TaqMan™-PCR for the detection of pathogenic E. coli strains
AssigneeBavarian Nordic Research Institute
InventorKlaus Pfeffer
Life of this patent
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Abstract
The present invention relates to a method for the detection of pathogenic E. coli in a sample comprising PCR amplification of DNA isolated from said sample using oligonucleotide primers specific for pathogenic E. coli.
Claims16
A method for detection and differentiation of pathogenic enterobacteria in a sample, said method comprising:
- isolating nucleic acid from said sample;
- adding a set of oligonucleotide primer pairs to said nucleic acid sample wherein said set of oligonucleotide primers comprises at least five oligonucleotide primer pairs wherein, wherein each primer pair specifically amplifies a DNA sequence of a virulence factor/toxin gene characteristic for one each of the subgroups of the pathogenic E. coli strains, said subgroups comprising enterotoxigenic, enteroaggregative, enteroinvasive, enteropathogenic and enterohemorrhagic E. coli strains and wherein for amplification of each subgroup at least one oligonucleotide primer pair is included in said set of oligonucleotide primer pairs, and wherein the set comprises a primer pair that hybridizes to an inv-plasmid of enteroinvasive E. coli and wherein said primer pair is El-1:
- 5″TTT CTG GAT GGT ATG GTG AGG 3′ (SEQ ID NO:
- 9) and E1-2:
- 5′CTT GAA CAT AAG GAA ATA AAC 3′ (SEQ ID NO:
- 10) which specifically amplifies a DNA sequence of an inv plasmid characteristic for enteroinvasive E. coli;
- and
- subjecting said nucleic acid and said set of primer pairs to an amplification process;
- and
- detecting the presence of at least one amplified product, wherein the presence of at least one amplified product indicates the presence of at least one pathogenic enterobactcria strain in said sample.
The method according to claim, wherein detecting the presence of at least one amplified product is performed using at least one oligonucleotide probe capable of hybridizing to the amplified product wherein said oligonucleotide probe is labeled at the 5′end with a fluorescent reporter dye and at the 3′ end with a fluorescent quencher dye and is susceptible to 5′-3′ exonuclease degradation by a polymerase, and wherein said amplification process uses a polymerase having 5′-3′ exonuclease degradation activity.
A set of oligonucleotide primer pairs useful for polymerase chain reaction (PCR) amplification of pathogenic enterobacteria allowing detection and differentiation of pathogenic enterobacteria in a sample wherein following amplification the presence of at least one amplified product indicates the presence of at least one pathogenic enterobacteria strain in said sample, wherein said set comprises at least five primer pairs, wherein each primer pair specifically amplifies a DNA sequence of a virulence factor/toxin gene characteristic for one each of the subgroups of the pathogenic E. coli strains, said subgroups comprising enterotoxigenic, enteroaggregative, enteroinvasive, enteropathogenic and enterohemorrhagic E. coli strains and wherein for amplification of each subgroup at least one oligonucleotide primer pair is included in said set of oligonucleotide primer pairs, and wherein the set comprises a primer pair that hybridizes to an inv-plasmid of enteroinvasive E. coli and wherein said primer pair is El-1:
- 5′TTT CTG GAT GGT ATG GTG AGG 3′ (SEQ ID NO:
- 9) and E1-2:
- 5′CTT GAA CAT AAG GAA ATA AAC 3′ (SEQ ID NO:
- 10).
A set of oligonucleotide primer pairs an a set of oligonucleotide primer probes useful for diagnosing an enterobacteria infection in samples derived from a living animal body including a human, by real time PCR method, wherein said sets of oligonucleotide primer pairs and oligonucleotide probes allow detection and differentiation of pathogenic enterobacteria in a sample, wherein said set of oligonucleotide primer pairs comprises at least five primer pairs, wherein each primer pair specifically amplifies a DNA sequence of a virulence factor/toxin gene characteristic for one each of the subgroups of the pathogenic E. coli strains, said subgroups comprising enterotoxigenic, enteroaggregative, enteroinvasive, enteropathogenic and enterohemorrhagic E. coli strains and wherein for amplification of each subgroup at least one oligonucleotide primer pair is included in said set of oligonucleotide primer pairs, and wherein the set comprises a primer pair that hybridizes to an inv-plasmid of enteroinvasive E. coli, wherein said primer pair is El-1:
- 5ΔTTT CTG GAT GGT ATG GTG AGG 3′ (SEQ ID NO:
- 9) and E1-2:
- 5′CTT GAA CAT AAG GAA ATA AAC 3′ (SEQ ID NO:
- 10).
Dependencies are inferred from the claim text.