Methods and oligonucleotide combinations for detecting the presence of monkeypox virus (MPXV) in a sample

Abstract

The present invention is directed to a method for detecting the presence or absence of monkeypox virus (MPXV) in a biological sample, the method comprising: a) contacting the sample or nucleic acid extracted from said sample with reagents and an oligonucleotide combination capable of amplifying a target region of a monkeypox viral genome, wherein said oligonucleotide combination is selected from the group consisting of: i) a B7R oligonucleotide primer set specific for amplifying a target sequence in the B7R gene of the MPXV genome generating an MPXV amplification product comprising the sequence of SEQ ID NO:65; and ii) a G2R oligonucleotide primer set specific for amplifying a target sequence in the G2R gene of the MPXV genome generating an MPXV amplification product comprising the sequence of SEQ ID NO:63; b) performing an in vitro nucleic acid amplification reaction, wherein any MPXV nucleic acid present in the sample is used as a template to generate the MPXV amplification product corresponding to the oligonucleotide combination of i) or ii); and c) detecting the presence or absence of the MPXV amplification product generated in step b), wherein the presence of the MPXV amplification product is indicative of the presence of MPXV in the biological sample. The present invention also provides oligonucleotides which can be used in the method of the invention as primers and probes.

Description

[0001] METHODS AND OLIGONUCLEOTIDE COMBINATIONS FOR DETECTING

[0002] THE PRESENCE OF MONKEYPOX VIRUS (MPXV) IN A SAMPLE

[0003] FIELD

[0004] The present disclosure relates to the field of molecular diagnostics, and more particularly to the detection of Monkeypox Virus (MPXV) by a polymerase chain reaction (PCR) assay.

[0005] BACKGROUND

[0006] 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. From 1 January 2022 through 31 March 2024, a cumulative total of 95,226 laboratory- confirmed cases of monkeypox, including 185 deaths, were reported to WHO from 117 countries. Severe cases can occur, especially in children, pregnant women, or people with suppressed immune systems, with a fatality ratio of approximately 3-10%.

[0007] SUMMARY

[0008] The present disclosure provides methods, oligonucleotide combinations and a kit for amplifying and detecting monkeypox virus in a biological sample. The disclosure provides methods for detecting monkeypox virus in a biological sample comprising: contacting the sample or nucleic acid extracted from said sample with reagents and an oligonucleotide combination capable of amplifying a target region of a monkeypox viral genome; performing an in vitro nucleic acid amplification reaction, wherein any MPXV nucleic acid present in the sample is used as a template to generate the MPXV amplification product; detecting the presence or absence of the MPXV amplification product, wherein the presence of the MPXV amplification product is indicative of the presence of MPXV in the biological sample. Certain embodiments in the present disclosure relate to methods for the rapid detection of the presence or absence of MPXV in a biological sample, for example, multiplex detection of MPXV by real-time PCR.

[0009] A method for detecting the presence or absence of monkeypox virus (MPXV) in a biological sample in accordance with the present disclosure generally includes, a) contacting the sample or nucleic acid extracted from said sample with reagents and an oligonucleotide combination capable of amplifying a target region of a monkeypox viral genome, wherein said oligonucleotide combination is selected from the group consisting of: i) a B7R oligonucleotide primer set specific for amplifying a target sequence in the B7R gene of the MPXV genome generating an MPXV amplification product comprising the sequence of SEQ ID NO: 65: catattacat aatatataca ggctatttac aagatgcacc g ( SEQ ID NO : 65 ) , and ii) a G2R oligonucleotide primer set specific for amplifying a target sequence in the G2R gene of the MPXV genome generating an MPXV amplification product comprising the sequence of SEQ ID NO: 63: cgtctcttcc acagataaat gcgaacccgt cgtaaccagc aatacattta actatatcga tgtggaaatt aacctgtatc cag ( SEQ ID NO : 63 ) b) performing an in vitro nucleic acid amplification reaction, wherein any MPXV nucleic acid present in the sample is used as a template to generate the MPXV amplification product corresponding to the oligonucleotide combination of i) or ii); and c) detecting the presence or absence of the MPXV amplification product generated in step b), wherein the presence of the MPXV amplification product is indicative of the presence of MPXV in the biological sample.

[0010] The disclosure also provides an oligonucleotide combination selected from the group consisting of: i) a B7R oligonucleotide primer set comprising a forward primer or forward primers selected from the group consisting of SEQ ID NOs:41 and 42, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:41 or 42, an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:41 or 42, and combinations thereof, wherein a reverse primer for the oligonucleotide primer set is SEQ ID NO:29, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:29, or an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:29, and wherein the B7R oligonucleotide primer set can be used for amplifying a target sequence in the B7R gene of a monkeypox virus (MPXV); and ii) a G2R oligonucleotide primer set comprising a forward primer selected from the group consisting of SEQ ID NOs:05 and 20, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:05 or 20, and an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO: 05 or 20, wherein a reverse primer for the oligonucleotide primer set is selected from the group consisting of SEQ ID NOs:23, 30, and 31, an oligonucleotide having at least 70% sequence identity with SEQ ID NO: 23, 30, or 31, or an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO: 23, 30, or 31, and wherein the G2R oligonucleotide primer set can be used for amplifying a target sequence in the G2R gene of a monkeypox virus (MPXV).

[0011] The disclosure also provides an oligonucleotide primer set comprising a forward primer comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs:07, 17, and 21, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:07, 17, or 21, and an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:07, 17, or 21, a reverse primer comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs:01, 12, and 40, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:01, 12, or 40, and an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:01, 12, or 40, and wherein the oligonucleotide primer pair can be used for amplifying a target sequence in the E9L gene of a non-variola orthopoxvirus (OPXV).

[0012] The disclosure further provides a kit for detecting monkeypox virus in a sample.

[0013] Representative embodiments of the method are further set forth below.

[0014] BRIEF DESCRIPTIONS OF THE DRAWINGS

[0015] Figures 1A-1C show primer / probe (PPR) sets PPR14, PPR15, PPR16, and PPR24 aligned to portion 56711-53691 (F8L) of GenBank Accession No. AF380138.1 of Monkeypox virus strain Zaire-96-1-16. SEQ ID NOs:06, 07, 17, 21, 39, & 61 are shown in antisense orientation.

[0016] Figures 2A-2C show primer / probe sets PPR1, PPR2, PPR3, PPR4, PPR17, PPR18, and PPR23 aligned to portion 165891-166421 (B7R) of GenBank Accession No. AF380138.1 of Monkeypox virus strain Zaire-96-1-16. SEQ ID NOs:02, 14, 29, & 58 are shown in antisense orientation. Figures 3A-3B show primer / probe sets PPR5, PPR6, and PPR20 aligned to portion 19710- 19060 (D14L) of GenBank Accession No. AF380138.1 of Monkeypox virus strain Zaire- 96-1-16. SEQ ID NOs:08, 34, 43, & 49 are shown in antisense orientation.

[0017] Figure 4 shows primer / probe set PPR7 aligned to portion 135481-135850 (intergenic) of GenBank Accession No. AF380138.1 of Monkeypox virus strain Zaire-96-1-16. SEQ ID NOs:03 & 24 are shown in antisense orientation.

[0018] Figure 5 shows primer / probe sets PPR21 and PPR25 aligned to portion 2786-1740 (J2L) of GenBank Accession No. AF380138.1 of Monkeypox virus strain Zaire-96-1-16. SEQ ID NOs:37 & 52 are shown in antisense orientation.

[0019] Figure 6A-6D show primer / probe sets PPR8, PPR9, PPR10, PPR11, PPR12, PPR19, PPR25, PPR26, PPR27, PPR28, PPR29, PPR30 and PPR31 aligned to a portion of the GenBank Accession No. U87845.1 reference sequence. SEQ ID NOs:22, 23, 30, 31, 37, & 46 are shown in antisense orientation.

[0020] Figure 7 shows primer / probe set PPR13 aligned to portion 134102-132954 of the GenBank Accession No. OP555662.1 reference sequence. SEQ ID NO:27 is shown in antisense orientation.

[0021] Figure 8 shows primer / probe set PPR22 aligned to portion 45683-45222 of the GenBank Accession No. OP555662.1 reference sequence. SEQ ID NOs:54 & 56 are shown in antisense orientation.

[0022] Figures 9A-9B show primer / probe sets PPR8, PPR9, PPR10, PPR11, PPR12, PPR19, PPR27, PPR28, PPR29, PPR30 and PPR31 aligned to a portion of the synthetic target nucleic acid of SEQ ID NO:64 representing the West Africa clade of monkeypox virus. SEQ ID NOs:23, 30, 31, & 46 are shown in antisense orientation.

[0023] EMBODIMENTS

[0024] Embodiment 1. A method for detecting the presence or absence of monkeypox virus (MPXV) in a biological sample, the method comprising: a) contacting the sample or nucleic acid extracted from said sample with reagents and an oligonucleotide combination capable of amplifying a target region of a monkeypox viral genome, wherein said oligonucleotide combination is selected from the group consisting of: i) a B7R oligonucleotide primer set specific for amplifying a target sequence in the B7R gene of the MPXV genome generating an MPXV amplification product comprising the sequence of SEQ ID NO: 65: catattacat aatatataca ggctatttac aagatgcacc g ( SEQ ID NO : 65 ) , and ii) a G2R oligonucleotide primer set specific for amplifying a target sequence in the G2R gene of the MPXV genome generating an MPXV amplification product comprising the sequence of SEQ ID NO: 63: cgtctcttcc acagataaat gcgaacccgt cgtaaccagc aatacattta actatatcga tgtggaaatt aacctgtatc cag ( SEQ ID NO : 63 ) b) performing an in vitro nucleic acid amplification reaction, wherein any MPXV nucleic acid present in the sample is used as a template to generate the MPXV amplification product corresponding to the oligonucleotide combination of i) or ii); and c) detecting the presence or absence of the MPXV amplification product generated in step b), wherein the presence of the MPXV amplification product is indicative of the presence of MPXV in the biological sample.

[0025] Embodiment 2. The method according to Embodiment 1, wherein a forward primer or forward primers for the B7R oligonucleotide primer set is / are selected from the group consisting of SEQ ID NOs:41 and 42, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:41 or 42, and an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:41 or 42.

[0026] Embodiment 3. The method according to Embodiment 2, wherein the B7R oligonucleotide primer set comprises two forward primers, the first forward primer having the sequence of SEQ ID NO:41, a sequence having at least 70% sequence identity with SEQ ID NO:41, or a sequence comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:41, and the second forward primer having the sequence of SEQ ID NO:42, a sequence having at least 70% sequence identity with SEQ ID NO:42, or a sequence comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:42. Embodiment 4. The method according to Embodiment 1, wherein a reverse primer for the B7R oligonucleotide primer set is selected from the group consisting of: SEQ ID NO:29, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:29, and an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:29.

[0027] Embodiment 5. The method according to Embodiment 1, wherein the B7R oligonucleotide primer set comprises an oligonucleotide probe and the presence of the target sequence amplified with the B7R oligonucleotide primer set is detected in step c) by a B7R probe capable of hybridizing to the amplified target sequence, wherein said B7R probe binds specifically to SEQ ID NO: 65 or a complement thereof.

[0028] Embodiment 6. The method according to Embodiment 5, wherein said B7R probe for the B7R oligonucleotide primer set is selected from the group consisting of SEQ ID NOs:09 and 35, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:09 or 35, and an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:09 or 35.

[0029] Embodiment 7. The method according to Embodiment 6, wherein said B7R probe for the B7R oligonucleotide primer set is selected from the group consisting of SEQ ID NO:35, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:35, and an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO 35.

[0030] Embodiment 8. The method according to Embodiment 6, wherein said B7R probe for the B7R oligonucleotide primer set is SEQ ID NO:09, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:09, or an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:09.

[0031] Embodiment 9. The method according to Embodiment 1, wherein the B7R oligonucleotide primer set comprises two forward primers, the first forward primer having the sequence of SEQ ID NO:41, the second forward primer having the sequence of SEQ ID NO:42, a reverse primer having the sequence of SEQ ID NO:29, and a B7R probe having the sequence of SEQ ID NO: 09 or 35. Embodiment 10. The method according to Embodiment 1, wherein a forward primer for the G2R oligonucleotide primer set is selected from the group consisting of SEQ ID NOs:05, and 20, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:05 or 20, and an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:05 or 20.

[0032] Embodiment 11. The method according to Embodiment 1, wherein a reverse primer for the G2R oligonucleotide primer set is selected from the group consisting of SEQ ID NOs:23, 30 and 31, an oligonucleotide having at least 70% sequence identity with SEQ ID NO: 23, 30, or 31, and an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO: 23, 30, or 31.

[0033] Embodiment 12. The method according to Embodiment 1, wherein the G2R oligonucleotide primer set comprises an oligonucleotide probe and the presence of the target sequence amplified with the G2R oligonucleotide primer set is detected in step c) by a G2R probe capable of hybridizing to the amplified target sequence, wherein said G2R probe binds specifically to SEQ ID NO: 63 or a complement thereof.

[0034] Embodiment 13. The method according to Embodiment 12, wherein said G2R probe for the G2R oligonucleotide primer set is selected from the group consisting of SEQ ID NOs: 15 and 47, an oligonucleotide having at least 70% sequence identity with SEQ ID NO: 15 or 47, and an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO: 15 or 47.

[0035] Embodiment 14. The method according to Embodiment 13, wherein said G2R probe for the G2R oligonucleotide primer set is selected from the group consisting of SEQ ID NO: 15, an oligonucleotide having at least 70% sequence identity with SEQ ID NO: 15, and an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO: 15.

[0036] Embodiment 15. The method according to Embodiment 13, wherein said G2R probe for the G2R oligonucleotide primer set is selected from the group consisting of SEQ ID NO:47, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:47, and an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:47. Embodiment 16. The method according to Embodiment 1, wherein the G2R oligonucleotide primer set comprises a forward primer having the sequence of SEQ ID NO:05, a reverse primer having the sequence of SEQ ID NO:30, and a G2R probe having the sequence of SEQ ID NO: 15 or 47.

[0037] Embodiment 17. The method according to Embodiment 1, further comprising a step of detecting the presence or absence of a non-variola orthopox virus (OPXV) in the biological sample.

[0038] Embodiment 18. The method according to Embodiment 17, wherein step a) further comprises contacting the sample with an E9L oligonucleotide primer set capable of amplifying an E9L gene target region of the OPXV, wherein at step b) any OPXV nucleic acid present in the sample is used as a template to generate an OPXV amplification product; and wherein at step c) the presence or absence of the OPXV amplification product generated in the step b) is detected, wherein the presence of the OPXV amplification product is indicative of the presence of OPXV in the biological sample.

[0039] Embodiment 19. The method according to Embodiment 18, wherein the E9L oligonucleotide primer set comprises a forward primer comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs:07, 17, 21, and 60, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:07, 17, 21, or 60, and an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:07, 17, 21, or 60.

[0040] Embodiment 20. The method according to Embodiment 18, wherein the E9L oligonucleotide primer set comprises a reverse primer comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs:01, 12, 40, and 61, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:01, 12, 40, or 61, and an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:01, 12, 40, or 61.

[0041] Embodiment 21. The method according to Embodiment 18, wherein the E9L oligonucleotide primer set comprises an oligonucleotide probe and the presence of the second target sequence amplified with the oligonucleotide primer is detected in step c) by an E9L probe capable of hybridizing to the amplified E9L target sequence. Embodiment 22. The method according to Embodiment 21, wherein said E9L probe is selected from the group consisting of SEQ ID NOs:06, 39, and 62, an oligonucleotide having at least 70% sequence identity with SEQ ID NO: 06, 39, or 62, an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO: 06, 39, or 62, and complements thereof.

[0042] Embodiment 23. The method according to Embodiment 18, wherein the E9L oligonucleotide primer set comprises a forward primer comprising a nucleic acid sequence of SEQ ID NO:60, a reverse primer comprising a nucleic acid sequence of SEQ ID NO:61, and an E9L probe comprising a nucleic acid sequence of SEQ ID NO:62.

[0043] Embodiment 24. The method according to Embodiment 1, wherein said B7R oligonucleotide primer set amplifies the target sequence providing an amplicon comprising at least the sequence of SEQ ID NO:66 of the B7R gene: tgtgtatgga agaagtaaca ccatattaca taatatatac aggctattta caagatgcac cgttatacag ttcgatgata tagtac ( SEQ ID NO : 66 ) .

[0044] Embodiment 25. The method according to Embodiment 18, wherein the B7R oligonucleotide primer set comprises two forward primers, the first forward primer having the sequence of SEQ ID NO:41, the second forward primer having the sequence of SEQ ID NO:42, a reverse primer having the sequence of SEQ ID NO:29, and a B7R probe having the sequence of SEQ ID NO:35, and wherein the E9L oligonucleotide primer set comprises a forward primer having the sequence of SEQ ID NO:60, a reverse primer having the sequence of SEQ ID NO:61, and an E9L probe having the sequence of SEQ ID NO:62.

[0045] Embodiment 26. The method according to Embodiment 1, wherein said G2R oligonucleotide primer set amplifies the first target sequence providing an amplicon comprising at least the sequence of SEQ ID NO:67 of the G2R gene: acatattctc acaccgtctc ttccacagat aaatgcgaac ccgtcgtaac cagcaataca tttaactata tcgatgtgga aattaacctg tatccagtca acgac ( SEQ ID NO : 67 ) .

[0046] Embodiment 27. The method according to Embodiment 18, wherein the G2R oligonucleotide primer set comprises a forward primer having the sequence of SEQ ID NO:05, a reverse primer having the sequence of SEQ ID NO:30, and a G2R probe having the sequence of SEQ ID NO: 15 or 47; and wherein the E9L oligonucleotide primer set comprises a forward primer having the sequence of SEQ ID NO:60, a reverse primer having the sequence of SEQ ID NO:61, and an E9L probe having the sequence of SEQ ID NO:62.

[0047] Embodiment 28. The method according to Embodiment 18, wherein the B7R oligonucleotide primer set comprises two forward primers, the first forward primer having the sequence of SEQ ID NO:41, the second forward primer having the sequence of SEQ ID NO:42, a reverse primer having the sequence of SEQ ID NO:29, and a B7R probe having the sequence of SEQ ID NO:35, wherein the G2R oligonucleotide primer set comprises a forward primer having the sequence of SEQ ID NO:05, a reverse primer having the sequence of SEQ ID NO: 30, and a G2R probe having the sequence of SEQ ID NO: 15 or 47, and wherein the E9L oligonucleotide primer set comprises a forward primer having the sequence of SEQ ID NO: 60, a reverse primer having the sequence of SEQ ID NO:61, and an E9L probe having the sequence of SEQ ID NO:62.

[0048] Embodiment 29. The method according to Embodiment 1, wherein said oligonucleotide primer sets comprise one or more oligonucleotide probes, each probe comprising a detectable label.

[0049] Embodiment 30. The method according to Embodiment 29, wherein the detectable label is a fluor ophore.

[0050] Embodiment 31. The method according to Embodiment 29, wherein each probe comprises a quencher moiety.

[0051] Embodiment 32. The method according to Embodiment 1, wherein the biological sample is skin swab, lesion swab, oropharyngeal swab, or a blood product.

[0052] Embodiment 33. The method according to Embodiment 1, wherein the in vitro nucleic acid amplification reaction is a multiplex amplification reaction.

[0053] Embodiment 34. An oligonucleotide combination selected from the group consisting of: i) a B7R oligonucleotide primer set comprising a forward primer or forward primers selected from the group consisting of SEQ ID NOs:41 and 42, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:41 or 42, an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:41 or 42, and combinations thereof, wherein a reverse primer for the oligonucleotide primer set is SEQ ID NO:29, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:29, or an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:29, and wherein the B7R oligonucleotide primer set can be used for amplifying a target sequence in the B7R gene of a monkeypox virus (MPXV); and ii) a G2R oligonucleotide primer set comprising a forward primer selected from the group consisting of SEQ ID NOs:05 and 20, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:05 or 20, and an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO: 05 or 20, wherein a reverse primer for the oligonucleotide primer set is selected from the group consisting of SEQ ID NOs:23, 30, and 31, an oligonucleotide having at least 70% sequence identity with SEQ ID NO: 23, 30, or 31, or an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO: 23, 30, or 31, and wherein the G2R oligonucleotide primer set can be used for amplifying a target sequence in the G2R gene of a monkeypox virus (MPXV).

[0054] Embodiment 35. The oligonucleotide combination according to Embodiment 34, wherein the B7R oligonucleotide primer set comprises two forward primers, the first forward primer having the sequence of SEQ ID NO:41, a sequence having at least 70% sequence identity with SEQ ID NO:41, or a sequence comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:41, and the second forward primer having the sequence of SEQ ID NO:42, a sequence having at least 70% sequence identity with SEQ ID NO:42, or a sequence comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:42.

[0055] Embodiment 36. The oligonucleotide combination according to Embodiment 34, wherein the B7R oligonucleotide primer set comprises a B7R oligonucleotide probe, wherein said B7R probe binds specifically to SEQ ID NO: 65 or a complement thereof.

[0056] Embodiment 37. The oligonucleotide combination according to Embodiment 36, wherein said B7R probe is selected from the group consisting of SEQ ID NOs:09 and 35, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:09 or 35, an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:09 or 35.

[0057] Embodiment 38. The oligonucleotide combination according to Embodiment 37, wherein said B7R probe is SEQ ID NO:35, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:35, or an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:35.

[0058] Embodiment 39. The oligonucleotide combination according to Embodiment 37, wherein said B7R probe is SEQ ID NO:09, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:09, or an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:09.

[0059] Embodiment 40. The oligonucleotide combination according to Embodiment 34, wherein the B7R oligonucleotide primer set comprises two forward primers, the first forward primer having the sequence of SEQ ID NO: 41, the second forward primer having the sequence of SEQ ID NO:42, a reverse primer having the sequence of SEQ ID NO:29, and a probe having the sequence of SEQ ID NO: 09 or 35.

[0060] Embodiment 41. The oligonucleotide combination according to Embodiment 34, wherein the B7R oligonucleotide primer set can be used for amplifying a target sequence in the B7R gene of a monkeypox virus (MPXV) providing an amplicon comprising at least the sequence of SEQ ID NO:66 of the B7R gene: tgtgtatgga agaagtaaca ccatattaca taatatatac aggctattta caagatgcac cgttatacag ttcgatgata tagtac ( SEQ ID NO : 66 ) .

[0061] Embodiment 42. The oligonucleotide combination according to Embodiment 34, wherein the G2R oligonucleotide primer set comprises a G2R oligonucleotide probe, wherein said G2R probe binds specifically to SEQ ID NO: 67 or a complement thereof.

[0062] Embodiment 43. The oligonucleotide combination according to Embodiment 42, wherein said G2R probe is selected from the group consisting of SEQ ID NOs:15 and 47, an oligonucleotide having at least 70% sequence identity with SEQ ID NO: 15 or 47, an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO: 15 or 47. Embodiment 44. The oligonucleotide combination according to Embodiment 43, wherein said G2R probe is SEQ ID NO: 15, an oligonucleotide having at least 70% sequence identity with SEQ ID NO: 15, or an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO: 15.

[0063] Embodiment 45. The oligonucleotide combination according to Embodiment 43, wherein said G2R probe is SEQ ID NO:47, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:47, or an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:47.

[0064] Embodiment 46. The oligonucleotide combination according to Embodiment 34, wherein the G2R oligonucleotide primer set comprises a forward primer having the sequence of SEQ ID NO:05, a reverse primer having the sequence of SEQ ID NO:30, and a probe having the sequence of SEQ ID NO: 15 or 47.

[0065] Embodiment 47. The oligonucleotide combination according to Embodiment 34, wherein the G2R oligonucleotide primer set comprises a second G2R oligonucleotide probe.

[0066] Embodiment 48. The oligonucleotide combination according to Embodiment 47, wherein said second G2R probe is selected from the group consisting of SEQ ID NOs:06, 39 and 62, an oligonucleotide having at least 70% sequence identity with SEQ ID NO: 06, 39, or 62, and an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO: 06, 39, or 62, and complements thereof.

[0067] Embodiment 49. The oligonucleotide combination according to Embodiment 34, wherein the G2R oligonucleotide primer set can be used for amplifying a target sequence in the G2R gene of a monkeypox virus (MPXV) providing an amplicon comprising at least the sequence of SEQ ID NO:67 of the G2R gene: acatattctc acaccgtctc ttccacagat aaatgcgaac ccgtcgtaac cagcaataca tttaactata tcgatgtgga aattaacctg tatccagtca acgac ( SEQ ID NO : 67 ) .

[0068] Embodiment 50. The oligonucleotide combination according to Embodiment 34 further comprising an E9L oligonucleotide primer set comprising a forward primer comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs:07, 17, 21, and 60, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:07, 17, 21, or 60, and an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO: 07, 17, 21, or 60, wherein the E9L oligonucleotide primer pair comprises a reverse primer comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs:01, 12, 40 and 61, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:01, 12, 40 or 61, and an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:01, 12, 40, or 61, and wherein the E9L oligonucleotide primer pair can be used for amplifying a target sequence in the E9L gene of a non-variola orthopoxvirus (OPXV).

[0069] Embodiment 51. The oligonucleotide combination according to Embodiment 50, wherein the oligonucleotide primer set comprises an E9L oligonucleotide probe.

[0070] Embodiment 52. The oligonucleotide combination according to Embodiment 51, wherein said E9L probe is selected from the group consisting of SEQ ID NOs:06, 39, and 62, an oligonucleotide having at least 70% sequence identity with SEQ ID NO: 06, 39, or 62, an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO: 06, 39, or 62, and complements thereof.

[0071] Embodiment 53. The oligonucleotide combination according to Embodiment 52, wherein the E9L oligonucleotide primer pair comprises a forward primer comprising a nucleic acid sequence of SEQ ID NO:60, a reverse primer comprising a nucleic acid sequence of SEQ ID NO:61, and the E9L probe comprising a nucleic acid sequence of SEQ ID NO:62.

[0072] Embodiment 54. The oligonucleotide combination according to Embodiment 34, wherein the combination comprises a B7R oligonucleotide primer set comprising two forward primers, the first forward primer having the sequence of SEQ ID NO:41, the second forward primer having the sequence of SEQ ID NO:42, a reverse primer having the sequence of SEQ ID NO:29, and a B7R probe having the sequence of SEQ ID NO:35 for amplifying a target sequence in the B7R gene of a monkeypox virus (MPXV); and wherein the oligonucleotide combination further comprises an E9L oligonucleotide primer set comprising a forward primer having the sequence of SEQ ID NO:60, a reverse primer having the sequence of SEQ ID NO:61, and an E9L probe having the sequence of SEQ ID NO:62 for amplifying a target sequence in the E9L gene of a non-variola orthopoxvirus (OPXV). Embodiment 55. The oligonucleotide combination according to Embodiment 34, wherein the combination comprises a G2R oligonucleotide primer set comprising a forward primer having the sequence of SEQ ID NO:05, a reverse primer having the sequence of SEQ ID NO: 30, and a G2R probe having the sequence of SEQ ID NO: 15 or 47 for amplifying a target sequence in the G2R gene of a monkeypox virus (MPXV); and wherein combination further comprises an E9L oligonucleotide primer set comprising a forward primer having the sequence of SEQ ID NO: 60, a reverse primer having the sequence of SEQ ID NO:61, and an E9L probe having the sequence of SEQ ID NO:62 for amplifying a target sequence in the E9L gene of a non-variola orthopoxvirus (OPXV).

[0073] Embodiment 56. The oligonucleotide combination according to Embodiment 34, wherein said oligonucleotide primer sets comprise one or more oligonucleotide probes, each probe comprising a detectable label.

[0074] Embodiment 57. The oligonucleotide combination according to Embodiment 56, wherein the detectable label is a fluorophore.

[0075] Embodiment 58. The oligonucleotide combination according to Embodiment 56, wherein each probe comprises a quencher moiety.

[0076] Embodiment 59. An oligonucleotide primer set comprising a forward primer comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs:07, 17, and 21, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:07, 17, or 21, and an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:07, 17, or 21, a reverse primer comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs:01, 12, and 40, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:01, 12, or 40, and an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:01, 12, or 40, and wherein the oligonucleotide primer pair can be used for amplifying a target sequence in the E9L gene of a non-variola orthopoxvirus (OPXV).

[0077] Embodiment 60. The oligonucleotide primer set according to Embodiment 59 comprising an oligonucleotide probe selected from the group consisting of SEQ ID NOs:06 and 39, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:06 or 39, an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO: 06 or 39, and complements thereof. Embodiment 61. The oligonucleotide primer set according to Embodiment 59 comprising a forward primer having the sequence of SEQ ID NO: 07, a reverse primer having a nucleic acid sequence of SEQ ID NO: 12, and a probe having a nucleic acid sequence of SEQ ID NO:06.

[0078] Embodiment 62. A kit for detecting the presence of monkeypox virus (MPXV) in a biological sample comprising at least one of the following: i) the oligonucleotide combination according to any one of Embodiments 34-58; and ii) the oligonucleotide primer set according to any one of Embodiments 59-61.

[0079] Embodiment 63. A method for detecting the presence of monkeypox virus (MPXV) in a biological sample, the method comprising: a) contacting the sample or nucleic acid extracted from said sample with reagents suitable for performing a nucleic acid amplification reaction, wherein said reagents comprise an oligonucleotide primer set specific for amplifying a target sequence in the monkeypox viral genome; b) performing said nucleic acid amplification reaction in order to amplify the target sequence; and c) detecting the presence or absence of the amplification products obtained in step b), wherein the presence of the amplification products amplified by the oligonucleotide primer set is indicative of the presence of MPXV in the sample and wherein the absence of the amplification products amplified by the oligonucleotide primer set is indicative of the absence of MPXV in the sample; wherein said oligonucleotide primer set comprises a forward primer having the sequence selected from the group consisting of SEQ ID NOs:10 and 26, a reverse primer having the sequence selected from the group consisting of sequence of SEQ ID NO:02 and 14, and a probe having the sequence selected from the group consisting of SEQ ID NOs:04, 19, 38, and 44.

[0080] Embodiment 64. The method of Embodiment 63, wherein i) said oligonucleotide primer set comprises a forward primer having the sequence of SEQ ID NO: 10, a reverse primer having the sequence of SEQ ID NO: 14, and a probe having the sequence of SEQ ID NO:04; wherein ii) said oligonucleotide primer set comprises a forward primer having the sequence of SEQ ID NO: 10, a reverse primer having the sequence of SEQ ID NO: 14, and two probes having the sequence of SEQ ID NOs:38 and 44, wherein iii) said oligonucleotide primer set comprises a forward primer having the sequence of SEQ ID NO:26, a reverse primer having the sequence of SEQ ID NO:02, and a probe having the sequence of SEQ ID NO: 19; or wherein said iv) said oligonucleotide primer set comprises a forward primer having the sequence of SEQ ID NO: 10, a reverse primer having the sequence of SEQ ID NO: 14, and a probe having the sequence of SEQ ID NO: 19.

[0081] Embodiment 65. An oligonucleotide primer set comprising a forward primer selected from the group consisting of SEQ ID NOs: 10 and 26, a reverse primer selected from the group consisting of SEQ ID NOs: 02 and 14.

[0082] Embodiment 66. The oligonucleotide primer set according to Embodiment 65 comprising a probe selected from the group consisting of SEQ ID NOs:04, 19, 38, and 44.

[0083] Embodiment 67. The oligonucleotide primer set according to Embodiment 66, wherein each probe comprises a detectable label.

[0084] Embodiment 68. The oligonucleotide primer set according to Embodiment 67, wherein the detectable label is a fluorophore.

[0085] Embodiment 69. The oligonucleotide primer set according to Embodiment 67, wherein each probe comprises a quencher moiety.

[0086] Embodiment 70. The oligonucleotide primer set according to Embodiment 64, wherein i) said oligonucleotide primer set comprises a forward primer having the sequence of SEQ ID NO: 10, a reverse primer having the sequence of SEQ ID NO: 14, and a probe having the sequence of SEQ ID NO: 04; wherein ii) said oligonucleotide primer set comprises a forward primer having the sequence of SEQ ID NO: 10, a reverse primer having the sequence of SEQ ID NO: 14, and two probes having the sequence of SEQ ID NOs:38 and 44, wherein iii) said oligonucleotide primer set comprises a forward primer having the sequence of SEQ ID NO:26, a reverse primer having the sequence of SEQ ID NO:02, and a probe having the sequence of SEQ ID NO: 19; or wherein said iv) said oligonucleotide primer set comprises a forward primer having the sequence of SEQ ID NO: 10, a reverse primer having the sequence of SEQ ID NO: 14, and a probe having the sequence of SEQ ID NO: 19.

[0087] Embodiment 71. A method for detecting the presence of monkeypox virus (MPXV) of Congo Basin clade in a biological sample, the method comprising: a) contacting the sample or nucleic acid extracted from said sample with reagents suitable for performing a nucleic acid amplification reaction, wherein said reagents comprise an oligonucleotide primer set specific for amplifying a target sequence in the monkeypox viral genome, wherein said target sequence is located in the genome of monkeypox virus (MPXV) of Congo Basin clade; b) performing said nucleic acid amplification reaction in order to amplify the target sequence; and c) detecting the presence or absence of the amplification products obtained in step b), wherein the presence of the amplification products amplified by the oligonucleotide primer set is indicative of the presence of MPXV of Congo Basin clade in the sample and wherein the absence of the amplification products amplified by the oligonucleotide primer set is indicative of the absence of MPXV of Congo Basin clade in the sample; wherein said oligonucleotide primer set amplifies the target sequence providing an amplicon comprising at least the sequence of SEQ ID NO:68 from the genome of MPXV of Congo Basin clade: tccacccacg tgtcagattg ttaaatgtcc acatcctata tcaaacggaa ( SEQ ID NO : 68 ) .

[0088] Embodiment 72. The method according to Embodiment 71, wherein a forward primer for the oligonucleotide primer set is selected from the group consisting of SEQ ID NO:43, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:43, and an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:43.

[0089] Embodiment 73. The method according to Embodiment 71, wherein a reverse primer for the oligonucleotide primer set is selected from the group consisting of SEQ ID NO:33, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:33, and an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:33.

[0090] Embodiment 74. The method according to Embodiment 71, wherein the oligonucleotide primer set comprises a first oligonucleotide probe and the presence of the target sequence amplified with the oligonucleotide primer is detected in step c) by a probe capable of hybridizing to the amplified target sequence, wherein said probe binds specifically to SEQ ID NO: 68 or a complement thereof.

[0091] Embodiment 75. The method according to Embodiment 74, wherein said probe for the oligonucleotide primer set is selected from the group consisting of SEQ ID NOs:08 and 34, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:08 or 34, and an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:08 or 34.

[0092] Embodiment 76. The method according to Embodiment 71, wherein the oligonucleotide primer set comprises a forward primer having the sequence of SEQ ID NO:43, a reverse primer having the sequence of SEQ ID NO:33, and a probe having the sequence of SEQ ID NO:08 or 34.

[0093] Embodiment 77. An oligonucleotide primer set comprising a forward primer for the oligonucleotide primer set is selected from the group consisting of SEQ ID NO:43, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:43, and an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:43, and a reverse primer for the oligonucleotide primer set is selected from the group consisting of SEQ ID NO:33, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:33, and an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:33.

[0094] Embodiment 78. The oligonucleotide primer set according to Embodiment 77, wherein the oligonucleotide primer set comprises an oligonucleotide probe and the presence of the target sequence amplified with the oligonucleotide primer is detected in step c) by a probe capable of hybridizing to the amplified target sequence, wherein said probe binds specifically to SEQ ID NO: 68 or a complement thereof. Embodiment 79. The oligonucleotide primer set according to Embodiment 78, wherein said probe for the oligonucleotide primer set is selected from the group consisting of SEQ ID NOs:08 and 34, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:08 or 34, and an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:08 or 34.

[0095] Embodiment 80. The oligonucleotide primer set according to Embodiment 78, wherein each probe comprises a detectable label.

[0096] Embodiment 81. The oligonucleotide primer set according to Embodiment 78, wherein the detectable label is a fluorophore.

[0097] Embodiment 82. The oligonucleotide primer set according to Embodiment 78, wherein each probe comprises a quencher moiety.

[0098] Embodiment 83. The oligonucleotide primer set according to Embodiment 77, wherein the oligonucleotide primer set comprises a forward primer having the sequence of SEQ ID NO:43, a reverse primer having the sequence of SEQ ID NO:33, and a probe having the sequence of SEQ ID NO:08 or 34.

[0099] Embodiment 84. A method for detecting the presence of monkeypox virus (MPXV) of Congo Basin clade in a biological sample, the method comprising: a) contacting the sample or nucleic acid extracted from said sample with reagents suitable for performing a nucleic acid amplification reaction, wherein said reagents comprise an oligonucleotide primer set specific for amplifying a target sequence in the monkeypox viral genome, wherein said target sequence is located in the genome of monkeypox virus (MPXV) of Congo Basin clade; b) performing said nucleic acid amplification reaction in order to amplify the target sequence; and c) detecting the presence or absence of the amplification products obtained in step b), wherein the presence of the amplification products amplified by the oligonucleotide primer set is indicative of the presence of MPXV of Congo Basin clade in the sample and wherein the absence of the amplification products amplified by the oligonucleotide primer set is indicative of the absence of MPXV of Congo Basin clade in the sample; wherein said oligonucleotide primer set amplifies the target sequence providing an amplicon comprising at least the sequence of SEQ ID NO:69 from the genome of MPXV of Congo Basin clade: agctcacgtt taagagattc aatttccagt ttgtacattg atttcattat tacgtccgca ( SEQ ID NO : 69 ) .

[0100] Embodiment 85. The method according to Embodiment 84, wherein a forward primer for the oligonucleotide primer set is selected from the group consisting of SEQ ID NO:24, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:24, and an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:24.

[0101] Embodiment 86. The method according to Embodiment 84, wherein a reverse primer for the oligonucleotide primer set is selected from the group consisting of SEQ ID NO:32, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:32, and an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:32.

[0102] Embodiment 87. The method according to Embodiment 84, wherein the oligonucleotide primer set comprises a first oligonucleotide probe and the presence of the target sequence amplified with the oligonucleotide primer is detected in step c) by a probe capable of hybridizing to the amplified target sequence, wherein said probe binds specifically to SEQ ID NO: 69 or a complement thereof.

[0103] Embodiment 88. The method according to Embodiment 87, wherein said probe for the oligonucleotide primer set is selected from the group consisting of SEQ ID NO:03, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:03, and an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:03.

[0104] Embodiment 89. The method according to Embodiment 84, wherein the oligonucleotide primer set comprises a forward primer having the sequence of SEQ ID NO:24, a reverse primer having the sequence of SEQ ID NO:32, and a probe having the sequence of SEQ ID NO:03.

[0105] Embodiment 90. An oligonucleotide primer set comprising a forward primer for the oligonucleotide primer set is selected from the group consisting of SEQ ID NO:24, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:24, and an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:24, and a reverse primer for the oligonucleotide primer set is selected from the group consisting of SEQ ID NO:32, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:32, or an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:32.

[0106] Embodiment 91. The oligonucleotide primer set according to Embodiment 90, wherein the oligonucleotide primer set comprises an oligonucleotide probe and the presence of the target sequence amplified with the oligonucleotide primer is detected in step c) by a probe capable of hybridizing to the amplified target sequence, wherein said probe binds specifically to SEQ ID NO: 69 or a complement thereof.

[0107] Embodiment 92. The oligonucleotide primer set according to Embodiment 91, wherein said probe for the oligonucleotide primer set is selected from the group consisting of SEQ ID NOs:03, an oligonucleotide having at least 70% sequence identity with SEQ ID NOs:03, and an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NOs:03.

[0108] Embodiment 93. The oligonucleotide primer set according to Embodiment 91, wherein each probe comprises a detectable label.

[0109] Embodiment 94. The oligonucleotide primer set according to Embodiment 93, wherein the detectable label is a fluorophore.

[0110] Embodiment 95. The oligonucleotide primer set according to Embodiment 93, wherein each probe comprises a quencher moiety.

[0111] Embodiment 96. The oligonucleotide primer set according to Embodiment 90, wherein the oligonucleotide primer set comprises a forward primer having the sequence of SEQ ID NO:24, a reverse primer having the sequence of SEQ ID NO:32, and a probe having the sequence of SEQ ID NO:03.

[0112] Embodiment 97. A method for detecting the presence of monkeypox virus (MPXV) of West African clade in a biological sample, the method comprising: a) contacting the sample or nucleic acid extracted from said sample with reagents suitable for performing a nucleic acid amplification reaction, wherein said reagents comprise an oligonucleotide primer set specific for amplifying a target sequence in the monkeypox viral genome, wherein said target sequence is located in the genome of monkeypox virus (MPXV) of West African clade; b) performing said nucleic acid amplification reaction in order to amplify the target sequence; and c) detecting the presence or absence of the amplification products obtained in step b), wherein the presence of the amplification products amplified by the oligonucleotide primer set is indicative of the presence of MPXV of West african clade in the sample and wherein the absence of the amplification products amplified by the oligonucleotide primer set is indicative of the absence of MPXV of West African clade in the sample; wherein said oligonucleotide primer set amplifies the target sequence providing an amplicon comprising at least the sequence of SEQ ID NO:70 from the genome of MPXV of West African clade: aatctcagat cttgaaatac agttgaacga ctgcgaacgt aataatgaaa tcaatgc ( SEQ ID NO : 70 ) .

[0113] Embodiment 98. The method according to Embodiment 97, wherein a forward primer for the oligonucleotide primer set is selected from the group consisting of SEQ ID NO:28, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:28, and an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:28.

[0114] Embodiment 99. The method according to Embodiment 97, wherein a reverse primer for the oligonucleotide primer set is selected from the group consisting of SEQ ID NO:27, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:27, or an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:27.

[0115] Embodiment 100. The method according to Embodiment 97, wherein the oligonucleotide primer set comprises a first oligonucleotide probe and the presence of the target sequence amplified with the oligonucleotide primer is detected in step c) by a probe capable of hybridizing to the amplified target sequence, wherein said probe binds specifically to SEQ ID NO:70 or a complement thereof. Embodiment 101. The method according to Embodiment 100, wherein said probe for the oligonucleotide primer set is selected from the group consisting of SEQ ID NO: 11, an oligonucleotide having at least 70% sequence identity with SEQ ID NO: 11, and an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NOT E

[0116] Embodiment 102. The method according to Embodiment 97, wherein the oligonucleotide primer set comprises a forward primer having the sequence of SEQ ID NO:28, a reverse primer having the sequence of SEQ ID NO:27, and a probe having the sequence of SEQ ID NOT E

[0117] Embodiment 103. An oligonucleotide primer set comprising a forward primer for the oligonucleotide primer set is selected from the group consisting of SEQ ID NO:28, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:28, and an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:28, and a reverse primer for the oligonucleotide primer set is selected from the group consisting of SEQ ID NO:27, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:27, and an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:27.

[0118] Embodiment 104. The oligonucleotide primer set according to Embodiment 103, wherein the oligonucleotide primer set comprises an oligonucleotide probe and the presence of the target sequence amplified with the oligonucleotide primer is detected in step c) by a probe capable of hybridizing to the amplified target sequence, wherein said probe binds specifically to SEQ ID NO:70 or a complement thereof.

[0119] Embodiment 105. The oligonucleotide primer set according to Embodiment 104, wherein said probe for the oligonucleotide primer set is selected from the group consisting of SEQ ID NO: 11, an oligonucleotide having at least 70% sequence identity with SEQ ID NO: 11, and an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NOT E

[0120] Embodiment 106. The oligonucleotide primer set according to Embodiment 105, wherein each probe comprises a detectable label. Embodiment 107. The oligonucleotide primer set according to Embodiment 103, wherein the detectable label is a fluorophore.

[0121] Embodiment 108. The oligonucleotide primer set according to Embodiment 106, wherein each probe comprises a quencher moiety.

[0122] Embodiment 109. The oligonucleotide primer set according to Embodiment 103, wherein the oligonucleotide primer set comprises a forward primer having the sequence of SEQ ID NO:28, a reverse primer having the sequence of SEQ ID NO:27, and a probe having the sequence of SEQ ID NO: 11.

[0123] Embodiment 110. A method for detecting the presence of monkeypox virus (MPXV) in a biological sample, the method comprising: a) contacting the sample or nucleic acid extracted from said sample with reagents suitable for performing a nucleic acid amplification reaction, wherein said reagents comprise an oligonucleotide primer set specific for amplifying a target sequence in the monkeypox viral genome; b) performing said nucleic acid amplification reaction in order to amplify the target sequence; and c) detecting the presence or absence of the amplification products obtained in step b), wherein the presence of the amplification products amplified by the oligonucleotide primer set is indicative of the presence of MPXV in the sample and wherein the absence of the amplification products amplified by the oligonucleotide primer set is indicative of the absence of MPXV in the sample; wherein said oligonucleotide primer set comprises a forward primer having the sequence of SEQ ID NO: 13, a reverse primer having the sequence of sequence of SEQ ID NO: 37, and a probe having the sequence of SEQ ID NO:25.

[0124] Embodiment 111. An oligonucleotide primer set comprising a forward primer having the sequence of SEQ ID NO: 13, a reverse primer having the sequence of SEQ ID NO: 37, and a probe having the sequence of SEQ ID NO:25.

[0125] Embodiment 112. The oligonucleotide primer set according to Embodiment 111, wherein each probe comprises a detectable label. Embodiment 113. The oligonucleotide primer set according to Embodiment 112, wherein the detectable label is a fluorophore.

[0126] Embodiment 114. The oligonucleotide primer set according to Embodiment 112, wherein each probe comprises a quencher moiety.

[0127] Embodiment 115. A kit for detecting the presence of monkeypox virus (MPXV) in a biological sample comprising at least one of the following: i) the oligonucleotide combination according to any one of Embodiments 34-58; ii) the oligonucleotide primer set according to any one of Embodiments 59-61; iii) the oligonucleotide primer set according to any one of Embodiments 65-70; iv) the oligonucleotide primer set according to any one of Embodiments 77-83; v) the oligonucleotide primer set according to any one of Embodiments 90-96; vi) the oligonucleotide primer set according to any one of Embodiments 103-109; and vii) the oligonucleotide primer set according to any one of Embodiments 111-114.

[0128] DEFINITIONS

[0129] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art pertinent to the methods and compositions described. As used herein, the following terms and phrases have the meanings ascribed to them unless specified otherwise.

[0130] The terms "a," "an," and "the" include plural referents, unless the context clearly indicates otherwise.

[0131] "Sample" includes any specimen that may contain an analyte. Samples include "biological samples," which include any tissue or material derived from a living or dead mammal (e.g., human).

[0132] A "nucleic acid" refers to a multimeric compound comprising nucleotides or analogs that have nitrogenous heterocyclic bases or base analogs linked together to form a polymer, including conventional RNA, DNA, mixed RNA-DNA, and analogs thereof. 1

[0133] "Oligonucleotide" refers to a nucleic acid of generally less than 1,000 nucleotides (nt), including those in a size range having a lower limit of about 5 nt and an upper limit of about 900 nt. The term oligonucleotide does not denote any particular function to the reagent; rather, it is used generically to cover all such reagents described herein. Oligonucleotides may be referred to by a functional name, e.g., capture probe, detection probe, primer, or promoter primer. In an amplification reaction, an oligonucleotide can hybridize to a nucleic acid analyte / template and participate in a nucleic acid amplification reaction, e.g., serving as a primer. In an amplification reaction, oligonucleotide primers can have 3' ends that are extended by polymerization as part of the nucleic acid amplification reaction. Amplification oligonucleotides may be optionally modified to include 5' non-target-specific sequences such as tags, or other sequences used or useful for manipulating or amplifying the primer or target oligonucleotide.

[0134] The term "target sequence" or "target nucleic acid sequence" as used herein refers to the particular nucleotide sequence of a nucleic acid analyte that is to be amplified and / or detected. The "target sequence" includes the complexing sequences to which oligonucleotides (e.g., priming oligonucleotides and / or promoter oligonucleotides) complex during an amplification processes (e.g, PCR, TMA). Unless the context clearly dictates otherwise, where the nucleic acid analyte is originally single-stranded, the term "target sequence" will also refer to the sequence complementary to the "target sequence" as present in the nucleic acid analyte, and where the nucleic acid analyte is originally double-stranded, the term "target sequence" refers to both the sense (+) and antisense (-) strands.

[0135] "Nucleic acid amplification" refers to any well-known in vitro procedure that produces multiple copies of a target nucleic acid sequence. Examples of such procedures include transcription-associated methods, e.g, transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA) and others (e.g., US Patent Nos. 5,399,491, 5,554,516, 5,437,990, 5,130,238, 4,868,105, and 5,124,246), replicase- mediated amplification (e.g., US Patent No. 4,786,600), polymerase chain reaction (PCR) (e.g., US Patent Nos. 4,683,195, 4,683,202, and 4,800,159), ligase chain reaction (LCR) (e.g., EP Patent No. 0320308), and strand-displacement amplification (SDA) (e.g., US Patent No. 5,422,252). “Amplification reagents” or “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). Particular amplification reagents, and combinations thereof, are dependent on the amplification method and conditions employed.

[0136] By "amplicon" is meant a nucleic acid molecule generated in a nucleic acid amplification reaction and which is derived from a nucleic acid analyte. An amplicon or amplification product contains a target nucleic acid sequence that may be of the same or opposite sense as the nucleic acid analyte.

[0137] "Detection probe oligomer", or "probe" refers to an oligomer that hybridizes specifically to a target sequence, including an amplified product, under conditions that promote nucleic acid hybridization, for detection of a nucleic acid analyte. Detection may either be direct (ie., probe hybridized directly to the target) or indirect (z.e., a probe hybridized to an intermediate structure that links the probe to the target). A probe's target-specific sequence generally refers to the specific sequence within a larger sequence which the probe hybridizes specifically. A detection probe may include target-specific sequence(s) and non-target-specific sequence(s). Such non-target-specific sequences can include sequences which will confer a desired secondary or tertiary structure, such as a hairpin structure, which can be used to facilitate detection and / or amplification.

[0138] DESCRIPTION

[0139] The present nucleic acid amplification-based method enables rapid and accurate detection of the MPXV virus in a biological sample. Primer and probe oligonucleotides and combinations thereof for detecting MPXV are provided, as is a kit containing such primer and probe oligonucleotides. In the present invention, the inventors have identified target regions with optimal inclusivity for all MPXV genomes. The first target region is located in the B7R gene of the MPXV genome. The second target region is located in the G2R gene of the MPXV genome. Other target regions have also been disclosed in the following Examples and Figures. The disclosed methods may include performing at least one cycling step that includes amplifying one or more portions of MPXV B7R gene nucleic acid target and MPXV G2R gene nucleic acid target from a sample using one or more pairs of B7R gene primers and / or one or more pairs of G2R gene primers. “B7R gene primers” or “G2R primers” as used herein refer to oligonucleotide primers that specifically anneal to nucleic acid sequence in the B7R gene and the G2R gene, respectively, and initiate DNA synthesis therefrom under appropriate conditions. Each of the discussed B7R gene or G2R gene primers anneals to a target within or adjacent to the respective target nucleic acid molecule such that at least a portion of each amplification product contains nucleic acid sequence corresponding to the target. The one or more of the B7R gene amplification products and / or the G2R gene amplification products are produced provided that one or more of the B7R gene nucleic acid and / or the G2R gene nucleic acid is present in the sample, thus the presence of these one or more of amplification products is indicative of the presence of MPXV in the sample. The amplification product should contain the nucleic acid sequences that are complementary to one or more detectable probes for the B7R gene or for the G2R gene. Each cycling step preferably includes an amplification step, a hybridization step, and a detection step, in which the sample is contacted with the one or more detectable probes for the B7R gene or for the G2R gene for detection of the presence or absence of MPXV in the sample.

[0140] In an embodiment, a B7R oligonucleotide primer set specific for amplifying a target sequence in the B7R gene of the MPXV genome generates an MPXV amplification product comprising the sequence of SEQ ID NO:65: catattacat aatatataca ggctatttac aagatgcacc g ( SEQ ID NO : 65 ) , and ii) a G2R oligonucleotide primer set specific for amplifying a target sequence in the G2R gene of the MPXV genome generates an MPXV amplification product comprising the sequence of SEQ ID NO: 63: cgtctcttcc acagataaat gcgaacccgt cgtaaccagc aatacattta actatatcga tgtggaaatt aacctgtatc cag ( SEQ ID NO : 63 ) .

[0141] In some embodiments, the presence of one or more signals from a detectable label of one or more detectable probes indicates hybridization of the one or more probe oligonucleotides to the one or more amplified monkeypox virus target nucleic acid sequences. "Label" or "detectable label" refers herein to a moiety or compound joined directly or indirectly to a probe that is detected or leads to a detectable signal. Direct joining may use covalent bonds or non-covalent interactions (e.g, hydrogen bonding, hydrophobic or ionic interactions, and chelate or coordination complex formation) whereas indirect joining may use a bridging moiety or linker (e.g., via an antibody or additional oligonucleotide(s), which amplify a detectable signal). Any detectable moiety may be used, e.g., radionuclide, ligand such as biotin or avidin, enzyme, enzyme substrate, reactive group, chromophore such as a dye or particle (e.g., latex or metal bead) that imparts a detectable color, luminescent compound (e.g., bioluminescent, phosphorescent, or chemiluminescent compound such as an acridinium ester ("AE") compound), and fluorescent compound ( . e. , fluorophore). Fluorophores may be used in combination with a quencher molecule that absorbs light when in close proximity to the fluorophore to diminish background fluorescence. Detectably labeled probes include, for example, hydrolysis (e.g, TaqMan™) probes, AE-labeled probes, molecular torches, and molecular beacons.

[0142] The disclosed amplification method is for detecting monkeypox in a biological sample. The biological sample can be any suitable sample obtained from any suitable subject, typically a mammal. Preferably, the subject is a human. In some embodiments, 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.

[0143] In some embodiments, the sample is a skin lesion specimen (e.g., fluid, tissue, or other material from a monkeypox lesion). In a preferred embodiment, the lesion specimen is a lesion swab.

[0144] The sample can be obtained from the subject using routine techniques known to those skilled in the art, and the sample may be used directly as obtained from the biological source or following a pretreatment to modify the character of the sample. Such pretreatment may include, for example, preparing plasma from blood, diluting viscous fluids, filtration, precipitation, dilution, mixing, concentration, inactivation of interfering components, the addition of reagents, lysing, extracting nucleic acids and the like. After the sample is obtained from a subject and preferably pre-treated, the sample or the pre-treated sample or a fraction thereof may be contacted with the set of oligonucleotides, comprising amplification oligonucleotides and probes as described herein, and amplification reagents to form a reaction mixture.

[0145] The disclosure also provides an oligonucleotide combination selected from the group consisting of: i) a B7R oligonucleotide primer set comprising a forward primer or forward primers selected from the group consisting of SEQ ID NOs:41 and 42, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:41 or 42, an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:41 or 42, and combinations thereof, wherein a reverse primer for the oligonucleotide primer set is SEQ ID NO:29, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:29, or an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:29, and wherein the B7R oligonucleotide primer set can be used for amplifying a target sequence in the B7R gene of a monkeypox virus (MPXV); and ii) a G2R oligonucleotide primer set comprising a forward primer selected from the group consisting of SEQ ID NOs:05 and 20, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:05 or 20, and an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO: 05 or 20, wherein a reverse primer for the oligonucleotide primer set is selected from the group consisting of SEQ ID NOs:23, 30, and 31, an oligonucleotide having at least 70% sequence identity with SEQ ID NO: 23, 30, or 31, or an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO: 23, 30, or 31, and wherein the G2R oligonucleotide primer set can be used for amplifying a target sequence in the G2R gene of a monkeypox virus (MPXV).

[0146] Further oligonucleotide combinations and primer sets are disclosed in Embodiments 65- 70, 77-83, 90-96, 103-109, and 111-114.

[0147] In some embodiments, the combination comprises oligonucleotides comprising a nucleic acid sequence with at least 70% (e.g., 75%., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any of the oligonucleotide primers or probes defined in Embodiments 1-114. In some embodiments, the combination comprises oligonucleotides comprising or consisting of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous nucleotides of any of the oligonucleotide primers or probes defined in Embodiments 1-114.

[0148] 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. In some embodiments, 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. Many such reagents are described herein or otherwise known in the art and commercially available. Examples of suitable reagents for inclusion in the kit (in addition to the oligonucleotides described herein) include conventional reagents employed in nucleic acid amplification reactions, such as, for example, one or more enzymes having polymerase activity, enzyme cofactors (such as magnesium or nicotinamide adenine dinucleotide (NAD)), salts, buffers, deoxyribonucleotide, or ribonucleotide triphosphates (dNTPs / rNTPs).

[0149] EXAMPLES

[0150] EXAMPLE 1A

[0151] Three primer and probe combinations (PPR14, PPR15 and PPR24) for amplification and detection of non-variola orthopoxvirus (OPX), and one primer and probe combination (PPR17) for amplification and detection of the Congo basin and West African clades of monkeypox (MPX) were designed and tested. Testing was done using real-time PCR amplification and detection, and the assays were run in multiplex formats. Primer and probe combinations are shown in Table A and the real-time PCR data is shown in Table B.

[0152] Amplification and detection reactions were performed using an ABI 7500 Fast Real-Time thermocycler (ThermoFisher Scientific, Waltham MA, cat no 4377355). 3 OPX Primer and probe sets (allowing the detection of non-variola orthopoxvirus) were tested in multiplex reactions with one BR7 primer and probe set (allowing the detection of the Congo basin and the West Africa MPX) for the detection of a synthetic monkeypox virus DNA (DNA ATCC, Manassas VA, Cat. No. VR-3270SD). The following multiplex combinations were tested: PPR24 / PPR17, PPR14 / PPR17, and PPR15 / PPR17. Stock concentration of the synthetic DNA was serially diluted in a Tris-EDTA buffer to provide reaction (rxn) concentrations of 5,000 copies / rxn, 500 copies / rxn, 50 copies / rxn, 5 copies / rxn, and 2.5 copies / rxn.

[0153] Amplification and detection reactions were prepared by combining in a well of a microwell plate 5 pl of a dilution condition with 20 pL of an amplification reaction mixture containing one or two of the primer and probe combinations according to Table A (see also Figs. 1 A-1C for PPR14, PPR15 and PPR24; and Figs. 2A-2C for PPR17). Each condition was run in a replicate of 4. SEQ ID NOs:06, 39, 62, & 35 each comprised fluorescein attached to the nucleotide at position 1. SEQ ID NOs:06, 39, 62, & 35 further comprised a black hole quencher at position 25, 22, 11, & 25, respectively. SEQ ID NO:62 comprised a 3’ reverse polarity C residue. The nucleotide sequences for SEQ ID

[0154] NOs:06, 39, & 35 contained propyne dU analogs and 5 methyl dC analogs.

[0155] Table A.

[0156] The amplification reaction was performed on the ABI 7500 Fast instrument programmed for 2 minutes at 95 degrees C followed by 45 cycles of 8 seconds at 95 degrees C and 30 seconds at 60 degrees C. Detection was performed in real time and cycle threshold (Ct) data was collected and is presented in Table B.

[0157] Table B.

[0158]

[0159] The data show that OPX primer / probe sets PPR14 and PPR24 work well in multiplex when associated with BR7 primers and probe. The synthetic monkeypox virus DNA is well detected by OPX and BR7 targets and gave similar Ct results (for OPX PPR mixes 14 and 24; and when comparing OPX results to BR7 results) and demonstrated a comparable sensitivity (100% detection down to 2.5 Copies per reaction). The OPX PPR mix 15 didn’t demonstrate the expected performance when tested in multiplex with the BR7 primers and probe (none of the DNA concentration tested gave a positive result). In conclusion OPX PPR14 and PPR24 are good candidates for the multiplex assay design. EXAMPLE IB

[0160] Two primer and probe sets (PPR19 and PPR27) targeting the West Africa clade of monkeypox virus were designed and tested in singleplex reactions. The primer and probe combinations are shown in Table C (see also Figs. 6A-6D). For both primer and probe combinations, the detection probe SEQ ID NO:47 was labeled with Cal Fluor Red 610 (CalRed) fluorophore at its 5’ end, an internal BHQ-2 quencher, and included a 3’ reverse polarity C residue.

[0161] Table C.

[0162] Target nucleic acid was SEQ ID NO:64 spiked into sample transport media at 360 copies per reaction. The PPRs were each tested at 8 replicates. Results show that PPR27 had earlier Cts and higher RFUs than did PPR19 (PPR19: 32.4 mean Ct and 4531 mean RFU; PPR27: 31.5 mean Ct and 7010 mean RFU). Thus, PPR27 showed better performance in this assay.

[0163] EXAMPLE 1C

[0164] Six sets of primers and probe targeting the West Africa clade of monkeypox virus were designed and tested in singleplex reactions. The primer and probe combinations are shown in Table D (see also Figs. 6A-6D). Both detection probes SEQ ID NO: 15 & 47 were labeled with FAM at the 5’ end and BHQ-1 at the 3’ end, and SEQ ID NO: 15 contained 5 methyl C modifications at positions 1-3, 6, 11, 12, 15, & 20 and propyne dU modifications at positions 5, 8, 18, & 22.

[0165] Table D.

[0166] Target nucleic acid was a synthetic MPX gDNA (DNA ATCC, Manassas VA, Cat. No. VR-3270SD) spiked into a Tris-EDTA buffer and serially diluted to provide concentrations of 5,000 copies / reaction, 500 copies / reaction, 50 copies / reaction, and 5 copies / reaction. Each primer and probe combination was tested in replicates of 4. Results from this example showed that PPR11 had the best performance at all concentrations (Table E).

[0167] Table E.

[0168] * Only 3 of 4 replicates amplified for PPR31 at 5 copies per reaction target concentration. EXAMPLE 2

[0169] A first primer and probe set targeting non-variola orthopox virus (PPR24) and a second primer and probe set (PPR27) targeting monkeypox virus were tested in singleplex and multiplex reactions to determine whether singleplex performance was impacted by a multiplex system. The primer and probe combinations are shown in Table F (see also Figs. 1 A-1C for PPR24 and Figs 6A-6D for PPR27). For this example, SEQ ID NO:62 was labeled with a 5’ FAM fluorophore, an internal BHQ-1 quencher, and a 3’ reverse polarity C residue; and SEQ ID NO:47 was labeled with a 5’ Quasar 705 and a 3’ BHQ-2 quencher.

[0170] Table F.

[0171] The performances of the selected oligos were evaluated in singleplex and multiplex. PCR was performed on plasmid DNA (single target) and on MPX synthetic gDNA (multi targets). DNAs were spiked into STM at four concentrations, ranging from 1E+02 c / mL to 1E+05 c / mL.

[0172] Multiplexing the oligos has no impact on Ct mean, fluorescence, or Signal / Noise, on single or multitarget DNA samples.

[0173] These results suggest that (i) combining oligos of all targets in multiplex does not decrease performance and (ii) amplifications of single-target or multi-targets samples have comparable performance.

[0174] EXAMPLE 3

[0175] A limit of detection (LoD) study was conducted using serial dilutions of a synthetic monkeypox gDNA (American Type Cell Culture, cat no. VR-3270SD) spiked into a negative clinical matrix. The monkeypox spiked matrix was diluted to 1000 copies / mL, 500 c / mL, 100 c / mL, 75 c / mL, and 50 c / mL. A target capture reaction was performed on each dilution, and the eluates were individually combined with an RT-PCR reaction mixture to provide reactions at 36 c / rxn, 18 c / rxn, 3.6 c / rxn, 2.7 c / rxn, and 1.8 c / rxn (10 replicates per concentration). The primer and probe combinations are shown in Table G. Elution buffer from the target capture of the sample transport medium was used as a negative control. Real-time PCR reactions were performed, and the results are shown in Table H.

[0176] Table G.

[0177] Table H. As shown in Table H, all sample replicates are detected at 100% (10 / 10) down to 18 copies per reaction (suggesting an LoD95% around that concentration), and at 30% (PPR24) and 40% (PPR27) at 1.8 copies per reaction.

[0178] To confirm the pre-LoD, a second serial dilution was prepared using the synthetic monkeypox gDNA (American Type Cell Culture, cat no. VR-3270SD) spiked into a negative clinical matrix, as described above. Monkeypox spiked matrix was diluted to 1400 copies / mL, 500 c / mL, and 250 c / mL. A target capture reaction was performed, and eluates were combined with an PCR reaction mixture to provide 50.4 c / rxn, 18 c / rxn, and 9 c / rxn, respectively. Reaction conditions were tested on 16 replicates and on 3 instruments (total of 48 replicates). Elution buffer from the target capture of the sample transport medium was used as a negative control. Real-time PCR reactions were performed, and the results are shown in Table J.

[0179] Table J. Table J confirms that the pre-LoD95% (limit of detection at 95%) for both targets (PPR

[0180] 24) is 18 copies per reaction as it gave >95% of positive calls. SEQUENCES

[0181] Table K provides nucleic acid sequences written 5’ to 3’ and using nucleotide symbols from WIPO Standard ST.26, Annex I, Section 1. MPXV means monkeypox virus. NV AR means Non-Variola. Table K.

[0182] Table L provides exemplary primer and probe combinations.

[0183] Table L.

[0184]

[0185]

Claims

CLAIMS1. A method for detecting the presence or absence of monkeypox virus (MPXV) in a biological sample, the method comprising: a) contacting the sample or nucleic acid extracted from said sample with reagents and an oligonucleotide combination capable of amplifying a target region of a monkeypox viral genome, wherein said oligonucleotide combination is selected from the group consisting of: i) a B7R oligonucleotide primer set specific for amplifying a target sequence in the B7R gene of the MPXV genome generating an MPXV amplification product comprising the sequence of SEQ ID NO: 65: catattacat aatatataca ggctatttac aagatgcacc g ( SEQ ID NO : 65 ) , and ii) a G2R oligonucleotide primer set specific for amplifying a target sequence in the G2R gene of the MPXV genome generating an MPXV amplification product comprising the sequence of SEQ ID NO: 63: cgtctcttcc acagataaat gcgaacccgt cgtaaccagc aatacattta actatatcga tgtggaaatt aacctgtatc cag ( SEQ ID NO : 63 ) b) performing an in vitro nucleic acid amplification reaction, wherein any MPXV nucleic acid present in the sample is used as a template to generate the MPXV amplification product corresponding to the oligonucleotide combination of i) or ii); and c) detecting the presence or absence of the MPXV amplification product generated in step b), wherein the presence of the MPXV amplification product is indicative of the presence of MPXV in the biological sample.

2. The method according to claim 1, wherein a forward primer or forward primers for the B7R oligonucleotide primer set is / are selected from the group consisting of SEQ ID NOs:41 and42, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:41 or 42, and an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:41 or 42.

3. The method according to claim 2, wherein the B7R oligonucleotide primer set comprises two forward primers, the first forward primer having the sequence of SEQ ID NO:41, a sequence having at least 70% sequence identity with SEQ ID NO:41, or a sequencecomprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:41, and the second forward primer having the sequence of SEQ ID NO:42, a sequence having at least 70% sequence identity with SEQ ID NO:42, or a sequence comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:42.

4. The method according to claim 1, wherein a reverse primer for the B7R oligonucleotide primer set is selected from the group consisting of: SEQ ID NO:29, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:29, and an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:29.

5. The method according to claim 1, wherein the B7R oligonucleotide primer set comprises an oligonucleotide probe and the presence of the target sequence amplified with the B7R oligonucleotide primer set is detected in step c) by a B7R probe capable of hybridizing to the amplified target sequence, wherein said B7R probe binds specifically to SEQ ID NO:65 or a complement thereof.

6. The method according to claim 5, wherein said B7R probe for the B7R oligonucleotide primer set is selected from the group consisting of SEQ ID NOs:09 and 35, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:09 or 35, and an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:09 or 35.

7. The method according to claim 6, wherein said B7R probe for the B7R oligonucleotide primer set is selected from the group consisting of SEQ ID NO:35, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:35, and an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:35.

8. The method according to claim 6, wherein said B7R probe for the B7R oligonucleotide primer set is SEQ ID NO:09, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:09, or an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:09.

9. The method according to claim 1, wherein the B7R oligonucleotide primer set comprises two forward primers, the first forward primer having the sequence of SEQ ID NO:41, the second forward primer having the sequence of SEQ ID NO:42, a reverse primer having the sequence of SEQ ID NO:29, and a B7R probe having the sequence of SEQ ID NO: 09 or 35.

10. The method according to claim 1, wherein a forward primer for the G2R oligonucleotide primer set is selected from the group consisting of SEQ ID NOs:05, and 20, anoligonucleotide having at least 70% sequence identity with SEQ ID NO:05 or 20, and an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:05 or 20.

11. The method according to claim 1, wherein a reverse primer for the G2R oligonucleotide primer set is selected from the group consisting of SEQ ID NOs:23, 30 and 31, an oligonucleotide having at least 70% sequence identity with SEQ ID NO: 23, 30, or 31, and an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO: 23, 30, or 31.

12. The method according to claim 1, wherein the G2R oligonucleotide primer set comprises an oligonucleotide probe and the presence of the target sequence amplified with the G2R oligonucleotide primer set is detected in step c) by a G2R probe capable of hybridizing to the amplified target sequence, wherein said G2R probe binds specifically to SEQ ID NO:63 or a complement thereof.

13. The method according to claim 12, wherein said G2R probe for the G2R oligonucleotide primer set is selected from the group consisting of SEQ ID NOs: 15 and 47, an oligonucleotide having at least 70% sequence identity with SEQ ID NO: 15 or 47, and an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO: 15 or 47.

14. The method according to claim 13, wherein said G2R probe for the G2R oligonucleotide primer set is selected from the group consisting of SEQ ID NO: 15, an oligonucleotide having at least 70% sequence identity with SEQ ID NO: 15, and an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO: 15.

15. The method according to claim 13, wherein said G2R probe for the G2R oligonucleotide primer set is selected from the group consisting of SEQ ID NO:47, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:47, and an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:47.

16. The method according to claim 1, wherein the G2R oligonucleotide primer set comprises a forward primer having the sequence of SEQ ID NO:05, a reverse primer having the sequence of SEQ ID NO:30, and a G2R probe having the sequence of SEQ ID NO: 15 or 47.

17. The method according to claim 1, further comprising a step of detecting the presence or absence of a non-variola orthopox virus (OPXV) in the biological sample.

18. The method according to claim 17, wherein step a) further comprises contacting the sample with an E9L oligonucleotide primer set capable of amplifying an E9L gene target region of the OPXV, wherein at step b) any OPXV nucleic acid present in the sample is used as a template to generate an OPXV amplification product; and wherein at step c) the presence or absence of the OPXV amplification product generated in the step b) is detected, wherein the presence of the OPXV amplification product is indicative of the presence of OPXV in the biological sample.

19. The method according to claim 18, wherein the E9L oligonucleotide primer set comprises a forward primer comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs:07, 17, 21, and 60, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:07, 17, 21, or 60, and an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:07, 17, 21, or 60.

20. The method according to claim 18, wherein the E9L oligonucleotide primer set comprises a reverse primer comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs:01, 12, 40, and 61, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:01, 12, 40, or 61, and an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:01, 12, 40, or 61.

21. The method according to claim 18, wherein the E9L oligonucleotide primer set comprises an oligonucleotide probe and the presence of the second target sequence amplified with the oligonucleotide primer is detected in step c) by an E9L probe capable of hybridizing to the amplified E9L target sequence.

22. The method according to claim 21, wherein said E9L probe is selected from the group consisting of SEQ ID NOs:06, 39, and 62, an oligonucleotide having at least 70% sequence identity with SEQ ID NO: 06, 39, or 62, an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO: 06, 39, or 62, and complements thereof.

23. The method according to claim 18, wherein the E9L oligonucleotide primer set comprises a forward primer comprising a nucleic acid sequence of SEQ ID NO:60, a reverse primer comprising a nucleic acid sequence of SEQ ID NO:61, and an E9L probe comprising a nucleic acid sequence of SEQ ID NO: 62.

24. The method according to claim 1, wherein said B7R oligonucleotide primer set amplifies the target sequence providing an amplicon comprising at least the sequence of SEQ ID NO:66 of the B7R gene: tgtgtatgga agaagtaaca ccatattaca taatatatac aggctattta caagatgcac cgttatacag ttcgatgata tagtac ( SEQ ID NO : 66 ) .

25. The method according to claim 18, wherein the B7R oligonucleotide primer set comprises two forward primers, the first forward primer having the sequence of SEQ ID NO:41, the second forward primer having the sequence of SEQ ID NO:42, a reverse primer having the sequence of SEQ ID NO:29, and a B7R probe having the sequence of SEQ ID NO:35, and wherein the E9L oligonucleotide primer set comprises a forward primer having the sequence of SEQ ID NO:60, a reverse primer having the sequence of SEQ ID NO:61, and an E9L probe having the sequence of SEQ ID NO: 62.

26. The method according to claim 1, wherein said G2R oligonucleotide primer set amplifies the first target sequence providing an amplicon comprising at least the sequence of SEQ ID NO: 67 of the G2R gene: acatattctc acaccgtctc ttccacagat aaatgcgaac ccgtcgtaac cagcaataca tttaactata tcgatgtgga aattaacctg tatccagtca acgac ( SEQ ID NO : 67 ) .

27. The method according to claim 18, wherein the G2R oligonucleotide primer set comprises a forward primer having the sequence of SEQ ID NO:05, a reverse primer having the sequence of SEQ ID NO:30, and a G2R probe having the sequence of SEQ ID NO: 15 or 47; and wherein the E9L oligonucleotide primer set comprises a forward primer having the sequence of SEQ ID NO:60, a reverse primer having the sequence of SEQ ID NO:61, and an E9L probe having the sequence of SEQ ID NO:62.

28. The method according to claim 18, wherein the B7R oligonucleotide primer set comprises two forward primers, the first forward primer having the sequence of SEQ ID NO:41, the second forward primer having the sequence of SEQ ID NO:42, a reverse primer having the sequence of SEQ ID NO:29, and a B7R probe having the sequence of SEQ ID NO:35, wherein the G2R oligonucleotide primer set comprises a forward primer having the sequence of SEQ ID NO:05, a reverse primer having the sequence of SEQ ID NO:30, and a G2R probe having the sequence of SEQ ID NO: 15 or 47, and wherein the E9L oligonucleotide primer setcomprises a forward primer having the sequence of SEQ ID NO:60, a reverse primer having the sequence of SEQ ID NO:61, and an E9L probe having the sequence of SEQ ID NO:62.

29. The method according to claim 1, wherein said oligonucleotide primer sets comprise one or more oligonucleotide probes, each probe comprising a detectable label.

30. The method according to claim 29, wherein the detectable label is a fluorophore.

31. The method according to claim 29, wherein each probe comprises a quencher moiety.

32. The method according to claim 1, wherein the biological sample is skin swab, lesion swab, oropharyngeal swab, or a blood product.

33. The method according to claim 1, wherein the in vitro nucleic acid amplification reaction is a multiplex amplification reaction.

34. An oligonucleotide combination selected from the group consisting of: i) a B7R oligonucleotide primer set comprising a forward primer or forward primers selected from the group consisting of SEQ ID NOs:41 and 42, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:41 or 42, an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:41 or 42, and combinations thereof, wherein a reverse primer for the oligonucleotide primer set is SEQ ID NO:29, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:29, or an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:29, and wherein the B7R oligonucleotide primer set can be used for amplifying a target sequence in the B7R gene of a monkeypox virus (MPXV); and ii) a G2R oligonucleotide primer set comprising a forward primer selected from the group consisting of SEQ ID NOs:05 and 20, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:05 or 20, and an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO: 05 or 20, wherein a reverse primer for the oligonucleotide primer set is selected from the group consisting of SEQ ID NOs:23, 30, and31, an oligonucleotide having at least 70% sequence identity with SEQ ID NO: 23, 30, or 31, or an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO: 23, 30, or 31, and wherein the G2R oligonucleotide primer set can be used for amplifying a target sequence in the G2R gene of a monkeypox virus (MPXV).

35. The oligonucleotide combination according to claim 34, wherein the B7R oligonucleotide primer set comprises two forward primers, the first forward primer having the sequence of SEQ ID NO:41, a sequence having at least 70% sequence identity with SEQ ID NO:41, or a sequence comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:41, and the second forward primer having the sequence of SEQ ID NO:42, a sequence having at least 70% sequence identity with SEQ ID NO:42, or a sequence comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:42.

36. The oligonucleotide combination according to claim 34, wherein the B7R oligonucleotide primer set comprises a B7R oligonucleotide probe, wherein said B7R probe binds specifically to SEQ ID NO:65 or a complement thereof.

37. The oligonucleotide combination according to claim 36, wherein said B7R probe is selected from the group consisting of SEQ ID NOs:09 and 35, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:09 or 35, an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:09 or 35.

38. The oligonucleotide combination according to claim 37, wherein said B7R probe is SEQ ID NO:35, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:35, or an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO 35.

39. The oligonucleotide combination according to claim 37, wherein said B7R probe is SEQ ID NO:09, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:09, or an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:09.

40. The oligonucleotide combination according to claim 34, wherein the B7R oligonucleotide primer set comprises two forward primers, the first forward primer having the sequence of SEQ ID NO:41, the second forward primer having the sequence of SEQ ID NO:42, a reverse primer having the sequence of SEQ ID NO:29, and a probe having the sequence of SEQ ID NO:09 or 35.

41. The oligonucleotide combination according to claim 34, wherein the B7R oligonucleotide primer set can be used for amplifying a target sequence in the B7R gene of a monkeypox virus (MPXV) providing an amplicon comprising at least the sequence of SEQ ID NO:66 of the B7R gene:tgtgtatgga agaagtaaca ccatattaca taatatatac aggctattta caagatgcac cgttatacag ttcgatgata tagtac ( SEQ ID NO : 66 ) .

42. The oligonucleotide combination according to claim 34, wherein the G2R oligonucleotide primer set comprises a G2R oligonucleotide probe, wherein said G2R probe binds specifically to SEQ ID NO:67 or a complement thereof.

43. The oligonucleotide combination according to claim 42, wherein said G2R probe is selected from the group consisting of SEQ ID NOs:15 and 47, an oligonucleotide having at least 70% sequence identity with SEQ ID NO: 15 or 47, an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:15 or 47.

44. The oligonucleotide combination according to claim 43, wherein said G2R probe is SEQ ID NO: 15, an oligonucleotide having at least 70% sequence identity with SEQ ID NO: 15, or an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO: 15.

45. The oligonucleotide combination according to claim 43, wherein said G2R probe is SEQ ID NO:47, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:47, or an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:47.

46. The oligonucleotide combination according to claim 34, wherein the G2R oligonucleotide primer set comprises a forward primer having the sequence of SEQ ID NO:05, a reverse primer having the sequence of SEQ ID NO: 30, and a probe having the sequence of SEQ ID NO: 15 or 47.

47. The oligonucleotide combination according to claim 34, wherein the G2R oligonucleotide primer set comprises a second G2R oligonucleotide probe.

48. The oligonucleotide combination according to claim 47, wherein said second G2R probe is selected from the group consisting of SEQ ID NOs:06, 39 and 62, an oligonucleotide having at least 70% sequence identity with SEQ ID NO: 06, 39, or 62, and an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO: 06, 39, or 62, and complements thereof.

49. The oligonucleotide combination according to claim 34, wherein the G2R oligonucleotide primer set can be used for amplifying a target sequence in the G2R gene of a monkeypoxvirus (MPXV) providing an amplicon comprising at least the sequence of SEQ ID NO:67 of the G2R gene: acatattctc acaccgtctc ttccacagat aaatgcgaac ccgtcgtaac cagcaataca tttaactata tcgatgtgga aattaacctg tatccagtca acgac ( SEQ ID NO : 67 ) .

50. The oligonucleotide combination according to claim 34 further comprising an E9L oligonucleotide primer set comprising a forward primer comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs:07, 17, 21, and 60, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:07, 17, 21, or 60, and an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:07, 17, 21, or 60, wherein the E9L oligonucleotide primer pair comprises a reverse primer comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs:01, 12, 40 and 61, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:01, 12, 40 or 61, and an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:01, 12, 40, or 61, and wherein the E9L oligonucleotide primer pair can be used for amplifying a target sequence in the E9L gene of a non-variola orthopoxvirus (OPXV).

51. The oligonucleotide combination according to claim 50, wherein the oligonucleotide primer set comprises an E9L oligonucleotide probe.

52. The oligonucleotide combination according to claim 51, wherein said E9L probe is selected from the group consisting of SEQ ID NOs:06, 39, and 62, an oligonucleotide having at least 70% sequence identity with SEQ ID NO: 06, 39, or 62, an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO: 06, 39, or 62, and complements thereof.

53. The oligonucleotide combination according to claim 52, wherein the E9L oligonucleotide primer pair comprises a forward primer comprising a nucleic acid sequence of SEQ ID NO:60, a reverse primer comprising a nucleic acid sequence of SEQ ID NO:61, and the E9L probe comprising a nucleic acid sequence of SEQ ID NO:62.

54. The oligonucleotide combination according to claim 34, wherein the combination comprises a B7R oligonucleotide primer set comprising two forward primers, the first forward primer having the sequence of SEQ ID NO:41, the second forward primer having thesequence of SEQ ID NO:42, a reverse primer having the sequence of SEQ ID NO:29, and a B7R probe having the sequence of SEQ ID NO:35 for amplifying a target sequence in the B7R gene of a monkeypox virus (MPXV); and wherein the oligonucleotide combination further comprises an E9L oligonucleotide primer set comprising a forward primer having the sequence of SEQ ID NO:60, a reverse primer having the sequence of SEQ ID NO:61, and an E9L probe having the sequence of SEQ ID NO: 62 for amplifying a target sequence in the E9L gene of a non-variola orthopoxvirus (OPXV).

55. The oligonucleotide combination according to claim 34, wherein the combination comprises a G2R oligonucleotide primer set comprising a forward primer having the sequence of SEQ ID NO:05, a reverse primer having the sequence of SEQ ID NO:30, and a G2R probe having the sequence of SEQ ID NO: 15 or 47 for amplifying a target sequence in the G2R gene of a monkeypox virus (MPXV); and wherein combination further comprises an E9L oligonucleotide primer set comprising a forward primer having the sequence of SEQ IDNO: 60, a reverse primer having the sequence of SEQ ID NO:61, and an E9L probe having the sequence of SEQ ID NO: 62 for amplifying a target sequence in the E9L gene of a non-variola orthopoxvirus (OPXV).

56. The oligonucleotide combination according to claim 34, wherein said oligonucleotide primer sets comprise one or more oligonucleotide probes, each probe comprising a detectable label.

57. The oligonucleotide combination according to claim 56, wherein the detectable label is a fluor ophore.

58. The oligonucleotide combination according to claim 56, wherein each probe comprises a quencher moiety.

59. An oligonucleotide primer set comprising a forward primer comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs:07, 17, and 21, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:07, 17, or 21, and an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:07, 17, or 21, a reverse primer comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs:01, 12, and 40, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:01, 12, or 40, and an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:01, 12, or 40, and wherein theoligonucleotide primer pair can be used for amplifying a target sequence in the E9L gene of a non-variola orthopoxvirus (OPXV).

60. The oligonucleotide primer set according to claim 59 comprising an oligonucleotide probe selected from the group consisting of SEQ ID NOs:06 and 39, an oligonucleotide having at least 70% sequence identity with SEQ ID NO:06 or 39, an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides of SEQ ID NO:06 or 39, and complements thereof.

61. The oligonucleotide primer set according to claim 59 comprising a forward primer having the sequence of SEQ ID NO: 07, a reverse primer having a nucleic acid sequence of SEQ ID NO: 12, and a probe having a nucleic acid sequence of SEQ ID NO:06.

62. A kit for detecting the presence of monkeypox virus (MPXV) in a biological sample comprising at least one of the following: i) the oligonucleotide combination according to any one of claims 34-58; and ii) the oligonucleotide primer set according to any one of claims 59-61.