacts the first amplification product with a promoter reagent to form a second phase amplification reaction mixture, wherein the promoter reagent comprises a second promoter primer and a probe oligomer, wherein the second promoter primer comprises a second RNA polymerase promoter sequence linked to the 5′ end of a third target hybridizing sequence, wherein the third target hybridizing sequence is 21-40 nucleobases in length and is at least 90% identical to a 21-40 contiguous nucleotide sequence present in SEQ ID NO: 3, and wherein the probe oligomer comprises a fourth target hybridizing sequence, wherein the fourth target hybridizing sequence comprises 24-35 contiguous nucleobases that hybridizes to SEQ ID NO:81; wherein (i) a second phase amplification is performed to form a second amplification product, wherein the second phase amplification comprises an isothermal, transcription-associated amplification reaction, (d) detects hybridization of the probe oligomer to the second amplification product.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. application Ser. No. 17/269,775, filled Feb. 19, 2021, is a U.S. National Stage entry of International Application No. PCT/US2019/047419, filed Aug. 21, 2019, which claims the benefit of priority to U.S. Provisional Application No. 62/720,658, filed Aug. 21, 2018, which are herein incorporated by reference in their entirety for all purposes. SEQUENCE LISTING The Sequence Listing written in filed DIA.0073.02_SeqListing.xml is 168,401 bytes in size, was created Jun. 18, 2024, and is hereby incorporated by reference. BACKGROUND Human Cytomegalovirus (CMV, also called human herpesvirus 5 (HHV5)) is part of a larger family of viruses that include Herpes simplex virus (HSV), Varicella-Zoster virus (VZV), and Epstein-Barr virus (EBV). CMV is an enveloped double-stranded DNA virus causing infections in humans. CMV is a common virus that can infect almost anyone. It is so common that almost all adults in developing countries and 50% to 85% of adults in the United States have been infected. CMV spreads from person to person through body fluids, such as blood, saliva, urine, semen, vaginal fluids, and breast milk. Similar to other herpes viruses, CMV establishes a lifelong latency that may reactivate intermittently. There is no cure. In the immunocompetent host, the CMV infection is generally asymptomatic and self-limited. CMV infection is cause for concern in pregnant women, infants, and immunocompromised individuals. Active CMV infection during pregnancy can pass the virus to the baby. For people with compromised immunity, especially due to organ transplantation, CMV infection is an important cause of morbidity and mortality. However, medications can help treat newborns and people with weak immune systems. In solid organ transplantation (SOT) recipients, CMV transmitted from the donor (D) organ to the recipient (R), may cause primary infection in CMV seronegative SOT recipients (R−) or re-infection in CMV seropositive SOT recipients (R+). In D−/R+ SOT recipients, the impaired CMV-specific immunity due to immunosuppression may result in re-activation of endogenous latent CMV. Since D+/R− SOT recipients lack the pre-existing host immunity, they are at high-risk for developing CMV disease, whereas 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 transplant and/or the development of CMV disease, by suppression of viral replication. Viral load testing has become the main method to diagnose active disease due to CMV infections and a routine component in the care of transplant recipients (Rychert J., et. al 2014). Testing is important in pregnant women and those with compromised or weakened immune systems. Current diagnostic tests look for anti-CMV antibodies. However, such testing requires multiple tests for accuracy and the person must be symptomatic. Additional diagnostic tests include culture, PCR, and the CMV pp65 antigenemia assay. The CMV pp65 antigenemia assay, which quantitates the number of CMV-infected leukocytes in peripheral blood, has been used in the detection and monitoring of CMV infection in immunocompromised patients. There is a need for compositions and methods that allow sensitive and specific detection and quantification of CMV. This disclosure aims to meet these needs, provide other benefits, or at least provide the public with a useful choice. SUMMARY Described are amplification oligomers, nucleic acids, methods, compositions, and kits for detecting and/or quantifying human cytomegalovirus (CMV) in a sample, or to amplify a CMV UL56 gene sequence. 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. Further described are probe oligomers and target capture oligomers (TCO) that facilitate detection of amplified sequence and isolation of CMV nucleotide sequence from a sample, respectively. The methods involve the amplification of viral nucleic acid to detect the CMV target sequence in the sample. The methods can advantageously provide for the sensitive detection CMV. The amplification oligomers can be used in the amplification, detection, and/or quantification of a CMV sequence using any nucleic amplification method known in the art. The nucleic acid amplification methods can use thermal cycling, or they can 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 QB-replicase-mediated amplification), ligase chain reaction (LCR), strand-displacement amplificat