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. A method for identifying a stx1 gene in a sample, the method comprising:
a) contacting the sample with a pair of amplification oligomers comprising a first amplification oligomer and a second amplification oligomer, wherein
the first amplification oligomer comprises a target hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO: 30, and
the second amplification oligomer comprises a target hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO: 31, and the pair of amplification oligomers amplify the stx1 gene at a concentration as low as 5.08 copies per microliter;
b) amplifying nucleic acid in the sample with the pair of amplification oligomers to obtain an amplification product; and
c) determining the sequence of the amplification product or detecting the amplification product using a detection probe oligomer comprising a fluorescent dye compound, wherein the detection probe oligomer comprises 10 to 40 contiguous nucleotides that specifically hybridize to the amplicon generated by the pair of amplification oligomers.
2. The method of claim 1 , wherein the sample comprises bacterial nucleic acid originating from Escherichia coli, Citrobacter freundii, Aeromononas hydrophila, Aeromononas caviae , or Enterobacter cloacae.
3. The method of claim 2 , wherein the detection probe oligomer further comprises a non-fluorescent quenching dye compound.
4. The method of claim 1 , wherein the detection probe oligomer comprises a length of contiguous nucleotides selected from the group consisting of: from 15 to 30 continuous nucleotides.
5. The method of claim 3 , wherein the detection probe oligomer comprises a length of contiguous nucleotides selected from the group consisting of: from 15 to 30 continuous nucleotides.
6. The method of claim 1 , wherein the sequence of the first amplification oligomer consists of SEQ ID NO: 30.
7. A method for identifying a stx1 gene in a sample, the method comprising:
a) contacting the sample with a pair of amplification oligomers comprising a first amplification oligomer and a second amplification oligomer, wherein
the sequence of the first amplification oligomer consists of SEQ ID NO: 30, and
the sequence of the second amplification oligomer consists of SEQ ID NO: 31;
b) amplifying nucleic acid in the sample with the pair of amplification oligomers to obtain an amplification product; and
c) determining the sequence of the amplification product or detecting the amplification product using a detection probe oligomer comprising a fluorescent dye compound, wherein the detection probe oligomer comprises 10 to 40 contiguous nucleotides that specifically hybridize to the amplicon generated by the pair of amplification oligomers.
8. The method of claim 1 , wherein the sequence of the second amplification oligomer consists of SEQ ID NO: 31.
9. The method of claim 7 , wherein the sequence of the detection probe oligomer consists of SEQ ID NO: 32.
10. A primer pair and a detection probe oligomer for generating from a stx1 gene an amplicon of approximately 130 bp useful for identifying the presence of a stx1 gene in a sample, the primer pair comprising a first amplification oligomer and a second amplification oligomer, wherein
the sequence of the first amplification oligomer consists of SEQ ID NO: 30, and
the sequence of the second amplification oligomer consists of SEQ ID NO:31;
the detection probe oligomer comprises 10 to 40 contiguous nucleotides that specifically hybridize to the amplicon generated by the pair of amplification oligomers, and further comprises a fluorescent dye compound.
11. A reaction mixture for generating and detecting from a stx1 gene an amplicon of approximately 130 bp useful for identifying the presence of a stx1 gene in a sample, wherein the reaction mixture comprises a first amplification oligomer, a second amplification oligomer and a detection probe oligomer, wherein
the sequence of the first amplification oligomer consists of SEQ ID NO: 30,
the sequence of the second amplification oligomer consists of SEQ ID NO: 31;
the detection probe oligomer comprises 10 to 40 contiguous nucleotides that specifically hybridize to the amplicon generated by the pair of amplification oligomers, and further comprises a fluorescent dye compound.
12. The reaction mixture of claim 11 , wherein the sequence of the detection probe oligomer consists of SEQ ID NO: 32.