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 suitable primer pairs for amplification of at least a portion of
(i) two or more E. coli O157:H7 genomic DNA regions within the pO157 portion of the E. coli O157:H7 genome, wherein said primer pairs are suitable for amplification of SEQ ID NO: 2 and SEQ ID NO: 3, wherein said primer pair for amplification of the nucleic acid region of SEQ ID NO: 2 comprises SEQ ID NO: 10 and SEQ ID NO: 12, wherein said primer pair for amplification of the nucleic acid region of SEQ ID NO: 3 comprises SEQ ID NO:15 and SEQ ID NO:16, and wherein each of said primers comprising SEQ ID NO:10 and SEQ ID NO:15 further comprise a detectable label, and
(ii) one or more E. coli O157:H7 genomic DNA regions outside the pO157 portion of the E. coli O157:H7 genome, wherein said primer pair is suitable for amplification of SEQ ID NO: 1, and wherein said primer pair for amplification of SEQ ID NO: 1 comprises SEQ ID NO:4 and SEQ ID NO:5, and wherein said primer comprising SEQ ID NO:4 further comprises a detectable label;
(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 in (a)(i) and (a)(ii) indicates the presence of E. coli O157:H7 in the sample.
2. The method of claim 1 , wherein said reaction mixture further comprises a blocking oligonucleotide capable of quenching said detectable label of said primer comprising SEQ ID NO:10, said blocking oligonucleotide comprising SEQ ID NO:11.
3. The method of claim 1 , wherein the sample comprises a food sample or a water sample.
4. A replication composition for use in performance of PCR, comprising:
(a) a primer pair comprising nucleic acid sequences SEQ ID NO:4 and SEQ ID NO:5;
(b) two or more primer pairs comprising nucleic acid sequences (i) SEQ ID NO:10 and SEQ ID NO:12 and (ii) SEQ ID NO:15 and SEQ ID NO:16; and
(b) thermostable DNA polymerase.
5. The replication composition of claim 4 further comprising a blocking oligonucleotide SEQ ID NO:11.
6. A replication composition for use in performance of PCR, comprising:
(a) a primer pair comprising nucleic acid sequences SEQ ID NO:5 and SEQ ID NO:6;
(b) two or more primer pairs comprising nucleic acid sequences (i) SEQ ID NO:12 and SEQ ID NO:13 and (ii) SEQ ID NO:16 and SEQ ID NO:17; and
(b) thermostable DNA polymerase.
7. The replication composition of claim 6 further comprising one nucleic acid probe for each primer pair selected, wherein said nucleic acid probes comprise SEQ ID NO:7, SEQ ID NO:14, and SEQ ID NO:18.
8. A kit for detection of E. coli O157:H7 in a sample, comprising the replication composition of claim 4 .
9. A kit for detection of E. coli O157:H7 in a sample, comprising the replication composition of claim 6 .
10. A tablet comprising the replication composition of claim 4 .
11. A tablet comprising the replication composition of claim 6 .