Sequences and their use for detection and characterization of
This invention relates to a rapid method for detection and characterization of Escherichia coli bacteria serotype O157:H7 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 E. coli O157:H7 in a food or water sample, such as a beef enrichment. The present invention further relates to replication compositions and kits for carrying out the method of the present invention.
1. A method for detecting the presence of E. coli O157:H7 in a sample, said sample comprising nucleic acids, said method comprising:
(a) providing a reaction mixture comprising a primer pair selected from the group consisting of primer pair SEQ ID NO:1 and SEQ ID NO:2, primer pair SEQ ID NO:3 and SEQ ID NO:4, primer pair SEQ ID NO:5 and SEQ ID NO:6, primer pair SEQ ID NO:7 and SEQ ID NO:8, and a combination thereof;
(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), whereby a positive detection of amplification indicates the presence of E. coli O157:H7 in the sample.
2. The method of claim 1 , wherein said reaction mixture further comprises a nucleic acid probe.
3. The method of claim 2 , wherein said nucleic acid probe comprises SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or a combination thereof.
4. The method of claim 3 , wherein said probe further comprises a detectable label.
5. The method of claim 4 , wherein said reaction mixture further comprises a blocking oligonucleotide capable of quenching said detectable label of said probe, said blocking oligonucleotide comprising SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or a combination thereof.
6. The method of claim 5 , wherein said reaction mixture comprises primer pair SEQ ID NO:1 and SEQ ID NO:2, probes SEQ ID NO:9 and SEQ ID NO:10, and blocking oligonucleotides SEQ ID NO:14 and SEQ ID NO:15.
7. The method of claim 1 , wherein the sample comprises a food sample or a water sample.
8. An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18, wherein said polynucleotide comprises a primer region and a probe region, and wherein said polynucleotide further comprises a non-amplifiable linker joining said primer region to said probe region.
9. The isolated polynucleotide of claim 8 , wherein said non-amplifiable linker is an 18-carbon non-amplifiable linker, and wherein said polynucleotide further comprises a detectable label.
10. A replication composition for use in performance of PCR, comprising:
(a) a primer pair selected from the group consisting of primer pair SEQ ID NO:1 and SEQ ID NO:2, primer pair SEQ ID NO:3 and SEQ ID NO:4, primer pair SEQ ID NO:5 and SEQ ID NO:6, primer pair SEQ ID NO:7 and SEQ ID NO:8, and a combination thereof; and
(b) a nucleic acid probe comprising a detectable label.
11. The replication composition of claim 10 further comprising a blocking oligonucleotide selected from the group consisting of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, and a combination thereof.
12. The replication composition of claim 11 , wherein said nucleic acid probe comprises SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or a combination thereof.
13. The replication composition of claim 12 , wherein said replication composition comprises primer pair SEQ ID NO:1 and SEQ ID NO:2, probes SEQ ID NO:9 and SEQ ID NO:10, and blocking oligonucleotides SEQ ID NO:14 and SEQ ID NO:15.
14. A kit for detection of E. coli O157:H7 in a sample, comprising the replication composition of claim 10 .
15. A tablet comprising the replication composition of claim 10 .