Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-08-04
2001-07-31
Brusca, John S. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007320, C435S091200
Reexamination Certificate
active
06268143
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the fields of microbiology and molecular biology of
E. coli
. More specifically, the present invention relates to the development and evaluation of the sensitivity and specificity of a PCR-based 5′ nuclease assay for presumptively detecting
E.coli
O157:H7 DNA and the uses of such assay.
2. Description of the Related Art
Enterohemorrhagic
Escherichia coli
O157:H7 is an important human pathogen that is predominantly associated with hemorrhagic colitis and the more severe complications of hemolytic uremic syndrome. Although human to human transmission of
E. coli
O157:H7 has been demonstrated (25), most infections have been associated with the consumption of contaminated ground beef, milk, water, produce, and apple juice products that have been improperly handled, stored, or cooked (1,5,7,15,18,19,25,43). The primary reservoir is believed to be cattle (24,27). However, a clear understanding of the farm ecology of
E. coli
O157:H7 is lacking, partly because of the low detected prevalence in individual cattle and herds (20,40,55) and the low infectious dosage required for human infections (25,53).
Established methods for recovering and identifying
E. coli
O157:H7 from foods and feces have been hindered by the inability to specifically and rapidly detect small numbers of organisms from complex matrices and background microflora. Although the inclusion of pre-enrichment incubations and immunomagnetic separation (IMS) (6,9,11,16,22,31,44,48) and additional selective subculturing or secondary enrichment incubations (12,14,30) have been reported to increase the detection rate of
E. coli
O157:H7 from foods and fecal specimens, these methods are dependent on isolating individual colonies from selective and/or indicator media and then characterizing them in immunological and biochemical/fermentation reactions. Immunological assays are used to determine if the O157 somatic and H7 flagellar antigens are present, while the biochemical/fermentation reactions determine the genus and species of the isolate in classical taxonomic fashion. Combined with the initial replication steps in the isolation process, the current
E. coli
O157:H7 identification process takes upwards of 5 or more days to complete. This adds considerably to the costs required to determine whether a sample contains
E. coli
O157:H7, and is a limiting factor in doing more
E. coli
O157:H7 tests.
Rapid methods for identifying
E. coli
O157:H7 in foods or fecal specimens have been directed at immunological or genetic targets. Antigenic targets have included the
E. coli
somatic (O157) or flagellar (H7) antigens (21,50); and two low-molecular-weight antigens (30,45); and the virulence-associated Shiga-like toxins (SLT) type I and II (3,17,33). However, these assays are occasionally unable to distinguish certain other
E. coli
strains from
E. coli
O157:H7 strains (30,49) and/or toxigenic from nontoxigenic
E. coli
O157 strains (46).
PCR-based detection procedures have been used to identify
E. coli
O157:H7 and have targeted the sltI and sltII genes (32,47,54); the EHEC uidA gene (10); and a portion of a 60-MDa plasmid (23). Because similar genes are present in some nonpathogenic
E. coli
and in other bacteria, individual PCRs that target these genes are unable to confirm the identity of an isolate as
E. Coli
O157:H7. Identification by PCR requires that multiple genes be targeted in separate PCRs on the DNA from a suspect organism or that the DNA from that organism be subjected to a multiplex PCR that targets the multiple genes simultaneously (10,23,42,51).
Presumptive identification of
E. coli
O157:H7 is possible in an individual, non-multiplexed PCR, if the reaction targets the EHEC eaeA gene. In separate studies targeting two different regions of the eaeA gene, every
E. coli
O157:H7 reference strain evaluated demonstrated the predicted PCR product. Louie et al. (36) targeted the 3′ end of EHEC eaeA gene, whereas Meng et al. (41) amplified a 633 bp product upstream of the 5′ end of the EHEC eaeA gene using PCR primers SZ-I and SZ-II. Both reactions were limited as confirmatory PCRs for identifying
E. coli
O157:H7, because similar PCR products were evident with some
E. coli
O157:NM strains and some enteropathogenic
E. coli
O55:H7 and 055:NM strains (36,41).
Although PCR can amplify DNA molecules thousands-fold, the specifically amplified product must be detected in order to prove its presence and a variety of methods have been developed. The most common research application is by gel electrophoresis, however, it does not show specificity of the PCR product and lacks sensitivity. Southern blots or dot blot hybridizations with probes will demonstrate specificity of the PCR, but they require multi-step processing and add considerable time and expense to the detection process. Neither of these PCR detection processes is conducive to rapid, high-throughput, automated PCR detection schemes.
Recently, 5′ nuclease assays (TaqMan™, PE Applied Biosystems, Foster City, Calif.) have been described that allow the automated PCR amplification, detection, and analysis of Salmonella spp. (13,38);
Listeria monocytogenes
(2,4); and SLT genes (28,54) in various foods. The 5′ nuclease assay exploits the 5′→43′ exonuclease activity of
Thermus aquaticus
DNA polymerase (29,37) to hydrolyze an internal TaqMan™ probe labeled with a fluorescent reporter dye and a quencher dye (34). For the intact probe, the quencher dye suppresses the fluorescent emission of the reporter dye because of its spatial proximity on the probe. During PCR, the probe anneals to the target amplicon and is hydrolyzed during extension by the Taq DNA polymerase. The hydrolysis reduces the quenching effect and allows for an increase in emission of the reporter fluorescence. This increase is a direct consequence of a successful PCR, whereas the emission of the quencher dye remains constant irrespective of amplification.
Because development of fluorogenic reporter signals occurs only with a successful PCR, detection of specific DNA sequences can be based on monitoring for an increase in reporter fluorescence following PCR with a fluorometer (ABI Prism™ Sequence Detection System, PE Applied Biosystems). The fluorometric data can be automatically read and interpreted using a 96-sample format and presented as “yes” or “no” conclusions as to the presence or absence of the DNA within 15 minutes of the completion of the PCR.
The prior art is deficient in the lack of fully automated and high sample throughput PCR-based
E. coli
O157:H7 amplification/detection system. The present invention fulfills this long-standing need and desire in the art.
SUMMARY OF THE INVENTION
The present invention describes the development and evaluation of the sensitivity and specificity of a 5′ nuclease assay for amplifying and presumptively detecting
E. coli
O157:H7 DNA. Evaluation of the 5′ nuclease assay included the comparison of two DNA extraction procedures for recovering
E. coli
O157:H7 DNA from pure broth cultures; from pure broth cultures immediately before IMS and then retrieved on sorbitol MacConkey agar supplemented with cefeximine and tellurite (CT-SMAC); from pure broth cultures following immunomagnetic separation step (IMS) plus 18 h secondary enrichment and then retrieved on CT-SMAC; and from spiked broth cultures containing ground beef.
The present invention is directed to a method of presumptively detecting
E. coli
O157:H7 using SZ-primed, eaeA-based 5′ nuclease assay, comprising the steps of collecting a sample suspected to contain
E. coli
O157:H7; preparing an enrichment broth culture of the sample; extracting DNA from the culture; amplifying the DNA by PCR using
E coli
O157:H7-specific primers; detecting the sample for
E. coli
O157:H7 by adding a fluorogenic probe specific for
E. coli
O157:H7 to the same reaction tube where the DNA was amplified; and determining the presence or absence
Adler Benjamin Aaron
Brusca John S.
Kansas State University Research Foundation
Kim Young
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