US2009068651A1PendingUtilityA1
Method for the detection of the antibiotic resistance spectrum of mycobacterium species
Est. expiryJun 9, 2014(expired)· nominal 20-yr term from priority
C12Q 1/689C12Q 2600/156
64
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Claims
Abstract
Method for the detection of the antibiotic resistance spectrum of Mycobacterium species present in a sample, possibly coupled to the identification of the Micobacterium species involved, comprising the steps of: (i) if need be releasing, isolating or concentrating the polynucleic acids present in the sample; (ii) if need be amplifying the relevant part of the antibiotic resistance genes present in said sample with at least one suitable primer pair; (iii) hybridizing the polynucleic acids of step (i) or (ii) with at least one of the rpo B gene probes, as specified in table 2, under the appropriate hybridization and wash conditions; (iv) detecting the hybrids formed in step (iii); (v) inferring the Mycobacterium antibiotic resistance spectrum, and possibly the Mycobacterium species involved from the differential hybridization signal(s) obtained in step (iv).
Claims
exact text as granted — not AI-modified1 - 27 . (canceled)
28 . An isolated oligonucleotide selected from the group consisting of:
P1
(SEQ ID NO: 30),
P5
(SEQ. ID NO: 33),
P3
(SEQ ID NO: 31),
P4
(SEQ ID NO: 32),
P7
(SEQ ID NO: 41),
P8
(SEQ ID NO: 42),
MT-POL-2
(SEQ ID NO: 24),
MT-POL-3
(SEQ ID NO: 25),
MT-POL-4
(SEQ ID NO: 26),
MT-POL-5
(SEQ ID NO: 27),
S55C
(SEQ ID NO: 44),
S55M
(SEQ ID NO: 45),
SIL-1
(SEQ ID NO: 13),
R1
(SEQ ID NO: 46),
R2B
(SEQ ID NO: 47),
R2C
(SEQ ID NO: 48),
R3
(SEQ ID NO: 49),
R4A
(SEQ ID NO: 15),
R44A
(SEQ ID NO: 16),
R4B
(SEQ ID NO: 18),
R44B
(SEQ ID NO: 19),
R4C
(SEQ ID NO: 50),
R4D
(SEQ ID NO: 51),
R4E
(SEQ ID NO: 52),
R5
(SEQ ID NO: 21),
R5B
(SEQ ID NO: 53),
R5C
(SEQ ID NO: 54),
MGRPO-1
(SEQ ID NO: 64),
MGRPO-2
(SEQ ID NO: 65),
MGRPO-3
(SEQ ID NO: 66),
MGRPO-4
(SEQ ID NO: 67),
MP-POL-1
(SEQ ID NO: 28),
MA-POL-1
(SEQ ID NO: 29),
MS-POL-1
(SEQ ID NO: 38),
MK-POL-1
(SEQ ID NO: 55),
MI-POL-1
(SEQ ID NO: 68),
ML-POL-1
(SEQ ID NO: 57),
ML-S1
(SEQ ID NO: 58),
ML-S2
(SEQ ID NO: 59),
ML-S3
(SEQ ID NO: 60),
ML-S4
(SEQ ID NO: 61),
ML-S5
(SEQ ID NO: 62),
ML-S6
(SEQ ID NO: 63),
any M. paratuberculosis specific probe derived from the sequence of the rpoB gene fragment of M. paratuberculosis represented by SEQ ID NO:35,
any M. avium specific probe derived from the sequence of the rpoB gene fragment of M. avium represented by SEQ ID NO:36,
any M. scrofulaceum specific probe derived from the sequence of the rpoB gene fragment of M. scrofulaceum represented by SEQ ID NO:37,
any M. kansasii specific probe derived from the sequence of the rpoB gene fragment of M. kansasii represented by SEQ ID NO:56,
any MAC-strain specific probe derived from the sequence of the rpoB gene fragment of a MAC-strain represented by SEQ ID NO:69, and the RNA form of said SEQ ID NOs where T is replaced by U, and the complementary form of said SEQ ID NOs.
29 . A composition comprising at least one isolated oligonucleotide selected from the group consisting of:
P1
(SEQ ID NO: 30),
P5
(SEQ. ID NO: 33),
P3
(SEQ ID NO: 31),
P4
(SEQ ID NO: 32),
P7
(SEQ ID NO: 41),
P8
(SEQ ID NO: 42),
MT-POL-2
(SEQ ID NO: 24),
MT-POL-3
(SEQ ID NO: 25),
MT-POL-4
(SEQ ID NO: 26),
MT-POL-5
(SEQ ID NO: 27),
S55C
(SEQ ID NO: 44),
S55M
(SEQ ID NO: 45),
SIL-1
(SEQ ID NO: 13),
R1
(SEQ ID NO: 46),
R2B
(SEQ ID NO: 47),
R2C
(SEQ ID NO: 48),
R3
(SEQ ID NO: 49),
R4A
(SEQ ID NO: 15),
R44A
(SEQ ID NO: 16),
R4B
(SEQ ID NO: 18),
R44B
(SEQ ID NO: 19),
R4C
(SEQ ID NO: 50),
R4D
(SEQ ID NO: 51),
R4E
(SEQ ID NO: 52),
R5
(SEQ ID NO: 21),
R5B
(SEQ ID NO: 53),
R5C
(SEQ ID NO: 54),
MGRPO-1
(SEQ ID NO: 64),
MGRPO-2
(SEQ ID NO: 65),
MGRPO-3
(SEQ ID NO: 66),
MGRPO-4
(SEQ ID NO: 67),
MP-POL-1
(SEQ ID NO: 28),
MA-POL-1
(SEQ ID NO: 29),
MS-POL-1
(SEQ ID NO: 38),
MK-POL-1
(SEQ ID NO: 55),
MI-POL-1
(SEQ ID NO: 68),
ML-POL-1
(SEQ ID NO: 57),
ML-S1
(SEQ ID NO: 58),
ML-S2
(SEQ ID NO: 59),
ML-S3
(SEQ ID NO: 60),
ML-S4
(SEQ ID NO: 61),
ML-S5
(SEQ ID NO: 62), and
ML-S6
(SEQ ID NO: 63).
30 . A composition of isolated oligonucleotides for the amplification of the rpoB gene fragment of M. tuberculosis comprising at least one isolated oligonucleotide selected from the group consisting of:
P1
(SEQ ID NO: 30),
P5
(SEQ ID NO: 33),
P3
(SEQ ID NO: 31),
P4
(SEQ ID NO: 32),
P7
(SEQ ID NO: 41), and
P8
(SEQ ID NO: 42)
31 . A composition for the amplification of the rpoB gene fragment in mycobacteria other than M. tuberculosis comprising at least one isolated oligonucleotide selected from the group consisting of:
MGRPO-1
(SEQ ID NO: 64),
MGRPO-2
(SEQ ID NO: 65),
MGRPO-3
(SEQ ID NO: 66), and
MGRPO-4
(SEQ ID NO: 67).
32 . A kit for the detection of the resistance to rifampicin of Mycobacterium tuberculosis present in a biological sample comprising the following components:
(i) at least one rpoB gene fragment selected from the group consisting of:
S55C
(SEQ ID NO: 44),
S55M
(SEQ ID NO: 45),
(ii) a hybridization buffer, or components necessary for producing said buffer; and
(iii) a wash solution, or components necessary for producing said solution.
33 . The kit according to claim 32 , further enabling Mycobacterium species determination, and further comprising at least one isolated oligonucleotide selected from the group consisting of:
MT-POL-2
(SEQ ID NO: 24),
MT-POL-3
(SEQ ID NO: 25),
MT-POL-4
(SEQ ID NO: 26),
MT-POL-5
(SEQ ID NO: 27),
MP-POL-1
(SEQ ID NO: 28),
MA-POL-1
(SEQ ID NO: 29),
MS-POL-1
(SEQ ID NO: 38),
MK-POL-1
(SEQ ID NO: 55),
MI-POL-1
(SEQ ID NO: 68), and
ML-POL-1
(SEQ ID NO: 57).
34 . The kit according to claim 32 , for the simultaneous detection of M. tuberculosis and its resistance to rifampicin, wherein
(i) said rpoB gene fragment of component (i) is selected from the group of consisting of:
S55C
(SEQ ID NO: 44),
S55M
(SEQ ID NO: 45),
wherein
(ii) said kit further comprises at least one additional rpoB gene fragment selected from the group consisting of:
MT-POL-2
(SEQ ID NO: 24),
MT-POL-3
(SEQ ID NO: 25),
MT-POL-4
(SEQ ID NO: 26), and
MT-POL-5
(SEQ ID NO: 27).
35 . A kit for the detection of the resistance to rifampicin of Mycobacterium leprae present in a biological sample comprising the following components:
(i) at least one rpoB gene fragment selected from the group consisting of:
ML-S1
(SEQ ID NO: 58),
ML-S2
(SEQ ID NO: 59),
ML-S3
(SEQ ID NO: 60),
ML-S4
(SEQ ID NO: 61),
ML-S5
(SEQ ID NO: 62), and
ML-S6
(SEQ ID NO: 63)
(ii) a hybridization buffer, or components necessary for producing said buffer; and
(iii) a wash solution, or components necessary for producing said solution.
36 . The kit according to claim 35 , further enabling Mycobacterium species determination, and further comprising at least one additional rpoB gene fragment selected from the group consisting of:
MT-POL-2
(SEQ ID NO: 24),
MT-POL-3
(SEQ ID NO: 25),
MT-POL-4
(SEQ ID NO: 26),
MT-POL-5
(SEQ ID NO: 27),
MP-POL-1
(SEQ ID NO: 28),
MA-POL-1
(SEQ ID NO: 29),
MS-POL-1
(SEQ ID NO: 38),
MK-POL-1
(SEQ ID NO: 55),
MI-POL-1
(SEQ ID NO: 68), and
ML-POL-1
(SEQ ID NO: 57).
37 . The kit according to claim 35 , for the simultaneous detection of M. leprae and its resistance to rifampicin, wherein the rpoB gene fragment of component (i) is selected from the group consisting of:
ML-S1
(SEQ ID NO: 58),
ML-S2
(SEQ ID NO: 59),
ML-S3
(SEQ ID NO: 60),
ML-S4
(SEQ ID NO: 61),
ML-S5
(SEQ ID NO: 62), and
ML-S6
(SEQ ID NO: 63),
in combination with the rpoB gene fragment ML-POL-1 (SEQ ID NO:57).
38 . A method for the identification of a mycobacterial species and the simultaneous detection of its resistance to rifampicin, comprising the simultaneous use of two rpoB gene fragments, wherein the first rpoB gene fragment is selected from the group consisting of:
S55C
(SEQ ID NO: 44),
S55M
(SEQ ID NO: 45),
SIL-1
(SEQ ID NO: 13),
R1
(SEQ ID NO: 46),
R2B
(SEQ ID NO: 47),
R2C
(SEQ ID NO: 48),
R3
(SEQ ID NO: 49),
R4A
(SEQ ID NO: 15),
R44A
(SEQ ID NO: 16),
R4B
(SEQ ID NO: 18),
R44B
(SEQ ID NO: 19),
R4C
(SEQ ID NO: 50),
R4D
(SEQ ID NO: 51),
R4E
(SEQ ID NO: 52),
R5
(SEQ ID NO: 21),
R5B
(SEQ ID NO: 53), and
R5C
(SEQ ID NO: 54),
and wherein the second rpoB gene fragment is selected from the group consisting of:
MT-POL-2
(SEQ ID NO: 24),
MT-POL-3
(SEQ ID NO: 25),
MT-POL-4
(SEQ ID NO: 26),
MT-POL-5
(SEQ ID NO: 27),
MP-POL-1
(SEQ ID NO: 28),
MA-POL-1
(SEQ ID NO: 29),
MS-POL-1
(SEQ ID NO: 38),
MK-POL-1
(SEQ ID NO: 55),
MI-POL-1
(SEQ ID NO: 68),
ML-POL-1
(SEQ ID NO: 57),
ML-S1
(SEQ ID NO: 58),
ML-S2
(SEQ ID NO: 59),
ML-S3
(SEQ ID NO: 60),
ML-S4
(SEQ ID NO: 61),
ML-S5
(SEQ ID NO: 62), and
ML-S6
(SEQ ID NO: 63).
39 . The isolated oligonucleotide according to claim 28 , wherein said isolated oligonucleotide is a probe or a primer.
40 . The composition according to claim 29 , wherein said isolated oligonucleotide is a probe or a primer.
41 . The composition according to claim 30 , wherein said isolated oligonucleotide is a primer.
42 . The kit according to claim 32 , wherein said at least one rpoB gene fragment is an S-probe.
43 . The kit according to claim 33 , wherein said isolated oligonucleotide is a probe or a primer.
44 . The kit according to claim 34 , wherein said rpoB gene fragment and said additional rpoB gene fragment are independently a probe or a primer.
45 . The kit according to claim 35 , wherein said rpoB gene fragment is a ML-S probe.
46 . The kit according to claim 36 , wherein said additional rpoB gene fragment is a probe or a primer.
47 . The kit according to claim 37 , wherein said rpoB gene fragment is a probe or a primer.
48 . The kit according to claim 31 , wherein said isolated oligonucleotide is a probe or a primer.Join the waitlist — get patent alerts
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