Detection of Shiga Toxin Genes in Bacteria
The disclosed invention is related to methods, compositions and kits for targeting nucleic acid derived from Shiga toxin-producing bacteria such as E. coli . Compositions include amplification oligomers and/or detection probe oligomers. Kits and methods comprise at least one pair of amplification oligomers.
1 - 47 . (canceled)
48 . A method for identifying at least one of a stx1 gene and a stx2 gene in a sample, said method comprising:
a) contacting said sample with a pair of stx1-specific amplification oligomers and a pair of stx2-specific amplification oligomers, said pair of stx1-specific amplification oligomers comprising an oligomer pair selected from the group consisting of
(1-i) a first amplification oligomer comprising a target hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:30 and a second amplification oligomer comprising a target hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:31;
(1-ii) a first amplification oligomer comprising a target hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:1 and a second amplification oligomer comprising a target hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:2;
(1-iii) a first amplification oligomer comprising a target hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO: 8 and a second amplification oligomer comprising a target hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO: 9;
(1-iv) a first amplification oligomer comprising a target hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:12 and a second amplification oligomer comprising a target hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:13; and
(1-v) a first amplification oligomer comprising a target hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:19 and a second amplification oligomer comprising a target hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:20; and
said pair of stx2-specific amplification oligomers comprising an oligomer pair selected from the group consisting of
(2-i) a first amplification oligomer comprising a target hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:33 and a second amplification oligomer comprising a target hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:34;
(2-ii) a first amplification oligomer comprising a target hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:40 and a second amplification oligomer comprising a target hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:41;
(2-iii) a first amplification oligomer comprising a target hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO: 36 and a second amplification oligomer comprising a target hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO: 37; and
(2-iv) a first amplification oligomer comprising a target hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:47 and a second amplification oligomer comprising a target hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:48;
b) amplifying nucleic acid in said sample with said stx1-specific and/or stx2-specific pairs of amplification oligomers to obtain at least one amplification product; and
c) determining the sequence of said at least one amplification product or detecting said at least one amplification product using a stx1-specific detection probe and a stx2-specific detection probe
wherein the stx1-specific pair of amplification oligomers have the property of amplifying the stx1 gene at a concentration as low as 1 copy/μL; or
the stx2-specific pair of amplification oligomers have the property of amplifying the stx2 gene at a concentration below 7.5 copies/μL with no or very limited cross reactivity.
49 . The method of claim 48 , wherein each of said stx1-specific and stx2-specific detection probes is an oligomer having a length of from about 15 to about 30 contiguous oligomer residues.
50 . The method of claim 49 , wherein
(I) for the amplification oligomer pair of (1-i), said stx1-specific probe has at least 90% sequence identity to SEQ ID NO:32;
for the amplification oligomer pair of (1-ii), said stx1-specific probe has at least 90% sequence identity to SEQ ID NO:5;
for the amplification oligomer pair of (1-iii), said stx1-specific probe has at least 90% sequence identity to SEQ ID NO: 11;
for the amplification oligomer pair of (1-iv), said stx1-specific probe has at least 90% sequence identity to SEQ ID NO:14; or
for the amplification oligomer pair of (1-v), said stx1-specific probe has at least 90% sequence identity to SEQ ID NO:22;
and
(II) for the amplification oligomer pair of (2-i), said stx2-specific probe has at least 90% sequence identity to SEQ ID NO:35;
for the amplification oligomer pair of (2-ii), said stx2-specific probe has at least 90% sequence identity to SEQ ID NO:42;
for the amplification oligomer pair of (2-iii), said stx2-specific probe has at least 90% sequence identity to SEQ ID NO: 39; or
for the amplification oligomer pair of (2-iv), said stx2-specific probe has at least 90% sequence identity to SEQ ID NO:49.
51 . The method of claim 48 , wherein the stx1-specific and stx2-specific pairs of amplification oligomers are selected from the following combinations of stx1-specific and stx2-specific oligomer pairs:
(A) the amplification oligomer pairs of (1-i) and (2-i);
(B) the amplification oligomer pairs of (1-i) and (2-ii);
(C) the amplification oligomer pairs of (1-ii) and (2-i);
(D) the amplification oligomer pairs of (1-iv) and (2-iv);
(E) the amplification oligomer pairs of (1-v) and (2-i); and
(F) the amplification oligomer pairs of (1-v) and (2-iv).
52 . The method of claim 51 , wherein
for the combination of stx1-specific and stx2-specific oligomer pairs of (A), the stx1-specific probe has at least 90% sequence identity to SEQ ID NO:32 and the stx2-specific probe has at least 90% sequence identity to SEQ ID NO:35;
for the combination of stx1-specific and stx2-specific oligomer pairs of (B), the stx1-specific probe has at least 90% sequence identity to SEQ ID NO:32 and the stx2-specific probe has at least 90% sequence identity to SEQ ID NO:42;
for the combination of stx1-specific and stx2-specific oligomer pairs of (C), the stx1-specific probe has at least 90% sequence identity to SEQ ID NO:5 and the stx2-specific probe has at least 90% sequence identity to SEQ ID NO:35;
for the combination of stx1-specific and stx2-specific oligomer pairs of (D), the stx1-specific probe has at least 90% sequence identity to SEQ ID NO:14 and the stx2-specific probe has at least 90% sequence identity to SEQ ID NO:49;
for the combination of stx1-specific and stx2-specific oligomer pairs of (E), the stx1-specific probe has at least 90% sequence identity to SEQ ID NO:22 and the stx2-specific probe has at least 90% sequence identity to SEQ ID NO:35; or
for the combination of stx1-specific and stx2-specific oligomer pairs of (F), the stx1-specific probe has at least 90% sequence identity to SEQ ID NO:22 and the stx2-specific probe has at least 90% sequence identity to SEQ ID NO:49.
53 . The method of claim 48 , wherein said sample comprises bacterial nucleic acid originating from Escherichia coli, Citrobacter freundii, Aeromononas hydrophila, Aeromononas caviae , or Enterobacter cloacae.
54 . The method of claim 53 , wherein said sample comprises bacterial nucleic acid originating from a strain of Escherichia coli.
55 . The method of claim 54 , wherein said strain of Escherichia coli is E. coli 0157:H7.
56 . The method of claim 48 , wherein said amplification step is performed using a polymerase chain reaction.
57 . The method of claim 56 , wherein said polymerase chain reaction is a real-time polymerase chain reaction.
58 . The method of claim 48 , wherein step c) comprises detecting said at least one amplification product using the stx1-specific and stx2-specific detection probes.
59 . The method of claim 58 , wherein each of the stx1-specific and stx2-specific detection probes is a fluorescence probe comprises a fluorescent dye compound.
60 . The method of claim 58 , wherein each of the stx1-specific and stx2-specific detection probes is a fluorescence probe comprises a fluorescent dye compound and a non-fluorescent quenching dye compound.
61 . A primer set for amplification of at least one of a stx1 gene and a stx2 gene in a sample, said primer set comprising:
a pair of stx1-specific amplification oligomers and a pair of stx2-specific amplification oligomers, said pair of stx1-specific amplification oligomers comprising an oligomer pair selected from the group consisting of
(1-i) a first amplification oligomer comprising a target hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:30 and a second amplification oligomer comprising a target hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:31;
(1-ii) a first amplification oligomer comprising a target hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:1 and a second amplification oligomer comprising a target hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:2;
(1-iii) a first amplification oligomer comprising a target hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO: 8 and a second amplification oligomer comprising a target hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO: 9;
(1-iv) a first amplification oligomer comprising a target hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:12 and a second amplification oligomer comprising a target hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:13; and
(1-v) a first amplification oligomer comprising a target hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:19 and a second amplification oligomer comprising a target hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:20; and
said pair of stx2-specific amplification oligomers comprising an oligomer pair selected from the group consisting of
(2-i) a first amplification oligomer comprising a target hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:33 and a second amplification oligomer comprising a target hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:34;
(2-ii) a first amplification oligomer comprising a target hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:40 and a second amplification oligomer comprising a target hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:41;
(2-iii) a first amplification oligomer comprising a target hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO: 36 and a second amplification oligomer comprising a target hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO: 37; and
(2-iv) a first amplification oligomer comprising a target hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:47 and a second amplification oligomer comprising a target hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:48.
62 . The primer set of claim 61 , wherein the stx1-specific and stx2-specific pairs of amplification oligomers are selected from the following combinations of stx1-specific and stx2-specific oligomer pairs:
(A) the amplification oligomer pairs of (1-i) and (2-i);
(B) the amplification oligomer pairs of (1-i) and (2-ii);
(C) the amplification oligomer pairs of (1-ii) and (2-i);
(D) the amplification oligomer pairs of (1-iv) and (2-iv);
(E) the amplification oligomer pairs of (1-v) and (2-i); and
(F) the amplification oligomer pairs of (1-v) and (2-iv).
63 . A primer-probe set for identification of at least one of a stx1 gene and a stx2 gene in a sample, said primer-probe set comprising:
a pair of stx1-specific amplification oligomers and a pair of stx2-specific amplification oligomers, said pair of stx1-specific amplification oligomers comprising an oligomer pair selected from the group consisting of
(1-i) a first amplification oligomer comprising a target hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:30 and a second amplification oligomer comprising a target hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:31;
(1-ii) a first amplification oligomer comprising a target hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:1 and a second amplification oligomer comprising a target hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:2;
(1-iii) a first amplification oligomer comprising a target hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO: 8 and a second amplification oligomer comprising a target hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO: 9;
(1-iv) a first amplification oligomer comprising a target hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:12 and a second amplification oligomer comprising a target hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:13; and
(1-v) a first amplification oligomer comprising a target hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:19 and a second amplification oligomer comprising a target hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:20; and
said pair of stx2-specific amplification oligomers comprising an oligomer pair selected from the group consisting of
(2-i) a first amplification oligomer comprising a target hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:33 and a second amplification oligomer comprising a target hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:34;
(2-ii) a first amplification oligomer comprising a target hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:40 and a second amplification oligomer comprising a target hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:41;
(2-iii) a first amplification oligomer comprising a target hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO: 36 and a second amplification oligomer comprising a target hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO: 37; and
(2-iv) a first amplification oligomer comprising a target hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:47 and a second amplification oligomer comprising a target hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:48;
a stx1-specific detection probe hybridizable to a stx1 gene region located between the regions of hybridization of said pair of stx1-specific amplification oligomers; and
a stx2-specific detection probe hybridizable to a stx2 gene region located between the regions of hybridization of said pair of stx2-specific amplification oligomers;
wherein the stx1-specific detection probe and/or the stx2-specific detection probe comprises a non-nucleotide detectable label; and
(i) the amplification oligomers have the property of amplifying the stx1 gene at a concentration as low as 1 copy/μL; or
(ii) the pair of amplification oligomers have the property of amplifying the stx2 gene at a concentration below 7.5 copies/μL with no or very limited cross reactivity.
64 . The primer-probe set of claim 63 , wherein each of said stx1-specific and stx2-specific detection probes is an oligomer having a length of from about 15 to about 30 or to about 40 contiguous oligomer residues.
65 . The primer-probe set of claim 64 , wherein
(I) for the amplification oligomer pair of (1-i), said stx1-specific probe has at least 90% sequence identity to SEQ ID NO:32;
for the amplification oligomer pair of (1-ii), said stx1-specific probe has at least 90% sequence identity to SEQ ID NO:5;
for the amplification oligomer pair of (1-iii), said stx1-specific probe has at least 90% sequence identity to SEQ ID NO: 11;
for the amplification oligomer pair of (1-iv), said stx1-specific probe has at least 90% sequence identity to SEQ ID NO:14; or
for the amplification oligomer pair of (1-v), said stx1-specific probe has at least 90% sequence identity to SEQ ID NO:22;
and
(II) for the amplification oligomer pair of (2-i), said stx2-specific probe has at least 90% sequence identity to SEQ ID NO:35;
for the amplification oligomer pair of (2-ii), said stx2-specific probe has at least 90% sequence identity to SEQ ID NO:42;
for the amplification oligomer pair of (2-iii), said stx2-specific probe has at least 90% sequence identity to SEQ ID NO: 39; or
for the amplification oligomer pair of (2-iv), said stx2-specific probe has at least 90% sequence identity to SEQ ID NO:49.
66 . The primer-probe set of claim 63 , wherein the stx1-specific and stx2-specific pairs of amplification oligomers are selected from the following combinations of stx1-specific and stx2-specific oligomer pairs:
(A) the amplification oligomer pairs of (1-i) and (2-i);
(B) the amplification oligomer pairs of (1-i) and (2-ii);
(C) the amplification oligomer pairs of (1-u) and (2-i);
(D) the amplification oligomer pairs of (1-iv) and (2-iv);
(E) the amplification oligomer pairs of (1-v) and (2-i); and
(F) the amplification oligomer pairs of (1-v) and (2-iv).
67 . The primer-probe set of claim 66 , wherein
for the combination of stx1-specific and stx2-specific oligomer pairs of (A), the stx1-specific probe has at least 90% sequence identity to SEQ ID NO:32 and the stx2-specific probe has at least 90% sequence identity to SEQ ID NO:35;
for the combination of stx1-specific and stx2-specific oligomer pairs of (B), the stx1-specific probe has at least 90% sequence identity to SEQ ID NO:32 and the stx2-specific probe has at least 90% sequence identity to SEQ ID NO:42;
for the combination of stx1-specific and stx2-specific oligomer pairs of (C), the stx1-specific probe has at least 90% sequence identity to SEQ ID NO:5 and the stx2-specific probe has at least 90% sequence identity to SEQ ID NO:35;
for the combination of stx1-specific and stx2-specific oligomer pairs of (D), the stx1-specific probe has at least 90% sequence identity to SEQ ID NO:14 and the stx2-specific probe has at least 90% sequence identity to SEQ ID NO:49;
for the combination of stx1-specific and stx2-specific oligomer pairs of (E), the stx1-specific probe has at least 90% sequence identity to SEQ ID NO:22 and the stx2-specific probe has at least 90% sequence identity to SEQ ID NO:35; or
for the combination of stx1-specific and stx2-specific oligomer pairs of (F), the stx1-specific probe has at least 90% sequence identity to SEQ ID NO:22 and the stx2-specific probe has at least 90% sequence identity to SEQ ID NO:49.
68 . The primer-probe set of claim 63 , further comprising an internal control system for verifying reaction conditions, said system comprising a control template polynucleotide, a pair of control amplification oligomers, and a control probe.
69 - 70 . (canceled)
71 . A kit for amplification of at least one of a stx1 gene and a stx2 gene, said kit comprising the primer set of claim 61 .
72 . A kit for identification of at least one of a stx1 gene and a stx2 gene, said kit comprising the primer-probe set of claim 63 .
73 . The method of claim 48 , wherein the stx1-specific pair of amplification oligomers have the property of amplifying the stx1 gene at a concentration as low as 1 copy/μL; and
the stx2-specific pair of amplification oligomers have the property of amplifying the stx2 gene at a concentration below 7.5 copies/μL with no or very limited cross reactivity.
74 . The primer-probe set of claim 63 , wherein the amplification oligomers have the property of amplifying the stx1 gene at a concentration as low as 1 copy/μL; and
wherein the oligomer probe comprises a non-nucleotide detectable label, wherein the pair of amplification oligomers have the property of amplifying the stx2 gene at a concentration below 7.5 copies/μL with no or very limited cross reactivity.