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WO2025049450A1 - Dosages pour détecter un virus bk (bkv) - Google Patents

Dosages pour détecter un virus bk (bkv) Download PDF

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Publication number
WO2025049450A1
WO2025049450A1 PCT/US2024/044000 US2024044000W WO2025049450A1 WO 2025049450 A1 WO2025049450 A1 WO 2025049450A1 US 2024044000 W US2024044000 W US 2024044000W WO 2025049450 A1 WO2025049450 A1 WO 2025049450A1
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Prior art keywords
bkv
amplification
microparticles
seq
well
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Dan R. TOOLSIE
Shihai Huang
Masakuni Mark SASAKI
Charles Ingersoll
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Abbott Molecular Inc
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Abbott Molecular Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

  • BKV BK virus
  • 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 BK virus (BKV) may occur within months after transplantation.
  • BKV infections may be managed by decreasing immunosuppression. Accordingly, methods, compositions, kits and systems for detecting nucleic acid sequences from BKV in samples including, for example, plasma, whole blood and urine samples are needed.
  • 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).
  • the set of oligonucleotides is for polymerase amplification and detection of BKV in a sample.
  • the reagents comprise a group of oligonucleotides comprising one or more sets of oligonucleotides.
  • the present invention provides dual-target PCR primer and probe sets for each of BKV nucleic acid sequences.
  • the present in invention provides methods for detecting BKV in a plasma or urine sample 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, 55 pL +/- 20% CuTi microparticles, and 50 pL +/- 20% elution buffer; pretreatment comprising addition of 500pL +/- 20% of a sample to a lysis well at 72°C +/- 20%; lysis comprising transfer of the pretreated sample to a lysis well comprising 1500pL +/- 20% lysis buffer and 3x CuTi microparticles with mixing by a magnetic plunger sheathed with a disposable plastic sheath during incubation; washing comprising transfer of the CuTi microparticles to a wash well comprising 900pL +/- 20% lysis buffer; transfer of the CuTi microparticles to 2 successive washes with 1400pL +/- 20% H2O and lOOpL +/
  • the present in invention provides methods for detecting BKV in a plasma or urine sample 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, CuTi microparticles, and an elution buffer; pretreatment comprising addition of the sample to a lysis well at 72°C; lysis comprising transfer of the pretreated sample to a lysis well comprising lysis buffer and CuTi microparticles with mixing by a magnetic plunger sheathed with a disposable plastic sheath during incubation; washing comprising transfer of the CuTi microparticles to a wash well comprising lysis buffer; transfer of the CuTi microparticles to 2 successive washes with H2O; capture of the CuTi microparticles by the plunger magnet; transfer of the CuTi microparticles to an elution well comprising elution buffer with elution of purified DNA at 80 °C; transfer of the e
  • the present invention provides a method for detecting BKV in a plasma or urine sample, comprising: a) transfer of the sample to a lysis well comprising lysis buffer and CuTi microparticles followed by mixing and incubation at 72°C in the well; b) transfer of the CuTi microparticles of step a) to a first wash well comprising lysis buffer; c) transfer of the CuTi microparticles of step b) to a second wash well and washing the microparticles with water; d) capture of the magnetic microparticles of step c) with a plunger magnet; e) transfer of the magnetic microparticles of step d) to an elution well comprising an elution buffer; f) elution of purified DNA from the CuTi microparticles of step e) at 80°C; g) transfer of the purified DNA to a reaction vessel comprising an activator, one or more amplification primers, one or more probes comprising
  • the one or more amplification primers comprise one or more of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6, and SEQ ID NO: 7.
  • the one or more probes comprise one or more of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 8, and SEQ ID NO: 9.
  • 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 lysis well comprising an internal control, lysis buffer, CuTi microparticles at 72°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 water: 5) a second wash well comprising water;
  • IRU integrated reaction unit
  • an elution well comprising elution buffer 2 at 80°C; and 7) a PCR reaction vessel comprising activator formulation, Mastermix, and one or more BKV primer pairs and probes; c) a thermocycler: and d) a fluorescence detector.
  • the disclosure also provides methods for detecting BKV in a sample.
  • the sample may comprise a nasal swab or brush, saliva, mucus, blood, serum, plasma, urine or feces.
  • kits for detecting BKV 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.
  • kits for detecting BKV 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.
  • Figure 1 shows the Alinity m BKV dual target VP2/VP3 and small-t antigen assay design.
  • Figures 2A and 2B show BKV in plasma and urine sensitivity results. Each panel member was tested in 10 replicates per day on 3 days with each of 3 AMP kit lots.
  • Figures 3 A and 3B show a BKV quantification range from 50 to IxlO 19 lU/mL (1.7 to 9 Log lU/mL). 24 replicates were tested at each target level.
  • Figures 4A and 4B show BKV in plasma and urine precision results. Each panel member was tested in multiple replicates twice each day for 5 days in Alinity m Systems by 3 operators using 3 AMP kit lots.
  • Figures 5A and 5B show BKV urine specimen quantification and correlation with a comparator. 165 quantified fresh (never frozen) urine samples from transplant recipients were collected at 4 sites.
  • Figures 6A and 6B show BKV plasma specimen quantification and correlation with a comparator. 171 quantified fresh and frozen specimens were collected at 6 sites.
  • Figure 7 shows BKV urine specimen agreement vs. a comparator. 453 fresh (never frozen) specimens were collected from transplant patients at 4 sites.
  • Figure 8 shows BKV plasma specimen evaluation agreement vs. a comparator. 521 fresh and frozen plasma samples were collected at 6 sites.
  • Figure 9 shows BKV plasma sample specimen analytical sensitivity evaluation agreement vs. a comparator. Analytical sensitivity was assessed using a panel prepared by diluting a verification panel member to 40 lU/mL (1.60 Log lU/mL) and 50 lU/mL (1.70 Log lU/mL) in negative plasma.
  • Figure 10 shows BKV plasma sample specimen linearity. Linearity was evaluated with a commercially available BKV panel in plasma with concentration ranging from 2.00 Log lU/mL to 7.30 Log lU/mL (ExactDx, Ft. Worth, Tx) that was tested at 2 sites.
  • Figure 11 shows BKV plasma sample specimen precision. Precision was evaluated by testing panels ranging from 2.00 to 7.30 Log lU/mL across 5 separate runs over 5 days at two sites.
  • Figure 12 shows BKV plasma sample specimen regression and mean bias evaluated by comparing the Alinity m BKV IUO with the ELITech MGV Alert BKV run on the Abbott 7712000 using DNA extraction.
  • PPA 98.6%
  • NPA 91.7%. 4 samples not detected by ELITech were quantitated ⁇ LLOQ by Alinity m BKV
  • Figure 13 shows regression analysis of Figure 12 samples.
  • Figure 14 shows mean bias analysis of Figure 12samples.
  • Figure 16 shows regression analysis of Figure 15 samples.
  • Figure 17 shows mean bias analysis of Figure 15 samples.
  • Figure 18 shows BKV urine sample specimen regression and mean bias evaluated by comparing the Alinity m BKV IUO with the ELITech MGV Alert BKV run on the Abbott ///2000 using total nucleic acid (TNA) extraction.
  • PPA 96.6%
  • NPA 81.3%.
  • Figure 19 shows regression analysis of Figure 18 samples.
  • Figure 20 shows mean bias analysis of Figure 18 samples.
  • BKV BK virus
  • BKV The BK virus
  • BKV The BK virus
  • BKV is a polyomavirus with seroprevalence over 90% by 4 years of age.
  • Immunocompromised individuals such as transplant recipients and those with other acquired immune system deficiencies are at risk of complications due to BKV reactivation or new infections from BKV positive donors
  • BKV causes BKV-associated nephropathy (BKV AN) occurring in up to 10% of kidney transplant recipients and ureteral stenosis.
  • BKV-associated hemorrhagic cystitis BK-HC
  • BK-HC BKV-associated hemorrhagic cystitis
  • BK viruria and viremia precede the histologic diagnosis of polyomavirus- associated nephropathy (PVAN) by 12 weeks and 8 weeks, respectively, screening for BKV replication in urine and plasma I recommended at least every 3 months during the first 2 years after transplantation, or if allograft dysfunction occurs prompting an allograft biopsy for workup of BKV-associated kidney injury.
  • PVAN polyomavirus- associated nephropathy
  • BKV loads persisting at levels greater than 4 log geq/ml for 4 weeks or longer defines presumptive PVAN for which intervention is considered even in the absence of positive histologic findings.
  • BKV genomic variants may emerge that are associated with higher replicative capacity and more extensive cytopathic damage. Because of earlier manifestations, biweekly or monthly screening may be used during the first 6 months as a presumptive intervention strategy.
  • 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.
  • each intervening number there between with the same degree of precision is explicitly contemplated.
  • 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.
  • oligonucleotide 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).
  • nucleic acid and polynucleotide refer to a polymeric form of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA).
  • RNA and DNA 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • the set of oligonucleotides described herein may be used to amplify and detect one or more target BKV sequences in a sample.
  • 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.
  • 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 single-stranded or double-stranded.
  • the target BKV sequence may be within any portion of the BKV genome.
  • the set comprises a first amplification oligonucleotide, a second amplification oligonucleotide, and a probe oligonucleotide.
  • 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.
  • an oligonucleotide sequence as described herein may comprise one or more modified oligonucleotide.
  • any of the sequences listed which include internal spacers or modifications may be used without the modifications or spacers.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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).
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • the detectable label may be directly detected.
  • 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.
  • 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.
  • a fluorescein-family dye 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
  • fluorophores examples include, but are not limited to, FAMTM, CAL-FLUOR®, QUASAR®, HEXTM, JOETM, NEDTM, PET®, ROXTM, TAMRATM, TETTM, TEXAS RED®, and VIC®.
  • FAMTM FAMTM
  • CAL-FLUOR® QUASAR®
  • HEXTM JOETM
  • NEDTM NEDTM
  • PET® ROXTM
  • TAMRATM TAMRATM
  • TETTM TEXAS RED®
  • VIC® VIC®
  • Methods for labeling oligonucleotides, such as probes are well-known in the art and described in, e.g., L. J. Kricka, Ann. Clin.
  • any one or more of the oligonucleotides described herein may also comprise a quencher moiety.
  • a detectable label e.g., a fluorophore
  • quencher moiety prevents detection of a signal (e.g., fluorescence) from the detectable label.
  • a signal e.g., fluorescence
  • 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.
  • the oligonucleotide probe may comprise a FAM fluorophore, CAL-FLUOR®, or QUASAR fluorophore and a BHQ-1 or BHQ-2 quencher.
  • the present disclosure provides a method for detecting BKV 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 BKV nucleic acid sequences present in the sample; hybridizing one or more of the oligonucleotide probes to one or more amplified target BKV nucleic acid sequences; and detecting hybridization of the one or more probe oligonucleotide sequences to the one or more amplified BKV 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.
  • 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).
  • 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.
  • 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.
  • a 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.
  • 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.
  • amplification conditions 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.
  • Amplifying a BKV nucleic acid sequence in the sample can be performed using any suitable nucleic acid sequence amplification method known in the art.
  • the amplification includes, but is not limited to, polymerase chain reaction (PCR), reversetranscriptase 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 (HD A), Loop mediated amplification (LAMP), Recombinase-Polymerase Amplification (RPA), and ligase chain reaction (LCR).
  • PCR polymerase chain reaction
  • RT-PCR reversetranscriptase PCR
  • TMA transcription-mediated amplification
  • NASBA nucleic acid sequence-based amplification
  • SDA strand displacement amplification
  • amplification of BKV nucleic acid sequences is performed using isothermal amplification(e.g., RPA or NEAR). In some embodiments, amplification and detection of BKV nucleic acid sequences is performed using a point of care device (e.g., the ID NOW system (Abbott)).
  • a point of care device e.g., the ID NOW system (Abbott)
  • amplification of BKV 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 double-stranded 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'.
  • m2000rt REALTLWETM PCR system (Abbott Molecular, Inc., Des Plaines, IL); CFX Real-Time PCR Detection Systems (Bio-Rad Laboratories, Inc., Hercules, CA); and TAQMANTM Real-Time PCR System (ThermoFisher Scientific, Waltham, MA)), any of which can be employed in the methods described herein.
  • sources e.g., m2000rt REALTLWETM PCR system (Abbott Molecular, Inc., Des Plaines, IL); CFX Real-Time PCR Detection Systems (Bio-Rad Laboratories, Inc., Hercules, CA); and TAQMANTM Real-Time PCR System (ThermoFisher Scientific, Waltham, MA)), any of which can be employed in the methods described herein.
  • 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.
  • nicking and extension amplification may include, for example, reactions that use only two amplification oligonucleotides, one or two nicking enzymes, and a polymerase, under isothermal conditions.
  • a target nucleic acid sequence having a sense and antisense strand
  • a pair of amplification oligonucleotides 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.
  • 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.
  • amplification of BKV 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.
  • RPA Recombinase-Polymerase Amplification
  • a recombinase agent is contacted with first and second amplification oligonucleotides to form nucleoproteins.
  • 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.
  • suitable recombinase agents include the E. 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-5, 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.
  • RPA Reactive protein kinase
  • ATP ATP
  • ATP analog ATP
  • ATP-P-S ATP-P-S
  • ddATP phosphate
  • 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.
  • the disclosed method may further comprise hybridizing one or more of the probe oligonucleotide sequences disclosed herein to the one or more amplified target BKV 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 BKV nucleic acid sequences and the presence of BKV in the sample, and (ii) the absence of signals indicates the absence of BKV 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.
  • a subject identified according to the methods described herein as having BKV may be treated, monitored (e.g., for the presence of a BKV nucleic acid determined in a sample from the subject), treated and monitored, and/or monitored and treated using routine techniques known in the art.
  • the methods described herein further include treating the subject when the presence of BKV nucleic acid is determined in one or more samples obtained from the subject using the present methods.
  • the disclosure also provides a kit for amplifying and detecting BKV in a sample.
  • the kit comprises at least one oligonucleotide as described herein.
  • 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 BKV. 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.
  • suitable reagents for inclusion in the kit 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.
  • enzyme cofactors such as magnesium or nicotinamide adenine dinucleotide (NAD)
  • NAD nicotinamide adenine dinucleotide
  • salts such as magnesium or nicotinamide adenine dinucleotide (NAD)
  • NAD nic
  • kits of the present invention comprise one or more of a calibrator, a positive control, a negative control and/or an internal control.
  • Activation Reagent was prepared by mixing molecular biology grade water, ProCiin 950, magnesium chloride, and tetramethyl ammonium chloride (TMAC). The activator solution provides reactions with the necessary salts to activate PCR enzymes and establish an ionic strength environment that is conducive to efficient amplification.
  • TMAC tetramethyl ammonium chloride

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Abstract

L'invention concerne des procédés, des compositions, des kits et des systèmes pour détecter des séquences d'acide nucléique cibles issues du virus BK (BKV).
PCT/US2024/044000 2023-08-28 2024-08-27 Dosages pour détecter un virus bk (bkv) Pending WO2025049450A1 (fr)

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