Assays for detecting cytomegalovirus (CMV) or epstein-bar virus (EBV)

Abstract

Provided herein are methods, compositions, kits and systems for detecting target nucleic acid sequences from cytomegalovirus (CMV) or Epstein-Bar virus (EBV).

Description

[0001] ASSAYS FOR DETECTING CYTOMEGALOVIRUS (CMV) OR EPSTEIN-BAR VIRUS (EBV)

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] The present invention claims priority to U.S. Provisional Patent Application Nos. 63 / 564,277, filed March 12, 2024, and 63 / 534,934, filed August 28, 2024, each of which is incorporated by reference in its entirety.

[0004] SEQUENCE LISTING

[0005] The text of the computer readable sequence listing filed herewith, titled “42341- 601_SEQUENCE_LISTING”, created August 27, 2024, having a file size of 28,785 bytes, is hereby incorporated by reference in its entirety.

[0006] FIELD

[0007] Provided herein are methods, compositions, kits and systems for detecting target nucleic acid sequences from cytomegalovirus (CMV) or Epstein-Bar virus (EBV).

[0008] BACKGROUND OF THE INVENTION

[0009] Viral infections are a major cause of morbidity and mortality in a diversity of clinical settings including, for example, care of immunocompromised patients and postoperative care of solid organ transplant recipients. Chronic immunosuppression required to maintain allograft function post-organ transplant predisposes transplant patients to viral infections. These may arise at each stage post-transplantation. Certain infections, such as cytomegalovirus (CMV) or Epstein Barr virus (EBV) may occur within months after transplantation. CMV infections may be prevented by prophylaxis therapy, and EVB infections may be managed by decreasing immunosuppression. Accordingly, methods, compositions, kits and systems for detecting nucleic acid sequences from CMV or EBV in samples including, for example, plasma, whole blood and urine samples are needed.

[0010] SUMMARY OF THE INVENTION

[0011] Provided herein are methods, compositions, kits and systems for detecting target nucleic acid sequences from cytomegalovirus (CMV) or Epstein-Bar virus (EBV). The present invention provides reagents, including oligonucleotides, for amplifying and detecting CMV or EBV in a sample. In some embodiments, a set of oligonucleotides comprises at least one first amplification oligonucleotide, at least one second amplification oligonucleotide, and at least one probe oligonucleotide. The probe oligonucleotide may comprise a detectable label (e.g., a fluorophore). In some embodiments, the set of oligonucleotides is for polymerase amplification and detection of CMV and / or EBV in a sample. In some embodiments, the reagents comprise a group of oligonucleotides comprising one or more sets of oligonucleotides. In some embodiments, the present invention provides dual-target PCR primer and probe sets for each of CMV or EBV nucleic acid sequences.

[0012] In some embodiments, whole blood is prepared for CMV and / or EBV detection by a method comprising one or more of: instrument preparation comprising filling an integrated reaction unit with one or more of a lysis buffer and a wash buffer, an Internal Control, Proteinase K, 30pL + / - 20% silica microparticles, and 70pL + / - 20% elution buffer; pretreatment comprising addition of 300pL + / - 20% sample plus 400pL + / - 20% lysis buffer to a pretreatment well and incubation of the sample in the pretreatment well with Proteinase K and lysis solution at 50°C + / - 20%; lysis comprising transfer of the pretreated sample to a lysis well comprising 1300pL + / - 20% lysis buffer and microparticles incubated at 68°C + / - 20% with mixing by a magnetic plunger sheathed with a disposable plastic sheath during incubation; washing comprising transfer of the microparticles to a wash well comprising 1200pL + / - 20% lysis buffer; transfer of the magnetic microparticles to 2 successive washes with 779pL + / - 20% ethanol and 221 pL + / - 20% H2O, and capture of the magnetic microparticles by the plunger magnet followed by evaporation of the ethanol; elution comprising transfer of the microparticles to an elution well containing 70pL + / - 20% elution buffer with elution of purified DNA at 72°C + / -20%; transfer of the purified DNA to a PCR reaction vessel comprising 5.8pL + / - 20% activator and 24.2pL + / - 20% of eluate to rehydrate a reaction mixture comprising PCR primers and detection probes, addition of the rehydrated solution to the PCR reaction vessel, layering 14pL + / - 20% of vapor barrier on top of the reaction mixture, and capping and transfer for thermocycling and signal detection.

[0013] In some embodiments, the present invention provides a method for detecting CMV, and / or EBV in a whole blood sample, comprising: a) incubation of the sample with Proteinase K in a pretreatment well at 50°C; b) transfer of the incubated sample of step a) to a lysis well comprising lysis buffer and magnetic microparticles followed by mixing and incubation at 68°C in the well; c) transfer of the magnetic microparticles of step b) to a first wash well comprising lysis buffer; d) transfer of the magnetic microparticles of step c) to a second wash well and washing the microparticles with a mixture of ethanol and water; e) capture of the magnetic microparticles of step d) with a plunger magnet and evaporation of the ethanol; f) transfer of the magnetic microparticles of step e) to an elution well comprising an elution buffer; g) elution of purified DNA from the magnetic microparticles of step f) at 72°C; h) transfer of the purified DNA to a reaction vessel comprising one or more amplification primers, one or more probes comprising a detectable label, and one or more reagents for amplification comprising one or more primer pairs and probes; i) thermocycling the reaction vessel to generate a signal from the detectable label; and j) detecting the CMV and / or EBV by measuring the signal from the detectable label. In some embodiments, the one or more amplification primers comprise one or more of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 5. In some embodiments, the one or more amplification primers comprise one or more of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 14. In some embodiments, the one or more probes comprise one or more of SEQ ID NO: 3 and SEQ ID NO: 6. In some embodiments, the one or more probes comprise one or more of SEQ ID NO: and SEQ ID NO: 15.

[0014] In some embodiments, the present invention provides a system comprising: a) a pipettor; b) an integrated reaction unit (IRU) comprising one or more lanes comprising in linear order of sample transfer: 1) a pretreatment well comprising Proteinase K and lysis solution at 50°C and an internal control; 2) a lysis well comprising lysis buffer and silica magnetic microparticles at 68°C and a magnetic microparticle transfer plunger sheathed with a disposable plastic sheath; 3) a wash well comprising lysis buffer; 4) a first wash well comprising ethanol and water; 5) a second wash well comprising ethanol and water; 6) an elution well comprising an elution buffer at 72°C; and 7) a PCR reaction vessel comprising activator formulation, Mastermix and one or more CMV or EBV primer pairs and probes; c) a thermocycler: and d) a fluorescence detector.

[0015] The disclosure also provides methods for detecting CMV or EBV in a sample. The sample may comprise a nasal swab or brush, saliva, mucus, blood, serum, plasma, urine or feces.

[0016] The disclosure further provides kits for detecting CMV or EBV in a sample comprising at least one set of oligonucleotides, any of the oligonucleotides as disclosed herein, reagents for amplifying and detecting nucleic acid sequences, and / or instructions for use.

[0017] Other aspects and embodiments of the disclosure will be apparent in light of the following detailed description and accompanying figures.

[0018] BRIEF DESCRIPTIONS OF THE DRAWINGS

[0019] Figures 1 A and IB show CMV and EBV sensitivity results with over 95% detection at 100 IU for Alinity m CMV and EBV in whole blood (WB).

[0020] Figures 2A and 2B show linearity results of CMV and EBV in WB. All genotypes were evaluated across multiple target levels: CMV : 80 - 2 x 108lU / mL and EBV 80 - 4 x 108lU / mL with 24 replicates at each target level.

[0021] Figures 3A and 3B show CMV and EBV in WB precision results. Each panel member was tested in multiple replicates twice each day for 5 days on 3 Alinity m systems by 3 operators using 3 AMP kits.

[0022] Figures 4A and 4B show CMV in WB clinical specimen quantitation vs. RealTime CMV. 108 quantified fresh and frozen specimens from hematopoietic stem cell transplantation (HSCT) and solid organ transplantation (SOT) patients and other immunocompromised patients were collected from 5 sites. Top plot: N = 108, slope: 1.05, Intercept: -0.14, r: 0.958. Bottom plot: Mean bias = 0.01 Log / mL.

[0023] Figure 5 shows concordance between Alinity m CMV in WB assays vs. RealTime CMV assays. 736 fresh and frozen samples from HSCT, SOT and other immunocompromised patients were collected at 5 sites.

[0024] Figures 6A and 6B show Alinity m EBV in WB specimen quantitation vs. RealTime EBV. 119 quantified fresh and frozen specimens from transplant patients and other immunocompromised patients were collected at 5 sites. Top plot: N=119, Slope: 0.92, Intercept: 0.33, r: 0.893. Botttom plot: Mean bias = 0.05 Log / mL.

[0025] Figure 7 shows concordance between Alinity m EBV in WB assays vs. RealTime EBV assays. 378 fresh and frozen samples from transplant and other immunocompromised patients were collected at 5 sites.

[0026] DETAILED DESCRIPTION OF THE INVENTION

[0027] Provided herein are methods, compositions, kits and systems for detecting target nucleic acid sequences from cytomegalovirus (CMV) or Epstein-Bar virus (EBV). Cytomegalovirus

[0028] Infection with cytomegalovirus (CMV), a double-stranded Herpesviridae DNA virus, is elevated among solid organ transplant recipients, and is a risk factor for graft failure with high morbidity and mortality after renal transplant. CMV infection among renal transplant recipients is caused by drug-induced T-cell immunosuppression leading to activation and replication of CMV after surgery. Between 20% and 60% of persons with a solid organ transplant develop CMV infection. CMV infection may comprise viral penetration of diverse tissues and organs including the liver, gastrointestinal organs, the renal graft, lungs, retina and bone marrow. Accordingly, CMV monitoring and prevention strategies are in frequent use in the post-operative care of transplant recipients. Preventive therapy reduces reactivation of latent CMV infection and acquisition of acute infection in CMV-negative recipients of seropositive grafts. However, CMV disease may still occur despite preventive therapies, especially when infections are not dosed adequately. CMV disease also occurs following discontinuation of preventive therapy. Post-transplant patients undergo PCR testing for 3 months or longer after surgery to measure viral load to assess CMV risk and severity, guide therapeutic interventions, rapidly diagnose CMV infection, and monitor the effectiveness of therapy detect antiviral resistance.

[0029] Epstein-Varr virus

[0030] The Epstein-Barr virus (EBV), also known as human herpesvirus 4, is one of the most common human-associated viruses with approximately 95% of the world’s adult population as lifelong carriers. Following primary infection, the virus establishes persistence in B cells. Other EBV-related infections are chronic active EBV infection and lymphoproliferative EBV-infections. Post-transplant lymphoproliferative disease (PTLD), that is EBV-associated in 90% of cases, is a significant illness that occurs in transplant recipients and other immunocompromised patients. In addition to PTLD, EBV is responsible for up to 1% of tumors worldwide and plays a role in the development of nasopharyngeal cancer (NPC). Diagnosis of EBV infection is immunocompetent individuals is done either by clinical diagnosis or by antibody testing in patient exhibiting characteristic signs and symptoms of infectious mononucleosis or other illnesses caused by EBV infection. In immunocompromised patients, rising EBV viral titers raise the suspicion of EBV-related PTLD with confirmation by biopsy. Accordingly, quantitative monitoring of EBV viral load is required in support of clinical management to accurately distinguish health low-level carriers from high-level infected patients. Before disease manifests, high risk individuals including transplant recipients benefit from regular screening to track possible disease progression. Traditional serological methods for EBV detection are constrained in immunosuppressed patients due to the high probability of varied serology profiles, consequently real-time PCR is preferred for viral load measurement. Treatment for EBV related illness varies based on immunocompetence as well as type of EBV related illness. For EBV-associated PTLD in transplant recipients, therapeutic strategies include a reduction in immunosuppression for 2-4 weeks, monoclonal antibody therapy, chemotherapy, EBV- specific cytotoxic T-cell therapy and steroids.

[0031] Definitions

[0032] As used herein, the terms “comprise(s)”, “include(s)”, “having”, “has”, “can”, “contain(s)”, and variants thereof are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a”, “and” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of’ and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0033] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6- 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0034] The terms “first” and “second” are used in this disclosure in their relative sense only. It will be understood that, unless otherwise noted, those terms are used merely as a matter of convenience in the description of one or more of the embodiments. The terms “first” and “second” are only used to distinguish one element from another element, and the scope of the rights of the disclosed technology should not be limited by these terms. For example, a first element may be designated as a second element, and similarly the second element may be designated as the first element.

[0035] The term “oligonucleotide,” as used herein, refers to a short nucleic acid sequence comprising from about 2 to about 100 nucleotides (e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100 nucleotides, or a range defined by any of the foregoing values). The terms “nucleic acid” and “polynucleotide” as used herein refer to a polymeric form of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA). These terms refer to the primary structure of the molecule, and thus include double- and single- stranded DNA, and double- and single-stranded RNA. The terms include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs and modified polynucleotides such as, for example, methylated and / or capped polynucleotides. Nucleic acids are typically linked via phosphate bonds to form nucleic acid sequences or polynucleotides, though many other linkages are known in the art (e.g., phosphorothioates, boranophosphates, and the like).

[0036] Oligonucleotides can be single- stranded or double-stranded or can contain portions of both double-stranded and single-stranded sequences. The oligonucleotide can be DNA, both genomic and complimentary DNA (cDNA), RNA, or a hybrid, where the nucleic acid can contain combinations of deoxyribo- and ribonucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine. Oligonucleotides can be obtained by chemical synthesis methods or by recombinant methods.

[0037] As used herein, the term “percent sequence identity” refers to the percentage of nucleotides or nucleotide analogs in a nucleic acid sequence, or amino acids in an amino acid sequence, that is identical with the corresponding nucleotides or amino acids in a reference sequence after aligning the two sequences and introducing gaps, if necessary, to achieve the maximum percent identity. Hence, in case a nucleic acid according to the technology is longer than a reference sequence, additional nucleotides in the nucleic acid, that do not align with the reference sequence, are not taken into account for determining sequence identity. Methods and computer programs for alignment are well known in the art, including BLAST, Align 2, and FASTA.

[0038] Unless otherwise defined herein, scientific, and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0039] Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting. The section headings used herein are for organizational purposes only and are not to be construed as limiting the described subject matter in any way.

[0040] Whole blood sample pretreatment

[0041] In some embodiments, the methods, steps, compositions, and kits described herein are used for whole blood sample pretreatment before CMV and / or EBV amplification, detection, identification, and quantification. In experiments conducted during the course of development of the present invention, it was discovered that addition of an aliquot of whole blood to a solution comprising proteinase K (PK) and a lysis buffer comprising, for example, guanidine isothiocyanate (GITC), in pretreatment surprisingly supports quantitative analysis of CMV and EBV viral load in whole blood using real-time PCR. During pretreatment, GITC lyses cells and viral particles and denatures proteins from whole blood samples. PK digests the proteins. During the course of development of the present invention, it was discovered that a high concentration of GITC inactivates PK by its denaturation, thereby resulting in under-quantification of the viral load in the whole blood sample. It was further discovered that a low concentration of GITC (including no GITC) allows full activity of PK in releasing cellular components from the whole blood samples, thereby resulting in undesirable formation of whole blood and magnetic microparticle aggregates during the lysis step i.e., “clumping”. In experiments conducted during the course of development of the present invention, it was discovered that a specific amount of GITC combined with PK resolves the pretreatment problems of both 1) inaccurate quantification of the viral load from whole blood and 2) clumping during the lysis step. In some embodiments, the concentration of GITC is 1.74 M to 2.80 M. In some embodiments, the concentration of GITC is 2.42 M to 2.69 M. In some embodiments, the concentration of GITC is 2.54 M. In some embodiments, the amount of PK is 0.4 U to 1.2 U per reaction. In some embodiments, the amount of PK is 0.6 U to 0.9 U per reaction. In some embodiments, the amount of PK is 0.76 U per reaction. In some embodiments, the pretreatment temperature is 40 °C to 65 °C. In some embodiments, the pretreatment temperature is 48°C to 52 °C. In some embodiments, the pretreatment temperature is 50 °C. In some embodiments, the lysis temperature is 50 °C to 72 °C. In some embodiments, the lysis temperature is 65 °C to 70 °C. In some embodiments, the lysis temperature is 68 °C.

[0042] Amplification and Probe Oligonucleotides

[0043] In some embodiments, the oligonucleotides described herein may be used for nucleic acid amplification (e. ., primers) or as probes for nucleic acid hybridization and detection. The terms “primer”, “primer sequence”, “primer oligonucleotide”, and “amplification oligonucleotide” as used herein, refer to an oligonucleotide which is capable of acting as a point of initiation of synthesis of an extension product that is a complementary strand of nucleic acid (all types of DNA or RNA) when placed under suitable amplification conditions (e.g., buffer, salt, temperature and pH) in the presence of nucleotides and an agent for nucleic acid polymerization (e.g., a DNA-dependent or RNA-dependent polymerase). The amplification oligonucleotides of the present disclosure can be of any suitable size, and desirably comprise, consist essentially of, or consist of about 15 to 50 nucleotides, preferably about 20 to 40 nucleotides. The oligonucleotides of the present disclosure can contain additional nucleotides in addition to those described herein. Depending on the type of amplification process employed, the amplification oligonucleotides can include, for example, a nicking enzyme site and a stabilizing region upstream (see, e.g., U.S. Patent Nos 9,689,031; 9,617,586; 9,562,264; and 9,562,263, each of which is incorporated herein by reference in its entirety).

[0044] The terms “probe”, “probe sequence”, and “probe oligonucleotide”, refer to an oligonucleotide that can selectively hybridize to at least a portion of a target sequence (e.g., a portion of a target sequence that has been amplified) under appropriate hybridization conditions. In general, a probe sequence is identified as being either “complementary” (e.g., complementary to the coding or sense strand (+)), or “reverse complementary” (e.g., complementary to the anti-sense strand (-)). The probes of the present disclosure can be of any suitable size, and desirably comprise, consist essentially of, or consist of about 10-50 nucleotides, preferably about 12-35 nucleotides.

[0045] As used herein, the terms “set”, “primer set”, “probe set”, and “primer and probe set” refer to two or more oligonucleotides which together are capable of priming the amplification of a target sequence or target nucleic acid of interest (e.g., a target sequence within CMV or EBV) and / or at least one probe which can detect the target sequence or target nucleic acid. In certain embodiments, the term “set” refers to a pair of oligonucleotides including a first oligonucleotide that hybridizes with the 5 ’-end of the target sequence or target nucleic acid to be amplified and a second oligonucleotide that hybridizes with the complement of the target sequence or target nucleic acid to be amplified.

[0046] The set of oligonucleotides described herein may be used to amplify and detect one or more target CMV or EBV sequences in a sample. The terms “target sequence” and “target nucleic acid” are used interchangeably herein and refer to a specific nucleic acid sequence, the presence or absence of which is to be detected by the disclosed method. In the context of the present disclosure, a target sequence preferably includes a nucleic acid sequence to which one or more oligonucleotides will hybridize and from which amplification will initiate. The target sequence can also include a probe-hybridizing region with which a probe may form a stable hybrid under appropriate amplification conditions. A target sequence may be singlestranded or double-stranded. The target CMV or EBV sequence may be within any portion of the CMV or EBV genome.

[0047] In some embodiments, the set comprises a first amplification oligonucleotide, a second amplification oligonucleotide, and a probe oligonucleotide.

[0048] Any of the oligonucleotides described herein may be modified in any suitable manner so as to stabilize or enhance the binding affinity of the oligonucleotide for its target. For example, an oligonucleotide sequence as described herein may comprise one or more modified oligonucleotide. Furthermore, any of the sequences listed which include internal spacers or modifications may be used without the modifications or spacers.

[0049] Any of the oligonucleotides described herein may include, for example, spacers, blocking groups, and modified nucleotides. Modified nucleotides are nucleotides or nucleotide triphosphates that differ in composition and / or structure from natural nucleotides and nucleotide triphosphates. Modifications include those naturally occurring that result from modification by enzymes that modify nucleotides, such as methyltransferases. Modified nucleotides also include synthetic or non-naturally occurring nucleotides. For example, modified nucleotides include those with 2' modifications, such as 2'-O-methyl and 2'-fluoro. Other 2'-modified nucleotides are known in the art and are described in, for example U.S. Pat. No. 9,096,897, which is incorporated herein by reference in its entirety. Modified nucleotides or nucleotide triphosphates used herein may, for example, be modified in such a way that, when the modifications are present on one strand of a double-stranded nucleic acid where there is a restriction endonuclease recognition site, the modified nucleotide or nucleotide triphosphates protect the modified strand against cleavage by restriction enzymes. Blocking groups or polymerase-arresting molecules are chemical moieties that inhibit target sequence-independent nucleic acid polymerization by the polymerase. The blocking group may render the oligonucleotide capable of binding a target nucleic acid molecule, but incapable of supporting template extension utilizing the detectable oligonucleotide probe as a target. For example, the presence of one or more moieties that do not allow polymerase progression likely causes polymerase arrest in non-nucleic acid backbone additions to the oligonucleotide or through stalling of a replicative polymerase. Oligonucleotides with these moieties may prevent or reduce illegitimate amplification of the probe during the course of the amplification reaction. Examples of blocking groups include, for example, alkyl groups, non-nucleotide linkers, phosphorothioate, alkane-diol residues, peptide nucleic acid, and nucleotide derivatives lacking a 3 '-OH, including, for example, cordycepin, spacer moieties, damaged DNA bases and the like. Examples of spacers include, for example, C3 spacers. Spacers may be used, for example, within the oligonucleotide, and also, for example, at the ends to attach other groups, such as, for example, labels.

[0050] Any of the oligonucleotide sequences described herein may comprise, consist essentially of, or consist of a complement of any of the sequences disclosed herein. The terms “complement” or “complementary sequence” as used herein, refer to a nucleic acid sequence that forms a stable duplex with an oligonucleotide described herein via Watson-Crick base pairing rules, and typically shares about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% greater identity with the disclosed oligonucleotide. Nucleic acid sequence identity can be determined using any suitable mathematical algorithm or computer software known in the art, such as, for example, CLUSTAL-W, T-Coffee, and ALIGN (for alignment of nucleic acid and amino acid sequences), BLAST programs (e.g., BLAST 2.1, BL2SEQ, and later versions thereof) and FASTA programs (e.g., FASTA3 '. FASTM, and SSEARCH) (for sequence alignment and sequence similarity searches). Sequence alignment algorithms also are disclosed in, for example, Altschul et al., J. Molecular Biol., 215(3): 403-410 (1990); Beigert et al., Proc. Natl. Acad. Sci. USA, 1 6(\U): 3770-3775 (2009), Durbin et al., eds., Biological Sequence Analysis: Probalistic Models of Proteins and Nucleic Acids, Cambridge University Press, Cambridge, UK (2009); Soding, Bioinformatics, 21(f): 951-960 (2005); Altschul et al., Nucleic Acids Res., 25(Y1): 3389-3402 (1997); and Gusfield, Algorithms on Strings, Trees and Sequences, Cambridge University Press, Cambridge UK (1997), each of which is incorporated herein by reference in its entirety).

[0051] The oligonucleotides described herein may be prepared using any suitable method, a variety of which are known in the art (see, for example, Sambrook et al., Molecular Cloning. A Laboratory Manual, 1989, 2. Supp. Ed., Cold Spring Harbour Laboratory Press: New York, N.Y.; M. A. Innis (Ed.), PCR Protocols. A Guide to Methods and Applications, Academic Press: New York, N.Y. (1990); P. Tijssen, Hybridization with Nucleic Acid Probes - Laboratory Techniques in Biochemistry and Molecular Biology (Parts I and II), Elsevier Science (1993); M. A. Innis (Ed.), PCR Strategies, Academic Press: New York, N.Y. (1995); and F. M. Ausubel (Ed.), Short Protocols in Molecular Biology, John Wiley & Sons: Secaucus, N.J. (2002); Narang et al., Meth. Enzymol., 68: 90-98 (1979); Brown et al., Meth. Enzymol., 68: 109-151 (1979); and Belousov et al., Nucleic Acids Res., 25: 3440-3444 (1997), each of which is incorporated herein by reference in its entirety). Oligonucleotide pairs also can be designed using a variety of tools, such as the Primer-BLAST tool provided by the National Center of Biotechnology Information (NCBI). Oligonucleotide synthesis may be performed on oligo synthesizers such as those commercially available from Perkin Elmer / Applied Biosystems, Inc. (Foster City, CA), DuPont (Wilmington, DE), or Milligen (Bedford, MA). Alternatively, oligonucleotides can be custom made and obtained from a variety of commercial sources well-known in the art, including, for example, the Midland Certified Reagent Company (Midland, TX), Eurofins Scientific (Louisville, KY), BioSearch Technologies, Inc. (Novato, CA), and the like. Oligonucleotides may be purified using any suitable method known in the art, such as, for example, native acrylamide gel electrophoresis, anion-exchange HPLC (see, e.g., Pearson et al., J. Chrom., 255: 137-149 (1983), incorporated herein by reference), and reverse phase HPLC (see, e.g., McFarland et al., Nucleic Acids Res., 7: 1067-1080 (1979), incorporated herein by reference).

[0052] The sequence of the oligonucleotides can be verified using any suitable sequencing method known in the art, including, but not limited to, chemical degradation (see, e.g., Maxam et al., Methods of Enzymology, 65: 499-560 (1980), incorporated herein by reference), matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (see, e.g., Pieles et al., Nucleic Acids Res., 21: 3191-3196 (1993), incorporated herein by reference), mass spectrometry following a combination of alkaline phosphatase and exonuclease digestions (Wu et al., Anal. Biochem., 290: 347-352 (2001), incorporated herein by reference), and the like. Detectable Label

[0053] Any one or more of the oligonucleotide sequences described herein may comprise a detectable label, such that the amplification oligonucleotide(s) and / or the probe oligonucleotide can be measured. In one embodiment, each of the probe oligonucleotide sequences described herein comprise a detectable label. The term “detectable label” as used herein, refers to a moiety or compound that generates a signal which can be measured and whose intensity is related to (e.g., proportional to) the amount of entity bound thereto. Any suitable detectable label that can be conjugated or linked to an oligonucleotide in order to detect binding of the oligonucleotide to a target sequence can be used, many of which are known in the art. In one embodiment, the detectable label may be detected indirectly. Indirectly detectable labels are typically specific binding members used in conjunction with a “conjugate” that is attached or coupled to a directly detectable label. Coupling chemistries for synthesizing such conjugates are well-known in the art and are designed such that the specific binding property of the specific binding member and the detectable property of the label remain intact. As used herein, “specific binding member” and “conjugate” refer to the two members of a binding pair, e.g., two different molecules, where the specific binding member binds specifically to the polynucleotide of the present disclosure, and the “conjugate” specifically binds to the specific binding member. Binding between the two members of the pair is typically chemical or physical in nature. Examples of such binding pairs include, but are not limited to, antigens and antibodies, avidin / streptavidin and biotin, haptens and antibodies specific for haptens, complementary nucleotide sequences, enzyme cofactors / substrates and enzymes, and the like.

[0054] Each of the probe oligonucleotide sequences desirably comprises a detectable label. Each of the probes may be labeled with the same detectable label or different detectable labels.

[0055] In some embodiments, the detectable label may be directly detected. Such directly detectable labels include, for example, radioisotopes, fluorophores, chemiluminophores, enzymes, colloidal particles, fluorescent microparticles, intercalating dyes (e.g., SYBR Green or ethidium bromide), and the like. In select embodiments, the detectable label may be a fluorophore, such as a fluorescein-family dye, polyhalofluorescein-family dye, hexachlorofluorescein-family dye, coumarin-family dye, rhodamine-family dye, cyanine- family dye, oxazine-family dye, thiazin-family dye, squaraine-family dye, chelated lanthanide-family dye, azo-family dye, triphenylmethane-family dye, or a BODIPY®-family dye. Examples of fluorophores include, but are not limited to, FAM™, CAL-FLUOR®, QUASAR®, HEX™, JOE™, NED™, PET®, ROX™, TAMRA™, TET™, TEXAS RED®, and VIC®. One skilled in the art will appreciate that directly detectable labels may require additional components, such as substrates, triggering reagents, light, and the like, to enable detection of the label. Methods for labeling oligonucleotides, such as probes, are well-known in the art and described in, e.g., L. J. Kricka, Ann. Clin. Biochem., 39: 114-129 (2002); van Gijlswijk et al., Expert Rev. Mol. Diagn., 1: 81-91 (2001); Joos et al., J. Biotechnol., 35: 135- 153 (1994); Smith et al., Nucl. Acids Res., 13: 2399-2412 (1985); Connoly et al., Nucl. Acids. Res., 13: 4485-4502 (1985); Broker et al., Nucl. Acids Res., 5: 363-384 (1978); Bayer et al., Methods ofBiochem. Analysis, 26: 1-45 (1980); Langer et al., Proc. Natl. Acad. Sci. USA, 78: 6633-6637 (1981); Richardson et al., Nucl. Acids Res., 11: 6167-6184 (1983); Brigati et al., Virol., 126: 32-50 (1983); Tchen et al., Proc. Natl. Acad. Sci. USA, 81: 3466-3470 (1984); Landegent et al., Exp. Cell Res., 15: 61-72 (1984); A. H. Hopman et al., Exp. Cell Res., 169: 357-368 (1987); and Temsamani et al., Mol. Biotechnol., 5: 223-232 (1996), each of which is incorporated herein by reference in its entirety.

[0056] In some embodiments, any one or more of the oligonucleotides described herein may also comprise a quencher moiety. When a detectable label (e.g., a fluorophore) and quencher moiety are held in close proximity, such as at the ends of a probe, the quencher moiety prevents detection of a signal (e.g., fluorescence) from the detectable label. When the two moieties are physically separated, the signal becomes detectable. The quencher may be selected from any suitable quencher known in the art, such as, for example, BLACK HOLE QUENCHER® 1 (BHQ-1®), BLACK HOLE QUENCHER® 2 (BHQ-2®), BLACK HOLE QUENCHER® 3 (BHQ-3®), IOWA BLACK® FQ, and IOWA BLACK® RQ. For example, the oligonucleotide probe may comprise a FAM fluorophore, CAL-FLUOR®, or QUASAR fluorophore and a BHQ-1 or BHQ-2 quencher.

[0057] The selection of a particular label and labeling technique will depend on several factors, such as the ease and cost of the labeling method, spectral spacing between different detectable labels used, the quality of sample labeling desired, the effects of the detectable moiety on the hybridization reaction (e.g., on the rate and / or efficiency of the hybridization process), the nature of the amplification method used, the nature of the detection system, the nature and intensity of the signal generated by the detectable label, and the like. Methods for Amplifying and Detecting CMV or EBV

[0058] The present disclosure provides a method for detecting CMV or EBV in a sample. The method comprises: contacting a sample with the set of oligonucleotides disclosed herein and reagents for amplification; amplifying one or more target CMV or EBV nucleic acid sequences present in the sample; hybridizing one or more of the oligonucleotide probes to one or more amplified target CMV or EBV nucleic acid sequences; and detecting hybridization of the one or more probe oligonucleotide sequences to the one or more amplified CMV or EBV target nucleic acid sequences by measuring a signal from the detectable labels. Descriptions of the oligonucleotides set forth herein with respect to the aforementioned set of oligonucleotides also are applicable to the disclosed method.

[0059] The sample can be any suitable sample obtained from any suitable subject, typically a mammal (e.g., dogs, cats, rabbits, mice, rats, goats, sheep, cows, pigs, horses, non-human primates, or humans). In some embodiments, the subject is a human. The sample may be obtained from any suitable biological source, such as, a nasal swab or brush, or a physiological fluid including, but not limited to, whole blood, serum, plasma, interstitial fluid, saliva, ocular lens fluid, cerebral spinal fluid, sweat, urine, milk, ascites fluid, mucous, synovial fluid, peritoneal fluid, vaginal fluid, menses, amniotic fluid, semen, feces, and the like.

[0060] In some embodiments, the present invention provides compositions, methods, systems and kits for isolation, detection, identification and / or quantification of CMV and EBV in whole blood. In experiments conducted in the course of development of the present invention, it was discovered that the addition of an aliquot of whole blood to a solution comprising Proteinase K and incubation at 50°C prior to transfer to a cell lysis well containing lysis buffer and microparticles surprisingly supports quantitative analysis of CMV and EBV viral load in whole blood using real-time PCR. Compositions, methods, systems and kits of the present invention configured for quantitative analysis of CMV and EBV viral load in whole blood samples fulfill a long felt unmet need essential to determination of the requirement, selection and dosing of immunosuppressive and antiviral medications in the care of immunocompromised patients and organ transplant recipients after surgery.

[0061] The sample can be obtained from the subject using routine techniques known to those skilled in the art, and the sample may be used directly as obtained from the biological source or following a pretreatment to modify the character of the sample. Such pretreatment may include, for example, preparing plasma from blood, diluting viscous fluids, filtration, precipitation, dilution, distillation, mixing, concentration, inactivation of interfering components, the addition of reagents, lysing, and the like.

[0062] After the sample is obtained from a subject, the sample may be contacted with the set of oligonucleotides comprising amplification oligonucleotides and probes as described herein to form a reaction mixture. The reaction mixture is then placed under amplification conditions. The term “amplification conditions,” as used herein, refers to conditions that promote annealing and / or extension of the amplification oligonucleotides. Amplification conditions encompass all reaction conditions including, but not limited to, temperature and / or temperature cycling, buffer, salt, ionic strength, pH, and the like.

[0063] Amplifying a CMV or EBV nucleic acid sequence in the sample can be performed using any suitable nucleic acid sequence amplification method known in the art. In some embodiments, the amplification includes, but is not limited to, polymerase chain reaction (PCR), reverse-transcriptase PCR (RT-PCR), real-time PCR, transcription-mediated amplification (TMA), rolling circle amplification, nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), Transcription-Mediated Amplification (TMA), Single Primer Isothermal Amplification (SPIA), Helicase-dependent amplification (HDA), Loop mediated amplification (LAMP), Recombinase-Polymerase Amplification (RPA), and ligase chain reaction (LCR). In some embodiments, amplification of CMV or EBV nucleic acid sequences is performed using isothermal amplification(e. ., RPA or NEAR). In some embodiments, amplification and detection of CMV or EBV nucleic acid sequences is performed using a point of care device (e.g., the ID NOW system (Abbott)).

[0064] In some embodiments, amplification of CMV or EBV nucleic acid sequences is performed using real-time PCR. “Real-time PCR,” as used herein, refers to a PCR method in which the accumulation of amplification product is measured as the reaction progresses, in real time, with product quantification after each cycle, in contrast to conventional PCR in which the amplified DNA product is detected in an end-point analysis. Real-time PCR also is known in the art as “quantitative PCR (qPCR).” Real-time detection of PCR products typically involves the use of non-specific fluorescent dyes that intercalate with any doublestranded DNA and sequence-specific fluorescently-labeled DNA probes. Real-time PCR techniques and systems are known in the art (see, e.g., Dorak, M. Tevfik, ed. Real-time PCR. Taylor & Francis (2007); and Fraga et al., “Real-time PCR,” Current protocols essential laboratory techniques'. 10-3 (2008), each of which is incorporated herein by reference in its entirety) and are commercially available from a variety of sources (e.g., m2000rt REALTIA7E™ PCR system (Abbott Molecular, Inc., Des Plaines, IL); CFX Real-Time PCR Detection Systems (Bio-Rad Laboratories, Inc., Hercules, CA); and TAQMAN™ Real-Time PCR System (ThermoFisher Scientific, Waltham, MA)), any of which can be employed in the methods described herein.

[0065] In select embodiments, the isothermal amplification methods may rely on nicking and extension reactions, “nicking and extension amplification,” to amplify shorter sequences in a quicker timeframe than traditional amplification reactions. These methods may include, for example, reactions that use only two amplification oligonucleotides, one or two nicking enzymes, and a polymerase, under isothermal conditions.

[0066] In nicking and extension amplification, a target nucleic acid sequence, having a sense and antisense strand, is contacted with a pair of amplification oligonucleotides. The first amplification oligonucleotide comprises a nucleic acid sequence comprising a recognition region at the 3' end that is complementary to the 3' end of the target sequence antisense strand, a nicking enzyme site upstream of said recognition region, and a stabilizing region upstream of said nicking enzyme site. The second amplification oligonucleotide comprises a nucleotide sequence comprising a recognition region at the 3' end that is complementary to the 3' end of the target sequence sense strand, a nicking enzyme site upstream of said recognition region, and a stabilizing region upstream of said nicking enzyme site. Two nicking enzymes are provided. One nicking enzyme is capable of nicking at the nicking enzyme site of the first amplification oligonucleotide but incapable of nicking within said target sequence. The other nicking enzyme is capable of nicking at the nicking enzyme site of the second amplification oligonucleotide but incapable of nicking within said target sequence. A DNA polymerase is employed under conditions for amplification which involves multiple cycles of extension of the amplification oligonucleotides thereby producing a double-stranded nicking enzyme site which are nicked by the nicking enzymes to produce the amplification product. For example, see U.S. Patent Nos: 10,851,406; 9,689,031; 9,617,586; 9,562,264; and 9,562,263, and U.S. Patent Application Nos: 15 / 467,893; 15 / 600,951; and 16 / 243,829, each of which is incorporated herein by reference in its entirety.

[0067] In some embodiments, amplification of CMV or EBV nucleic acid sequences is performed using Recombinase-Polymerase Amplification (RPA), which relies on the properties of recombinases and related proteins, to invade double-stranded DNA with single stranded homologous DNA permitting sequence specific priming of DNA polymerase reactions. In RPA, a recombinase agent is contacted with first and second amplification oligonucleotides to form nucleoproteins. These nucleoproteins contact the target sequence to form a first double stranded structure at a first portion of said first strand and form a double stranded structure at a second portion of said second strand so the 3' ends of said first amplification oligonucleotide and the second amplification oligonucleotide are oriented towards each other on the DNA comprising the target sequence. The 3' end of the amplification oligonucleotides in the nucleoprotein are extended by DNA polymerases to generate first and second double stranded nucleic acids, and first and second displaced strands of nucleic acid. The steps are repeated until the desired level of amplification is achieved.

[0068] Methods and materials useful for RPA of a target nucleic acid sequence are known in the art. See U.S. Patent Nos: 7,270,981; 8,460,875; 7,399,590; 7,666,598; 8,030,000; 8,426,134; 8,945,845; 9,663,820; 10,329,603; 10,329,602; 8,017,339; 8,574,846; 8,962,255; 10,036,057; 8,071,308; 10,093,908; 11,339,382; 10,947,584: and 8,637,253, and U.S. Patent Application Nos: 15 / 099,754; and 14 / 705,150 each of which is incorporated herein by reference in its entirety. For example, suitable recombinase agents include the A’, coli RecA protein, the T4 uvsX protein, or any homologous protein or protein complex from any phyla. Other non-homologous recombinase agents may be utilized in place of RecA, for example as RecT, or RecO. Suitable recombinase loading proteins may include, for example, T4uvsY, E. coli recO, E. coli recR and derivatives and combinations of these proteins. Suitable single stranded DNA binding proteins may be the E. coli SSB or the T4 gp32 or a derivative or a combination of these proteins. The DNA polymerase may be a eukaryotic or prokaryotic polymerase. Examples of eukaryotic polymerases include pol-a, pol-P, pol-8, pol-s and derivatives and combinations thereof. Examples of prokaryotic polymerase include E. coli DNA polymerase I Klenow fragment, bacteriophage T4 gp43 DNA polymerase, Bacillus stearothermophilus polymerase I large fragment, Phi-29 DNA polymerase, T7 DNA polymerase, Bacillus subtilis Pol I, E. coli DNA polymerase I, E. coli DNA polymerase II, E. coli DNA polymerase III, E. coli DNA polymerase IV, E. coli DNA polymerase V and derivatives and combinations thereof. Other components of RPA include ATP, an ATP analog, or a system for ATP regeneration (convert ADP to ATP). Such system may utilize, for example, phosphocreatine and creatine kinase. The ATP or ATP analog may be any of ATP, ATP-y-S, ATP-P-S, ddATP or a combination thereof. The RPA reaction may also include a system to regenerate ADP from AMP and a to convert pyrophosphate to phosphate (pyrophosphate). Suitable crowding agents used in RPA include polyethylene glycol (PEG), dextran and ficoll.

[0069] Following amplification of one or more CMV or EBV virus nucleic acid sequences present in the sample, the disclosed method may further comprise hybridizing one or more of the probe oligonucleotide sequences disclosed herein to the one or more amplified target CMV or EBV nucleic acid sequences.

[0070] Following hybridization of the one or more of the probe oligonucleotide sequences to the one or more amplified target nucleic acid sequences, the method comprises detecting hybridization of the one or more probe oligonucleotide sequences to the one or more amplified target nucleic acid sequences by assessing a signal from each of the detectable labels, whereby (i) the presence of one or more signals indicates hybridization of the one or more probe oligonucleotide sequences to the one or more target CMV or EBV nucleic acid sequences and the presence of CMV or EBV in the sample, and (ii) the absence of signals indicates the absence of CMV or EBV in the sample. Detection of signals from the one or more probe oligonucleotide sequences may be performed using a variety of well-known methodologies, depending on the type of detectable label. For example, detection may be done using solution real-time fluorescence or using a solid surface method.

[0071] In some embodiments, the present invention provides compositions, methods, systems and kits for multiplex isolation, detection, identification and / or quantification of CMV and EBV in whole blood wherein CMV and EBV are simultaneously isolated, detected, identified and / or quantified in a single sample and / or reaction vessel.

[0072] Treatment and Monitoring of Subjects Identified as having CMV or EBV

[0073] A subject identified according to the methods described herein as having CMV or EBV may be treated, monitored (e.g., for the presence of a CMV or EBV nucleic acid determined in a sample from the subject), treated and monitored, and / or monitored and treated using routine techniques known in the art. In some embodiments, the methods described herein further include treating the subject when the presence of CMV or EBV nucleic acid is determined in one or more samples obtained from the subject using the present methods. Kits

[0074] The disclosure also provides a kit for amplifying and detecting CMV or EBV in a sample. The kit comprises at least one oligonucleotide as described herein. In some embodiments, the kit comprises a set or group of oligonucleotides described herein. The kit may further comprise reagents for amplifying and detecting nucleic acid sequences, and instructions for amplifying and detecting CMV or EBV. Descriptions of the oligonucleotides and sets of oligonucleotides set forth herein with respect to the aforementioned methods also are applicable to those same aspects of the kit described herein. Many such reagents are described herein or otherwise known in the art and commercially available. Examples of suitable reagents for inclusion in the kit (in addition to the oligonucleotides described herein) include conventional reagents employed in nucleic acid amplification reactions, such as, for example, one or more enzymes having polymerase activity, enzyme cofactors (such as magnesium or nicotinamide adenine dinucleotide (NAD)), salts, buffers, deoxyribonucleotide, or ribonucleotide triphosphates (dNTPs / rNTPs; for example, deoxyadenosine triphosphate, deoxyguanosine triphosphate, deoxycytidine triphosphate, and deoxythymidine triphosphate) blocking agents, labeling agents, and the like. Other reagents used in amplification reactions include nicking enzymes, single-strand binding proteins, helicases, resolvases, and the like. In some embodiments, kits of the present invention comprise one or more of a calibrator, a positive control, a negative control and / or an internal control.

[0075] The kit may comprise instructions for using the amplification reagents and oligonucleotides described herein, e.g., for processing the test sample, extracting nucleic acid molecules, and / or performing the test; and for interpreting the results obtained. The instructions may be printed or provided electronically (e.g., DVD, CD, or available for viewing or acquiring via internet resources).

[0076] The kit may be supplied in a solid (e.g., lyophilized) or liquid form. The various components of the kit of the present disclosure may optionally be contained within different containers (e.g., vial, ampoule, test tube, flask, or bottle) for each individual component (e.g., amplification oligonucleotides, probe oligonucleotides, or buffer). Each component will generally be suitable as aliquoted in its respective container or provided in a concentrated form. Other containers suitable for conducting certain steps of the amplification / detection assay may also be provided. The individual containers are preferably maintained in close confinement for commercial sale. The kit may further comprise a swab for obtaining a biological sample. In some embodiments, the kit comprises reagents for gaining access to and / or extracting / isolating nucleic acid from a biological sample.

[0077] Systems

[0078] In some embodiments, the present invention provides systems for amplifying and detecting CMV or EBV in a sample. In some embodiments, the system performs sample preparation, amplification and detection of target nucleic acid sequences using, for example, real-time PCR. In some embodiments, a system operator interacts with a touch screen to select icons, buttons, menu commands, and other screen elements. In some embodiments, a monitor displays a user interface of the system and accepts on-screen / touchscreen selections from the operator. In some embodiments, optional components include a keyboard, a pointing mouse, system information, software function and updates and online help. In some embodiments, the two or more sample preparation steps of the system are executed automatically. In some embodiments, the system comprises sample preparation protocols that are shared between plasma and urine specimens.

[0079] In some embodiments, systems of the present invention perform continuous and random-access sample processing using multiple sample processors and thermocyclers in parallel. In some embodiments, an individual sample occupies a single sample lane followed by an associated PCR amplification-detection lane. In some embodiments, parallel lanes provide access to 300 or more samples per day in 2-8 hours from first aspiration to test result. In some embodiments, the system’s capacity incorporates all required reagents in integrated reaction units (IRUs). Samples and controls for a walk away time of 2-4 hours with continuous operator access to remove or replenish amplification reagents, ancillary reagents and commodities without interfering with system processing. In some embodiments, the system comprises a plurality of Assay Processing Units configured for sample preparation and amplification-detection.

[0080] In some embodiments, the system provides individual fluorescent dye intensity changes over PCR cycles. In some embodiments, data reduction comprises a maxRatio algorithm. The maxRatio algorithm evaluates 2 features of assay results: 1) When the Reduced Fluorescence Intensity curves crosses a threshold, the algorithm determines whether a signal corresponds to PCR amplification of the true target’s existence (i.e., whether the PCR is reactive) based on maxRatio’s “peak PCR efficiency” analysis; 2) For rising signals, the algorithm determines whether there is an interference to the PCR (e.g., inhibition of noise) that potentially affects the accuracy of the assay result based on the maxRatio’s “curve shape” analysis. The cycle number (CN) corresponding to the peak maxRatio value (MR) is identified as the Fractional Cycle Number (FCN). Cycle threshold (CT) and FCN are inversely proportional to the Log of the target concentration in the sample prior to amplification. The algorithm provides an additional confirmation of the data’s quality and provides confidence for the reported results.

[0081] In some embodiments, systems and kits of the present invention comprise positive controls, negative controls, internal controls (for example, rehydrated from a lypholized unit dose), and calibrators. In some embodiments, systems, kits and methods comprise reagents and components configured to remove PCR inhibitors from sample DNA extract.

[0082] EXPERIMENTAL EXAMPLES Example 1 - CMV whole blood assay

[0083] Table 1. CMV target primers and probes aFAM = CarboxyfluoresceinbBHQ1 = Black Hole Quencher 1. The 3 terminal (T) is part of the BHQ1 structure and is not complementary to thepProbe target sequence. Note Modified bases: pdU= 5-Propynyl dU, pdC=5-Propynyl dC

[0084] Table 2. Internal control primers and probes a Q670 = fluorophore Quasar 670. b BHQ2 = Black Hole Quencher 2. The 3' terminal (T) is part of the BHQ2 structure and is not complementary to the IC target sequence.

[0085] Note Modified bases: pdU= 5-Propynyl dU, pdC=5-Propynyl dC

[0086] RT-PCR Mastermix (MMX) reagent formulation

[0087] The PCJR Mastermix reagent formulation is compatible with lyophilization and enables fast PCR (completion of cycling in less than one hour). Mastrermix Reagent was prepared by combining KAPA 2G DNA Polymerase, MT9 complex excipient, and CMV oligonucleotide mix (consisting of molecular biology grade water, PCR buffer components, dNTPs, oligonucleotide primers and probes, and Cal Fluor Red 610 passive reference dye). The Mastermix reagent formulation is shown in Table 3.

[0088] Table 3. RT-PCR CMV Mastermix reagent formulation

[0089] *KAPA2G Polymerase is an engineered enzyme for higher processivity and speed through directed evolution. It provides significantly faster extension rates than wild-type Taq DNA polymerase. It has a highly processive 5’-3’ DNA polymerase but lacking 3 ’-5’ exonuclease activity.

[0090] Activator solution was prepared by mixing molecular biology grade water, ProCiin 950, magnesium chloride, potassium chloride and tetramethyl ammonium chloride (TMAC). The activator solution provides the reaction with the necessary salts to activate PCR enzymes and establish an ionic strength environment that is conducive to efficient amplification. The Activator Reagent Formulation is shown in Table 4.

[0091] Table 4. Activation reagent formulation

[0092] The internal control contains the pumpkin hydroxypyruvate reductase cDNA sequences, which is unrelated to CMV target sequences. Internal Control / Proteinase K Reagent is prepared by mixing trehalose with water, 2X Internal Control Bulk (consisting of Internal Control plasmid DNA and Tris-based buffer containing salmon testes DNA), Proteinase K and an IC Diluent (consisting of Tris-based buffer containing salmon testes DNA). The formulation of the internal control is shown in Table 5.

[0093] Table 5. Internal control formulation PCR cycling conditions used in the CMV WB assay are provided in Table 6.

[0094] Table 6. PCR cycling conditions

[0095] CMV whole blood sample preparation

[0096] Whole blood specimen, calibrator and control samples were first pretreated with Proteinase K rehydrated from a lyophilized unit dose. The samples, along with Proteinase K, Internal Control (IC), and lysis solution were pipetted by instrument into an IRU (Integrated Reaction Unit) well for an initial pretreatment incubation. Once incubation was complete, the instrument transferred the volume of the pretreatment well to a second lysis well containing magnetic silica microparticles (Alinity m Microparticles 1) and Alinity m Lysis Solution. Alinity m Lysis Solution inactivates Proteinase K, lyses cells and denatures proteins in the sample, and promotes nucleic acid binding to the magnetic microparticles during the lysis incubation step. At the conclusion of the lysis step, magnetic microparticles with bound sample DNA were captured by a magnetic plunger sheathed with a disposable plastic sleeve. The magnetic microparticles were then successively transferred to wells within the IRU containing a series of wash solutions with ethanol. After wash steps were complete, the magnetic silica microparticles were captured by the plunger magnet and transferred into an elution well within the IRU where the purified DNA is eluted from the microparticles into Alinity m Elution Buffer 1. Upon completion of the sample preparation process, the purified DNA eluate and unit-dose Alinity m EBV WB real-time PCR reagents were combined by the Alinity m System and transferred to a real-time PCR reaction vessel (RV). A layer of Alinity m Vapor Barrier Solution was then automatically added to the RV. The RV was then capped and transferred to a thermal cycling position on the APU for amplification / detection.

[0097] Table 7. Workflow of CMV whole blood sample preparation

[0098] Example 2 - EBV whole blood assay

[0099] Table 8. EBV target primers and probes aFAM = CarboxyfluoresceinbBHQ1 = Black Hole Quencher 1. The 3’ terminal (T) is part of the BHQ1 structure and is not complementary to the Probe target sequence. Note Modified bases: pdU= 5-Propynyl dU, pdC=5-Propynyl dC

[0100] Table 9. Internal control primers and probes a Q670 = fluorophore Quasar 670. b BHQ2 = Black Hole Quencher 2. The 3' terminal (T) is part of the BHQ2 structure and is not complementary to the IC target sequence.

[0101] RT-PCR EBV Mastermix reagent formulation The PCR Mastermix reagent formulation is compatible with lyophilization and enables fast PCR (completion of cycling in less than one hour). Mastermix reagent was prepared by combining KAPA 2G DNA Polymerase, MT9 complex excipient, and EBV oligonucleotide mix (consisting of molecular biology grade water, PCR buffer components, dNTPs, oligonucleotide primers and probes, and Cal Fluor Red 610 passive reference dye). The formulation of the Mastermix Reagent is shown in Table 10. Table 10. PCR EBV Mastermix reagent formulation *KAPA2G Polymerase is an engineered enzyme for higher processivity and speed through directed evolution. It offers significantly faster extension rates than wild-type Taq DNA polymerase. It has a highly processive 5’-3’ DNA polymerase but lacking 3 ’-5’ exonuclease activity.

[0102] Activation Reagent was prepared by mixing molecular biology grade water, ProCiin 950, magnesium chloride, potassium chloride and tetramethyl ammonium chloride (TMAC). The activator solution provides reaction with the necessary salts to activate PCR enzymes and establish an ionic strength environment that is conducive to efficient amplification. The formulation of the Activator Reagent Formulation is shown in Table 11.

[0103] Table 11. Activation reagent formulation

[0104] The EBV Internal Control / Proteinase K Reagent is prepared by mixing trehalose with water, 2X Internal Control Bulk (consisting of Internal Control plasmid DNA and Tris-based buffer containing salmon testes DNA), Proteinase K and an IC Diluent (consisting of Trisbased buffer containing salmon testes DNA). The Internal Control Formulation is shown in Table 12.

[0105] Table 12. Internal control formulation

[0106] PCR cycling conditions used by the Alinity m EBV assay are shown in Table 13. Table 13. PCR cycling conditions

[0107] EBV whole blood sample preparation

[0108] Whole blood specimen, calibrator and comparator samples were first pretreated with Proteinase K rehydrated from a lyophilized unit dose. The samples, along with Proteinase K, Internal Control (IC), and lysis solution were pipetted by instrument into an IRU (Integrated Reaction Unit) well for an initial pretreatment incubation. Once incubation was complete, the instrument transferred the entire volume of the pretreatment well to a second lysis well containing magnetic silica microparticles (Alinity m Microparticles 1) and Alinity m Lysis Solution. Alinity m Lysis Solution inactivates the Proteinase K, lyses cells and denatures proteins in the sample, and promotes nucleic acid binding to the magnetic microparticles during the lysis incubation step. At the conclusion of the lysis step, magnetic microparticles with bound sample DNA were captured by a magnetic plunger sheathed with a disposable plastic sleeve. The magnetic microparticles were then successively transferred to wells within the IRU containing a series of wash solutions with ethanol. After the wash steps were complete, the magnetic silica microparticles were captured by the plunger magnet and transferred into an elution well within the IRU where the purified DNA was eluted from the microparticles into Alinity m Elution Buffer 1.

[0109] Upon completion of the sample preparation process, the purified DNA eluate and unit-dose Alinity m EBV WB real-time PCR reagents were combined by the Alinity m System and transferred to a real-time PCR reaction vessel (RV). A layer of Alinity m Vapor Barrier Solution was then automatically added to the RV. The RV was then capped and transferred to a thermal cycling position on the APU for amplification / detection.

[0110] Table 14. Workflow of EBV WB sample preparation process

[0111] Example 3 - Pretreatment with GITC and PK

[0112] Fresh whole blood samples were collected from 5 control healthy donors to test pretreatment with and without GITC pretreatment. 200uL of GITC was added to the pretreatment well (final GITC concentration 1.88M). Samples were tested with and without 0.8U and 1.2U of PK. 300uL of whole blood sample was aspirated in each case. Whole blood samples were also tested on m2000 EBV WB assay for comparison. Samples were not spiked with exogenous EBV virus or DNA to test detection of endogenous donor EBV as a more challenging target than spiked material.

[0113] Table 15. Test conditions

[0114] Table 16. shows the number of detected / valid replicates for 5 normal donors tested on the m2000 platform compared to 4 different sample preparation conditions on the Alinity mEBV whole blood (WB) platform.

[0115] Table 16. Detection of endogenous EBY in normal donors Nine replicates per donor were tested on the m2000 system and 10 replicates were tested per donor on the Alinity system for each condition. Condition 3 comprising GITC pretreatment with 0.8U PK had the highest detection on Alinity and matched m2000 detection. This detection was significantly higher than pretreatment with no PK. Higher PK levels had a similar if lower level of detection, but also had 2 fluorescence errors which could indicate that there were contaminants in the eluate (e.g., microparticle carryover, and / or whole blood proteins). Extraction without pretreatment, or preteatment with no PK, had lower overall detection of EBV. Example 4 - Pretreatment GITC concentration

[0116] Specifications conditions were generated to add different volumes of GITC to the pretreatment well. In one condition lOOuL of diluent (H2O) was added to the pretreatment well together with GITC. The final GITC concentration was calculated based on the addition of 150uL of a whole blood sample to the pretreatment well. AMP plates with / without 0.4U PK were used with each condition. The MMx formulation, sample prep and PCR conditions were identical. Samples tested comprised inactivated EBV virus in whole blood. Table 17. Shows the tested conditions.

[0117] Table 17. Conditions tested Table 18. shows the mean and standard deviation (SD) for Target FAM and IC Q670 Ct and MR for each condition tested. Increasing GITC concentration in pretreatment trended to early FAM Cts. The highest GITC level tested had increased SD indicating inconsistent target recovery. Addition of 0.4U of PK further decreased sample variability at 2.69M GITC and also has earlier IC Ct.

[0118] Table 18. Target and internal control (IC) Cts and MR

[0119] Table 19 shows the target FAM and IC Q670 Ct for each condition tested. For FAM target, increasing GITC concentration in pretreatment trended to early FAM Cts, but increased variability at higher levels. For IC increasing GITC levels also lead to earlier Cts up to 2.69M. The addition of PK further decreased the IC Ct. These data indicate that 2.59 GITC + 0.4 U PK had early target, and IC Cts and least variability. Table 19. Target and IC Cts

[0120] Example 5 - Pretreatment conditions

[0121] Fresh whole blood was collected from 40 normal donors. Blood from each donor was divided into 2 pools. One pool was tested for endogenous EBV, and one pool was spiked to 1E3 lU / mL with EBV inactivated virus. Samples were tested in a with different sample input and pretreatment conditions i.e., No PK, with PK, with PK and PEG, with PK, PEG and SDS.

[0122] Table 20. Pretreatment conditions Addition of PEG did not improve target recovery. (Table 21.) Addition of SDS in pretreatment did not improve target recovery and further increased error rates. (Table 21.) Error rates decreased when the ratio of sample to lysis volume in pretreatment decreased. (Table 22.) Addition of PK improved target recovery. (Table 21.) Table 21. 1E3 lU / mL EBV inactivated virus panel test results

[0123] Table 22. Ratio of sample volume to lysis volume effects on error rates

[0124] It is understood that the foregoing detailed description and accompanying examples are merely illustrative and are not to be taken as limitations upon the scope of the disclosure, which is defined solely by the appended claims and their equivalents. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art and may be made without departing from the spirit and scope thereof.

Claims

CLAIMSWhat is claimed is:

1. A method for detecting CMV and / or EB V in a whole blood sample, comprising: a) incubation of said sample with Proteinase K in a pretreatment well at 50°C; b) transfer of said incubated sample of step a) to a lysis well comprising lysis buffer and magnetic microparticles followed by mixing and incubation at 68°C in said well; c) transfer of said magnetic microparticles of step b) to a first wash well comprising lysis buffer; d) transfer of said magnetic microparticles of step c) to a second wash well and washing said microparticles with a mixture of ethanol and water; e) capture of said magnetic microparticles of step d) with a plunger magnet and evaporation of said ethanol; f) transfer of said magnetic microparticles of step e) to an elution well comprising an elution buffer; g) elution of purified DNA from said magnetic microparticles of step f) at 72°C; h) transfer of said purified DNA to a reaction vessel comprising an activator, one or more amplification primers, one or more probes comprising a detectable label, and one or more reagents for amplification; i) thermocycling said reaction vessel to generate a signal from said detectable label; and j) detecting said CMV and / or EBV by measuring said signal from said detectable label.

2. The method of claim 1, wherein said one or more amplification primers comprise one or more of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 5.

3. The method of claim 1, wherein said one or more amplification primers comprise one or more of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 14.

4. The method of claim 1, wherein said one or more probes comprise one or more of SEQ ID NO: 3 and SEQ ID NO: 6.

5. The method of claim 1, wherein said one or more probes comprise one or more of SEQ ID NO: 12 and SEQ ID NO: 15.

6. A system, comprising: a) a pipettor; b) an integrated reaction unit (IRU) comprising one or more lanes comprising in linear order of sample transfer:1) a pretreatment well comprising Proteinase K and lysis solution at 50°C and an internal control;2) a lysis well comprising lysis buffer and silica magnetic microparticles at 68°C and a magnetic microparticle transfer plunger sheathed with a disposable plastic sheath;3) a wash well comprising lysis buffer;4) a first wash well comprising ethanol and water;5) a second wash well comprising ethanol and water;6) an elution well comprising an elution buffer at 72°C; and7) a PCR reaction vessel comprising activator formulation, Mastermix and one or more CMV or EBV primer pairs and probes; c) a thermocycler: and d) a fluorescence detector.

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