Sequences and their use for detection and characterization of <i>E. coli </i>O157:H7

摘要

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.

主权项

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.