GENETIC MARKERS AND METHODS FOR Similarly. Tommassen et al. (WO 9011370) disclose poly
THE DETECTION OF ESCHERICHIA COLI nucleotide probes and methods for the identification and
SEROTYPE-0157:H7 detection of gram-positive bacteria. The method of Tommassen et al. relies on probes corresponding to relatively
FIELD OF INVENTION 5 short fragments of the outer membrane protein OmpA.
known to be highly conserved throughout gram-positive
The invention relates to the field of molecular biology and genera Aflas et al. (EP 517154) teach a nucleic acid hybrid
the use of randomly amplified nucleic acid fragments for the ization method for me detection of Giardia sp. based on
selection of genetic markers useful in the identification of designing probes with sequences complementary to regions
bacteria. More specifically, the invention relates to a specific of me gene encoding the giardin protein. Webster et al. (U.S.
DNA marker sequence useful for the detection of E. coli Pat No 4,7x7,653) has expanded upon the use of rRNA in
serotype 0157:H7 and use of that diagnostic marker to disclosing a method for the characterization of bacteria
determine if an unknown bacterium is a member of the based on ,he comparison 0f the chromatographic pattern of
0157:H7 serotype. restriction endonuclease-digested DNA from the unknown
BACKGROUND 15 or8anism witn equivalent chromatographic patterns of at
least 2 known different organism species. The digested DNA
Central to the field of microbiology is the ability to has been hybridized or reassociated with ribosomal RNA positively identify microorganisms at the level of genus, information-containing nucleic acid from, or derived from a species or serotype. Correct identification is not only an known probe organism. The method of Webster et al. essential tool in the laboratory, but it plays a significant role 2Q effectively establishes a unique bacterial nucleic acid "finin the control of microbial contamination in the processing gerprint" corresponding to a particular bacterial genus of food stuffs, the production of agricultural products, and against which unknown "fingerprints" are compared, the monitoring of environmental media such as ground Similar methods have been use for the detection of E. coli water. Increasing stringency in regulations which apply to 0157:H7. For example. Samadour (/. Clin. Microbiol. microbial contamination have resulted in a corresponding 2J (1995), 33(8). 2150-4) teaches the detection of E. coli increase in industry resources which must be dedicated to 0157:H7 by restriction fragment length polymorphism using contamination monitoring. Shiga-like toxin genes which are conserved between the
Of greatest concern is the detection and control of patho- 0157:H7 serotype and shigella. Similarly. Ramotar et al. (J.
genie microorganisms. Although a broad range of microor- Clin. Microbiol. (1995), 33(3). 519-24) and Fratamico et al.
ganisms have been classified as pathogenic, attention has 30 (J. Clin. Microbiol. (1995). 33(8), 2188-91) teach PGR
primarily focused on a few bacterial groupings such as basedmethods for the detection of conserved 0157 :H7 genes
Escherichia, Salmonella, Listeria and Clostridia. Typically. encoding either shiga-like toxins or verotoxins.
pathogen identification has relied on methods for distin- The methods described above are useful for the detection
guishing phenotypic aspects such as growth or motility 0f bacteria, but each relies upon knowledge of a gene,
characteristics, and for immunological and serological char- 35 protein, or other specific sequence known a priori to be
acteristics. Selective growth procedures and immunological highly conserved throughout a specific bacterial group. An
methods are the traditional methods of choice for bacterial alternative method would involve a nontargeted analysis of
identification and these can be effective for the presumptive bacterial genomic DNA for specific non-phenotypic genetic
detection of a large number of species within a particular markers common to all species of that bacteria. For example,
genus. However, these methods are time consuming and are 40 genetic markers based on single point mutations may be
subject to error. Selective growth methods require culturing detected by differentiating DNA banding patterns from
and subculturing in selective media, followed by subjective restriction enzyme analysis. As restriction enzymes cut DNA
analysis by an experienced investigator. Immunological at specific sequences, a point mutation within this site results
detection (e.g., ELISA) is more rapid and specific, however, in the loss or gain of a recognition site, giving rise in that
it still requires growth of a significant population of organ- 45 region to restriction fragments of different length. Mutations
isms and isolation of the relevant antigens. For these reasons caused by the insertion, deletion or inversion of DNA
interest has turned to detection of bacterial pathogens on the stretches will also lead to a length variation of DNA restric
basis of nucleic acid sequence. tion fragments. Genomic restriction fragments of different
It is well known, for example, that nucleic acid sequences lengths between genotypes can be detected on Southern
associated with the ribosomes of bacteria are often highly 50 blots (Southern. J. MoL Biol. 98, 503, (1975)). The genomic
conserved across genera and are therefore useful for iden- DNA is typically digested with any restriction enzyme of
tification (Webster, U.S. Pat. No. 4,717,653 and U.S. Pat choice, the fragments are electrophoretically separated, and
No. 5.087,558; Enns, Lab. Med., 19, 295, (1988); then hybridized against a suitably labelled probe for detec
Mordarski. Soc. Appl. Bacteriol. Tech. Ser., 20 (Chem. tion. The sequence variation detected by this method is
Methods Bad. Syst), 41. (1985)). Weisburg et al. (EP 55 known as restriction length polymorphism or RFLP
51736) disclose a method for the detection and identification (Botstein et al.. Am. J. Hum. Genet. 342,314, (1980)). RFLP
of pathogenic microorganisms involving the PCR amplifi- genetic markers are particularly useful in detecting genetic
cation and labeling of a target nucleotide for hybridization to variation in phenotypically silent mutations and serve as
16S rDNA of E. coli. Lane et al. (WO 9015157) teach highly accurate diagnostic tools.
universal nucleic acid probes that hybridize to conserved «) Another method of identifying genetic polymorphic
regions of 23S or 16S rRNA of eubacteria. markers employs DNA amplification using short primers of
Although bacterial ribosomal nucleic acids contain highly arbitrary sequence. These primers have been termed "ran
conserved sequences, they are not the only sources of base dom amplified polymorphic DNA" or "RAPD" primers (see
sequence conservation that is useful for microorganism Williams et al., Nucl. Acids. Res., 18,6531 (1990) and U.S.
identification. Wheatcroft et al. (CA 2055302) describe the 65 Pat. No. 5,126,239; also EP 0 543 484 A2, WO 92/07095,
selection of transposable elements, flanked by unique DNA WO 92/07948. WO 92/14844, and WO 92/03567). The
sequences, for the detection of various Rhizobium strains. RAPD method amplifies either double or single stranded
