TaqMan™-PCR for the detection of pathogenic E. coli...

Details TaqMan™-PCR for the detection of pathogenic E. coli... TaqMan™-PCR for the detection of pathogenic E. coli...

2000-01-26

2003-12-16

Benzion, Gary (Department: 1634)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

C435S006120, C435S091200, C536S024320, C536S024300, C536S023100

Reexamination Certificate

active

06664080

ABSTRACT:

The present invention relates to a rapid, high performance assay for the detection of pathogenic
E. coli
which is based on TaqMan™ PCR technique, and to specific optimised oligonucleotide primers and labelled oligonucleotide probes useful in the assay.
BACKGROUND OF THE INVENTION
Enterohemorrhagic, shiga-like toxin (sit) producing
Escherchia coli
(EHEC) have recently been recognized as an important human and animal pathogen (1-7). EHEC has been responsible for several food-borne outbreaks (8). The most notable were a multistate outbreak associated with a fast food chain in the western states of the USA with more than 600 individuals affected and 3 deaths in Washington (9), and an epedemic occurence in Japan with more than 6000 patients and approx. 8 fatal cases (10). Infection with EHEC causes diarrhea, hemorrhagic colitis, thrombotic thrombocytopenic purpura, and hemolytic uremic syndrome (HUS) that is characterised by acute renal failure, thrombocytopenia, and microangiopathic hemolytic anemia. HUS ultimately can result in a fatal outcome in affected children and immunocompromised individuals (3,11-17). Recently, in the South-Eastern parts of Germany (Bavaria) an increase of EHEC cases was reported during October 1995 and July 1996 with at least 45 severe infections leading to HUS accompanied by 7 deaths (18). Estimating that approx. 1 out of 15 EHEC infections results in HUS approx. 600-700 affected individuals might be assumed.
In most outbreaks reported, consumption of contaminated ground beef has been the source of infection (5,8,19-22), whereas in Japan raddish sprouts are suspected (10). EHEC has been isolated from cow milk (6,19,23), water (19), chicken, pork, and apple cider (19,24,25), but also human horizontal smear infections have been reported (15). Cattle appear likely to be the reservoir (22,26). Cross contamination, improper handling, and inadequate cooking all contribute to food-borne infections caused by EHEC. EHEC produce Shiga-like toxins (slt), also known as verotoxins or cytotoxins (12,27). A large proportion of EHEC have been found to belong to the serogroup O157:H7, but notably, also a variety of EHEC belonging to other serogroups (O22, O26, O55, O111, O114, O145) have been reported especially in Europe (12,15,28-32).
Besides EHEC, certain other strains of
E. coli
can cause enteritis or gastroenteritis and are grouped in enterotoxigenic strains (ETEC) (33-36), enteropathogenic strains (EPEC) (37), enteroinvasive strains (EIEC) (38,39), and enteroaggregative strains (EaggEC) (40,41). These strains are important pathogens and also pose severe public health problems. The diagnosis of these pathogens is vastly neglegted due to the lack of specific and sensitive routine test methods. ETEC synthesize heat labile and/or heat stable enterotoxins that can cause a secretory diarrhea (“traveller's diarrhea”) resembling that of
Vibrio cholerae
(36,42,43). Surface attachment of the ETEC organisms to the intestinal epithelial cell is a prerequisite to toxin production. Toxin production is plasmid mediated and most commonly involves
E. coli
serogroups O6, O15, O124, O136, O143, O145, and 0147 (32). EPEC cause diarrheal symptoms primarily in infants (32). Although the pathogenesis is unclear, the epithelial degradation of the gut, and the inflammatory response that are observed in tissue sections may be a consequence due to the adhesive properties of the bacterium. Specific attachment factors of EPEC are plasmid encoded (EAF=EPEC adherence factor) (37,44). EHEC often contain an adherence factor closely related to EAF that is known as ene (EHEC attaching and effacing gene) (45,46). EPEC most often belong to serogroups O6, O8, O25, O111, O119, and O142 (32).
EIEC strains are capable of penetrating and invading the intestinal epithelial cells and produce an inflammatory diarrhea similar to that caused by Shigella bacteria (38,47,48). Fecal smears contain blood, mucus and segmented neutrophils. EIEC contain virulence plasmids coding for additional pathogenic factors (48). Serogroups O28, O112, O115, O124, O136, O143, O145, and O147 are most commonly found on EIEC (32).
EaggEC are associated with persistent diarrhea in children and with traveller's diarrhea. EaggEC are characterized by their adherence capacity that leads to aggregation of Hep-2 cells. This effect is associated with the presence of a virulence plasmid (pCVD432). EaggEC are supected to also produce a heat stable enterotoxin (EAST1) (49-53). They can belong to serogroups O44 and O126 (32).
Conventional detection methods for EHEC encompass enrichment and isolation with selective and/or indicator media such as
E. coli
broth, lauryl sulfate tryptose 4-methylumbelliferyl-b-acid broth, eosin methylene blue agar, McConkey sorbitol agar, and enterohemolysin agar (28,32,54-59). All of these assays, unfortunately, are indirect and lack the ability to identify EHEC or the other pathogenic
E. coli
strains specifically. Several methods for biochemical identification and immunological detection of EHEC have been put forward (54,60-63), however, it is well recognized that pathogenic
E. coli
strains neither posess nor lack unique fermentation pathways (58,64). Serotyping is not conclusive since no absolute correlation between serotype and pathogenic
E. coli
group can be established (12,27,32,58,65). DNA hybridization techniques have been established for experimental research but are not applicable for large scale routine diagnostic procedures (66,67). DNA amplification based assays, using PCR have been reported (68-72). Limitations to these methods include cumbersome post-PCR detection methods (agarose gel electrophoresis, Biotin/Avidin based ELISA detection systems).
To overcome these problems, a PCR assay which allows the specific determination of virulence factors characteristic for EHEC, ETEC, EPEC, EIEC, and EaggEC that is based on a fluorigenic detection method of PCR amplification has been developed.
This assay exploits the 5′→3′ exonuclease activity of Taq-DNA polymerase (73) to cleave an internal oligonucleotide probe that is covalently conjugated with a fluorescent reporter dye (e.g. 6-carboxy-fluorescein [FAM]; &lgr;
em
=518 nm) and a fluorescent quencher dye (6-carboxytetram-ethyl-rhodamine [TAMRA]; &lgr;
em
=582 nm) at the 5′ and 3′ end, respectively (74,75). Fluorescence from FAM is efficiently quenched by TAMRA on the same, intact probe molecule (76). In the case that cognate PCR amplification occurs, Taq polymerase extends from the specific PCR primer and cleaves the internal, fluorigenic oligonucleotide probe annealed to the template strand. Thus, the reporter dye and the quencher dye get spatially separated. As a consequence of oligonucleotide hydrolysis and physical separation of the reporter and the quencher dyes, a measurable increase in fluoresecence intensity at 518 nm can be observed. PCR cycling leads to exponential amplification of the PCR product and consequently of fluorescence intensity.
TAQMAN™-PCR is performed in optical tubes that allow measurements of fluorescence signals without opening the PCR tubes. This dramatically minimizes post-PCR processing time and almost completely eliminates cross-PCR contamination problems. Employing this approach, simultaneous testing of biological materials for the presence of virulence genes of Lcoh strains and other enterobacteria, harboring virulence genes can be semiautomated and performed within 18 h.
According to the present invention Real Time PCR (e.g., TAQMAN™ PCR) for the detection of pathogenic
E. coli
is provided, enabling for the first time the specific, rapid and high throughput routine detection of EHEC, ETEC, EPEC, EIEC, and EaggEC and related enterobacteria that harbor these virulence genes in routine bacteriological laboratories.
OBJECT OF THE INVENTION
It is an object of the present invention to provide a rapid, high performance assay for the detection and identification of pathogenic
E. coli
in biological samples.
It is a further object of

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