[go: up one dir, main page]

WO2024030985A1 - Dosages pour la détection virale de la variole du singe - Google Patents

Dosages pour la détection virale de la variole du singe Download PDF

Info

Publication number
WO2024030985A1
WO2024030985A1 PCT/US2023/071560 US2023071560W WO2024030985A1 WO 2024030985 A1 WO2024030985 A1 WO 2024030985A1 US 2023071560 W US2023071560 W US 2023071560W WO 2024030985 A1 WO2024030985 A1 WO 2024030985A1
Authority
WO
WIPO (PCT)
Prior art keywords
nucleic acid
amplification
oligonucleotide
seq
sample
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2023/071560
Other languages
English (en)
Inventor
Gavin Cloherty
Ka-Cheung Luk
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Abbott Laboratories
Original Assignee
Abbott Laboratories
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Abbott Laboratories filed Critical Abbott Laboratories
Priority to EP23764783.9A priority Critical patent/EP4565720A1/fr
Publication of WO2024030985A1 publication Critical patent/WO2024030985A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • 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
    • C12Q2545/00—Reactions characterised by their quantitative nature
    • C12Q2545/10—Reactions characterised by their quantitative nature the purpose being quantitative analysis
    • C12Q2545/101—Reactions characterised by their quantitative nature the purpose being quantitative analysis with an internal standard/control
    • 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
    • C12Q2561/00—Nucleic acid detection characterised by assay method
    • C12Q2561/113—Real time assay

Definitions

  • the present disclosure relates to methods for amplifying target nucleic acid sequences from monkeypox virus and detecting monkeypox infection.
  • Monkeypox is a viral zoonotic disease caused by monkeypox virus (MPXV), a member of the Orthopoxvirus genus in the family Poxviridae, endemic in West and Central Africa. With the eradication of smallpox and subsequent cessation of smallpox vaccination, monkeypox has emerged as the most important Orthopoxvirus for public health. As of July of 2022, more than 16,000 cases of monkeypox have been reported across more than 70 countries. Severe cases can occur, especially in children, pregnant women, or people with suppressed immune systems, with a fatality ratio of approximately 3-10%. The recent monkeypox outbreaks demonstrated the ability of the virus to exploit new hosts and move globally and quickly.
  • MPXV monkeypox virus
  • Monkeypox viruses are divided into two clades or branches, having unique genomic signatures and cause disease of different severity. Viruses sequenced in the current outbreak broadly match those in the typically milder clade. However, methods for detecting the totality of the known genomic signatures of monkeypox viruses are vital for accurately monitoring and detecting present and future monkeypox outbreaks.
  • the disclosure provides a set of reagents, including oligonucleotides, for amplifying and detecting monkeypox virus in a sample.
  • the set can comprise a first amplification oligonucleotide, a second amplification oligonucleotide, and a probe oligonucleotide.
  • the set may comprise a first amplification oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 1, a second amplification oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 2, and a probe oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 3; or a first amplification oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 4, a second amplification oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 5, and a probe oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 6; or a combination thereof.
  • the probe oligonucleotides may comprise a detectable label.
  • the detectable label is a fluorophore.
  • the probe oligonucleotides may comprise a quencher moiety.
  • the disclosure also provides methods for detecting monkeypox virus in a sample comprising: contacting a sample with the set of oligonucleotides disclosed herein and reagents for amplification; amplifying one or more target monkeypox virus nucleic acid sequences present in the sample; hybridizing one or more of the oligonucleotide probes to one or more amplified target monkeypox virus nucleic acid sequences; and detecting hybridization of the one or more probe oligonucleotide sequences to the one or more amplified monkeypox virus target nucleic acid sequences by measuring a signal from the detectable labels.
  • the sample comprises a blood product, an oropharyngeal swab, a rectal swab, a genital swab, saliva, urine, or semen. In some embodiments, the sample comprises a skin lesion specimen.
  • the reagents for amplification comprise one or more or each of a nicking enzyme, a polymerase, a single-strand binding protein, a recombinase, a helicase, a resolvase, an enzyme cofactor, a buffer, deoxyribonucleotide or ribonucleotide triphosphates, or combinations thereof.
  • the presence of one or more signals from the detectable label indicates hybridization of the one or more probe oligonucleotides to the one or more amplified monkeypox virus target nucleic acid sequences.
  • the disclosure provides a system for detecting monkeypox virus in a sample.
  • the system comprises a set of oligonucleotides, as disclosed herein; one or more nucleic acid amplification reagents; and a nucleic acid amplification reaction system configured to amplify one or more target monkeypox virus nucleic acid sequences in the sample.
  • the nucleic acid amplification reaction system is further configured to facilitate hybridization of one or more of the probe oligonucleotides to amplified target monkeypox virus nucleic acid sequences.
  • the nucleic acid amplification reaction system is further configured to detect hybridization of one or more of the probe oligonucleotides to amplified target monkeypox virus nucleic acid sequences. In some embodiments, the nucleic acid amplification reaction system is further configured to facilitate hybridization of one or more of the probe oligonucleotides to amplified target monkeypox virus nucleic acid sequences and detect hybridization of one or more of the probe oligonucleotides to amplified target monkeypox virus nucleic acid sequences. In some embodiments, the nucleic acid amplification reaction system is further configured to measure a signal from one or more detectable label.
  • the system further comprises an internal control sequence, primer and probe oligonucleotide sequences for amplifying and detecting an internal control (IC) sequence, or a combination thereof.
  • IC internal control
  • the one or more nucleic acid amplification reagents is selected from the group consisting of: a nicking enzyme, a polymerase, a single-strand binding protein, a recombinase, a helicase, a resolvase, an enzyme cofactor, a buffer, deoxyribonucleotide or ribonucleotide triphosphates, and combinations thereof.
  • the sample comprises a blood product, an oropharyngeal swab, a rectal swab, a genital swab, saliva, urine, or semen.
  • the sample comprises a skin lesion specimen.
  • the disclosure also provides a reaction mixture comprising one or more nucleic acid amplification reagents; a set of oligonucleotides as disclosed herein; and a sample suspected of containing monkeypox virus.
  • the reaction mixture further comprises an internal control sequence, primer and probe oligonucleotide sequences for amplifying and detecting an internal control (IC) sequence, or a combination thereof.
  • IC internal control
  • the one or more nucleic acid amplification reagents is selected from the group consisting of: a nicking enzyme, a polymerase, a single-strand binding protein, a recombinase, a helicase, a resolvase, an enzyme cofactor, a buffer, deoxyribonucleotide or ribonucleotide triphosphates, and combinations thereof.
  • the sample comprises a blood product, an oropharyngeal swab, a rectal swab, a genital swab, saliva, urine, or semen.
  • the sample comprises a skin lesion specimen.
  • the disclosure further provides kits for detecting monkeypox virus in a sample.
  • the set may comprise a first amplification oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to any of SEQ ID NOs: 7-9, a second amplification oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to any of SEQ ID NOs: 10-11 and a probe oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to any of SEQ ID NOs: 12-15; or a first amplification oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to any of SEQ ID NOs: 16-20, a second amplification oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to any of SEQ ID NOs: 21-25, and a probe oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to any of SEQ ID NOs: 26-
  • the probe oligonucleotides may comprise a detectable label.
  • the detectable label is a fluorophore.
  • the probe oligonucleotides may comprise a quencher moiety.
  • the disclosure also provides methods for detecting monkeypox virus in a sample comprising: contacting a sample with the set of oligonucleotides disclosed herein and reagents for amplification; amplifying one or more target monkeypox virus nucleic acid sequences present in the sample; hybridizing one or more of the oligonucleotide probes to one or more amplified target monkeypox virus nucleic acid sequences; and detecting hybridization of the one or more probe oligonucleotide sequences to the one or more amplified monkeypox virus target nucleic acid sequences by measuring a signal from the detectable labels.
  • the sample comprises a blood product, an oropharyngeal swab, a rectal swab, a genital swab, saliva, urine, or semen. In some embodiments, the sample comprises a skin lesion specimen.
  • the reagents for amplification comprise one or more or each of a nicking enzyme, a polymerase, a single-strand binding protein, a recombinase, a helicase, a resolvase, an enzyme cofactor, a buffer, deoxyribonucleotide or ribonucleotide triphosphates, or combinations thereof.
  • the presence of one or more signals from the detectable label indicates hybridization of the one or more probe oligonucleotides to the one or more amplified monkeypox virus target nucleic acid sequences.
  • the disclosure provides a system for detecting monkeypox virus in a sample.
  • the system comprises a set of oligonucleotides, as disclosed herein; one or more nucleic acid amplification reagents; and a nucleic acid amplification reaction system configured to amplify one or more target monkeypox virus nucleic acid sequences in the sample.
  • the nucleic acid amplification reaction system is further configured to facilitate hybridization of one or more of the probe oligonucleotides to amplified target monkeypox virus nucleic acid sequences.
  • the nucleic acid amplification reaction system is further configured to detect hybridization of one or more of the probe oligonucleotides to amplified target monkeypox virus nucleic acid sequences. In some embodiments, the nucleic acid amplification reaction system is further configured to facilitate hybridization of one or more of the probe oligonucleotides to amplified target monkeypox vims nucleic acid sequences and detect hybridization of one or more of the probe oligonucleotides to amplified target monkeypox virus nucleic acid sequences. In some embodiments, the nucleic acid amplification reaction system is further configured to measure a signal from one or more detectable label.
  • the system further comprises an internal control sequence, primer and probe oligonucleotide sequences for amplifying and detecting one or more internal control (IC) sequences, or a combination thereof.
  • IC internal control
  • the one or more nucleic acid amplification reagents is selected from the group consisting of: a nicking enzyme, a polymerase, a single-strand binding protein, a recombinase, a helicase, a resolvase, an enzyme cofactor, a buffer, deoxyribonucleotide or ribonucleotide triphosphates, and combinations thereof.
  • the sample comprises a blood product, an oropharyngeal swab, a rectal swab, a genital swab, saliva, urine, or semen.
  • the sample comprises a skin lesion specimen.
  • the disclosure also provides a reaction mixture comprising one or more nucleic acid amplification reagents; a set of oligonucleotides as disclosed herein; and a sample suspected of containing monkeypox virus.
  • the reaction mixture further comprises an internal control sequence, primer and probe oligonucleotide sequences for amplifying and detecting one or more internal control (IC) sequences, or a combination thereof.
  • IC internal control
  • the one or more nucleic acid amplification reagents is selected from the group consisting of: a nicking enzyme, a polymerase, a single-strand binding protein, a recombinase, a helicase, a resolvase, an enzyme cofactor, a buffer, deoxyribonucleotide or ribonucleotide triphosphates, and combinations thereof.
  • the sample comprises a blood product, an oropharyngeal swab, a rectal swab, a genital swab, saliva, urine, or semen.
  • the sample comprises a skin lesion specimen.
  • the disclosure further provides kits for detecting monkeypox virus in a sample.
  • FIG. 1 is qPCR results for MPXV plasmid assay (left) and the internal control (right).
  • FIG. 2 is qPCR results for MPXV genomic DNA assay (left) and the internal control
  • FIG. 3A is a schematic of a dual target design to detect B7R and J2R genes of the MPXV genome.
  • FIG. 3B shows detection of MPXV and internal controls in an exemplary RT-PCR based method.
  • the present disclosure is predicated, at least in part, on the development of a collection of oligonucleotide sequences that facilitate rapid detection of known genomic signatures for monkeypox viruses.
  • Many current monkeypox nucleic acid assays are not specific as they cross-react with other orthopoxviral DNA.
  • the methods described herein do not cross react with other orthopox viral DNA (e.g., variola, cowpox, camelpox, taterapox, ectromelia virus and vaccinia).
  • the disclosed methods employ internal controls, for example, an endogenous cellular target control and an exogenous control, which validate the assays for sample adequacy, sample extraction and amplification efficiency.
  • 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.
  • first,” “second,” “third,” etc. may be used herein to describe various steps, elements, compositions, components, regions, layers, and/or sections, these steps, elements, compositions, components, regions, layers, and/or sections should not be limited by these terms, unless otherwise indicated. These terms are used to distinguish one step, element, composition, component, region, layer, and/or section from another step, element, composition, component, region, layer, and/or section. Terms such as “first,” “second,” and other numerical terms when used herein do not imply a sequence or order unless clearly indicated by the context. Thus, a first step, element, composition, component, region, layer, or section discussed herein could be termed a second step, element, composition, component, region, layer, or section without departing from technology.
  • amplifying or “amplification” in the context of nucleic acids refers to the production of multiple copies of a polynucleotide, or a portion of the polynucleotide, typically starting from a small amount of the polynucleotide (e.g., a single polynucleotide molecule), where the amplification products or amplicons are generally detectable. Amplification of polynucleotides encompasses a variety of chemical and enzymatic processes.
  • 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, which 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 oligonucleotides described herein may be used for nucleic acid amplification (e.g., primers) or as probes for nucleic acid hybridization and detection.
  • primer e.g., primers
  • primer sequence e.g., primer sequence
  • primer oligonucleotide e.g., primer sequence
  • amplification oligonucleotide refers 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).
  • suitable amplification conditions e.g., buffer, salt, temperature and pH
  • 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.
  • probe refers 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.
  • 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.
  • 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 the monkeypox virus) and/or at least one probe which can detect the target sequence or target nucleic acid.
  • a target sequence or target nucleic acid of interest e.g., a target sequence within the monkeypox virus
  • 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.
  • the set of oligonucleotides described herein may be used to amplify and detect one or more target monkeypox virus 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 monkeypox sequence may be within the gene encoding DNA polymerase (E9L) or envelope protein (B6R).
  • the set comprises a first amplification oligonucleotide, a second amplification oligonucleotide, and a probe oligonucleotide. In some embodiments, the set comprises two or more first amplification oligonucleotides, two or more second amplification oligonucleotides, and two or more probe oligonucleotides.
  • the set comprises a first amplification oligonucleotide comprising a nucleic acid sequence with at least 70% (e.g., 75%., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) similarity to SEQ ID NO: 1, a second amplification oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 2 and a probe oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 3.
  • a first amplification oligonucleotide comprising a nucleic acid sequence with at least 70% (e.g., 75%., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) similarity to SEQ ID NO: 1
  • the set comprises a first amplification oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 4, a second amplification oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 5, and a probe oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 6.
  • the set comprises a first amplification oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 1, a second amplification oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 2 a probe oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 3, a first amplification oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 4, a second amplification oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 5, and a probe oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 6.
  • the set comprises a first amplification oligonucleotide comprising a nucleic acid sequence with at least 70% (e.g., 75%., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) similarity to any of SEQ ID NOs: 7-9, a second amplification oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to any of SEQ ID NOs: 10-11 and a probe oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to any of SEQ ID NOs: 12-15; or a first amplification oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to any of SEQ ID NOs: 16-20, a second amplification oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to any of SEQ ID NOs: 16-20,
  • the set comprises a first amplification oligonucleotide comprising a nucleic acid sequence with at least 70% (e.g., 75%., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) similarity to SEQ ID NO: 7, a second amplification oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 10 and a probe oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 12; or a first amplification oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 16, a second amplification oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 21, and a probe oligonucleotide comprising a nucleic acid sequence with at
  • the set comprises a first amplification oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 7.
  • the set comprises a second amplification oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 10.
  • the set comprises a probe oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 12.
  • the set comprises a first amplification oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 16. In select embodiments, the set comprises a second amplification oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 21. In select embodiments, the set comprises a probe oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 26.
  • the set comprises a first amplification oligonucleotide comprising a nucleic acid sequence of any of SEQ ID NOs: 7-9, a second amplification oligonucleotide comprising a nucleic acid sequence of any of SEQ ID NOs: 10-11 and a probe oligonucleotide comprising a nucleic acid sequence of any of SEQ ID NOs: 12-15; or a first amplification oligonucleotide comprising a nucleic acid sequence of any of SEQ ID NOs: 16-20, a second amplification oligonucleotide comprising a nucleic acid sequence of any of SEQ ID NOs: 21-25, and a probe oligonucleotide comprising a nucleic acid sequence of any of SEQ ID NOs: 26-39; or a combination thereof.
  • the set of oligonucleotides for detecting monkeypox virus described above may further comprise primer and probe oligonucleotide sequences for amplifying and detecting one or more internal control (IC) sequences.
  • the internal controls may be any suitable non- monkeypox nucleic acid sequence, including, for example, a nucleic acid sequence encoding GAPDH, beta globulin, beta-globin, beta-actin, R18, or 16S RNA.
  • the internal control may comprise an endogenous cellular control target and an exogenous control target.
  • the internal control may be an endogenous human DNA sequence.
  • the internal control may comprise an DNA sequence derived or obtained from the hydroxypyruvate reductase gene of the pumpkin plant, Curcurbita pepo.
  • 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 bases.
  • 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 which does 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 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., FASTA3x, 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.
  • 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).
  • 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. 2. Detectable Label
  • 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.
  • each of the probe oligonucleotide sequences described herein comprise a detectable label.
  • detectable label 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.
  • 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,
  • 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.
  • the fluorescent dye may be conjugated to either end of the probe oligonucleotide, or to an internal site (e.g., within the oligonucleotide sequence).
  • the probe oligonucleotide sequence comprises a fluorescent dye at the 5 ’ end of the oligonucleotide.
  • the probe oligonucleotide sequence comprises a fluorescein-based dye at the 5’ end of the oligonucleotide.
  • the probe oligonucleotide sequence comprises a fluorescent dye at the 3’ end of the oligonucleotide.
  • the probe oligonucleotide sequence comprises a fluorescein-based dye at the 3’ end of the oligonucleotide.
  • any one or more of the oligonucleotides described herein may also comprise a quencher moiety.
  • a quencher moiety is any molecule or part of a molecule that decreases the light emitted by the detectable label (e.g., fluorophore) at the wavelength at which signal is measured or serves to shift the wavelength of light emitted by the detectable label (e.g., fluorophore).
  • the detectable label e.g., a fluorophore
  • quencher moiety prevents detection of a signal (e.g., fluorescence) from the detectable label.
  • 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®), 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 quencher moiety may be conjugated to either end of the probe oligonucleotide, or to an internal site (e.g., within the oligonucleotide sequence).
  • the probe oligonucleotide sequence comprises a quencher moiety at the 3 ’ end of the oligonucleotide.
  • the probe oligonucleotide sequence comprises a quencher moiety at the 5’ end of the oligonucleotide.
  • the quencher moiety is distant from the detectable label (e.g., at the opposite end of the oligonucleotide).
  • the internal control probe also desirably comprises a detectable label.
  • the internal control probe may comprise a different label than the probes used to detect monkeypox virus, which allows for simultaneous detection and differentiation of internal control and monkeypox virus-amplified products within the same reaction.
  • the internal control probe may also comprise a quencher moiety.
  • 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.
  • the present disclosure provides methods for detecting monkeypox in a sample.
  • the methods comprise one or more of: contacting a sample with one or more sets of oligonucleotides disclosed herein and reagents for amplification; amplifying one or more target monkeypox virus nucleic acid sequences present in the sample; hybridizing one or more of the oligonucleotide probes to one or more amplified target monkeypox virus nucleic acid sequences; and detecting hybridization of the one or more probe oligonucleotide sequences to the one or more amplified monkeypox 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., squirrels, mice, rats, non-human primates, or humans).
  • the subject is a human.
  • the sample may be obtained from any suitable biological source, such as, 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 is obtained from a blood product (e.g., whole blood, serum, plasma, or other blood source), an oropharyngeal swab, a rectal swab, a genital swab, saliva, urine, or semen.
  • a blood product e.g., whole blood, serum, plasma, or other blood source
  • an oropharyngeal swab e.g., whole blood, serum, plasma, or other blood source
  • rectal swab e.g., a rectal swab
  • a genital swab e.g., saliva, urine, or semen.
  • the sample is a skin lesion specimen (e.g., fluid, tissue, or other material from a monkeypox lesion).
  • Skin lesion specimens include but are not limited to swabs of lesion surface and/or lesion fluid or exudate, roofs from one or more lesions, or lesion crusts.
  • the lesion specimen is a lesion swab.
  • 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 refrigerated (2- 8 °C) or frozen (-20 °C or lower) following collection and prior to analysis.
  • the sample may be contacted with the set of oligonucleotides, comprising amplification oligonucleotides and probes as described herein, and amplification reagents to form a reaction mixture.
  • primer and probe oligonucleotide sequences for amplifying and detecting an internal control (IC) sequence(s), as described herein are added to each sample.
  • the internal control(s) are then processed through the entire sample preparation and amplification procedure along with the test samples, to demonstrate proper sample processing and assay validity.
  • internal control primer and probe oligonucleotide sequences may be added to the reaction mixture after sample processing.
  • the set of oligonucleotides useful for amplification may comprise a first amplification oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 1, a second amplification oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 2, and a probe oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 3 and/or a first amplification oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 4, a second amplification oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 5, and a probe oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 6.
  • the set of oligonucleotides useful for amplification may comprise a first amplification oligonucleotide comprising a nucleic acid sequence with at least 70% (e.g., 75%., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) similarity to any of SEQ ID NOs: 7-9, a second amplification oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to any of SEQ ID NOs: 10-11 and a probe oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to any of SEQ ID NOs: 12-15; or a first amplification oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to any of SEQ ID NOs: 16-20, a second amplification oligonucleotide comprising
  • the set comprises a first amplification oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 7.
  • the set comprises a second amplification oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 10.
  • the set comprises a probe oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 12.
  • the set comprises a first amplification oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 16. In select embodiments, the set comprises a second amplification oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 21. In select embodiments, the set comprises a probe oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 26.
  • the set comprises a first amplification oligonucleotide comprising a nucleic acid sequence of any of SEQ ID NOs: 7-9, a second amplification oligonucleotide comprising a nucleic acid sequence of any of SEQ ID NOs: 10-11 and a probe oligonucleotide comprising a nucleic acid sequence of any of SEQ ID NOs: 12-15; or a first amplification oligonucleotide comprising a nucleic acid sequence of any of SEQ ID NOs: 16-20, a second amplification oligonucleotide comprising a nucleic acid sequence of any of SEQ ID NOs: 21-25, and a probe oligonucleotide comprising a nucleic acid sequence of any of SEQ ID NOs: 26-39; or a combination thereof.
  • amplification conditions refers to conditions that promote annealing and/or extension of the amplification oligonucleotides. Such conditions are well-known in the art and depend on the selected amplification method.
  • PCR amplification conditions generally comprise thermal cycling, e.g., cycling of the reaction mixture between two or more temperatures. In isothermal amplification reactions, amplification occurs without thermal cycling although an initial temperature increase may be required to initiate the reaction.
  • Amplification conditions encompass all reaction conditions including, but not limited to, temperature and/or temperature cycling, buffer, salt, ionic strength, pH, and the like.
  • amplification reagents refers to reagents used in amplification reactions and may include, but is not limited to, buffers, reagents, enzymes having reverse transcriptase and/or polymerase activity or exonuclease activity; enzyme cofactors such as magnesium or manganese; salts; and deoxynucleotide triphosphates (dNTPs) such as deoxy adenosine triphosphate (dATP), deoxy guanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP), deoxythymidine triphosphate (dTTP) and deoxyuridine triphosphate (dUTP).
  • dNTPs deoxynucleotide triphosphates
  • dATP deoxy adenosine triphosphate
  • dGTP deoxy guanosine triphosphate
  • dCTP deoxycytidine triphosphate
  • dTTP deoxythymidine triphosphate
  • Amplifying a monkeypox vims 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), reverse-transcriptase PCR (RT-PCR), real-time PCR, digital PCR, transcription-mediated amplification (TMA), rolling circle amplification, nucleic acid sequence-based amplification (NASBA), self-sustained sequence replication (3SR), 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).
  • PCR polymerase chain reaction
  • RT-PCR reverse-transcriptase PCR
  • RT-PCR reverse-transcriptase PCR
  • PCR
  • amplification of monkeypox virus nucleic acid sequences is performed using PCR.
  • Any suitable PCR methodology, combination of PCR methodologies, or combination of PCR with other amplification techniques may be used.
  • Exemplary PCR methodologies include, but are not limited to, digital PCR (dPCR), competitive dPCR (cdPCR), dPCR LATE (linear-after-the-exponential), allele- specific PCR, assembly PCR, asymmetric PCR, endpoint PCR, hot-start PCR, in situ PCR, intersequence-specific PCR, inverse PCR, linear after exponential PCR, ligation-mediated PCR, methylation-specific PCR, miniprimer PCR, multiplex ligation-dependent probe amplification, multiplex PCR, nested PCR, overlap-extension PCR, polymerase cycling assembly, qualitative PCR, quantitative PCR, real-time PCR, RT-PCR, singlecell PCR, solid-phase PCR, thermal asymmetric interlaced PCR,
  • amplification of monkeypox virus 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.
  • m2000rt REALTIMETM 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 REALTIMETM 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 amplification may be performed using isothermal amplification.
  • Strand-displacement amplification combines the ability of a restriction endonuclease to nick the unmodified strand of its target DNA and the action of an exonuclease-deficient DNA polymerase to extend the 3' end at the nick and displace the downstream DNA strand at a fixed temperature (See, Walker et al., Proc. Natl. Acad. Sci. USA, 1992).
  • Primers used in SDA include a restriction endonuclease recognition at site 5' to the target binding sequence (See, U.S. Pat. Nos. 5,270,184 and 5,344,166, each of which is incorporated herein by reference).
  • Nucleic acid sequence based amplification uses three enzymes (e.g., RNase H, avian myeloblastosis vims (AMV) reverse transcriptase and T7 RNA polymerase) working in concert at a low isothermal temperature, generally 41° C. (See, Compton, Nature, 1991, 350: 91-92; Chan et al., Rev. Med. Microbiol., 1999, 10: 185-196).
  • the self-sustained sequence replication (3SR) reaction is a very efficient method for isothermal amplification of target DNA or RNA sequences.
  • a 3SR system involves the collective activities of AMV reverse transcriptase, E.
  • T7 RNA polymerase DNA-dependent RNA polymerase
  • Transcription-mediated amplification uses an RNA polymerase to make RNA from a promoter engineered in the primer region, a reverse transcriptase to produce complementary DNA from the RNA templates and RNase H to remove the RNA from cDNA (See, Guatelli et al., Proc. Natl. Acad. Sci. USA, 1990).
  • NASBA, 3SR, and TMA primers comprise an RNA polymerase promoter linked to the target binding sequence of the primer. Promoters or promoter sequences for incorporation in the primers are nucleic acid sequences (either naturally occurring, produced synthetically or a product of a restriction digest) that are specifically recognized by an RNA polymerase that recognizes and binds to that sequence and initiates the process of transcription whereby RNA transcripts are generated.
  • useful promoters include those which are recognized by certain bacteriophage polymerases such as those from bacteriophage T3, T7 or SP6 or a promoter from E. coli.
  • 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.
  • 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.
  • RPA Recombinase-polymerase amplification
  • 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.
  • Loop-mediated isothermal amplification uses 4-6 primers recognizing multiple distinct regions of target DNA for a highly specific amplification reaction.
  • a strand-displacing DNA polymerase initiates synthesis and two specially designed primers form “loop” structures to facilitate subsequent rounds of amplification through extension on the loops and additional annealing of primers.
  • LCR Ligation chain reactions
  • HD A Helicase-dependent amplification
  • Rolling circle amplification is described by Lisby, Mol. Biotechnol. 12(l):75-99 (1999)), Hatch et al., Genet. Anal. 15(2):35-40 (1999).
  • Rolling circle replication is a process of unidirectional nucleic acid replication that can rapidly synthesize multiple copies of circular molecules of DNA or RNA.
  • Two primers can be used with a small (less than 150 bp) circularized padlock DNA probe to produce hyperbranched amplification of the entire small circle (Lizardi, et al., Nature Genetics 19:225-232 (1998), Zhang, et al., Gene 211:277-285 (1998)).
  • the disclosed methods may further comprise hybridizing one or more of the probe oligonucleotide sequences disclosed herein to the one or more amplified target monkeypox virus nucleic acid sequences.
  • the methods comprise 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 monkeypox virus nucleic acid sequences and the presence of monkeypox virus in the sample, and (ii) the absence of signals indicates the absence of monkeypox virus 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.
  • the probes described herein are used to detect amplification products generated by the amplification reaction.
  • the probes described herein can be employed using a variety of well-known homogeneous or heterogeneous methodologies.
  • Homogeneous detection methods include, but are not limited to, the use of FRET labels that are attached to the probes and that emit a signal in the presence of the target sequence, Molecular Beacons (See, Tyagi et al., Nature Biotechnol., 1996, 14: 303-308; Tyagi et al., Nature Biotechnol., 1998, 16: 49-53; Kostrikis et al., Science, 1998, 279: 1228-1229; Sokol et al., Proc.
  • homogeneous detection methods including hybridization protection assays (HP A).
  • the probes are labeled with acridinium ester (AE), a highly chemiluminescent molecule (See, Weeks et al., Clin. Chem., 1983, 29: 1474-1479; Berry et al., Clin. Chem., 1988, 34: 2087-2090), using a non-nucleotide-based linker arm chemistry (See, U.S. Pat. Nos. 5,585,481 and 5,185,439).
  • AE acridinium ester
  • AE highly chemiluminescent molecule
  • Chemiluminescence is triggered by AE hydrolysis with alkaline hydrogen peroxide, which yields an excited N-methyl acridone that subsequently deactivates with emission of a photon.
  • AE hydrolysis is rapid.
  • the rate of AE hydrolysis is greatly reduced when the probe is bound to the target sequence.
  • Heterogeneous detection systems are also well-known in the art and generally employ a capture agent to separate amplified sequences from other materials in the reaction mixture.
  • Capture agents typically comprise a solid support material (e.g., microtiter wells, beads, chips, and the like) coated with one or more specific binding sequences.
  • a binding sequence may be complementary to a tail sequence added to oligonucleotide probes of the invention.
  • a binding sequence may be complementary to a sequence of a capture oligonucleotide, itself comprising a sequence complementary to a tail sequence of a probe.
  • the amplification product/probe hybrids can be detected using any detection methods, such as those described herein.
  • a subject identified according to the methods described above as having monkeypox may be treated and/or monitored using routine techniques known in the art.
  • the methods further comprise: monitoring the subject for monkeypox virus infection symptoms, treating the subject for monkeypox virus infection, or monitoring the subject for monkeypox virus infection symptoms prior to or after treating the subject for monkeypox virus infection.
  • Treatment for monkeypox may include symptom management (e.g., use of pain relievers, fever reducers, oatmeal baths, rest, hydration) and antiviral agents, such as tecovirimat (TPOXX), brincidofovir (Tembexa) and cidofovir (Vistide), particularly for those who are at risk of severe illness (e.g., children, pregnant women, and those individuals with compromised immune systems).
  • Treatment may also include antibiotics to treat secondary bacterial infections, if observed.
  • a subject may be monitored prior to or after being treated for monkeypox virus infection. Such monitoring involves observing symptoms, or changes in symptoms, including presence of fever, chills, swelling of the lymph nodes, and development or exacerbations of skin rashes.
  • the disclosure also provides systems for amplifying and detecting monkeypox virus in a sample.
  • the system comprises a set of oligonucleotide sequences described herein.
  • the system comprises a first amplification oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 1, a second amplification oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 2 and a probe oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 3.
  • the system comprises a first amplification oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 4, a second amplification oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 5, and a probe oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 6.
  • the system comprises a first amplification oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 1, a second amplification oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 2 a probe oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 3, a first amplification oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 4, a second amplification oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 5, and a probe oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 6.
  • the system comprises a first amplification oligonucleotide comprising a nucleic acid sequence with at least 70% (e.g., 75%., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) similarity to any of SEQ ID NOs: 7-9, a second amplification oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to any of SEQ ID NOs: 10-11 and a probe oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to any of SEQ ID NOs: 12-15; or a first amplification oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to any of SEQ ID NOs: 16-20, a second amplification oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to any
  • the system comprises a first amplification oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 7.
  • the system comprises a second amplification oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 10.
  • the system comprises a probe oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 12.
  • the system comprises a first amplification oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 16. In select embodiments, the system comprises a second amplification oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 21. In select embodiments, the system comprises a probe oligonucleotide comprising a nucleic acid sequence with at least 70% similarity to SEQ ID NO: 26.
  • the system comprises a first amplification oligonucleotide comprising a nucleic acid sequence of any of SEQ ID NOs: 7-9, a second amplification oligonucleotide comprising a nucleic acid sequence of any of SEQ ID NOs: 10-11 and a probe oligonucleotide comprising a nucleic acid sequence of any of SEQ ID NOs: 12-15; or a first amplification oligonucleotide comprising a nucleic acid sequence of any of SEQ ID NOs: 16-20, a second amplification oligonucleotide comprising a nucleic acid sequence of any of SEQ ID NOs: 21- 25, and a probe oligonucleotide comprising a nucleic acid sequence of any of SEQ ID NOs: 26-39; or a combination thereof.
  • the system may further comprise one or more nucleic acid amplification reagents.
  • nucleic acid amplification 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, deoxy adenosine triphosphate, deoxy guanosine triphosphate, deoxycytidine triphosphate, and deoxythymidine triphosphate) blocking agents, labeling agents, and the like.
  • enzyme cofactors such as magnesium or nicotinamide adenine dinucleotide (NAD)
  • reagents used in amplification reactions include nicking enzymes, single-strand binding proteins, helicases, resolvases, and the like.
  • the one or more nucleic acid amplification reagents is selected from the group consisting of: a nicking enzyme, a polymerase, a single-strand binding protein, a recombinase, a helicase, a resolvase, an enzyme cofactor, a buffer, deoxyribonucleotide or ribonucleotide triphosphates, and combinations thereof.
  • the system may further comprise a nucleic acid amplification reaction system configured to amplify one or more target monkeypox virus nucleic acid sequences in the sample.
  • the nucleic acid amplification reaction system may further be configured to facilitate hybridization of one or more of the probe oligonucleotides to amplified target monkeypox virus nucleic acid sequences and/or detect hybridization of one or more of the probe oligonucleotides to amplified target monkeypox virus nucleic acid sequences.
  • the nucleic acid amplification reaction system is further configured to measure a signal from one or more detectable label.
  • the nucleic acid amplification reaction system may comprise a single component which is configured to carry out the above listed roles.
  • the nucleic acid amplification reaction system may comprise multiple components, which in combinations are capable of carrying out the above listed roles.
  • the nucleic acid amplification reaction system may comprise one or more of: a thermocycler, an incubator or incubation chamber, a fluorometer, a spectrophotometer, a colorimeter, and the like.
  • the disclosure also provides a kit for amplifying and detecting monkeypox virus in a sample.
  • the kit comprises at least one oligonucleotide as described herein.
  • the kit comprises a set of oligonucleotide sequences described herein.
  • the kit may further comprise reagents for amplifying and detecting nucleic acid sequences, and instructions for amplifying and detecting monkeypox virus. 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, deoxy guanosine 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
  • 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).
  • 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.
  • Inclusivity To evaluate the conservation of oligos designed to detect monkeypox or VZV targets in prototype assays, a sequence analysis pipeline was developed. First, all available monkeypox complete genome sequences from the recent outbreak were downloaded from GISAID and added to all Genbank monkeypox complete genomes under taxon ID 10244, which includes both Central and Western African lineages (date of accession 7/15/22). Likewise, all available complete genomes for VZV were downloaded from Genbank (date of accession 6/27/22). The BLAST aligner was utilized in a python pipeline to produce alignments with each appropriate oligo and complete percent identity was calculated from the local percent identity.
  • Exclusivity To evaluate the potential cross reactivity of all monkeypox oligo sets with off target pathogens, an exclusivity analysis was conducted using BLAST. First, sequences were downloaded from Genbank for each taxon ID and subnodes (date of accession 6/29/22) to assemble a database that was segregated to 28 individual servers for parallel BLAST alignment processing. Duplicate sequences were removed before BLAST alignment was conducted. Results were annotated by cross-indexing to the oligo table and the group taxonomy name. Results were sorted by bit scores and the corrected percent identity was calculated by normalizing the length of the alignment to the length of the query oligo. Likewise, the total number of mismatches was corrected to account for end positions that did not align.
  • the resulting metadata and BLAST results were then processed to generate the ‘midline’ result - the position dots (match) and dashes (mismatch), resulting in >2.4 million records.
  • Hits were then parsed to unique oligo target accession values to remove hits that were in the same taxon ID as the intended target.
  • the individual hit with the highest corrected percent identity for each unique oligo/taxon group combination was collected. Results with 79% identity or higher were selected for further manual examination.
  • the individual oligos with 80% identity or higher were entered into a summary table (see appendix summary tables file). If an organism is not listed in the summary table for an oligo set, it is because there were no hits for any oligos with >80% identity.
  • Hits where an amplicon was not possible due to only a single oligo having >80% identity were discarded, as well as hits where a forward and reverse oligo had >80% identity but the probe had ⁇ 80%.
  • Hits where a forward, reverse, and probe all had >80% identity were further examined for a mismatch position in the 3 nucleotides on the 3 ’ end, for forward and reverse primer positions on the same strand, distance between the forward and reverse oligos, and inclusion of probe in between forward and reverse primers in an investigation table (see appendix investigation tables file).
  • B6R and E9L oligo sets had >90% sequence identity to >99% of all monkeypox sequences analyzed, which included both Central and West African genomes.
  • B6R oligos were predicted to detect all Central and Western African monkeypox infections.
  • Orthopoxviruses vaccinia, akhmeta virus, camelpox, cowpox, horsepox, rabbitpox, and taterapox
  • No cross reactivity is predicted outside of the orthopoxvirus genus.
  • E9L oligos were predicted to detect all Central and Western African monkeypox infections.
  • Orthopoxviruses may have cross reactivity. No cross reactivity is predicted outside of the orthopoxvirus genus.
  • monkeypox virus nucleic acid sequences were performed by real-time PCR using the described amplification and probe oligonucleotides.
  • the monkeypox virus sample was provided in the reaction as plasmids carrying either E9L or B6R gene (FIG. 1) or monkeypox (MPXV) genomic DNA (FIG. 2).
  • the PCR reaction contained equal amounts of each of the amplification and probe oligonucleotides (SEQ ID NOs: 1-6). An internal control nucleic acid and a primer set to the internal control were also included as a positive control.
  • the reaction mixture also included reaction buffers, dNTPs, reference dyes, DNA polymerase (TaqGold), and cofactors in concentrations commonly used in the art.
  • NR- 27 was serially diluted into UTM and amplified using the amplification and probe oligonucleotides described herein. Amplified products were detected at virus dilutions down to 2.8 x 10 A 0 TCIDso/mL. Monkeypox Virus, USA-2003 (Catalog No. NR-2500) was serially diluted into either UTM or a 1 : 1 mixture of UTM and saliva and amplified using the Monkeypox real-time PCR assay. Amplified products were detected at virus dilutions down to 3.2 x 10 A 0 pfu/mL in both UTM and UTM/saliva.
  • monkeypox virus nucleic acid sequences were performed by real-time PCR using the described amplification and probe oligonucleotides.
  • the monkeypox virus sample was provided in the reaction as plasmids carrying B7R and J2R gene (FIG. 3A)
  • the PCR reaction contained the amplification and probe oligonucleotides (SEQ ID NOs: 7, 10, 12, 16, 21 and 26).
  • An internal control nucleic acid, human genomic DNA and sets of amplification and probe oligonucleotides to the internal control and a cellular control were also included as a validity control.
  • the reaction mixture also included reaction buffers, dNTPs, reference dyes, DNA polymerase, and cofactors in concentrations commonly used in the art.
  • the PCR cycling parameters used in this Example 2 are shown below:
  • monkeypox complete genome sequences were downloaded from GISAID. All available monkeypox complete genome sequences (678) included in the current release of the standard GISAID MSA (multiple sequence alignment) file were downloaded from GISAID and extracted into a valid fasta file. This download included both Central and Western African lineages (date of download 8/10/22). BLASTn was utilized to produce alignments with each oligo and corrected sequence alignment percent identity was calculated from the local alignment percent identity.
  • Oligos directed to B7R are capable of detecting >99% of all Central and Western African monkeypox infections.
  • Oligos directed to J2R are capable of detecting all Central and Western African monkeypox infections.
  • a dual-target assay utilizing both B7R and J2R oligos are capable of detecting all Central and Western African monkeypox infections.
  • GISAID data are subject to GISAID’s Terms and Conditions that can be accessed via gisaid.org. Data cannot be disclosed externally.
  • Results were annotated by cross-indexing to the oligo table and the group taxonomy name. Results were sorted by bit scores and the corrected percent identity was calculated by normalizing the length of the alignment to the length of the query oligo. Likewise, the total number of mismatches was corrected to account for end positions that did not align.
  • Hits where a forward, reverse, and probe all had >80% identity were further examined for a mismatch position in the 3 nucleotides on the 3 ’ end, for forward and reverse primer being positioned on the same strand, for distance between the forward and reverse oligos, and for probe binding position relative to forward and reverse primers, in an investigation table.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Virology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente divulgation concerne des matériaux et des procédés d'amplification et de détection virale de la variole du singe dans un échantillon, comprenant une variété de combinaisons d'oligonucléotides d'amplification et de sondes oligonucléotidiques. La divulgation concerne également des séquences oligonucléotidiques, des kits et des procédés de détection virale de la variole du singe.
PCT/US2023/071560 2022-08-04 2023-08-03 Dosages pour la détection virale de la variole du singe Ceased WO2024030985A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP23764783.9A EP4565720A1 (fr) 2022-08-04 2023-08-03 Dosages pour la détection virale de la variole du singe

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US202263395007P 2022-08-04 2022-08-04
US63/395,007 2022-08-04
US202263403162P 2022-09-01 2022-09-01
US63/403,162 2022-09-01

Publications (1)

Publication Number Publication Date
WO2024030985A1 true WO2024030985A1 (fr) 2024-02-08

Family

ID=87929311

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2023/071560 Ceased WO2024030985A1 (fr) 2022-08-04 2023-08-03 Dosages pour la détection virale de la variole du singe

Country Status (2)

Country Link
EP (1) EP4565720A1 (fr)
WO (1) WO2024030985A1 (fr)

Citations (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0320308A2 (fr) 1987-12-11 1989-06-14 Abbott Laboratories Procédé pour détecter une séquence cible d'acide nucléique
US5185439A (en) 1987-10-05 1993-02-09 Gen-Probe Incorporated Acridinium ester labelling and purification of nucleotide probes
US5210015A (en) 1990-08-06 1993-05-11 Hoffman-La Roche Inc. Homogeneous assay system using the nuclease activity of a nucleic acid polymerase
US5270184A (en) 1991-11-19 1993-12-14 Becton, Dickinson And Company Nucleic acid target generation
US5344166A (en) 1993-09-30 1994-09-06 Microcentric Corporation Jaw assembly for chucks
US5487792A (en) 1994-06-13 1996-01-30 Midwest Research Institute Molecular assemblies as protective barriers and adhesion promotion interlayer
US5585481A (en) 1987-09-21 1996-12-17 Gen-Probe Incorporated Linking reagents for nucleotide probes
US5846726A (en) 1997-05-13 1998-12-08 Becton, Dickinson And Company Detection of nucleic acids by fluorescence quenching
US5925517A (en) 1993-11-12 1999-07-20 The Public Health Research Institute Of The City Of New York, Inc. Detectably labeled dual conformation oligonucleotide probes, assays and kits
US6235504B1 (en) 1999-01-11 2001-05-22 The Rockefeller University Methods for identifying genomic equivalent markers and their use in quantitating cells and polynucleotide sequences therein
US6277581B1 (en) 1999-03-01 2001-08-21 Lankenau Medical Research Center ODC allelic analysis method for assessing carcinogenic susceptibility
WO2001086001A1 (fr) 2000-05-09 2001-11-15 Biosearch Technologies, Inc. Extincteurs non-phosphorescents pour transfert d'energie donneur-accepteur
US7270981B2 (en) 2002-02-21 2007-09-18 Asm Scientific, Inc. Recombinase polymerase amplification
US7399590B2 (en) 2002-02-21 2008-07-15 Asm Scientific, Inc. Recombinase polymerase amplification
US8030000B2 (en) 2002-02-21 2011-10-04 Alere San Diego, Inc. Recombinase polymerase amplification
US8071308B2 (en) 2006-05-04 2011-12-06 Alere San Diego, Inc. Recombinase polymerase amplification
US9096897B2 (en) 2012-04-09 2015-08-04 Envirologix Inc. Compositions and methods for quantifying a nucleic acid sequence in a sample comprising a primer oligonucleotide with a 3′-terminal region comprising a 2′-modified nucleotide
US20150240298A1 (en) 2009-06-05 2015-08-27 Alere San Diego Inc. Recombinase polymerase amplification reagents and kits
US9562264B2 (en) 2007-07-14 2017-02-07 Ionian Technologies, Inc. Nicking and extension amplification reaction for the exponential amplification of nucleic acids

Patent Citations (40)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5585481A (en) 1987-09-21 1996-12-17 Gen-Probe Incorporated Linking reagents for nucleotide probes
US5185439A (en) 1987-10-05 1993-02-09 Gen-Probe Incorporated Acridinium ester labelling and purification of nucleotide probes
EP0320308A2 (fr) 1987-12-11 1989-06-14 Abbott Laboratories Procédé pour détecter une séquence cible d'acide nucléique
US5210015A (en) 1990-08-06 1993-05-11 Hoffman-La Roche Inc. Homogeneous assay system using the nuclease activity of a nucleic acid polymerase
US6214979B1 (en) 1990-08-06 2001-04-10 Roche Molecular Systems Homogeneous assay system
US5804375A (en) 1990-08-06 1998-09-08 Roche Molecular Systems, Inc. Reaction mixtures for detection of target nucleic acids
US5270184A (en) 1991-11-19 1993-12-14 Becton, Dickinson And Company Nucleic acid target generation
US5344166A (en) 1993-09-30 1994-09-06 Microcentric Corporation Jaw assembly for chucks
US5925517A (en) 1993-11-12 1999-07-20 The Public Health Research Institute Of The City Of New York, Inc. Detectably labeled dual conformation oligonucleotide probes, assays and kits
US5487792A (en) 1994-06-13 1996-01-30 Midwest Research Institute Molecular assemblies as protective barriers and adhesion promotion interlayer
US5846726A (en) 1997-05-13 1998-12-08 Becton, Dickinson And Company Detection of nucleic acids by fluorescence quenching
US6235504B1 (en) 1999-01-11 2001-05-22 The Rockefeller University Methods for identifying genomic equivalent markers and their use in quantitating cells and polynucleotide sequences therein
US6277581B1 (en) 1999-03-01 2001-08-21 Lankenau Medical Research Center ODC allelic analysis method for assessing carcinogenic susceptibility
WO2001086001A1 (fr) 2000-05-09 2001-11-15 Biosearch Technologies, Inc. Extincteurs non-phosphorescents pour transfert d'energie donneur-accepteur
US8426134B2 (en) 2002-02-21 2013-04-23 Alere San Diego, Inc. Recombinase polymerase amplification
US8460875B2 (en) 2002-02-21 2013-06-11 Alere San Diego, Inc. Recombinase polymerase amplification
US7666598B2 (en) 2002-02-21 2010-02-23 Twistdx, Inc. Recombinase polymerase amplification
US8017339B2 (en) 2002-02-21 2011-09-13 Alere San Diego, Inc. Compositions and kits for recombinase polymerase amplification
US8030000B2 (en) 2002-02-21 2011-10-04 Alere San Diego, Inc. Recombinase polymerase amplification
US10036057B2 (en) 2002-02-21 2018-07-31 Alere San Diego, Inc. Recombinase polymerase amplification
US7270981B2 (en) 2002-02-21 2007-09-18 Asm Scientific, Inc. Recombinase polymerase amplification
US20170044598A1 (en) 2002-02-21 2017-02-16 Alere San Diego, Inc. Compositions for recombinase polymerase amplification
US8574846B2 (en) 2002-02-21 2013-11-05 Alere San Diego, Inc. Recombinase polymerase amplification
US7399590B2 (en) 2002-02-21 2008-07-15 Asm Scientific, Inc. Recombinase polymerase amplification
US8945845B2 (en) 2002-02-21 2015-02-03 Alere San Diego Inc. Recombinase polymerase amplification
US8962255B2 (en) 2002-02-21 2015-02-24 Alere San Diego, Inc. Recombinase polymerase amplification
US9663820B2 (en) 2002-02-21 2017-05-30 Alere San Diego Inc. Recombinase polymerase amplification
US10947584B2 (en) 2002-02-21 2021-03-16 Abbott Diagnostics Scarborough, Inc. Recombinase polymerase amplification
US10329602B2 (en) 2002-02-21 2019-06-25 Alere San Diego, Inc. Recombinase polymerase amplification
US10329603B2 (en) 2002-02-21 2019-06-25 Alere San Diego Inc. Recombinase polymerase amplification
US8071308B2 (en) 2006-05-04 2011-12-06 Alere San Diego, Inc. Recombinase polymerase amplification
US10093908B2 (en) 2006-05-04 2018-10-09 Alere San Diego, Inc. Recombinase polymerase amplification
US11339382B2 (en) 2006-05-04 2022-05-24 Abbott Diagnostics Scarborough, Inc. Recombinase polymerase amplification
US8637253B2 (en) 2006-05-04 2014-01-28 Alere San Diego, Inc. Recombinase polymerase amplification
US9689031B2 (en) 2007-07-14 2017-06-27 Ionian Technologies, Inc. Nicking and extension amplification reaction for the exponential amplification of nucleic acids
US9617586B2 (en) 2007-07-14 2017-04-11 Ionian Technologies, Inc. Nicking and extension amplification reaction for the exponential amplification of nucleic acids
US9562263B2 (en) 2007-07-14 2017-02-07 Ionian Technologies, Inc. Nicking and extension amplification reaction for the exponential amplification of nucleic acids
US9562264B2 (en) 2007-07-14 2017-02-07 Ionian Technologies, Inc. Nicking and extension amplification reaction for the exponential amplification of nucleic acids
US20150240298A1 (en) 2009-06-05 2015-08-27 Alere San Diego Inc. Recombinase polymerase amplification reagents and kits
US9096897B2 (en) 2012-04-09 2015-08-04 Envirologix Inc. Compositions and methods for quantifying a nucleic acid sequence in a sample comprising a primer oligonucleotide with a 3′-terminal region comprising a 2′-modified nucleotide

Non-Patent Citations (47)

* Cited by examiner, † Cited by third party
Title
"Biological Sequence Analysis: Probalistic Models of Proteins and Nucleic Acids", 2009, CAMBRIDGE UNIVERSITY PRESS
"PCR Strategies", 1995, ACADEMIC PRESS
"Real-time PCR", 2007, TAYLOR & FRANCIS
A. H. HOPMAN ET AL., EXP. CELL RES., vol. 169, 1987, pages 357 - 368
ALTSCHUL ET AL., J. MOLECULAR BIOL., vol. 215, no. 3, 1990, pages 403 - 410
ALTSCHUL ET AL., NUCLEIC ACIDS RES, vol. 25, no. 17, 1997, pages 3389 - 3402
BAYER ET AL., METHODS OF BIOCHEM. ANALYSIS, vol. 26, 1980, pages 1 - 45
BEIGERT ET AL., PROC. NATL. ACAD. SCI. USA, vol. 106, no. 10, 2009, pages 3770 - 3775
BELOUSOV ET AL., NUCLEIC ACIDS RES., vol. 25, 1997, pages 3440 - 3444
BERRY ET AL., CLIN. CHEM., vol. 34, 1988, pages 2087 - 2090
BRIGATI ET AL., VIROL, vol. 126, 1983, pages 32 - 50
BROKER ET AL., NUCL. ACIDS RES, vol. 5, 1978, pages 363 - 384
BROWN ET AL., METH. ENZYMOL, vol. 68, 1979, pages 109 - 151
CHAN ET AL., REV. MED. MICROBIOL, vol. 10, 1999, pages 185 - 196
COMPTON, NATURE, vol. 350, 1991, pages 91 - 92
CONNOLY ET AL., NUCL. ACIDS. RES, vol. 13, 1985, pages 4485 - 4502
FRAGA ET AL., CURRENT PROTOCOLS ESSENTIAL LABORATORY TECHNIQUES, 2008, pages 10 - 3
GHATE SUDEEP D. ET AL: "Molecular detection of monkeypox and related viruses: challenges and opportunities", 2 July 2022 (2022-07-02), XP093100666, Retrieved from the Internet <URL:https://osf.io/jtdnz/> [retrieved on 20231113], DOI: 10.1007/s11262-023-01975-3 *
GUATELLI ET AL., PROC. NATL. ACAD. SCI. USA, 1990
JOOS ET AL., J. BIOTECHNOL, vol. 35, 1994, pages 135 - 153
KOSTRIKIS ET AL., SCIENCE, vol. 279, 1998, pages 1228 - 1229
L. J. KRICKA, ANN. CLIN. BIOCHEM., vol. 39, 2002, pages 114 - 129
LANDEGENT ET AL., EXP. CELL RES., vol. 15, 1984, pages 61 - 72
LANDEGREN ET AL., SCIENCE, vol. 241, 1988, pages 1077
LANGER ET AL., PROC. NATL. ACAD. SCI. USA, vol. 78, 1981, pages 6633 - 6637
LI Y ET AL: "Detection of monkeypox virus with real-time PCR assays", JOURNAL OF CLINICAL VIROLOGY, ELSEVIER, AMSTERDAM, NL, vol. 36, no. 3, 1 July 2006 (2006-07-01), pages 194 - 203, XP028037992, ISSN: 1386-6532, [retrieved on 20060701], DOI: 10.1016/J.JCV.2006.03.012 *
LISBY, MOL. BIOTECHNOL, vol. 12, no. 1, 1999, pages 75 - 99
MARRAS ET AL., GENET. ANAL, vol. 14, no. 2, 1999, pages 151 - 156
MAXAM ET AL., METHODS OF ENZYMOLOGY, vol. 65, 1980, pages 499 - 560
MCFARLAND ET AL., NUCLEIC ACIDS RES., vol. 7, 1979, pages 1067 - 1080
NATURE GENETICS, vol. 19, 1998, pages 225 - 232
PEARSON, J. CHROM, vol. 255, 1983, pages 137 - 149
PIELES ET AL., NUCLEIC ACIDS RES., vol. 21, 1993, pages 3191 - 3196
RICHARDSON ET AL., NUCL. ACIDS RES, vol. 11, 1983, pages 6167 - 6184
SMITH ET AL., NUCL. ACIDS RES, vol. 13, 1985, pages 2399 - 2412
SODING, BIOINFORMATICS, vol. 21, no. 7, 2005, pages 951 - 960
SOKOL ET AL., PROC. NATL. ACAD. SCI. USA, vol. 95, 1998, pages 11538 - 11543
TCHEN ET AL., PROC. NATL. ACAD. SCI. USA, vol. 81, 1984, pages 3466 - 3470
TEMSAMANI ET AL., MOL. BIOTECHNOL, vol. 5, 1996, pages 223 - 232
TYAGI ET AL., NATURE BIOTECHNOL., vol. 14, 1996, pages 303 - 308
TYAGI ET AL., NATURE BIOTECHNOL., vol. 16, 1998, pages 49 - 53
VAN GIJLSWIJK ET AL., EXPERT REV. MOL. DIAGN, vol. 1, 2001, pages 81 - 91
WALKER ET AL., PROC. NATL. ACAD. SCI. USA, 1992
WEEKS ET AL., CLIN. CHEM., vol. 29, 1983, pages 1474 - 1479
WU ET AL., ANAL. BIOCHEM., vol. 290, 2001, pages 347 - 352
WU ET AL., GENOMICS, vol. 4, 1989, pages 560
ZHANG ET AL., GENE, vol. 211, 1998, pages 277 - 285

Also Published As

Publication number Publication date
EP4565720A1 (fr) 2025-06-11

Similar Documents

Publication Publication Date Title
EP1583949B1 (fr) Compositions et methodes de detection du virus du nil occidental
US20210324487A1 (en) Method for detecting chikungunya virus
CN113508182B (zh) 用于检测人乳头状瘤病毒(hpv)的测定
JP2024032995A (ja) 水痘帯状疱疹ウイルスを増幅または検出するための組成物および方法
WO2009155103A2 (fr) Compositions destinées à être utilisées dans l’identification de pathogènes transmis par les tiques
WO2021204642A1 (fr) Kit de diagnostic à base de pcr, compositions et procédés d&#39;amplification et de détection de sras-cov-2
US11028439B2 (en) Assay for detecting hepatitis B virus (HBV)
WO2024030985A1 (fr) Dosages pour la détection virale de la variole du singe
US20230313323A1 (en) Assays for detecting coronavirus disease 2019 (covid-19)
EP2975139A1 (fr) Dosage de détection et de quantification du vih-1
JP7542546B2 (ja) ヒトポリオーマウイルスｂｋウイルスを増幅、検出または定量化するための組成物および方法
JP2019524123A (ja) 核酸の増幅及び検出／定量の効率を改良するためのヘルパーオリゴヌクレオチド
CN119193791A (zh) 用于核酸检测的引物探针及其应用
JP2023181438A (ja) アデノウイルス、メタニューモウイルス、および／またはライノウイルス核酸を検出するための組成物、方法、およびキット
EP4077729A1 (fr) Compositions et procédés pour détecter l&#39;acide nucléique de coronavirus

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23764783

Country of ref document: EP

Kind code of ref document: A1

REG Reference to national code

Ref country code: BR

Ref legal event code: B01A

Ref document number: 112025002066

Country of ref document: BR

WWE Wipo information: entry into national phase

Ref document number: 2023764783

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2023764783

Country of ref document: EP

Effective date: 20250304

WWP Wipo information: published in national office

Ref document number: 2023764783

Country of ref document: EP

ENP Entry into the national phase

Ref document number: 112025002066

Country of ref document: BR

Kind code of ref document: A2

Effective date: 20250131