Detection of Shiga Toxin Genes in Bacteria

Abstract

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.