Sequences and their use for detection and characterization of STEC bacteria
This invention relates to a rapid method for detection and characterization of STEC bacteria based on the presence of nucleic acid sequences, in particular, to a PCR-based method for detection, and to oligonucleotide molecules and reagents and kits useful therefore. This method is preferably employed to detect STEC bacteria in a food or water sample, such as a beef enrichment. The present invention further relates to isolated polynucleotides, replication compositions, kits, and reagent tablets for carrying out the method of the present invention.
1. A method for detecting the presence of STEC bacteria in a sample, said sample comprising nucleic acids, said method comprising:
(a) providing a reaction mixture comprising a first primer, a second primer, and a probe for amplification and detection of at least a portion of:
(i) SEQ ID NO: 688; wherein each of said first primer for the amplification of SEQ ID NO: 688, second primer for the amplification of SEQ ID NO: 688, and probe for the detection of SEQ ID NO: 688 comprises a 5′ end and a 3′ end; wherein said first primer for the amplification of SEQ ID NO: 688 comprises at least 15 contiguous nucleotides of SEQ ID NO: 145 or a sequence complementary thereto; and wherein said probe for the detection of SEQ ID NO: 688 comprises at least 15 contiguous nucleotides of SEQ ID NO: 160 or a sequence complementary thereto; and
(ii) one or more nucleic acid sequences selected from the group consisting of:
(A) SEQ ID NO: 686; wherein each of said first primer for the amplification of SEQ ID NO: 686, second primer for the amplification of SEQ ID NO: 686, and probe for the detection of SEQ ID NO: 686 comprises a 5′ end and a 3′ end; and wherein said first primer for the amplification of SEQ ID NO: 686 and probe for the detection of SEQ ID NO: 686 are selected from the group consisting of:
(I) a first primer comprising at least 15 contiguous nucleotides of SEQ ID NO: 1 or a sequence complementary thereto and a probe comprising at least 15 contiguous nucleotides of SEQ ID NO: 16 or a sequence complementary thereto;
(II) a first primer comprising at least 15 contiguous nucleotides of SEQ ID NO: 1 or a sequence complementary thereto and a probe comprising at least 15 contiguous nucleotides of SEQ ID NO: 49 or a sequence complementary thereto; and
(III) a first primer comprising SEQ ID NO: 55 or a sequence complementary thereto and probe comprising SEQ ID NO: 56 or a sequence complementary thereto; and
(B) SEQ ID NO: 687; wherein each of said first primer for the amplification of SEQ ID NO: 687, second primer for the amplification of SEQ ID NO: 687, and probe for the detection of SEQ ID NO: 687 comprises a 5′ end and a 3′ end; and wherein said first primer for the amplification of SEQ ID NO: 687 and probe for the detection of SEQ ID NO: 687 are selected from the group consisting of:
(I) a first primer comprising at least 15 contiguous nucleotides of SEQ ID NO: 74 or a sequence complementary thereto and a probe comprising at least 15 contiguous nucleotides of SEQ ID NO: 89 or a sequence complementary thereto;
(II) a first primer comprising SEQ ID NO: 120 or a sequence complementary thereto and a probe comprising SEQ ID NO: 121 or 122 or a sequence complementary thereto; and
(III) a first primer comprising SEQ ID NO: 125 or a sequence complementary thereto and probe comprising SEQ ID NO: 126 or a sequence complementary thereto; and
(b) performing PCR amplification of said nucleic acids of said sample using the reaction mixture of step (a); and
(c) detecting the amplification of step (b).
2. The method of claim 1 , wherein said second primer for the amplification of SEQ ID NO: 688 comprises a nucleic acid sequence comprising at least 15 contiguous nucleotides of SEQ ID NO: 191 or a sequence complementary thereto.
3. The method of claim 2 , wherein said first primer for the amplification of SEQ ID NO: 688 comprises a sequence selected from the group consisting of SEQ ID NOs: 146-159, said second primer for the amplification of SEQ ID NO: 688 comprises a sequence selected from the group consisting of SEQ ID NOs: 192-205, and said probe for the detection of SEQ ID NO: 688 comprises a sequence selected from the group consisting of SEQ ID NOs: 161-175.
4. The method of claim 1 , wherein the 3′ end of said probe for the detection of SEQ ID NO: 688 is directly or indirectly attached to the 5′ end of said first primer for the amplification of SEQ ID NO: 688 forming a primer-probe complex for the amplification and detection of SEQ ID NO: 688, and wherein said primer-probe complex for the amplification and detection of SEQ ID NO: 688 is detectably labeled.
5. The method of claim 4 , wherein said reaction mixture for the amplification and detection of SEQ ID NO: 688 further comprises a quencher oligonucleotide comprising at least 15 contiguous nucleotides of SEQ ID NO: 176.
6. The method of claim 1 , wherein said second primer for the amplification of SEQ ID NO: 686 comprises a nucleic acid sequence comprising at least 15 contiguous nucleotides of SEQ ID NO: 58 or comprising SEQ ID NO: 73.
7. The method of claim 6 , wherein said first primer for the amplification of SEQ ID NO: 686 comprises a sequence selected from the group consisting of SEQ ID NOs: 2-15, 48; said second primer for the amplification of SEQ ID NO: 686 comprises a sequence selected from the group consisting of SEQ ID NOs: 59-72; and said probe for the detection of SEQ ID NO: 686 comprises a sequence selected from the group consisting of SEQ ID NOs: 17-30, 49-52.
8. The method of claim 1 claim, wherein the 3′ end of said probe for the detection of SEQ ID NO: 686 is directly or indirectly attached to the 5′ end of said first primer forming a primer-probe complex for the amplification and detection of SEQ ID NO: 686, and wherein said primer-probe complex for the amplification and detection of SEQ ID NO: 686 is detectably labeled.
9. The method of claim 8 , wherein said reaction mixture for the amplification and detection of SEQ ID NO: 686 further comprises a quencher oligonucleotide comprising SEQ ID NO: 53, 54, or 57, or comprising at least 15contiguous nucleotides of SEQ ID NO: 31.
10. The method of claim 1 , wherein said second primer for the amplification of SEQ ID NO: 687 comprises a nucleic acid sequence comprising at least 15 contiguous nucleotides of SEQ ID NO: 128 or comprising SEQ ID NO: 143 or 144.
11. The method of claim 10 , wherein said first primer for the amplification of SEQ ID NO: 687 comprises a sequence selected from the group consisting of SEQ ID NOs: 75-88; said second primer for the amplification of SEQ ID NO: 687 comprises a sequence selected from the group consisting of SEQ ID NOs: 129-144; and said probe for the detection of SEQ ID NO: 687 comprises a sequence selected from the group consisting of SEQ ID NOs: 90-104.
12. The method of claim 1 , wherein the 3′ end of said probe for the detection of SEQ ID NO: 687 is directly or indirectly attached to the 5′ end of said first primer for the amplification of SEQ ID NO: 687 forming a primer-probe complex for the amplification and detection of SEQ ID NO: 687, and wherein said primer-probe complex for the amplification and detection of SEQ ID NO: 687 is detectably labeled.
13. The method of claim 12 , wherein said reaction mixture for the amplification and detection of SEQ ID NO: 687 further comprises a quencher oligonucleotide comprising SEQ ID NO: 123, 124, or 127, or comprising at least 15 contiguous nucleotides of SEQ ID NO: 105.
14. The method of claim 1 , wherein the sample comprises a food sample or a water sample.